BLOCKADE OF THE OGF-OGFR AXIS ENHANCES REPAIR …
Transcript of BLOCKADE OF THE OGF-OGFR AXIS ENHANCES REPAIR …
The Pennsylvania State University
The Graduate School
Department of Neural and Behavioral Sciences
BLOCKADE OF THE OGF-OGFR AXIS ENHANCES REPAIR PROCESSES
IN DIABETIC SKIN AND BONE
A Dissertation in
Anatomy
by
Michelle B. Titunick
©2018 Michelle B. Titunick
Submitted in Partial Fulfillment of the Requirements
for the Degree of
Doctor of Philosophy
December 2018
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The dissertation of Michelle B. Titunick was reviewed and approved* by the following: Patricia J. McLaughlin Professor of Neural and Behavioral Sciences Director of the Graduate Program in Anatomy Dissertation Advisor Chair of Committee Ian S. Zagon Distinguished Professor of Neural and Behavioral Sciences Gregory S. Lewis Assistant Professor of Orthopaedics and Rehabilitation Christopher Niyibizi Associate Professor of Orthopaedics and Rehabilitation *Signatures are on file in the Graduate School.
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ABSTRACT
Almost 9.5% of the U.S. population had diabetes in 2015 with 5-10% having type 1
diabetes. In addition to diabetic complications such as retinopathy, nephropathy,
neuropathy, and cardiovascular disease, there is also poor wound healing and delayed
fracture healing. Preclinical studies from our laboratory have demonstrated that topical
naltrexone (NTX) is an effective and safe treatment for diabetic complications such as
corneal keratopathy, dry eye, and non-healing ulcers. Naltrexone is an opioid receptor
antagonist that, at an appropriate dosage, blocks the opioid growth factor (OGF)-opioid
growth factor receptor (OGFr) axis resulting in increased cell proliferation. [Met5]-
enkephalin, or OGF, is a highly conserved endogenous opioid peptide that tonically
regulates cell proliferation by delaying the G0/G1 phase of the cell cycle.
Both systemic and topical NTX have been shown to enhance corneal epithelial wound
healing in normal and diabetic rats, diabetic rabbits, as well as the type 2 diabetic model,
db/db. Topical NTX reverses dry eye in diabetic animal models, and increases collagen
formation and angiogenesis in cutaneous wounds of diabetic rats and mice. Phase 1
clinical trials with topical NTX administered to the eye have reported tolerability and
safety of the therapy; preclinical pathology studies confirm the lack of toxicity following
sustained topical application.
The underlying cause of delayed healing of bone fractures in diabetes is unclear. Both
preclinical and clinical studies report elevated levels of enkephalins in diabetics
suggesting that the increase in OGF, an inhibitory growth factor, may suppress cell
replication and contribute to poor wound and fracture healing. There is limited
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information on the presence and role of the OGF-OGFr pathway in normal bone growth
or in the process of fracture repair. Furthermore, the ability to manipulate the OGF-OGFr
axis with naltrexone during the process of bone fracture repair is unknown.
This dissertation hypothesizes that topical naltrexone to block the OGF-OGFr
regulatory pathway is an effective modulator of diabetic complications related to delays
cutaneous wound healing and bone fracture repair. The first aim tested the hypothesis
that topical naltrexone enhances closure of full-thickness cutaneous wounds in type 1
diabetic rats at a rate comparable to standard of care- Regranex®. In aims 2 and 3, studies
were conducted to investigate the role of the OGF-OGFr axis in diabetic bone. Studies
were designed to determine whether diabetes is associated with a dysregulation of the
OGF-OGFr pathway that subsequently changes bone composition in a type 1 diabetic rat
model, and whether blockade of the pathway alters the rate of repair of diabetic bone.
Lastly, studies were conducted to test the hypothesis that systemic naltrexone blockade
of the OGF-OGFr pathway in diabetic animal models may protect against the
complications related to bone fracture and repair.
Male Sprague Dawley rats (Charles River Laboratories) were used throughout all the
experiments and type 1 diabetes was induced by injections of streptozotocin. In aim 1
topical application of NTX accelerated the rate of closure of 6mm full thickness cutaneous
wounds at a rate comparable to a daily application of the standard of care Regranex®.
Analyses of the skin revealed that naltrexone treatment increased DNA synthesis, as well
as expression of platelet-derived growth factor and vascular endothelial growth factor,
required for the granulation tissue formation and angiogenesis.
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In the second series of experiments, OGF expression was detected at a comparable
level in bones from normal rats aged 1 day to adult. OGFr expression was detected in the
femur at all ages, and expression was elevated at days 1 and 21. OGF and OGFr expression
were increased in type 1 diabetic rat bone relative to normal, non-diabetic bone. Serum
levels of OGF were also increased in diabetic rats.
Histological analyses of femurs from normal and type 1 diabetic rats revealed no
significant differences in the number of osteoclasts, but did indicate with safranin O
staining a decrease in calcified cartilage in diabetic rats. In comparison to normal bone
composition, Ki67 staining showed a decrease in proliferative cells, and VEGF staining
revealed a decrease in vascularity in diabetic bone. Seven days following fracture, the
callus was examined by radiography and histology. Calluses in normal femurs had more
cartilage than in diabetic bones, and more granulation tissue was evident in diabetic bones
in comparison to diabetic bones treated with naltrexone. Bone tissue treated with
naltrexone from diabetic rats displayed elevated levels of Ki67 staining relative to tissue
treated with vehicle suggesting that topical application of naltrexone may accelerate early
phases of bone repair. Serum OGF levels increased in DB rats with fractures treated with
vehicle compared to the serum of DB rats without a fracture, suggesting fracture or
fracture repair may increase serum enkephalin levels. NTX-treated rats had decreased
serum OGF levels relative to that of diabetic vehicle-treated rats.
Assessment of diabetic bone composition in animals treated systemically with
either saline or naltrexone revealed that naltrexone-treated bones appeared to be stronger
and absorbed more energy than vehicle-treated diabetic bones. No significant differences
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between the 3 groups were seen in trabecular μCT measurements. Diabetic bone treated
with either vehicle or naltrexone had significantly lower cross-sectional areas and cortical
areas. No gross morphological differences were detected after 21 days of naltrexone
therapy.
In summary, naltrexone is effective in enhancing full-thickness cutaneous wound
healing and early phases of fracture repair, and is able to change mechanical properties of
diabetic bone. Naltrexone is comparable to the current standard of care for non-healing
wounds, Regranex®, and has been proven to be safe in multiple tolerability studies. Local
NTX can decrease granulation tissue in diabetic fracture calluses and increase cartilage and
bone volume. Systemic naltrexone can increase diabetic bone strength and reduce OGFr
expression.
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TABLE OF CONTENTS LIST OF FIGURES ………………………………..……………………………………………………………. xi LIST OF TABLES …………………………………..………………………………………………………….. xii ABBREVIATIONS .………………………………..………………………………………………………….. xiii ACKNOWLEDGEMENTS .……………………….………………………………………………………… xvi
CHAPTER 1: INTRODUCTION …………………………………………………………………………. 1 1.1. Diabetes ……………….………………………………………………………………………… 2
1.2. Animal Models ……………………………………………………………………………….. 2 1.3. Cutaneous Wound Healing ……………………….……………………………………. 5
1.3.1. Cellular and Molecular Basis of Full-Thickness Wound Healing ……… 5 1.3.1.1. Hemostasis ....………………………………………………………………. 5 1.3.1.2. Inflammation ..……………….…………………………………………….. 6 1.3.1.3. Proliferation ....……………………………………………………………… 7 1.3.1.4. Remodeling ….……………………………………………………………… 8 1.3.2. Cutaneous Healing Complications, Amputation, and Diabetes ……… 8 1.3.3. Current Treatments in Wound Healing …………………………………………. 9 1.4. Bone Growth and Remodeling ……………………………………………………….. 12 1.4.1. Cellular and Molecular Basis of Fracture Healing …………………………… 12 1.4.1.1. Inflammation ……………………………………………………………….. 12 1.4.1.2. Soft Callus Formation …………………………………………………… 13 1.4.1.3. Hard Callus Formation …………….…………………………………… 14 1.4.1.4. Bone Remodeling ……………………………………….………………… 15 1.4.2. Bone Healing Complications and Fractures in the Diabetic
Population ………………………………………………………………………………………………. 15
1.4.3. Current Treatments in Fracture Repair ………………………………………….. 18 1.5. Opioids …………………………………………..………………………………………………. 19 1.6. The OGF-OGFr Axis ..………………………………………..……………………………… 21 1.7. Opioid Receptor Antagonists ..……………………………………………………..… 21 1.7.1. Naltrexone …………………………………………………………………………………….. 21 1.7.1.1. Function of Naltrexone Blockade Actions ……………………… 22 1.7.2. Naloxone ……………………………………………………………………………………….. 24 1.8. Current Gap in Knowledge ..……………………………………………………………. 26 1.9. Hypothesis and Specific Aims .………………………………………………………… 26 1.10. References ………..………………………..…………………………………………………… 28
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CHAPTER 2: TOPICAL NALTREXONE IS A SAFE AND EFFECTIVE ALTERNATIVE TO STANDARD TREATMENT OF DIABETIC WOUNDS ………………………………………
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2.1. Abstract ……………………………………………………………………………………………….. 54 2.1.1. Objective ……………………………………………………………………………………….. 54 2.1.2. Approach ………………………………………………………………………………………. 54 2.1.3. Results …………………………………………………………………………………………… 54 2.1.4. Innovation and Conclusion ……..…………………………………………………….. 55 2.2. Introduction ………………………………………………………………..……………………… 55 2.3. Clinical Problem Addressed ……………………………………………………..…………. 57 2.4. Materials and Methods ……………………………..………………………………………… 58 2.4.1. Animals and Induction of Diabetes ………………………………………………… 58 2.4.2. Cutaneous Wound Surgery ……………………………………………………………. 59 2.4.3. Wound Treatment and Closure ……………………………………………………… 59 2.4.4. BrdU-Labeling and DNA Synthesis …………………………………………………. 60 2.4.5. Histological Analysis and Immunohistochemistry ………………………….. 60 2.4.6. Statistical Analysis …………………………………………………………………………. 61 2.5. Results …………………………………………………………………………………………………. 61 2.5.1. Body Weight and Blood Glucose Measurements …………………………… 61 2.5.2. Full-Thickness Wound Closure .……………………………………………………… 62 2.5.3. Histological Analysis of Skin .………………………………………………………….. 64 2.5.4. DNA Labeling Indexes .…………………………………………….…………………….. 64 2.5.5. Tissue Pathology and Immunohistochemistry .………………………………. 66 2.5.6. PDGF Expression ….………………………………………………………………………… 66 2.5.7. VEGF Expression .…………………………………………………………………………… 67 2.5.8. FGF-2 Staining .………………………………………………………………………………. 68 2.6. Discussion .……………………..……………………………………………………………………. 69 2.7. Innovation .………………………………………………………………………………………….. 71 2.8. Key Findings …….………………………………………………………………………………….. 72 2.9. Acknowledgements and Funding Sources …………………………………………… 72 2.10. Author Disclosure and Ghostwriting ………………………………………….……… 72 2.11. Abbreviations and Acronyms ……………………………………………………..……… 72 2.12. References ..………………………………………………………………………………………. 74 2.13. About the Authors …………………………………………………………………………….. 79
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CHAPTER 3: THE OPIOID GROWTH FACTOR-OPIOID GROWTH FACTOR RECEPTOR AXIS AND DIABETIC BONE COMPOSITION …………………………………….
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3.1. Abstract ……………………………………………………………………………………………….. 81 3.1.1. Background …………………………………………………………………………………… 81 3.1.2. Methods ………………………………………………………………………………………… 81 3.1.3. Results …………………………………………………………………………………………… 81 3.1.4. Conclusions …………………………………………………………………………………… 82 3.1.5. Keywords ………………………………………………………………………………………. 82 3.2. Abbreviations …………..…………………………………………………………………………. 82 3.3. Introduction ………………………………………………………………………………………… 83 3.4. Materials and Methods ……………………………..………………………………………… 85 3.4.1. Animals and induction of diabetes .……………………………………………….. 85 3.4.2. Bone fracture surgical model ………………………………………………………… 85 3.4.3. Immunohistochemistry …..…………………………………………………………….. 86 3.4.4. Histomorphometry …..…………………………………………………………………… 87 3.4.5. Measurement of serum enkephalin ..…………………………………………….. 87 3.4.6. Statistical analyses ..………………………………………………………………………. 87 3.5. Results …………………………………………………………………………………………………. 88 3.5.1. Expression levels of OGF and OGFr during bone development ………. 88 3.5.2. Levels of OGF and OGFr in bone and serum of T1D animals ..………… 89 3.5.3. Cellular composition of T1D bone …………………………………………………. 91 3.5.4. Cellular composition of T1D bone following fracture …………………….. 92 3.6. Discussion ……..……………………..……………………………………………………………… 97 3.7. Conclusion …………………………………………………………………………………………… 99 3.8. Acknowledgements .…………………………………….……………………………………… 99 3.9. Author Contributions .………………………………….……………………………………… 99 3.10. Funding …..………………..……………………………………………………………………….. 99 3.11. Competing Interests ………………………………………………………………………….. 99 3.12. References …………………………………………………………………………………………. 100
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CHAPTER 4: THE OPIOID ANTAGONIST NALTREXONE PREVENTS BONE DEFECTS IN A DIABETIC RAT MODEL ………………………………………………………………
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4.1. Abstract …………………………………………………………………………………………………… 106 4.1.1. Background ……………………………………………………………………………………….. 106 4.1.2. Methods ……………………………………………………………………………………………. 106 4.1.3. Results ………………………………………………………………………………………………. 106 4.1.4. Conclusions ……………………………………………………………………………………….. 106 4.1.5. Keywords ………………………………………………………………………………………….. 107 4.3. Introduction …………………………………………………………………………………………….. 108 4.4. Materials and Methods …………………………………………………………………………… 109 4.4.1. Animals, induction of diabetes, and treatment …………………………………. 109 4.4.2. Three-point bending …………………………………………………………………………. 110 4.4.3. MicroCT …………………………………………………………………………………………….. 110 4.4.4. Immunohistochemistry ……………………………………………………………………… 111 4.4.5. Histomorphometry ……………………………………………………………………………. 112 4.4.6. Data analyses ……………………………………………………………………………………. 112 4.5. Results …………………………………………………………………………………………………….. 112 4.5.1. Three-point bending …………………………………………………………………………. 112 4.5.2. MicroCT …………………………………………………………………………………………….. 113 4.5.3. Immunohistochemistry ……………………………………………………………………… 115 4.5.4. Histomorphometry ……………………………………………………………………………. 118 4.6. Discussion ……………………………………………………………………………………………….. 118 4.7. Conclusion ………………………………………………………………………………………………. 120 4.8. Acknowledgements …………………………………………………………………………………. 120 4.9. Author Contributions ………………………………………………………………………………. 120 4.10. Funding …………………………………………………………………………………………………. 120 4.11. Competing Interests ………………………………………………………………………………. 120 4.12. References …………………………………………………..………………………………………… 121 CHAPTER 5: DISCUSSION …………………………………………………..…………………………… 126 5.1. Discussion …………………………………..…………………………………………………………… 127 5.2. References …………………………………..………………………………………………………….. 140
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LIST OF FIGURES
Figure 1.1. Temporal overview of phases of wound healing, prominent cell
types, and actions …………………………………………………………………………
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Figure 1.2. Temporal overview of phases of fracture repair with prominent
cell types ………………………………………………………………………………………
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Figure 2.1. Residual wounds over time ………………………………..………………………… 63
Figure 2.2. BrdU labeling of cutaneous wounds ………………..…………………………… 65
Figure 2.3. PDGF labeling of cutaneous wounds ……………..…………………………….. 67
Figure 2.4. VEGF labeling of cutaneous wounds ……………..………………………….…. 68
Figure 3.1. OGF and OGFr staining in developing bone ..………………………………… 89
Figure 3.2. OGF and OGFr staining in normal and diabetic bone ………….………….. 90
Figure 3.3. Comparison of normal and diabetic bone .……………………..……………. 92
Figure 3.4. Fracture callus composition …………………………………………….……….….. 93
Figure 3.5. Fracture callus composition subperiosteum …………………….………….. 95
Figure 3.6. Ki67 labeling in fracture calluses .…………………………………………………. 96
Figure 4.1. Force, energy, and stiffness histograms ……………………………………….. 113
Figure 4.2. OGF and OGFr staining in bone ……………………………………………………. 116
Figure 4.3. Osteocalcin staining in bone ………………………………………………………… 117
Figure 4.4. Ki67 staining in bone ……………………………………………………………………. 118
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LIST OF TABLES 4.1. Trabecular μCT measurements ………………………………………………………………….. 114
4.2. Cortical μCT measurements ………………………………………………………………………. 114
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ABBREVIATIONS
AAALAC, Association for Assessment & Accreditation of Laboratory Animal Care
ACTH, adrenocorticotropic hormone
AGE, advanced glycation end products
ANOVA, analysis of variance
ATP, adenosine triphosphate
BB, biobreeding
β-end, β-endorphin
BMD, bone mineral density
BMP, bone morphogenetic proteins
BrdU, 5-bromo-2’-deoxyuridine
BV/TV, bone volume/total volume
cDNA, complementary deoxyribose nucleic acid
CLIP, corticotropin-like intermediate lobe peptide
CN, Charcot neuroarthropathy
DAPI, 4’,6-diamidino-2-phenylindole
DB, type 1 diabetic
DB/R, diabetic Regranex®-treated
DB/NTX, diabetic naltrexone-treated
DFU, diabetic foot ulcer
DKK1, dickkopf-related protein 1
Dlx5, distal-less homeobox 5
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DNA, deoxyribose nucleic acid
ER, endoplasmic reticulum
ELISA, enzyme-linked immunosorbent assay
FDA, Food and Drug Administration
FGF, fibroblast growth factor
FOXO, forkhead box O
GK, Goto-Kakizaki
HDN, high dose naltrexone
hIAPP, human islet amyloid polypeptide
IACUC, Institutional Animal Care and Use Committee
IGF, insulin like growth factor
IL-1, interleukin-1
INSR, insulin receptor
ip, intraperitoneal
LCMV, lymphocytic choriomeningitis virus
LDN, low dose naltrexone
M-CSF, macrophage-colony stimulating factor
MMP, matrix metallopeptidase
mRNA, messenger RNA
MSC, mesenchymal stem cells
MSH, melanocyte stimulating hormone
NAD+, nicotinamide adenine dinucleotide
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NOD, non-obese diabetic
NTX, naltrexone
N, non-diabetic
OGF, opioid growth factor
OGFr, opioid growth factor receptor
PARP, poly(ADP-ribose) polymerase
PDGF, platelet derived growth factor
PENK, proenkephalin
PlGF, placental growth factor
POMC, proopiomelanocortin
RANKL, receptor activator of NFκB ligand
ROS, reactive oxygen species
RUNX2, runt-related transcription factor 2
SEM, standard error of the mean
SOX9, SRY-box 9
STZ, streptozotocin
TGF, transforming growth factor
TNF-α, tumor necrosis factor alpha
TRAP, tartrate resistant alkaline phosphatase
T1D, type 1 diabetic
T2D, type 2 diabetes
VEGF, vascular endothelial growth factor
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ACKNOWLEDGEMENTS I would like to express my gratitude to my dissertation advisor, Dr. Patricia McLaughlin,
for her guidance and support throughout the dissertation process. I would also like to
thank my committee for their helpful input on this project.
