The Effect of Macroporous Biphasic Calcium Phosphate Block as Carrier of Recombinant Human Bone Morphogenetic Protein-2 on Bone Formation in Rat Calvarial Defects. Yong-Jun Lee The Graduate School Yonsei University Department of Dental Science
The Effect of Macroporous Biphasic Calcium Phosphate Block as Carrier of Recombinant Human Bone Morphogenetic Protein-2 on Bone Formation in Rat Calvarial Defects. Submitted to the Department of Dental Science and the Graduate School of Yonsei University In partial fulfillment of the Requirements for the degree of Doctor of Philosophy of Dental Science Yong-Jun Lee December 2005
This certifies that the dissertation thesis of Yong-Jun Lee is approved Thesis Supervisor: Kyoo-Sung Cho Jung-Kiu Chai Jong-In Yook Hyung-Jun Kim Chang-Sung Kim The Graduate School Yonsei University December 2005
먼저이논문이나올수있게끔부족한저를지도해주시고이끌어주셨던존경하는조규성교수님의큰가르침에깊은감사를드립니다. 그리고많은관심과격려를해주신김종관교수님, 채중규교수님, 최성호교수님, 김창성교수님에게도진심으로감사드립니다. 연구내내많은도움을준조익현선생님과한동관선생님그리고치주과교실원여러분께도고마움을전합니다. 또항상저에게인생의비전을제시해주시는신흥의이용익사장님께도깊은감사의말씀을전합니다. 깊은사랑과염려로저를키워주시고보살펴주시는아버지, 어머님, 또부족한남편에게큰사랑과아낌없는도움으로큰의지가되어주는사랑하는나의아내와나에게항상빛이되어주고따뜻함이되어주는사랑하는나의두딸다인, 다윤에게진정으로사랑과고마움을전합니다. 다시한번모든분들께고개숙여감사의말씀을올립니다. 2005 년 12 월 저자씀
Table of Contents Abstract (English) iii I. Introduction 1 II. Materials and Methods 5 1. Animals 5 2. rhbmp-2 Implant Construction 6 3. Surgical Procedures 6 4. Histologic and Histometric Procedures 8 5. Statistical Analysis 9 III. Results 10 1. Clinical Observations 10 2. Histologic Observations 10 3. Histometric Analysis 11 IV. Discussion 13 V. Conclusion 16 References 17 Legends 22 Figures 24 Abstract (Korean) 27 i
List of Figures Figure 1 Schematic drawing of calvarial osteotomy defect showing the histometric analysis 9 Figure 2. Representative photomicrographs of MBCP block carrier control at 2 weeks. 24 Figure 3. Representative photomicrographs of MBCP block carrier control at 8weeks. 24 Figure 4. Representative photomicrographs of rhbmp-2/mbcp block group at 2weeks. 25 Figure 5. Representative photomicrographs of rhbmp-2/mbcp block group at 8weeks. 25 Figure 6. Representative photomicrographs of rhbmp-2/mbcp block group at 8weeks (base of MBCP block, x100) 26 Figure 7. Representative photomicrographs of rhbmp-2/mbcp block group at 8weeks (top area of MBCP block, x100) 26 List of Tables Table 1. Experimental design 5 Table 2. Augmented area. 12 Table 3. Bone density 12 ii
Abstract The effect of macroporous biphasic calcium phosphate Blocks as carrier of recombinant human bone morphogenetic protein-2 on bone formation in Rat Calvarial Defects Bone morphogenetic proteins (BMPs) are currently being evaluated as potential candidates for periodontal and bone regenerative therapy. In spite of the good prospect of BMP applications, an ideal carrier system for BMPs has not been identified. The macroporous biphasic calcium phosphate (MBCP) block can be used as a bone augmentation material because of its hardness and ability to generate space. The purpose of this study is to evaluate the effect of the MBCP block as an rhbmp-2 carrier in the rat calvarial defect model. Eight-mm critical-size calvarial defects were created in 40 male Sprague-Dawley rats. The animals were divided into 2 groups of 20 animals each. The defects were treated with MBCP blocks alone or rhbmp-2/ MBCP blocks. Defects were evaluated by histological and histometric parameters following a 2- or 8-week healing interval (10 animals/group/healing intervals). The new bone area of the MBCP/rhBMP-2 group was significantly greater than the MBCP control group at both 2 and 8 weeks (p< 0.01).The new bone area of the 8 week group was greater than the 2 week group in all treatment conditions. iii
