Effect of local hemostatics on bone induction in rats: A comparative study of bone wax, fibrin-collagen paste, and bioerodible polyorthoester with and without gentamicin Eirik Solheim,* Else Marie Pinholt, Gisle Bang,+ and Einar Sudmand lnstitute for Surgical Research, Rikshospitalet, University of Oslo, ‘Department of Oral Pathology and Forensic Odontology and #Hagavik Orthopaedic Hospital, University of Bergen, Norway Local hemostatics for osseous tissue should preferably be absorbable and biocompatible and should not inhibit osteogenesis. The tissue response and effect on demineralized bone-induced heterotopic osteogenesis in the abdominal muscle of 120 male Wistar rats by different local hemostatics were evaluated by light microscopy and 85Sruptake analyses. Nonabsorbable bone wax of 88% beeswax and absorbable bovine fibrin-collagen paste both significantly in hi bi ted o st eoinduc-
tion, whereas a bioerodible polyorthoester drug delivery system with or without 4% gentamicin did not. Bone wax was not absorbed and induced a chronic foreign body reaction. Fibrin-collagen paste induced less inflammation with numerous monocytes and macrophages with e n g u l f e d material. Bioerodible polyorthoester caused a very moderate tissue reaction a n d was mostly resorbed at week 4.
INTRODUCTION
In bone surgery, nonabsorbable bone wax’,* of 88% beeswax and 12% isopropylpalmitat is being used for local hemo~tasis.”~ Bone wax may, however, produce a chronic inflammation with foreign-body r e a ~ t i o n ,retard ~ bone healing6 predispose for infections,7.*impair bacterial clearance: and cause wax embolization.’” These complications have spurred the development of absorbable local hemostatics, like fibrin,“ oxidized cellulose,’2 gelatin ~ p o n g e , ’microcrys~ talline collagen,14 fibrin sealant,” fibrin-collagen paste,16 fibrillar collagen,” and bioerodible polyorthoester sustained drug release systems,” which may be formulated with physical properties similar to that of bone wax, i.e., soft and moldable, and can be used as a local hemostatic in the same way.” The purpose of the present study was to investigate host-tissue response and effect on demineralized bone-induced heterotopic osteogenesis by three *To whom correspondence should be addressed at Hagavik Orthopaedic Hospital, N-5220 Hagavik, Norway.
Journal of Biomedical Materials Research, Vol. 26, 791-800 (1992) 0 1992 John Wiley & Sons, Inc. CCC 0021-9304/92/060791-10$4.00
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local hemostatics for osseous tissue: (a) nonabsorbable bone wax and the absorbable alternatives, (b) fibrin-collagen paste, and (c) polyorthoester with and without gentamicin.
MATERIALS A N D METHODS
Implants Demineralized bone matrix (DBM) was prepared by sterile technique from the long bones of male Wistar rats of the same weight as the recipients. Dissected diaphyses were crushed and the marrow was removed. The cortex was cut into chips and demineralized in 0.2N HC1 for 48 h at 4OC,Z"flushed by saline, suspended in liquid nitrogen, and lyophilized for 22 h. The DBM was kept at 4°C and implanted within 48 h. Each DBM-chip measured 0.5 X 2.0 X 2.0 mm and weighed about 0.7 mg. The following local hemostatics were used: (1) Nonabsorbable bone wax (Ethicon Ltd., Edinburgh) of 88% purified beeswax and 12% isopropylpalmitate. (2) Absorbable bone sealant paste (fibrin-collagen paste) (AbseleB, Ethicon Ltd., Edinburgh) containing stabilized ox fibrin 17.5%,solubilized ox collagen 17.5%,dextran 70 8.0%, glycerol B.P. 30.0%),and water to 100 w/w. (3) Bioerodible polyorthoester poly(2,2-dioxy-cis,trans-1,4-cyclohexanedimethylene tetrahydrofuran) (AlzamerB, Alza Corporation, Palo Alto, CA) with and without 4% gentamicin. The polyorthoester results from condensation of 2,2-diethoxytetrahydrofuran and cis,trans-1,4-bis(hydroxymethyl)cyclohexane. The biodegradation takes place by hydrolysis to the ultimate products 4-hydroxybutyrate (4HB) and cis,trans-1,4-bis(hydroxymethyl)cyclohexane (CHDM). 4HB is further metabolized in the tricarboxylic acid cycle, with COz and HzO as the end products. CHDM is excreted in urine." The polyorthoester may be formulated with different physical properties, in the present study soft and moldable. Spherical composite implants were made by mixing approximately equal volumes of DBM (4 chips) and local hemostatic (15 mg) manually at room temperature under sterile conditions immediately before implantation. The DBM chips were partly exposed and partly embedded in the hemostatic.
