组织工程骨—软骨复合组织的构建及体内外形态学观察的实验研究
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摘要
骨-软骨复合组织多数存在于运动关节处,其发生、发育是在非常复杂的生物信号调控下完成的。一旦发育完成其将会保持相对恒定的解剖形态并发挥稳定的功能,同时也就失去了再生功能,但会发生微小的生理性组织改建以满足功能需要。此外,如果在发育过程中受到外来因素的影响,这一复杂组织的形成就会受到阻碍,导致发育不全甚至发育停止。这就提示我们,如果关节组织发生了损伤尤其是大面积缺损,由于其缺乏再生能力,仅依靠机体自身难以恢复。目前,现有的医疗技术还无法对这类疾病进行非常有效的治疗。
本研究的目的就是利用组织工程技术寻找一种科学的方法,在体外模拟骨-软骨形成的条件,构建出具有生物活性的组织工程骨-软骨复合组织,用于修复关节缺损从而恢复其外形与功能。本研究通过对种子细胞、支架的筛选,选择出适宜的种子细胞与支架,然后构建出具有解剖形态的复合组织,并进行体内移植的初步探索。基于这一目的我们主要进行了以下几方面的研究。
一、组织工程骨-软骨复合组织种子细胞的筛选
1.软骨细胞、骨髓间充质干细胞的获取及分离培养以2W龄新西兰大白兔的耳软骨、关节软骨和骨髓为组织来源,采用组织块酶消化法和全骨髓贴壁法进行不同细胞的分离培养、增殖与纯化。同时采用高密度和低密度细胞培养方式,对原代及第三代细胞的增殖能力、细胞表型、细胞分泌功能等生物学特性进行分析。结果表明高密度培养的软骨细胞尤其是纯化的原代细胞具有良好的生物学性能,具有较强的增殖及分泌细胞外基质的能力。而且耳软骨及关节软骨细胞在实验中没有表现出明显的生物学差别,为利用不同部位软骨细胞并获得大量原代细胞提供了依据。实验结果同时提示,采用贴壁筛选法进行骨髓间充质干细胞的扩增纯化可提供大量纯化的骨髓间充质干细胞
2.骨髓间充质干细胞的诱导分化为了进一步筛选适宜的种子细胞,分别采用细胞非接触式共培养诱导方式、条件培养液诱导方式对骨髓间充质干细胞进行体外诱导。结果显示不论采用何种诱导方式均可以将骨髓间充质干细胞诱导分化为所希望的细胞,通过MTT、细胞周期、糖胺多糖分泌能力、碱性磷酸酶活性以及细胞染色等检测手段都证实诱导后的细胞具有良好的增殖分化能力,证实所培养的骨髓间充质干细胞具有干细胞的特性。
通过对结果的进一步分析可以看出,骨髓间充质干细胞向软骨细胞分化后,共培养诱导的细胞其各项生物学特性似乎要优于条件培养液诱导的细胞,这可能与软骨细胞分泌更多的细胞因子和基质有关,但与原代软骨细胞相比其细胞状态及分泌功能等还有一定差距。而向成骨方向诱导则取得了明确的结果,其成骨能力是肯定的,同时在对照组可以观察到骨髓间充质干细胞本身就具有成骨趋势,这可能与其来源于骨髓有关。因此,为了对后续实验产生尽量少的干扰因素,最终确定将原代软骨细胞和成骨诱导的第三代骨髓间充质干细胞作为种子细胞。
二、组织工程骨-软骨复合组织支架的筛选
1.构建骨-软骨复合组织支架的形态学和物理性能检测选用4组多孔支架,分别进行表面形态学、力学性能、体外降解等相关检测,结果表明四组支架中明胶-硫酸软骨素-透明质酸钠与明胶-陶瓷化骨支架基本符合实验要求,并最终选定为复合组织的支架。
2.组织工程骨-软骨复合组织的体外构建及生物学性能为了观察支架与种子细胞间生物相容性以及能否促进细胞增殖分化等性能,本实验进行了支架复合细胞的研究。此外为了增强细胞在三维培养条件下增殖分化能力,还在支架内复合了生长因子控释系统,同时将支架构建为含有双层结构的整体支架,分别在成骨与非成骨诱导条件下观察体外组织工程骨-软骨复合组织的形成情况。结果表明采用含有双层结构的整体支架分层复合两种细胞能够在成骨诱导条件下形成较为理想的组织工程骨-软骨复合组织。
三、组织工程骨-软骨复合组织的构建及体内移植实验
1.组织工程骨-软骨复合组织体内异位移植采用荧光小鼠肋软骨细胞和骨髓间充质干细胞为种子细胞,参照前期实验方法与整体双层支架复合,并在复合后第1、7、14d植入裸鼠皮下。观察其体内异位形成组织情况。经过8W的连续观察可以发现移植组织逐渐形成与天然组织类似的骨-软骨结构,而且通过荧光观察新形成的组织系来自植入的复合组织。通过对实验各组的比较观察发现细胞与支架复合后早期植入体内更有助于组织的成熟,这可能与体内环境含有更多的营养成分以及复合组织允许血管结构的早期长入有关。
2.组织工程骨-软骨复合组织修复兔膝关节大部分缺损的实验研究为了探讨具有解剖外形的骨-软骨复合组织修复关节大部分缺损的能力。实验利用灌注快速成形技术并复合生长因子控释系统构建出具有三维解剖形态的骨-软骨复合组织,然后进行自体原位移植。通过术后24W连续观察及相关检测,观察到在体内关节微环境内,植入的组织生长良好并与周围组织融合,而且通过功能改建不但在形态上恢复了缺损区外形而且具有良好的运动功能。可能与局部存在着多种生理刺激,包括生长因子、生物力学刺激等有关。
综上所述,本研究在种子细胞及支架进行多次筛选的基础上,成功构建了具有解剖形态的组织工程骨-软骨复合组织,并用于修复大面积的关节组织缺损,取得了较为理想的修复效果。为治疗临床相关疾病奠定了良好的实验基础,同时也为构建其他复杂组织和器官提供了新思路。
In the natural joint, articular cartilage and subchondral bone form theload-bearing system, which was regulated by the biological signals. After theaccomplishment of development, relative invariable anatomical configuration andstable function was maintained, while regenerative ability was lost even thoughexiguity physiological tissue rebuilding. Furthermore, articular cartilage andsubchondral bone would undergo agenesis and undevelopment as the result ofexotic obstacle. It suggested that joint deformity and loss of joint could notrecover by itself, thus requiring surgical procedures for treatment ofosteochondral defects. However, present medical treatments have not yetsucceeded in this field.