The total augmented area did not change when all groups were compared. In conclusion, the bone regenerative effect of MBCP/ rhbmp-2 was superior to MBCP blocks alone in the rat calvarial critical-sized defect model. Surgical implantation of rhbmp-2/ MBCP blocks may be able to regenerate bone in the rat calvarial critical sized defects without any side effects. In addition, MBCP blocks may be considered effective carriers of rhbmp-2. Key Words : rhbmp-2, critical sized defect, macroporous biphasic calcium phosphate block, augmentation, carrier. iv
The Effect of Macroporous Biphasic Calcium Phosphate Block as Carrier of Recombinant Human Bone Morphogenetic Protein-2 on Bone Formation in Rat Calvarial Defects. Yong-Jun Lee, D.D.S. Department of Dental Science Graduate School, Yonsei University (Directed by Prof. Kyoo-sung cho, D.D.S., M.S.D., PhD.) Ⅰ. Introduction Bone morphogenetic protein-2 (BMP-2) is a member of the transforming growth factor- superfamily of multifunctional cytokines. It induces bone formation 5,24,16 and plays an important role in development 32. Implantation of BMPs alone does not induce bone formation because the protein rapidly diffuses from the site of implantation. Use of an appropriate carrier material is essential for the delivery, retention, and release of BMPs at defect site 15,21,22,3. A carrier material should have the following qualities: biocompatible in order to minimize local tissue response, easy to mold to the desired shape, biodegradable for 1
its replacement by newly formed bone, enable the sustained release of BMP, and have mechanical stability in bone defects 29,3,30,28,12,14,20,23. Many materials, such as tricalcium phosphate 29,30,9,33 polylactic acid polymer 11, absorbable collagen sponge (ACS) 1,8,31, demineralized bone matrix 11, hydroxyapatite (HA) 17,21, fibrin sealant 10, polylactic-polyglycolic polymer 4,19, and composites of these materials 23 have been used and evaluated as a BMP carriers for the healing of bone defects. Until now, rhbmp-2/acs has been effective when used as an inlay, but it has the great limitation of onlayindication 6,26,27. Many studies have shown that the rhbmp carrier maintains augmented space firmly and has good bone regeneration. In our previous study, we tried to find the proper rhbmp dose 25 that could be compared to rhbmp-2,4,7. 13 We also continued to search for excellent carriers of rhbmp such as ACS, β-tcp, the fibro-fibronectin sealing system 10, and their combination. However, no rhbmp carriers were hard enough to endure the compressive force. Apparently, with the appropriate space-providing carriers or adjunctive devices, rhbmp-2 may not only induce clinically relevant bone formation for alveolar ridge augmentation, but has also been shown to have compressive forces in the craniofacial complex 7. 2
In recent years, studies by Daculsi et al, have demonstrated the stability and effectiveness of the mixture of hydroxyapaptite (HA) and beta-tricalcium phosphate ( -TCP). HA provides a good scaffold for the new bone to grow, but has poor regeneration potential. -TCP has good bone regeneration potential, but is not able to provide sufficient space for bone growth. Mixing HA and TCP permits the association between the physico-chemical properties of each compound. This process allows the manufacturing of materials with controllable resorption and bone substitution depending on the proportion of HA and β-tcp. According to studies conducted by Lynch and Nery, Legeros et al, and Daculsi et al, the mixture of 60% HA and 40% -TCP constitutes the ideal mixture for using macroporous biphasic calcium phosphate (MBCP) as a bone substitute. MBCP TM has a porous form required for the biological exchanges that are particularly essential for bone in growth and mineralization. A MBCP block has a 70% global porosity. The MBCP macropore structure will help ionic change, BMP diffusion, and the movement and retention of osteogenic cells. In addition, HA provides sufficient firmness to the MBCP block and maintains the bone augmented space. Furthermore, the MBCP block can be molded into any desirable shape. Therefore, the MBCP block can be used as bone augmentation material due to its hardness and space-generating ability. 3
