多孔硅酸钙生物活性陶瓷的生物性能研究
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摘要
当患者有大面积骨缺损或经历多次骨重建后,自体骨往往不足或不适合。这时,应当考虑具有良好生物相容性、安全性和骨传导性的人工骨替代物。生物活性陶瓷可以与骨自主结合,已经用作骨移植替代物超过30年。现在,应当考虑使用更生物性的方法-具有生物活性和可降解性的“第三代生物医学材料”。作为第三代生物材料之一,近年来硅酸钙陶瓷开始引起研究者的重视。因此,本课题试图探讨硅酸钙陶瓷在生物学领域应用,特别是临床前期动物实验方面的表现。材料特性和制备工艺是骨组织工程用多孔支架的两个最重要的影响因素。传统制备方法的主要缺点是孔道结构的不可控性,尤其是对于微观参数,如孔道尺寸、孔道连通率等是不能控制的。据我们所知,目前为止国际上还没有关于使用快速成型技术制备可控结构多孔硅酸钙陶瓷的相关研究报道。因此,本课题还探索了利用快速成型技术制备具有可控结构多孔硅酸钙陶瓷支架的可能性,并初步探讨其体外、体内生物学表现。
本课题主要从以下四个方面进行具体研究:
1.多孔硅酸钙生物陶瓷体内筋膜下植入研究
采用泡沫浸渍法制备得到多孔硅酸钙(CS)和β-磷酸三钙(β-TCP)生物陶瓷材料,将其植入家兔皮下筋膜组织中以研究其非骨性环境下的生物学行为。分别植入1、2、4周后取材,采用SPECT、Micro-CT、V-G染色、SEM、EDX等方法进行样品的观察分析。研究表明,多孔硅酸钙生物陶瓷植入后未见明显毒性反应、且表面沉积了一层类骨羟基磷灰石层,说明材料具有良好的生物相容性和生物活性。植入4周时SPECT扫描表明,CS和β-TCP的ROI值分别为53.95±15.14和9.81±3.64(p<0.01),表明CS的血管化程度明显高于β-TCP。植入4周时Micro-CT分析表明,CS和β-TCP的残余材料占总体积百分比分别为16.41%±1.96%和30.72%±0.69%(p<0.05),组织学半定量分析也表明,CS的残余面积明显小于β-TCP(p<0.01),说明CS的降解性明显优于β-TCP。与β-磷酸三钙相比较,多孔硅酸钙陶瓷材料在早期血管化、新生组织形成、材料降解性方面均具有明显优势。研究结果显示多孔硅酸钙生物陶瓷有望用作硬组织修复和组织工程用支架材料。
2.五种多孔生物玻璃/陶瓷支架体内肌肉植入研究
利用添加造孔剂方法成功制备五种多孔支架,包括β-硅酸钙、α-硅酸钙、β-磷酸三钙、羟基磷灰石和生物玻璃。将其植入兔背部肌肉后4、8、12、16周取材分析。结果表明,所制备硅酸钙陶瓷具有良好的孔隙率和力学性能。通过动物试验,借助图像分析、组织学观察分析、Micro-CT分析、扫描电镜观察和X线能谱分析、反转录PCR检测等方法,发现硅酸钙具有良好体内生物活性,可以沉积羟基磷灰石层;具有良好降解性,明显优于其它三种生物玻璃/陶瓷;没有观察到其具有诱导成骨能力,但可以引起周围组织BMP的mRNA表达。因此,很有希望用作骨缺损修复和骨组织工程研究领域。
3.多孔硅酸钙生物活性陶瓷修复家兔颅骨缺损的实验研究
为了进一步研究多孔硅酸钙在体内骨性环境的表现,将制备的多孔β-CS和β-TCP陶瓷植入兔颅骨缺损处。术后4、8、16周取材,利用显微CT、组织学分析等方法评价其生物学表现。结果表明,与β-TCP相比,β-CS在体内具有更快的降解性和更好的骨再生能力。β-CS表面出现TRAP染色阳性的多核细胞,提示细胞介导过程参与β-CS的体内降解。骨组织可以长入β-CS陶瓷的多孔结构内部,同时表面还出现骨样磷灰石层。电镜观察和能谱分析表明,骨与β-CS通过这一磷灰石层相结合。磷灰石层的形成对于与骨的结合可能起关键作用。因此,多孔β-CS陶瓷可以作为具有生物活性和生物可降解性的材料用于硬组织修复和组织工程领域。
4.基于快速成型技术的可控结构多孔硅酸钙陶瓷支架的制备及体外体内研究
利用快速成型技术和凝胶铸模工艺,成功制备了可控结构多孔硅酸钙陶瓷。体外模拟体液浸置表明,RP-CS可以体外沉积羟基磷灰石层,具有生物活性。与兔骨髓细胞共培养发现,RP-CS具有良好细胞相容性,可以促进骨髓细胞增殖并向成骨细胞分化。将其植入兔桡骨12mm缺损最长至24周发现,与RP-TCP相比,RP-CS有较高的成骨速率和降解性。此研究为今后可控结构硅酸钙的制备、体内研究提供了可能性。
Autogenous bone is not always available or suitable for large areas of bone defect or in patients who have already had multiple procedures for bone reconstruction. In these cases, artificial bone substitutes can be chosen, with improved biocompatibility, safety, and osteoconductivity. Bioactive ceramics, which can bond with bone spontaneously, have been considered for use as bone graft substitutes for over 30 years. It is time to consider a shift towards a more biologically based method termed as“Third-Generation Biomedical Materials”designed to be both bioactive and resorbable. As one of the most important part