灌注性三维动态细胞接种和培养方法构建组织工程骨的实验研究
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摘要
骨缺损的修复和重建是骨科临床面临的问题之一。自体骨是骨移植的金标准,但其来源有限且易引发供区并发症;同种异体骨和异种骨也可用于治疗骨缺损,但存在免疫排斥反应和病毒传播等潜在危险。利用组织工程技术将成骨性种子细胞与多孔支架材料复合后体外构建组织工程骨为骨缺损的临床治疗提供了新途径。但是,传统的静态细胞接种和培养方法存在细胞接种率低和分布均匀性差、支架内气体交换及营养供应不足等缺陷,降低了组织工程骨的成骨活性。本研究利用自行设计的灌注式生物反应器进行成骨细胞的灌注性三维动态接种和培养,以提高细胞的接种率和分布均匀性,促进支架内细胞的均匀增殖,增强组织工程骨的成骨活性和对节段性骨缺损的修复效果。
1.成骨细胞在四种多孔支架材料内的灌注性三维动态接种研究
目的:检验自行设计的灌注式生物反应器用于成骨细胞灌注接种的可行性,对比四种支架材料的细胞接种效果,筛选较优的支架材料。方法:选取异体松质骨粒、多孔磷酸三钙(β-TCP)、明胶海绵、胶原蛋白海绵四种支架材料作为载体,使用自行设计的生物反应器进行人胚胎成骨细胞的灌注接种,以静态接种方法为对照,通过细胞活力、活细胞接种率检测及组织学观察和计量,比较两种接种方法和四种支架材料对细胞接种效果的影响。结果:β-TCP和明胶海绵:灌注接种优于静态接种(P<0.05);松质骨粒:两接种组间无统计学差异;胶原蛋白海绵:灌注接种组的细胞活力、活细胞接种率和细胞计数均低于静态接种组(P<0.05),细胞分布均匀性则无显著差异。灌注接种时,β-TCP、明胶海绵的接种效果优于松质骨粒和胶原蛋白海绵,静态接种时,胶原蛋白海绵的细胞接种率和细胞计数最高。结论本研究自行设计的灌注式生物反应器提高了β-TCP和明胶海绵支架内成骨细胞的接种效率和分布均匀性。松质骨粒和胶原蛋白海绵不适于灌注接种方法,但胶原蛋白海绵适于静态接种方法。
2.成骨细胞在多孔β-TCP支架内灌注性接种和培养的优化研究
目的:探讨自行设计的灌注式生物反应器用于成骨细胞灌注性接种和培养的可行性,对比不同条件对细胞接种和培养效果的影响,筛选较优的细胞接种和培养条件。方法:以多孔β-TCP支架为载体,采用自行设计的灌注式生物反应器进行人胚胎成骨细胞的灌注接种和短期灌注培养,以静态接种方法为对照,比较细胞接种时间、细胞悬液密度、灌注接种及灌注培养速率的变化对活细胞接种率和细胞活力的影响。结果:灌注接种12h接种率显著高于8h和4h(P<0.05),但与24h间无统计学差异;灌注接种细胞悬液密度为2×10~5/mL时接种率显著高于1×10~5/mL(P<0.05),但与5×10~5/mL相比无统计学意义;灌注速率为1mL/min时灌注接种率最高。灌注培养24h内、灌注速率0.5mL/min～2mL/min间细胞活力无显著变化,但至4mL/min时细胞活力显著下降(P<0.05);24h至4d内灌注速率2mL/min时细胞活力最高。结论:细胞接种时间、细胞悬液密度、灌注接种和灌注培养速率等因素均影响细胞接种和培养效果;在本研究实验条件下,细胞灌注性接种和培养的最优条件为:接种时间12h～24h,细胞悬液密度2×10~5/mL～5×10~5/mL,灌注接种速率1mL/min,灌注培养速率0.5mL/min～2mL/min(24h内)和2mL/min(24h～4d)。
3.利用灌注性细胞接种/培养系统体外构建组织工程骨
目的:利用灌注性细胞接种和培养系统体外构建组织工程骨,以静态接种和静态培养(SSSC)以及静态接种和灌注培养(SSPC)为对照,对比三种方法的构建效果。方法:以多孔β-TCP支架为载体,采用自行设计的生物反应器进行人胚胎成骨细胞的灌注接种和灌注培养(PSPC),以SSSC法和SSPC法为对照,通过葡萄糖日耗量、细胞活力检测、扫描电镜(SEM)以及硬组织切片观察和组织形态学计量,比较三种方法的细胞增殖和分布情况。结果:三组的葡萄糖日耗量均随培养时间的延长而增加,SSPC组和PSPC组的葡萄糖日耗量高于SSSC组(P<0.05),培养6d内PSPC组的葡萄糖日耗量高于SSPC组(P<0.05),培养8d时两组的葡萄糖日耗量接近一致。SSPC组和PSPC组细胞活力无显著差异,但均高于SSSC组细胞活力(P<0.05)。SEM及组织学观察显示SSSC组细胞仅分布在支架周缘而SSPC组和PSPC组细胞在支架内部及周边均有分布且两组的细胞占孔率均高于SSSC组(P<0.05)。结论:PSPC法体外构建组织工程骨的效果优于SSSC法;培养8d后SSPC法和PSPC法的构建效果相似,但PSPC法可显著促进培养初期细胞增殖从而加快组织构建速度,随着支架载体体积和孔隙空间的增加这一优势可能更加明显。
4.灌注法结合可控微结构β-TCP支架体外构建大体积组织工程骨
目的:探讨采用灌注培养方法和可控微结构β-TCP支架体外构建大体积组织工程骨的构建效果以及支架微结构在其中的作用。方法:以快速成形(RP)技术制备的大体积可控微结构β-TCP支架为载体,采用自行设计的灌注式生物反应器进行人胚胎成骨细胞的灌注性三维培养,以静态培养方法为对照,通过葡萄糖日消耗量、细胞活力检测、组织学观察和计量、SEM观察及X射线能谱(EDX)分析,比较两种培养方法对支架内细胞增殖和分布的影响。结果:葡萄糖日耗量和细胞活力均随培养时间延长而升高(P<0.05),灌注培养组的葡萄糖日耗量和细胞活力显著高于静态培养组(P<0.05)。组织学及SEM观察显示静态培养条件下细胞仅在支架边缘存活而灌注培养条件下细胞在支架内部和边缘均能正常生长,细胞数量明显多于静态培养组;灌注培养支架内形成了以正交管道结构为模板的新生三维组织。EDX分析证实胞外类球体基质为磷酸钙结节。结论:灌注培养方法结合可控微结构β-TCP支架促进大体积支架内细胞均匀扩增和成骨分化;可控正交管道结构利于支架内形成均匀一致的营养供应和流体应力刺激环境;可控微结构可能影响新生组织的形态和分布。