Surgical procedure
A total of 120 male Wistar rats weighing 215 g (SD 10) was used. The rats were randomized in eight groups, A-H, of 15 rats each. The animals were fed on standard laboratory food and water ad libitum. The Norwegian national guidelines for the care and use of laboratory animals were observed. Anesthesia was induced with 0.15 mL/100 g HypnormB-DormicumB intramuscularly. The abdomen was shaved and washed with 3% chlorhexidine solution. The fascia was exposed through a median incision. One pouch in each
LOCAL HEMOSTATICS A N D BONE INDUCTION
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rat was created between the right oblique abdominal muslces by blunt dissection. The pouch was filled with one of the following: (a) Group A, 4 DBM chips; (b) Group B, 4 DBM chips and 15 mg polyorthoester; (c) Group C, 4 DBM chips and 15 mg polyorthoester with 4% gentamicin; (d) Group D, 4 DBM chips and 15 mg fibrin-collagen paste; (e) Group E, 4 DBM chips and 15 mg bone wax; (f) Group F, 15 mg polyorthoester; (g) Group G, 15 mg fibrin-collagen paste; (h) Group H, 15 mg bone wax. The incision was closed in layers.
Evaluation
All animals were killed at 4 weeks after having received 10 microci of "Sr as SrClz per 100 g body weight intraperitoneally 4 days earlier. The implant and the right 0s ilium of each rat were dissected, weighed and fixed in 4% neutral formalin. "Sr uptake was determined in a Packard gamma counter. The ratio between the total activity of the implant and the specific activity of the 0s ilium ((counts/min implant)/(counts/min/mg ilium)) was computed and the ratio is called osteoquantum index." After 85Sruptake determination, the implants were demineralized in 17% formic acid, dehydrated, and embedded in paraffin. Serial sections were cut at 5-pm interval and stained with Harris' hernatoxylin and eosin. Qualitative histological evaluation was performed. Osteoinduction was defined as bone and bone marrow f ~ r m a t i o n . ~ ~ ' ~ ~
Statistics
The statistical evaluation was carried out by SPSS/PC + statistical software. The Bartlett's test for the homogeneity of variances showed that the variance of the osteoquantum index was significantly different among the groups, also after rescaling the data using log transformation, invalidating the use of the overall F test for one-way analyses of variance. Thus, pairs of means were compared with two-sample t test for independent samples with unequal or equal variances. The osteoquantum indices of the composites of local hemostatics and DBM (Groups B-E) and the local hemostatics (Groups F-H) were compared with that of DBM alone (Group A). As comparing multiple groups introduces greater probability of type I error, the required p value (0.05) was divided by the number of comparisons (Bonferroni's correction); p < 0.007 (0.05/7) was regarded as significant.
RESULTS
There were no perioperative or postoperative deaths. The animals gained weight and showed no sign of unhealthiness.
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Histological evaluation
By qualitative histology, no differences in bone induction could be identified between DBM alone (Group A) (Figs. 1 and 2) and composite of DBM and polyorthoester without drug (Group B) or with gentamicin (Group C) (Figs. 3 and 4). At the time of evaluation ossicles with bone marrow had formed in the implants of the three groups. Little or no inflammation was evident and only traces of the polyorthoester were seen in some implants of composite of DBM and polyorthoester with or without gentamicin and polyorthoester alone (Group F). In the implants of fibrin-collagen paste alone (Croup G) and composite of fibrin-collagen paste and DBM (Group D) (Fig. 5) reactive changes with numerous macrophages containing engulfed material, a few multinuclear giant cells and proliferation of fibroblasts were seen. Part of the paste was still present and little sign of osteoinduction was seen in the composite implants. Histological evaluation of the implants of bone wax alone (Group H) and composite of bone wax and DBM (Group E) (Fig. 6) showed an inflammation with numerous multinuclear giant cells and large amounts of bone wax. In the composite implants inflammatory cells were seen uniformly through all parts of the implants interspersed between bone wax and partly resorbed DBM generally without signs of osteoinduction.
