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Surgical Neurology xx (2009) xxx xxx www.surgicalneurology-online.com

Spine

Systemic and local reactions of a water-soluble copolymer bone on a bony defect of rabbit model
Tao-Chen Lee, MD a , Nyuk-Kong Chang, DVM a , Feng-Wen Su, MD a , Yu-Lin Yang, PhD b , Thung-Ming Su, MD a , Yu-Jun Lin, MD a , Wan-Ching Lin, BS a , Hsiu-Yu Huang, MD c,
Departments of aNeurosurgery and cPathology, Chang Gung Memorial Hospital-Kaohsiung Medical Center, Chang Gung University Collage of Medicine, Kaohsiung 83301, Taiwan b Department of Biological Science and Technology, Chung Hwa University of Medical Technology, Tainan 71703, Taiwan Received 19 May 2009; accepted 4 June 2009

Abstract

Background: Ostene, a synthetic water-soluble bone hemostatic agent, is commercially available. In the current study, we evaluated the systemic and local effects of this copolymer in a rabbit model. Methods: Eighteen rabbits underwent creation of a bony defect at right iliac crest. These rabbits were then evenly divided into 3 groups. In group 1, the defect surfaces were treated with bone wax; in group 2, the defect surfaces were treated with Ostene; in group 3, the defect surfaces were not treated with anything. Then, the animals underwent blood examinations, including WBC count, CRP, and ESR at 0, 1, 3, and 6 weeks, and were killed at 6 weeks for histologic examination. Another 6 rabbits (group 4) underwent the same surgical treatment of group 2 animals but had blood examinations of BUN and creatinine. Results: The blood examinations showed that the WBC count, CRP, and ESR of all the animals in the first 3 groups were within normal limits in the postoperative periods. Microscopic examinations demonstrated residual bone wax and fibrotic tissue at the defect surfaces in group 1 animals. However, there was no Ostene at the defect surfaces in group 2 animals. The groups 2 and 3 animals showed no fibrotic tissue at the defect surfaces. The group 4 animals showed normal serum levels of BUN and creatinine in the postoperative periods. Conclusion: Ostene is absorbable and induces no systemic inflammation (including acute renal damage) and local inflammation in animal bodies. 2009 Elsevier Inc. All rights reserved.
Bone hemostatic agent; Bone wax; Ostene

Keywords:

1. Introduction Ostene, a synthetic bone hemostatic agent made of water-soluble alkylene oxide copolymers, is now commercially available [11,12]. It has been demonstrated that this material does not elicit a foreign body response at the site of the bone defect surface [12]. Furthermore, the use of
Abbreviations: BUN, blood urea nitrogen; CRP, C-reactive protein; ESR, erythrocyte sedimentation rate; WBC, white blood cell. Corresponding author. Tel.: +886 7 7317123 8746; fax: +886 7 7354309. E-mail address: fakefionna@yahoo.com.tw (H.-Y. Huang). 0090-3019/$ - see front matter 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.surneu.2009.06.007

this material as an alternative to bone wax decreases the rates of postoperative bone infections [11]. A similar alkylene oxide copolymer material used by Wang et al [10] early in 2001 has proved effective in allowing hemostasis at the bleeding sites of bone and can be absorbed within 24 to 48 hours. Studies have shown that water-soluble alkylene oxide copolymers are eliminated via the kidney of human subjects [3-5,14]. Therefore, their systemic toxicity (including renal toxicity) should be carefully evaluated before they are widely used. In this animal experiment, we studied systemic inflammatory reactions (including renal damage),