The aim of this study is to show potential of using a tissue engineeringapproach for regeneration of osteochondral defects. In vitro bone and cartilageengineering via combining a novel composite, biphasic scaffold technology withcells has been shown a high potential in the defect regeneration in the animal models. The fitable seeding cells and scaffolds were chosen to construct theanatomical osteochondral composites and relative studies of scaffolds systemloaded with seeding cells in the repair of large osteochondral defects was alsoprovided as follows:
I Selection of seeding cells of osteochondral composites
1. Obtainment and culture of bone mesenchymal stem cells (BMSCs) andchondrocytes in vitro
Two-week-old New Zealand White rabbits were selected for BMSCs andchondrocytes isolation and culture. Trypsin-collagenase digested method,adhesive culture method, high or low-density culture method were adopted andbiological characters such as proliferative capability, cell phenotype and cellexcretion of primitive cells and third-generation cells were inveatigated. Theresults showed that high-density culture chondrocytes, especially purifiedprimitive cells, have well proliferative capability and cell excretion. Moreover, alarge quantity of primitive cells obtained from different parts could be guaranteed,because the biological characters of BMSCs from ears or joints were almost same.Adhesive culture method was proved to be an effective method to provide a largenumber of cells. All above was the favorable base of future study.
2. Inducement and differentiation of BMSCsCelluar non-contact co-culture induced method and conditioned mediuminduced method were taken to futher choose the seeding cells in this study. Theresults suggested that BMSCs induced by different methods could bedifferentiated into desired cells and the induced cells had the well proliferativecapability evaluated by MTT, mitotic cycle, GAG secretion, ALP, cell-stainingmethods.
The results also displayed that the biological characters of co-cultureinduced cells was seemed to be better than conditioned medium induced cells,which was related with the growth factors and matrices from chondrocytes, eventhough poorer than primitive cells. Osteoinductive ability and osteogenetictendency of BMSC was definite. Therefore, primitive chondrocytes andthird-generation osteoinductive BMSC were comfirmed as the ultimate seedingcells in this study.