of“Third-Generation Biomedical Materials”, calcium silicate has been in study in recent years. This research focused on the in vivo performance of calcium silicate in the biomedical application, especially in the preclinical experiment. Materials and fabrication technologies are critically important in designing temporary scaffolds for bone tissue engineering. The methods manual-based fabrication techniques produce poorly controlled architecture. Especially, the microscopic parameters, such as pore size and interconnectivity, are fully uncontrollable. To our knowledge, no reports about porous calcium silicate scaffolds fabricated by rapid prototyping have been published until now. So, much attention has been paid in this research on fabricating porous calcium silicate scaffolds with controlled architecture and on evaluating the in vitro and in vivo performance of the prepared scaffolds.
Four main experiments were involved in this research as follows:
1. In Vivo Study of Porous Calcium Silicate Bioceramic in Subfascial Sites
Porous calcium silicate (CS) andβ-tricalcium phosphate (β-TCP) bioceramics were obtained by sintering polymeric sponges infiltrated with ceramic slurry. They were implanted in rabbit subfascial sites and the biological characteristics were investigated. At 1, 2 and 4 weeks after implantation, specimens were harvested and analyzed by SPECT, Von Gieson staining, Micro-CT, SEM and EDX. There is no obvious toxic reaction in porous CS ceramics, showing the excellent biocompatibility of CS ceramics. In SPECT scanning, the ROI of CS andβ-TCP is 53.95±15.14 and 9.81±3.64 respectively (p<0.01), showing higher vascularization for CS. In Micro-CT analysis, the percentage of residual material volume fraction of CS andβ-TCP after 4 weeks is 16.41%±1.96% and 30.72%±0.69% respectively (p<0.01). In semi-quantitative analysis of histological observation, the percentage of residual material in CS is obviously lower than that inβ-TCP. These results show that the biodegradation of CS is higher than that ofβ-TCP. The deposition of the bone-like hydroxyapatite layer at 2 week after implantation show good bioactivity of CS in vivo. In conclusion, compared withβ-TCP, porous CS bioceramics have superiority in vascularization, ingrowth of new tissue and degradation in early stage. Therefore, porous CS bioceramics may be potential candidates as biocompatible, bioactive and biodegradable scaffolds for hard tissue repair and tissue engineering applications.