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in addition to local inflammatory reactions, of rabbits treated with Ostene. 2. Materials and methods The Ostene used in this study was a gift from the Ceremed Inc (Los Angeles, Calif). The US Food and Drug Administration has cleared this material for use as an implant and for control of bleeding from bone surfaces. Eighteen male New Zealand white rabbits, each weighing approximately 3.0 kg, were anesthetized by intramuscular injection of Rompun (an anesthetic and muscle relaxant for animals; Bayer, Leverkusen, Germany) (50 mg/kg) and Ketalar (ketamine hydrochloride; Parke-Davis, Taipei, Taiwan) (50 mg/kg). The anesthesia methods were generally the same as those used in our previous rabbit experiments [6]. The back of the rabbits were shaved and prepared with Betadine solution (Povidone iodine, Sinphar Inc., Taipei, Taiwan). After infiltration with Xylocaine (1% lidocaine; Fujisawa, Osaka, Japan), a transverse 4-cm skin incision was made just above the right iliac crest. It was followed by gluteal muscular dissection down to the periosteum of the right iliac crest. After the periosteum was stripped from the bone, a rectangular-shaped surgical defect (0.5 1 2 cm3) was made in the iliac crest using a rongeur. The rabbits were then divided into 3 groups. For the animals from group 1 (numbering 6 rabbits), the cut surfaces of the defects were treated with bone wax (Ethicon, Inc, Johnson & Johnson, Edinburgh, UK). For the animals from group 2 (numbering 6 rabbits), the cut surfaces of the defects were treated with Ostene (Ceremed). For the animals from group 3 (numbering 6 rabbits), the cut surfaces of the defects were not treated with any hemostatic agent and served as controls. The blood loss from the bone defect in each animal was measured by weighing the bloodstained gauze that had been inserted into the defect to control bleeding with an electronic balance (Precisa 3100C; Precisa Instruments, Switzerland). The wounds were then closed with 4-0 absorbable sutures. For each rabbit, sodium aminobenzyl penicillin (500 mg) was administered intramuscularly daily for 7 days postoperatively. From each rabbit in groups 1, 2, and 3, blood samples were drawn at 0 (before surgery), 1, 3, and 6 weeks for evaluating the systemic inflammation markers. These markers included WBC counts, CRP, and ESR. Animals were killed at 6 weeks, and the right iliac bones were harvested and fixed in 10% formalin. These specimens were decalcified in 5% acetic acid for 7 days; they were then embedded in paraffin wax, sectioned, and stained with hematoxylin and eosin. All sections were examined by a single pathologist according to the single-blind method. For us to assess the renal toxicity of Ostene, another 6 rabbits (group 4) underwent the same surgical treatment of group 2 animals (Ostene group), but had blood examinations of 2 renal toxicity markers: BUN and creatinine, at 0 (before surgery), 1, 3, and 6 weeks.

All the animals were cared for in accordance with the regulations of the National Institutes of Health of the Republic of China (Taiwan), and this study protocol was approved by the Committee of Animal Experimentation of the Chang Gung Memorial Hospital, Kaohsiung, Taiwan. No authors in this research have a financial interest in Ceremed Inc, and none have received grant support from Ceremed Inc. 3. Results Ostene is waxy and similar to conventional bone wax in its physical properties. The mean blood loss at the bone defect in group 1 animals was 0.110 0.009 g. The mean blood loss at the bone defect in group 2 animals was 0.118 0.008 g. The mean blood loss at the bone defect in group 3 animals was 0.437 0.101 g. Statistical analysis using Student t test to compare the blood loss between groups 1 and group 2 showed that group 2 animals (Ostene group) had slightly more blood loss than group 1 animals (bone wax group), but it was not statistically significant at a P level of .111. However, statistical analysis using Student t test to compare the blood loss between groups 2 and group 3 showed that the group 2 animals had significantly less blood loss than group 3 animals (control group) at a level of P less than .0001. All statistical results were declared significant if P is less than .05. The blood WBC counts (normal range for rabbit, between 3.0 and 12.5 103/CMM-cubic milimeter) of all the first 3 groups were within normal limit in the pre- and postoperative stages. All the animals in group 2 (Ostene group) showed blood WBC counts less than 10.11 103/CMM in the preand postoperative stages (Table 1). The serum levels of CRP of all the first 3 groups were 0.2 mg/L or less in the preoperative stage. They were also 0.2 mg/L or less in the early and late postoperative stages. Table 2 showed the details of serum CRP levels of the group 2 (Ostene group) in the pre- and postoperative stages. The ESR values of all the first 3 groups were 2 mm/HR(hour) or less in the preoperative stage. They were also 2 mm/HR or less in the early and late postoperative stages. Table 3 showed the details of ESR values of group 2 (Ostene group) in the

Table 1 The WBC counts (103 per CMM) of the group 2 (Ostene-treated group, O1-O6) animals in the pre- and postoperative stages Preoperative O1 O2 O3 O4 O5 O6 5.79 6.34 8.66 8.60 9.50 9.10 1 wk postoperative 8.26 8.13 7.89 8.16 8.62 10.11 3 wks postoperative 8.60 8.70 9.74 8.90 8.58 9.94 6 wks postoperative 8.70 8.90 8.34 8.10 9.12 9.80

The normal range of WBC count for rabbit is between 3.0 and 12.5 103/CMM.

