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Osteoinduction within PEO/PBT copolymer implants in cranial defects using demineralized bone matrix

Osteoinduction within PEO/PBT copolymer implants in cranial defects using demineralized bone matrix JOURNAL OF MATERIALS SCIENCE: MATERIALS IN MEDICINE 5(1994) 764-769 Osteoinduction within PEO/PBT copolymer implants in cranial defects using demineralized bone matrix R. M. VAN HAASTERT, J. J. GROTE, C. A. VAN BLITTERSWlJK ENT-department, University Hospital Leiden, and Biomaterials Research Group, University of Leiden, Rijnsburgerweg 10, 2333 AA Leiden, The Netherlands A. B. PREWETT Osteotech Inc., 1151 Shrewsbury Avenue, Shrewsbury N.J. 07702, USA This study was undertaken to assess the osteoinductive effect addition of demineralized bone matrix (DBM) gel has, on the behaviour of osteoconductive bone-bonding PEO/PBT copolymer (Polyactive R) implants. Cranial defects in rats were filled with these composites to study bone formation in comparison with several controls after 2 and 8 weeks survival time. Osteogenesis was qualitatively evaluated by using light- and transmission electron microscopy as well as backscatter electron imaging. Quantification of the amount of bone ingrowth was performed by using a computerized image analysis system. Initially, rapid calcification was observed in the polymer and DBM, followed by formation of new trabecular bone around the demineralized bone fragments. Bone ingrowth in implants consisting of plain copolymer was less than expected based on previous research, but the addition of demineralized bone matrix gel resulted in a significantly greater amount of new bone formation in the defects. We concluded that the application of DBM-gel to Polyactive R implants had a beneficial effect on the amount of new bone formation in this material. This procedure combines the osteoinductive potential of DBM with the mechanical and bone-bonding properties of a copolymer, thus opening the way to the development of a line of osteoactive composite implants with good surgical handling properties. 1. Introduction Bone-bonding biomaterials like calcium phos- Bone grafts are widely used by orthopaedic, cranio- phates [6] and glass ceramics [7] have shown convin- facial and dental surgeons in the repair of osseous cing osteoconductive capabilities. Although recently defects due to trauma, birth defects, tumor removal or some calcium phosphates have been seen to induce pathological processes like osteomyelitis [1]. Auto- osteogenesis after intramuscular or subcutaneous im- graft bone, because of its osteogenic potential and plantation [8], these biomaterials are not considered inherent biocompatibility, remains the material of to have a significant osteoinductive potential. Fur- choice. Apart from problems related to the limited thermore, their use in surgery has been limited due to availability of autogenous bone, specific morbidity non-optimal mechanical properties. These materials may arise as a consequence of the harvesting proced- are stiffer than bone and relatively brittle, which limits ure including donor site pain, infection, blood loss and their use to non-loadbearing sites [9]. Polymers other post-operative complications [2]. An alternative possess much better elastomeric properties but un- fortunately their osteogenic capacity is mostly poor for autografts is the use of human donor bone (allo- graft). This bone, however, shows a higher resorption without the addition of growth factors like bone mor- rate, potential host rejection as well as the possibility phogenetic protein 1-10-14] or ceramic coatings. Re- of disease transmission. These disadvantages of tradi- cently, however, a polyethylene oxide/polybutylene tional bone grafting have stimulated research into terephthalate (PEO/PBT) segmented copolymer (PolyactiveR), which was originally investigated for use potential bone graft substitutes. Such materials should as an artificial tympanic membrane [15], was found to be osteoactive, which means that they are able to enhance new bone formation [3-5]. This can take bond mechanically tight to bone without prior addi- place through osteoinduction whereby mesenchymal tion of bone-bonding substrates [16-18]. The exact cells will be stimulated to differentiate into osteogenic mechanism behind this bonding remains to be elucid- cells, and by osteoconduction in which the implanted ated but it does seem to be related to calcium absorb- material acts like a scaffold along which new bone tion and hydrogel behaviour, characteristics which are formation can take place. directly related to the soft PEO segment of this mater- 0957-4530 © 1994 Chapman & Hall and cartilage the bone was morselized to yield cortical ial. Although research has shown that Polyactive R has chips which were washed, soaked in ethanol and osteocondaac l-fi~Tand bone-bonding properties it does freeze-dried. They were ground further in a water- not seem to be osteogenic in itself. The addition of an osteoinductive substrate will therefore be a promising cooled bone mill, sieved to a particle size of 100-500 ~tm and decalcified in a solution containing procedure which might optimize the osteoactivity of 0.6 N HCL and a non-ionic detergent. After washing these implants. and freeze-drying, 50% v/v glycerol was added to act The osteoinductive effect of demineralized bone has as a carrier and preservative. been described since 1889 when Senn [19] used decal- cified bone