可控微结构电子束熔化成形钛合金支架作为成骨细胞载体修复兔骨缺损的研究

Objective To evaluate effects of titanium alloy scaffolds with controlled internal architecture as an osteoblast carrier on bone response in models of rabbit defects. Methods Electron beam melting process was utilized to fabricate porous titanium alloy scaffolds with fully interconnected and controlled internal pore architecture. After osteoblasts were seeded on the scaffolds and cultured for up to 7 days, the growth of rabbit osteoblasts on the scaffolds was observed by scanning electron microscopy. The experiment was conducted in 4 groups to evaluate the bone formation in vivo: group A (cell/scaffold composite), group B (scaffold only), group C (left empty) and group D (autogenous bone implant) . The scaffolds were transplanted into the rabbit defects after cultured in vitro for 7 days. The animals were sacrificed at 4, 8, and 12 weeks after implantation. Bone formation in the scaffolds was investigated by gross observation, histology and histomorphometry of non-decalcified sections and fluorochrome markers. Results Confluent cell layers could be observed on the scaffold surface and in the internal pores after 7 days of incubation in vitro. New bone growth and revascularization could be observed not only at the margins of the scaffolds, but also inside the central pores of the scaffolds after 12 weeks. New bone formed along the controlled internal channels of the scaffolds. The scaffolds were filled fully with the new bone tissue and blood vessels. More extensive new bone formation was found to originate from the host bone towards the implant in group A than in group B (P ＜0. 05) . Conclusions The controlled scaffolds are well biocompatible enough to accelerate healing of rabbit defects and new bone formation. The controlled honeycomb-like architecture may guide and promote the formation of mineralized tissue.     

增强型生物活性玻璃-胶原复合支架材料的成骨效能研究

Objective To study the in vivo and vitro biocompatibility and osteogenetic capacity of enhanced bioactive glass/collagen composite scaffold. Methods Bone marrow stromal cells(BMSCs)were collected and induced to osteoblast-like cells.The growth rate of BMSCs was detected and compared progressively through Alamar Blue.The RNAs of the cells were collected and detected for bone morphogenetic protein-2(BMP-2),alkaline phosphatase(ALP),collagen Ⅰ(Col-Ⅰ)through qRT-PCR on the fourth and seventh days.Scaffolds with induced osteoblasts were embedded into 3 nude mice subcutaneously in vivo and detected after 6 weeks.X-ray,qRT-PCR and tissue staining were used to detect the mRNA expressions of BMP-2,Col Ⅰ,osteocalcin(OCN)and ostcopontin(OPN)and bone formation. Results SEM(scanning electronic microscopy)showed BMSCs attached to the scaffold tightly and viably and proliferated actively on the scaffold.The growth rate in the experimental group was significantly higher after 7 days(P<0.05)than in the control group.qRT-PCR showed that the mRNA expressions of BMP-2,ALP and Col-Ⅰ in the experimental group were significantly higher than in the control group on the seventh day(P<0.05).X-ray showed that the dense images of embedded scaffolds were locally similar to those of normal bone after 6 weeks.qRT-PCR showed that the mRNA expressions of BMP-2,Col Ⅰ,OCN and OPN in the experimental group were significantly higher than those of normal bone(P<0.05).HE and Massort staining of the paraffin sections showed the scaffolds degraded generally and osteoblasts and chondrocytes proliferated abundantly and distributed irregularly.Bone formation could be observed obviously. Conclusion Enhanced bioactive glass/collagen composite scaffolds have good biocompatibility and osteogenetic capacity in vitro and vivo.     

Repair of the radial defect of rabbit with polyester/ tricalcium phosphate scaffolds prepared by rapid prototyping technology

Objective： To evaluate the effects of repairing rabbit radial defects with polyester/tricalcium phosphate scaffolds prepared by rapid prototyping technology loaded with bovine bone morphogenetic protein （ bBMP）, and find new carriers for growth factors. Methods： Polyester/tricalcium phosphate scaffolds prepared by rapid prototyping technology loaded with and without bovine BMP were used to repair the 15 mm radial defect in rabbit. Then the results of radiography, histology, scaffolds degrade rates and bone mineral density （BMD） were appraised to examine the effects at the 12th week. Results ： At the 12th week postoperatively, all defects treated with bBMP were radiographically repaired. No radius implanted polyester/tricalcium phosphate scaffolds without bBMP showed radiographic and histological union. At experimental groups, longitudinal alignment of lamellar structure was observed histologically at the 12th week,indicating that remodeling of regenerated bone was complete in different degree. Of the three experimental groups, the bony regeneration and remodeling of callus in poly lactide-co-glycolide/tricalcium phosphate （PLGA/ TCP） group was the best. The BMD values were beyond 70% of normal value at the 12th week while the PLGA/ TCP scaffolds group was the highest, and no abnormalities were observed in the surrounding soft tissue in all groups. Conclusions - Polyester/tricalcium phosphate scaffolds prepared by rapid prototyping technology loaded with bovine BMP can repair a 15 mm radial defect of rabbit. As for the results, the PLGA/TCP scaffold is ideal and better than poly L-lactide-co-D, L-lactide （ PDLLA/TCP ） scaffold, but the ploy L-lactic acid （PLLA/TCP） is not so good for its low degradation rates.     