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T.-C. Lee et al. / Surgical Neurology xx (2009) xxxxxx Table 2 The serum CRP (milligrams per liter) levels of the group 2 (Ostene-treated group, O1-O6) animals in the pre- and postoperative stages Preoperative O1 O2 O3 O4 O5 O6 0.2 0.2 b0.2 b0.2 b0.2 b0.2 1 wk postoperative b0.2 b0.2 b0.2 b0.2 0.2 0.2 3 wk postoperative b0.2 b0.2 0.2 0.2 0.2 0.2 6 wk postoperative 0.2 0.2 b0.2 b0.2 b0.2 b0.2 3

They were 0.2 mg/L or less in the preoperative stage. They were also 0.2 mg/L or less in the postoperative stages.

pre- and postoperative stages. Considering the preoperative CRP levels and ESR values of the animals (numbering 18 rabbits) as normal (we could not obtain the data of normal range of CRP and ESR for rabbit), we defined the normal range of these 2 markers for rabbit as 0.2 mg/L or less and 2 mm/HR or less, respectively. Accordingly, our study results indicated that none of the animals showed abnormally high blood inflammatory markers (including WBC count, CRP level, and ESR value) after treatment with conventional bone wax or Ostene. In each animal from group 1 (bone waxtreated group), the postmortem gross examination showed a large amount of residual bone wax at the surgical site, and histologic examination showed small particles of bone wax surrounded by fibrotic tissue and no new bone formation at the iliac bone defect surface (Fig. 1). In each animal from group 2 (Ostene-treated group), the postmortem gross examination showed no residual Ostene substance at the surgical site, and histologic examination showed new bone formation but no visible Ostene particles or fibrotic tissue at the iliac bone defect surface (Fig. 2). In each animal from group 3 (control group), the histologic examination showed new bone formation but no fibrotic tissue at the iliac bone defect surface (Fig. 3). In group 4 animals (which had the same Ostene treatment of group 2 animals), the serum levels of BUN were 15 mg/dL or less in the preoperative stage. They were also 15 mg/dL or less in the early and late postoperative stages (Table 4). In the same group of animals, the serum

Fig. 1. Microphotography of the iliac bone defect surface of a group 1 (bone wax treated) rabbit demonstrating residual particles of bone wax (small arrow) and surrounding inflammatory reaction with fibrous tissue formation (large arrow) (hematoxylin and eosin stain, original magnification 100).

levels of creatinine were 0.8 mg/dL or less in the preoperative stage. They were also 0.8 mg/dL or less in the early and late postoperative stages (Table 5). Considering the preoperative serum levels of BUN and creatinine of this group of animals (numbering 6 rabbits) as normal, we defined the normal range of these 2 markers for rabbit as 15 mg/dL or less and 0.8 mg/ dL or less, respectively. Accordingly, our results indicated that none of this group of animals had abnormally high serum levels of renal toxicity markers (BUN and creatinine) after treatment with Ostene. 4. Discussion Bone wax, comprising beeswax softened with paraffin, has been widely introduced to control bleeding from bone

Table 3 The ESR (millimeter per HR) of the group 2 (Ostene-treated group, B1-B6) animals in the pre- and postoperative stages Preoperative O1 O2 O3 O4 O5 O6 1 1 1 2 1 1 1 wk postoperative 1 2 2 2 1 1 3 wk postoperative 2 2 1 2 1 1 6 wk postoperative 1 1 2 2 1 1

They were 2 mm/HR or less in the preoperative stage. They were also 2 mm/ HR or less in the postoperative stages.

Fig. 2. Microphotography of the iliac bone defect surface of a group 2 (Ostene treated) rabbit demonstrating new bone formation (arrow) with no focal fibrotic tissue and no residual Ostene (hematoxylin and eosin stain, original magnification 100).