Figure 1. Allogeneic, demineralized bone matrix, DBM, inducing bone, B, in the shape of an ossicle containing bone marrow, BM, 4 weeks after implantation to the rat abdominal muscles (hematoxylin & eosin, original magnification X100).
LOCAL HEMOSTATICS AND BONE INDUCTION
Figure 2. Higher magnification of framed area in Figure 1 exhibiting host osteoprogenitor cells, OPC, induced new bone, B, with osteocytes, bone marrow, BM, and implanted demineralized bone matrix, DBM (hematoxylin & eosin, original magnification X715).
Figure 3. Composite of allogeneic, demineralized bone matrix, DBM, and polyorthoester with gentamicin inducing bone, B, in the shape of an ossicle containing bone marrow, BM, 4 weeks after implantation to the rat abdominal muscles (hematoxylin & eosin, original magnification X100).
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Figure 4. Higher magnification of framed area in Figure 3 showing osteoblast, 0, induced bone, B, and implanted demineralized bone matrix, DBM (hematoxylin & eosin, original magnification X400).
Figure 5. Composite of fibrin-collagen paste and partly resorbed demineralized bone matrix, DBM, 4 weeks after implantation to the rat abdominal muscles. No bone induction is evident. Reactive changes with inflammatory cells and macrophages, M, are seen (hematoxylin & eosin, original magnification X250).
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Figure 6. Composite of bone wax, W, and partly resorbed demineralized bone matrix, DBM, 4 weeks after impIantation to the rat abdominal muscles. No bone induction is evident. Reactive changes with many multinucleated giant cells, GC, are seen (hematoxylin & eosin, original magnification X250).
"Sr uptake
No significant difference was found between the osteoquantum index of DBM alone and composites of DBM and polyorthoester ( p = 0.90) or DBM and polyorthoester with 4% gentamicin ( p = 0.89) (Table I). The osteoquanturn indices of the composites of fibrin-collagen paste or bone wax and DBM, and the local hemostatics alone was significantly lower than that of DBM alone (p < 0.001). TABLE I Osteoquantum Index. Mean, SD Implant
Group
DBM DBM and polyorthoester DBM and polyorthoester-gentamicin DBM and fibrin-collagen paste DBM and bone wax Polyor thoester Fibrin collagen paste Bone wax **Significantly different from group A ( p < 0.001).
Osteoquantum index 6.88 6.77 6.70 2.05 1.83 0.31 0.62 0.58
I .26 3.04 4.88 1.30** 1.85** 0.40"" 0.99** 0.31**
SOLHEIM ET AL.
798 DISCUSSION
Local hemostatics for osseous tissue should preferably be absorbable and bicompatible and should not inhibit osteogenesis. DBM regularly induces heterotopic bone in rodent^^^"^ and DBM-induced heterotopic osteogenesis in rodents has been used as an experimental model for evaluating host tissue response and the effect of biomaterials on oste~genesis.~~-'~ Ordinary nonabsorbable bone wax may induce a foreign-body reaction both in animal^','^,^^ and in human^.'^,^"^^ In addition, bone wax inhibits the healing of experimentally created bone defects in rats6,'' and rabbits3' and the union of a median sternotomy in This inhibition of bone healing has been thought to be caused by unabsorbed bone wax acting as a physical barrier.6 The previous reports regarding absorption and biocompatibility of fibrincollagen paste are conflicting as in one study it was found that 30 mg fibrincollagen paste implanted in the lumbar muscles of rats was totally absorbed with minimal tissue reaction in approximately 3 weeks," while others have reported delayed absorption of the hemostatic, chronic inflammation and retarded bone healing in rabbit mandibular cavities.33 In the present study, bone wax and fibrin-collagen paste were incompletely absorbed, the two materials inhibited osteoinduction and caused a chronic inflammation. The bone wax caused an inflammation with numerous multinuclear giant cells, while fibrin-collage paste caused an inflammation with accumulation of macrophages and proliferation of fibroblasts. As connective tissue and inflammatory cells were seen uniformly through all parts of the composite implants of the bone wax or fibrin-collagen paste and DBM, interspersed between local hemostatic and partly resorbed DBM, the lack of osteoinduction cannot be explained solely as an effect of a physical barrier of the local hemostatic. Previous studies have indicated that while an unspecific transient inflammation is part of the osteoinduction,2" a chronic inflammation may inhibit o s t e ~ i n d u c t i o n . ~Ox ~ , ~fibrin ~ , ~ ~and , ~ ~ox collagen of fibrincollagen paste are xenoimplants and may elicit immunological reactions that could inhibit o s t e o i n d ~ c t i o n . ~ ~ The polyorthoester did not inhibit osteoinduction histologically or by "Sr evaluation, little or no inflammation was seen and the polyorthoester was mostly resorbed. The polyorthoester adheres to bone surfaces and provides local hemostasis by plugging of spongy bone and secondarily promoting concentration of platelets and coagulation factor^.'^*^^ We thank Katrine Hove, Inger Liv Nordli and Gunnvor Bjordsbakken for technical assistance and Alza Corporation, Palo Alto and Johnson & Johnson AB, Sollentuna for providing the polyorthoester (Alzamerm) and the fibrin-collagen paste (Abselem, Ethicon Ltd., Edinburgh), respectively.