Reconstruction of caprine mandibular segmental defect by tissue engineered bone reinforced by titanium reticulum

Objective: To investigate the feasibility of using natural poritos as scaffolds in bone tissue engineering (TE) and repair of caprine mandibular segmental defect with titanium reticulum reinforced. Methods: Natural poritos with a pore of 190-230 μn in size and porosity of about 50% -65% was molded into the shape of granules 5 mm×5 mm×5 mm in size. Expanded autologous caprine marrow mesenchymal stem cells were induced by recombinant human morphogenetic protein-2 ( rhBMP2 ) to improve osteoblastic phenotype. Then marrow derived osteoblasts were seeded into poritos in density of 4×107/ml and incubated in vitro for 48 hours prior to implantation. Then osteoblastic cells/poritos complexes were implanted into mandibular defect and the defect was reinforced by titanium reticulum. Implantation of poritos alone acted as the control. Bone regeneration was assessed 4, 8, 16 weeks after implantation using roentgenographic analysis and histological observation was done after 16 weeks. Results: New bone could be observed histologically on the surface and in the pores of natural coral in all specimens in the cell-seeding group, whereas in the control group there was no evidence of osteogenesis process in the center of the construction. The results showed that new bone grafts were successfully restored 16 weeks after implantation. Conclusions: This study suggests the feasibility of using porous coral as scaffold material transplanted with marrow derived osteoblasts by TE method. By means of titanium reticulum reinforcement, mandibular defect could be successfully restored. It shows the potentiality of using this method for the reconstruction of bone defect in clinic.     

灌注培养对骨髓基质干细胞在大段材料上增殖与分布的影响

Objective To study the role of perfusion bioreactor in proliferation and distribution of rat bone marrow stromal cells (BMSCs) in a large-scale scaffold. Methods SD rat BMSCs transfected with enhanced green fluorescent protein (eGFP) (eGFP-BMSCs) were planted in large-scale porous β-tricalcium phosphate (β-TCP) scaffolds. In the dynamic perfusion culture group, the scaffold with eGFP-BMSCs was continuously cultured in our self-designed three-dimensional perfusion bioreactor for 7, 14 and 28 days. In the static culture group, the scaffold was put into a medium reservoir without perfusion for 7, 14 and 28 days.Proliferation and distribution of the cells in the scaffold were examined by scanning electronic microscopy (SEM), fluorescence microscopy (FM), measuring daily glucose consumption, and counting eGFP-BMSCs in each layer. Results SEM and FM showed that eGFP-BMSCs distributed and proliferated throughout the scaffold in dynamic perfusion culture, but distributed and proliferated only in the peripheral pores of the scaffold in static culture. The daily glucose consumption in both groups increased with time. Cell proliferation reached the plateau phase after culture for 14 days in the static culture group, but after culture for 21 days in the perfusion culture group. The rate and margin of increase were much more evident in the perfusion culture group. On day 28, glucose consumption in the perfusion culture group was 36. 33 ± 3. 14 mg/d, 3. 7 times as large as that in the static culture group (9. 82 ± 1. 33 mg/d). The eGFP-BMSCs counting revealed there were no significant differences in cell number between layers of the scaffold on days 7 and 28 d ( P > 0. 05), but there were significant differences on day 14 in the perfusion culture group ( P < 0. 05); while in the static culture group, there were significant differences in cell number between layers of the scaffold ( P < 0. 05),with most of the cells assembled at the bottom of the scaffold.Conclusion Our self-designed three-dimensional perfusion bioreactor may help eGFP-BMSCs proliferate and distribute uniformly in a large-scale porous scaffold.     