for implantation in human osseous defects. This process of bone induction has been attributed to 2.3. Surgical procedure and experimental the presence of polypeptide factors in demineralized bone belonging to the TGF-13 superfamily called bone design morphogenetic proteins. Demineralized bone matrix Forty Long-Evans rats (weight range 250-300 g) were has been used in craniomaxillofacial reconstruction in operated on to create 8 mm cranial defects. The ani- mals were anesthetized intraperitoneally using a com- the form of blocks or particulates [20], which have a bination of 1% ketamine, 0.1% xylazine and 0.02% tendency to migrate in the surgical site. Recently acepromazine. After shaving the skin overlying the glycerol has been explored as a carrier vehicle for preservation, storage and wetting of DBM [21]. Addi- parietal bone, a midline incision was made along the tion of glycerol produces a gel-like material with saggital suture of the skull and an 8 mm defect was handling properties far superior to those of partic- created under copious irrigation, using a trephine ulates. mounted in a dental handpiece. After removal of the In this study we qualitatively and quantitatively calvarial disc the defect was filled using the selected assessed the effect addition of DBM has on bone implant material. 12 defects were treated with circular formation in porous Polyactive R which was implanted porous Polymer implants; to another 12 Polyactive R implants we added 100 mgr DBM-gel under sterile in cranial defects in rats, to see whether the osteocon- conditions. For controls we filled eight defects with ductive properties of this polymer can be supple- mented with the osteoinductive potential of DBM. just 150 mg of DBM-gel whereas eight defects were left unfilled (see Table I). The healing response was exam- ined after 2 and 8 week periods. 2. Materials and methods 2.1. Implants The PEO/PBT copolymer (provided by HC Implants, 2.4. Microscopy The Netherlands), used in this study was porous (pore After sacrifice the implants with a surrounding bone size 150-400 lam) with a PEO/PBT ratio of 80/20 and margin were removed from the skulls. For every Poly- a molecular weight of the PEO segment of 1000 D. active R group two implants were fixed in 1.5% glutar- aldehyde and post-fixed in 1% osmium ferro (osmium This material was produced as rods with a length of tetraoxide/potassium ferrocyanate 1:1) in 0.14 M 40 mm and a diameter of 10 mm out of which, after sodium cacodylate buffer after which they were em- gamma-irradiation, implants were fabricated to fill the bedded in Spurr's resin in order to be processed for 8 mm cranial defects. Because of its hydrogel proper- ties Polyactive R will increase in volume after uptake of transmission electron microscopy (TEM). The other aqueous solutions, a phenomenon which can be useful implants were fixed in 10% neutrally buffered for- in obtaining a tight fit for these implants in defect sites. malin followed by dehydration in a graded series of The rat parietal bone, however, is thin (0.8-1.2 mm), ethanol and subsequent embedding in polymethyl which results in a relatively small contact area be- methacrylate. tween the implant and bone. If this fact is not taken With the use of a histological diamond saw the into account prior to surgery, the polymer might be defects were first sectioned medially. Next, one half of pushed out of the operation site due to swelling after each sample was sectioned in the coronal plane (Fig. 1) soaking in saline or body fluids. Based on results from thus yielding four undecalcified sections (10 ~tm thick) which were stained using methylene blue and basic a pilot study, it was decided that implants with a pre- fuchsine. These four sections were studied with a light operative diameter of 7.3 mm and 2 mm thickness microscope coupled to a Vidas Image Analysis System would be best suited for use in an 8 mm defect. After to determine the amount of bone ingrowth into the soaking, the diameter can theoretically increase to about 9.4 mm, which will secure the implant in the implants, which was expressed as a percentage of the defect site without it immediately being pushed out by the swelling pressure. TABLE I Experimental design Implant material Number of animals 2.2. DBM-gel 2 weeks 8 weeks For the preparation of rat demineralized bone matrix, Polyactive R 6 6 tibiae and femora were harvested from Long-Evans PolyaetiveR/DBM 6 6 rats (250-300 g) and placed in an iced antibiotic solu- DBM 4 4 tion (500 000 U of Polymyxin B sulfate and 50 000 U Unfilled 4 4 of Bacitracin). After removal of adherent soft tissue 765 scopical evaluation of the copolymer implants after 2 weeks, showed the presence of loosely organized fibrous tissue and some phagocytes as well as in- growth of new trabecular bone from the edges of the implant into the pores. Some intimate contact be- tween the polymer and bone was observed, but fre- quently an interposed cellular layer consisting mainly of fibroblasts and collagen was present. At this time we could also see numerous globular structures lo- cated within the implant surface. These spots clearly reflected in BSE (Fig. 2), which is suggestive of calci- fication, and at times showed intimate contact with newly formed bone (Fig. 3). Transmission electron Coronal section Horizontal section micrographs of decalcified sections of these areas Figure 1 Illustration representing the different directions in which showed the presence of