a r ch i v e s o f o r a l b i o l o g y 5 3 ( 2 0 0 8 ) 9 2 8 9 3 6

avai lable at www.sc iencedi rec t .comHuman osteogenic protein-1 induces osteogenicdifferentiation of adipose-derived stem cells harvestedfrom mice

Fahd Al-Salleeh a, Mark W. Beatty a,b,*, Richard A. Reinhardt a,c, Thomas M. Petro a,Larry Crouch a

aDepartment of Oral Biology, University of Nebraska Medical Center College of Dentistry, Lincoln, NE 68583-0750, USAbDepartment of Adult Restorative Dentistry, University of Nebraska Medical Center College of Dentistry, Lincoln, NE 68583-0750, USAcDepartment of Surgical Specialties, University of Nebraska Medical Center College of Dentistry, Lincoln, NE 68583-0740, USA

a r t i c l e i n f o

Article history:

Accepted 16 May 2008

Keywords:

Adipose-derived stem cells

Osteogenic protein-1

Osteogenic differentiation

Calcification

Osteopontin

a b s t r a c t

Objective: Osteogenic protein-1 (OP-1) has been shown to stimulate undifferentiated cells to

produce mineralized tissue. Adipose tissue is a rich source of undifferentiated cells for

tissue engineering purposes. The purpose of this study was to investigate the effect of OP-1

on osteogenic differentiation of adipose-derived stem cells and the production of bony

tissue in vitro.

Design: Adipose-derived stem cells (ADSCs) were isolated from inguinal fat pads of adult

mice. Following cell expansion the cells were plated in 8-well chambered slides. The cells

received one of four treatments: Group 1 cells were maintained in control medium, Group 2

cells were cultured in a common osteogenic medium, Group 3 cells were cultured in

osteogenic medium supplemented with 250 ng/mL of OP-1, and Group 4 cells were cultured

with 250 ng/mL of OP-1 added to control medium. Osteogenic differentiation of ADSCs was

determined by estimating the number and size of mineralized nodules, and the amount of

extracellular osteopontin secreted into cell culturemedium. Mineralized nodule production

was assessed at day 21 with von Kossa staining. Extracellular osteopontin release was

measured after 8 and 21 days by enzyme-linked immunosorbant assay (ELISA). ANOVA/

Tukey tests were used to identify differences among the four treatment groups for miner-

alized nodule production and osteopontin release ( p 0.05).Results: Deposition of calcified nodules and osteopontin secretion was significantly greater

for cell cultures incubated with OP-1 ( p 0.05). At day 21, no significant differences inosteopontin secretion were noted among groups incubated with osteogenic nutrients and/

or OP-1 ( p > 0.05), which were significantly higher than the group incubated in cell growth

medium only ( p 0.05). No significant differences in osteopontin secretion were notedbetween 8 and 21 days for any group ( p > 0.05). Linear regression analysis demonstrated a

linear relationshipwas present between the presence of calcified nodules and the amount of

osteopontin released ( p 0.05).Conclusions: OP-1 is a powerful inducer of osteogenic differentiation of adult adipose-

derived stem cells.

# 2008 Elsevier Ltd. All rights reserved.

* Corresponding author at: Department of Adult Restorative Dentistry, 40th & Holdrege Streets, Lincoln, NE 68583-0740, USA.Tel.: +1 402 472 1261; fax: +1 402 472 6681.

E-mail address: mbeatty@unmc.edu (M.W. Beatty).

journa l homepage: www. int l .e lsev ierhea l th .com/ journals /arob

00039969/$ see front matter # 2008 Elsevier Ltd. All rights reserved.doi:10.1016/j.archoralbio.2008.05.014

Committee of the University of Nebraska at Lincoln. The

of Dulbeccos Modified Eagle Medium (DMEM) containing 10%

a r c h i v e s o f o r a l b i o l o g y 5 3 ( 2 0 0 8 ) 9 2 8 9 3 6 9291. Introduction