2. Comprative study of in vivo performance of five porous bioactive glass/ceramics after implantation in muscle
Five porous scaffolds were fabricated by the addition of porogens, including porousβ-calcium silicate,α-calcium silicate,β-tricalcium phosphate, hydroxyapatite and Bioglass. After 4, 8, 12 and 16 weeks of implantation into muscles of rabbit back, specimens were harvested and analyzed. Characterization of porous ceramic showed that the resulting porous calcium silicates had suitable porosity and mechanical properties. By Micro-CT, Histomorphometric analysis, RT-PCR, SEM and EDX, it was found that the fabricated calcium silicates were bioactive in mucle and can formed hydroxyapatite layer on the surface. Compared with other three porous scaffolds, the porous calcium silicates in the study showed higher in vivo resorption. No bone tissue formation could be observed in all five scaffols. However, the mRNA expression of BMP in calcium silicate group should be emphasized via Reverse-Transcription Polymerase Chain Reaction. Therefore, it is promising for calcium silicate for application on bone defect reconstruction and bone tissue engineering.
3. Reconstruction of calvarial defect using porous calcium silicate bioactive ceramics in Rabbits
The porousβ-CS andβ-TCP ceramics were implanted in rabbit calvarial defects and the specimens were harvested after 4, 8 and 16 weeks, and evaluated by Micro-CT and histomorphometric analysis. The Micro-CT and histomorphometric analysis showed that the resorption ofβ-CS was much higher than that ofβ-TCP. The TRAP-positive multinucleated cells were observed on the surface ofβ-CS, suggested a cell-mediated process involved in the degradation ofβ-CS in vivo. The amount of newly formed bone was also measured, and more bone formation was observed withβ-CS as compared withβ-TCP (p<0.05).
Histological observation demonstrated that newly formed bone tissue grew into the porousβ-CS, and a bone-like apatite layer was identified between the bone tissue andβ-CS materials. This study showed that the porousβ-CS ceramics could stimulate bone regeneration and may be used as bioactive and biodegradable materials for hard tissue repair and tissue engineering applications.
4. Fabrication, in vitro and in vivo evaluation of a novel calcium silicate scaffolds with controlled architecture by rapid prototyping
Porous calcium silicate scaffolds with controlled architecture (RP-CS) were fabricated by rapid prototyping and gel-casting. After immersion into SBF, a hydroxyapatite layer was precipitated on the surface of RP-CS, which showed its bioactivity. Co-cultured with rabbit bone marrow cells, RP-CS demonstrated suiiable biocompratibility. Futhermore, MTT tests and ALP activity tests showed the ability of RP-CS on bone marrow cells to osteogenic differentiation. Implanted into 12 mm bone defect of rabbit radius up to 24 weeks, RP-CS showed faster mineral apopsitional rate and higher resorption than RP-TCP. The study demonstrated the possibility of fabricating CS scaffolds with controlled architecture and further in vivo research.