The purpose of this study is to evaluate the effectiveness of the MBCP block as an rhbmp-2 carrier. 4
Ⅱ. MATERIALS & METHODS 1. Animals A total of 40 male Sprague-Dawley rats (weight 200-300 g) were used. Animals were maintained in plastic cages in a room with 12 h-day/night cycles, an ambient temperature of 21 o C, and ad libitum access to water and a standard laboratory pellet diet. Animal selection and management, surgical protocol, and preparation followed routines approved by the Institutional Animal Care and Use Committee, Yonsei Medical Center, Seoul, Korea. Table-1 Experimental design Group week Number of rat MBCP block only rhbmp-2/ MBCP block 2 weeks 10 rats 8 weeks 10 rats 2 weeks 10 rats 8 weeks 10 rats 5
2. rhbmp-2 implant construction rhbmp-2 Π was diluted to a concentration of 0.025mg/ml. For the rhbmp-2/ MBCP block implant, a sterile MBCP block Ω was loaded with 0.5ml of the rhbmp-2 solution (100 micron litter per 1 block). Following a 5-minute binding time, the implant was prepared to fit the defect. 3. Surgical procedure The animals were generally anaesthetized with an intramuscular injection (5mg/kg body wt.) consisting of ketamine hydrochloride θ. During surgery, routine infiltration anaesthesia δ was used at the surgical site.the surgical site was shaved and scrubbed with iodine. An incision was made in the sagittal plane across the cranium. A full thickness flap including periosteum was reflected, exposing the calvarial bone. Then, a standardized, round, transosseous defect 8 mm in diameter was created similarly on the cranium with the use of a saline cooled trephine drill in the same Π R&D Systems Inc., Minneapolis, MN, U.S.A Ω Biomatlante Inc, France θ Ketalar, Yuhan Co., Seoul, Korea δ 2% lidocaine, 1:100,000 epinephrine, Kwangmyung Pharm., Seoul, Korea 3i, Palm Beach Gardens, FL, USA 6
manner as described by Schmitz and Hollinger. Then, each animal received one of two experimental conditions: thembcp block alone or the rhbmp-2/ MBCP block. The skin was sutured for primary closure with 4-0 coated Vicryl sutures Polyglactin 910, braided absorbable suture, Ethicon, Johnson & Johnson Int., Edinburgh, UK 7
4. Histological and histometrical procedures The animals were sacrificed by CO 2 asphyxiation at 2 and 8 weeks post surgery. Block sections including the surgical sites were removed. The Samples were placed immediately into vials and fixed in 10% neutral buffered formalin solution for 10 days. All samples were decalcified in EDTA-HCl for 7 days, and embedded in paraffin. Three µm thick coronal sections through the center of the augmented area were stained with hematoxylin-eosin. After conventional microscopic examination, computer-assisted histometric measurements of the newly formed bone were obtained using an automated image analysis system ƒ coupled with a video camera on a light microscope. Sections were examined at 20x magnification. And then Three parameters were measured (figure-1). 1) Augmented area (mm 2 ) was measured including new bone, the residual biomaterials, mineralized bone, fatty marrow and fibrovascular tissue. 2) New bone area (mm 2 ) was determined by the newly formed bone area within the total augmented area. 3) Bone density was calculated as follows: Bone density (%) = New bone area / Augmented area x 100 ƒ Image-Pro Plus, Media Cybernetics, Silver Spring, MD, USA Olympus BX50, Olympus Optical Co., Tokyo, Japan 8
