骨髓间充质干细胞复合β-TCP生物陶瓷修复山羊骨软骨缺损的实验研究
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摘要
背景
关节软骨缺乏血供,软骨自身修复能力低下,一旦发生缺损,往往导致不可逆性的关节退变。而这种自身修复能力的先天不足,在合并有软骨下骨的复合型缺损时,则显得尤为突出。作为关节软骨的坚强支撑,软骨下骨对关节内的应力传导发挥着重要作用。骨软骨缺损一旦发生,由于失去了坚强的软骨下骨支撑,软骨面局部的应力异常分布及塌陷则难以避免,由此引发的恶性循环将造成正常关节软骨的连锁破坏,最终导致骨性关节炎甚至骨性强直。
目前临床上较为成熟的软骨修复技术包括骨髓刺激和移植两大类:前者如软骨下钻孔术和微骨折技术;后者有自体骨软骨镶嵌成型术(Mosaicplasty)和自体软骨细胞移植(ACI)术。但上述技术本身均有其各自的局限性。组织工程学的发展为骨软骨缺损的修复提供了新的思路,复合器官及组织构建是当今组织工程学发展的方向。理想的组织工程化修复应提供软骨及软骨下骨宿主组织的良好结合,已知骨—骨结合远远优于软骨—软骨以及软骨—骨的结合,而理想的组织工程化复合骨软骨,则能够首先满足骨—骨的结合,一期恢复对软骨的力学支撑,并且避免软骨与骨结合不良的问题。组织工程骨和组织工程软骨分别构建成功的报道早已不胜枚举,这也使得骨软骨复合组织构建成为研究者们关注的热点。
目前,生物反应器是组织工程领域的研究热点及重要发展方向,其目的在于体外模拟动物机体组织、器官的生理环境,以促进组织的再生。体内正常的骨及软骨组织,其所处生理环境不仅仅限于物理及化学因素,正常的应力刺激是维持关节软骨生理功能的关键。在组织工程学研究中,适当的力学载荷作为一个必要的生理性刺激因素,可以在一定程度上克服传统细胞培养条件的不足。力学因素对于软骨细胞生物学行为的影响过程极其复杂,涉及到细胞类型、形态、生长周期及分布部位等诸多方面。有学者研究表明:流体剪切力可增加矿物质的沉积,增强成骨细胞表型的表达,并使成骨细胞分泌的细胞外基质在支架材料内获得良好的分布。还有研究发现,一定范围的剪切力可促进种子细胞与载体的贴附,并维持成骨细胞在体外的表型。另外,压力负荷可以调节体外构建的组织工程化软骨中细胞的增殖、基质代谢及合成。由生物反应器模拟体内的力学载荷进行组织构建,研究其对于种子细胞的基质合成、形态、力学信号传导机制及电机械性能等,对于以组织工程学方法进行骨软骨损伤的修复具有重要意义。
磷酸钙生物陶瓷作为一种优秀的骨组织工程支架材料已被广泛接受,其中应用最多的是羟基磷灰石(HA)和β-磷酸三钙(β-TCP)。β-TCP具有良好生物相容性和降解性、骨传导能力佳、机械强度适中、微结构和体内降解速率可操控等优点。磷酸钙生物陶瓷在被用作骨支架材料的同时,也已被用于组织工程软骨的构建,已有学者使用β-TCP复合软骨细胞或成软骨诱导的干细胞成功地进行了软骨构建。
本研究以山羊BMSCs作为种子细胞,β-TCP生物陶瓷作为组织工程骨及软骨的构建材料。首先进行山羊BMSCs的成骨成软骨方面的诱导,以鉴定其多向诱导分化的能力;进而采用5-溴脱氧尿嘧啶核苷(BrdU)标记山羊BMSCs,研究其对山羊BMSCs标记的可行性及其最佳标记浓度和时间,以及对标记对细胞的增殖的影响,为确认下一步山羊体内实验形成的新生软骨组织来源提供研究基础;最后,利用负压抽吸的方法,使山羊BMSCs结合于β-TCP生物陶瓷,设计并制作双腔生物反应器,在其中分别进行成骨诱导分化和成软骨诱导分化;并制作山羊膝关节骨软骨缺损,将在双腔生物反应器中分别诱导2周的β-TCP-细胞复合物移植于山羊膝关节骨软骨缺损部位,观察其对骨软骨缺损的治疗效果,为临床利用自体BMSCs进行组织工程修复骨软骨缺损提供寻找一种新的方法。