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4 T.-C. Lee et al. / Surgical Neurology xx (2009) xxxxxx Table 5 The serum levels of creatinine (milligrams per deciliter) of the group 4 (R1-R6) animals in the pre- and postoperative stages Preoperative R1 R2 R3 R4 R5 R6 0.7 0.6 0.7 0.6 0.8 0.7 1 wk postoperative 0.6 0.7 0.7 0.7 0.7 0.6 3 wk postoperative 0.7 0.8 0.6 0.6 0.7 0.8 6 wk postoperative 0.6 0.6 0.7 0.7 0.6 0.6

They were 0.8 mg/dL or less in the preoperative stage. They were also 0.8 mg/dL or less in the postoperative stages.

Fig. 3. Microphotography of the iliac bone defect surface of a group 3 (control) rabbit demonstrating new bone formation (arrow) with no focal fibrotic tissue (hematoxylin and eosin stain, original magnification 100).

defects [9]. However, residual bone wax could be found up to 10 years after surgery, and some patients required a repeat surgery to remove bone wax granulomas [2]. Furthermore, bone wax forms a barrier to bone healing, and the use of bone wax increased infection rates at surgical sites [1]. Our study showed that Ostene is as effective as bone wax in achieving bone hemostasis. The meticulously obtained data showed that group 2 animals (Ostene group) had slightly more blood loss than group 1 animals (bone wax group), but it was not statistically significant. However, the group 2 animals had significantly less blood loss than group 3 animals (control group). The WBC count is a well-known indicator of infection, inflammation, or allergy. CRP is one of the acute phase proteins that increase in the blood of animals or human subjects during systemic inflammation [8]. As for ESR, it is a nonspecific measure of inflammation [13]. The serum BUN and creatinine level are well-known indicators for renal function. Serial serum creatinine measurement can provide useful insights to the cause of acute renal failure [7]. We introduced the normal range for rabbit WBC count (between 3.0 and 12.5 103/CMM) from Guide to the care

and use of experimental animals, volume 1, 1980 edited by the Canadian Council on Animal Care (no publisher). Our results showed that the blood WBC counts of the first 3 group rabbits were within normal limit in the postoperative stages. As for the normal range of CRP and ESR for rabbit, we cannot find them in books or manuals. Introducing preoperative data of CRP and ESR levels as normal range for rabbit, we found that all of the rabbits in the first 3 groups had normal serum CRP and ESR levels in the postoperative stages. From the data obtained in the current study, we found that neither conventional bone wax nor Ostene elicited a systemic inflammatory response in the rabbits. The study of group 4 was specific for renal toxicity of Ostene. We found that all the rabbits in this group had serum BUN and creatinine values within normal limit in postoperative stages. These results imply that Ostene does not cause acute renal damage although it is eliminated via the kidney. Despite having no systemic inflammation, the Ostenetreated rabbits showed no residual Ostene particles and no surrounding fibrosis at the osteotomy site under the microscope. Moreover, this group of animals showed new bone formation at the iliac bone defect surface. In conclusion, our study showed that Ostene is effective in achieving bone hemostasis and is absorbable in the body of rabbits. It does not inhibit new bone formation at the cut surface of the bone and does not cause local inflammation or systemic (including acute renal damage) inflammation in rabbits. 5. Conclusion Ostene has the potential to replace bone wax for bone hemostasis in the future. References
[1] Alberius P, Klinge B, Sjogren S. Effects of bone wax on rabbit cranial bone lesions. J Craniomaxillofac Surg 1987;15(2):63-7. [2] Allison RT. Foreign body reactions and an associated histological artifact due to bone wax. Br J Biomed Sci 1994;51:14-7. [3] Danielson GK, Dubilier LD, Bryant LR. Use of Pluronic F-68 to diminish fat emboli and hemolysis during cardiopulmonary bypass: a controlled clinical study. J Thorac Cardiovasc Surg 1970;59:178-84.

Table 4 The serum levels of BUN (milligrams per deciliter) of the group 4 (R1-R6) animals in the pre- and postoperative stages Preoperative 1 wk postoperative R1 R2 R3 R4 R5 R6 15 14 15 15 13 14 13 15 12 15 13 14 3 wk postoperative 14 13 13 12 14 11 6 wk postoperative 12 12 12 13 11 13

They were 15 mg/dL or less in the preoperative stage. They were also 15 mg/ dL or less in the postoperative stages.