an amorphous electron dense every implant was sectioned for light microscopy. layer with a general thickness of around 200 gm (Fig. 4), which is characteristic of the interface between bone and bone-bonding biomaterials like hydroxy- total defect area in the coronal plane. The remaining apatite. MMA-blocks (from which the four sections were Globular structures, which stained red with basic taken) were polished, carbon coated and examined by fuchsine, were also observed at the surface of many backscatter electron imaging (BSE) using a Philips DBM fragments. These spheres, which closely re- $525 scanning electron micrscope. The other half of semble mineral deposits, were seen to merge, thus each implant was sectioned in the horizontal plane forming large areas of apparent re-mineralization. which gives more information about the distribution Occasionally groups of spherical cells with large cent- of new bone in the defect. rally placed nuclei, closely resembling chondrocytes, were seen in proximity of the demineralized bone blocks (Fig. 5). 3. Results After 8 weeks the presence of more bone tissue in 3.1. Morphology the pores was observed when compared to 2 weeks On first macroscopical observation it was noticed that post-operatively, although the overall amount of new five Polyactive R discs had been partly pushed out of bone formation was very variable. It was also seen the defect due to swelling pressure. Closer light micro- how many fragments of demineralized bone were Figure 2 (a) Backscatter electron micrograph of Polyactive R after 8 weeks displaying extensive calcification (C) within the implant surface which is in close contact with adjacent bone tissue (B). (b) A similar appearance to that in (a) showing the intimate contact (arrows) between globular calcifications (C) and bone (B). 766 Figure 3 (a) Light micrograph of Polyactive R implant after 8 weeks implantation time showing bone (B) ingrowth into the pores. Note the extensive calcification (C) of this material in the shape of multiple globular structures. (b) Detail, showing the calcified polymer (P) in close contact with surrounding bone tissue (B). ingrowth). This difference was statistically significant notwithstanding considerable standard deviations, which were due to large variations in individual bone ingrowth. Defects which were treated with just DBM- gel or were left unfilled showed 29.2% and 11% bone ingrowth, respectively. 4. Discussion This study was undertaken to investigate the potential osteoinductive effect of the addition of demineralized bone matrix gel to an osteoconductive bone-bonding copolymer. Apart from some direct bone ingrowth extending from the edges of the bony defect, osteo- genesis in the pores of these composite implants took place as has been described for demineralized bone matrix in the literature [22]. In short, acellular min- eral deposits [23] were seen on the DBM fragments after 2 weeks which gradually grew and fused together. After 8 weeks these remineralized areas occurred mostly in close contact with newly formed trabecular Figure 4 Transmission electron micrograph of the bone (B)- bone tissue, by which they frequently were incorpor- polymer (P) interface showing an electron dense layer (arrows) indicative of bone-bonding. ated. Sometimes groups of cells, closely resembling chondrocytes, were seen surrounding the DBM blocks which might indicate endochondral ossification taking place, a chain of events triggered by the action of bone recalcified and surrounded by trabecular bone, where- morphogenetic protein present in the DBM. These as sometimes they were completely incorporated in phenomena were observed in the combined PolyactiveR-DBM implants as well as in defects filled newly formed bone (Fig. 6). with just DBM-gel, showing that the presence of this polymer did not compromise the process of osteo- 3.2. Histomorphometry induction. The amount of bone ingrowth expressed as a percent- The PEO/PBT copolymer qualitatively interacted age of the total defect area (the area that had to be with bone tissue, as has recently been observed in filled with bone), is graphically represented in Fig. 7. other research [24]. This material underwent rapid After a 2-week period no significant differences in the and extensive calcification and locally exhibited intim- amount of bone formation were observed between the ate contact with newly formed bone. Transmission different treatment groups. After 8 weeks, however, we electron micrographs of the interface showed an elec- observed less bone formation in plain Polyactive R tron dense layer, a structure which is usually seen at implants (13.5% ingrowth) when compared to poly- the bone-hydroxyapatite interface and is often re- mer implants to which DBM-gel was added (29% ferred to as morphological indication of bone-bonding 767 Figure 5 (a)Histology of demineralized bone gel at the 2 week survival time. At the centre a fragment of demineralized bone is visible with multiple acellular deposits (D) on its surface. At some places the formation of new trabecular bone (T) can be seen. (b) Higher magnification of the same section. Fusing acellular deposits on DBM-fragment forming an area of re-mineralization. On the left large cartilage-like cells (*) are clearly visible. 