II Selection of scaffolds of osteochondral composites
1. Morphology and physical characters of scaffolds of osteochondral compositesFour kinds of scaffolds were made by different biomaterials, and the surfacemorphology, mechanical propertyand in vitro degradation of them wasinvestigated. Gelatin–chondortin–hyaluronan hybrid scaffold and Gelatin–CBBscaffold were the ultimate scaffolds.
2. Construction and biocompatibility of osteochondral compositesTo investigate the cytotoxicity and the effect on cell proliferation of thescaffolds, the cells were seeded on them. And the novel three-dimensional specialscaffold was developed into a biphasic scaffolds with a controlled release ofbFGF, which provided structural support and stimulated repair. The in vitroformation of osteochondral composites was observed under the condition ofosteoinduction or un-osteoinduction. The results testified the formation ofosteochondral composite tissue on the biphasic scaffolds by morphological andmatrix secretion assay.
Construction of osteochondral composites and in vivo study
1. Heterotopic transplantation of osteochondral composites in vivoThree-dimensional biphasic scaffolds seeded with cultured fluorescence mouse costal chondrocytes and BMSCs were transplanted subcutaneously intonude mice after 1day, 7day and 14day, and analyzed histologically during 8weeks after the operation. Our findings showed that chondral and boneregenerations of the subchondral phases were observed via fluorescent analysisand the results demonstrated the newel tissue was derived from transplantedosteochondral composites. On the other hand, the earlier transplantedosteochondral composites were contributed to tissue maturation, which impliedthe nutrient content and blood vessel ingrowth in the early stage.
2. Repair and regeneration of osteochondral defects in rabbit articular jointsThe aim of this study was to discuss the regenerative capability ofanatomical osteochondral composites in in repairing large articular joint defects.The scaffolds were fabricated via Perfusion Rapid Prototyping technology andcontrolled release system of growth factors ,and the resulted anatomicalosteochondral composites were orthotopic transplantated. In the study, hyalinecartilage-like regeneration tissue was observed at the border of the normalcartilage with histologically 24 weeks postoperatively, for physiologicalstimulation,such as growth factors and biomechanics, were existed in jointmicroenvironment. Both morphology and motor function were recovered byfunctional rebuilding.
In conclusion, osteochondral composites were constructed successfully invitro after selection of seeding cells and scaffolds. The in vivo bone and cartilageengineering via combining a novel composite, biphasic scaffold technology withtwo kinds of seed cells has been shown a high potential in the large defectregeneration in the animal models. The present study implied the great potentialof the novel strategy as a new way to promote clinical relative diseases and it might serve as a desirable approach to contruct other tissue engineering organs.
