Analysis of Bone Healing with a Novel Bone Wax Substitute … · 4 67 Since Bone wax is comprised...

1

Analysis of Bone Healing with a Novel Bone Wax Substitute Compared to Bone Wax in a 1

Porcine Bone Defect Model 2

3

Tristan Tham, MD1; Keith Roberts

2; John Shanahan, PhD

2; John Burban, PhD

2; Peter 4

Costantino, MD FACS1

5 6

1 - New York Head & Neck Institute, Lenox Hill Hospital, New York, USA 7

2 – Hemostasis LLC 8

9 10

Lenox Hill Hospital 11 (Black Hall Building) 12

130 East 77th Street 13 10th Floor 14

New York, NY 10075 15

16 Corresponding author: 17 Tristan Tham, MD – [email protected] 18

19

Senior author: 20 Peter Costantino, MD – [email protected] 21

22 Co-authors: 23 Keith Roberts – [email protected] 24

John Shanahan – [email protected] 25 John Burban – [email protected] 26

27

.CC-BY-NC-ND 4.0 International licenseunder anot certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available

The copyright holder for this preprint (which wasthis version posted December 20, 2017. ; https://doi.org/10.1101/236430doi: bioRxiv preprint

mailto:[email protected]





https://doi.org/10.1101/236430

http://creativecommons.org/licenses/by-nc-nd/4.0/

2

Abstract: 250 words 28

Background: Bone wax is used in surgery as a hemostatic device for bone. Despite its good 29

functional capacity as a bone hemostat, Bone wax materials often have very poor long-term 30

interactions with bone. This study describes a novel composite of hydroxyapatite (HA) and 31

biodegradable poly-lactic acid (PLA) with wax-like handling properties (OsteoStat). The goal 32

was to compare qualitative and quantitative measures between OsteoStat versus Bone wax. 33

Methods: The porcine critical size defect model was chosen in this study. OsteoStat and Bone 34

wax were introduced into separate critical size defects located in the femur and humerus of a 35

single porcine specimen. After a duration of 6 weeks, the defect sites were harvested for clinical, 36

histological, and histomorphometric analysis. 37

Results: Both groups had effective hemostatic action when introduced into the defects. Analysis 38

of the histomorphometric data revealed that the amount of new bone was significantly greater at 39

6 weeks in the OsteoStat group (38.05%) versus the Bone wax group (11.88%), p=0.028. 40

OsteoStat also demonstrated less soft tissue and less test material remaining in the defect sites; 41

however, this was not statistically significant. 42

Conclusions: We speculate that the incomplete biodegradation of Bone wax as well as its 43

intrinsic inflammatory properties may have retarded osseous regeneration and promoted fibrosis. 44

In contrast, well known biodegradation pathways for PLA combined with the HA component of 45

OsteoStat may have accounted for the positive results of OsteoStat compared to Bone wax. It is 46



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important that bone hemostat substances have biocompatible, osteoconductive, hemostatic, as 47

well as good handling properties. 48

49

Keywords 50

Biomaterials, hydroxyapatite, biocompatibility, osteoconduction, bone wax, porcine model, 51

hemostasis 52

53

Background 54

Bone wax, first described by Sir Victor Horsley as an antiseptic in 1892, is now 55

commonly used in surgery as a hemostatic device.1,2

In cardiothoracic surgery, the main 56

indication for Bone wax is extensive bleeding from the sternal bone marrow. During sternotomy 57

procedures, intraoperative exposure of the cut surfaces of the sternum, together with concurrent 58

anticoagulant therapies increases the patients’ risk of bleeding into the sternal wound. Surgeons 59

try to minimize such bleeding as the eventual intrasternal hematoma collection could serve as a 60

nidus for bacterial infection, an uncommon but serious sequalae of cardiothoracic surgery.3 In 61

order to prevent extensive hematoma formation, surgeons apply topical hemostatic agents, such 62