Lastly, I would like to thank Gary, Elaine, and Marci Titunick for their unconditional
support and encouragement throughout my studies.
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CHAPTER 1: INTRODUCTION
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1.1. Diabetes
In 2015 an estimated 9.4% of the U.S. population had diabetes, 5-10% of whom
had type 1 diabetes. The total estimated healthcare cost of both types of diabetes in the
U.S. in 2017 was $327 billion[1]. Type 1 diabetes is defined by a lack of insulin production.
This autoimmune disease causes the insulin-producing beta cells of the pancreas to be
degraded and individuals with diabetes cannot control their blood glucose levels. A
fasting glucose level over 126 mg/dL with symptoms of hyperglycemia such as polydipsia,
polyphagia, and polyuria is sufficient for a diagnosis of diabetes[2]. In type 2 diabetes
individuals cannot utilize the insulin made by the pancreas leading to cells lacking energy.
Often type 2 diabetes can be controlled by lifestyle changes; eating well and exercise[3].
Complications arising from both types of diabetes include retinopathy, nephropathy,
neuropathy, cardiovascular disease[4], poor wound healing[5], and delayed fracture
healing[6].
1.2 Animal Models
Animals can be induced to have either type 1 or type 2 diabetes. Type 1 diabetes
models are preferentially used to eliminate some of the complications caused by the
comorbidities of type 2 diabetes such as increased adiposity. Chemically induced type 1
diabetes can be produced by injections of either streptozotocin (STZ) or alloxan at least
5-7 days prior to detection of hyperglycemia[7]. The disadvantage of using chemically
induced diabetes is that it can be toxic at other organs including the liver, kidney, lung,
intestines, and brain[8], and does not mimic human pathology.
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STZ, [2-deoxy-2-(3-(methy-3-nitrosoureido)-D-glucopyranose], is synthesized
from Streptomycetes achromogenes and is administered either intraperitoneally or
intravenously. It enters pancreatic beta cells by way of the Glut-2 transporter, alkylates
the DNA[9] activating PARP leading to NAD+ depletion, a reduction in cellular ATP, and
finally a decrease in insulin production[10]. STZ also creates free radicals causing DNA
damage and cell death[7]. It can be injected as either a single high dose or multiple low
doses. STZ has been used successfully to test cutaneous wound healing in numerous
studies[11–13].
Alternatively, alloxan (2,4,5,6-tetraoxypyrimidine; 5,6-dioxyuracil) is quickly taken
up by pancreatic beta cells where free radicals are formed to which the beta cells are
especially susceptible to damage[14]. It can be administered intraperitoneally,
subcutaneously, or at a lower dose intravenously. The disadvantage to alloxan is that a
slight overdose can lead to general toxicity[9].
The most common autoimmune models of type 1 diabetes are the non-obese
diabetic (NOD) mouse and the Biobreeding (BB) rat. NOD mice have a prediabetic phase
that can be used to examine preventative treatments. These mice require insulin
treatment and many drugs that work in diabetes prevention in NOD mice do not translate
to humans. Additional limitations to this model include the requirement of a specific
pathogen-free space, an unpredictable onset of diabetes, and high maintenance
expenses[7]. BB rats derived from Wistar rats also develop diabetes spontaneously. They
require insulin therapy and develop lymphopenia which is not characteristic of type 1
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diabetes in humans. These rats have been used for transplant and interventional studies
as well as studies on diabetic neuropathy[7].
Type 1 diabetes can be genetically induced in the AKITA mouse. In this mouse
model beta cells are destroyed due to ER stress and the animals are insulin dependent.
This model is useful for testing new insulin, transplantation, and treatments involving ER
stress. Virally-induced type 1 diabetes utilizes coxsackie B, encephalomyocarditis, or
Kilham rat viruses. Lymphocytic choriomeningitis virus (LCMV) can also be used under
the rat insulin promoter. In this case, beta cells are destroyed due to viral infection or are
involved in an immune response when exposed to LCMV. This model is complex and is
used mainly to detail the extent to which viruses can cause diabetes. In large animals,
pancreatectomy is the primary technique for inducing diabetes[7].
Type 2 diabetes can be induced genetically or by controlling food intake. Obese
monogenetic models like ob/ob mice deficient in leptin or db/db mice and Zucker diabetic
fatty rats deficient in leptin receptors are commonly used. They develop obesity-induced
hyperglycemia and can be used in studies to improve insulin resistance and beta cell
function[7]. db/db mice have also been used in studying corneal complications related to
diabetes[15]. Obese polygenic models are also available and have obesity-induced
hyperglycemia. Obesity can be induced by feeding rodents high fat diets or using animals
like the Nile grass rat and desert gerbil which naturally become obese in captivity. Non-
obese models of type 2 diabetes include the Goto-Kakizaki (GK) rat where hyperglycemia
is induced by insufficient beta cell mass or function. Genetically induced models of beta
cell dysfunction include the hIAPP mouse and the AKITA mouse. hIAPP mice produce
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human islet amyloid polypeptide which creates amyloids in pancreatic beta cells. AKITA
mice as mentioned earlier have beta cell destruction due to ER stress[7].
The STZ-induced type 1 diabetic rat model has been successfully used in studies
looking at complications resulting from type 1 diabetes. In addition, the model is less
expensive than breeding colonies of genetically induced mice or mice that are
immunocompromised.
1.3 Cutaneous Wound Healing
1.3.1 Cellular and Molecular Basis of Full-Thickness Wound Healing
1.3.1.1 Hemostasis
Understanding the cellular and molecular basis of full-thickness wound healing
allows for investigation into complications associated with diabetes. Wound healing
begins at the time of injury with hemostasis (figure 1.1.)[16]. Vascular injury during
wounding initiates the formation of a fibrin-fibronectin clot. Fibrillar collagens type I and
II promote aggregation of platelets into a clot with platelets embedded among the fibers.
The clot acts as a hemostatic plug protecting the wounded region, facilitates cell
migration, and stores growth factors and cytokines which aid in inflammatory cell
recruitment[17].
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Figure 1.1.: Temporal overview of phases of wound healing, prominent cell types, and
actions; from Baltzis et al. 2014[16]
1.3.1.2 Inflammation
In the second phase of wound healing, neutrophils and monocytes are recruited
by various chemotactic signals (e.g. cytokines and growth factors) including tumor
necrosis factor alpha (TNF-α), interleukin (IL)-1, and platelet derived growth factor
(PDGF). Neutrophils attack bacteria while monocytes differentiate into macrophages and
phagocytose pathogenic organisms and debris[17]. Adherence to the extracellular matrix
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promotes colony-stimulating factor 1, tumor necrosis factor α, and platelet-derived
growth factor secretion by monocytes and macrophages. Colony-stimulating factor 1 is
necessary for monocyte and macrophage survival. Tumor necrosis factor α is an
inflammatory cytokine. Platelet-derived growth factor (PDGF) is a chemoattractant and
supports proliferation of fibroblasts[18]. Macrophages secrete transforming growth
factor alpha (TGF-α), fibroblast growth factor (FGF), and vascular endothelial growth
factor (VEGF). These factors initiate the proliferation process[17].
1.3.1.3 Proliferation
Reepithelialization begins within hours of injury[18] and is stimulated by cytokines
released by activated fibroblasts and keratinocytes[17]. There is an increase in cell
proliferation and migration. Migration is made possible by the dissolution of
hemidesmosomes[18] and concludes when there is a single layer of keratinocytes
covering the wound area and the stratified epidermis is re-established[17].
Granulation tissue infiltrates the wound area around day 4 post-injury[18] and
contains capillaries which grow into the wounded region and undergo angiogenesis[17].
Angiogenesis is promoted by cytokines such as FGF-2, VEGF, PDGF, and TGF-β1,2.
Fibronectin and hyaluronan are deposited. Fibronectin allows for initiation of collagen
fibrillogenesis and acts as an anchor for wound contraction. Hyaluronan forms a matrix
that allows for cell migration. This preliminary matrix is then replaced by collagen, and
the cellularity decreases as the granulation tissue matures[17].
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1.3.1.4 Remodeling
Fibroblasts deposit and remodel the extracellular matrix. Collagen is deposited
causing an increase in strength. Contraction involves pulling normal dermal and adipose
tissue into the wound region[17]. Remodeling may last up to two years or more[19].
1.3.2 Cutaneous Wound Healing Complications, Amputation, and Diabetes
Five out of 1,000 diabetics require a lower extremity amputation[20]. More than
half of non-traumatic limb amputations occur in patients with diabetes, usually following
a non-healing wound or ulcer[21]. Neuropathy and vascular issues contribute to the
production of non-healing wounds and ulcers. Peripheral neuropathy is a leading cause
of foot ulcers as patients do not feel the discomfort or pain associated with an injury[22].
Autonomic neuropathy results in reduced sweating causing dry skin and fissures in
diabetics[23], creating an entry point for bacterial and fungal infections.
Diabetics are at twice the risk for peripheral vascular disease compared to non-
diabetics[5]. Red blood cells in diabetics are less deformable causing complications in
capillaries[24] and high blood glucose levels increase blood viscosity[25]. These two
conditions cause blood to remain stagnant in vessels. Peripheral arterial disease is caused
by atherosclerosis where fatty deposits form plaques within arteries which eventually
occlude the lumen[22]. Calcification leading to stroke and cardiovascular disease is also
common in diabetes[26]. Resultant reduced mobility may put patients at greater risk for
ulcers. Diabetic microangiopathy exhibits thickened capillary basement membranes
leading to decreased oxygen and nutrient delivery to tissues[5]. Although hypoxia is
necessary for vascularization, when oxygen levels do not eventually elevate fibroblasts
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cannot produce collagen[27]. Leukocytes have difficulty reaching the wound site due to
narrowed vessels[28,29] and may also be less active[28].
Diabetics have a prolonged inflammatory phase preventing granulation tissue
formation[22]. Proteases that remain can cause damage to the new extracellular
matrix[5]. Inflammatory cytokines also cause a reduction in proliferative factors[22] and
make fibroblasts less responsive to growth factors[30]. It is suggested that bacteria thrive
in wounds with high blood glucose levels and that the increase in glucose may also be
detrimental to neutrophil action[31,32]. Often the classic signs of infection are absent or
diminished in diabetics due to neuropathy and ischemia[33].
1.3.3 Current Treatments in Wound Healing
Standard care of diabetic ulcers includes debridement, dressing, pressure off
loading, and infection management. Debridement removes callus, necrotic dermal tissue,
foreign debris, and bacteria; all of which retard wound healing. Sharp debridement is the
gold standard of care and allows the wound to respond better to topical treatments[16].
Wound dressings must protect the wound from secondary infections, keep the wound
moist, remove exudates, and promote tissue regeneration. Research has not shown a
specific type of dressing to be significantly better than another[16]. Removing pressure
from the ulcer is another important aspect to adequate wound care. Casts can be used
to redistribute pressure or surgical methods such as Achilles tendon lengthening,
metatarsal-phalangeal joint arthroplasty, metatarsal head resection, and liquid silicone
injections plantar to metatarsal heads may be utilized. Research suggests there is very
little gained by surgical methods compared to casting methods[16].
10
To assist in adequate vascularization surgical therapies including angioplasty,
endarterectomy, grafting, or bypass may be considered[16]. Infection is managed by
debridement and antibiotics. Improvements of the patient’s general health will also
contribute to better outcomes. Keeping blood glucose levels under control, managing
dyslipidemia, smoking cessation, and appropriate diet will aid healing[34,35].
Adjunctive therapies include bioengineered skin substitutes, growth factors,
oxygen therapy, negative pressure wound therapy, and electrical stimulation and
shockwaves. Currently, there are two US Food and Drug Administration (FDA) approved
bioengineered skin substitutes. Apligraf® which is composed of cultured living dermis and
epidermis from neonatal foreskin with extracellular matrix, dermal fibroblasts, epidermal
keratinocytes, and a stratum corneum has an extracellular matrix lattice made of bovine
type I collagen. The dermal fibroblasts and keratinocytes produce growth factors. The
stratum corneum is a physical barrier against mechanical damage and infection.
Dermagraft® is composed of neonatal-derived dermal fibroblasts on bioabsorbable
polyglactin mesh. The fibroblasts secrete extracellular matrix and along with growth
factors such as PDGF-A, insulin-like growth factor, keratinocyte growth factor, heparin-
binding epidermal growth factor, transforming growth factors, and VEGF[16] is used to
promote healing.
Platelet-derived growth factor (PDGF), or becaplermin, has been approved by the
FDA for treatment of diabetic foot ulcers under the name Regranex®. PDGF reportedly
activates inflammatory cells, stimulates cell proliferation and migration, and enhances
11
protein and extracellular matrix synthesis[16]. However, there is a black box warning on
Regranex® indicating an increased risk of cancer mortality[36].
Another, more experimental method of wound healing utilizes hyperbaric oxygen
therapy that alters microbial balance, soft tissue infection, and angiogenesis[16].
Negative pressure wound therapy exposes the wound to subatmospheric pressure[37] to
assist in the removal of excess fluid[38,39], reduce bacterial load, reduce mechanical load,
or increase granulation tissue production[40]. The results have been inconclusive as to
its effects[16]. Pulsed electromagnetic field stimulation may decrease fibroblast and
endothelial cell doubling time in culture, have bacteriostatic and bactericidal properties,
increase migration of neutrophils and macrophages[41], and increase expression of
angiogenesis-related growth factors[42]. There are few clinical trials on electrical
stimulation[43].
Future directions in wound therapy include stem cell transplants, gene therapy,
neuropeptide-based treatments, and cytokine inhibition. Intramuscular injections of
peripheral blood mononuclear cells, bone marrow mesenchymal stem cells, or bone
marrow-derived mononuclear cells have increased wound healing[44,45]. Local
application of both bone marrow cells[46] and bone marrow mesenchymal stem cells[47]
have improved wound healing. Topical application of a replication-defective adenovirus,
encoding PDGF, accelerated wound healing in diabetic patients[48]. VEGF has been
administered using adenovirus in animal models with promising results[49].
Studies have suggested angiotensin II and angiotensin (1-7) play a role in wound
healing and have used norleu3-angiotensin (1-7), an angiotensin (1-7) analog, to enhance
12
wound healing in patients[50–52]. Topical substance P accelerated wound healing,
increased contraction, and increased levels of VEGF, TGF-β1 and TNF-α[53]. Another
treatment being explored is recruitment and activation of anti-inflammatory
macrophages. This will promote an anti-inflammatory environment conducive for growth
factors and wound healing[54].
1.4. Bone Repair and Remodeling
1.4.1 Cellular and Molecular Basis of Fracture Healing
1.4.1.1 Inflammation
Figure 1.2.: Temporal overview of phases of fracture repair with prominent cell types,
from Einhorn and Gerstenfeld, 2015[55]
Fracture repair greatly mirrors the wound repair process. Both processes begin
with an inflammatory phase and rely healivy on vascularzation. Immediately following
traumatic bone fracture, bleeding causes the formation of a hematoma (figure 1.2.) which
is invaded by platelets and macrophages. These inflammatory cells secrete factors that
13
foster clotting. Capillaries advance into the clot which is now becoming granulation
tissue. Multiple factors, including platelet-derived growth factor (PDGF), fibroblast
growth factor-2 (FGF-2), and vascular endothelial growth factor (VEGF), are released to
recruit more inflammatory cells and mesenchymal stem cells[56]. PDGF is secreted by
platelets, monocytes, macrophages, endothelial cells, and osteoblasts, and stimulates
proliferation and migration of osteoblasts and mesenchymal stem cells (MSCs). Fibroblast
growth factor is secreted by monocytes, macrophages, mesenchymal cells, osteoblasts,
and chondrocytes, and supports angiogenesis and mesenchymal cell proliferation. Acidic
FGF regulates chondrocyte proliferation[57] while basic FGF enhances proliferation of
osteoblasts and prevents their apoptosis[58]. Insulin like growth factor, secreted by bone
matrix, endothelial cells, osteoblasts, and chondrocytes, promotes bone matrix formation
and cell proliferation[57]. Bone morphogenetic proteins (BMPs) are secreted by
osteoprogenitor cells, mesenchymal cells, osteoblasts, and chondrocytes. BMPs are
chemotactic factors that also regulate mesenchymal and osteoprogenitor cell
proliferation and differentiation, enhance angiogenesis by activating endothelial cells,
promote extracellular matrix synthesis, and stimulate the synthesis and secretion of
insulin like growth factor (IGF) and VEGF to support angiogenesis[57].
1.4.1.2 Soft Callus Formation
Fractures heal through the process of endochondral ossification when there is
mechanical instability. A callus is formed first from cartilage and later by bone giving
stability to the fractured ends of the bone. The invading mesenchymal cells differentiate
into chondrocytes. The cartilage bridges the fracture increasing support. Chondrogenesis
14
is supported by bone morphogenetic proteins. Chondrocytes secrete extracellular matrix
proteins, especially collagen II. The cartilaginous callus is then invaded by vasculature
stimulated by pro-angiogenic factors[56]. Proliferating chondrocytes increase levels of
SOX9 (SRY-box9)[59]. Hypertrophic chondrocytes increase levels of runt-related
transcription factor 2 (RUNX2), alkaline phosphatase, collagen I and X, matrix
metallopeptidase 13 (MMP13), VEGF, osteocalcin, osterix, osteopontin, PDGF, BMPs, and
placental growth factor (PIGF). RUNX2 is associated with osteoblast differentiation from
MSC to preosteoblast[60]. Alkaline phosphatase correlates to osteoblast activity.
MMP13 degrades collagen II and aggrecan in the extracellular matrix[59]. Osterix is
required for differentiation from preosteoblast to mature osteoblast, activating DKK1, a
WNT antagonist[60]. Osteocalcin is pro-osteoblastic and stimulates matrix
mineralization. Osteopontin is a non-collagenous component of the extracellular
matrix[61].