Bone loss is one of the major health concerns in the United

States and Europe. There are roughly more than one million

cases of bony defects each year, and this number is projected

to increase dramatically due the ageing of their populations.13

Bone serves several important functions in the craniofacial

region and throughout the body, and there is a significant need

for bone replacement therapy due to congenital defects,

disease and trauma. Current treatments for repairing cranio-

facial bony defects are based on autologous and autogenous

bone grafts. Allografts, or bone taken from another human,

and autologous bone grafts, or bone taken fromanother region

of ones own body, have been the gold standards of bone

replacement for many years. More than 250,000 bone grafts

are performed annually in the United States alone in an

attempt to assist the body in regenerating new bone lost by

trauma or disease.4 However, despite the usefulness of these

reparative strategies, each method has inherent limitations,

primarily the limited osteoinductive potential that restrict

their universal application.57 As a result, many bony defects

are inadequately treated. Tissue engineering offers an alter-

native to existing treatments for restoring bone loss. A tissue

engineering approach utilizes an extracellularmatrix scaffold,

osteogenic or stem cells, bone morphogenic signals, or a

combination of the three.8 This approach offers the possibility

of rapid bone regeneration.

Cell-based therapy incorporates the direct delivery of cells

and bone morphogenic signals into an anatomic site to yield

bone regeneration. This approach is advantageous because

cells may directly participate in the regenerative process, and

the signalling molecules exert both autocrine and paracrine

effects, which may render the regenerative process more

robust.9 The source and type of cells are important considera-

tions when planning a cell-based therapeutic approach. Stem

cells, in particular, are characterized as being clonogenic,

having the capacity for self-renewal, and having the ability to

terminally differentiate into cells of various lineages.10

Because of the controversy surrounding the derivation and

procurement of embryonic stem cells, many regenerative

approaches have relied on the use of adult stem cells. Recent

studies have reported the widespread distribution of adult

stem cells in various tissues and organs.1115 Adipose tissue is

particularly desirable as a source of stem cells because of its

abundance and the ease of cell procurement through local

excision or liposuction.

Bone morphogenic proteins (BMPs) are important morpho-

gens needed for bone tissue engineering. They form a unique

group of proteins within the transforming growth factor beta

(TGF-b) superfamily.16 There is extensive evidence supporting

their role as regulators of bone induction, maintenance and

repair. The clinical applications of recombinant BMP-2 and

BMP-7 (also known as osteogenic protein-1, or OP-1) have been

studied extensively. Recombinant human OP-1 has been

shown to induce bone formation in animals by stimulating

osteogenic progenitor cells.17 The efficacy of OP-1 for treating

orthopaedic patients is being evaluated in clinical trials.8

Many studies have been conducted to clarify the regulatory

mechanisms of OP-1 that underlie the differentiation processof mesenchymal stem cells to osteogenic cells. These studiesventral surface of eachmouse was scrubbedwith a germicidal

disinfectant and a midline incision was made with sterile

scissors in the epigastric region of the animal, exposing the

abdominal cavity and its contents. Visceral fat encasing the

bladder and epididymus were carefully dissected and tissues

were transferred to a centrifuge tube containing Hanks

Balanced Salt Solution with penicillin (100 IU/mL), strepto-

mycin (100 mg/mL), and amphotericin B (2.5 mg/mL) (HBSS-P/S/

A). After transferring the centrifuge tube to a laminar flow

hood, the tissues were removed and minced into smaller

pieceswith sterile scissors, allowing theminced pieces to drop

directly into a new 50 mL sterile centrifuge tube. Minced

tissueswere rinsed three times by adding 25 mLHBSS-P/S/A to

the centrifuge tube, shaking, and evacuating the solution. The

tissues were resuspended in 25 mL HBSS-P/S/A and centri-

fuged at 500 times gravity for 5 min. Free floating tissue was

removed with sterile forceps and placed into a new centrifuge

tube containing 25 mL of 0.05% collagenase dissolved in HBSS-

P/S/A. The centrifuge tube was allowed to roll on its side on a

circular shaker at 37 8C for 45 min to digest the tissue and

release the cells. The collagenase was neutralized with 25 mLhave focused on the phenotypic characteristics of osteogenic

cells, such as alkaline phosphatase activity, extracellular

matrix mineralization, and the expression of various extra-

cellular matrix proteins such as collagen type II, osteopontin,

or osteocalcin.18,19 To date, however, it is unknown whether

OP-1 can stimulate differentiation of adipose-derived stem

cells into an osteogenic lineage.