as Bone wax, to the cut sternal marrow.4 Surgical Bone wax consists of a naturally occurring 63

substance, sterilized Cera Alba (honeybees wax), and is usually mixed with paraffin wax which 64

acts as a softening agent to enhance handling characteristics. 65

66



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Since Bone wax is comprised of paraffin wax and esterified fatty acids, it is highly 67

hydrophobic. This enables Bone wax to serve as a physical barrier against blood, occluding the 68

bleeding channels and achieving hemostasis by a tamponade and blood stasis effect. However, 69

this hydrophobic property in conjunction with limited enzymatic degradation of waxes in the 70

human body prevents appreciable rates of absorption and/or excretion post-surgical application.. 71

It is well documented that Bone wax impairs optimal bone formation and healing of 72

sternotomies, 3,5

which could be due to, in part, physical inhibition of osteoblast and osteocyte 73

migration to the site of bony injury. Furthermore, the nature of Bone wax which makes it highly 74

resistant to degradation has also been linked with infection,6,7

although large randomized studies 75

have found the infection link to be inconclusive.8 Since intraoperative bone bleeding can be 76

heavy, physicians must weigh the benefits of bone hemostasis using Bone wax versus the risk of 77

decreased bone healing and other complications such as infection. 78

79

It is important that Bone wax like substances have biocompatible, osteoconductive, 80

hemostatic, as well as good handling properties. Several alternative materials have been reported 81

in the literature such as PEG/collagen,9 polyorthoester,

10 fibrin-collagen,

11 chitin-based 82

material,12

, and gelfoam.13

However, none of these alternative materials have yet seen 83

widespread adoption, suggesting that a material which meets the effective hemostatic qualities of 84

Bone wax together with good osseous integration and affordability has not been met. 85

86

Herein we describe OsteoStat (Hemostasis LLC, MN, USA), a novel composite of 87

hydroxyapatite (HA) and biodegradable poly-lactic acid (PLA) with wax-like handling 88

properties. HA (Ca10(PO4)6(OH)2) is a biomaterial similar to the mineral component of natural 89



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bone, and exhibits good osteoconductivity.14

Similar to Bone wax, the OsteoStat creates a 90

physical barrier which is its primary mechanism of hemostasis. Unlike Bone wax, we 91

hypothesized that the OsteoStat would, due to its HA content, have an enhanced bone healing 92

profile in addition to hemostatic qualities. The goal of this experiment was to compare the widely 93

used cardiothoracic surgery hemostatic agent, Bone wax, versus OsteoStat. We investigated 94

qualitative and quantitative measures of bone healing between the two materials. 95

Materials & Methods 96

Details of animal husbandry, diet, care, monitoring, health, and well-being as well as 97

measures to alleviate suffering were all performed in accordance with International Organization 98

for Standardization document ISO 10993-11:2006: Biological evaluation of medical devices. 99

This experiment was conducted in a Sichuan Province Experimental Animal Management 100

Committee Accredited Facility in China. Ethics approval for animal experimentation was 101

approved by the animal ethics board of the Sichuan Province Experimental Animal Management 102

Committee of China. 103

A porcine bone defect model was used for this study. One female young adult Landrace pig 104

weighing 52.8 kg was used for this study, with an acclimation period of 7 days. Two groups of 105

holes were drilled into the femur and humerus and filled with either OsteoStat or Bone wax. 106

After a period of 6 weeks, qualitative (clinical and histology) and quantitative 107

(histomorphometry) analyses were performed on the drilled sites. 108

Surgical Procedure 109

The animal was prepared for operation under general anesthesia. Intraperitoneal 110

pentobarbital sodium was administered and the field of operation was then sterilized and selected 111



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at the right humerus and contralateral left femur. Tissue dissection was performed, exposing the 112

underlying periosteum at these respective sites. Four test sites were chosen for the bone defect, 2 113

holes located in the right diaphyseal humerus and 2 holes located in the left diaphyseal femur. 114