1.4.1.3 Hard Callus Formation
High osteoblast activity and mineralized bone matrix formation are key
characteristics of hard callus formation[56]. MMP9 degrades collagen[59] and the soft
callus is removed and replaced by irregular bone[56]. This initial woven bone matrix is
created by mature osteoblasts. BMPs play an important role in differentiation from
osteoprogenitor cell to mature osteoblast. High levels of BMPs inhibit osteocalcin and
osteopontin[59]. Although stem cells from the periosteum and bone marrow can
produce bone, they are not necessary for bone formation. This indicates that
15
osteoprogenitors may originate from other locations such as circulation[62],
vasculature[63], and local tissue[56,64].
1.4.1.4 Bone Remodeling
In the final stage of fracture repair woven bone is formed into organized lamellar
bone. The osteoclast, a large multinucleated cell originating from a hemopoietic
precursor, resorbs bone. A border is sealed off and acid and proteinases are released
allowing for demineralization and degradation of organic components. The debris is
accumulated in vesicles, and the osteoclasts are apoptosed or resume a non-resorbing
form. Osteoblasts migrate to the region and lay down new bone. Osteoblasts secrete
macrophage-colony stimulating factor (M-CSF) and receptor activator of NFκB ligand
(RANKL) to promote osteoclasts survival and activity. RANKL is vital in coordinating the
balance of bone formation and resorption[56].
1.4.2 Bone Healing Complications and Fractures in the Diabetic Population
Diabetes can decrease linear bone growth in adolescence[65], reduce bone
mineral density (BMD)[66], increase fracture risk[67], and result in poor osseous
healing[68]. Type 1 diabetes has been associated with decreased osteoblast recruitment
and activity. Studies have shown unaltered or decreased bone resorption in experimental
diabetes. Experiments have also shown “poor trabecular connectivity, increased porosity
and lower bone spicule/marrow space ratio[69].” Collagen reduction was noted within 2
weeks of diabetes induction. The fracture callus maturation time is prolonged by 87%,
with bone bridging delayed by 40%[69]. The callus showed reduced undifferentiated
mesenchymal cells at 4 days with delayed differentiation[69]. DNA content within a
16
diabetic callus was 40% less compared to normal calluses indicating decreased cell
proliferation[70].
Diabetics are at risk for developing Charcot neuroarthropathy (CN) whereby
bones, joints, and soft tissue of the foot and ankle are susceptible to inflammation and
eventual deformation. The Charcot foot is subject to fractures and dislocations as the
bone deteriorates. Loss of protective sensation in diabetes prevents patients from
noticing a plantar foot injury resulting in prolonged weight-bearing insult, perpetuating
inflammation. Motor neuropathy causes tendon contractures exacerbating deforming
forces on the foot. Autonomic neuropathy prevents regulation of peripheral circulation,
increasing blood flow, and resulting in a bounding pulse. A common feature of CN is
“rocker bottom foot,” a collapse of the midfoot joint[71].
It is possible that increased levels of tumor necrosis factor-α (TNF-α), a
proinflammatory mediator, may prevent diabetics from restricting levels of other
regulatory factors[72]. Glycation of type I collagen decreases osteoblast ability to adhere
to extracellular matrix and lowers alkaline phosphatase activity[73]. Serum alkaline
phosphatase and osteocalcin, indicators of osteoblasts and osteoblastic activity, are lower
in T1D animals than in non-diabetic animals. Both STZ-induced diabetic mice and
nonobese spontaneously diabetic mice have decreased trabecular bone in the tibia[74].
In vitro studies suggest high glucose and advanced glycation end products (AGEs)
are associated with inhibited osteoblast function and decreased mineralized matrix
formation[75]. Experimentally AGE treatment caused a dose-dependent inhibition in
bone healing amongst non-diabetic animals[76,77]. Reactive oxygen species (ROS) are
17
formed due to either high glucose levels and/or insulin insufficiency. High ROS levels
reportedly inhibit differentiation of osteoblasts[78]. Bone may also be affected by poor
expression of genes such as Cbfa1/Runx2 and Dlx5 which regulate osteoblast
differentiation[79]. High levels of Forkhead box O (FOXO), induced by oxidative stress,
antagonizes Wnt signaling and decreases bone formation[80]. A lack of insulin can also
have an effect on proliferation. Insulin acts through the insulin receptor (INSR) to increase
cell proliferation and cell growth through mitogen-activated protein kinases[81].
During fracture repair there is a decrease in growth and angiogenic factors,
reduced proliferation, and increased apoptosis[82]. Long bone fractures in STZ animal
models form smaller calluses with reduced bone composition (less bone per area of
callus) and decreased mechanical strength[70,83,84]. Biomechanical strength was
decreased in animal models by 20% in femurs and tibias[85,86]. A study[87] involving
ectopic bone formation in rats noted that cell proliferation in diabetic rats was 35% that
of non-diabetic controls. Systemic insulin raised cell proliferation to 81% of controls. It
is important to note that systemic administration of insulin may result in
hypoglycemia[81] and may not be a preferred treatment.
Often low oxygen levels are associated with diabetes. Ischemia can lead to cell
death, delayed chondrocyte and osteoblast differentiation, and poor fracture healing[88].
Oxygen is necessary for aerobic metabolism, enzymatic activities, collagen synthesis, as a
signaling molecule, and potentially for stem cell maintenance[89]. Hyperbaric oxygen
enhances cell proliferation and mineralization of osteoblasts[90]. Oxygen “decreases
18
sclerostin expression, increases Wnt signaling, and increases BMP2, IGF, and VEGF
expression[89].”
1.4.3 Current Treatments in Fracture Repair
Maintaining healthy insulin levels does not completely ameliorate bone loss[91].
At this time, standard of care for diabetic patients receiving surgical treatment for a
fracture include internal fixation and prolonged off-loading. Biologics have begun to be
applied in cases were a patient is at higher risk of non-union such as diabetics and
smokers. These include platelet-derived growth factor, platelet-rich plasma, bone
morphogenetic proteins 2 and 7, and demineralized bone matrix. As these treatments
are not fully integrated into standard practice there is still room for improvement and for
a disease modifying treatment. Experimentally, local delivery of insulin[92],
vanadium[93], and manganese chloride[94] have all shown to improve fracture healing in
a rat model. Local insulin may inhibit FOXO1 and therefore prevent Wnt signaling
antagonism. Vanadium[93] and manganese chloride[94] are insulin-mimetic and
osteogenic. Basic fibroblast growth factor applied to fractures normalized diabetic repair
and enhanced healing in non-diabetic animals[95]. In another study using STZ-induced
diabetic rats there was a dose-dependent effect on callus formation 3 weeks post-
fracture. Increases in mechanical properties mirrored the histological and radiological
changes[95]. Low intensity pulsed ultrasound treatment improved levels of growth
factors and increased callus cartilage and blood vessel density[96]. Injection of platelet-
rich plasma which contains high levels of mitogenic factors normalized fracture repair by
increasing cell proliferation in diabetic rats[97]. Recombinant human bone
19
morphogenetic protein-2[98] and PDGF-BB[99] have both been used to enhance diabetic
fracture healing in rat models.
Most of the new platforms for treatment are focused on treating the diabetic
complications only and not altering the disease. Disease modifying treatments would be
beneficial and focus on multiple complications of diabetes. It is thought that the
dysregulation of the OGF-OGFr axis may cause disruption in bone healing and that
blockade of the pathway would treat the underlying pathophysiology of diabetes.
1.5. Opioids
Opioids function in many capacities including analgesia, cardiovascular control,
behavior, and cell division and growth[100], and can be endogenous or exogenous in
nature. Endogenous opioids regulate stress responses and are found in the
hypothalamus, pituitary, and adrenal medulla. Precursors for endogenous opioid
peptides include beta-endorphin/ACTH precursor (also known as
proopiomelanocortin/POMC), proenkephalin/proenkephalin A, or dynorphin/neo-
endorphin precursor (also known as prodynorphin/proenkephalin B). The beta-
endorphin (β-END)/ACTH precursor gives rise to beta-endorphin and adrenocorticotropic
hormone (ACTH). ACTH (1-39) can be cleaved into melanocyte stimulating hormone
(MSH) and CLIP (Corticotropin-Like Intermediate Lobe Peptide) or ACTH (18-39).
Proenkephalin produces several copies of met-enkephalin and leu-enkephalin, whereas
prodynorphin gives rise to α/β-neo-endorphin, dynorphin A, and dynorphin B. The
pituitary is the major site of β-END/ACTH synthesis and secretion, but proenkephalin is
localized in a wide variety of tissues spanning all three germ layers. Dynorphin is
20
produced in the gut, posterior pituitary, and brain[101]. The endogenous opioid of
interest in this dissertation is [met5]-enkephalin, a pentapeptide with the sequence Tyr-
Gly-Gly-Phe-Met[100]. [Met5]-enkephalin expression appears to be conserved from
bacteria to humans[102,103]. It also goes by the name opioid growth factor (OGF) and
decreases cell proliferation without increasing apoptosis or necrosis and does not affect
cell differentiation[100].
Insulin and glucagon secretion is stimulated by β-END and inhibited by [met5]-
enkephalin[104]. Plasma [met5]-enkephalin levels were reported to be significantly
higher in type 1 diabetics compared to normal individuals[105].
Proenkephalin (PENK), the precursor to [met5]-enkephalin, is expressed in embryonic
mesenchymal tissues of cartilage and bone[106]. Fetal rat calvaria-derived cells and
osteoblast-like cells grown in culture also tested positive for PENK mRNA[107]. Elhassan
et al.[108] demonstrated the presence of PENK-derived peptides in bone using
immunohistochemistry. [Met5]-enkephalin appeared in both mature bone matrix and in
osteoblasts. PENK declines with age[109]. There is an inverse relationship between
osteoblastic PENK mRNA and cell differentiation. As levels of PENK decrease, alkaline
phosphatase activity increases. [Met5]-enkephalin reduced alkaline phosphatase activity
in ROS17/2.8, an osteoblastic cell line[107]. Enkephalinase is expressed in
osteoblasts[110] and may moderate the levels of enkephalin to allow for activity during
particular stages of fracture healing[106].
21
1.6. The OGF-OGFr Axis
[Met5]-enkephalin, also termed opioid growth factor (OGF), is a negative growth
regulator delaying the G0/G1 phase of the cell cycle through interactions at the opioid
growth factor receptor (OGFr), originally termed opioid receptor zeta (ζ). It has been
shown to be the most potent endogenous opioid associated with growth, with
concentrations as low as 10-10M inhibiting cell proliferation[111]. The peptide-receptor
interaction is reversible with the peptide released in an autocrine manner that is tissue
and species non-specific[111]. OGF binds to OGFr creating a complex that translocates
into the nucleus resulting in an increase of the cell cycle inhibitors p16 and p21 and a
decrease in cell proliferation[111,112].
Biochemical and molecular studies of OGFr indicate that OGFr is present on a variety
of tissues and located on the nuclear membrane where it then translocates into the
nucleus. Molecular comparison of classical opioid recpetors and OGFr indicate that there
is no homology between the classical opioid receptors and OGFr. Fluorescence in situ
hybridization determined that OGFr is at human chromosome location 20q13.3. OGFr is
found predominantly in the perinuclear region of developing and renewing cells[111].
1.7. Opioid Receptor Antagonists
1.7.1. Naltrexone
Naltrexone (NTX) is a general opioid receptor antagonist that has no intrinsic
abilities except to block ligands from opioid receptors including μ, δ, and κ receptors. NTX
is a synthetic congener of oxymorphone[113,114]. The duration of receptor blockade by
naltrexone is important for determining the biological outcome. Intermittent blockade
22
by naltrexone causes feedback mechanisms to upregulate production and/or secretion of
more endogenous opioids that subsequently interact with the receptors allowing for
enhanced decreased cell replication. This paradigm is activated following low dose
naltrexone (LDN) therapy. LDN treatment is effective for cancer and inflammatory
disorders whereby the circulating levels of OGF are diminished[115,116]. Continuous
receptor blockade, often invoked by higher dosages of naltrexone, results in blockade of
the OGF inhibition of the cell cycle, and the result is an increase in cell replication. This
paradigm has been explored for the treatment of epithelial complications associated with
diabetes[117].
1.7.1.1 Function of Naltrexone Blockade Actions
Corneal explants grown in culture were used to examine growth of epithelium in
the presence of OGF and high dose naltrexone (HDN). Exogenous OGF decreased cell
division and led to disorganized outgrowth of the epithelium while NTX accelerated cell
division without changing the normal growth pattern[118,119]. OGF decreased DNA
synthesis and NTX increased DNA synthesis[119]. Corneal epithelial wound healing in rats
was increased by both systemic and topical NTX[120,121]. Rabbit corneal wounds also
healed more rapidly with blockade of the OGF-OGFr axis[122]. In a study using a Helios®
gene gun system to deliver sense cDNA or antisense cDNA for OGFr into corneal epithelial
cells, sense cDNA increased OGFr production and antisense decreased OGFr production.
Sense cDNA caused a delay in wound healing of rat corneas while those corneas treated
with antisense cDNA resulted in increased wound healing[123,124]. Animals were
treated with NTX for 1 week to determine its safety. Cells having undergone DNA
23
synthesis were increased by 69-85% with NTX treatment. Epithelial thickness and cellular
packing density also increased. There were no negative effects on the tissue. Organ
cultured human corneas with epithelial wounds were exposed to OGF and NTX resulting
in decreased cell proliferation in those treated with OGF and increased proliferation in
those treated with NTX[125].
Humans with both type 1 and type 2 diabetes often have complications associated
with the corneal surface include keratopathy and delayed corneal healing[126–128],
decreased corneal sensitivity[127–131], and dry eye[129,130,132,133]. Diabetic animal
models for type 1 diabetes were established, and rats received standardized corneal
epithelial wounds and a subset were treated intraperitoneally with NTX twice daily. There
was a significant increase in corneal reepithelialization in type 1 diabetic rats receiving
NTX compared to diabetic rats receiving saline. NTX normalized the rate of repair in
diabetic rats to the level of normal non-diabetic rats[134]. A similar study was used to
show that intensive insulin therapy would also be able to enhance the repair
process[135]. In another study, diabetic animals treated with topical insulin had wounds
19-60% smaller than those treated with vehicle only[136]. Topical NTX also increased the
rate of reepithelialization in diabetic rats with corneal wounds[137]. Investigations
combining topical NTX and topical insulin demonstrated that combined treatment was
not more effective than either insulin or NTX used alone[138] suggesting a similar
pathway or maximum potential to increase wound healing[100]. Investigations
examining use of NTX for treatment of corneal surface wound in the type 2 diabetic
model, db/db, revealed that the OGF-OGFr axis was present in this model and if
24
completely blocked by NTX, enhanced reepithelialization[15]. No pathology was
associated with NTX treatment[139].
Diabetes is often accompanied by peripheral neuropathy[140–145] and dry
eye[129,131,146]. Using von Frey hairs to test sensitivity, corneal sensitivity was
normalized in diabetic rats by treatment with topical NTX[147]. Treatment with NTX
normalized tear production for up to 48h. When treating one eye only there is no effect
seen in the contralateral eye, suggesting there is no cross over[147]. NTX did not affect
tear production in normal rats with normal tear volume.
Diabetes is also associated with slow cutaneous wound healing. Both normal and
type 1 diabetic rats treated three times daily with NTX had increased DNA synthesis[148].
Normal wounded rats treated with NTX had smaller residual wounds than control
animals. Diabetic rats treated with NTX also had smaller wounds than diabetic controls.
Moreover, there was no difference in skin histology between those animals treated with
NTX and control animals[148]. NTX helps improve cutaneous wound healing by
enhancing angiogenesis. There is a delay in FGF-2 and VEGF expression in wounded
diabetic animals[100]. Naltrexone increases formation and maturation of collagen and is
able to normalize tensile strength of healed skin[13]. The changes imparted by NTX in
type 1 diabetes have been seen in the db/db mouse model of type 2 diabetes as well[149].
1.7.2. Naloxone
Naloxone, also known as Narcan®, is most often used to treat opioid overdose and
improve respiratory depression[150]. Naloxone is shorter acting[117] and less
potent[151–153] relative to naltrexone. Naloxone, like naltrexone, has duration
25
dependent actions[154]. Studies determined that developing rats receiving naloxone for
less than 12 hours per day had decreased body and organ weight compared to
controls[154], suggesting a relationship between naloxone and organ growth.
Chick embryos received naloxone hydrochloride on multiple days[155]. Chicks
were sacrificed and femora were collected. Injection of naloxone resulted in a 2-fold
increase in thickness of the perichondrial bone cuff compared to controls. There was no
change in the density of osteoblasts in the diaphysis or mitotic activity in the growth plate.
There was a decrease in diaphyseal osteocyte density. Chicks treated during week 3 of
embryogenesis had an increase in the number of dividing cells in the growth plate and
the density of cells in the proliferating cartilage. Findings suggest that osteogenesis is
stimulated by naloxone[155].
A study utilizing a “drill hole” model examined the use of naloxone to enhance
mineralization and callus remodeling in sheep[156]. Treatment was administered
intramuscularly over 24h for four weeks. Naloxone enhanced mineralization and
remodeling especially when combined with calcium gluconate. Sheep receiving naloxone
had more radiodense drill holes than controls[156].
Mesenchymal stem cells (MSCs) and osteoblasts from human bone marrow were
cultured[157]. MSCs and osteoblasts did not express mRNA for μ- or κ-opioid receptor
nor POMC. They did express δ-opioid receptor, OGFr, and PENK. MTT assay was used to
determine proliferation in culture. Addition of [met5]-enk had no effect on cell
proliferation compared to control. Treatment with 1mM naloxone decreased the rate of
MSC proliferation and increased mineralization. There were no signs of apoptosis.
26
Osteocalcin gene expression increased after MSC culture treatment with naloxone. When
utilizing a unicortical defect model in mice researchers discovered treatment with
naloxone increased bone within the defect. There was an increase in relative bone
volume (BV/TV) corresponding to a 1.2-fold increase in trabecular number. Defect
diameter decreased 20.31% in naloxone treated defects compared to control. Treatment
with [met5]-enk resulted in no change compared to controls[157]. While studies have
shown how naloxone increases bone growth, testing has not been done using naltrexone.
1.8. Current Gap in Knowledge
The underlying causes of delayed fracture healing in diabetes are not known. Studies
in humans and animals, have reported that enkephalin levels are elevated in subjects with
diabetes[105,158]. Given that OGF (i.e. methionine enkephalin) is an inhibitory growth
factor known to depress cell replication, the dysregulation of the OGF-OGFr axis in
diabetics may be a factor in the delayed repair processes. Moreover, there is a potential
to modulate the axis to accelerate cell proliferation. Studies in animal models of type 1
and type 2 diabetes have reported that blockade of the OGF-OGFr axis with naltrexone
accelerates repair mechanisms in corneal epithelial wounds and full-thickness cutaneous
wounds. However, there is no information on the activity of this pathway in bone, or
whether the OGF-OGFr regulatory axis can be manipulated by naltrexone in bone growth
and repair.