8O o~ 6O t- 2 weeks 8 weeks Figure 7 Graphical representation of bone ingrowth expressed as a Figure 6 Light micrograph of Polyactive a implant(P) with DBM- percentage of the total defect area after 2 and 8 weeks (11 unfilled; gel after 8 week survival time. A fragment of DBM (D) is [] PA; [] PA-DBM; [] DBM), incorporated in newly formed bone. Note the residual acellular mineral deposits (arrows). The addition of DBM-gel to the Polyactive R does [16, 17, 25]. Quantitative data concerning the Poly- result in a significantly greater amount of new bone active R differed, however, from previous results. After formation (29% ingrowth) in the implants after 8 8 weeks plain polymer implants showed 14% bone weeks. This effect must be partly due to the osteoin- ingrowth as compared to 11% in unfilled defects. This ductive properties of DBM. Defects filled with just is less than expected based on results by Radder [24] DBM-gel also showed 29% bone ingrowth, which is which showed union of 5 mm transcortical defects less than reported by Prewett et al. [21] who showed after 6 weeks implantation time of this polymer in 85% bone ingrowth after 8 weeks. It has to be stressed, goat femora. Furthermore, Bulstra [26] described fas- however, that in the above-mentioned study the mass ter repair of cortical defects in rabbit femora which of de'mineralized bone particles was included in the were filled with Polyactive R as opposed to untreated calculation of new bone ingrowth, whereas we chose defects. These conflicting findings, besides resulting merely to quantify the amount of new trabecular bone from differences between test animals and implant formation surrounding the DBM-fragments, thus locations, could be largely due to the fact that a tight yielding a relatively smaller percentage of bone in- fit between the copolymer and bone, which seems to growth. be a prerequisite for optimal bone-bonding, could not By adding DBM to a PEO/PBT copolymer, we reproducibly be obtained using this experimental have combined the osteoinductive potential of DBM model. with the mechanical and bone-bonding properties of 768 this polymer, thus contributing to the development of 9. K. de GROOT, C. de PUTTER, P. A. E. SILLEVIS SMIT, A. A. DRIESSEN, Sci. Ceram. (1981) 33. composite implants with optimal osteoactive behavi- 10. M.R. URIST, Science 150 (1965) 893. our. These composites could serve as optimal replace- 11. M.R. URIST and B. S. STRATES, J. Dent. Res. 50 (1971) ments for autogenous bone in bone graft surgery thereby eliminating the morbidity associated with har- 12. M.R. URIST, K. SATO, A. G. BROWNELL et al., Proc. Soc. vest surgery. Exp. Bid. Med. 173 (1983) 194. 13. M. WOZNEY, Prog. Growth Factor Res. 1 (1981) 267. 14. K. ELIMA, Ann. Med. 25 (1993) 395. 15. D. BAKKER, C. A. van BLITTERSWIJK, S. C. HESSELING, H. K. KOERTEN, W. KUYPERS and J. J. Acknowledgements GROTE, J. Biomed. Mater. Res. 24 (1990) 489. The authors thank Albert Manrique and Suzanne 16. C.A. van BLITTERSWlJK, D. BAKKER, H. LEENDERS, J. van den BRINK, S. C. HESSELING, Y. P. BOVELL, A. Collier for their excellent technical assistance, advice M. RADDER, R. J. B. SAKKERS, M. L. GAILLARD, P. H. and support during this work. Also, thanks to Henk HEINZE and G. J. BEUMER, in "Bone-bonding biomater- Leenders and Ineke Klamer for their help with the ials", edited by P. Duchenne, T. Kokubo and C. A. van histological work and to Lambert Verschragen for the Blitterswijk (Reed Healthcare Communications, Leiderdorp, preparation of the micrographs. 1992) p. 13. 17. C.A. van BLITTERSWlJK, J. van den BRINK, H. LEEN- DERS and D. BAKKER, Cells Mater. 3 (1993) 23. 18. A.M. RADDER, H. LEENDERS and C. A. van BLITTER- SWIJK, J. Biomed. Mater. Res. 28 (1994) 141. References 19. N. SENN, Amer. J. Med. Sci. 98 (1889) 219. 1. G.F. MUSCHLER and J. M. LANE, in "Bone graft substi- 20. J.B. MULLIKEN, J. GLOWACKI, L. B. KABAN, J. FOL- tutes" (W.B. Saunders, New York, 1992) p. 375. KMAN and J. E. MURRAY, Ann. Surg. 194 (1981) 366. 2. E.M. YOUNGER and M. W. CHAPMAN, J. Orthop. Trauma 21. A. B. PREWETT, J. R. PARSONS, C. J. DAMIEN and 3 (1989) 192. L. W. MEEKS, in "Bone-bonding biomaterials", edited by 3. A. KENLEY, K. YIM, J. ABRAMS, E. RON, T. TUREK, L. P. Duchenne, T. Kokubo and C. A. van Blitterswijk (Reed J. MARDEN and J. O. HOLLINGER, Pharmaceutical Res. Healthcare Communications, Leiderdorp, 1992) p. 139. 10 (1993) 1393. 22. J.W. HAGEN, J. M. SEMMELINK, C. P. A. T. KLEIN, 4. C.J. DAMIEN and J. R. PARSONS, J. Appl. Biomater. 2 B. PRAHL-ANDERSEN and E. H. BURGER, J. Biomed. (1991) 187. Mater. Res. 26 (1992) 897. 5. C.A. EAST and W. F. LARRABEE, J. Long-term effects Med. 23. K. YAMASHITA and T. TAKAGI, Arch. Histol. Cytol. 55 Implants 1 (1991) 53. (1992) 31. 6. M. JARCHO, Clin. Orthop. 157 (1981) 259. 24. A.M. RADDER and C. A. van BLITTERSWIJK, J. Mater. 7. T. KOKUBO, in "Bone-bonding biomaterials', edited by Sci. Mater. Med. (in press). P. Duchenne, T. Kokubo and C. A. van Blitterswijk (Reed 25. C. A. van BLITTERSWIJK, D. BAKKER, S. C. Healthcare Communications, Leiderdorp, 1992) p. 31. HESSELING and H. K. KOERTEN, Biomaterials 12 (1991) 8. J.M. TOTH, K. L. LYNCH and D. A. HACKBARTH, in 87. "Bioceramics", Vol. 6, edited by P. Duchenne and D. Chris- 26. S.K. BULSTRA, M. M. W. F. DREES, R. KUYPER, C. A. tiansen (Butterworth-Heinemann Ltd., Philadelphia, 1993) van BLITTERSWIJK, G. HEIDENDAL and A. J. van der p. 9. LINDEN (submitted). http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Materials Science: Materials in Medicine Springer Journals

Osteoinduction within PEO/PBT copolymer implants in cranial defects using demineralized bone matrix