5.灌注法构建组织工程骨修复兔桡骨节段性缺损研究
目的:探讨灌注性细胞接种和培养方法体外构建组织工程骨以及修复兔桡骨节段性缺损的可行性和效果。方法:以RP技术制备的可控微结构β-TCP支架为载体,采用自行设计的生物反应器进行兔成骨细胞的灌注接种和灌注培养(PSPC),以静态接种和静态培养(SSSC)以及静态接种和灌注培养(SSPC)方法为对照,通过葡萄糖日耗量、细胞活力检测和组织学观察,对比三种方法的细胞增殖和分布情况;将组织工程骨植入兔桡骨节段性骨缺损,以缺损旷置、自体骨和单纯β-TCP支架为对照,通过影像学和组织学检查比较骨缺损的修复效果。结果:PSPC组的葡萄糖日耗量和细胞活力高于SSPC和SSSC组(P<0.05),SSSC组细胞仅在支架边缘分布而PSPC组和SSPC组细胞在支架内部和边缘均有分布,PSPC组的细胞占孔率高于SSPC和SSSC组(P<0.05)。植入兔桡骨缺损12周后自体骨组6例全部愈合(6/6),SSPC组有2例愈合(2/6),PSPC组有4例愈合(4/6),其余各组缺损均未愈合。PSPC组的新骨生成量高于SSPC组、SSPC组和单纯β-TCP组(P<0.05)。β-TCP残余量与SSPC组相比无显著性差异但低于单纯β-TCP和SSSC组(P<0.05)。结论:PSPC法可促进支架内细胞增殖和均匀分布,构建的组织工程骨可修复兔桡骨节段性缺损,修复效果优于SSSC法和SSPC法但仍低于自体骨。RP支架的降解性有待改进。体内血管化和营养供应障碍可能导致支架内部分细胞死亡从而减弱了组织工程骨的修复效果。
综上所述,以上研究结果说明,本研究自行设计的灌注式生物反应器可用于多孔支架材料上的成骨细胞灌注性接种和培养;与传统的静态细胞接种和培养方法以及采用静态接种的灌注培养方法相比,灌注性细胞接种和培养系统显著提高了支架内接种细胞的数量和分布均匀性,增强了支架内部营养物质的传输和气体交换,从而促进细胞在整个支架内均匀扩增,增强了组织工程骨的成骨活性和对兔桡骨节段性缺损的修复效果。灌注性细胞接种和培养方法优于显著增强了组织工程骨的成骨性能,构建的组织工程骨可修复兔桡骨节段性缺损。
Reconstructing large bone defects is still a major concern in the orthopedic field. Multiple approaches have been attempted using autografts, allografts, and xenografts. Autografts such as iliac crest bone grafts still remain the gold standard in bone replacement, but the available autologous bone supplies are limited and their harvesting may induce donor site morbidity. Surgeons often use allografts and xenografts to overcome the drawbacks of autografts. However, such grafts had limited success because of slow integration, poor remodeling, immunoreactions, and disease transmission. For these reasons, tissue engineering approaches that incorporate osteoblastic cells and porous biocompatible scaffolds are currently being explored as potential alternatives. Although traditional static seeding and static culture have been the most widely used methods of cell seeding and cell/scaffold construct culture, these techniques suffer from poor cellular distribution, low seeding efficiencies and limited diffusion, confining the majority of the cells to the outer surface of the scaffold. In this work, we explored a novel method of three-dimensional cell seeding and growth in a flow perfusion bioreactor which allowed for improved efficiency and uniformity of seeding, enhanced proliferation and homogenous distribution of osteoblasts, and reinforced osteogenic potential of tissue engineered bone to heal the large segmental bone defect.