材料与方法
1.山羊BMSCs的分离、培养和鉴定及向成骨细胞、成软骨细胞诱导分化
抽取10月龄健康中国青山羊骨髓,全骨髓培养法培养BMSCs,体外培养至第4代(P4)时行流式细胞术鉴定,并加入成骨诱导培养基和成软骨诱导培养基。成骨诱导培养基成分为:终浓度为100nmol/l地塞米松、10mmol/lβ-磷酸甘油、50ug/ml抗坏血酸的10%FBS高糖DMEM;成软骨诱导培养基成分:终浓度为6.25μg/ml胰岛素、6.25μg/ml转铁蛋白、50ug/ml抗坏血酸、100nmol/L地塞米松及10 ng/ml TGF-β1的10%FBS高糖DMEM。诱导两周后分别行细胞化学染色、免疫细胞化学染色,并采用逆转录-聚合酶链反应(RT-PCR)和蛋白印迹试验(Western blot)检测0、1、2、4周的Ⅰ型胶原、Ⅱ型胶原和Aggrecan的表达。采用SPSS 13.0统计软件分析,结果用(?)±s表示,p值＜0.05为有统计学差异。第4代和第8代的细胞倍增时间的比较,采用两独立样本t检验;各时间点mRNA和蛋白的相对表达量的比较,应用多个样本均数比较的方差分析(One-way ANOVA),组间多重比较采用LSD法。
2、BrdU体外标记山羊骨髓间充质干细胞的研究
利用BrdU标记山羊BMSCs,检测其最佳标记时间、标记剂量及毒性作用,探讨其作为山羊BMSCs标记示踪方法的可行性。培养山羊BMSCs,取第4代细胞以浓度分别为5、10、15和20gmol/L的BrdU进行标记,分别记为A、B、C、D组;另1孔不含BrdU,作为空白对照(E组)。分别标记12、24、48和72h后,采用免疫荧光法检测各组细胞阳性率,并用苔盼蓝拒染法测定标记后细胞活率。并观察山羊BMSCs标记后向骨和软骨细胞的分化;使用MTT法检测标记后细胞的增殖能力。细胞阳性标记率和标记前后存活率的均值比较采用析因设计资料的方差分析;细胞标记前、后的细胞倍增时间的比较采用两独立样本的t检验,以P＜0.05为有显著性差异。
3、双腔搅拌式生物反应器的制作及组织工程骨软骨修复山羊膝关节缺损的体内研究
制作双腔搅拌式生物反应器。取第4代山羊BMSCs,以5×10~7/ml的细胞密度与β-TCP生物陶瓷相复合。在双腔生物反应器中诱导2周后,将BMSCs—β-TCP生物陶瓷复合物植入山羊膝关节缺损区域内。于山羊的双后肢股骨内髁负重区制造直径6mm和深度12mm的缺损,使用外径为6mm的环锯,在负重位置的软骨上钻孔,取出骨和软骨碎屑后,压配方式植入直径6mm,长度12mm的BMSCs—β-TCP生物陶瓷复合物,缺损区域表面用复合山羊BMSCs的1.2%藻酸钠与102mM氯化钙交联形成的凝胶覆盖。根据植入物在生物反应器中是否经力学刺激,将山羊分为三组:A组:力学刺激+双向诱导组;B组:单纯双向诱导组;C组:空白对照组。每组山羊各4只,共12只24个膝关节。术后圈养,不限活动,于术后12周、24周分别处死各组2只山羊。进行如下项目检测:大体观察;组织学及组织化学检测:HE染色;甲苯胺蓝染色;Masson染色,Ⅱ型胶原免疫组化,并观察Brdu的表达。进行O'Driscoll,Keeley and Salter组织形态学评分。评分后的数据以(?)±s表示,应用两因素方差分析,组间比较用LSD法,以P＜0.05为有显著性差异。