An important treatment goal is to develop methods for

bone repair that are more predictable, rapid, and reliable than

those presently in existence. The purpose of this study is to

focus on the effect OP-1 renders on adipose-derived stem cells

(ADSCs) toward the production of bony tissue in vitro. Two

markers for bone production are chosen, namely, the amount

of mineralized tissue and the amount of extracellular

osteopontin (a marker of osteoblast differentiation) produced

in cell culture. Comparisons are made with cells exposed to a

standard osteogenic environment, with and without the

addition of OP-1, and with an unstimulated control cell

population. The hypothesis tested in this study is that OP-1

can enhance the ability of ADSCs to differentiate into

osteoblast-like cells and secrete mineralized matrix.

2. Materials and methods

Unless otherwise stated, all chemicals were procured from

Sigma Chemical, St. Louis, Missouri.

2.1. Stem cell harvesting procedures

Since a goal of this study was to develop tissue engineering

techniques that could succeed with the use of adult donor

tissues, 20-week-old mice were chosen for study. Five male

Mus musculus mice, 20 weeks of age, were handled and

euthanized by carbon dioxide inhalation according to the

procedures approved by the Institutional Animal Care andUseof fetal bovine serum (FBS) and 1% P/S/A. The neutralized

a r c h i v e s o f o r a l b i o l o g y 5 3 ( 2 0 0 8 ) 9 2 8 9 3 6930solution was poured through a 250 mm mesh into a sterile

600 mL beaker, then transferred to a new centrifuge tube and

centrifuged at 500 times gravity for 10 min. Floating tissue

fragments were removed with a sterile tongue blade and

discarded, the supernatant evacuated from the tube, the cell

pellet resuspended in 10 mL DMEM containing erythrocyte

lysing buffer (160 mMNH4Cl), and the centrifuge tube allowed

to roll on a rotating shaker at room temperature. After 10 min,

the tube was removed from the shaker and it was centrifuged

at 500 times gravity for 10 min. The solution was removed, the

cell pellet resuspended in 10 mL of DMEM-P/S/A + 10% FBS,

and the suspension poured through a 100 mmmesh to remove

lysed red blood cells. A 2 mL aliquot was placed into each of

five 75 cm2 tissue culture flasks, and 9 mL of control medium

(DMEM-P/S/A + 10%FBS) was added to each flask. The cells

were incubated at 37 8C in the presence of 5% CO2 and 95%

humidity. Following the plating procedures, medium was

changed after 34 days of incubation to remove nonadherent

cells, and thrice weekly thereafter. Cells were maintained at

subconfluent levels and passaged with 0.05% trypsin/EDTA

(GibcoBRL, Carlsbad, CA) upon reaching 8590% confluency.

Cell expansion continued through the second passage.

2.2. Confirmation of stem cell population

In order to verify that cells isolated from adipose tissue were

truly derived from a stem cell population, cells were exposed

to cell-growth conditions that promoted either chondrogen-

esis or osteogenesis. Once this was verified, the study focused

on osteogenic differentiation.

2.2.1. Verification of chondrogenic potentialAfter culture expansion to two passages, the cells were

trypsinized and placed into 6-well well culture plates at

approximately 106 cells/well. The Trypan blue exclusion test

was used to estimate the number of viable cells in each well.

Cells were incubated in 2 mL of the control medium per well

for 24 h. To induce chondrogenic differentiation, chondro-

genic medium consisting of DMEM, 1% FBS, 10 ng/mL

transforming growth factor-b1 (R&D Systems, Minneapolis,

Minnesota), and 6.25 mg/mL insulinwas used. A second group

was incubatedwith controlmedium consisting of DMEMplus

10% FBS and 1% P/S/A. Themedia were changed every 3 days.