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T.-C. Lee et al. / Surgical Neurology xx (2009) xxxxxx [4] Grindel JM, Jaworski T, Emanuele RM, et al. Pharmacokinetics of a novel surface-active agent, purified Poloxamer 188, in rat, rabbit, dog and man. Biopharm Drug Dispos 2002;23:87-103. [5] Jewell RC, Khor SP, Kisor DF, et al. Pharmacokinetics of RheothRx injection in healthy male volunteers. J Pharm Sci 1997;86:808-12. [6] Lee TC, Ho JT, Hung KS, et al. Bone morphogenetic protein gene therapy using a fibrin scaffold for a rabbit spinal-fusion experiment. Neurosurgery 2006;58:373-80. [7] Liu KD, Chertow GM. Acute renal failure. In: Fauci AS, Braunwald E, Kasper DL, Hauser SL, Longo DL, Jameson JL, Loscalzo J, editors. Harrison's principles of internal medicine. 17th ed. New York: MacGraw-Hill Co.; 2008. p. 1752-61. [8] Pepys MB, Hirschfield GM. C-reactive protein: a critical update. J Clin Invest 2003;111(12):1805-12. [9] Sudmann B, Bang G, Sudmann E. Histology verified bone wax (beeswax) after median sternotomy in 17 of 18 autopsy cases. Pathology 2006;38:138-41. [10] Wang MY, Armstrong JK, Fisher TC, et al. A new, Pluronic-based, bone hemostatic agent that does not impair osteogenesis. Neurosurgery 2001;49:962-8. [11] Wellisz T, An YH, Wen X, et al. Infection rates and healing using bone wax and a soluble polymer material. Clin Orthop Relat Res 2008;466: 481-6. [12] Wellisz T, Armstrong JK, Cambridge J, et al. Ostene, a new watersoluble bone hemostasis agent. J Craniofac Surg 2006;17:420-5. [13] Wetteland P, Rger M, Solberg HE, et al. Population-based erythrocyte sedimentation rates in 3910 subjectively healthy Norwegian adults. A statistical study based on men and women from the Oslo area. J Intern Med 1996;240(3):125-31. [14] Willcox ML, Newman MM, Paton BC. A study of labeled Pluronic F-68 after intravenous injection into the dog. J Surg Res 1978;25:349-56. 5

Commentary I congratulate the authors on a well-done study involving Ostene. Ostene (water-soluble alkylene oxide copolymers), a synthetic bone hemostatic agent, was evaluated in a rabbit model (18 rabbits) where it was applied to a bone defect in

the iliac crest. The animals were placed into 3 groups: (1) bone wax, (2) treated with Ostene, (3) no treatment. Laboratory studies sequentially evaluated WBC, CRP, and ESR obtained at 0, 1, 3, and 6 weeks after the procedures; animals were killed at 6 postoperative weeks. A group 4 was also created (6 rabbits) and was evaluated following the application of Ostene alone, and BUN and creatinine studies were obtained to rule out systemic nephrotoxicity. Laboratory studies (WBC, CRP, and ESR) in the animals in groups 1 to 3 remained normal postoperatively. The amount of postoperative blood loss at the surgical sites in group 2 rabbits was slightly but not significantly greater than for group 1 rabbits, whereas those in group 3, where no hemostatic agent was used, sustained a greater local blood loss. Histologic evaluations revealed that group 1 subjects exhibited fibrosis at the site where bone wax was used, group 2 animals demonstrated no Ostene residua, and groups 2 and 3 demonstrated no fibrotic tissue at the bone defect sites. Furthermore, there were no systemic changes in BUN and CRP in group 4 animals. The authors concluded that Ostene reabsorbs and is not associated with any systemic toxicity or inflammatory local tissue reaction as tested in a rabbit animal model. In summary, Ostene would appear to be an extremely useful alternative to bees wax, because the latter is responsible for a significant inflammatory response (remains in situ for more than a 10-year period and becomes encapsulated with fibrotic tissue), and inhibits new bone formation. This is an excellent study, is clear, and is well thought of. Furthermore, it should prompt spinal surgeons to choose Ostene instead of bone (bees) wax in the future. Nancy Epstein, MD Long Island Neurosurgical Associates New Hyde Park, NY 11042-1101, USA