本研究的目的就是利用组织工程技术寻找一种科学的方法,在体外模拟骨-软骨形成的条件,构建出具有生物活性的组织工程骨-软骨复合组织,用于修复关节缺损从而恢复其外形与功能。本研究通过对种子细胞、支架的筛选,选择出适宜的种子细胞与支架,然后构建出具有解剖形态的复合组织,并进行体内移植的初步探索。基于这一目的我们主要进行了以下几方面的研究。
一、组织工程骨-软骨复合组织种子细胞的筛选
1.软骨细胞、骨髓间充质干细胞的获取及分离培养以2W龄新西兰大白兔的耳软骨、关节软骨和骨髓为组织来源,采用组织块酶消化法和全骨髓贴壁法进行不同细胞的分离培养、增殖与纯化。同时采用高密度和低密度细胞培养方式,对原代及第三代细胞的增殖能力、细胞表型、细胞分泌功能等生物学特性进行分析。结果表明高密度培养的软骨细胞尤其是纯化的原代细胞具有良好的生物学性能,具有较强的增殖及分泌细胞外基质的能力。而且耳软骨及关节软骨细胞在实验中没有表现出明显的生物学差别,为利用不同部位软骨细胞并获得大量原代细胞提供了依据。实验结果同时提示,采用贴壁筛选法进行骨髓间充质干细胞的扩增纯化可提供大量纯化的骨髓间充质干细胞
2.骨髓间充质干细胞的诱导分化为了进一步筛选适宜的种子细胞,分别采用细胞非接触式共培养诱导方式、条件培养液诱导方式对骨髓间充质干细胞进行体外诱导。结果显示不论采用何种诱导方式均可以将骨髓间充质干细胞诱导分化为所希望的细胞,通过MTT、细胞周期、糖胺多糖分泌能力、碱性磷酸酶活性以及细胞染色等检测手段都证实诱导后的细胞具有良好的增殖分化能力,证实所培养的骨髓间充质干细胞具有干细胞的特性。
通过对结果的进一步分析可以看出,骨髓间充质干细胞向软骨细胞分化后,共培养诱导的细胞其各项生物学特性似乎要优于条件培养液诱导的细胞,这可能与软骨细胞分泌更多的细胞因子和基质有关,但与原代软骨细胞相比其细胞状态及分泌功能等还有一定差距。而向成骨方向诱导则取得了明确的结果,其成骨能力是肯定的,同时在对照组可以观察到骨髓间充质干细胞本身就具有成骨趋势,这可能与其来源于骨髓有关。因此,为了对后续实验产生尽量少的干扰因素,最终确定将原代软骨细胞和成骨诱导的第三代骨髓间充质干细胞作为种子细胞。
二、组织工程骨-软骨复合组织支架的筛选
1.构建骨-软骨复合组织支架的形态学和物理性能检测选用4组多孔支架,分别进行表面形态学、力学性能、体外降解等相关检测,结果表明四组支架中明胶-硫酸软骨素-透明质酸钠与明胶-陶瓷化骨支架基本符合实验要求,并最终选定为复合组织的支架。
2.组织工程骨-软骨复合组织的体外构建及生物学性能为了观察支架与种子细胞间生物相容性以及能否促进细胞增殖分化等性能,本实验进行了支架复合细胞的研究。此外为了增强细胞在三维培养条件下增殖分化能力,还在支架内复合了生长因子控释系统,同时将支架构建为含有双层结构的整体支架,分别在成骨与非成骨诱导条件下观察体外组织工程骨-软骨复合组织的形成情况。结果表明采用含有双层结构的整体支架分层复合两种细胞能够在成骨诱导条件下形成较为理想的组织工程骨-软骨复合组织。
三、组织工程骨-软骨复合组织的构建及体内移植实验
1.组织工程骨-软骨复合组织体内异位移植采用荧光小鼠肋软骨细胞和骨髓间充质干细胞为种子细胞,参照前期实验方法与整体双层支架复合,并在复合后第1、7、14d植入裸鼠皮下。观察其体内异位形成组织情况。经过8W的连续观察可以发现移植组织逐渐形成与天然组织类似的骨-软骨结构,而且通过荧光观察新形成的组织系来自植入的复合组织。通过对实验各组的比较观察发现细胞与支架复合后早期植入体内更有助于组织的成熟,这可能与体内环境含有更多的营养成分以及复合组织允许血管结构的早期长入有关。
2.组织工程骨-软骨复合组织修复兔膝关节大部分缺损的实验研究为了探讨具有解剖外形的骨-软骨复合组织修复关节大部分缺损的能力。实验利用灌注快速成形技术并复合生长因子控释系统构建出具有三维解剖形态的骨-软骨复合组织,然后进行自体原位移植。通过术后24W连续观察及相关检测,观察到在体内关节微环境内,植入的组织生长良好并与周围组织融合,而且通过功能改建不但在形态上恢复了缺损区外形而且具有良好的运动功能。可能与局部存在着多种生理刺激,包括生长因子、生物力学刺激等有关。
综上所述,本研究在种子细胞及支架进行多次筛选的基础上,成功构建了具有解剖形态的组织工程骨-软骨复合组织,并用于修复大面积的关节组织缺损,取得了较为理想的修复效果。为治疗临床相关疾病奠定了良好的实验基础,同时也为构建其他复杂组织和器官提供了新思路。
In the natural joint, articular cartilage and subchondral bone form theload-bearing system, which was regulated by the biological signals. After theaccomplishment of development, relative invariable anatomical configuration andstable function was maintained, while regenerative ability was lost even thoughexiguity physiological tissue rebuilding. Furthermore, articular cartilage andsubchondral bone would undergo agenesis and undevelopment as the result ofexotic obstacle. It suggested that joint deformity and loss of joint could notrecover by itself, thus requiring surgical procedures for treatment ofosteochondral defects. However, present medical treatments have not yetsucceeded in this field.