All test sites were drilled to have standardized intraosseous defects with a circumference of 3cm 115

and equal depth. Care was taken to drill the cortical bone down to a similar depth in all the drill 116

sites. One defect on each bone (humerus or femur) was filled with OsteoStat (Hemostasis LLC, 117

MN, USA) and the other defect was filled with Bone wax W31G (Ethicon, NJ, USA). The 118

surgical sites were closed in multiple layers. 119

The animal was given a healing period of 6 weeks before it was sacrificed. During the 120

healing period, the animal displayed no signs of local infection and surgical site incisions were 121

well healed. The animal was fully ambulant during the entire period and was effectively load 122

bearing on all four limbs. After the healing period, the animal was sacrificed with an overdose of 123

pentobarbital sodium. The right humerus and left femur diaphysis were cut to harvest the test 124

sites as discrete blocks. On harvesting of the test sites, no signs of gross inflammation or necrosis 125

were observed in any of the sites. 126

Histology Preparation 127

The harvested blocks of tissue containing the test sites were fixed with a 10% neutral 128

buffered solution of formalin for a period of 7 days. The blocks were then decalcified by a 129

solution of mixed acid decalcification agent for 6 weeks. Next, the sections were dehydrated with 130

ethanol gradient and prepared with paraffin. Hematoxylin and Eosin (H&E) stain was applied to 131

the sections for histological observation at x40, x100, and x 400 magnification. Two sections 132

were prepared from each of the 4 drilled sites, for a total of 8 sections for 133



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histological/histomorphometric analysis (4 sections for the OsteoStat defect and 4 sections for 134

the Bone wax defect). 135

Histomorphometric Evaluation 136

Two sections per drilled site were used for histomorphometric analysis, for a total of 4 137

sections per test material. Analysis was done at the x40 power magnification level, as this would 138

give a balance between tissue resolution and reduction in section variability, which would be 139

greatly biased at higher power magnification. Histomorphometric data was obtained by 140

analyzing the magnified cross sections of the defect sites using National Institute of Health 141

program software, ImageJ 1.48v. Areas of interest were measured in terms of pixel counts. 142

Parameters delineated in the histomorphometric evaluation were % bone area, % soft tissue area, 143

% test material, all of which combined would add up to approximately 100% of the defect size. 144

Statistical Analysis 145

Histomorphometric data was analyzed by comparing the mean between the two test materials 146

using Student’s t-test. Results were considered significant with a p value of less than 0.05. 147

Results 148

Clinical Evaluation 149

Upon drilling defects into the femoral and humeral diaphysis, bone bleeds were observed in 150

all the bone defects. There was effective hemostasis in both materials. The animal remained 151

healthy for the duration of the study with no post-operative or surgical site complications. The 152

rest of the 6 week healing period was uneventful. 153

Histology 154



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A selection of images which are representative of the qualitative findings are included below. 155

X40 power magnification: In the OsteoStat sites, new bone formation was observed, in 156

addition to the soft tissue stroma. Furthermore, new vessel formation was observed in some of 157

the sections (Fig. 1). In the Bone wax sites, fibrous soft tissue stroma was the predominating 158

component. Some new bone can also be seen, and there is also the presence of undegraded test 159

material. The amount of new bone appears visually less than in the OsteoStat sites (Fig. 2). 160

161

Fig. 1 H&E stained view of the bony defect containing OsteoStat at x40 power 162 magnification. 163 Formation of new bone (B) can be seen on the lateral and inferior borders of the image. 164 Haversian/volkman-like vessels are present in the central portion of the image. Fibrous stroma 165 (F) is also present in the middle of the section at the interface of the newly formed bone. 166