1.9. Hypothesis and Specific Aims
The hypothesis of this research is that the OGF-OGFr axis is dysregulated in type 1
diabetes and that sustained blockade of the axis using naltrexone will reverse diabetic
27
complications including delayed repair of full-thickness wounds and bone fractures.
Specific Aim 1 will test the hypothesis that naltrexone administered topically to a full-
thickness cutaneous wound in type 1 diabetic rats will enhance the rate of wound closure
in a manner comparable to standard of care- Regranex®. Specific Aim 2 will test the
hypothesis that the dysregulation of the OGF-OGFr pathway alters markers within bone
in diabetic animal models, and that blockade of the OGF-OGFr pathway modulates the
repair of fractured femurs. Specific Aim 3 will test the hypothesis that systemic treatment
with naltrexone to block the OGF-OGFr pathway with the antagonist naltrexone may
prevent or change bone composition in diabetes that ultimately results in complications
related to fracture and repair.
28
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[3] Facts About Type 2 http://www.diabetes.org/diabetes-basics/type-2/facts-about-
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[5] Falanga, V. Wound Healing and Its Impairment in the Diabetic Foot. The Lancet,
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[8] Lee, J.; Yang, S.; Oh, J.; Lee, M. Pharmacokinetics of Drugs in Rats with Diabetes
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29
[10] Sandler, S.; Swenne, I. Streptozotocin, but Not Alloxan, Induces DNA Repair
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[14] Nerup, J.; Mandrap-Poulsen, T.; Helqvist, S.; Andersen, H.U.; Pociot, F.; Reimers,
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IDDM. Diabetologia, 1994, 37, S82–S89.
[15] Zagon, I.S.; Sassani, J.W.; Immonen, J.A.; McLaughlin, P.J. Ocular Surface
Abnormalities Related to Type 2 Diabetes Are Reversed by the Opioid Antagonist
Naltrexone: NTX Repairs Corneal Surface Epithelium. Clin. Experiment.
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CHAPTER 2: TOPICAL NALTREXONE IS A SASFE AND EFFECTIVE ALTERNATIVE TO
STANDARD TREATMENT OF DIABETIC WOUNDS1
Patricia J. McLaughlin, Jarrett D. Cain, Michelle B. Titunick, Ian S. Zagon __________________________________ 1Chapter 2 consists of a previously published paper approved by the dissertation committee for the use of this dissertation. The paper has been reformatted to fit into this dissertation. The citation for the original paper is: McLaughlin, P., Cain, J., Titunick, M., Sassani, J., & Zagon, I. (2017). Topical naltrexone is a safe and effective alternative to standard treatment of diabetic wounds. Advances in Wound Care, 6(9), 279-288. doi:10.1089/wound.2016.0725
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2.1. Abstract
2.1.1. Objective:
Diabetes affects more than 29 million individuals in the United States resulting in
$245 billion in healthcare costs. Approximately 15% of these individuals will develop a
chronic, non-healing foot ulcer (DFU), and if untreated, may lead to amputation of part
or the entire lower limb. The current treatments for DFU are expensive, have significant
side-effects, and result in non-compliant patients. A new topical treatment is described
that enhances wound repair and more importantly, focuses on underlying diabetic
pathways.
2.1.2. Approach:
The efficacy of topical naltrexone (NTX), an opioid receptor antagonist, and
Regranex® was compared in preclinical studies using type 1 diabetic (T1D) rats. Dorsal
cutaneous wounds were treated topically with 0.03% NTX, Regranex®, or moisturizing
cream alone. Wound closure, DNA synthesis, and cytokine production were monitored.
2.1.3. Results:
Wound closure rates with topical NTX in diabetic rats were comparable to
Regranex®. Topical NTX accelerated DNA synthesis as measured by BrdU incorporation,
enhanced platelet derived growth factor (PDGF) expression, and increased angiogenesis
as measured by vascular endothelial growth factor (VEGF) expression. Regranex® had
little effect on DNA synthesis and VEGF expression relative to vehicle-treated wounds,
and only temporarily increased PDGF expression. Neither treatment altered fibroblast
growth factor expression over the four day observation period.
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2.1.4. Innovation and Conclusion:
The data suggest that blockade of the OGF-OGFr axis utilizing 0.03% NTX cream is
a safe, inexpensive and effective alternative for treatment for diabetic wounds. NTX also
increased cell replication and angiogenesis that are two important components of wound
healing. In head-to-head comparisons with Regranex, NTX was equally effective.
2.1. Introduction
More than 29 million individuals in the United States are diagnosed with diabetes,
with type 1 diabetes accounting for approximately 5-10% [1]. It is estimated that an
additional 37% of the population have signs of pre-diabetes
[https://www.sciencedaily.com/releases/2015/09/150908112428.htm]. The estimated
annual healthcare costs exceed $245 billion annually [1-3]. In addition to treatment of the
disease itself, diabetes is accompanied by complications that increase healthcare costs
and reduce the quality of life [3-5]. Several non-life threatening complications involve
delayed epithelialization and/or impaired sensitivity such as dry eye, corneal keratopathy
and delayed cutaneous wound closure, often manifested as diabetic foot ulcers (DFU).
Impaired cutaneous wound healing, even if treated, can result in chronic lesions, ulcers,
epithelial erosion, and amputation of the extremities [1-7]. Approximately 50% of the
170 million people worldwide with diabetes experience complications associated with
delayed cutaneous wound healing [1,2,4,5].
The processes underlying wound healing are dynamic and culminate in the
restoration of proper anatomical function of the tissue [8,9]. Wound closure occurs as a
continuum of overlapping phases that involve re-epithelialization, inflammation,
56
proliferation, and formation of granulation tissue during remodeling. Each phase is
initiated by cytokines and growth factors [7], and the secretion and expression of these
cytokines appears to be compromised in hyperglycemic individuals and animals leading
to delayed cell replication [9-15]. Many of the current therapies used for treatment of
DFU provide topical application of a cytokine or growth factor [10-12], but are not disease
modifying treatments that address underlying defects related to diabetes. Regranex® is
an FDA-approved standard of care for DFUs [16]. Preclinical and clinical studies have
demonstrated that the active ingredient of platelet-derived growth factor is effective in
wound restoration following topical application [17-19]. However, this therapy is
expensive and associated with side-effects and warnings that prevent extended usage.
There remains a need for new alternative treatments for chronic, non-healing wounds.
The novel therapy discussed in this study is safe, inexpensive, effective, and
targets one of the underlying dysregulated pathways in diabetes. The therapy utilizes
topical application of the opioid antagonist naltrexone (NTX) to block the opioid growth
factor (OGF) – OGF receptor (OGFr) regulatory pathway. Preclinical studies have shown
that this small molecular weight compound (377 MW) can be dissolved in a carrier and
topically applied to full-thickness wounds to accelerate full-thickness cutaneous wounds
in type 1 diabetic rats, as well as type 2 genetically diabetic mice (db/db), for accelerated
wound repair [13-15]. As an opioid receptor antagonist, NTX lacks intrinsic biological
activity and works by blocking interactions between an inhibitory peptide and its receptor
[20,21]. The mechanism of action involves extended blockade of the OGF-OGFr
regulatory pathway [22] that has been reported to be dysregulated in diabetes leading to
57
overexpression of the inhibitory peptide, OGF, chemically termed [Met5 enkephalin], is
an endogenous neuropeptide that inhibits cell replication. OGF levels are elevated in
human and animal models of diabetes leading to downregulation of cell proliferation and
renewal processes in wound healing [27-29]. Total opioid receptor blockade by NTX
restores the proliferating homeostasis required for tissue repair [13-15, 22-26].
In this preclinical study, a comparison of effectiveness was made between the new
NTX formulation (0.03%) and Regranex® applied once daily for treatment of cutaneous
wounds in type 1 diabetic rats. In addition to cell replication and cytokine production of
VEGF, PDGF, and FGF were evaluated in order to determine mechanism of action for both
therapies.
2.2. Clinical Problem Addressed
A major complication of diabetes is delayed closure of full thickness cutaneous
wounds. The standard of care therapy, Regranex, works by increasing PDGF, is
expensive and is associated with severe side-effects including cancer, and even death.
An alternative topical therapy that has been shown preclinically to be safe and effective
is NTX. NTX targets the underlying pathophysiology of diabetes and enhances cell
replication at all stages of wound remodeling. NTX is safe, inexpensive, and effectively
targets the disease-modifying pathways in this comparison study of wound closure in
Type 1 diabetic rats. These data support large clinical trials and FDA-approval for this
topical treatment.
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2.3. Materials and Methods
2.4.1. Animals and Induction of Diabetes
Adult male Sprague-Dawley rats (Charles River Laboratories, Wilmington, MA)
were purchased at 6 weeks of age and housed in AAALAC approved facilities with water
and food (2018 Global Rodent Diet, Teklad®, Indianapolis, IN) provided ad libitum. All
protocols were approved by the IACUC at Penn State University College of Medicine, and
conformed to guidelines of the National Institutes of Health.
At six weeks of age, rats were rendered hyperglycemic by two consecutive
injections (i.p.) of 40 mg/kg streptozotocin (STZ, Sigma, St. Louis, MO) dissolved in citrate
buffer (pH 4.5). Normal rats received only i.p. injections of citrate buffer. This regimen
produced insulin-dependent type 1 diabetes (T1D) within 4-5 days. Although this model
of hyperglycemia and subsequent delayed wound healing does not mimic the long-term
chronic, pressure wounds associated with human diabetes, the model lends itself to study
of mechanism and therapeutic response [30].
Body weights were recorded periodically. Blood glucose measurements were
taken using a True Track Smart System glucometer (Home Diagnostics, Ft. Lauderdale, FL).
Three independent experiments were performed over a period of 9 months; each
experiment had approximately 15 rats rendered hyperglycemic. In each experiment, rats
remained hyperglycemic (blood glucose >350 mg/dL) for 6 weeks prior to study without
insulin supplementation. At the start of each experiment if the rat had blood glucose
measurements greater than 600 mg/dl or appeared lethargic and unwilling to eat, the
animal was not included in the experiment.
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2.4.2. Cutaneous wound surgery
Full thickness cutaneous wounds were created as described previously [13,14]. In
brief, on the day prior to surgery, the dorsum of each rat was shaved using an electric
razor followed by application of Nair® to remove all hair. Rats were immobilized by i.p.
injection of a mixture of ketamine (60 mg/kg; Ketaject®), xylazine (10 mg/kg,
TranquiVed®, Vedco), and acepromazine (1 mg/kg; acepromazine maleate; Vedco). Four
excisional circular (6 mm diameter) skin wounds were created 1 cm off the midline under
sterile conditions on each rat. Wounds were created to the level of the panniculus muscle
using disposable biopsy punches (Accuderm). After surgery, wounds were swabbed with
an antiseptic surgical scrub and left without dressing. All surgeries were conducted
between 0800-1100 h to alleviate the potential effects of diurnal rhythm.
2.4.3. Wound Treatment and Closure
Topical treatment of wounds with NTX, Regranex®, or vehicle was randomized for
each rat in order to control for placement of wound and treatment response. NTX was
dissolved in sterile saline (v/v) for a final dosage of 0.03% NTX in Neutrogena moisturizing
cream, 0.1 ml sterile saline was dissolved in Neutrogena cream, and Regranex® was used
directly from the pharmaceutical-grade tube. Each formulation was applied once daily (1X)
at 0900 h. The fourth wound received one of the treatments in a random manner.
Residual wound sizes were visualized with 20x magnification on an Olympus BH2-
RFCA microscope, and photographed using Spot Advanced software; the digital camera
was placed on a stable tripod located approximately 15 cm above the animal.
Measurements were calibrated with a ruler photographed adjacent to the wounds.
60
Animals were sedated in a regulated vaporizer with a 3% isoflurane- oxygen mixture and
photographs taken immediately after surgery (day 0) and every other day for two weeks.
Areal analysis of each wound was performed using Image J software, and the percent area
of residual defect was calculated.
2.4.4 BrdU-labeling and DNA synthesis
To assess cell replication, rats were injected with bromodeoxyuridine (100 mg/kg,
0.2ml BrdU, Sigma-Aldrich) at 6 hr and 3 hr prior to euthanasia on day 4. Tissue sections
were stained with anti-BrdU antibody (1:50, Invitrogen) followed by monoclonal
secondary antibody (1:2000, Invitrogen) and counterstained with hematoxylin. BrdU
labeling indexes were determined as the percentage of positive BrdU-labeled basal
epithelial cells per total basal epithelial cells superficial to the wound site.
2.4.5 Histological analysis and immunohistochemistry
On days 1, 2, and 4 following initial surgery, skin was harvested from rats. A 3-cm2
region of skin encompassing the original wound was removed for at least 6
wounds/treatment group, bisected, fixed in 10% neutral buffered formalin and processed
for paraffin embedding. Skin sections (10 μm thick) were stained with hematoxylin-eosin
for general assessment of toxicity and pathology. Angiogenesis monitored by expression
of VEGF was performed by immunohistochemical staining with polyclonal antibodies to
VEGF (1:200, sc-152, Santa Cruz) [31,32]. Other cytokine markers for wound repair were
evaluated by immunohistochemical staining with polyclonal antibodies to FGF-2 (1:500,
Ab106245, Abcam, Cambridge, MA) and PDGF (1:75, Ab21234, Abcam, Cambridge, MA);
all primary reactions were followed by staining with goat anti-rabbit (1:1000;
61
ThermoFisher) secondary antibodies. Antibody retrieval was completed by placing slides
in a pressure cooker for 3 min in sodium citrate buffer or EDTA buffer.
2.4. Statistical Analysis
Body weights and glucose measurements, as well as epithelial thicknesses in
unwounded animals, were evaluated using the Student’s two-tailed t-test. Residual
wound areas, epithelial thicknesses, and BrdU labeling indexes were analyzed using
analysis of variance (ANOVA) with subsequent planned comparisons made using
Newman-Keuls tests. A statistical power analysis for the number of wounds treated, as
well as the number of sections per wound required for morphological reliability was based
on previous experiments. Cytokine expression levels were semi-quantitatively assessed
by either measuring the area of staining or by counting the number of positively stained
cells within a grid.
2.5. Results
2.6.1. Body Weight and Blood Glucose Measurements
The mean weight of all male rats was 170 ± 2 g at the start of experimentation.
Approximately six weeks after STZ injection, the diabetic animals weighed 324 ± 7 g in
comparison to normal controls weighing 399 ± 18 g. Diabetic rats had significantly
elevated blood glucose levels within 2-3 days of STZ injections (~540 mg/dL), and by the
initiation of wound surgery, glucose levels in the DB rats were 563 ±12 mg/dL in
comparison to normal, non-diabetic with glucose levels of 124 ± 6 mg/dL.
62
2.6.2. Full-thickness Wound Closure
Overall analyses of wound closure indicated that wounds in diabetic rats healed
more slowly than those in normal animals. Comparison of Regranex® and NTX treatments
in T1D rats revealed that both treatments accelerated wound closure (Figure 2.1.A, B).
Evaluation of residual wound size over a 14 day period demonstrated that within 48 hr of
treatment, wound sizes in the DB/R and DB/NTX groups were smaller than those in the
DB/Vehicle group, with the mean DB/R group reaching statistical significance. At 4, 8 and
10 days, both treatment groups had wound areas that were significantly smaller than
those in the DB/Vehicle group. By day 12, the DB/NTX group was statistically smaller,
although the wound size was virtually negligible. At no time did the DB/NTX and DB/R
groups demonstrate differences in residual wound size or rates of closure.
63
Figure 2.1. Wound closure is accelerated by treatment of topical NTX or Regranex®. (A)
Photographs of full-thickness wounds following application of topical NTX, Regranex®, or Vehicle
to type 1 diabetic (DB) rats. Wounds (5 mm diameter) were created on the dorsum and treated
once daily (0.05 ml) with 10-5 M NTX (DB/NTX) or saline (DB/Vehicle) dissolved in Neutrogena
moisturizing cream, or Regranex® (DB/R). Photographs were taken immediately after surgery
(day 0) and on days 4, 8, and 12. Bar = 5 mm. (B) Residual wound areas (%) following surgical
wounding on the dorsum of DB mice receiving saline dissolved in Neutrogena moisturizing cream
(Normal + Vehicle), 10-5 M NTX dissolved in Neutrogena moisturizing cream (DB/NTX), or
Regranex® (DB/R). Significantly different between DB/Vehicle and NTX or Regranex® treatment
at *P<0.05, **P<0.01. Rate of healing or residual wound size did not differ between NTX-
treatment and Regranex®.
64
2.6.3. Histological Analysis of Skin
Tissue sections of wounds receiving all 3 treatments and stained with hematoxylin
and eosin revealed that neither Regranex® nor NTX distorted wound closure or appeared
to have abnormal pathology.
2.5.4. DNA Labeling Indexes
Given that most of the processes involved with wound closure require cell
replication, levels of DNA synthesis were evaluated to determine mechanism of action for
NTX and Regranex® (Figure 2.2.). Evaluation of wounds from normal rats treated with
vehicle revealed BrdU labeling indexes of 47.6 ± 3.5% in comparison to those with 29.3
±1.9% DNA incorporation in diabetic rats receiving vehicle. However, BrdU incorporation
in wounds of diabetic rats receiving NTX demonstrated 41.9 ± 2.3% labeling, a 43%
increase over DB/Vehicle rats (p<0.001), and comparable to that of normal animals.
Regranex® treatment had little effect on cell proliferation as the DNA synthesis rates were
21.0 ± 3.6, nearly 50% less than naltrexone treatment.
65
Figure 2.2. Cell proliferation in the basal epithelial layer in renewed skin. (A) Photomicrographs
of BrdU labeling in the basal layer of epithelium positioned over the granulation tissue of closed
wounds. Type 1 diabetic (DB) rats were treated with 10-5 M NTX (DB/NTX) or saline (DB/Vehicle)
dissolved in Neutrogena moisturizing cream, or Regranex® (DB/R) for 4 days. A separate group
of Normal rats received daily application of vehicle (N/Vehicle). Bar = 50 µm. (B) BrdU labeling
indexes (%) were calculated from the number of BrdU positive basal cells relative to the total
number of basal cells in sections of skin treated topically with either NTX or saline dissolved in
cream, or Regranex®; N/Vehicle rats received vehicle only. Values represent mean ± SEM.
Significantly different from N/Vehicle group at ***P<0.001; significantly different from
DB/Vehicle at ++P<0.01 and +++P<0.001.
66
2.5.5. Tissue Pathology and Immunohistochemical Studies
Hematoxylin and eosin stained sections of wounds did not show significant cell
death or structural pathology in response to treatment with either 0.03% NTX or
Regranex®.