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Materials Science; Biomaterials; Biomedical Engineering and Bioengineering; Regenerative Medicine/Tissue Engineering; Polymer Sciences; Ceramics, Glass, Composites, Natural Materials; Surfaces and Interfaces, Thin Films
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Abstract

JOURNAL OF MATERIALS SCIENCE: MATERIALS IN MEDICINE 5(1994) 764-769 Osteoinduction within PEO/PBT copolymer implants in cranial defects using demineralized bone matrix R. M. VAN HAASTERT, J. J. GROTE, C. A. VAN BLITTERSWlJK ENT-department, University Hospital Leiden, and Biomaterials Research Group, University of Leiden, Rijnsburgerweg 10, 2333 AA Leiden, The Netherlands A. B. PREWETT Osteotech Inc., 1151 Shrewsbury Avenue, Shrewsbury N.J. 07702, USA This study was undertaken to assess the osteoinductive effect addition of demineralized bone matrix (DBM) gel has, on the behaviour of osteoconductive bone-bonding PEO/PBT copolymer (Polyactive R) implants. Cranial defects in rats were filled with these composites to study bone formation in comparison with several controls after 2 and 8 weeks survival time. Osteogenesis was qualitatively evaluated by using light- and transmission electron microscopy as well as backscatter electron imaging. Quantification of the amount of bone ingrowth was performed by using a computerized image analysis system. Initially, rapid calcification was observed in the polymer and DBM, followed by formation of new trabecular bone around the demineralized bone fragments. Bone ingrowth in implants consisting of plain copolymer was less than expected based on previous research, but the addition of demineralized bone matrix gel resulted in a significantly greater amount of new bone formation in the defects. We concluded that the application of DBM-gel to Polyactive R implants had a beneficial effect on the amount of new bone formation in this material. This procedure combines the osteoinductive potential of DBM with the mechanical and bone-bonding properties of a copolymer, thus opening the way to the development of a line of osteoactive composite implants with good surgical handling properties. 1. Introduction Bone-bonding biomaterials like calcium phos- Bone grafts are widely used by orthopaedic, cranio- phates [6] and glass ceramics [7] have shown convin- facial and dental surgeons in the repair of osseous cing osteoconductive capabilities. Although recently defects due to trauma, birth defects, tumor removal or some calcium phosphates have been seen to induce pathological processes like osteomyelitis [1]. Auto- osteogenesis after intramuscular or subcutaneous im- graft bone, because of its osteogenic potential and plantation [8], these biomaterials are not considered inherent biocompatibility, remains the material of to have a significant osteoinductive potential. Fur- choice. Apart from problems related to the limited thermore, their use in surgery has been limited due to availability of autogenous bone, specific morbidity non-optimal mechanical properties. These materials may arise as a consequence of the harvesting proced- are stiffer than bone and relatively brittle, which limits ure including donor site pain, infection, blood loss and their use to non-loadbearing sites [9]. Polymers other post-operative complications [2]. An alternative possess much better elastomeric properties but un- fortunately their osteogenic capacity is mostly poor for autografts is the use of human donor bone (allo- graft). This bone, however, shows a higher resorption without the addition of growth factors like bone mor- rate, potential host rejection as well as the possibility phogenetic protein 1-10-14] or ceramic coatings. Re- of disease transmission. These disadvantages of tradi- cently, however, a polyethylene oxide/polybutylene tional bone grafting have stimulated research into terephthalate (PEO/PBT) segmented copolymer (PolyactiveR), which was originally investigated for use potential bone graft substitutes. Such materials should as an artificial tympanic membrane [15], was found to be osteoactive, which means that they are able to enhance new bone formation [3-5]. This can take bond mechanically tight to bone without prior addi- place through osteoinduction whereby mesenchymal tion of bone-bonding substrates [16-18]. The exact cells will be stimulated to differentiate into osteogenic mechanism behind this bonding remains to be elucid- cells, and by osteoconduction in which the implanted ated but it does seem to be related to calcium absorb- material acts like a scaffold along which new bone tion and hydrogel behaviour, characteristics which are formation can take place. directly related to the soft PEO segment of this mater- 0957-4530 © 1994 Chapman & Hall and cartilage the bone was morselized to yield cortical ial. Although research has shown that Polyactive R has chips which were washed, soaked in ethanol and osteocondaac l-fi~Tand bone-bonding properties it does freeze-dried. They were ground further in a water- not seem to be osteogenic in itself. The addition of an osteoinductive substrate will therefore be a promising cooled bone mill, sieved to a particle size of 100-500 ~tm and decalcified in a solution containing procedure which might optimize the osteoactivity of 0.6 N HCL and a non-ionic detergent. After washing these implants. and freeze-drying, 50% v/v glycerol was added to act The osteoinductive effect of demineralized bone has as a carrier and preservative. been described