Chondrogenesis was assessed using Alcian blue staining at

day 21 according to a procedure described previously.20

Briefly, culture plates were rinsed with phosphate-buffered

saline and fixed in a 4% paraformaldehyde solution for

15 min.After fixation theywere rinsedwith0.1NHCl for 5 min

to decrease the pH to 1.0. The plates were stained with 1%

Alcian blue dissolved in 0.1N HCl (pH 1.0), which stained

highly sulfated proteoglycans that were characteristic of

cartilaginous matrices. After staining overnight, the plates

were washed twice with 0.1N HCl for 5 min to remove any

nonspecific staining of the plates, followed by a 5 min rinse

with tap water. To verify results, both groups were run in

triplicate.

2.2.2. Verification of osteogenic potential

After culture expansion to two passages, the cells were

trypsinized and placed into 6-well well culture plates atapproximately 1 106 cells/well. The Trypan blue exclusiontest was used to estimate the number of viable cells in each

well. Cells were incubated in 2 mL of the control medium per

well for 24 h. To induce osteogenic differentiation, osteogenic

medium consisting of the controlmedium supplementedwith

0.1 mM dexamethasone, 50 mM ascorbate-2-phosphate and

10 mM b-glycerophosphate was prepared. Osteogenesis was

assessed using the von Kossa staining procedure according to

the procedure described below. To verify results, both groups

were run in triplicate.

2.3. Study protocols

Following the second passage, the cells were trypsinized and

placed into 8-well chambered slides (Lab-Tek, Nunc. Naper-

ville, IL) at approximately 2 104 cells/well. The Trypan blueexclusion testwas used to estimate the number of cells in each

well. Cells were incubated in 300 mL of control medium per

well for 24 h, which was designated as day 0. Then the cells

received one of four different treatment protocols (Table 1).

Group (1): Cellsweremaintained in controlmedium,DMEM

plus 10% FBS and 1% P/S/A. This served as a negative

control.

Group (2): Cells were cultured in an osteogenic medium

consisting of the negative control medium supplemented

with 0.1 mM dexamethasone, 50 mM ascorbate-2-phos-

phate, and 10 mM b-glycerophosphate. This served as a

positive control.

Group (3): Cells were cultured with the same osteogenic

medium used for group (2), supplemented with 250 ng/mL

of osteogenic protein-1 (OP-1, R&D System, Minneapolis,

MN).

Group (4): Cellswere culturedwith 250 ng/mLofOP-1 added

to the negative controlmedium. This groupwas included to

examine the effect of OP-1 in the production ofmineralized

nodules and expression of OPN in the absence of an

osteogenic environment.

In both groups containing OP-1 (groups 3 and 4), a

concentration of 250 ng/mL was chosen based on previous

research that demonstrated this amount was required to

induce osteogenesis.19,21

Mediumwas changed every 4 days for all groups. At days 8

and 21, the supernatant was collected from 3 wells for each

group and frozen until analyses could be performed.

2.4. Histochemical staining

Threewells fromeach groupwere chosen at day 21 to examine

extracellular matrix calcification through a modified von

Kossa staining technique.22 First, the cells were rinsed with

phosphate-buffered saline and fixed to the well bottoms for

1 h with 4% paraformaldehyde. Then the cells were rinsed

with distilled water and incubated in 5% silver nitrate in

darkness for 30 min. The silver nitrate solution was rinsed

away several times with distilled water and exposed to

ultraviolet light for 60 min. Finally, the cells were counter-stained with 0.1% eosin in ethanol. Following the von Kossa

staining procedure, a histomorphometric analysis was per-

a r c h i v e s o f o r a l b i o l o g y 5 3 ( 2 0 0 8 ) 9 2 8 9 3 6 931formed on each well using a Nikon Labophot-2 light micro-