2.5.6. PDGF Expression
Positive stained PDGF cells were located at the edge of the wound and deep in the
wound matrix, embedded within the granulation tissue. Cells were counted within a grid
and presented as the number of cells per mm2. Within 1 day of treatment, groups of DB
animals receiving either Regranex® or NTX had more than 1.5-fold (p<0.01) more PDGF+
stained cells than vehicle treated wounds. The elevation in PDGF+ cells continued on day
2 and day 4, whereas Regranex® treatment had no further effect, and levels returned to
those observed in the DB/Vehicle rats (Figure 2.3.).
67
Figure 2.3. PDGF expression in full-thickness skin wounds from type 1 diabetic (DB) rats receiving
daily treatment of a single drop of 0.03% NTX (DB/NTX) or saline (DB/Vehicle) dissolved in
moisturizing cream, or Regranex® (DB/R) for 1, 2, and 4 days. (A) Representative
photomicrographs of granulation tissue positively stained for PDGF on day 4 following wounding
(bar = 25 μm). (B) Values represent mean ± SEM number of PDGF positive stained cells within
one mm2. Significantly different from the DB/Vehicle group at *P<0.05 and **P<0.01;
significantly different between the DB/NTX and DB/R groups at +P<0.05.
2.5.7. VEGF Expression
The percentage of VEGF-positive stained blood vessels on day 4 was 43% more for
the NTX-treated group relative to diabetic controls (Figure 2.4.). Nearly 60% of all vessels
in the NTX-treated group were positive for VEGF, relative to less than 40% of the vessels
being VEGF-positive in either the control of Regranex®-treated rats.
68
Figure 2.4. VEGF expression in full-thickness skin wounds from type 1 diabetic (DB) rats receiving
daily treatment of a single drop of topical NTX or saline dissolved in moisturizing cream, or
Regranex® (DB/R) for 1, 2, and 4 days. (A) Representative photomicrographs of granulation tissue
with microvessels positively stained for VEGF on day 4 following wounding (bar = 25 μm). (B)
Values represent the percentage (± SEM) of VEGF-positive vessels within one mm2. Significantly
different from the DB/Vehicle group at ***P<0.001; significantly different between the DB/NTX
and DB/R groups at +++P<0.001.
2.5.8. FGF-2 Staining
Following wounding and initial mast cell infiltration, cytokines including FGF-2,
PDGF and VEGF are found circulating in the blood. FGF-2 cytokines are at the highest
levels within 24 hr of wounding. Examination of tissue surrounding the wound on days 1,
2 and 4 revealed that FGF staining was comparable among all treatment groups.
69
2.6. Discussion
Delayed wound healing is a major complication associated with diabetes,
particularly uncontrolled and/or chronic diabetes such as that associated with T2D.
Pressure-related wounds and diabetic foot ulcers affect nearly half of all persons with
diabetes at some point in their lifetime. In addition to the disabling side-effects of
unhealed wounds, the diabetic patients become more vulnerable to infections that may
in fact lead to more serious medical conditions requiring amputation [1-3]. Previous
studies in our laboratory have defined mechanisms that relate to reversing delayed
wound repair, particularly in T1D rats [13-15]. These preclinical studies have
demonstrated that topical application of NTX enhances epithelial cell replication,
angiogenesis, and increases cytokine profiles related to formation of skin [13,14].
Treatment with NTX enhanced wound closure by XXXX in T1D rats relative to rats
receiving three applications of vehicle only. DNA synthesis experiments revealed that
epithelialization was accelerated, as was blood vessel formation. These preclinical studies
were also confirmed in a mouse model of T2D [33]. The db/db mouse was surgically
wounded and 10-5M NTX dissolved in Vigamox® was applied three times daily. Wound
size was markedly reduced relative to diabetic mice receiving saline in Vigamox®. This
work corroborates studies on corneal surface epithelialization where topical NTX
enhanced repair of corneal abrasions in type 1 diabetic rats and rabbits, as well as type 2
diabetic mice [34].
In the present study, a novel formulation of NTX was used - 0.03% NTX dissolved
in moisturizing cream and the application was reduced to one time daily. This regimen
70
was consistent with, or even less, than that prescribed for Vigamox® therapy. The results
with one application of 0.03% NTX correlated well with those reported for 3 daily
applications of 10-5 M NTX [13-15]. In comparison to Regranex®, wound healing was
comparable in the T1D rat and both treatments accelerated wound closure relative to rats
receiving vehicle. Moreover, NTX targeted an underlying mechanisms associated with
diabetic complications by blocking the interfacing of OGF and its overexpression in
diabetes and OGFr. This overexpression of OGF inhibits cell replication and thus delays
many of the phases of wound closure. BrdU-labeling of the rats demonstrated that topical
0.03% NTX accelerated DNA synthesis and epithelial replication relative to both
Regranex® treatment and vehicle controls. Evaluation of cytokine expression
demonstrated that NTX also elevated VEGF expression whereas Regranex® had no effect
of this angiogenesis marker. Both Regranex® and NTX increased PDGF expression, and
NTX extended the enhanced expression for 48 hr relative to the effects seen following
Regranex® treatment that lasted only one day. FGF-2 expression did not change, at least
at the time points evaluated in these experiments.
The present investigation extends the work demonstrating that continuous
blockade of the OGF and OGFr regulatory pathway using NTX is an effective novel therapy
for non-healing ulcers in individuals with diabetes. The new formulation of 0.03% NTX
applied topically once daily substantiates an alternative, effective therapy for DFUs, and
lends support for clinical trials. Given that the population of diabetes is approaching 29
million in the United States [1], there is an urgent need to development safe, inexpensive,
71
and effective treatments for one of the many complications associated with the chronic
disorder.
2.7. Innovation
Topical application of an opioid antagonist for treatment of cutaneous wounds is
as effective as the standard of care and provides a safe, inexpensive, and effective
therapeutic for wound care in diabetes.
72
2.8. Key Findings
• Topical application of NTX accelerates closure of full thickness cutaneous wounds in
T1D rats
• Topical application of NTX heals full thickness cutaneous wounds at a rate comparable
to that of the standard care, Regranex®
• Topical NTX increases DNA synthesis in basal epithelial cells of the skin, whereas
Regranex® has little or no effect on cell replication
• Topical NTX increases PDGF+ stained cell number
• Topical NTX increases angiogenesis based on VEGF+ stained vessels
• Blockade of the OGF-OGFr axis with NTX targets an underlying pathway in diabetes
• Preclinical data support proof-of-concept clinical trials using topical NTX as a safe,
effective, and inexpensive method of treatment of diabetic foot ulcers
2.9. Acknowledgments and Funding Sources
Work was supported in part by private funding to the laboratories of Drs Zagon and
McLaughlin.
2.10. Author Disclosure and Ghostwriting: None
2.11. Abbreviations and Acronyms
BrdU, 5-bromo-2’-deoxyuridine
FGF-2, fibroblast growth factor -2
NTX, naltrexone
OGF, opioid growth factor
OGFr, opioid growth factor receptor
73
PDGF, platelet-derived growth factor
VEGF, vascular endothelial growth factor
74
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[7] Werner S, Grose R. Regulation of wound healing by growth factors and cytokines. Physiol.
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[8] Stadelmann WK, Digenis AG, Tobin GR. Physiology and healing dynamics of chronic
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[9] Gurtner GCX, Werner S, Barrandon Y, Longaker MT. Wound repair and regeneration.
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[10] Lim Y-C, Bhatt MP, Kwon M-H, Park D, Na SH, Kim YM, Ha KS. Proinsulin C-peptide prevents
impaired wound healing by activating angiogenesis in diabetes. J Invest Dermatol 135:269-
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signaling prevents delayed wound healing in diabetes by attenuating the production of IL-
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[13] McLaughlin PJ, Pothering CA, Immonen JA, Zagon IS. Topical treatment with the opioid
antagonist naltrexone facilitates closure of full-thickness wounds in diabetic rats. Exp Biol
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[14] McLaughlin PJ, Immonen JA, Zagon IS. Topical naltrexone accelerates full-thickness wound
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[15] Immonen, JA, McLaughlin PJ, Zagon IS. Topical treatment with the opioid antagonist
naltrexone accelerates the remodeling phase of full-thickness wound healing in type 1
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[16] http://www.regranex.com/ Faster Healing of Diabetic Neuropathic Ulcers. Accessed
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[17] Chan RK, Liu PH, Pietramaggiori G et al. Effect of recombinant platelet-derived growth
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[18] Niezgoda JA, Van Gils CC, Frykberg RG et al. Randomized clinical trial comparing OASIS
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[19] Howard JD, Sarojini H, Wan R, Chien S. Rapid granulation tissue regeneration by
intracellular ATP delivery-A comparison with Regranex. PLoS One 9:e91787 2014.
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[24] Zagon IS, McLaughlin PJ. Naltrexone modulates tumor response in mice with
neuroblastoma. Science 1983;221:671–3.
[25] Zagon IS, McLaughlin PJ. Duration of opiate receptor blockade determines tumorigenic
response in mice with neuroblastoma: a role for endogenous opioid systems in cancer. Life
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[26] Donahue RD, McLaughlin PJ, Zagon IS. Cell proliferation of human ovarian cancer is
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[27] Fallucca F, G Tonnarini, N Di Biase, M D'Alessandro, M Negri. Plasma met-enkephalin levels
in diabetic patients: Influence of autonomic neuropathy. Metabolism 45:1065-1068, 1996.
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[28] Negri M, G Tonnarini, M D'Alessandro, F Fallucca. Plasma met-enkephalin in type 1
diabetes. Metabolism 41:460-461, 1992. PMID:1588823
[29] Timmers K, NR Voyles, C Zalenski, S Wilkins, L Recant. Altered β-endorphin, met- and leu-
enkephalins, and enkephalin-containing peptides in pancreas and pituitary of genetically
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[30] King A, Bowe J. Animal models for diabetes: understanding the pathogenesis and finding
new treatments. Biochem Pharmacol 99:1-10, 2016.
[31] Van der Loos CM, Meijer-Jorna LB, Broekmans MEC, Pleogmakers HPHM, Teeling P, de
Boer OJ, van der Wal AC. Anti-human vascular endothelial growth factor (VEGF) antibody
selection for immunohistochemical staining of proliferating blood vessels. J Histochem
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[32] Lim Y-C, Bhatt MP, Kwon M-H, et al. Preinsulin C-peptide prevents impaired wound healing
by activating angiogenesis in diabetes. J Invest Dermatol 135:269-278, 2015.
[33] Klocek MS, Sassani JW, McLaughlin PJ, Zagon IS. Topically applied naltrexone restores
corneal reepithelialization in diabetic rats. J Ocul Pharmacol Ther 2007;23:89-102.
[34] Sassani J.W., McLaughlin PJ, Zagon IS. The Yin and Yang of the opioid growth regulatory
system: Focus on diabetes: The Lorenz E. Zimmerman Tribute Lecture. J. Diabetes Res.,
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[35] Zagon I.S., J.W. Sassani, J.A. Immonen and P.J. McLaughlin. 2014. Ocular surface
abnormalities related to Type 2 diabetes are reversed by the opioid antagonist naltrexone.
Clin. Exp. Ophthalmol. 42:159-168. PMID: 23777539.
[36] Immonen, J.A., I.S. Zagon, and P.J. McLaughlin. 2014. Topical naltrexone as treatment for
type 2 diabetic cutaneous wounds. Advances Wound Care 3: 419-427. PMID:24940556
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pathway by naltrexone accelerates fibroblast proliferation and wound healing. Exp. Biol.
Med. 239:1300-1309. PMID:25050485
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2.13. About the authors
Patricia J. McLaughlin, M.S., D.Ed., is Professor of Neural and Behavioral Science at Penn
State University College of Medicine.
Jarrett Cain, DPM is a podiatric surgeon at Penn State Hershey and co-Director of the Foot
Clinic. Dr. Cain has an active service treating diabetic foot ulcers and other complications
from diabetes.
Michelle Titunick, BA completed these experiments as a portion of her doctoral research
in the Anatomy Graduate Program at Penn State University College of Medicine.
Ian S. Zagon, M.S., Ph.D, is Distinguished University Professor at Penn State University
College of Medicine.
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CHAPTER 3: THE OPIOID GROWTH FACTOR-OPIOID GROWTH FACTOR RECEPTOR
AND DIABETIC BONE COMPOSITION
M.B. Titunick, J.D. Cain, I.S. Zagon, P.J. McLaughlin
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3.1. Abstract
3.1.1. Background:
Complications from diabetes, a disease affecting more than 29 million individuals
in the United States, include increased fracture rates and delays in bone fracture repair
that contribute to the healthcare costs of $322 billion. Treatments to accelerate bone
healing are needed.
3.1.2. Methods:
Type 1 diabetes was induced in rats by injection of streptozotocin. Open fractures
were created in the right femur of 5 week hyperglycemic animals and fixed internally.
Naltrexone (5x10-5 M) was dissolved in calcium sulfate solution used for fixation and
applied generously to the fracture site. Animals were allowed to recover for 7 days prior
to euthanasia. Callus tissue and bone were prepared for morphological studies. Tissues
were immunohistochemically stained for expression of opioid growth factor (OGF), OGF
receptor (OGFr), as well as for osteoclasts, cartilage and bone by staining with tartrate
resistant alkaline phosphatase and safranin O, respectively. Antibodies to osteocalcin and
Ki-67 were used to assess osteoblasts and proliferation rates.
3.1.3. Results:
Semiquantitative analyses of OGF and OGFr indicated the presence of both
peptide and receptor throughout development in rat bone tissue, and increased
expression levels in diabetic rat bone. Importantly, serum levels of OGF, chemically
termed [Met5]-enkephalin, by ELISA indicated elevated serum OGF levels in diabetic rats
relative to values in non-diabetic rats. Diabetic bone was diminished with respect to area
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of cartilage, proliferative cells, and VEGF+ cells. Analysis of callus tissue revealed an
increase in cartilage and bone, and compensatory decrease in granulation tissue in NTX-
treated diabetic fractures compared to vehicle-treated diabetic fractures.
3.1.4. Conclusions:
Diabetes is associated with defective levels of the inhibitory peptide OGF leading
to delayed proliferation. Bone healing processes were supported by addition of NTX to
block the interaction of OGF and OGFr. These data suggest that additional studies are
warranted to assess the value of NTX as a therapeutic in diabetic bone repair.
3.1.5. Keywords:
Naltrexone, type 1 diabetes, bone composition, enkephalin, femur fracture
3.2. Abbreviations:
DAPI, 4’,6- diamidino-2-phenylindole
DB, type 1 diabetes
ELISA, enzyme-linked immunosorbent assay
NTX, naltrexone hydrochlorid
OGF, opioid growth factor
OGFr, opioid growth factor receptor
PBS, phosphate buffered saline
SEM, standard error of the mean
T1D, type 1 diabetes
TRAP, tartrate resistant alkaline phosphatase
83
3.3. Introduction
An estimated 30.3 million people in the U.S. have diagnosed or undiagnosed
diabetes, of which 5-10% is type 1 diabetes (T1D) [1]. According to the 2017 report by
the Center for Disease Control, healthcare costs for diabetes costs in the U.S. are
estimated to be $322 billion [2]. Complications arising from diabetes include poor wound
healing, increased risk of bone fracture, and delayed fracture healing [1-4]. In human
studies, diabetes has been associated with decreased linear bone growth, lower bone
mineral density, increased fracture risk, and poor bone healing [5-9]. Preclinical changes
in bone resorption, trabecular connectivity, bone porosity, and spicule/marrow space
ratio, as well as decreased osteoblastic recruitment have been reported in animal models
of diabetes [10, 11]. Collagen reduction was noted within two weeks post diabetes
induction in a rat model of diabetes followed by prolonged fracture callus maturation and
delayed bone bridging [10]. Complications associated with diabetes in mice have also
been shown to disrupt normal bone formation by reducing cellular processes required for
osteoblast formation and differentiation [11]. Specific causes of these diabetic
complications are unclear, but the high cellular glucose levels that increase reactive
oxygen species and pro-inflammatory cytokine release may be contributing factors to
defects in bone repair [12, 13].
Current therapies to increase bone quality are not completely effective [14-16].
Despite the lack of a direct correlation between bone density and risk for fracture in type
2 diabetes, calcium supplementation, strict regulation of insulin and glucose, diet, and
exercise are often recommended to limit the risk of osteopenia and osteoporosis [14,15].
84
These health initiatives are secondary to the primary complication of delayed bone repair;
thus, there remains an unmet medical need to identify specific treatments for bone
healing in diabetic individuals.
Another modifier of the healing process is the neuropeptide methionine
enkephalin or opioid growth factor (OGF) [17]. OGF is an endogenous opioid peptide and
negative growth regulator that binds selectively to OGFr to maintain cellular homeostasis.
The mechanism of action involves alteration of the p16 or p21 cyclin-dependent inhibitory
kinases that alter the G0/G1 phase of the cell cycle and inhibit DNA synthesis [18]. In
preclinical and clinical studies, increased plasma levels of [Met5]-enkephalin have been
reported in diabetic animals and humans [19-21], suggesting that the tonic interaction
impacting cell homeostasis is disrupted favoring inhibited cell replication. Blockade of the
OGF-OGFr pathway with opioid antagonists such as naltrexone (NTX) that provide a
continuous receptor blockade results in enhanced cell proliferation [22]. Studies in our
laboratory using sufficient dosages of NTX to produce a continuous blockade of OGFr have
shown efficacy in the treatment of diabetic corneal wounds [23,24] and full-thickness
cutaneous wounds in diabetic (type 1 diabetes) [25-27] and db/db mice (type 2 diabetes)
[28]. The mechanism of action in these studies indicated increased proliferation of
epithelial cells [27, 29], endothelial cells contributing to angiogenesis [25], and
granulation tissue formation [26].
In this study, we investigated the hypothesis that excess OGF ([Met5]-enkephalin)
in diabetes may contribute to changes in bone quality, and that blockade of the OGF-OGFr
axis will restore diabetic bone quality, and fracture repair processes.
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3.4. Materials and Methods
3.4.1. Animals and induction of diabetes
Male 6-week-old Sprague Dawley rats (Charles River Laboratories, Wilmington,
MA) were housed under controlled environmental conditions with food and water ad
libitum. All protocols were approved by Penn State College of Medicine Institutional
Animal and Use Committee. Type 1 diabetes was induced in a subset of rats by
intraperitoneal injection of streptozotocin (40 mg/kg; Sigma) on two consecutive days;
control rats received sodium citrate buffer only [23,27]. Serum glucose levels were
measured with a TRUETrack® glucometer (Nipro Diagnostics). Rats with a blood glucose
reading of at least 250 mg/dL were considered to be hyperglycemic.
3.4.2. Bone fracture surgical model
Under sterile conditions 37 male rats were anesthetized using a cocktail of
ketamine (50mg/kg), xylazine (5mg/kg), and acepromazine (1mg/kg). Animals were
shaved between the thigh and knee, sprayed with 70% ethanol and swabbed with iodine.