since 1889 when Senn [19] used decal- cified bone for implantation in human osseous defects. This process of bone induction has been attributed to 2.3. Surgical procedure and experimental the presence of polypeptide factors in demineralized bone belonging to the TGF-13 superfamily called bone design morphogenetic proteins. Demineralized bone matrix Forty Long-Evans rats (weight range 250-300 g) were has been used in craniomaxillofacial reconstruction in operated on to create 8 mm cranial defects. The ani- mals were anesthetized intraperitoneally using a com- the form of blocks or particulates [20], which have a bination of 1% ketamine, 0.1% xylazine and 0.02% tendency to migrate in the surgical site. Recently acepromazine. After shaving the skin overlying the glycerol has been explored as a carrier vehicle for preservation, storage and wetting of DBM [21]. Addi- parietal bone, a midline incision was made along the tion of glycerol produces a gel-like material with saggital suture of the skull and an 8 mm defect was handling properties far superior to those of partic- created under copious irrigation, using a trephine ulates. mounted in a dental handpiece. After removal of the In this study we qualitatively and quantitatively calvarial disc the defect was filled using the selected assessed the effect addition of DBM has on bone implant material. 12 defects were treated with circular formation in porous Polyactive R which was implanted porous Polymer implants; to another 12 Polyactive R implants we added 100 mgr DBM-gel under sterile in cranial defects in rats, to see whether the osteocon- conditions. For controls we filled eight defects with ductive properties of this polymer can be supple- mented with the osteoinductive potential of DBM. just 150 mg of DBM-gel whereas eight defects were left unfilled (see Table I). The healing response was exam- ined after 2 and 8 week periods. 2. Materials and methods 2.1. Implants The PEO/PBT copolymer (provided by HC Implants, 2.4. Microscopy The Netherlands), used in this study was porous (pore After sacrifice the implants with a surrounding bone size 150-400 lam) with a PEO/PBT ratio of 80/20 and margin were removed from the skulls. For every Poly- a molecular weight of the PEO segment of 1000 D. active R group two implants were fixed in 1.5% glutar- aldehyde and post-fixed in 1% osmium ferro (osmium This material was produced as rods with a length of tetraoxide/potassium ferrocyanate 1:1) in 0.14 M 40 mm and a diameter of 10 mm out of which, after sodium cacodylate buffer after which they were em- gamma-irradiation, implants were fabricated to fill the bedded in Spurr's resin in order to be processed for 8 mm cranial defects. Because of its hydrogel proper- ties Polyactive R will increase in volume after uptake of transmission electron microscopy (TEM). The other aqueous solutions, a phenomenon which can be useful implants were fixed in 10% neutrally buffered for- in obtaining a tight fit for these implants in defect sites. malin followed by dehydration in a graded series of The rat parietal bone, however, is thin (0.8-1.2 mm), ethanol and subsequent embedding in polymethyl which results in a relatively small contact area be- methacrylate. tween the implant and bone. If this fact is not taken With the use of a histological diamond saw the into account prior to surgery, the polymer might be defects were first sectioned medially. Next, one half of pushed out of the operation site due to swelling after each sample was sectioned in the coronal plane (Fig. 1) soaking in saline or body fluids. Based on results from thus yielding four undecalcified sections (10 ~tm thick) which were stained using methylene blue and basic a pilot study, it was decided that implants with a pre- fuchsine. These four sections were studied with a light operative diameter of 7.3 mm and 2 mm thickness microscope coupled to a Vidas Image Analysis System would be best suited for use in an 8 mm defect. After to determine the amount of bone ingrowth into the soaking, the diameter can theoretically increase to about 9.4 mm, which will secure the implant in the implants, which was expressed as a percentage of the defect site without it immediately being pushed out by the swelling pressure. TABLE I Experimental design Implant material Number of animals 2.2. DBM-gel 2 weeks 8 weeks For the preparation of rat demineralized bone matrix, Polyactive R 6 6 tibiae and femora were harvested from Long-Evans PolyaetiveR/DBM 6 6 rats (250-300 g) and placed in an iced antibiotic solu- DBM 4 4 tion (500 000 U of Polymyxin B sulfate and 50 000 U Unfilled 4 4 of Bacitracin). After removal of adherent soft tissue 765 scopical evaluation of the copolymer implants after 2 weeks, showed the presence of loosely organized fibrous tissue and some phagocytes as well as in- growth of new trabecular bone from the edges of the implant into the pores. Some intimate contact be- tween the polymer and bone was observed, but fre- quently an interposed cellular layer consisting mainly of fibroblasts and collagen was present. At this time we could also see numerous globular structures lo- cated within the implant surface. These spots clearly reflected in BSE (Fig. 2), which is suggestive of calci- fication, and at times showed intimate contact with newly formed bone (Fig. 3). Transmission electron Coronal section Horizontal section micrographs of decalcified sections of these areas Figure 1 Illustration representing the different