167

Fig. 2. H&E section containing Bone wax at x40 power magnification. 168 This section is dominated by fibrous stroma (F) in the middle of the image, and a small amount 169

of new bone (B) formation at the peripheries. 170

171

X100 power magnification: In the OsteoStat sites, new bone formation can be seen which 172

make up a large portions of the images (Fig. 3). Bone wax sites show a large amount of fibrous 173

tissue and some new bone is also present, but as immature bony trabeculae (Fig. 4). 174

175

Fig. 3 Section of OsteoStat site at x100 power magnification. 176 New bone (B) can be seen on the right margins, with the left margin composed of fibrous stroma 177

(F). 178 179

Fig. 4 Section of the Bone wax site at x100 power magnification. 180



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Immature bony trabeculae (B) are seen at the inferior margins. The rest of the image is mainly 181

composed of fibrous stroma (F). 182 183

X400 power magnification: OsteoStat sites shows active osteoblast activity lining the bony 184

trabeculae. Osteocytes are also present in the bony matrix. No osteoclasts are seen (Fig. 5). 185

Similarly, the Bone wax site (Fig. 6) shows osteoblast activity lining the bony trabeculae, with 186

no osteoclasts. The variation in sections at the x400 magnification is inherently greater. 187

188

Fig. 5 OsteoStat site at x400 power magnification. 189 Osteoblasts (OB) are seen lining the newly formed bony trabeculae (B), with osteocytes (OCY) 190 also present in the bony matrix. 191

192

Fig. 6 Bone wax site at x400 power magnification. 193 Similar to figure 5, osteoblasts (OB) are seen lining the newly formed bony trabeculae (B), with 194 osteocytes (OCY) also present in the bony matrix. The bottom portion of the image consists of 195

soft tissue 196 197

Histomorphometry 198

Histomorphometric analysis of the sections at x40 power magnification was performed. 199

Parameters measured were mean volume fraction of test material remaining, soft tissue area and 200

bone area. The mean area fractions of test material, soft tissue and bone in the OsteoStat 201

composite were 12.31%, 49.64%, and 38.05% respectively. In the Bone wax group, the mean 202

area fractions of test material, soft tissue and bone were 16.08%, 72.04%, 11.88% and 203

respectively (Table 1). The difference in bone area fraction between the OsteoStat group 204

(38.05%) and the Bone wax group (11.88%) was significant (p = 0.028) (Table 2). The soft 205

tissue area fraction in the OsteoStat group (49.64%) was also less than the Bone wax group 206



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(72.04%); however, this result was not statistically significant (p = 0.089). Similarly, the amount 207

of test material remaining in the OsteoStat group (12.31%) was less than the Bone wax group 208

(16.08%) and was also not statistically significant (p = 0.421). 209

210

211



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Table 1. Histomorphometric Analysis of OsteoStat versus Bone wax 212

213

Histomorphometric analysis of the sections at x40 power magnification. Parameters measured 214 were mean volume fraction of test material remaining, soft tissue and bone. All area fractions 215

add up to make 100% of the total volume. The difference in areas of bone (p=0.028) between the 216 two groups are statistically significant. The difference in areas of soft tissue (p=0.089) and test 217 material remaining (p=0.421) were not statistically significant. 218

219

220

38.05

11.88

49.64

72.04

12.31 16.08

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

OsteoStat Bone wax

% M

ean

Are

a

Histomorphometric Analysis

Test materialremaining

Soft tissue

Bone area



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Table 2. Bone area seen in histomorphometric analysis in OsteoStat versus Bone wax 221

OsteoStat bone area (%) Bone wax bone area (%)

Sample 1 52.49 Sample 1 9.65




Mean *38.05 Mean *11.88

Standard deviation 17.6 Standard deviation 4.61

Histomorphometric analysis of 4 samples of bone defects for OsteoStat and Bone wax. The mean 222 amount of bone observed in histomorphometric analysis was significantly greater in OsteoStat 223 compared to bone wax. * p<0.05 224