References 1. R. Parker, "Aural pyaemia successfully treated by removing putrid thrombus of jugular vein and lateral sinus," Br. Med. J, 1, 1076-1077 (1892).
LOCAL HEMOSTATICS AND BONE INDUCTION 2. 3. 4. 5.
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10. 11. 12. 13. 14. 15. 16. 17. 18.
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22. 23.
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V. Horsley, “Antiseptic wax,” Br. Med. J., 1, 1165 (1892). R. Baldauf and 1.0. Kanat, “The use of bone wax,” J. Foot Surg., 25,456458 (1986). A. H. Crenshaw, ”Surgical techniques,” in Campbell’s Operative Orthopaedics, A . H . Crenshaw (ed.), The C.V. Mosby Company, St. Louis, 1987, pp. 3-22. J. R. Geary and V. K. Frantz, ”New absorbable bone wax surgery,” Ann. Surg., 132, 1128-1137 (1950). T.C. Howard and R. R. Kelley, ”The effect of bone wax on the healing of experimental rat tibia1 lesions,” Clin. Ortkop. Rel. Res., 63, 226-231 (1969). A.T. Culliford, J. N. Cunningham, R.H. Zeff, O.W. Isom, P. Teiko, and E C. Spencer, ”Sternal and costochondral infections following openheart surgery. A review of 2,594 cases,“ ]. Thoruc. Cardiovasc. Surg., 72, 714-726 (1976). F. Robicsek, H. K. Daugherty, J.W. Cook, J.G. Selle, T.N. Masters, P.R. O’Bar, C. R. Fernandez, C.U. Mauney, and D. M. Calhoun, ”Mycobacterium fortuitum epidemics after open-heart surgery,” Thoruc. Cardiovasc. Surg., 1, 91-96 (1978). P. Johnson and D. Fromm, ”Effects of bone wax on bacterial clearance,” Surgery, 89, 206-209 (1981). F. Robicsek, T. N. Masters, L. Littman, and G.V. Born, “The embolization of bone wax from sternotomy incisions,” Ann. Tkorac. Surg., 31, 357-359 (1981). S. Bergel, ”Ueber Wirkungen des Fibrins,” Dtsch. Med. Wockenschr., 35, 663-665 (1909). V. K. Frantz, H.T. Clarke, and R. Lattes, ”Hemostasis with absorbable gauze (oxidized cellulose),” Ann. Surg., 120, 181-198 (1944). J.T. Correll and E.C. Wise, “Certain properties of a new physiologically absorbable sponge,” Proc. Soc. E x p . Biol. Med., 58, 233-235 (1945). 0.A. Battista, N. Z. Erdi, and C. F. Ferraso, ”Colloidal macromolecular phenomena: 11. Novel microcrystals of polymer,” 1. Appl. Polymer. Sci., 11, 481-498 (1967). H. Matras, H. P. Dinges, H. Lassman, and B. Mamoli, ”Zur nathlosen interfaszikularen Nerventransplantation im Tierexperiment,” Wien. Med. Wochensckr., 122, 517-523 (1972). P. Harris and I. Capperauld, “A new absorbable haemostatic bone sealant,” ]. R. Coll. Surg. Edinb., 23, 285-291 (1978). M. E. Silverstein, K. Keown, J. A. Owen, and M. Chvapil, ”Collagen fibers as a fleece hemostatic agent,” 1. Trauma, 20, 688-694 (1980). R. Capozza, L. Sendelbeck, and W. Balkenhol, ”Preparation and evaluation of bioerodible naltrexone delivery system,” in Polymeric Delivery Systems, R. J. Kostelnick (ed.), Gordon and Breach Science Publishers, New York, 1978, pp. 59-73. B. Sudmann, 0.-G. Anfinsen, M. Rait, G. Bang, and E. Sudmann, “Use of a new hemostatic, bioerodible polymer versus bone wax made of beeswax- a clinical and experimental study,” Acta Orthop. Scand. (SUPPI. 237), 61, 63-64 (1990). G. Bang, “Induction of heterotopic bone formation by demineralized dentin: an experimental model in guinea pigs,” Scund. J. Dent. Res., 81, 240-250 (1973). S. L. Sendelbeck and C. L. Girdis, “Disposition of a 14C-labeled bioerodible polyorthoester and its hydrolysis products, 4-hydroxybuturate and cis,truns-1,4-bis(hydroxymethyl)cyclohexane,in rats,” Drug Metab. Dispos., 13, 291-295 (1985). E. Solheim, E. M. Pinholt, G. Bang, and E. Sudmann, ”Comparison of histomorphometry and 85Sr uptake in induced heterotopic bone in rats,“ Actu Ortkop. Scund., in press. M. R. Urist, “Bone: Formation by autoinduction,” Science, 150, 893-899 (1965).