directions in which showed the presence of an amorphous electron dense every implant was sectioned for light microscopy. layer with a general thickness of around 200 gm (Fig. 4), which is characteristic of the interface between bone and bone-bonding biomaterials like hydroxy- total defect area in the coronal plane. The remaining apatite. MMA-blocks (from which the four sections were Globular structures, which stained red with basic taken) were polished, carbon coated and examined by fuchsine, were also observed at the surface of many backscatter electron imaging (BSE) using a Philips DBM fragments. These spheres, which closely re- $525 scanning electron micrscope. The other half of semble mineral deposits, were seen to merge, thus each implant was sectioned in the horizontal plane forming large areas of apparent re-mineralization. which gives more information about the distribution Occasionally groups of spherical cells with large cent- of new bone in the defect. rally placed nuclei, closely resembling chondrocytes, were seen in proximity of the demineralized bone blocks (Fig. 5). 3. Results After 8 weeks the presence of more bone tissue in 3.1. Morphology the pores was observed when compared to 2 weeks On first macroscopical observation it was noticed that post-operatively, although the overall amount of new five Polyactive R discs had been partly pushed out of bone formation was very variable. It was also seen the defect due to swelling pressure. Closer light micro- how many fragments of demineralized bone were Figure 2 (a) Backscatter electron micrograph of Polyactive R after 8 weeks displaying extensive calcification (C) within the implant surface which is in close contact with adjacent bone tissue (B). (b) A similar appearance to that in (a) showing the intimate contact (arrows) between globular calcifications (C) and bone (B). 766 Figure 3 (a) Light micrograph of Polyactive R implant after 8 weeks implantation time showing bone (B) ingrowth into the pores. Note the extensive calcification (C) of this material in the shape of multiple globular structures. (b) Detail, showing the calcified polymer (P) in close contact with surrounding bone tissue (B). ingrowth). This difference was statistically significant notwithstanding considerable standard deviations, which were due to large variations in individual bone ingrowth. Defects which were treated with just DBM- gel or were left unfilled showed 29.2% and 11% bone ingrowth, respectively. 4. Discussion This study was undertaken to investigate the potential osteoinductive effect of the addition of demineralized bone matrix gel to an osteoconductive bone-bonding copolymer. Apart from some direct bone ingrowth extending from the edges of the bony defect, osteo- genesis in the pores of these composite implants took place as has been described for demineralized bone matrix in the literature [22]. In short, acellular min- eral deposits [23] were seen on the DBM fragments after 2 weeks which gradually grew and fused together. After 8 weeks these remineralized areas occurred mostly in close contact with newly formed trabecular Figure 4 Transmission electron micrograph of the bone (B)- bone tissue, by which they frequently were incorpor- polymer (P) interface showing an electron dense layer (arrows) indicative of bone-bonding. ated. Sometimes groups of cells, closely resembling chondrocytes, were seen surrounding the DBM blocks which might indicate endochondral ossification taking place, a chain of events triggered by the action of bone recalcified and surrounded by trabecular bone, where- morphogenetic protein present in the DBM. These as sometimes they were completely incorporated in phenomena were observed in the combined PolyactiveR-DBM implants as well as in defects filled newly formed bone (Fig. 6). with just DBM-gel, showing that the presence of this polymer did not compromise the process of osteo- 3.2. Histomorphometry induction. The amount of bone ingrowth expressed as a percent- The PEO/PBT copolymer qualitatively interacted age of the total defect area (the area that had to be with bone tissue, as has recently been observed in filled with bone), is graphically represented in Fig. 7. other research [24]. This material underwent rapid After a 2-week period no significant differences in the and extensive calcification and locally exhibited intim- amount of bone formation were observed between the ate contact with newly formed bone. Transmission different treatment groups. After 8 weeks, however, we electron micrographs of the interface showed an elec- observed less bone formation in plain Polyactive R tron dense layer, a structure which is usually seen at implants (13.5% ingrowth) when compared to poly- the bone-hydroxyapatite interface and is often re- mer implants to which DBM-gel was added (29% ferred to as morphological indication of bone-bonding 767 Figure 5 (a)Histology of demineralized bone gel at the 2 week survival time. At the centre a fragment of demineralized bone is visible with multiple acellular deposits (D) on its surface. At some places the formation of new trabecular bone (T) can be seen. (b) Higher magnification of the same section. Fusing acellular deposits on DBM-fragment forming an area of re-mineralization. On the left large cartilage-like cells (*) are clearly visible. 