1. Three-dimensional cell seeding of four kinds of porous scaffolds in a flow perfusion bioreactor
The purpose of the present study was to develop a novel flow perfusion bioreactor for the cell seeding of three-dimensional (3D) scaffolds, designed to induce continuous fluid flow of cell suspension through the scaffold pores. Using quantitative biochemical and image analysis techniques, we have evaluated the effects of flow perfusion on seeding efficiency and spatial distribution of human fetal osteoblasts in four kinds of porous scaffolds includingβ-tricalcium phosphate (β-TCP), human allogenic cancellous bone, gelatin sponge and collagen sponge, using statically seeded scaffolds as controls. The effects of scaffold was also evaluated. Perfusion seeding ofβ-TCP and gelatin sponge yielded higher seeding efficiencies and more homogeneous distribution as compared with those of static seeding. There were no significant differences in cell load and cell distribution in cancellous bone scaffolds between the perfusion group and static group. Cell viability, cell seeding efficiency and cell counting in collagen sponge were significantly higher in static group than in perfusion group, but the uniformity of seeding was similar for both groups. It was found that perfusion seeding ofβ-TCP and gelatin sponge was effective. In addition, cancellous bone and collagen sponge did not facilitate perfusion seeding but collagen sponge did facilitate static seeding.
2. Flow perfusion bioreactor improves three-dimensional cell seeding and growth in porousβ-TCP scaffolds
The purpose of the present study was to create a new perfusion bioreactor in which the full process of tissue regeneration, from cell seeding to cell growth, can be performed in successive steps. The effect of different conditions such as seeding time, density of cell suspension and flow rate on the efficiency of cell seeding and cell growth were also investigated. Human fetal osteoblasts were dynamically seeded and cultured in this system in relevant volumes (1.5cc) of small sized porousβ-TCP scaffolds (3.5～5mm). We have evaluated the effect of different conditions (seeding time, density of cell suspension, rate of fluid flow) on cell seeding and growth by the cell viability, cell seeding efficiency and histological study, using statically seeded scaffolds as controls. It was found that seeding efficiency and cell viability could be alternated by several conditions including seeding time, density of cell suspension, flow rate of cell suspension and culture medium. It was demonstrated that the seeding time of 12～24 hours and the density of cell suspension of 2×10~5/mL～5×10~5/mL was effective, and that the optimal flow rate for cell seeding was 1mL/min. Moreover, the optimal flow rate for cell growth was 0.5mL/min～2mL/min in initial 24 hours and 2mL/min subsequently. These results demonstrated the feasibility and benefit of combing high efficiency cell seeding and cell/scaffold constructs cultivation culture in a flow perfusion bioreactor for bone tissue engineering applications, and which can be improved by optimization and control of several conditions such as seeding time, cell density and flow rate.