Bone regeneration with osteogenic matrix cell sheet and tricalcium phosphate:An experimental study in sheep

AIM To determine the effects of a cell sheet created from sheep bone marrow and tricalcium phosphate(TCP) on osteogenesis.METHODS Bone marrow cells were harvested from a sheep and cultured in a minimal essential medium(MEM) containing ascorbic acid phosphate(AscP) and dexamethasone(Dex). After 2 wk, the formed osteogenic matrix cell sheet was lifted from the culture dish using a scraper. Additionally, harvested bone marrow cells were cultured in MEM only as a negative control group, and in MEM with AscP, Dex, and β-glycerophosphate as a positive control group. For in vitro evaluation, we measured the alkaline phosphatase(ALP) activity and osteocalcin(OC) content in the media of the cultured cells from each group. For in vivo analysis, a porous TCP ceramic was used as a scaffold. We prepared an experimental group comprising TCP scaffolds wrapped with the osteogenic matrix cell sheets and a control group consisting of the TCP scaffold only. The constructs wereimplanted subcutaneously into athymic rats and the cell donor sheep, and bone formation was confirmed by histology after 4 wk.RESULTS In the in vitro part, the mean ALP activity was 0.39 ± 0.03 mg/well in the negative control group, 0.67 ± 0.04 mg/well in the sheet group, and 0.65 ± 0.07 mg/well in the positive control group. The mean OC levels were 1.46 ± 0.33 ng/well in the negative control group, 3.92 ± 0.16 ng/well in the sheet group, and 4.4 ± 0.47 ng/well in the positive control group, respectively. The ALP activity and OC levels were significantly higher in the cell sheet and positive control groups than in the negative control group(P 0.05). There was no significant difference in ALP activity or OC levels between the cell sheet group and the positive control group(P 0.05). TCP constructs wrapped with cell sheets prior to implantation showed bone formation, in contrast to TCP scaffolds alone, which exhibited poor bone formation when implanted, in the subcutaneous layer both in athymic rats and in the sheep. CONCLUSION This technique for preparing highly osteoinductive TCP may promote regeneration in large bone defects.     

新型多孔可吸收骨替代材料修复兔股骨缺损的实验研究

Objective To test the osteogenetic ability and biocompatibility of a new type of porous morsel blocks of resorbable bioglass bone graft substitute. Methods Thirty healthy New Zealand adult white rabbits were selected to establish bone defect models and randomly assigned into 3 even groups(with 20hind legs in each group)for 3 methods of repairing the defects.The 6 mm×10 to 12 mm defeets were made at the bilateral fenloral condyles of each rabbit.In group A,a porous morsel block of resorbable bioactive glass bone substitute was implanted;in group B,commonly used bioactive glass was implanted;in group C,nothing was implanted.Specimens were collected at 6 and 12 weeks after operation to evaluate the biodegradation and osteogenic properties by gross observation, radiography, Micro-CT and histopathological examinations.ResulIs No local adverse reaction was observed in each group.At 6 weeks.gradual growth of the new bone was observed inside the implants in groups A and B.The amount of new bone in group A was significantly higher than in group B.In group A,the porous morsel material was almost completely degraded,bone defects were completely repaired,and bone trabecula moulding reconstruction was completed.In group B,a great amount of material was not completely degraded in the central area of the graft,while bone defects were not healed in the group C.The percentages of new bone area were 0.30±0.02 in group A,0.17±0.03 in group B and 0.06±0.01 in group C,with significant differences(F=374.202,P<0.001).At 12weeks.the percentages of new bone area were 0.53 ±0.05 in group A,0.39±0.05 in group B and 0.07±0.01 in group C,with significant differences(F=317.243,P<0.001). Conclusion New porous morsel blocks of resorbable bioglass bone graft substitute show superior osteogenetic properties and biodegradability because of their good three dimensional structure.     

增强型生物活性玻璃-胶原复合支架材料的成骨效能研究

目的 探讨增强型生物活性玻璃-胶原复合支架材料的体内外成骨效能.方法取第3代BMSCs种植于支架材料(支架组),以等量细胞常规培养作为非支架组,培养1、3、5、7、9、11 d采用阿尔玛蓝法动态检测细胞的增殖率.取第3代BMSCs种植于支架材料(体外实验组),以等量细胞常规培养作为体外对照组,培养4、7 d采用实时定量逆转录聚合酶链式反应(qRT-PCR)检测细胞骨形态发生蛋白-2(BMP-2)、碱性磷酸酶(ALP)、Ⅰ型胶原的mRNA表达.裸鼠皮下植入复合成骨样细胞的支架材料(体内实验组),取正常骨组织作为体内对照组,6周后以X线片、qRT-PCR、绀织学染色评估成骨情况.结果培养7～11 d支架组细胞增殖率显著高于非支架组,差异均有统计学意义(P<0.05).体外实验组培养7 d BMP-2、ALP、Ⅰ型胶原的mRNA表达显著高于体外对照组,差异均有统计学意义(P<0.05).体内实验组X线片示植入区域有密度增高影,支架材料形成白色硬性组织;BMP-2、Ⅰ型胶原、骨钙素、骨桥蛋白的mRNA表达较体内埘照组均增高,差异有统计学意义(P<0.05);组织学染色示支架材料大部分降解,新生骨形成明显.结论增强型生物活性玻璃-胶原复合支架材料具有良好的生物相容性,体内外均具有成骨效应.