scope (Nikon, Tokyo, Japan) at 100 magnification. Thisprocedure was performed to quantify the percentage of

culture area that was covered by black stain, which indicated

the presence of calcified nodules. A grid with 100 intersections

was held within the light microscope over the culture field,

and five adjacent areas were identified in each well using a

uniform method. Points of intersection covered by the black

stain were counted to yield percentage of calcified extra-

cellularmatrix. To prevent bias, the experimental groupswere

masked and an independent observer preformed the counting

procedure. The first field chosen was positioned at the centre

of the culture well. The microscope stage was then moved 1/4

turn of a stage knob toward 12 o clock, into field 2. The next

three fields were taken by first moving the stage by 1/4 knob

turn to centre position, then toward the 3, 6, and 9 o clock

positions sequentially. The average percentage of staining for

each well was calculated by averaging the five counted fields.

2.5. Enzyme-linked immunosorbant assay (ELISA)

A commercially available mouse osteopontin assay kit was

chosen (Immuno-Biological Laboratories, Gunma, Japan) to

measure secreted osteopontin. The assay was performed as

per instructions provided by the kitmanufacturer for a 96-well

plate. Absorbency was measured at 450 nm with an EL312e

microplate reader (Bio-Tek Instruments, Inc., Winooski, VT).

The sensitivity of the assay was 1 ng/mL.

2.6. Data analysis

For each experimental group, four wells were included for

each experiment and the experiment was conducted twice.

For histomorphometric analysis, the average percentage area

stained within a cell culture well served as the dependent

variable. A one-way analysis of variance (ANOVA) was

Table 1 Medium composition for each experimentalgroup

Group Medium composition

1 DMEM + 10% FBS + 1% P/S/A

2 DMEM + 10% FBS + 0.1 mM dexamethasone

+ 50 mM ascorbate-2 phosphate

+ 10 mM b-glycerophosphate + 1% P/S/A

3 Group 2 Medium + 250 ng/mL OP-1

4 Group 1 Medium + 250 ng/mL OP-1conducted to determine whether differences existed among

test groups. If results from ANOVA were significant (p 0.05),a Tukeys Least Significant Difference test (LSD) was per-

formed for pairwise comparisons ( p 0.05).For ELISA analysis, osteopontin concentration served as

the dependent variable. A repeated measures analysis of

variance was conducted, where each well of the ELISA plate

wasmeasured at 8 and 21 days to determine the effect of OP-1

presence or absence on osteopontin release. If results were

significant (p 0.05), a Turkey LSD post hoc test wasconducted (p 0.05).

To better understand the relationship between osteopontin

secretion in cell culture and the amount of calcified nodule3.3. Osteopontin secretion in cell culture

Results from ELISA showed that after 8 days, osteopontin

secretionwas significantly higher for cell cultures treatedwith

OP-1 (groups 3 and 4) compared to those not treated with OP-1

(groups 1 and 2, p 0.05, Fig. 4). No significant differenceswerenoted between groups 3 and 4, which contained OP-1 and

differed by the presence or absence of osteogenic nutrientsproduction, a scatter plot was constructed from histomorpho-

metric and ELISA data. Pearsons correlation coefficient (r) and

the coefficient of determination (r2) were calculated. A linear

regression analysis was applied to the data, and a least

squares relationship determined the slope of the regression

line. AnANOVA tested the null hypothesis that no relationship

existed between the histomorphometric and ELISA results

(slope = 0, p 0.05).

3. Results

3.1. Stem cell differentiation

Following excision, visceral fat pads yielded approximately

5 107 nucleated cells per mouse. When plated, the cellsexpanded readily in culture and exhibited a fibroblast-like

morphology within a week.

Cells incubated in chondrogenic medium for 21 days

stained positively for Alcian blue, whereas cells cultured in

control medium demonstrated a light, background staining,

indicating a negative reaction to Alcian blue (Fig. 1A and B).

This provided evidence that the adipose-derived cells pos-

sessed a capacity to differentiate towards a chondrogenic

lineage.