3. Integrated three-dimensional seeding and culture of osteoblasts in a perfusion bioreactor system for in vitro tissue engineered bone reconstruction
In the present study, a perfusion bioreactor system with integrated seeding and long-term culture functions was utilized for producing 3D engineered bone constructs. In the perfusion seeding and perfusion culture (PSPC) group, human fetal osteoblasts were dynamically seeded in porousβ-TCP scaffolds and the cell/scaffold constructs were cultured under flow perfusion conditions for 8 days. Cell proliferation and distribution were assessed by daily D-glucose consumption, cell viability (MTT assay), histological evaluation and scanning electron microcopy (SEM) observation as compared to the conventional method of static seeding and static culture (SSSC) and the method of static seeding and perfusion culture (SSPC). The daily glucose consumption was much higher in the SSPC and PSPC group than in the SSSC group (P<0.05), and the results of cell viability via MTT colorimetry after 8 days of incubation coincided with those of glucose consumption. Although the daily glucose consumption was significantly higher in PSPC group than in SSPC group after 2, 4 and 6 days of incubation, the results of the 8th day were similar for both groups. SEM and histological analysis showed that cells were distributed throughout the entire scaffold by 8 days of perfusion culture in both the SSPC and PSPC groups whereas they were located only along the scaffold perimeter in the SSSC group. The results of histomorphometry showed a significantly higher cell quantity in SSPC and PSPC group as compared to SSSC group. In summary, we demonstrated that the PSPC method was superior to the SSPC method in tissue engineered bone reconstruction. both SSPC and PSPC were effective for 8 days of construct cultivation, but the perfusion seeding resulted in a high efficiency and a uniform distribution of cells throughout the scaffold, which should accelate engineered tissue reconstruction. Moreover, this superiority would become more obvious as the size and porosity volume of the scaffold increased.
4. The development of constructing large tissue engineered bone by flow perfusion culture of osteoblasts inβ-TCP scaffolds with controlled architecture
Large-scaleβ-TCP scaffolds with tightly controlled architectures were fabricated using rapid prototyping (RP)technique and a custom designed perfusion bioreactor was developed in the present study. Human fetal osteoblasts were seeded onto the scaffolds, cultured for up to 16 days in static or flow perfusion conditions. After 4, 8, 16 days of incubation, proliferation and distribution of osteoblasts were determined by daily D-glucose consumption, cell viability (MTT assay), histological evaluation and SEM observation. Sphere like structures observed in the SEM images were assessed by energy dispersive X-ray (EDX) analysis. The daily D-glucose consumption and cell viability were significantly higher in the perfusion culture than in static culture (P<0.05). Flow perfused constructs demonstrated improved cell proliferation and a homogeneous layer composed of cells and extracellular matrix in channels throughout the whole scaffold. However, cells were biased to periphery in scaffolds culture statically. Sphere like structures present in the matrix were identified as calcium phosphate nodules via EDX analysis. It was found that flow perfusion culture combined with well-defined 3D interconnected channel environments mitigated nutrient transport limitation, enhanced the proliferation and improved the distribution of osteoblasts in large scaffolds, and provided mechanical stimulation to seeded cells in the form of fluid shear stress, resulting in improved cell osteodifferentiation. The interconnected channel geometry of the scaffold may facilitate uniform media flow and cell feeding and the internal architecture appears to induce de novo tissue modeling and affect the morphology and distribution of the newly formed tissue.
5. Repair of radius segmental defect in rabbits using tissue engineered bone graft obtained by a combined perfusion seeding/culture system
The aim of this study was to investigate if in vitro pre-culture period in a combined perfusion seeding/culture system of rabbit osteoblasts influence the tissue engineered bone ability to regenerate bone when implanted in a long segmental radius defect in rabbits.β-TCP scaffolds with controlled architectures were fabricated using RP technique. Three groups were assessed over 8 days in vitro:β-TCP scaffolds with cells that were dynamically seeded and proliferated in a perfusion seeding/culture bioreactor system (PSPC group);β-TCP scaffolds with cells that were statically seedied and then proliferated in a perfusion culture system (SSPC group); andβ-TCP scaffolds with cells that were statically seeded and proliferated under static conditions (SSSC group). Cell load and cell distribution were shown using daily D-glucose consumption, cell viability (MTT assay) and histological analysis. These constructs were implanted in 1.5 cm segmental bone defects in radius of New-Zealand white rabbits for 12 weeks, using scaffolds alone, autografts, and empty defects as controls. The daily D-glucose consumption and cell viability were significantly higher in PSPC group than in SSPC group and SSSC group (P<0.05). Histological study showed that PSPC and SSPC group produced a more uniform distribution of cells throughout the scaffold than SSSC group did, but the cell quantity analysis indicated that PSPC group yielded more cells than SSPC group did. The healing rates of autografts, PSPC group, SSPC group, SSSC group and scaffolds without cells were 6/6, 4/6, 2/6, 0/6 and 0/6 respectively at the 12th week. Significantly more new bone formation was found in PSPC group than in SSPC group. The residual material percentage in PSPC group was similar to SSPC group but significantly lower than SSSC group and simple scaffolds. In conclusion, tissue engineered bone graft obtained by the perfusion seeding/culture system is feasible in treating long segmental bone defects. However, although the rate of bone healing and new bone formation were strikingly improved when PSPC was used as compared with SSPC and SSSC group, it was still less satisfactory than that obtained with autografts.