An open mid-diaphyseal fracture was created by opening the skin superficial to the femur
and cutting through the quadricep muscles to expose the distal portion of the femur. A
bore hole was created by a 22 G needle and a 25 G spinal needle was used as internal
fixation. The spinal needle was retracted from the marrow cavity to create the fracture
using a Dremel saw. The two ends were attached using the spinal needle, fixing the
needle’s end into the proximal portion of the femur. Surgical Simplex® P calcium sulfate
with either 5x10-5M NTX or sterile water was applied to the fracture bone. The calcium
sulfate degraded over time and released naltrexone in situ. The fracture site was closed
86
and skin sutured; animals were given carprofen (0.2ml/100g) during surgery and as
needed following the procedure. The rats were euthanized 1 week after surgery with
Euthasol (Virbac Animal Health).
3.4.3. Immunohistochemistry
Femurs were fixed in 10% neutral buffered formalin for 3 days and placed in 14%
ethylenediaminetetra-acetic acid for 2-3 weeks to decalcify. Tissue was dehydrated in
increasing dilutions of ethanol and xylene, embedded in paraffin, and sectioned at 5 µm.
The sections were rehydrated and incubated with pepsin for 10 minutes for antigen
retrieval. Following washes, tissue was blocked with bovine serum albumin for 1 hour.
Primary antibodies (1:200 OGF, 1:200 OGFr, 1:500 osteocalcin) were incubated overnight
at 4°C; Ki67 (1:300) and VEGF (1:100) were incubated for 30 min at room temperature.
Anti-OGF was generated in our laboratory [30], and all other antibodies were purchased
from the following sources: OGFr (Bethyl Laboratories, Montgomery, TX), Ki67 (Merck
Millipore, Burlington, MA), osteocalcin (Santa Cruz Biotechnology, Inc, Dallas TX), and
VEGF from Neo Markers (ThermoFisher Scientific; Waltham, MA). Following primary
antibody incubation, tissues were washed and incubated with goat anti-rabbit secondary
antibody (Alexa Fluor® 568, A11011, ThermoFisher) for 2 hours. Coverslips were
mounted using 50:50 glycerol:PBS with DAPI (1:2000). Intact bone was assessed at the
mid-shaft, whereas fractured bone was evaluated within the callus. Fluorescent images
were taken with an Olympus IX81 and Spot RT3 camera, and cell number evaluated in 3-
5 sections per bone, 3-6 bones per treatment group. Semiquantitative measurements of
TRITC intensity were assessed using Slidebook 5.0 (Silicon Graphics).
87
3.4.4. Histomorphometry
To assess composition of the callus, bones were processed as described above for
paraffin embedding, and sections stained with hematoxylin, fast green, and safranin O
[31] to determine the percent area of granulation tissue (where present), cartilage, and
bone. Adjacent sections were stained with tartrate resistant alkaline phosphatase (TRAP)
by staining with naphthol AS-MX phosphate, Fast Red (>1 hour) and counterstained with
methyl green to evaluate the number of osteoclasts [32]. The percentage of cartilage or
bone was assessed in intact femur, as well as in the callus region of fractured bone by
placing a grid adjacent to the fracture edge. Area measurements were collected to
determine the percentage of cartilage per grid, as well as calculation of cartilage for the
entire area of the callus. Stained sections were photographed on an Olympus BX51 with
a Spot RTKE camera. Areal analysis of safranin O and TRAP were assessed using ImageJ
[33].
3.4.5. Measurement of serum enkephalin
Blood samples from T1D and normal, non-diabetic rats were collected
immediately post-mortem and allowed to coagulate on ice; serum was separated and
frozen at -80oC until assayed. Levels of [Met5]-enkephalin were measured in duplicate
using an ELISA kit (MyBioSource.com; MBS9342519).
3.4.6. Statistical analyses
Data were analyzed using GraphPad Prism 7.0 software (Graph Pad). In most
cases, data were analyzed with one-way analysis of variance (ANOVA) followed by
88
Newman-Keuls post-hoc test. Student’s two-tailed unpaired t-test was used where
indicated. Significance was determined at α-value of 0.05 for all tests.
3.5. Results
3.5.1. Expression levels of OGF and OGFr during bone development
Throughout development (1 day to 4 months), there were no significant
differences in bone OGF levels (Fig.1A) with mean intensity ranging from 384.1 on day 21
to 487.5 on day 5. In contrast, OGFr levels were significantly higher immediately following
birth (1 day) in comparison to all other time points through 4 months of age. OGFr
expression was 1201 ± 127.6 on day 1 in comparison to levels of 266 ± 36.5 on day 5 and
643 ± 118.5 on day 21 (Fig. 1B). Mean values on days 5, 10, 30, and 120 were significantly
less than those recorded on days 1 and 21.
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3.5.2. Levels of OGF and OGFr in bone and serum of T1D animals
Figure 3.1.: Expression of opioid growth factor (OGF) (A) and opioid growth factor
receptor (OGFr) (B) in long bone of developing rats on days 1, 5, 10, 21, 30, and 4 months
of age. Histograms represent mean ± SEM intensity of TRITC. Significantly different from
day 1 at **** p<0.0001; significantly different from day 21 at + p<0.05 and ++ p<0.01.
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Figure 3.2.: Expression of OGF and OGFr in long bone of type 1 diabetic (DB) and normal
(N) rats. Immunofluorescence images of OGF (A) and OGFr (C) stained bone with merged
DAPI staining. Scale bar=10um. Inset shows secondary only stained tissue.
Semiquantitative immunohistochemical assessment of OGF (B) and OGFr (D) in sections
of femur from DB and N rats. Histogram represents mean ± SEM intensity TRITC.
Significantly different from N values at *p<0.05. (E) Serum levels of [Met5]-enkephalin
(ng/ml) in DB and N rats assayed by ELISA. Values represent mean ± SEM. Diabetic serum
levels of OGF differed from normal serum levels at * p=0.0327
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Immunohistochemical staining for OGF and OGFr in intact femur tissue of 14-
week-old T1D (hyperglycemic for 6 weeks) and normoglycemic male Sprague-Dawley rats
demonstrated specific staining (Figs 2A, C). Semiquantitative analysis of TRITC
fluorescence in intact cortical bone revealed that T1D femurs exhibited a mean intensity
of OGF-TRITC staining of approximately 495 units in comparison to normal femurs with a
mean intensity of OGF-TRITC staining of 253.6 units, with a significant difference p<0.05
(Fig 2B). OGFr staining in T1D cortical bone was assessed at approximately 645 units, an
84% increase (p<0.05) from the TRITC intensity measured in cortical bone of
normoglycemic rats (Fig. 2D).
Serum levels of OGF were 3.5 ± 0.2 ng/ml in normal non-fractured rats and 4.3 ±
0.2 ng/ml in the serum of T1D rats; this represented a 23% increase over baseline or
normal values (Fig. 2E). Blood collected 7 days after fracture surgery was assayed, and
there were no differences in OGF levels in the serum.
3.5.3. Cellular composition of T1D bone
The cellular composition of intact bone tissue from T1D and normal male rats was
assessed following TRAP and revealed no significant differences in the number of
osteoclasts. Evaluation of the bone tissues with safranin O indicated a 73% decrease in
the area of calcified cartilage in diabetic rats (Fig. 3A). Tissue stained for proliferation
revealed a 66% decrease in Ki67 positive cells in diabetic bones compared to normal
bones (20%) (Fig. 3B). VEGF staining to assess the number of endothelial cells associated
with blood vessels decreased to approximately 30% in diabetic bones compared to
approximately 41% in normal bones (Fig. 3C).
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Figure 3.3.: Analyses of bone tissue and markers in DB and N adult male rats. (A) Safranin
O stained sections were analyzed for the area (percent) of cartilage. Cortical bone sections
were evaluated for the percentage of Ki67+ cells (B) and VEGF+ vessels (C). Values
represent means ± SEM for 1-5 sections/rat and 6 rats per group. Significantly different
from N values at *p<0.05 and **p<0.01.
3.5.4. Cellular composition of T1D calluses following fracture
Experiments were conducted twice with a total of 7 normal and 13 diabetic rats
monitored. Femurs collected 7 days following fracture were analyzed to assess cellular
composition in the entire callus area, as well as within a smaller grid adjacent to the
fracture edge which may represent a more actively dividing region. Tissue from intact
femurs exhibited a 73% increase in cartilage of normal femurs compared to diabetic
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femurs. There is a significant 2% increase in bone of diabetic femurs (99%) compared to
normal femurs (97%) (Fig. 4).
Figure 3.4.: Callus in N and DB rats following femur fracture. Fractures were treated with
sterile water (DB/V) or 5x10-5M NTX (DB/NTX) dissolved in calcium sulfate bone cement
and topically applied to the fracture site. (A) Photographs of safranin O stained fracture
calluses at 7 days post-fracture. Arrowheads indicate the location of a 1mm2 counting
grid. (B) Histogram represents composition (percentage) of total callus tissue 7 days post-
fracture for normal vehicle-treated (N/Vehicle), type 1 diabetic vehicle-treated
(DB/Vehicle), and type 1 diabetic naltrexone-treated (DB/NTX) fractures. No differences
were noted. Values were collected from 9-13 sections/rat and 4-6 rats per treatment
group.
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With regard to granulation tissue in fractured bone, there is 84% more granulation
tissue remaining in the callus of DB vehicle-treated rats at 7 days post-fracture than in
N/Vehicle calluses, and 52% more than in DB NTX-treated calluses (Fig. 5A). NTX-treated
DB rat calluses had 88% more cartilage than DB vehicle-treated calluses (Fig. 5B). There
was 24% less bone in DB vehicle-treated calluses than in N/V calluses and 35% more bone
in DB NTX-treated calluses than in DB vehicle-treated calluses (Fig. 5C). DB rats treated
with vehicle showed significantly less Ki67 staining than normal rat bones (Fig. 6). NTX
treatment of DB rats increased Ki67 staining of the callus by 35% compared to DB rats
treated with vehicle only (32%) (Fig. 6).
Blood was collected 7days after fracture. OGF serum levels increased 28% in DB
rats with fractures treated with vehicle compared to the serum of DB rats without a
fracture (4.3 ng/ml); OGF serum levels were 13% less in DB rats treated with NTX relative
to that recorded for diabetic vehicle-treated rats.
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Figure 3.5.: Composition of callus tissue measured at the fracture site. Areas (percent)
of granulation tissue (A), cartilage (B), and (C) bone within 1mm2 grids of callus tissue
adjacent to fracture site as indicated by the arrowheads in Figure 3.3. at 7 days post-
fracture for normal vehicle-treated (N/Vehicle), type 1 diabetic vehicle-treated
(DB/Vehicle), and type 1 diabetic naltrexone-treated (DB/NTX) fractures. Fractures were
treated with H2O or 5x10-5M NTX within calcium sulfate bone cement. Histograms
represent means ± SEM; values were collected from multiple sections/rat and 4-6 rats per
treatment group. Significantly different from N/Vehicle at **p<0.01 or *** p<0.001;
significantly different between DB treatment groups at +p<0.05 (Student’s t-test) or +++
p<0.001.
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Figure 3.6.: Proliferative cells in callus tissue at 7 days following femur fracture in DB and
N rats. (A) Photomicrographs of Ki67+ cells in normal vehicle-treated (N/Vehicle), type 1
diabetic vehicle-treated (DB/Vehicle), and type 1 diabetic naltrexone-treated (DB/NTX)
fractured rat femurs; Bar = 10 µm. (B) Histogram represents the percent Ki67+ cells within
1mm2 grids of callus tissue adjacent to fracture site as indicated by the arrowheads in
Figure 3.3. Values represent means ± SEM. Significantly different from N/Vehicle at
*p<0.05 and significantly different between DB treatment groups at +p<0.05.
97
3.6. Discussion
While T1D is more often associated with reduced bone mass and increased risk of
fracture, type 2 diabetes is associated with increased risks of hip and foot fractures [34].
The impact of diabetes on the normal stages of bone healing has been extensively
documented to show increased required healing times that can lead to delayed union [4].
This study investigated the presence and functioning of the OGF-OGFr axis and
diabetic bone quality. OGF and OGFr are present in bone throughout development and
into adulthood. There was an increased expression of OGF in diabetic bone as well as in
the serum of diabetic rats. The OGF-selective receptor, OGFr, was also reported to be
elevated in T1D rats suggesting that this pathway, involved with homeostatic cellular
replication, is aberrant in diabetes. These findings in bone are not unique, as OGF and
OGFr levels are elevated in epithelial tissues.
Utilizing NTX to block OGFr limits the activity of OGF providing a potential
therapeutic to increase fracture healing in diabetes. In this study we began to examine
callus composition in the presence of NTX following fracture. With a follow-up of only 7
days, it was evident that topical application of NTX may have an effect on the proportion
of cartilage and bone within the callus. Histomorphometric analyses indicated that NTX-
treated diabetic fracture calluses contained more cartilage and bone and less granulation
tissue than vehicle-treated diabetic fractures. This would support our hypotheses that
NTX blocks the inhibitory action of OGF and therefore enhance the timing of bone repair.
Reports on well controlled diabetes demonstrate there is delayed fracture healing
suggesting that hyperglycemia alone is not responsible for the delay in bone healing.
98
Elevated serum levels of the inhibitory growth factor OGF may contribute to the delayed
cellular renewal required for bone fracture repair.
One of the limitations of the study is the evaluation of the treatment after only 7
days. This study was limited to 7 days post-fracture. While the initial phases of bone
healing involve stimulation by inflammatory responses of recruitment of mesenchymal
stem cells that lead to differentiation, a further study will be necessary to investigate the
time frame of bone bridging, and the mechanical properties of the bone once fully healed.
Future studies also need to measure the release rate of NTX from the calcium sulfate to
determine effective dosage level. In addition, damage to the muscle and periosteum, two
of three major blood supplies to the bone, are damaged in the open fracture model, and
potentially limit vascularity to the wound.
Despite the experimental limitations, the current data support and extend our
knowledge about the modulation of the OGF-OGFr axis and complications of diabetes. In
preclinical studies utilizing both type 1 and type 2 diabetes animal models, delayed wound
closure was reversed following topical application of NTX [26, 28]. The mechanism of
action appeared to be enhanced cell replication, with the mechanism of action utilizing
the specific OGF receptor action site [29]. The growing evidence of an increase in OGF in
diabetes and the potential for dysregulation of the OGF-OGFr regulatory axis that plays a
role in cellular homeostasis supports the need for further study on NTX as a therapeutic
agent for diabetic fracture repair. Moreover, research is encouraged to examine the use
of systemic NTX to thwart other bone diseases including osteopenia or osteoporosis.
99
3.7. Conclusions
The OGF-OGFr regulatory axis is present in long bone throughout rat
development. However, in type 1 diabetic models, OGFr is overexpressed in tissue and
OGF is overexpressed in tissue and serum possibly contributing to the decreased quality
of long bone, and the changes observed in a small window of time in callus tissue
following femur fracture. Blockade of the OGF-OGFr pathway with NTX appears to
increase cell replication and capillary formation, leading to accelerated bone repair in
diabetes.
3.8. Acknowledgements
Special thanks to Michael D. Ludwig, PhD who helped throughout the surgeries.
3.9. Author contributions
Study design: MBT, PJM; Study conduct: MBT, JDC; Data collection and analyses: MBT,
PJM
Data interpretation: MBT, PJM, JDC; Drafting, editing and approving final manuscript:
MBT, JDC, ISZ, PJM
3.10. Funding
Research was supported in part by discretionary gift funds to ISZ and PJM, as well
as a grant with the Pennsylvania Department of Health using Tobacco CURE Funds (PJM).
The Department specifically disclaims responsibility for any analyses, interpretations or
conclusions.
3.11. Competing interests - None.
100
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CHAPTER 4: THE OPIOID ANTAGONIST NALTREXONE PROTECTS
BONE IN A DIABETIC RAT MODEL
M.B. Titunick, G.S. Lewis, I.S. Zagon, P.J. McLaughlin
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4.1 ABSTRACT
4.1.1. Aims:
Type 1 diabetes (DB) may result in a dysregulated Opioid Growth Factor (OGF)-
Opioid Growth Factor Receptor (OGFr) regulatory pathway. This research investigated
whether modulation of this axis by naltrexone (NTX) alters bone characteristics in DB rats.
4.1.2. Methods:
DB was induced by streptozotocin (n=16 male rats); 4 control rats received buffer.
Hyperglycemic animals were injected i.p. daily for 3 weeks with either 30 mg/kg NTX or
phosphate buffered saline; normoglycemic rats were untreated. At 21 days, bones were
processed for immunohistochemistry, μCT scanning, or 3-point bending to failure.
4.1.3. Results:
Relative to normal bone, DB decreased the strength (26%), osteocalcin expression
(17%), and Ki67 staining (33%) of femurs, and increased OGFr levels (126%). Systemic NTX
treatment increased the strength (21%) and energy absorbed (105%) in bone tissue relative
to tissues from saline-treated rats. Cortical and cross-sectional areas of femurs decreased
with diabetes.
4.1.4. Conclusions:
Bones from rats with diabetes were weaker and absorbed less energy than normal
bones, had increased OGFr expression, and decreased expression of osteocalcin and Ki67.
Three weeks of systemic NTX decreased OGFr levels and restored strength and energy
absorption, suggesting that NTX may protect bone quality in diabetes.
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4.1.5. Keywords: Naltrexone, type 1 diabetes, bone composition, enkephalin
108
4.2. INTRODUCTION
More than 9% of the U.S. population has diabetes with 5-10% of the individuals
diagnosed with type 1 diabetes[1]. The estimated healthcare cost of diabetes in the U.S.
exceeds $325 billion[2]. Complications associated with diabetes include neuropathy,
retinopathy, nephropathy, cardiovascular disease[3], poor wound healing[4], and delayed
fracture healing[5].
Over time, and particularly if uncontrolled, diabetes can decrease adolescent
linear bone growth[6], reduce bone mineral density (BMD)[7], result in poor osseous
healing[8], and increase fracture risk[9]. Type 1 diabetes is associated with decreased
osteoblast recruitment and activity. Studies have shown bone resorption to be unaltered
or decreased in experimental diabetes. Loss of collagen can be measured within 2 weeks
of diabetes induction in animals with type 1 diabetes[10], and serum alkaline phosphatase
and osteocalcin levels are lower in T1D animals than in control animals. Studies have
shown decreased trabecular bone in the tibia in both streptozotocin (STZ)-induced
diabetic mice and nonobese spontaneously diabetic mice[11].