本课题主要从以下四个方面进行具体研究:
1.多孔硅酸钙生物陶瓷体内筋膜下植入研究
采用泡沫浸渍法制备得到多孔硅酸钙(CS)和β-磷酸三钙(β-TCP)生物陶瓷材料,将其植入家兔皮下筋膜组织中以研究其非骨性环境下的生物学行为。分别植入1、2、4周后取材,采用SPECT、Micro-CT、V-G染色、SEM、EDX等方法进行样品的观察分析。研究表明,多孔硅酸钙生物陶瓷植入后未见明显毒性反应、且表面沉积了一层类骨羟基磷灰石层,说明材料具有良好的生物相容性和生物活性。植入4周时SPECT扫描表明,CS和β-TCP的ROI值分别为53.95±15.14和9.81±3.64(p<0.01),表明CS的血管化程度明显高于β-TCP。植入4周时Micro-CT分析表明,CS和β-TCP的残余材料占总体积百分比分别为16.41%±1.96%和30.72%±0.69%(p<0.05),组织学半定量分析也表明,CS的残余面积明显小于β-TCP(p<0.01),说明CS的降解性明显优于β-TCP。与β-磷酸三钙相比较,多孔硅酸钙陶瓷材料在早期血管化、新生组织形成、材料降解性方面均具有明显优势。研究结果显示多孔硅酸钙生物陶瓷有望用作硬组织修复和组织工程用支架材料。
2.五种多孔生物玻璃/陶瓷支架体内肌肉植入研究
利用添加造孔剂方法成功制备五种多孔支架,包括β-硅酸钙、α-硅酸钙、β-磷酸三钙、羟基磷灰石和生物玻璃。将其植入兔背部肌肉后4、8、12、16周取材分析。结果表明,所制备硅酸钙陶瓷具有良好的孔隙率和力学性能。通过动物试验,借助图像分析、组织学观察分析、Micro-CT分析、扫描电镜观察和X线能谱分析、反转录PCR检测等方法,发现硅酸钙具有良好体内生物活性,可以沉积羟基磷灰石层;具有良好降解性,明显优于其它三种生物玻璃/陶瓷;没有观察到其具有诱导成骨能力,但可以引起周围组织BMP的mRNA表达。因此,很有希望用作骨缺损修复和骨组织工程研究领域。
3.多孔硅酸钙生物活性陶瓷修复家兔颅骨缺损的实验研究
为了进一步研究多孔硅酸钙在体内骨性环境的表现,将制备的多孔β-CS和β-TCP陶瓷植入兔颅骨缺损处。术后4、8、16周取材,利用显微CT、组织学分析等方法评价其生物学表现。结果表明,与β-TCP相比,β-CS在体内具有更快的降解性和更好的骨再生能力。β-CS表面出现TRAP染色阳性的多核细胞,提示细胞介导过程参与β-CS的体内降解。骨组织可以长入β-CS陶瓷的多孔结构内部,同时表面还出现骨样磷灰石层。电镜观察和能谱分析表明,骨与β-CS通过这一磷灰石层相结合。磷灰石层的形成对于与骨的结合可能起关键作用。因此,多孔β-CS陶瓷可以作为具有生物活性和生物可降解性的材料用于硬组织修复和组织工程领域。
4.基于快速成型技术的可控结构多孔硅酸钙陶瓷支架的制备及体外体内研究
利用快速成型技术和凝胶铸模工艺,成功制备了可控结构多孔硅酸钙陶瓷。体外模拟体液浸置表明,RP-CS可以体外沉积羟基磷灰石层,具有生物活性。与兔骨髓细胞共培养发现,RP-CS具有良好细胞相容性,可以促进骨髓细胞增殖并向成骨细胞分化。将其植入兔桡骨12mm缺损最长至24周发现,与RP-TCP相比,RP-CS有较高的成骨速率和降解性。此研究为今后可控结构硅酸钙的制备、体内研究提供了可能性。
Autogenous bone is not always available or suitable for large areas of bone defect or in patients who have already had multiple procedures for bone reconstruction. In these cases, artificial bone substitutes can be chosen, with improved biocompatibility, safety, and osteoconductivity. Bioactive ceramics, which can bond with bone spontaneously, have been considered for use as bone graft substitutes for over 30 years. It is time to consider a shift towards a more biologically based method termed as“Third-Generation Biomedical Materials”designed to be both bioactive and resorbable. As one of the most important part of“Third-Generation Biomedical Materials”, calcium silicate has been in study in recent years. This research focused on the in vivo performance of calcium silicate in the biomedical application, especially in the preclinical experiment. Materials and fabrication technologies are critically important in designing temporary scaffolds for bone tissue engineering. The methods manual-based fabrication techniques produce poorly controlled architecture. Especially, the microscopic parameters, such as pore size and interconnectivity, are fully uncontrollable. To our knowledge, no reports about porous calcium silicate scaffolds fabricated by rapid prototyping have been published until now. So, much attention has been paid in this research on fabricating porous calcium silicate scaffolds with controlled architecture and on evaluating the in vitro and in vivo performance of the prepared scaffolds.