Discussion 225

Histomorphometric data revealed that the amount of new bone was significantly greater at 6 226

weeks in the OsteoStat group (38.05%) versus the Bone wax group (11.88%), p=0.028. This data 227

is consistent with the published literature in that Bone wax is a potent inhibitor of bone healing 228

4,15. Histological examinations in animal models as well as human autopsies demonstrate that the 229

application of Bone wax not only prevents bone healing, but also promotes granuloma formation, 230

chronic inflammation, and fibrotic scar tissue.3,5,16

Furthermore, Bone wax is resistant to 231

degradation and remains in the implanted sites indefinitely.5,16

In a case series of 18 post-mortem 232

examinations, histologically verified Bone wax granulomas were found, in one case as long as 10 233

years after implantation.5 Because of these potential complications, good surgical practices 234

minimize the amount of Bone wax used, whenever needed. Its use is avoided altogether when 235



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fusion of bone is critical for post-operative function, for example, in most orthopedic surgery 236

procedures. 237

Bone wax hemostatic activity is purely mechanical. It physically occludes the bleeding haversian 238

canals in cortical and medullary bone and activates coagulation via the stasis component of 239

Virchow’s Triad. The OsteoStat composite used in this experiment has similar handling 240

characteristics to Bone wax, and was designed to promote hemostasis via a similar tamponade-241

like mechanism. Despite that no objective measures of hemostasis were designed as part of this 242

study, both materials are subjectively reported to have equal hemostatic efficacy. 243

244

Our results showed that the amount of fibrous tissue remaining in the OsteoStat group (49.64%) 245

was less than in the Bone wax group (72.04%), though this was not statistically significant 246

(p=0.089). Furthermore, amount of test material remaining for the OsteoStat group (12.31%), 247

though less than Bone wax (16.08%), was also not significant (p=0.421). One possible 248

explanation for these findings could be that the timeframe of 6 weeks was too short for all the 249

HA particles to take part in resorption and osteointegration. As described earlier, it would be 250

reasonable to assume that the Bone wax particles would be resistant to degradation and 251

resorption. However, HA particles have been shown to have complete osseous integration over a 252

period of time, especially when blended with PLA oligomer.17

253

254

Subjectively, both OsteoStat and Bone wax had effective hemostatic action on the bleeding bone. 255

However, in this experiment, we noted that OsteoStat has the added advantage of having a higher 256



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bone healing capacity compared to Bone wax. One other hemostatic Bone wax substitute that 257

does not inhibit healing is water soluble Bone wax (WSW). WSW was first reported as 258

‘pluronic-based’ wax by Wang et al in 2001, and similar to this study, demonstrated superior 259

bone healing to Bone wax.18

A subsequent study by Vestergaard et al of a randomized trial in 260

humans comparing WSW versus Bone wax found no differences in infection rates, but radiologic 261

bone measurements indicated lower levels of bone healing in Bone wax. Additionally, the study 262

surgeons commented the WSW had some drawbacks, specifically the need to reapply due to 263

dissolution of the WSW and the need to heat the WSW product before application to make it 264

more pliable to smear on trabecular bone surfaces.19

265

Whether or not the lower levels of radiologic bone healing in Bone wax compared to WSW 266

translate into real clinical effects such as sternal bone strength is a matter for debate, as 267

demonstrated in long-term animal trials. A study comparing WSW and Bone wax in porcine 268

sternotomies after a period of 6 months showed that although Bone wax had poorer histological 269

and radiological outcomes, bone mechanical properties were similar. Sternal wounds closed with 270

Bone wax were found to be weaker compared to a negative control, but no difference in sternal 271

strength was observed between Bone wax and WSW.20 272

Similar to WSW, OsteoStat has several advantages over Bone wax and other reported Bone wax 273

substitutes. OsteoStat used in this study has demonstrated a higher amount of bone growth, is 274

easily sterilizable, is biocompatible and would theoretically fully integrate in bony architecture 275

over a longer period of time.21

Furthermore, since it has no biological components, the material 276

would be immunogenic and allergen free. Physical handling characteristics which are similar to 277