Osteopenia accompanies the onset of type 1 diabetes[12]. Levin et al. revealed
decreased skeletal mass in 50% of type 1 and type 2 diabetes patients[13]. Guo et al.[14]
examined STZ-induced diabetic Sprague Dawley rats after 6 months of hyperglycemia and
reported that saline-treated diabetic rats had a lower relative bone volume, trabecular
number, and trabecular thickness in comparison to normoglycemic controls. Load-to-
failure, energy absorption, and stiffness were also significantly decreased in diabetic
vehicle-treated rats. In some reports insulin therapy for 8 weeks was found to be
109
ineffective at reversing defects in diabetic bone[15]. STZ-induced diabetic Wistar-Albino
rats treated with insulin for 8 weeks had significant decreases in bone mineral density,
load, and energy relative to normal rats suggesting that insulin alone cannot restore the
biomechanical properties of bone in STZ-induced T1D rats.
Naltrexone (NTX), an opioid receptor antagonist, blocks the interaction of OGF
with OGFr, and continuous blockade of the receptor leads to increased cell
proliferation[16]. Preclinical studies in our labs investigating NTX modulation of the OGF-
OGFr regulatory pathway have documented that both systemic and topical NTX enhanced
corneal epithelial wound healing in rats[17,18] without adverse effects [19]. NTX
treatment also enhanced full-thickness cutaneous wound healing in diabetic rats by
accelerating wound closure, increasing angiogenesis, and collagen production [20,21].
Based on these observations, it was hypothesized that type 1 diabetes is
associated with a dysregulated OGF-OGFr axis and that modulation of this axis by high
dose NTX may alter bone composition. This study examines the effects of a continuous
blockade of the OGF-OGFr pathway by NTX on bone composition.
4.3. MATERIALS AND METHODS
4.3.1. Animals, induction of diabetes, and treatment
Twenty 6-week-old male Sprague Dawley rats (Charles River Laboratories,
Wilmington, MA) were housed under controlled environmental conditions with food and
water available ad libitum. All protocols were approved by Penn State College of
Medicine Institutional Animal and Use Committee. Type 1 diabetes was induced in 16
rats by intraperitoneal (i.p.) injection of streptozotocin (40 mg/kg; Sigma) on two
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consecutive days; control rats (n=4) received sodium citrate buffer only [19,21]. Serum
glucose measurements were obtained using a TRUETrack® glucometer (Nipro
Diagnostics). Animals with serum glucose levels of at least 250 mg/dL were considered
hyperglycemic. Hyperglycemic rats were injected i.p. daily for 3 weeks with either 30
mg/kg naltrexone (NTX; 8 animals) or phosphate buffered saline (PBS; 8 animals); normal
animals received no treatment.
4.3.2. Three-point bending
A 225N load cell was used in conjunction with the ElectroForce LM1 Testbench®
system (Bose/TA Instruments, New Castle, DE) with standard three-point bend fixture.
The two supports were spread 1.5 cm apart, and femurs were oriented for an anterior to
posterior bend to failure. Maximum force (failure point), stiffness, and energy-to-failure
were recorded.
4.3.3. MicroCT
High-resolution images of the femur were acquired with a μCT imaging system
(vivaCT 40, Scanco, Brüttisellen, Switzerland) and analyzed based on our previously
described protocol[22]. Briefly, soft tissue was removed and the femurs were stored in
PBS until time of scan. Femurs were scanned with a voxel size of 10.5 μm. Analysis
regions were selected proximal to the distal femur growth plate (trabecular) and at the
midshaft (cortical). Standard parameters[23] calculated include bone volume (BV), total
volume (TV), relative bone volume (BV/TV), trabecular number, trabecular thickness,
trabecular separation, bone mineral density (BMD), tissue mineral density (TMD), cross-
sectional area, cortical area, cortical area fraction, cortical thickenss, and cortical tissue
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mineral density (BMD) were calculated using the Scanco software at the distal and mid-
shaft scanning regions. Seventy-five slices were used for trabecular calculations proximal
to the distal growth plate and 20 slices were used for cortical calculations at the
midshaft[22].
4.3.4. Immunohistochemistry
Bones were fixed in 10% neutral buffered formalin for 3 days and allowed to
decalcify in 14% ethylenediaminetetra-acetic acid for 2-3 weeks. The bones were
dehydrated in ethanol and xylene, embedded in paraffin, and sectioned at 5 µm. Sections
were rehydrated and underwent antigen retrieval with pepsin for 10 minutes. Following
washes, tissue was blocked using bovine serum albumin for 1 hour. Primary antibodies
requiring overnight incubation at 4°C included 1:200 OGF, 1:200 OGFr (Bethyl
Laboratories, Montgomery, TX), and 1:500 osteocalcin (Santa Cruz Biotechnology, Inc,
Dallas, TX). Ki67 (1:300; Merck Millipore, Burlington, MA) and VEGF (1:100; Neo Markers
ThermoFisher Scientific, Waltham, MA) were incubated for half an hour at room
temperature. Anti-OGF was generated in our laboratory[24]. Following washes, sections
were incubated with goat anti-rabbit secondary antibody (Alexa Fluor® 568, A11011,
ThermoFisher) for 2 hours, washed and mounted with 50:50 glycerol:PBS with DAPI
(1:2000). Double labeling of bone tissues for OGFr and osteocalcin were performed by
incubation with both primary antibodies overnight at 4°C followed by washes and
incubation with Alexa Fluor® 488 goat anti-mouse (1:1000) and Alexa Fluor® 568 goat
anti-rabbit (1:1000) for 2 hours. An Olympus IX81 and Spot RT3 camera were used to
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obtain fluorescent images. Intensity of TRITC staining was obtained semiquantitatively
using Slidebook software.
4.3.5. Histomorphometry
Bones were processed as described above for paraffin embedding, sectioned, and
stained with fast green and safranin O[26] to differentiate between bone (green) and
cartilage (red). Adjacent sections were stained using hematoxylin and eosin (H & E).
Cartilage and bone were assessed in femora using photographs taken on an Olympus
BX51 with a Spot RTKE camera. Analysis was performed using ImageJ[26].
4.3.6. Data Analyses
Data were analyzed using GraphPad Prism 7.0 software (Graph Pad) with one-way
analysis of variance (ANOVA) followed by Newman-Keuls post-hoc test. Student’s two-
tailed unpaired t-test was used where indicated; significance was determined at α-
value of 0.05.
4.4. RESULTS
4.4.1. Three-point bending
The force necessary to break normal bones averaged 95.4N (figure 1). There was
a 26% decrease in strength of diabetic vehicle-treated bones compared to normal bones,
whereas NTX increased the strength of diabetic bones by 21%. The energy-to-failure of
normal bones averaged 52.4±5.7Nmm. Diabetic vehicle-treated bones required 37% less
energy to break. In our samples, naltrexone-treated diabetic bones had a trend of being
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37% stiffer than those in diabetic rats receiving vehicle, but this did not reach significance
(p=0.064).
Figure 4.1: Diabetes altered the responses to 3-point bending to failure. Bars (means ±
SEM) represent the force (A) necessary to break bones by 3-point bending, the energy (B)
absorbed by the bones prior to breaking, and the stiffness (C) of the bones. Significantly
different from N at * p<0.05, ** p<0.01, and between diabetes treatment groups at +
p<0.05. N = normal rats; DB/V = diabetic rat receiving saline; DB/NTX = diabetic rats
receiving 30mg/kg NTX for 21 days. At least 3 specimens were tested per group
4.4.2. MicroCT
There were no significant differences between the 3 groups in trabecular μCT
measurements (table 1). Relative to normal rats, cortical μCT measurements (table 2)
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revealed significant decreases of 21% and 18% in cross-sectional area (inside periosteal
envelope) of vehicle-treated rats with diabetes and NTX-treated diabetic rats,
respectively, as well as significant decreases of 22% and 16% in cortical area of diabetic
vehicle-treated rats and NTX-treated diabetic rats, respectively.
TV (mm3) BV (mm3) BV/TV (%)
trabecular number (1/mm)
trabecular thickness (mm)
trabecular separation (mm)
BMD (mgHA/ cm3)
TMD (mgHA/ cm3)
N 11.01±
0.58 3.06±
0.46 31.07±
0.04 7.15±
0.64 0.071±
0.002 0.16±
0.01 287±
22 753±
6
DB/V 9.95±
0.40 2.06±
0.84 29.50±
0.07 6.26±
1.12 0.067±
0.004 0.20±
0.03 273±
47 774±
12
DB/NTX 10.38±
0.53 4.63±
0.78 44.45±
0.07 8.38±
1.45 0.070±
0.002 0.15±
0.04 368±
45 764±
8 Table 4.1: Mean ± SEM trabecular measurements from μCT evaluation. N = normal rats;
DB/V = diabetic rats receiving saline; DB/NTX = diabetic rats receiving 30mg/kg NTX for
21 days. TV = total volume; BV = bone volume; BV/TV = relative bone volume; BMD =
bone mineral density; TMD = tissue mineral density. n ≥ 5 per group.
total cross-sectional area (mm)
cortical area (mm2)
cortical area fraction (%)
cortical thickness (mm)
TMD (mgHA/cm3)
N 2.12±0.05 1.34±0.04 63.45±0.02 0.50±0.02 1099±7.3DB/V 1.68±0.06a 1.05±0.05b 62.61±0.02 0.49±0.02 1112±4.3DB/NTX 1.74±0.06a 1.13±0.04b 64.68±0.01 0.51±0.03 1111±8.7
Table 4.2: Mean ± SEM cortical measurements from μCT evaluation. Significantly
different from N/V at ap<.001 and bp<.01. N = normal rats; DB/V = diabetic rats receiving
saline; DB/NTX = diabetic rats receiving 30mg/kg NTX for 21 days. TMD = tissue mineral
density. n ≥ 4 per group.
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4.4.3. Immunohistochemistry
There was a significant increase (126%) in OGFr expression (figures 2A and 2B) in
diabetic femurs compared to that recorded in normal bone that had a mean TRITC
intensity of 84.8 units. Naltrexone-treated diabetic rats had a reduced amount (57%) of
OGFr immunostaining in comparison to diabetic vehicle-treated rats. In normal femur
tissue 31.5% of cells stained positive for osteocalcin (figures 3A and 3B), an osteoblast
marker, whereas only 26% of the cells in the femurs of diabetic rats receiving vehicle.
Although not reaching significance, NTX-treated diabetic rat bones had increased (32%)
osteocalcin staining compared to vehicle-treated bone.
Tissue sections of femora from normal rats had 48% of cells stained positive for
Ki67 (figure 4), a proliferation marker. Relative to normal values, there was a 33%
decrease in Ki67 staining in diabetic vehicle-treated rat bones and a 31% decrease in
diabetic NTX-treated bones.
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Figure 4.2: OGF and OGFr immunohistochemistry. OGF immunohistochemistry (A) and
histogram representing staining intensity of OGF in an individual cell (B); OGFr
immunohistochemistry (C) and histogram representing staining intensity of OGFr in an
individual cell (D); insets show secondary antibody only staining. Scale bar = 10μm.
Values represent means ± SEM for at least 3 specimens per group; significantly different
from N at *** p<0.001 and between different treatment groups of diabetic rats at ++
p<0.01. N = normal rats; DB/V = diabetic rats receiving saline; DB/NTX = diabetic rats
receiving 30mg/kg NTX for 21 days.
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Figure 4.3: The presence of OGFr and osteocalcin in bone tissue from normal and diabetic
rats. Immunohistochemistry double-labeling of osteocalcin and OGFr (A) and histogram
representing the percentage of osteocalcin-positive cells within a field of view (B). Scale
bar = 10μm. Values represent means ± SEM; significantly different from N at * p<0.05
(Student’s t-test). N = normal rats; DB/V = diabetic rats receiving saline; DB/NTX = diabetic
rats receiving 30mg/kg NTX for 21 days. n ≥ 7 measurements per group.
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Figure 4.4: Ki67 staining. Histogram representing the percentage of Ki67-positive cells in
a field of view. Values represent means ± SEM for at least 3 fields from at least 3 bones
per group. Significantly different from N at * p<0.05, ** p<0.01. N = normal rats; DB/V =
diabetic rats receiving saline; DB/NTX = diabetic rats receiving 30mg/kg NTX for 21 days.
4.4.4 Histomorphometry
Analyses of safranin O stained sections revealed no significant differences in the
percentage of cartilage and bone between the 3 groups, and hematoxylin-eosin stained
tissues showed no difference in the morphology, suggesting that systemic NTX treatment
did not result in any gross pathology of the bone tissue.
4.5. DISCUSSION
Diabetes affects more than 30 million Americans[1] and of those with type 1
diabetes, 50-60% have osteopenia[27,28] and 14-20% have osteoporosis[28,29]. The
mechanism connecting poor bone quality to diabetes is still unclear[30]. The results of
this study support current literature that there is a decrease in osteoblast
119
activity/maturity. There was a decrease in osteocalcin staining in vehicle-treated diabetic
rat bones compared to normal bones, suggesting a decrease in the number of mature
osteoblasts; however, systemic NTX treatment appeared to be reestablishing the
osteoblast population. Histomorphological studies using the proliferative marker Ki67
showed decreased staining in diabetic bones from animals treated with either saline or
NTX compared to normal bones. The lack of change in Ki67 may be due to the relative
lack of dynamics occurring in an intact bone. Important to the work on modulation of the
OGF-OGFr axis in bone fracture repair, OGFr staining was significantly increased in
vehicle-treated diabetic bone, whereas systemic treatment with NTX reduced the number
of receptors in diabetic femurs to that of normal bone.
Studies involving STZ-induced diabetic mice showed significant decreases in tibia,
femur, and vertebrae trabecular bone volume fraction[11,31]. Although we did not see
any significant change in trabecular bone composition it may be that the rats did not have
diabetes long enough for these changes to become significant, and a longer period of
hyperglycemia may be required to see changes in bone composition. Hamada et al. did
not see significant differences in bone mineral density between STZ-induced diabetes and
normal mice at 4 weeks post STZ injection[32]. It is also predicted that longer periods of
NTX treatment, as well as beginning NTX treatment 4-5 weeks after rats become
hyperglycemic may be important for ascertaining the role of systemic NTX on bone
composition. Guo et al.[14] and Erdal et al.[15] observed decreases in bone quality at 6
months and 8 weeks of hyperglycemia, respectively.
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4.6. CONCLUSION
In conclusion systemic NTX treatment for 21 days in hyperglycemic rats reversed
certain important deficiencies in the femur. Bone tissue in diabetes rats receiving saline
had more OGFr present suggesting the greater availability of receptors to interact with
the inhibitory peptide OGF and thus reduce cell proliferation. Ki67 staining was decreased
in diabetic vehicle-treated bones compared to normal bones indicating a decrease in
proliferative cells. Importantly, diabetic bone required less force to break under 3-point
bending, a parameter which was rescued by NTX, suggesting that blockade of the OGF-
OGFr axis may increase bone strength in diabetes.
4.7. Acknowledgements
Special thanks to Hwa Bok Wee for assistance with μCT scanning.
4.8. Author contributions
Study design: MBT, PJM; Study conduct: MBT, GSL; Data collection and analyses: MBT,
PJM
Data interpretation: MBT, PJM, GSL; Drafting, editing and approving final manuscript:
MBT, GSL, ISZ, PJM
4.9. Funding
Research was supported in part by discretionary gift funds to ISZ and PJM.
4.10. Competing interests - None.
121
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Naltrexone Restores Corneal Reepithelialization in Diabetic Rats. J. Ocul.
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with the Opioid Antagonist Naltrexone Facilitates Closure of Full-Thickness
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Naltrexone Is a Safe and Effective Alternative to Standard Treatment of Diabetic
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[29] MunozTorres, M.; Jodar, E.; EscobarJimenez, F.; LopezIbarra, P.; Luna, J. Bone
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CHAPTER 5: DISCUSSION
127
5.1. Discussion
Over 30 million Americans have diabetes, 5-10% of that population have type 1
diabetes[1,2]. Diabetes costs the U.S. over $300 billion each year[1] including the cost of
complications associated with diabetes such as non-healing foot ulcers. Diabetic foot
ulcers (DFU) are also associated with peripheral neuropathy. DFUs are often undetected
by individuals due to sensory neuropathy in addition to the fact that the initial wound is
small. Eighty-five percent of amputations in diabetics were preceded by a non-healing
ulcer[3]. High incidences of diabetic foot ulceration is not unique to North America,
although it does have the highest prevalence at 13%[3]. Non-healing foot ulcers affect
6.3% of the world’s population[3]. There needs to be research on treating not just the
complications on a superficial level but at a mechanistic level as well.
Type 1 diabetic (T1D) bone has a lower bone mineral density (BMD) at the neck of
the femur than normal bone [4]. However, the same difference is not observed at in the
lumbar spine. This slight decrease in BMD is not great enough to account for the highly
increased fracture risk among T1D patients. Microvascular complications such as
retinopathy and neuropathy, coincide with low BMD. Trabecular bone score (TBS) is an
index used to evaluate pixel variation in lumbar spine DXA images and correlates with the
trabecular bone volume to tissue ratio, trabecular number, and connectivity. Osteocalcin,
an osteoblast marker, and CTX, an osteoclast marker, were both lower in diabetic bone
than normal bone. No differences were discerned among calcium, phosphorus, and
parathyroid hormone levels[4]. A study examining trabecular bone indicated no
differences in bone formation or resorption parameters between the two groups[5].
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Computed tomography suggested lower cortical thickness and cross-sectional area of the
femur in T1D patients. There was also a larger endosteal circumference amongst the T1D
patients. Studies suggest that type 1 diabetes affects cortical bone to a greater extent
than trabecular bone[4].
Although this dissertation is focused on type 1 diabetes, it is important to briefly
note the relationship of type 2 diabetes and bone. Type 1 diabetes has lower insulin-like
growth factor-1 and higher insulin-like growth factor binding protein-1 serum levels than
type 2 diabetes and normal controls[6]. Levels of 25OHD are significantly lower in both
forms of diabetes[7]. Circulating levels of sclerostin, a protein opposing bone building,
are greater in type 2 diabetes than in either type 1 diabetes or control. Osteocalcin and
amino-terminal propeptide of pro-collagen type 1 are lower in type 2 diabetes compared
to normal controls[8]. Hyperglycemia also increases the levels of anti-osteoblastic
cytokines. Studies indicate that osteoblast precursor cells are decreased in type 2
diabetes while osteoclast precursor cells are increased. Both osteoblast and osteoclast
expression is reduced in type 2 diabetes. Type 2 diabetes medications known as
thiazolidinediones which include rosiglitazone may put patients at a higher risk of
fracture. The drug promotes peroxisome proliferator-activated receptor gamma (PPARγ)
which favors mesenchymal differentiation to adipose rather than osteoblasts[9].
In vivo and in vitro studies of rat and rabbit corneas demonstrated that modulation
of the OGF-OGFr regulatory pathway with naltrexone (NTX) effectively enhanced wound
healing and reepithelialization[10]. Both systemic and topical naltrexone can enhance
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reepithelialization of the normal rat cornea[11–14]. Topical NTX can also enhance
reepithelialization of full-thickness cutaneous wound healing[15–19] in diabetic rats.