In summary, the results of above studies indicated that the perfusion bioreactor system developed in our lab is useful for the dynamical seeding of porous biomaterials and subsequent cell/scaffold constructs cultivation. The perfusion seeding/culture system generated constructs with remarkably uniform cell distribution at high efficiencies, and permitted the persistent nutrition supply and gas exchange into the centre of 3D scaffold, resulting in cells proliferation throughout the whole scaffold. In addition, this system significantly enhanced the osteogenic ability of the engineered bone grafts which could repair the long segmental radius defect efficiently in the rabbit.
1.成骨细胞在四种多孔支架材料内的灌注性三维动态接种研究
目的:检验自行设计的灌注式生物反应器用于成骨细胞灌注接种的可行性,对比四种支架材料的细胞接种效果,筛选较优的支架材料。方法:选取异体松质骨粒、多孔磷酸三钙(β-TCP)、明胶海绵、胶原蛋白海绵四种支架材料作为载体,使用自行设计的生物反应器进行人胚胎成骨细胞的灌注接种,以静态接种方法为对照,通过细胞活力、活细胞接种率检测及组织学观察和计量,比较两种接种方法和四种支架材料对细胞接种效果的影响。结果:β-TCP和明胶海绵:灌注接种优于静态接种(P<0.05);松质骨粒:两接种组间无统计学差异;胶原蛋白海绵:灌注接种组的细胞活力、活细胞接种率和细胞计数均低于静态接种组(P<0.05),细胞分布均匀性则无显著差异。灌注接种时,β-TCP、明胶海绵的接种效果优于松质骨粒和胶原蛋白海绵,静态接种时,胶原蛋白海绵的细胞接种率和细胞计数最高。结论本研究自行设计的灌注式生物反应器提高了β-TCP和明胶海绵支架内成骨细胞的接种效率和分布均匀性。松质骨粒和胶原蛋白海绵不适于灌注接种方法,但胶原蛋白海绵适于静态接种方法。
2.成骨细胞在多孔β-TCP支架内灌注性接种和培养的优化研究
目的:探讨自行设计的灌注式生物反应器用于成骨细胞灌注性接种和培养的可行性,对比不同条件对细胞接种和培养效果的影响,筛选较优的细胞接种和培养条件。方法:以多孔β-TCP支架为载体,采用自行设计的灌注式生物反应器进行人胚胎成骨细胞的灌注接种和短期灌注培养,以静态接种方法为对照,比较细胞接种时间、细胞悬液密度、灌注接种及灌注培养速率的变化对活细胞接种率和细胞活力的影响。结果:灌注接种12h接种率显著高于8h和4h(P<0.05),但与24h间无统计学差异;灌注接种细胞悬液密度为2×10~5/mL时接种率显著高于1×10~5/mL(P<0.05),但与5×10~5/mL相比无统计学意义;灌注速率为1mL/min时灌注接种率最高。灌注培养24h内、灌注速率0.5mL/min～2mL/min间细胞活力无显著变化,但至4mL/min时细胞活力显著下降(P<0.05);24h至4d内灌注速率2mL/min时细胞活力最高。结论:细胞接种时间、细胞悬液密度、灌注接种和灌注培养速率等因素均影响细胞接种和培养效果;在本研究实验条件下,细胞灌注性接种和培养的最优条件为:接种时间12h～24h,细胞悬液密度2×10~5/mL～5×10~5/mL,灌注接种速率1mL/min,灌注培养速率0.5mL/min～2mL/min(24h内)和2mL/min(24h～4d)。
3.利用灌注性细胞接种/培养系统体外构建组织工程骨
目的:利用灌注性细胞接种和培养系统体外构建组织工程骨,以静态接种和静态培养(SSSC)以及静态接种和灌注培养(SSPC)为对照,对比三种方法的构建效果。方法:以多孔β-TCP支架为载体,采用自行设计的生物反应器进行人胚胎成骨细胞的灌注接种和灌注培养(PSPC),以SSSC法和SSPC法为对照,通过葡萄糖日耗量、细胞活力检测、扫描电镜(SEM)以及硬组织切片观察和组织形态学计量,比较三种方法的细胞增殖和分布情况。结果:三组的葡萄糖日耗量均随培养时间的延长而增加,SSPC组和PSPC组的葡萄糖日耗量高于SSSC组(P<0.05),培养6d内PSPC组的葡萄糖日耗量高于SSPC组(P<0.05),培养8d时两组的葡萄糖日耗量接近一致。SSPC组和PSPC组细胞活力无显著差异,但均高于SSSC组细胞活力(P<0.05)。SEM及组织学观察显示SSSC组细胞仅分布在支架周缘而SSPC组和PSPC组细胞在支架内部及周边均有分布且两组的细胞占孔率均高于SSSC组(P<0.05)。结论:PSPC法体外构建组织工程骨的效果优于SSSC法;培养8d后SSPC法和PSPC法的构建效果相似,但PSPC法可显著促进培养初期细胞增殖从而加快组织构建速度,随着支架载体体积和孔隙空间的增加这一优势可能更加明显。
4.灌注法结合可控微结构β-TCP支架体外构建大体积组织工程骨