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800 24. 25. 26. 27. 28. 29. 30.
31. 32. 33. 34. 35.
36. 37.
A. H. Reddi and C. Huggins, ”Biochemical sequences in the transformation of normal fibroblasts in adolescent rats,” Proc. Natl. Acad. Sci. USA, 69, 1601-1605 (1972). J. J. Vandersteenhoven and M. Spector, ”Osteoinduction within porous polysulfone implants at extraosseous sites using demineralized allogeneic bone matrix,” J. Biomed. Muter. Res., 17, 793-806 (1983). G. Uretzky, J. Appelbaum, and J. Sela, ”Inhibition of the inductive activity of demineralized bone matrix by different percutaneous implants,’’ Biomaterials, 9, 195-197 (1988). U. Ripamonti, C. M. Schnitzler, and P.C. Cleaton-Jones,”Bone induction in a composite allogeneic bone/alloplastic implant,” J. Oral Maxillofac. Surg., 47, 963-969 (1989). D. Wolter, W. Mohr, and L. Kind, “Die Reaktion des Knochen- und Weichteilgewebes auf implantiertes Knochenwachs beim Schaf,” Chirurg, 46, 459-462 (1975). S. J. Sorrenti, W. J. Cumming, and D. Miller, ”Reaction of the human tibia to bone wax,” Clin. Orthop. Rel. Res., 182, 293-296 (1984). J. Aurelio, B. Chenail, and H. Gerstein, ”Foreign-body reaction to bone wax. Report of a case,” Oral Surg., 58, 98-100 (1984). P. Alberius, B. Klinge, and S. Sjogren, “Effects of bone wax on rabbit cranial bone lesions,” J. Craniomaxillofac. Surg., 15, 63-67 (1987). T.G. Brightmore, P. Hayes, J. Humble, and A. D. Morgan, “Haemostasis and healing following median sternotomy,” Langenbecks Arch. Chir., Suppl, 39-41 (1975). P. A. Kondell, A. Nordenram, G. Anneroth, and T. Mattsson, ’Rbsele in bone hemostasis- a clinical and experimental investigation,” Swed. Dent. J, 12, 85-90 (1988). J. Sela, J. Applebaum, and G. Uretzky, ”Osteogenesis induced by bone matrix is inhibited by inflammation,” Biomater. Med. Devi. Artif. Organs, 14,227-237 (1986). R.M. Nathan, H. Bentz, R.M. Armstrong, K.A. Piez, T.L. Smestad, L. R. Ellingsworth, J. M. McPherson, and S. M. Seyedin, ”Osteogenesis in rats with an inductive bovine composite,” 1. Orthop. Kes., 6, 324-334 (1988). M. R. Urist, 8. F. Silverman, K. Buring, EL. Dubuc, and J. M. Rosenberg, “The bone induction principle,” Clin. Orfhop. Rel. Res., 53, 243283 (1967). E. Solheim, 0.-G. Anfinsen, H. Holmsen, and E. Sudmann, “Effect of local hemostatics on platelet aggregation,” Eur. Surg. Res., 23, 45-50 (1991).