Four main experiments were involved in this research as follows:
1. In Vivo Study of Porous Calcium Silicate Bioceramic in Subfascial Sites
Porous calcium silicate (CS) andβ-tricalcium phosphate (β-TCP) bioceramics were obtained by sintering polymeric sponges infiltrated with ceramic slurry. They were implanted in rabbit subfascial sites and the biological characteristics were investigated. At 1, 2 and 4 weeks after implantation, specimens were harvested and analyzed by SPECT, Von Gieson staining, Micro-CT, SEM and EDX. There is no obvious toxic reaction in porous CS ceramics, showing the excellent biocompatibility of CS ceramics. In SPECT scanning, the ROI of CS andβ-TCP is 53.95±15.14 and 9.81±3.64 respectively (p<0.01), showing higher vascularization for CS. In Micro-CT analysis, the percentage of residual material volume fraction of CS andβ-TCP after 4 weeks is 16.41%±1.96% and 30.72%±0.69% respectively (p<0.01). In semi-quantitative analysis of histological observation, the percentage of residual material in CS is obviously lower than that inβ-TCP. These results show that the biodegradation of CS is higher than that ofβ-TCP. The deposition of the bone-like hydroxyapatite layer at 2 week after implantation show good bioactivity of CS in vivo. In conclusion, compared withβ-TCP, porous CS bioceramics have superiority in vascularization, ingrowth of new tissue and degradation in early stage. Therefore, porous CS bioceramics may be potential candidates as biocompatible, bioactive and biodegradable scaffolds for hard tissue repair and tissue engineering applications.
2. Comprative study of in vivo performance of five porous bioactive glass/ceramics after implantation in muscle
Five porous scaffolds were fabricated by the addition of porogens, including porousβ-calcium silicate,α-calcium silicate,β-tricalcium phosphate, hydroxyapatite and Bioglass. After 4, 8, 12 and 16 weeks of implantation into muscles of rabbit back, specimens were harvested and analyzed. Characterization of porous ceramic showed that the resulting porous calcium silicates had suitable porosity and mechanical properties. By Micro-CT, Histomorphometric analysis, RT-PCR, SEM and EDX, it was found that the fabricated calcium silicates were bioactive in mucle and can formed hydroxyapatite layer on the surface. Compared with other three porous scaffolds, the porous calcium silicates in the study showed higher in vivo resorption. No bone tissue formation could be observed in all five scaffols. However, the mRNA expression of BMP in calcium silicate group should be emphasized via Reverse-Transcription Polymerase Chain Reaction. Therefore, it is promising for calcium silicate for application on bone defect reconstruction and bone tissue engineering.
3. Reconstruction of calvarial defect using porous calcium silicate bioactive ceramics in Rabbits
The porousβ-CS andβ-TCP ceramics were implanted in rabbit calvarial defects and the specimens were harvested after 4, 8 and 16 weeks, and evaluated by Micro-CT and histomorphometric analysis. The Micro-CT and histomorphometric analysis showed that the resorption ofβ-CS was much higher than that ofβ-TCP. The TRAP-positive multinucleated cells were observed on the surface ofβ-CS, suggested a cell-mediated process involved in the degradation ofβ-CS in vivo. The amount of newly formed bone was also measured, and more bone formation was observed withβ-CS as compared withβ-TCP (p<0.05).
Histological observation demonstrated that newly formed bone tissue grew into the porousβ-CS, and a bone-like apatite layer was identified between the bone tissue andβ-CS materials. This study showed that the porousβ-CS ceramics could stimulate bone regeneration and may be used as bioactive and biodegradable materials for hard tissue repair and tissue engineering applications.
4. Fabrication, in vitro and in vivo evaluation of a novel calcium silicate scaffolds with controlled architecture by rapid prototyping
Porous calcium silicate scaffolds with controlled architecture (RP-CS) were fabricated by rapid prototyping and gel-casting. After immersion into SBF, a hydroxyapatite layer was precipitated on the surface of RP-CS, which showed its bioactivity. Co-cultured with rabbit bone marrow cells, RP-CS demonstrated suiiable biocompratibility. Futhermore, MTT tests and ALP activity tests showed the ability of RP-CS on bone marrow cells to osteogenic differentiation. Implanted into 12 mm bone defect of rabbit radius up to 24 weeks, RP-CS showed faster mineral apopsitional rate and higher resorption than RP-TCP. The study demonstrated the possibility of fabricating CS scaffolds with controlled architecture and further in vivo research.
引文
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