目的:探讨采用灌注培养方法和可控微结构β-TCP支架体外构建大体积组织工程骨的构建效果以及支架微结构在其中的作用。方法:以快速成形(RP)技术制备的大体积可控微结构β-TCP支架为载体,采用自行设计的灌注式生物反应器进行人胚胎成骨细胞的灌注性三维培养,以静态培养方法为对照,通过葡萄糖日消耗量、细胞活力检测、组织学观察和计量、SEM观察及X射线能谱(EDX)分析,比较两种培养方法对支架内细胞增殖和分布的影响。结果:葡萄糖日耗量和细胞活力均随培养时间延长而升高(P<0.05),灌注培养组的葡萄糖日耗量和细胞活力显著高于静态培养组(P<0.05)。组织学及SEM观察显示静态培养条件下细胞仅在支架边缘存活而灌注培养条件下细胞在支架内部和边缘均能正常生长,细胞数量明显多于静态培养组;灌注培养支架内形成了以正交管道结构为模板的新生三维组织。EDX分析证实胞外类球体基质为磷酸钙结节。结论:灌注培养方法结合可控微结构β-TCP支架促进大体积支架内细胞均匀扩增和成骨分化;可控正交管道结构利于支架内形成均匀一致的营养供应和流体应力刺激环境;可控微结构可能影响新生组织的形态和分布。
5.灌注法构建组织工程骨修复兔桡骨节段性缺损研究
目的:探讨灌注性细胞接种和培养方法体外构建组织工程骨以及修复兔桡骨节段性缺损的可行性和效果。方法:以RP技术制备的可控微结构β-TCP支架为载体,采用自行设计的生物反应器进行兔成骨细胞的灌注接种和灌注培养(PSPC),以静态接种和静态培养(SSSC)以及静态接种和灌注培养(SSPC)方法为对照,通过葡萄糖日耗量、细胞活力检测和组织学观察,对比三种方法的细胞增殖和分布情况;将组织工程骨植入兔桡骨节段性骨缺损,以缺损旷置、自体骨和单纯β-TCP支架为对照,通过影像学和组织学检查比较骨缺损的修复效果。结果:PSPC组的葡萄糖日耗量和细胞活力高于SSPC和SSSC组(P<0.05),SSSC组细胞仅在支架边缘分布而PSPC组和SSPC组细胞在支架内部和边缘均有分布,PSPC组的细胞占孔率高于SSPC和SSSC组(P<0.05)。植入兔桡骨缺损12周后自体骨组6例全部愈合(6/6),SSPC组有2例愈合(2/6),PSPC组有4例愈合(4/6),其余各组缺损均未愈合。PSPC组的新骨生成量高于SSPC组、SSPC组和单纯β-TCP组(P<0.05)。β-TCP残余量与SSPC组相比无显著性差异但低于单纯β-TCP和SSSC组(P<0.05)。结论:PSPC法可促进支架内细胞增殖和均匀分布,构建的组织工程骨可修复兔桡骨节段性缺损,修复效果优于SSSC法和SSPC法但仍低于自体骨。RP支架的降解性有待改进。体内血管化和营养供应障碍可能导致支架内部分细胞死亡从而减弱了组织工程骨的修复效果。
综上所述,以上研究结果说明,本研究自行设计的灌注式生物反应器可用于多孔支架材料上的成骨细胞灌注性接种和培养;与传统的静态细胞接种和培养方法以及采用静态接种的灌注培养方法相比,灌注性细胞接种和培养系统显著提高了支架内接种细胞的数量和分布均匀性,增强了支架内部营养物质的传输和气体交换,从而促进细胞在整个支架内均匀扩增,增强了组织工程骨的成骨活性和对兔桡骨节段性缺损的修复效果。灌注性细胞接种和培养方法优于显著增强了组织工程骨的成骨性能,构建的组织工程骨可修复兔桡骨节段性缺损。
Reconstructing large bone defects is still a major concern in the orthopedic field. Multiple approaches have been attempted using autografts, allografts, and xenografts. Autografts such as iliac crest bone grafts still remain the gold standard in bone replacement, but the available autologous bone supplies are limited and their harvesting may induce donor site morbidity. Surgeons often use allografts and xenografts to overcome the drawbacks of autografts. However, such grafts had limited success because of slow integration, poor remodeling, immunoreactions, and disease transmission. For these reasons, tissue engineering approaches that incorporate osteoblastic cells and porous biocompatible scaffolds are currently being explored as potential alternatives. Although traditional static seeding and static culture have been the most widely used methods of cell seeding and cell/scaffold construct culture, these techniques suffer from poor cellular distribution, low seeding efficiencies and limited diffusion, confining the majority of the cells to the outer surface of the scaffold. In this work, we explored a novel method of three-dimensional cell seeding and growth in a flow perfusion bioreactor which allowed for improved efficiency and uniformity of seeding, enhanced proliferation and homogenous distribution of osteoblasts, and reinforced osteogenic potential of tissue engineered bone to heal the large segmental bone defect.