Received April 10, 1991 Accepted November 20, 1991
tion, whereas a bioerodible polyorthoester drug delivery system with or without 4% gentamicin did not. Bone wax was not absorbed and induced a chronic foreign body reaction. Fibrin-collagen paste induced less inflammation with numerous monocytes and macrophages with e n g u l f e d material. Bioerodible polyorthoester caused a very moderate tissue reaction a n d was mostly resorbed at week 4.
INTRODUCTION
In bone surgery, nonabsorbable bone wax’,* of 88% beeswax and 12% isopropylpalmitat is being used for local hemo~tasis.”~ Bone wax may, however, produce a chronic inflammation with foreign-body r e a ~ t i o n ,retard ~ bone healing6 predispose for infections,7.*impair bacterial clearance: and cause wax embolization.’” These complications have spurred the development of absorbable local hemostatics, like fibrin,“ oxidized cellulose,’2 gelatin ~ p o n g e , ’microcrys~ talline collagen,14 fibrin sealant,” fibrin-collagen paste,16 fibrillar collagen,” and bioerodible polyorthoester sustained drug release systems,” which may be formulated with physical properties similar to that of bone wax, i.e., soft and moldable, and can be used as a local hemostatic in the same way.” The purpose of the present study was to investigate host-tissue response and effect on demineralized bone-induced heterotopic osteogenesis by three *To whom correspondence should be addressed at Hagavik Orthopaedic Hospital, N-5220 Hagavik, Norway.
Journal of Biomedical Materials Research, Vol. 26, 791-800 (1992) 0 1992 John Wiley & Sons, Inc. CCC 0021-9304/92/060791-10$4.00
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SOLHEIM ET AL.
local hemostatics for osseous tissue: (a) nonabsorbable bone wax and the absorbable alternatives, (b) fibrin-collagen paste, and (c) polyorthoester with and without gentamicin.
MATERIALS A N D METHODS
Implants Demineralized bone matrix (DBM) was prepared by sterile technique from the long bones of male Wistar rats of the same weight as the recipients. Dissected diaphyses were crushed and the marrow was removed. The cortex was cut into chips and demineralized in 0.2N HC1 for 48 h at 4OC,Z"flushed by saline, suspended in liquid nitrogen, and lyophilized for 22 h. The DBM was kept at 4°C and implanted within 48 h. Each DBM-chip measured 0.5 X 2.0 X 2.0 mm and weighed about 0.7 mg. The following local hemostatics were used: (1) Nonabsorbable bone wax (Ethicon Ltd., Edinburgh) of 88% purified beeswax and 12% isopropylpalmitate. (2) Absorbable bone sealant paste (fibrin-collagen paste) (AbseleB, Ethicon Ltd., Edinburgh) containing stabilized ox fibrin 17.5%,solubilized ox collagen 17.5%,dextran 70 8.0%, glycerol B.P. 30.0%),and water to 100 w/w. (3) Bioerodible polyorthoester poly(2,2-dioxy-cis,trans-1,4-cyclohexanedimethylene tetrahydrofuran) (AlzamerB, Alza Corporation, Palo Alto, CA) with and without 4% gentamicin. The polyorthoester results from condensation of 2,2-diethoxytetrahydrofuran and cis,trans-1,4-bis(hydroxymethyl)cyclohexane. The biodegradation takes place by hydrolysis to the ultimate products 4-hydroxybutyrate (4HB) and cis,trans-1,4-bis(hydroxymethyl)cyclohexane (CHDM). 4HB is further metabolized in the tricarboxylic acid cycle, with COz and HzO as the end products. CHDM is excreted in urine." The polyorthoester may be formulated with different physical properties, in the present study soft and moldable. Spherical composite implants were made by mixing approximately equal volumes of DBM (4 chips) and local hemostatic (15 mg) manually at room temperature under sterile conditions immediately before implantation. The DBM chips were partly exposed and partly embedded in the hemostatic.
Surgical procedure
A total of 120 male Wistar rats weighing 215 g (SD 10) was used. The rats were randomized in eight groups, A-H, of 15 rats each. The animals were fed on standard laboratory food and water ad libitum. The Norwegian national guidelines for the care and use of laboratory animals were observed. Anesthesia was induced with 0.15 mL/100 g HypnormB-DormicumB intramuscularly. The abdomen was shaved and washed with 3% chlorhexidine solution. The fascia was exposed through a median incision. One pouch in each
LOCAL HEMOSTATICS A N D BONE INDUCTION
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rat was created between the right oblique abdominal muslces by blunt dissection. The pouch was filled with one of the following: (a) Group A, 4 DBM chips; (b) Group B, 4 DBM chips and 15 mg polyorthoester; (c) Group C, 4 DBM chips and 15 mg polyorthoester with 4% gentamicin; (d) Group D, 4 DBM chips and 15 mg fibrin-collagen paste; (e) Group E, 4 DBM chips and 15 mg bone wax; (f) Group F, 15 mg polyorthoester; (g) Group G, 15 mg fibrin-collagen paste; (h) Group H, 15 mg bone wax. The incision was closed in layers.