Bone wax would make OsteoStat easy to use for surgeons familiar with working with traditional 278

Bone wax. 279



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Further studies are needed to ascertain the efficacy of OsteoStat in the human sternotomy and/or 280

cranial/spine surgery models, particularly comparing it against Bone wax and other common 281

hemostatic agents. Other useful avenues for investigation would be longer-term studies to 282

ascertain the rate of HA integration in bone after application as a hemostat. 283

Conclusion 284

This experiment has demonstrated that at 6 weeks, porcine bone defects will have higher 285

amounts of new bone if filled with OsteoStat than with Bone wax. OsteoStat test sites also 286

demonstrated less soft tissue and test material remaining than the Bone Wax, though the results 287

for these parameters did not meet the threshold for statistical significance. This might be because 288

the time frame for eventual HA osseous integration lasts many months to years. Subjectively, 289

both OsteoStat and Bone wax have effective hemostatic properties. It is important that bone 290

hemostat substances or its substitutes have biocompatible, osteoconductive, as well as hemostatic 291

properties. 292

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Figure 1. 348

349



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Figure 2. 350

351



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Figure 3.352

353



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Figure 4.354

355



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Figure 5.356

357



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Figure 6.358

359

360

List of abbreviations 361

PEG – polyethylene glycol, HA –hydroxyapatite, PLA- polylactic acid, mm – millimeter, 362

H&E – hematoxylin & eosin, 363

Acknowledgements 364

365

We would like to acknowledge the assistance and expertise of Liao WenJun, Wang Wei, Bao 366

WenTao and Cheng JingWen for the animal surgery and histology preparation. We would also 367



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like to acknowledge the expertise of Molly Speltz, DVM, in the histomorphometric analysis. 368

Funding Statement 369

The funder provided support in the form of salaries for authors JS JB KR, but did not have 370

any additional role in the study design, data collection and analysis, decision to publish, or 371

preparation of the manuscript. The specific roles of these authors are articulated in the ‘author 372

contributions’ section. 373

Author Contributions 374

Ideas; formulation or evolution of overarching research goals and aims – TT; Development 375

or design of methodology; creation of models – TT; Verification, whether as a part of the activity 376

or separate, of the overall replication/reproducibility of results/experiments and other research 377

outputs - TT PC JS JB KR; Application of statistical, mathematical, computational, or other 378

formal techniques to analyse or synthesize study data – TT; Conducting a research and 379

investigation process, specifically performing the experiments, or data/evidence collection – TT; 380

Provision of study materials, reagents, materials, patients, laboratory samples, animals, 381

instrumentation, computing resources, or other analysis tools - TT PC JS JB KR; Management 382

activities to annotate (produce metadata), scrub data and maintain research data (including 383

software code, where it is necessary for interpreting the data itself) for initial use and later re-use 384

– TT; Preparation, creation and/or presentation of the published work, specifically writing the 385

initial draft (including substantive translation) – TT; Preparation, creation and/or presentation of 386

the published work by those from the original research group, specifically critical review, 387

commentary or revision – including pre- or post-publication stages – TT; Preparation, creation 388

and/or presentation of the published work, specifically visualization/data presentation - TT PC JS 389



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JB KR; Acquisition of the financial support for the project leading to this publication - TT PC JS 390

JB KR 391

Conflict of Interest 392

The lead author Tristan Tham has no competing interests. Authors Keith Roberts, John 393

Shanahan, and John Burban are employees of Hemostasis LLC. The author Peter Costantino is 394

the co-developer of OsteoStat and has royalty position with Hemostasis LLC. This does not alter 395

our adherence to PLOS ONE policies on sharing data and materials. 396



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Analysis of Bone Healing with a Novel Bone Wax Substitute … · 4 67 Since Bone wax is comprised...

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