1. Three-dimensional cell seeding of four kinds of porous scaffolds in a flow perfusion bioreactor
The purpose of the present study was to develop a novel flow perfusion bioreactor for the cell seeding of three-dimensional (3D) scaffolds, designed to induce continuous fluid flow of cell suspension through the scaffold pores. Using quantitative biochemical and image analysis techniques, we have evaluated the effects of flow perfusion on seeding efficiency and spatial distribution of human fetal osteoblasts in four kinds of porous scaffolds includingβ-tricalcium phosphate (β-TCP), human allogenic cancellous bone, gelatin sponge and collagen sponge, using statically seeded scaffolds as controls. The effects of scaffold was also evaluated. Perfusion seeding ofβ-TCP and gelatin sponge yielded higher seeding efficiencies and more homogeneous distribution as compared with those of static seeding. There were no significant differences in cell load and cell distribution in cancellous bone scaffolds between the perfusion group and static group. Cell viability, cell seeding efficiency and cell counting in collagen sponge were significantly higher in static group than in perfusion group, but the uniformity of seeding was similar for both groups. It was found that perfusion seeding ofβ-TCP and gelatin sponge was effective. In addition, cancellous bone and collagen sponge did not facilitate perfusion seeding but collagen sponge did facilitate static seeding.
2. Flow perfusion bioreactor improves three-dimensional cell seeding and growth in porousβ-TCP scaffolds
The purpose of the present study was to create a new perfusion bioreactor in which the full process of tissue regeneration, from cell seeding to cell growth, can be performed in successive steps. The effect of different conditions such as seeding time, density of cell suspension and flow rate on the efficiency of cell seeding and cell growth were also investigated. Human fetal osteoblasts were dynamically seeded and cultured in this system in relevant volumes (1.5cc) of small sized porousβ-TCP scaffolds (3.5～5mm). We have evaluated the effect of different conditions (seeding time, density of cell suspension, rate of fluid flow) on cell seeding and growth by the cell viability, cell seeding efficiency and histological study, using statically seeded scaffolds as controls. It was found that seeding efficiency and cell viability could be alternated by several conditions including seeding time, density of cell suspension, flow rate of cell suspension and culture medium. It was demonstrated that the seeding time of 12～24 hours and the density of cell suspension of 2×10~5/mL～5×10~5/mL was effective, and that the optimal flow rate for cell seeding was 1mL/min. Moreover, the optimal flow rate for cell growth was 0.5mL/min～2mL/min in initial 24 hours and 2mL/min subsequently. These results demonstrated the feasibility and benefit of combing high efficiency cell seeding and cell/scaffold constructs cultivation culture in a flow perfusion bioreactor for bone tissue engineering applications, and which can be improved by optimization and control of several conditions such as seeding time, cell density and flow rate.
3. Integrated three-dimensional seeding and culture of osteoblasts in a perfusion bioreactor system for in vitro tissue engineered bone reconstruction
In the present study, a perfusion bioreactor system with integrated seeding and long-term culture functions was utilized for producing 3D engineered bone constructs. In the perfusion seeding and perfusion culture (PSPC) group, human fetal osteoblasts were dynamically seeded in porousβ-TCP scaffolds and the cell/scaffold constructs were cultured under flow perfusion conditions for 8 days. Cell proliferation and distribution were assessed by daily D-glucose consumption, cell viability (MTT assay), histological evaluation and scanning electron microcopy (SEM) observation as compared to the conventional method of static seeding and static culture (SSSC) and the method of static seeding and perfusion culture (SSPC). The daily glucose consumption was much higher in the SSPC and PSPC group than in the SSSC group (P<0.05), and the results of cell viability via MTT colorimetry after 8 days of incubation coincided with those of glucose consumption. Although the daily glucose consumption was significantly higher in PSPC group than in SSPC group after 2, 4 and 6 days of incubation, the results of the 8th day were similar for both groups. SEM and histological analysis showed that cells were distributed throughout the entire scaffold by 8 days of perfusion culture in both the SSPC and PSPC groups whereas they were located only along the scaffold perimeter in the SSSC group. The results of histomorphometry showed a significantly higher cell quantity in SSPC and PSPC group as compared to SSSC group. In summary, we demonstrated that the PSPC method was superior to the SSPC method in tissue engineered bone reconstruction. both SSPC and PSPC were effective for 8 days of construct cultivation, but the perfusion seeding resulted in a high efficiency and a uniform distribution of cells throughout the scaffold, which should accelate engineered tissue reconstruction. Moreover, this superiority would become more obvious as the size and porosity volume of the scaffold increased.