Evaluation
All animals were killed at 4 weeks after having received 10 microci of "Sr as SrClz per 100 g body weight intraperitoneally 4 days earlier. The implant and the right 0s ilium of each rat were dissected, weighed and fixed in 4% neutral formalin. "Sr uptake was determined in a Packard gamma counter. The ratio between the total activity of the implant and the specific activity of the 0s ilium ((counts/min implant)/(counts/min/mg ilium)) was computed and the ratio is called osteoquantum index." After 85Sruptake determination, the implants were demineralized in 17% formic acid, dehydrated, and embedded in paraffin. Serial sections were cut at 5-pm interval and stained with Harris' hernatoxylin and eosin. Qualitative histological evaluation was performed. Osteoinduction was defined as bone and bone marrow f ~ r m a t i o n . ~ ~ ' ~ ~
Statistics
The statistical evaluation was carried out by SPSS/PC + statistical software. The Bartlett's test for the homogeneity of variances showed that the variance of the osteoquantum index was significantly different among the groups, also after rescaling the data using log transformation, invalidating the use of the overall F test for one-way analyses of variance. Thus, pairs of means were compared with two-sample t test for independent samples with unequal or equal variances. The osteoquantum indices of the composites of local hemostatics and DBM (Groups B-E) and the local hemostatics (Groups F-H) were compared with that of DBM alone (Group A). As comparing multiple groups introduces greater probability of type I error, the required p value (0.05) was divided by the number of comparisons (Bonferroni's correction); p < 0.007 (0.05/7) was regarded as significant.
RESULTS
There were no perioperative or postoperative deaths. The animals gained weight and showed no sign of unhealthiness.
SOLHEIM ET AL.
794
Histological evaluation
By qualitative histology, no differences in bone induction could be identified between DBM alone (Group A) (Figs. 1 and 2) and composite of DBM and polyorthoester without drug (Group B) or with gentamicin (Group C) (Figs. 3 and 4). At the time of evaluation ossicles with bone marrow had formed in the implants of the three groups. Little or no inflammation was evident and only traces of the polyorthoester were seen in some implants of composite of DBM and polyorthoester with or without gentamicin and polyorthoester alone (Group F). In the implants of fibrin-collagen paste alone (Croup G) and composite of fibrin-collagen paste and DBM (Group D) (Fig. 5) reactive changes with numerous macrophages containing engulfed material, a few multinuclear giant cells and proliferation of fibroblasts were seen. Part of the paste was still present and little sign of osteoinduction was seen in the composite implants. Histological evaluation of the implants of bone wax alone (Group H) and composite of bone wax and DBM (Group E) (Fig. 6) showed an inflammation with numerous multinuclear giant cells and large amounts of bone wax. In the composite implants inflammatory cells were seen uniformly through all parts of the implants interspersed between bone wax and partly resorbed DBM generally without signs of osteoinduction.
Figure 1. Allogeneic, demineralized bone matrix, DBM, inducing bone, B, in the shape of an ossicle containing bone marrow, BM, 4 weeks after implantation to the rat abdominal muscles (hematoxylin & eosin, original magnification X100).
LOCAL HEMOSTATICS AND BONE INDUCTION
Figure 2. Higher magnification of framed area in Figure 1 exhibiting host osteoprogenitor cells, OPC, induced new bone, B, with osteocytes, bone marrow, BM, and implanted demineralized bone matrix, DBM (hematoxylin & eosin, original magnification X715).
Figure 3. Composite of allogeneic, demineralized bone matrix, DBM, and polyorthoester with gentamicin inducing bone, B, in the shape of an ossicle containing bone marrow, BM, 4 weeks after implantation to the rat abdominal muscles (hematoxylin & eosin, original magnification X100).
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Figure 4. Higher magnification of framed area in Figure 3 showing osteoblast, 0, induced bone, B, and implanted demineralized bone matrix, DBM (hematoxylin & eosin, original magnification X400).