Cells cultured in osteogenic medium for 21 days were

tested for the presence of a calcified extracellularmatrix using

von Kossa staining. Consistent with osteogenesis, black

regionswere observed in cells cultured in osteogenicmedium,

whereas no calcification was observed for cells cultured in

control medium (Fig. 1C and D). This suggested that the cells

possessed a capacity to differentiate toward an osteogenic

lineage. The results from these two experiments suggested

that the adipose-derived cells exhibited differentiation beha-

viours that were characteristic of a stem cell population.

3.2. Histomorphometric analyses

Deposition of calcified extracellular matrix, as measured by

histomorphometry, was significantly greater for cell cultures

incubatedwithOP-1 (p 0.05). For groups 3 and 4, the calcifiedregions were larger and more numerous (Fig. 2). Interestingly,

medium containing OP-1 without osteogenic nutrients pro-

duced significantly greater calcification than did the medium

containing OP-1 with osteogenic nutrients (p 0.05, Fig. 3).The presence of OP-1 increased the production of calcified

extracellular matrix by a factor of 2.32.8 times more than the

osteogenic environment without OP-1, and 2733 times more

than the control environment.(p > 0.05). At day 21, no significant differences in osteopontin

secretion were noted among the three treatment groups

a r c h i v e s o f o r a l b i o l o g y 5 3 ( 2 0 0 8 ) 9 2 8 9 3 6932(p > 0.05). Cells maintained in control medium secreted

significantly lesser amounts of osteopontin at day 21 com-

pared to treatment groups (p > 0.05). For all groups, no

significant differences in osteopontin secretion were noted

between 8 and 21 days ( p > 0.05, Fig. 4).

3.4. Relationship between calcification and osteopontinsecretion

Results from the ANOVA demonstrated that a relationship

was present between the calcification and osteopontin results

(p 0.05). The value of Pearsons correlation coefficient (r) was0.78, and the coefficient of determination value (r2) was 0.61.

This meant that 61% of the observed variation in percent

calcification was explained by osteopontin values (Fig. 5).

4. Discussion

This study demonstrated the effect OP-1 rendered on

adipose-derived stem cells toward the production of bony

tissue. Cell-based therapy incorporates the direct delivery of

cells and bone morphogenic signals into an anatomic site to

yield bone regeneration. This is advantageous because the

cells directly participate in the regenerative process and the

signalling molecules exert both autocrine and paracrine

Fig. 1 (A and B) Alcian blue staining performed to detect high

cells cultured in control medium (A) and chondrogenic medium

was observed only for cells cultured in chondrogenic medium (B

presence of calcified extracellular matrix produced by adipose-d

and osteogenic medium (D). Black staining, indicative of a calcifie

in osteogenic medium (D) (scale bar length = 95 mm).effects, which should render the bone regenerative process

more robust.9

Initially it was confirmed that adipose-derived stem cells

exhibited differentiation behaviours that were characteristic

of a stem cell population. Cells incubated in chondrogenic

medium stained positively for Alcian blue, a common marker

of chondrogenic differentiation. Induction of osteogenesis

was observed when cells incubated in a standard osteogenic

environment formed islands of mineralized extracellular

matrix, as detected with von Kossa staining. These findings

were similar to those reported previously.15,20,23,24

Using two markers for osteogenic differentation, namely

the amount of mineralized tissue and extracellular osteopon-

tin produced in cell culture, results from this study demon-

strated that a 250 ng/mL dose of OP-1 induced osteogenesis in

adipose-derived stem cells harvested from adult mice.

