| Bone defects have been clinically treated by the implantation of autogenous and allogenous bone grafts. Although autografting is a popular procedure for reconstructive surgery, it has several disadvantages, such as the shortage of donor supply, and the risk of reoperation. On the other hand, allografting has some clinical risks including disease transmission and immunological reaction. In order to overcome the problems, bone tissue engineering has been attracted much attention as a new therapeutic technology which restore and augment osseous structures by making use of osteoinductive growth factors, osteogenic cells, and scaffolds or their combination. The design of biomaterials as scaffolds for bone tissue engineering is a foundation for bone tissue engineering. The ideal scaffolds for bone tissue engineering should meet certain criteria to serve this function, including good biocompatibility, biodegradability at a rate commensurate with remodeling, osteoconductivity, osteoinductive and mechanical properties similar to those of the bone repair site. Additionnally,scaffolds can serve as delivery vehicles for cytokines such as bone morphogenetic proteins (BMPs) that transform recruited precursor cells from the host into bone matrix producing cells, thus providing osteoinduction.Calcium phosphate cements (CPC) with the bone-like chemical composition have been extensively explored as grafts for bone regeneration and carrier for BMP. Their popularity stems from the fact that they are biodegradable, biocompatible and injectable. Furthermore, CPCs are easy to shape and can be maintained locally. However, CPCs per se have been limited to use as scaffolds in tissue engineering applications because of their lack of porous structure and very slow resorption in vivo,which is unfavourable for bone formation. In fact, porous structure has emerged as one of the key requirements for the materials design to act as substrates for tissue engineering and regenerative medicine. Previous studies have indicated that porous structure plays a critical role in the mechanical and biological properties of biomaterials. But the optimal geometry for bone substitutes has not yet been determined.The effect of pore size of scaffolds as an important structural parameter on initial cell attachment and subsequently in the guidance of bone tissue formation has been addressed , but few literatures about role of the important morphological feature in mechanical property and degradation of biomaterials can be available. Additionally, considering the combination of BMP and scaffolds, it is necessary to study the role of pore size of scaffolds in osteoinductive of BMP and synergistic effect of pore size and BMP in osteogenesis. In the present study, we fabricated porous CPC with well-interconneced pores and different pores size by using pressure shaping and sodium chloride crystal as porogen agent. Then the rhBMP-2 was soak-loaded onto the porous CPC scaffolds with different pores with. Under the successful preparation of the combination of rhBMP-2 and porous CPC, we investigated the effect of pore size on the degradation and osteogenesis of the rhBMP-2-loaded porous CPC in order to study the possibility to the degradation rate and osteoinductive of biomaterials with BMP can be modulated by varying the pore size, and determine optimal macropore size of porous CPC for application in bone tissue engineering.Four main experiments were involved in this research as follows:1. Comprative Study of Ectopic Bone Formation of Porous CPC/ rhBMP-2 and Dense CPC/ rhBMP-2 in vivoPorous calcium phosphate cements (CPC) prepared by using sodium chloride crystal as porogen agent and dense ones were implanted into muscle pouches in the right and left thigh of mice, respectively. At everytime after implantation, specimens were harvested and the ectopic bone formation and degradation of two different CPCs were analyzed by Micro-CT, histology and fluorochrome labels in order to investigate the role of porous structure in the ossification and degradation of biomaterials. The results showed that better ossification was observed in porous CPC/rhBMP-2 compared to dense CPC including more newly formed bone tissue and higher rate of bone formation. The reason may be that porous structure is more favourable for the release of rhBMP-2, which can induced more bone formed. Moreover, porous structure can promote the ingrowth of bone tissue. In addition to better ossification, faster degradation was observed in the porous CPC compare to dense one. These results indicated porous structure can regulate the effectiveness of osteoinductive growth factors loaded scaffolds and degradation of biomaterials.2.Role of Macropore Size in the Degradation of Porous Calcium Phosphate CementsThe goal of the present study was to assess the effect of macropore size on the degradation of calcium phosphate cements (CPC) and investgated the possibility to the degradation rate of biomaterials can be modulated by varying the pore size. For that purpose, we prepared CPC with three different macropore sizes (200-300,300-450 and 450-600μm) but unchangeable porosity, and the study of degradation behavior was carried out