Connective Tissue New Bone Carrier Histomorphometric parameters - Augmented area - New bone area - New Bone density= New bone area/ Augmented areaⅹ100 Figure 1. Schematic drawing of calvarial osteotomy defect showing the histometric analysis 5. Statistical Analysis Histomorphometric recordings from the samples were used to calculate group means and standard deviation values (m±sd). To compare the 2 and 8week values in the same rhbmp, statistical significance was determined by a paired t-test. A twoway analysis of variance was used to analyze the effect of time and experimental conditions. The post hoc Scheffe s test was used to analyze the difference between the groups (P<0.01). 9
Ⅲ. Results 1. Clinical observation Wound healing was generally uneventful, Two exposed MBCP block were observed which were excluded from the analysis. 2. Histologic observation 1) MBCP block group At 2 and 8 weeks post surgery, the augmented areas were covered with dense, fibrous connective tissue. A small amount of new bone formation was observed adjacent to the margins of the defect at 2 weeks (Figure2). In addition, at 2 weeks a large number of residual MBCP particles were observed within the new bone. However, at 8 weeks, in the downside of augmented area, many MBCP particles were embedded in or surrounded by newly formed bone and it was possible to observe the close contact between graft particles and the newly formed bone trabecules (Figure3). Newly formed bone was characterized by lacunae containing osteoblasts, which appeared to be osteocytes, and had abundant medullary space filled with a wellvascularized connective tissue with no histological markers of inflammation or foreign body reactions. 10
2) MBCP/ BMP group At 2 and 8 weeks post surgery, the augmented areas were more filled with new bone in the MBCP/BMP group than the control group. At 2 weeks, many osteoblasts and osteocytes were observed in the bottom of the MBCP block. The quantity of new bone in the 8 week group was more than the quantity in the 2 weeks group, and the appearance of the new bone was more lamellar at 8 weeks than at 2 weeks. Concentric rings of the Haversian system, Cement lines, and fatty marrow were all observed in the new bone area (Figure 6). Newly formed bone grew from the bottom to the top of the MBCP block, and moved from the outside to the inside of the pore (Figure 5). In particular, a great deal of new bone was observed at 8weeks in the upside of augmented area (Figure7). In addition, in some cases the cranial bone of defect margin appeared to be covering the augmented material. 3. Histometric analysis Two exposed block were excluded from the analysis and another 6 samples were excluded from the analysis because of technical problems. The total augmented area did not change when all group were compared to each other (Table-2). 11
The new bone area of the rhbmp-2/mbcp block group was significantly greater than the MBCP block group at both 2 and 8 weeks (p< 0.01).The new bone area of the 8 week group was greater than the 2 week group in all treatment condition (Table- 3). A two-way ANOVA revealed that there was an interaction between the healing interval and treatment condition in the new bone area (p <0.01). Table-2. Total augmented area (group means ±SD, mm2 ) Total augmented area MBCP (2weeks) 19.859 ± 3.8 MBCP (8weeks) 20.346 ± 2.7 MBCP+rhBMP-2 (2weeks) 22.419 ± 3.4 MBCP+rhBMP-2 (8weeks) 22.373 ± 4.1 No significant difference when compared to all groups (P<0.01) Table-3. Bone density (group means±sd, N=8,%) New bone area/ augmented area * 100 MBCP (2 weeks) 7.27% ± 3.7 MBCP (8 weeks) 14.74% ± 5.2 MBCP+rhBMP-2 (2 weeks) 12.82% ± 5.0 * MBCP+rhBMP-2 (8 weeks) 23.64% ± 8.4 * : Statistically significant difference when compared to 2 weeks (P<0.01) *: Statistically significant difference when compared to the surgical control group (P<0.01) 12