Topical NTX is safe to use on the cornea[20]. Sprague Dawley rats received eye
drops of NTX at concentrations of 10-3, 10-4, 10-5, 10-6, or 10-7 M 4 times daily for 7 days.
No gross or histological pathology was detected. Topical NTX is also well tolerated by
humans [21]. Volunteers administered NTX eye drops of concentrations 1x10-6 M (1 drop
or 4 drops daily), 5x10-6 M (4 drops daily), 1x10-5 M (4 drops daily), 5x10-5 M (4 drops daily)
for 7 days and reported no adverse effects.
Moreover, NTX has been shown to be a safe and effective treatment for full-
thickness wound healing[19]. Specific Aim 1 showed that wounds treated with NTX
healed faster than vehicle-treated wounds surgically created on the dorsum of type 1
diabetic rats. The rate of wound closure was non-inferior to Regranex®-treated wounds,
demonstrating comparable efficacy with the standard of care. Mechanistically, NTX
increased BrdU labeling, indicative of cell proliferation, in the wound matrix, in
comparison to vehicle-treated wounds. NTX also increased expression of VEGF, required
for angiogenesis – necessary for wound healing – relative to both vehicle-treated and
Regranex®-treated wounds. The increased vascularization in NTX-treated wounds is an
important component of skin repair, as diabetes is often associated with poor vascularity
which can inhibit wound healing. A 1.5-fold increase in platelet derived growth factor
(PDGF) staining was seen at day 1 in both NTX-treated and Regranex®-treated wounds
compared to vehicle-treated wounds. The active ingredient in Regranex® is PDGF so it
may be expected that tissue treated with Regranex® would show an increase in PDGF
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expression. However, by day 2 the tissue levels of PDGF were comparable between
Regranex®-treated and vehicle-treated wounds. NTX-treated wounds at day 2 still
showed an increase in PDGF expression compared to vehicle-treated wounds.
The Regranex® study supports previous studies on corneal defect repair and full-
thickness cutaneous healing, indicating that naltrexone is an effective treatment for
diabetic healing. Naltrexone was also shown to be as effective as the standard of care,
Regranex®[19]. Benefits of using naltrexone include the safety of naltrexone as evidenced
by previous studies in the cornea and skin of rats and rabbits, as well as the decreased
cost of naltrexone. Regranex® comes with a black box warning stating that there is a
higher risk of mortality secondary to malignancy when using more than three tubes of the
gel[22]. The price differential would bring down the cost of diabetic foot and perhaps
make patients more compliant with the treatment.
The Specific Aim 1 study on full-thickness cutaneous wounds [19] lends
justification for a clinical trial to begin. There have been multiple studies performed on
both rats[15,16,19] and mice[23] indicating naltrexone is an effective treatment for full-
thickness wound healing. Safety studies on topical naltrexone have been performed on
the cornea[11] of both rats and rabbits (unpublished) indicating the safety of naltrexone.
A clinical study would be warranted for testing NTX as a topical therapy for non-healing
foot ulcers.
Specific Aim 2 established the presence of the OGF-OGFr axis in bone and the
potential for dysregulation of the pathway in diabetes. While OGF and OGFr have been
identified in bone throughout rat development and into adulthood, there is an increase
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in OGFr at days 1 and 21. The increase at day 21 may be biologically significant as this is
the point at which pups ween. There may be other demands that take priority at this
stage over bone growth causing an increase in OGFr and a decrease in bone proliferation.
There is a 95% increase in the inhibitory peptide OGF in diabetic rat bone relative to OGF
levels in normal bone. Diabetic bone showed an 84% increase in OGFr staining compared
to normal bone. Serum levels of OGF indicated a 23% increase in diabetic rats over normal
controls. Thus it appears that the OGF-OGFr regulatory pathway is dysregulated in
diabetes leading to elevated peptide and receptor interactions that may contribute to
bone loss and delayed bone healing.
The rat studies documented a 66% decrease in Ki67 staining in type 1 diabetic rat
bone compared to normal bone. Ki67 is a nuclear protein expressed during the active
phases of the cell cycle[24] thus indicating levels of cell proliferation. Vascularization was
also decreased in diabetic bone compared to normal controls, tissue with a difference of
30% in VEGF staining. During fracture repair the callus of vehicle-treated diabetic rats
retained more granulation tissue at day 7 than either diabetic naltrexone-treated and
normal controls. Diabetic vehicle-treated fractures had less cartilage within the callus
compared to normal fractures. There was also a decrease in bone seen in the callus of
the vehicle-treated diabetic fractures compared to those treated with naltrexone. When
examining the region closest to the periosteum there was 84% more granulation tissue in
diabetic vehicle-treated fracture calluses compared to normal control and 52% more than
diabetic naltrexone-treated fractures. There was 88% more cartilage in diabetic
naltrexone-treated fractures than in diabetic vehicle-treated fractures. Diabetic calluses
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had 24% less bone than normal calluses. Diabetic naltrexone-treated fractures had 35%
more bone than diabetic vehicle-treated calluses. Proliferation was also altered by
naltrexone with 35% more Ki67 staining compared to vehicle-treated diabetic fractures.
The present study corroborates that of Zagon, Wu, and McLaughlin who examined
fetal rat tissue from pregnant dams injected with NTX or sterile water [25]. Indexes of
radiolabeled thymidine were higher in fetuses from NTX-treated dams in comparison to
saline-treated controls, suggesting that prenatal NTX increased proliferation in bone; OGF
treatment was documented to decrease proliferation. Immunocytochemistry indicated
the presence of OGF and OGFr in both vertebrae and ribs of normal rat offspring. Further
evidence of the OGF-OGFr pathway reported in tissue culture studies where NTX and OGF
modulated cellular proliferation in the HT-1080 and SK-ES-1 sarcoma cell lines[26].
Data from the second aim supports the work of Liskov et al.[27], Thakur,
DeBoyace, and Margulies[28], and Petrizzi et al.[29], indicating a role for an opioid
antagonist in osteogenesis. Liskov et al. found a 2-fold increase in the thickness of the
perichondral bone cuff as well as over 6.5 times the number of dividing cells in the growth
plate of femurs. This paper neglected to look at OGFr and utilized a non-mammalian
model[27]. Thakur, DeBoyace, and Margulies used a mouse model to examine cortical
defects in tibiae. Again, naloxone was used. As in our fracture project, animals were
euthanized 7 days post-fracture. There was a 1.5-fold increase in relative bone volume
of naloxone-treated defects compared to control as well as an increase in trabecular
number. Naloxone was able to decrease the defect diameter by 20%. OGF and OGFr
were only studied in the cell culture experiments not in the in vivo experiments[28].
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Petrizzi et al. augmented naloxone treatment with calcium gluconate allowing for 4 weeks
of treatment of a metacarpal defect in sheep. There were more remodeling spaces and
fewer osteoblasts in the area of the defect, indicating more advanced stages of healing.
The authors did not look into the role of OGF and OGFr in the study[29].
In the fracture study there was significantly more bone and cartilage in diabetic
fracture calluses treated with NTX compared to those treated with vehicle only. At this
time point, 7 days post-fracture, there should still be a significant amount of cartilage. If
we were to have examined the bones at a much later timepoint and there was still an
increase in cartilage, then there would be a delay in the healing process. In addition, the
increase in bone may be from a combination of endochondral ossification and
intramembranous ossification, thus having increased cartilage and bone is reasonable.
The fracture study was limited by the short survival period of the rats. Healing
normally required 4-6 weeks, and the studies should be repeated to evaluate rate of bone
repair and strength (mechanical testing) of the bone following topical NTX application. In
addition, different methods of NTX application should be evaluated. Including micelles
or beads coated with NTX. Coating the wire in a slow-releasing material would be
beneficial in scenarios where internal stabilization or pinning is necessary. Future
research should examine angiogenesis in fracture repair and utilize db/db type 2 diabetic
mice. Type 2 diabetic mice may show differences in preference for differentiation of
newly proliferated progenitor cells toward adipose instead of osteoblasts. This could
further complicate the experiment however determining if NTX affects differentiation
would be an interesting observation. Differentiation may be increased by NTX as the
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entire process of proliferation is enhanced. Since cells are either dividing or
differentiating having a faster proliferation rate would allow cells to enter differentiation
sooner. The argument against this notion is that cells are stimulated by NTX to divide and
therefore must wait until division is finished to differentiate. This would decrease cell
differentiation overall.
Osteoclast numbers in diabetes has been reported to be increased, decreased, or
unchanged[30]. There was no significant difference in TRAP staining between diabetic
and normal bone (unpublished). It is unknown if hematopoietic cells react to NTX.
Macrophages replicate infrequently[31], and early osteoblast precursors may not
necessarily differentiate into osteoclasts. Therefore, NTX may not have as substantial an
effect on osteoclasts as it does osteoblasts. This would allow for the use of NTX to
increase osteoblastic cells without much of an increase in osteoclasts, resulting in greater
bone deposition.
Although bone loss is not prevented by maintenance of a healthy insulin level[32],
the Lin lab has used local delivery of insulin[33], vanadium[34], and manganese
chloride[35] to improve fracture healing in a rat model. Local insulin inhibits FOXO1
preventing Wnt inhibition. Vanadium[34] and manganese chloride[35] mimic insulin and
may also inhibit FOXO1. This helps to increase the number of osteoblasts. Naltrexone
also attempts to increase osteoblast numbers. Basic fibroblast growth factor normalized
diabetic fracture repair and enhanced healing in non-diabetic animals[36]. FGF was also
able to increase callus formation 3 weeks post-fracture[36]. Platelet-rich plasma high in
135
levels of mitogenic factors increased cell proliferation in diabetic rats and normalized
healing[37]. Naltrexone also has the ability to increase cell proliferation.
Kawaguchi et al.[36], also using Sprague Dawley rats, induced diabetes using STZ
and waited 2 weeks before commencing with fibula fractures. bFGF was introduced to
the fracture site within a fibrin gel. Rats were euthanized 1, 3, and 5 weeks after surgery.
Similar to our findings, Kawaguchi et al. saw intramembranous ossification under the
periosteum within the first week. The lab did not statistically compare the diabetic bone
values to those of non-diabetic rats although there appears to be a difference. bFGF
increased breaking strength and energy absorbed compared to diabetic vehicle-treated
rat bones. As seen in our study there was a decrease in breaking strength and energy
absorbed in diabetic vehicle-treated rats compared to non-diabetic rats.
Wang et al.[38] used STZ-induced diabetic Wistar rats and a closed femoral
fracture model. Fracture healing was evaluated 4 weeks post-fracture. At 4 weeks there
was no significant difference between total volume, bone volume, relative bone volume,
or trabecular thickness between diabetic and normal bones. There were significant
differences between trabecular number and trabecular separation. Load, stiffness, and
absorbed energy were decreased in diabetic bones compared to non-diabetic bones. This
is supported by results from Specific Aim 3.
Wang, Jiang, and Du[39] used rats to examine insulin therapy on Transforming
Growth Factor-Beta 1 (TGF-β1) expression in fracture calluses. Calluses were examined
1, 2, 4, 6, and 8 weeks post-fracture. As in our experiments the lab utilized an open
fracture model. Changes in TGFβ1 were present in the first week following fracture.
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Biomechanical properties were tested at 6 and 8 weeks post-fracture and showed
significant decreases in diabetic bone.
Cignachi et al.[40] used C57/BL6 mice and induced diabetes using injections of STZ.
Fractures were formed 7 days after the end of diabetes induction, creating a uni-cortical
defect. The RANKL/RANK/OPG system was evaluated using immunohistochemistry 21
days after the surgery. Similar to our results, the lab saw a significant decrease in bone
regeneration in STZ-induced diabetic animals. There was no significant difference
between the two groups in TRAP staining, RANK, OPG, or RANKL expression.
Ko et al.[41] used CD-1 mice to evaluate the mesenchymal stem cell population in
diabetic fracture healing. Diabetes was induced using STZ then mice underwent surgery
3 weeks later. Diabetic mice had 53% less new bone in fracture calluses compared with
non-diabetic mice 16 days after surgery.
Park et al.[42] used BB Wistar rats to examine femoral fracture healing treated
with insulin. Rats were euthanized 7 and 14 days post-fracture showing little change in
fracture repair. Mechanical testing at 4 weeks post-fracture showed an increase in
maximum torque to failure.
Yee et al.[43] utilized C57BL6/J mice injected with STZ as a diabetic model. Two
weeks after STZ injection closed fractures were generated and rats were euthanized 21
and 42 days later. At 21 days there was a decrease in bone volume and total volume, and
at 42 days there was a decrease in cortical bone mineral density.
Hreha et al.[35] used BB Wistar rats with a closed femoral fracture model.
Manganese chloride or saline were injected into the intramedullary canals. Femora were
137
collected at 7, 10, and 14 days post-fracture. Changes in maximum torque were seen at
4 weeks post-fracture. No differences in callus composition were seen at 7 days post-
fracture which resembles our findings. There was an increase in percent new mineralized
tissue in manganese chloride-treated bones at 10 days compared to saline controls. VEGF
and PECAM changes were not seen until day 10.
Specific Aim 3 addressed the ability to modulate the OGF-OGFr axis to prevent bone
loss associated with diabetes. The force necessary to break diabetic bones was significantly
less compared to normal bones. Naltrexone increased the strength of diabetic bones by
21% relative to vehicle-treated diabetic bones. Diabetic vehicle-treated bones absorbed
79% less energy prior to breaking than non-diabetic bones. Naltrexone increased the
bone’s integrity, requiring 105% more energy to break compared to vehicle-treated bones.
Although not statistically significant, normal and diabetic NTX-treated bones were stiffer
than diabetic vehicle-treated bones. No significant differences were seen between the 3
groups in trabecular μCT measurements. There was a decrease in cross-section and cortical
area in diabetic bone compared to non-diabetic bone. There was a 126% increase in OGFr
staining of diabetic bone compared to normal bone. Naltrexone reduced OGFr levels in
diabetic bone by 57%. There was a 26% decrease in osteocalcin staining of diabetic vehicle-
treated bones compared to normal controls. Naltrexone increased staining by 32% over
vehicle-treated bone. Diabetic vehicle-treated bone had 33% less Ki67 staining compared
to normal bone. Naltrexone did not adversely affect the morphology of the tissue, as
indicated by H & E staining.
138
The preventative study indicates there is a potential benefit to systemic
naltrexone in regards to glucose regulation. Additional studies should be conducted to
examine this phenomenon. If the study is allowed to continue past 3 weeks there may
be a more significant difference between diabetic and normal femurs, making the effect
of naltrexone more substantial. Also using systemic NTX as a treatment for bone
complications instead of a preventative by waiting a month to begin the treatment would
be another route to consider. This may result in fewer changes in the cortical bone but
may show a greater discrepancy between trabecular measurements in diabetic bone
relative to normoglycemic controls since there would be a longer duration of diabetes
and more chance for bone turnover to be slowed down. Without a trauma to spur
proliferation and repair there may be little change in NTX-treated groups. In addition, it
would be interesting to use systemic NTX as a treatment in an osteoporosis animal model.
Insulin therapy has been evaluated as a treatment for osteopenia in diabetes.
Hough et al.[44] induced diabetes in Wistar-Lewis rats with STZ and began insulin therapy
5 days later. Treatment was continued until day 48 when the rats were euthanized. There
was an increase in percent relative osteoid volume in insulin-treated tibiae compared to
that of diabetic saline-treated rats.
Guo et al.[45] injected Sprague Dawley rats with STZ to induce diabetes. Animals
were treated with a hydrogen-rich saline lavage for 3 months and rats were collected 12
weeks after the last injection. Diabetic saline-treated rats had a lower relative bone
volume, trabecular number, and trabecular thickness compared to non-diabetic controls.
There was an increase in trabecular separation as well. Ultimate load, energy absorption,
139
and stiffness were significantly decreased in diabetic vehicle-treated animals compared
to non-diabetic rats.
Erdal et al.[46] used Wistar-Albino rats injected with STZ as a model for diabetes.
Insulin treatments extended for 8 weeks. There was a significant decrease in length, cross
sectional area, bone mineral density, load, and energy in diabetic saline-treated bones
compared to non-diabetic bones. The study concluded that insulin is unable to restore
biomechanical deterioration of bone in STZ-induced T1D.
Treatments may be developed to work at various phases of the healing process.
Increasing cell proliferation and increasing vascularity are two such targets the lab looks
to utilize. Another phase prime for exploitation is the inflammatory phase. Much of the
delay in both full-thickness cutaneous wound healing and fracture repair in diabetes
relates to a prolonged inflammatory phase. There is over activation of the immune
system and this inflammatory process cannot be easily reduced to allow the subsequent
phases to begin.
In summary, NTX is an effective and safe treatment for full-thickness cutaneous
wounds and bone fractures. There appears to be merit to further exploration of
naltrexone’s protective effect in bone. The experiments supported work by various labs.
Further literature searches suggest an increase in treatment time for Specific Aims 2 and
3 may yield better results. Overall, NTX is a likely candidate as treatment for multiple
diabetic complications associated with skin and bone.
140
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Vita Michelle B. Titunick
EDUCATION:
• Ph.D. Candidate, Human Anatomy December 2018 Pennsylvania State University, College of Medicine, Hershey, PA
• Graduate School Teaching Certificate March 2018 Pennsylvania State University, College of Medicine, Hershey, PA
• Charter Foundation Program December 2010 St. George’s University, Grenada, West Indies
• Bachelor of Arts, Cell Biology & Neuroscience and Evolutionary Anthropology May 2010 Rutgers University, New Brunswick, NJ
PUBLICATIONS:
• McLaughlin, P.J.; Cain, J.D.; Titunick, M.B.; Sassani, J.W.; Zagon, I.S. Topical Naltrexone Is a Safe and Effective Alternative to Standard Treatment of Diabetic Wounds. Adv. Wound Care, 2017, 6, 279–288.
• Stockdale, D.P.; Titunick, M.B.; Biegler, J.M.; Reed, J.L.; Hartung, A.M.; Wiemer, D.F.; McLaughlin, P.J.; Neighbors, J.D. Selective Opioid Growth Factor Receptor Antagonists Based on a Stilbene Isostere. Bioorg. Med. Chem., 2017, 25, 4464–4474.
AWARDS:
• Graduate Education Travel Award Fall 2016 TEACHING EXPERIENCE:
• Dissection- and lecture-based gross anatomy o Penn State Hershey Medical School 2016-2018 o Penn State Hershey Physician’s Assistant Program 2015-2018 o Penn State Hershey Graduate Program in Anatomy 2016-2018
• Dissection-based neuroanatomy o Penn State Hershey Medical School 2016-2017
• Lecture-based neuroanatomy o Penn State Hershey Neurology Residents 2015-2018