Abstract：

Objective To study the in vivo and vitro biocompatibility and osteogenetic capacity of enhanced bioactive glass/collagen composite scaffold. Methods Bone marrow stromal cells(BMSCs)were collected and induced to osteoblast-like cells.The growth rate of BMSCs was detected and compared progressively through Alamar Blue.The RNAs of the cells were collected and detected for bone morphogenetic protein-2(BMP-2),alkaline phosphatase(ALP),collagen Ⅰ(Col-Ⅰ)through qRT-PCR on the fourth and seventh days.Scaffolds with induced osteoblasts were embedded into 3 nude mice subcutaneously in vivo and detected after 6 weeks.X-ray,qRT-PCR and tissue staining were used to detect the mRNA expressions of BMP-2,Col Ⅰ,osteocalcin(OCN)and ostcopontin(OPN)and bone formation. Results SEM(scanning electronic microscopy)showed BMSCs attached to the scaffold tightly and viably and proliferated actively on the scaffold.The growth rate in the experimental group was significantly higher after 7 days(P<0.05)than in the control group.qRT-PCR showed that the mRNA expressions of BMP-2,ALP and Col-Ⅰ in the experimental group were significantly higher than in the control group on the seventh day(P<0.05).X-ray showed that the dense images of embedded scaffolds were locally similar to those of normal bone after 6 weeks.qRT-PCR showed that the mRNA expressions of BMP-2,Col Ⅰ,OCN and OPN in the experimental group were significantly higher than those of normal bone(P<0.05).HE and Massort staining of the paraffin sections showed the scaffolds degraded generally and osteoblasts and chondrocytes proliferated abundantly and distributed irregularly.Bone formation could be observed obviously. Conclusion Enhanced bioactive glass/collagen composite scaffolds have good biocompatibility and osteogenetic capacity in vitro and vivo.     

软骨细胞外基质源性取向支架与骨髓基质干细胞体外软骨组织工程的初步研究

Objective To fabricate cartilage extracellular matrix (ECM) oriented scaffolds and investigate the attachment, proliferation, distribution and orientation of bone marrow mesenchymal stem cells (BMSCs) cultured within the scaffolds in vitro. Methods Cartilage slices were shattered in sterile phosphate-buffered saline (PBS) and the suspension were differentially centrifugated untill the micro- fiber of the cartilage extracellular matrix was disassociated from the residue cartilage fragments. At last the supernatant were centrifugated, the precipitation were collected and were made into 2%-3% suspension. Using unidirectional solidification as a freezing process and freeze-dried method, the cartilage extracellular matrix derived oriented scaffolds was fabricated. The scaffolds were then cross-linked by exposure to ultraviolet radiation and immersion in a carbodiimide solution. By light microscope and scan electron microscope (SEM) observation, histological staining, and biomechanical test, the traits of scaffolds were studied. After being labelled with PKH26 fluorescent dye, rabbit BMSCs were seeded onto the scaffolds. The attachment, proliferation and differentiation of the cells were analyzed using inverted fluorescent microscope. Results The histological staining showed that toluidine blue, safranin O, alcian blue and anti-collagen Ⅱ immunohistochemistry staining of the scaffolds were positive. A perpendicular pore-channel structures which has a diameter of 100 μm were verified by light microscope and SEM analysis. The cell-free scaffolds showed the compression moduli were (2.02±0.02) MPa in the mechanical testing. Inverted fluorescent microscope showed that most of the cells attached to the scaffold. Cells were found to be widely distributed within the scaffold, which acted as a columnar arrangement. The formation of a surface cells layer was found on the surface of the scaffolds which resembled natural cartilage. Coclusion The cartilage extracellular matrix derived oriented scaffolds have promising biological, structural, and mechanical properties.     

Effect of recombinant human basic fibroblast growth factor on angiogenesis during mandible fracture healing in rabbits

Objective: To investigate the effect of recombinant human basic fibroblast growth factor ( rhbFGF) on angiogenesis during mandible fracture healing in rabbit.Methods: Fifty adult white rabbits were used for animal model and randomly divided into a control group (25 rabbits) and an experimental group (25 rabbits). The membranous complex of rhbFGF and bovine type I collagen was prepared and implanted into the rabbit mandible fracture site under periosteum. The animals were sacrificed on 7, 14, 28, 56 and 84 days respectively after operation and the whole mandibles were harvested. The expression of factor Vm related antigen (F8-RA) in callus was examined with immimohistochemical staining.Results: The amounts of microvascular formation in calluses in the rhbFGF-treating group on days 7, 14, 28 and 56 were more than those of the control group (P < 0.01).Conclusions: The results indicated that rhbFGF could stimulate microvascular formation during mandible fracture healing in rabbits.