Previous reports showed that treatment of the non-osteogenic

mouse pluripotent cell line, C3H10T1/2 cells, with 200 or

250 ng/mL of OP-1 induced osteogenesis.19,25 However, a

80 ng/mL dose of OP-1 induced adipogenesis, whereas a high

dose (500 ng/mL) of OP-1 caused the same cells to exhibit

chondrocytic properties.18 More recently, a very low dose

(10 ng/mL) of OP-1 stimulated a chondrogenic phenotype in

adipose tissue-derived mesenchymal stem cells.26 Taken

together, changing OP-1 concentration may alter the differ-

entiation pathway in certain cell types, and the 250 ng/mL

ly sulfated proteoglycans present in adipose-derived stem

(B) for 21 days. Blue staining, indicative of chondrogenesis,

). (C and D) Von Kossa staining was performed to detect the

erived stem cells cultured for 21 days in control medium (C)

d extracellular matrix, was observed only for cells cultured

Fig. 2 Von Kossa staining of adipose-derived stem cells culture

osteogenic media + OP-1 (C) and control medium + OP-1 (D). Mo

observed by an increased black stain for cells cultured with OP

Fig. 3 Bar graphs of percent calcification area produced by

cells in each treatment group. Error bars represent

standard deviations. Groups with different letters are

significantly different at a = 0.05 confidence level.

a r c h i v e s o f o r a l b i o l o g y 5 3 ( 2 0 0 8 ) 9 2 8 9 3 6 933dose used in the current study seemed appropriate to induce

osteogenesis.

Previous research demonstrated that OP-1 induced chon-

drogenic cell differentiation prior to osteogenesis. Exposure of

mesenchymal cell lines to OP-1 for more than 8 days was

necessary to maintain a commitment to an osteoblast line-

age.18,19 It is possible that a similar pattern of differentiation

occurred in the present study. However, Alcian blue staining

was not performed at early stages of differentiation to confirm

such an occurrence.

The primary objective of the present study was to measure

extracellular mineralization and osteopontin production when

adipose-derived stem cells were incubated in control medium,

osteogenic medium, osteogenic medium supplemented with

OP-1, and control medium supplemented with OP-1. It was

clearly observed that cells exposed to OP-1 secreted a calcified

extracellular matrix that was greater than from cells cultured

without OP-1. Similarly, OP-1 caused the cells to produce

significantly higher quantities of osteopontin at both 8 and 21

days of cell culture. These findings were consistent with those

reported using fetal stem cell line.18,21 However, a critical factor

was the differentiation state of the cell. In a committed

d for 21 days in control medium (A), osteogenic medium (B),

re numerous and larger regions of calcified matrix were

-1 (C and D) (scale bar length = 95 mm).

Fig. 4 Bar graphs of mean osteopontin release into cell

a r c h i v e s o f o r a l b i o l o g y 5 3 ( 2 0 0 8 ) 9 2 8 9 3 6934osteoblast cell line, OP-1 increased the number of osteoblasts

and increasedextracellularmatrixmineralization, but it didnot

culturemediumasmeasured by ELISA. Error bars represent

standard deviations. Capital letters represent comparisons

among groups at 8 days; small letters represent

comparisons among groups at 21 days. Groups denoted

with the same letter are not significantly different at a = 0.05

confidence level. Brackets show comparisons between 8

and 21 days for each treatment group. All comparisons are

denoted as n.s., or non-significant (p > 0.05).increase osteopontin expression. However, in a primary

osteoblast cell line, OP-1 caused both mineralization and

osteopontin secretion to increase.27 In the present study,

osteopontin production for the two OP-1 containing groups

was significantly higher than that measured for the osteogenic

medium alone group at day 8, but by day 21 the differences

among the three groups were not significant. This implies that

the cells in osteogenic media alone were in a pre-osteoblastic

state at day 8, whereas OP-1 sped the differentiation process

earlier in culture.