Ⅳ. Discussion Currently clinicians need bone augmentation on the alveolar ridge, due to alveolar ridge loss resulting from early tooth loss, surgical and accidental trauma, or pathologic processes. To resolve such problems, osteoconductive biomaterials or block implantation, autogenous bone grafts, and guided bone regeneration (GBR) were developed. Until now, autogenous bone grafts were considered to be the best. Autogenous bone grafts have many advantages, such as great osteogenic properties, histocompatibility, and the elimination of disease transmission. However harvesting enough autogenous bone from an extraoral donor site requires general anesthesia, donor site operation, and hospitalization. For this reason, many researchers have been interested in rh-bmp. The object of this study is to evaluate the effects of MBCP blocks as rhbmp-2 carriers when they are used in bone augmentation. The critical-size rat calvarial defect used in this study compared to other experimental bone defects is a convenient model for evaluating bone regenerative effects of biomaterials. This model is relatively accessible, simple. and reproducible because spontaneous healing does not occur in the control specimens (Frame, 1980; Schmitz et al., 1986). In addition, after bone augmentation, this model has some compressive force, which is similar to intraoral conditions. 13
Recently, the use of β-tcp has developed as an osteoconductive bone substitute and a biodegradable delivery system for rhbmp 1,2,9,18. β-tcp is porous and able to entrap rhbmp within its micropores, thus allowing the intrinsically diffusible rhbmp to be retained and its action prolonged 29. The porous structure of β-tcp allows cells and newly forming tissues to migrate into it, and also provides sufficient firmness against soft tissue pressure. However there are some limitations to the use of β-tcp. β-tcp is quickly resorbed, therefore making the maintenance of space difficult. In addition, manipuration is not easily achieved in β-tcp without any assistance. MBCP blocks (consisting of a 60% HA and 40% β-tcp mixture) were expected to function better than the β-tcp material because of the addition of HA. Porous by nature, MBCP blocks would entrap rhbmp-2 within its micropores and macropores. In our histomorphometric analysis, there were statistically significant differences between the results obtained at 2 weeks and those obtained at 8 weeks in all groups. These results may be explained by the fact that new bone formation in the augmented area increased from 2 weeks to 8 weeks and because much of the carrier materials were resorbed at 8 weeks. There were statistically significant differences between the rhbmp-2/ MBCP group and the MBCP group. These findings showed that rhbmp-2 induced new bone formation in the augmented area. In our study, the total augmented area had no change in all groups, meaning that the MBCP block is better than other materials as rhbmp carrier. 14
In the rhbmp-2/mbcp block group at 8 weeks, we saw new bone in the upside of the MBCP block, thus establishing that rhbmp is associated with osteoinduction. In conclusion, the bone regenerative effect of the rhbmp-2/mbcp block was superior to the MBCP block in the rat calvarial critical-sized defect model. These findings may offer additional clinical uses, including dental implants, alveolar ridge augmentation, and other conservative treatments. Nevertheless, more research is necessary on the sustenance of HA particles without resorption, and their residual influence. 15
V. Conclusion The purpose of this study was to evaluate the osteogenic effects of macroporous biphasic calcium phosphate (MBCP) as a carrier system for rhbmp-2 in the rat calvarial defect model. The bone regenerative effect of the rhbmp-2/mbcp blocks was superior to the MBCP blocks alone. Surgical implantation of rhbmp-2/mbcp blocks may be able to regenerate bone in the rat calvarial critical-sized defects without any side effects. In conclusion, MBCP blocks may be considered effective carriers of rhbmp. 16