8O o~ 6O t- 2 weeks 8 weeks Figure 7 Graphical representation of bone ingrowth expressed as a Figure 6 Light micrograph of Polyactive a implant(P) with DBM- percentage of the total defect area after 2 and 8 weeks (11 unfilled; gel after 8 week survival time. A fragment of DBM (D) is [] PA; [] PA-DBM; [] DBM), incorporated in newly formed bone. Note the residual acellular mineral deposits (arrows). The addition of DBM-gel to the Polyactive R does [16, 17, 25]. Quantitative data concerning the Poly- result in a significantly greater amount of new bone active R differed, however, from previous results. After formation (29% ingrowth) in the implants after 8 8 weeks plain polymer implants showed 14% bone weeks. This effect must be partly due to the osteoin- ingrowth as compared to 11% in unfilled defects. This ductive properties of DBM. Defects filled with just is less than expected based on results by Radder [24] DBM-gel also showed 29% bone ingrowth, which is which showed union of 5 mm transcortical defects less than reported by Prewett et al. [21] who showed after 6 weeks implantation time of this polymer in 85% bone ingrowth after 8 weeks. It has to be stressed, goat femora. Furthermore, Bulstra [26] described fas- however, that in the above-mentioned study the mass ter repair of cortical defects in rabbit femora which of de'mineralized bone particles was included in the were filled with Polyactive R as opposed to untreated calculation of new bone ingrowth, whereas we chose defects. These conflicting findings, besides resulting merely to quantify the amount of new trabecular bone from differences between test animals and implant formation surrounding the DBM-fragments, thus locations, could be largely due to the fact that a tight yielding a relatively smaller percentage of bone in- fit between the copolymer and bone, which seems to growth. be a prerequisite for optimal bone-bonding, could not By adding DBM to a PEO/PBT copolymer, we reproducibly be obtained using this experimental have combined the osteoinductive potential of DBM model. with the mechanical and bone-bonding properties of 768 this polymer, thus contributing to the development of 9. K. de GROOT, C. de PUTTER, P. A. E. SILLEVIS SMIT, A. A. DRIESSEN, Sci. Ceram. (1981) 33. composite implants with optimal osteoactive behavi- 10. M.R. URIST, Science 150 (1965) 893. our. These composites could serve as optimal replace- 11. M.R. URIST and B. S. STRATES, J. Dent. Res. 50 (1971) ments for autogenous bone in bone graft surgery thereby eliminating the morbidity associated with har- 12. M.R. URIST, K. SATO, A. G. BROWNELL et al., Proc. Soc. vest surgery. Exp. Bid. Med. 173 (1983) 194. 13. M. WOZNEY, Prog. Growth Factor Res. 1 (1981) 267. 14. K. ELIMA, Ann. Med. 25 (1993) 395. 15. D. BAKKER, C. A. van BLITTERSWIJK, S. C. HESSELING, H. K. KOERTEN, W. KUYPERS and J. J. Acknowledgements GROTE, J. Biomed. Mater. Res. 24 (1990) 489. The authors thank Albert Manrique and Suzanne 16. C.A. van BLITTERSWlJK, D. BAKKER, H. LEENDERS, J. van den BRINK, S. C. HESSELING, Y. P. BOVELL, A. Collier for their excellent technical assistance, advice M. RADDER, R. J. B. SAKKERS, M. L. GAILLARD, P. H. and support during this work. Also, thanks to Henk HEINZE and G. J. BEUMER, in "Bone-bonding biomater- Leenders and Ineke Klamer for their help with the ials", edited by P. Duchenne, T. Kokubo and C. A. van histological work and to Lambert Verschragen for the Blitterswijk (Reed Healthcare Communications, Leiderdorp, preparation of the micrographs. 1992) p. 13. 17. C.A. van BLITTERSWlJK, J. van den BRINK, H. LEEN- DERS and D. BAKKER, Cells Mater. 3 (1993) 23. 18. A.M. RADDER, H. LEENDERS and C. A. van BLITTER- SWIJK, J. Biomed. Mater. Res. 28 (1994) 141. References 19. N. SENN, Amer. J. Med. Sci. 98 (1889) 219. 1. G.F. MUSCHLER and J. M. LANE, in "Bone graft substi- 20. J.B. MULLIKEN, J. GLOWACKI, L. B. KABAN, J. FOL- tutes" (W.B. Saunders, New York, 1992) p. 375. KMAN and J. E. MURRAY, Ann. Surg. 194 (1981) 366. 2. E.M. YOUNGER and M. W. CHAPMAN, J. Orthop. Trauma 21. A. B. PREWETT, J. R. PARSONS, C. J. DAMIEN and 3 (1989) 192. L. W. MEEKS, in "Bone-bonding biomaterials", edited by 3. A. KENLEY, K. YIM, J. ABRAMS, E. RON, T. TUREK, L. P. Duchenne, T. Kokubo and C. A. van Blitterswijk (Reed J. MARDEN and J. O. HOLLINGER, Pharmaceutical Res. Healthcare Communications, Leiderdorp, 1992) p. 139. 10 (1993) 1393. 22. J.W. HAGEN, J. M. SEMMELINK, C. P. A. T. KLEIN, 4. C.J. DAMIEN and J. R. PARSONS, J. Appl. Biomater. 2 B. PRAHL-ANDERSEN and E. H. BURGER, J. Biomed. (1991) 187. Mater. Res. 26 (1992) 897. 5. C.A. EAST and W. F. LARRABEE, J. Long-term effects Med. 23. K. YAMASHITA and T. TAKAGI, Arch. Histol. Cytol. 55 Implants 1 (1991) 53. (1992) 31. 6. M. JARCHO, Clin. Orthop. 157 (1981) 259. 24. A.M. RADDER and C. A. van BLITTERSWIJK, J. Mater. 7. T. KOKUBO, in "Bone-bonding biomaterials', edited by Sci. Mater. Med. (in press). P. Duchenne, T. Kokubo and C. A. van Blitterswijk (Reed 25. C. A. van BLITTERSWIJK, D. BAKKER, S. C. Healthcare Communications, Leiderdorp, 1992) p. 31. HESSELING and H. K. KOERTEN, Biomaterials 12 (1991) 8. J.M. TOTH, K. L. LYNCH and D. A. HACKBARTH, in 87. "Bioceramics", Vol. 6, edited by P. Duchenne and D. Chris- 26. S.K. BULSTRA, M. M. W. F. DREES, R. KUYPER, C. A. tiansen (Butterworth-Heinemann Ltd., Philadelphia, 1993) van BLITTERSWIJK, G. HEIDENDAL and A. J. van der p. 9. LINDEN (submitted).

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Journal of Materials Science: Materials in Medicine – Springer Journals

Published: May 19, 2004

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