4. The development of constructing large tissue engineered bone by flow perfusion culture of osteoblasts inβ-TCP scaffolds with controlled architecture
Large-scaleβ-TCP scaffolds with tightly controlled architectures were fabricated using rapid prototyping (RP)technique and a custom designed perfusion bioreactor was developed in the present study. Human fetal osteoblasts were seeded onto the scaffolds, cultured for up to 16 days in static or flow perfusion conditions. After 4, 8, 16 days of incubation, proliferation and distribution of osteoblasts were determined by daily D-glucose consumption, cell viability (MTT assay), histological evaluation and SEM observation. Sphere like structures observed in the SEM images were assessed by energy dispersive X-ray (EDX) analysis. The daily D-glucose consumption and cell viability were significantly higher in the perfusion culture than in static culture (P<0.05). Flow perfused constructs demonstrated improved cell proliferation and a homogeneous layer composed of cells and extracellular matrix in channels throughout the whole scaffold. However, cells were biased to periphery in scaffolds culture statically. Sphere like structures present in the matrix were identified as calcium phosphate nodules via EDX analysis. It was found that flow perfusion culture combined with well-defined 3D interconnected channel environments mitigated nutrient transport limitation, enhanced the proliferation and improved the distribution of osteoblasts in large scaffolds, and provided mechanical stimulation to seeded cells in the form of fluid shear stress, resulting in improved cell osteodifferentiation. The interconnected channel geometry of the scaffold may facilitate uniform media flow and cell feeding and the internal architecture appears to induce de novo tissue modeling and affect the morphology and distribution of the newly formed tissue.
5. Repair of radius segmental defect in rabbits using tissue engineered bone graft obtained by a combined perfusion seeding/culture system
The aim of this study was to investigate if in vitro pre-culture period in a combined perfusion seeding/culture system of rabbit osteoblasts influence the tissue engineered bone ability to regenerate bone when implanted in a long segmental radius defect in rabbits.β-TCP scaffolds with controlled architectures were fabricated using RP technique. Three groups were assessed over 8 days in vitro:β-TCP scaffolds with cells that were dynamically seeded and proliferated in a perfusion seeding/culture bioreactor system (PSPC group);β-TCP scaffolds with cells that were statically seedied and then proliferated in a perfusion culture system (SSPC group); andβ-TCP scaffolds with cells that were statically seeded and proliferated under static conditions (SSSC group). Cell load and cell distribution were shown using daily D-glucose consumption, cell viability (MTT assay) and histological analysis. These constructs were implanted in 1.5 cm segmental bone defects in radius of New-Zealand white rabbits for 12 weeks, using scaffolds alone, autografts, and empty defects as controls. The daily D-glucose consumption and cell viability were significantly higher in PSPC group than in SSPC group and SSSC group (P<0.05). Histological study showed that PSPC and SSPC group produced a more uniform distribution of cells throughout the scaffold than SSSC group did, but the cell quantity analysis indicated that PSPC group yielded more cells than SSPC group did. The healing rates of autografts, PSPC group, SSPC group, SSSC group and scaffolds without cells were 6/6, 4/6, 2/6, 0/6 and 0/6 respectively at the 12th week. Significantly more new bone formation was found in PSPC group than in SSPC group. The residual material percentage in PSPC group was similar to SSPC group but significantly lower than SSSC group and simple scaffolds. In conclusion, tissue engineered bone graft obtained by the perfusion seeding/culture system is feasible in treating long segmental bone defects. However, although the rate of bone healing and new bone formation were strikingly improved when PSPC was used as compared with SSPC and SSSC group, it was still less satisfactory than that obtained with autografts.
In summary, the results of above studies indicated that the perfusion bioreactor system developed in our lab is useful for the dynamical seeding of porous biomaterials and subsequent cell/scaffold constructs cultivation. The perfusion seeding/culture system generated constructs with remarkably uniform cell distribution at high efficiencies, and permitted the persistent nutrition supply and gas exchange into the centre of 3D scaffold, resulting in cells proliferation throughout the whole scaffold. In addition, this system significantly enhanced the osteogenic ability of the engineered bone grafts which could repair the long segmental radius defect efficiently in the rabbit.
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