Figure 5. Composite of fibrin-collagen paste and partly resorbed demineralized bone matrix, DBM, 4 weeks after implantation to the rat abdominal muscles. No bone induction is evident. Reactive changes with inflammatory cells and macrophages, M, are seen (hematoxylin & eosin, original magnification X250).
LOCAL HEMOSTATICS AND BONE INDUCTION
797
Figure 6. Composite of bone wax, W, and partly resorbed demineralized bone matrix, DBM, 4 weeks after impIantation to the rat abdominal muscles. No bone induction is evident. Reactive changes with many multinucleated giant cells, GC, are seen (hematoxylin & eosin, original magnification X250).
"Sr uptake
No significant difference was found between the osteoquantum index of DBM alone and composites of DBM and polyorthoester ( p = 0.90) or DBM and polyorthoester with 4% gentamicin ( p = 0.89) (Table I). The osteoquanturn indices of the composites of fibrin-collagen paste or bone wax and DBM, and the local hemostatics alone was significantly lower than that of DBM alone (p < 0.001). TABLE I Osteoquantum Index. Mean, SD Implant
Group
DBM DBM and polyorthoester DBM and polyorthoester-gentamicin DBM and fibrin-collagen paste DBM and bone wax Polyor thoester Fibrin collagen paste Bone wax **Significantly different from group A ( p < 0.001).
Osteoquantum index 6.88 6.77 6.70 2.05 1.83 0.31 0.62 0.58
I .26 3.04 4.88 1.30** 1.85** 0.40"" 0.99** 0.31**
SOLHEIM ET AL.
798 DISCUSSION
Local hemostatics for osseous tissue should preferably be absorbable and bicompatible and should not inhibit osteogenesis. DBM regularly induces heterotopic bone in rodent^^^"^ and DBM-induced heterotopic osteogenesis in rodents has been used as an experimental model for evaluating host tissue response and the effect of biomaterials on oste~genesis.~~-'~ Ordinary nonabsorbable bone wax may induce a foreign-body reaction both in animal^','^,^^ and in human^.'^,^"^^ In addition, bone wax inhibits the healing of experimentally created bone defects in rats6,'' and rabbits3' and the union of a median sternotomy in This inhibition of bone healing has been thought to be caused by unabsorbed bone wax acting as a physical barrier.6 The previous reports regarding absorption and biocompatibility of fibrincollagen paste are conflicting as in one study it was found that 30 mg fibrincollagen paste implanted in the lumbar muscles of rats was totally absorbed with minimal tissue reaction in approximately 3 weeks," while others have reported delayed absorption of the hemostatic, chronic inflammation and retarded bone healing in rabbit mandibular cavities.33 In the present study, bone wax and fibrin-collagen paste were incompletely absorbed, the two materials inhibited osteoinduction and caused a chronic inflammation. The bone wax caused an inflammation with numerous multinuclear giant cells, while fibrin-collage paste caused an inflammation with accumulation of macrophages and proliferation of fibroblasts. As connective tissue and inflammatory cells were seen uniformly through all parts of the composite implants of the bone wax or fibrin-collagen paste and DBM, interspersed between local hemostatic and partly resorbed DBM, the lack of osteoinduction cannot be explained solely as an effect of a physical barrier of the local hemostatic. Previous studies have indicated that while an unspecific transient inflammation is part of the osteoinduction,2" a chronic inflammation may inhibit o s t e ~ i n d u c t i o n . ~Ox ~ , ~fibrin ~ , ~ ~and , ~ ~ox collagen of fibrincollagen paste are xenoimplants and may elicit immunological reactions that could inhibit o s t e o i n d ~ c t i o n . ~ ~ The polyorthoester did not inhibit osteoinduction histologically or by "Sr evaluation, little or no inflammation was seen and the polyorthoester was mostly resorbed. The polyorthoester adheres to bone surfaces and provides local hemostasis by plugging of spongy bone and secondarily promoting concentration of platelets and coagulation factor^.'^*^^ We thank Katrine Hove, Inger Liv Nordli and Gunnvor Bjordsbakken for technical assistance and Alza Corporation, Palo Alto and Johnson & Johnson AB, Sollentuna for providing the polyorthoester (Alzamerm) and the fibrin-collagen paste (Abselem, Ethicon Ltd., Edinburgh), respectively.
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Received April 10, 1991 Accepted November 20, 1991