Fig. 5 Scatter plot with linear regression line of

osteopontin release versus calcified matrix production at

21 days of cell culture.A question arises as to why osteopontin was present at day

8 and 21 in all groups, including the cellsmaintained in control

medium. The fact that osteopontin was present in the control

group likely indicates that more differentiated mesenchymal

cells were mixed with the adipose-derived stem cell popula-

tion. Previous research demonstrated that osteopontin

expression was observed in osteogenically-induced and

non-induced adipose-derived stem cells containing both

undifferentiated and differentiated mesenchymal cells.20,24,28

Results from this study confirmed the fact that different

nutrients added to cell growth medium exerted differential

effects on osteogenesis, which agreed with previous

research.14,17,18,20,24,29,30 For example, when compared to cells

cultured in control medium (group 1), extracellular matrix

calcification at 21 days was significantly higher for cells grown

in osteogenic medium (group 2). However, when OP-1 was

added to the osteogenic medium (group 3), calcified extra-

cellular matrix was significantly lower than OP-1 alone (group

4). This brings into question whether certain osteogenic

nutrients may have exerted an inhibitory effect on OP-1. In

consideration of the roles played by osteogenic nutrients,

previously it was reported that when b-glycerophosphate was

added to osteoblast culture medium, ascorbic acid functioned

as a cofactor in hydroxylation reactions that produced

hydroxyapatite.31 Glucocorticoids have been shown to exert

both stimulatory and inhibitory effects on osteogenic differ-

entiation depending upon dose, duration, cell type and stage

of cell differentiation. For example, synthetic glucocorticoid

dexamethasone was shown to be an absolute requirement for

in vitro bone nodule formation and mineralization in rat

marrow stroma-derived cell cultures.32 However, dexametha-

sone exerted inhibitory effects on osteogenesis in adipose-

derived stem cells, andwhen dexamethasonewas substituted

with dihydroxyvitamin D3, osteogenesis increased.24 Osteo-

genesis was reduced when both dexamethasone and dihy-

droxyvitamin D3 were added to an osteogenic medium.33 The

effect of OP-1, when added mouse calvarial cell cultures

containing ascorbic acid and b-glycerophosphate, was to

increase osteocalcin production.19 Based on information from

these studies, it is postulated that certain osteogenic nutri-

ents, possibly dexamethasone, may have reduced the osteo-

genic capabilities of OP-1 observed in this study.

The bone nodule formation pathway in vitro has been

subdivided into three stages: proliferation, extracellular matrix

development, and maturation which includes mineralization.

Each phase is accompanied by characteristic changes in gene

expression. It has been shown that osteopontin is expressed by

pre-osteoblasticcellsearly inboneformation,withhigher levels

expressed after mineralization has been initiated.34,35 A strong

relationship between osteopontin secretion and calcified

extracellular matrix production was demonstrated in this

study. This supports the contention that when osteopontin is

upregulated, the amount of calcified extracellular matrix will

increase. Since a proportional relationship was demonstrated

(Fig. 5), it is possible to estimate the amount of calcification

based on osteopontin release, through substitution into a

linear regression equation. Under the conditions of this study,

each increase of 1 ng/mL osteopontin release extracellularlyresulted in an additional 1.6% calcified area within a cell

culture well.

2004;11:41726.

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15. Zuk PA, Zhu M, Mizuno H, Huang J, Futrell JW, Katz AJ, et al.10. Korbling M, Estrov Z, Champlin R. Adult stem cells andtissue repair. Bone Marrow Transplant 2003;32(Suppl. 1):S234.In conclusion, the results of the study indicate that OP-1

was a powerful inducer of terminal osteogenic differentiation

and mineralization, over and above growth factors present in

osteogenic medium.

Acknowledgments

The authors gratefully acknowledge Merlyn Nielsen for

supplying mice used in the study, Bobby Simetich for his

technical assistance and Kim Theesen for his work on graphic

arts.

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a r c h i v e s o f o r a l b i o l o g y 5 3 ( 2 0 0 8 ) 9 2 8 9 3 6936

Human osteogenic protein-1 induces osteogenic differentiation of adipose-derived stem cells harvested from miceIntroductionMaterials and methodsStem cell harvesting proceduresConfirmation of stem cell populationVerification of chondrogenic potentialVerification of osteogenic potential

Study protocolsHistochemical stainingEnzyme-linked immunosorbant assay (ELISA)Data analysis

ResultsStem cell differentiationHistomorphometric analysesOsteopontin secretion in cell cultureRelationship between calcification and osteopontin secretion

DiscussionAcknowledgmentsReferences