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22. Marden LJ, Hollinger JO, Chaudhari A, Turek T, Schaub RG, Ron E. Recombinant human bone morphogenetic protein-2 is superior to demineralized bone matrix in repairing craniotomy defects in rats. J Biomed Mater Res 1994; 28: 1127-1138. 23. Ohura K, Hamanishi C, Tanaka S, Matsuda N. Healing of segmental bone defects in rats induced by a β TCP-MCPM cement combine with rhbmp-2. J Biomed Mater Res 1999; 44: 168-175. 24. Ozkaynak E, Rueger DC, Drier EA, et al. OP-1 cdna encodes an osteogenic protein in the TGF-β family. EMBOJ 1990; 9; 2085-2093. 25. Pang EK, Im SU, Kim CS, et al. Effect of recombinant human bone morphogenetic protein-4 dose on bone formation in rat calvarial defects. J Periodontol 2004; 75: 1364-1370. 26. Sigurdsson TJ, Fu E, Tatakis DN, Rohrer MD, WikesjÖ UME. Bone regeneration and osseointegration. Clin Oral lmplants Res 1997; 8;367-374. 27. Tatakis DN, Koh A, Jin L, Wozney JM, Rohrer MD, WikesJÖUME. Periimplant bone regeneration using rhbmp-2/acs in a canine model: a doseresponse study. J Periodontol Res 2001; 37: 93-100. 28. Uludag H, Gao T, Porter TJ, Friess W, Wozney JM. Delivery systems for BMPs: factors contributing to protein retention at an application site. J Bone Joint Surg 2001; 83: 128S-135S. 29. urist MR, Lietze A, Dawson E. Beta-tricalcium phosphate delivery system for bone morphogenetic protein. Clin Orthop Rel Res 1984; 187: 277-280. 20
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Legends Figure 1. Schematic drawing of calvarial osteotomy defect showing the histometric analysis. Figure 2. Representative photomicrographs of MBCP block carrier control at 2 weeks. At 2 weeks, the augmented area were covered with dense connective tissue and particles of residual MBCP. Minimal new bone formation was observed. (x20) Figure 3. Representative photomicrographs of MBCP block carrier control at 8 weeks. At 8 weeks, more bone formation was observed in the base area comparing to 2 weeks (x20) Figure 4. Representative photomicrographs of rhbmp-2/mbcp block group at 2 weeks (x20). At 2 weeks, the augmented area are filled with new bone more than control group. A lot of osteoblast, osteocyte were observed in the bottom of MBCP block. Figure 5. Representative photomicrographs of rhbmp-2/mbcp block group at 8 weeks (x20). Concentric rings of the haversian system, cement lines and fatty marrow were observed in the new bone area. At 8weeks, in the upside of augmented area, a lot of new bone was observed, and the appearance of the new bone was more lamellar at 8 weeks. 22
Figure 6. Representative photomicrographs of rhbmp-2/mbcp block group at 8 weeks (base of MBCP block, x100). Figure 7. Representative photomicrographs of rhbmp-2/mbcp block group at 8 weeks (Top of MBCP block, x100). A lot of new bone was observed in top of MBCP block. 23
FiguresⅠ Figure 2 Figure 3 24
FiguresⅡ Figure 4 Figure 5 25
FiguresⅢ Figure 6 Figure 7 26
국문요약 bone morphogenetic protein-2 macroporous biphasic calcium phosphate-block < 지도교수조규성 > 연세대학교대학원치의학과 이용준 골형성유도단백질 (bone morphogenetic protein, BMP) 은성장이나골형성과정에서중요한역할을한다고입증되었고그것의운반체에대한연구가이뤄져왔다. 하지만수직압이존재하는곳에서골증대술에적용할수있을만큼강한공간유지능력이있는운반체에대한연구는그리많지않았다. macroporous biphasic calcium phosphate Block (MBCP block) 은공간유지능력이뛰어나며강한수직압을견딜수있는골대체물질이다. 이연구의목적은 MBCP block을골형성유도단백질 (rhbmp-2) 의운반체로사용하여백서두개골결손부에적용하였을때, 골형성효과를평가하는것이다. 27
36 마리의웅성백서에서 8 mm지름을갖는임계크기의두개부결손을형성하였다. 20 마리씩 2 개의군으로나누어 MBCP block 만이식한군, MBCP block 을운반체로사용하여농도 0.025mg/ml rhbmp-2 를이식한군으로나누어술후 2 주와 8 주에치유결과를조직학적, 조직계측학적으로비교관찰하였다. 조직계측학적관찰결과, rhbmp-2/mbcp block군에서 MBCP block군에서보다 2,8주모두골밀도 (bone density) 가유의성있게증가하였다 (P<0.01). 각군에서도 8주째가 2주째보다골밀도가유의성있게증가하였다 (P<0.01). 총조직형성량 (augmented area) 에서는변화가없었다. 백서두개골결손부에서 MBCP block은 rhbmp의운반체로사용하였을때신생골형성에유의한효과가있을뿐아니라공간유지능력이우수해서수직압이존재하는골증대술 (bone augmentation) 시 rhbmp의운반체로가능성이있다. 핵심되는말 : 골형성유도단백질, macroporous biphasic calcium phosphate Block, 운반체, 골증대술, 백서두개골결손부. 28