The aim of this study is to show potential of using a tissue engineeringapproach for regeneration of osteochondral defects. In vitro bone and cartilageengineering via combining a novel composite, biphasic scaffold technology withcells has been shown a high potential in the defect regeneration in the animal models. The fitable seeding cells and scaffolds were chosen to construct theanatomical osteochondral composites and relative studies of scaffolds systemloaded with seeding cells in the repair of large osteochondral defects was alsoprovided as follows:
I Selection of seeding cells of osteochondral composites
1. Obtainment and culture of bone mesenchymal stem cells (BMSCs) andchondrocytes in vitro
Two-week-old New Zealand White rabbits were selected for BMSCs andchondrocytes isolation and culture. Trypsin-collagenase digested method,adhesive culture method, high or low-density culture method were adopted andbiological characters such as proliferative capability, cell phenotype and cellexcretion of primitive cells and third-generation cells were inveatigated. Theresults showed that high-density culture chondrocytes, especially purifiedprimitive cells, have well proliferative capability and cell excretion. Moreover, alarge quantity of primitive cells obtained from different parts could be guaranteed,because the biological characters of BMSCs from ears or joints were almost same.Adhesive culture method was proved to be an effective method to provide a largenumber of cells. All above was the favorable base of future study.
2. Inducement and differentiation of BMSCsCelluar non-contact co-culture induced method and conditioned mediuminduced method were taken to futher choose the seeding cells in this study. Theresults suggested that BMSCs induced by different methods could bedifferentiated into desired cells and the induced cells had the well proliferativecapability evaluated by MTT, mitotic cycle, GAG secretion, ALP, cell-stainingmethods.
The results also displayed that the biological characters of co-cultureinduced cells was seemed to be better than conditioned medium induced cells,which was related with the growth factors and matrices from chondrocytes, eventhough poorer than primitive cells. Osteoinductive ability and osteogenetictendency of BMSC was definite. Therefore, primitive chondrocytes andthird-generation osteoinductive BMSC were comfirmed as the ultimate seedingcells in this study.
II Selection of scaffolds of osteochondral composites
1. Morphology and physical characters of scaffolds of osteochondral compositesFour kinds of scaffolds were made by different biomaterials, and the surfacemorphology, mechanical propertyand in vitro degradation of them wasinvestigated. Gelatin–chondortin–hyaluronan hybrid scaffold and Gelatin–CBBscaffold were the ultimate scaffolds.
2. Construction and biocompatibility of osteochondral compositesTo investigate the cytotoxicity and the effect on cell proliferation of thescaffolds, the cells were seeded on them. And the novel three-dimensional specialscaffold was developed into a biphasic scaffolds with a controlled release ofbFGF, which provided structural support and stimulated repair. The in vitroformation of osteochondral composites was observed under the condition ofosteoinduction or un-osteoinduction. The results testified the formation ofosteochondral composite tissue on the biphasic scaffolds by morphological andmatrix secretion assay.
Construction of osteochondral composites and in vivo study
1. Heterotopic transplantation of osteochondral composites in vivoThree-dimensional biphasic scaffolds seeded with cultured fluorescence mouse costal chondrocytes and BMSCs were transplanted subcutaneously intonude mice after 1day, 7day and 14day, and analyzed histologically during 8weeks after the operation. Our findings showed that chondral and boneregenerations of the subchondral phases were observed via fluorescent analysisand the results demonstrated the newel tissue was derived from transplantedosteochondral composites. On the other hand, the earlier transplantedosteochondral composites were contributed to tissue maturation, which impliedthe nutrient content and blood vessel ingrowth in the early stage.
2. Repair and regeneration of osteochondral defects in rabbit articular jointsThe aim of this study was to discuss the regenerative capability ofanatomical osteochondral composites in in repairing large articular joint defects.The scaffolds were fabricated via Perfusion Rapid Prototyping technology andcontrolled release system of growth factors ,and the resulted anatomicalosteochondral composites were orthotopic transplantated. In the study, hyalinecartilage-like regeneration tissue was observed at the border of the normalcartilage with histologically 24 weeks postoperatively, for physiologicalstimulation,such as growth factors and biomechanics, were existed in jointmicroenvironment. Both morphology and motor function were recovered byfunctional rebuilding.
In conclusion, osteochondral composites were constructed successfully invitro after selection of seeding cells and scaffolds. The in vivo bone and cartilageengineering via combining a novel composite, biphasic scaffold technology withtwo kinds of seed cells has been shown a high potential in the large defectregeneration in the animal models. The present study implied the great potentialof the novel strategy as a new way to promote clinical relative diseases and it might serve as a desirable approach to contruct other tissue engineering organs.
引文
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