in vitro and vivo. The results showed that the increase of macropore size of CPC resulted in a decrease in the compressive strength but increase in the degradation rate and porosity of CPC significantly in vitro simulation. But the rate of degradation of all porous CPC was lower in vitro simulation than that in vivo. In contrast to degradation in vitro, the decrease of macropore size of CPC resulted in an increase in the degradation rate of CPC significantly in vivo. These results suggest the possibility to the degradation rate and compressive strength of biomaterials can be modulated by varying the macropore size while maintaining porosity unchanged. But the role of macropore size in degradation of porous CPC scaffold in vitro simulation is different compare to that in vivo.3. Bone inductive properties of rhBMP-2 loaded porous calcium phosphate cement with different macropores size implanted in muscleThe goal of the present study was to assess the effect of macropore size on the osteoinductive properties of porous CPC loaded with rhBMP-2 in vivo. For that purpose, porous CPC cylinders with three different macropore sizes (200-300,300-450 and 450-600μm) were prepared by using same technique above. The release of rhBMP-2 loaded to porous CPC with different macropores size was investigated in vitro simulation. The results showed that the rhBMP-2 loaded into the scaffold with larger macropore showed a larger initial burst release than that in scaffold with smaller macropores. By contrast, the CPC delivery system with smaller macropores releases rhBMP-2 in a bioactive form for a prolonged period, which indicating that macropore size plays an important role in the kinetics of rhBMP-2 liberation from porous CPC carries. Then the CPC/ rhBMP-2 with three different macropores size were implanted into muscle pouches created in the thigh of mouse. At everytime after implantation, specimens were harvested and the ectopic bone formation and degradation of three different CPCs were analyzed by Micro-CT, histology and histomorphometry. The results indicated the bone tissue formation in implants with different macropores size showed difference in distribution and amount of newly formed bone at different period after implantation. Bone formation outside the implant was observed frequently in the initial stage of implantation. The increase of the macropore size resulted in a statistically significant increase of the total amounts of newly formed bone mainly from outside scaffolds. In the later stage, the increase of macropore size resulted in a statistically significant decrease of the amounts of newly formed bone in pores of scaffolds. Compared to porous CPC/rhBMP-2 with larger macropores, the scaffold with smaller macropores showed higher rate of bone formation. These results suggested macropores size can regulate the release and bone inductive properties of rhBMP-2 loaded to scaffolds. Compared with porous CPC/rhBMP-2 with larger macropores, the CPC/rhBMP-2 with smaller macropores showed better bone inductive properties in vivo.4. Role of macropores size in the osteoinductive properties and biodegradation of porous CPC loaded with rhBMP-2 in femoral condyles defect model of rabbit.The goal of the present study was to assess the effect of macropore size on the osteoinductive properties and biodegradation of porous CPC loaded with rhBMP-2 in femoral condyles defect model in rabbit. For that purpose, porous CPC cylinders with three different macropore sizes (200-300,300-450 and 450-600μm) were prepared by using same technique above. The scaffolds with or without rhBMP-2 were implanted into drill hole defects in cancellous bone of rabbit. At 4, 12 and 20 weeks after implantation, specimens were harvested and the bone formation and degradation of different CPCs were analyzed by DR-X ray, Micro-CT, histology and histomorphometry . The results showed that the amount of newly formed bone tissue and materials loss in all implants increased with time. The porous CPC with different macropores size showed difference in biodegradation and amount of newly formed bone at different period after implantation. Compared to the scaffolds without rhBMP-2, the amount of newly formed bone and the rate of biodegradation was more higher in porous CPC loaded with rhBMP-2. Moreover, the decrease of the macropore size resulted in a statistically significant increase of the total amounts of newly formed bone and biodegradation. Samples with 200-300μm of macropore size were resorbed significantly faster showed more bone formation than other samples. At 20 weeks after implantation, the percentage area of materials loss in scaffolds with 200-300μm of macropore size was about 52.5%. However, the area of bone formation in porous CPC loaded with rhBMP-2 decreased after 12 weeks , which may be caused by bone resorption and reconstitution. These results suggested macropores size can regulate bone inductive properties of rhBMP-2 loaded to scaffolds and the rate of degradation of scaffolds in vivo. Moreover, the macropores size in coordination with rhBMP-2 contributes to the new bone formation and resorption of scaffolds.
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