结果
1.原代培养的山羊BMSCs形态呈纺锤状或梭状,折光性好,贴壁生长,随着培养时间延长,细胞呈集落状生长,集落中细胞形态为典型的梭形细胞,并逐渐融合成片。培养7-8d约80%融合,9～10d可形成90%融合的细胞单层。传代细胞大部分为长梭形,细胞生长速度较原代细胞明显增快,潜伏期为2～3d后开始进入对数生长期,7～8d进入平台期。流式细胞术检测细胞表面抗原,可见表达BMSCs标志物CD29,而不表达传代造血干细胞标志物CD34。在成骨细胞诱导分化培养基、成软骨细胞诱导分化培养基的作用下,能分化出成熟的成骨细胞和软骨细胞;2周后分别行成骨细胞鉴定可见碱性磷酸酶染色、茜素红染色呈阳性,行成软骨细胞鉴定可见甲苯胺蓝和阿利新蓝染色阳性。RT-PCR、Western blot及免疫细胞染色结果均证实其向成骨和成软骨细胞的分化。第4代和第8代细胞的形态及生长增殖状况无明显差别(t＝0.342,p＝0.741),并且,在成骨及成软骨诱导2周时,CollagenⅠ、Ⅱ和Aggrecan mRNA和蛋白的表达量与诱导后4周无统计学差异(p＞0.05),与未诱导组及诱导1周组有统计学差异(p＜0.05)。
2.第4代山羊BMSCs经BrdU标记后行免疫荧光染色,在荧光显微镜下胞核呈绿色荧光。随着标记时间的延长和BrdU剂量的增加,标记率逐渐增高,BrdU终浓度为15μmol/L且标记48h后细胞标记率＞90%,但标记时间继续延长和BrdU浓度继续增加,细胞标记率无明显增高,表明BrdU标记山羊BMSCs的最佳浓度为15μmol/L,最佳标记时间为48h。与正常未标记细胞比较,标记后细胞的形态及生长增殖状况无明显差别(t＝0.178,p＝0.862),活细胞数＞98%。15gmol/LBrdU标记山羊BMSCs48h后不影响向成骨和成软骨细胞分化的能力。
3.术后12周取材,A组修复区可见部分软骨样组织,关节软骨无磨损,修复的软骨呈白色半透明外观,与周围正常关节软骨有连续性,可见一明显的凹陷,无明显软骨下骨外露;B组修复区也可见部分软骨样组织,关节软骨在修复区可见磨损,软骨呈白色不透明外观,软骨下骨形成较好;空白对照组术区关节凹陷,无关节软骨组织形成,关节下骨缺如。BrdU免疫荧光证实,新生软骨中部分细胞来源于植入的山羊自体BMSCs。组织学检查示,β-TCP生物陶瓷已基本吸收降解。术后24周取材,见A组山羊手术区关节表面较为光滑,与周边正常软骨自然连续平齐,透明的新生软骨组织形成,软骨下骨形成完好;B组山羊手术区修复的软骨组织基本完整,中心部位仍未完全融合,有微小凹陷;Ⅱ型胶原免疫组化示新生软骨组织呈棕黄色。C组术区关节凹陷,无关节软骨组织形成。A组和B组,软骨下骨的生长及与周围组织的结合均较好,无植入物脱落现象的发生。
组织学检测结果显示:A组在山羊体内形成的软骨质量优于B组,O'DriscollKeeley and Salter组织形态学评分为:A组12周(n＝4)16.00±0.816,24周(n＝4)18.75±0.957;B组(n＝4)12周11.00±0.816,24周(n＝4)14.75±0.957;C组未形成软骨,12周及24周时各组比较,差异有统计学意义(P＜0.05)。
结论
1、髂骨骨髓穿刺可分离出BMSCs。全骨髓培养法培养的BMSCs生长至第4代时,纯度较高,活力旺盛。本实验证明,山羊BMSCs具有向成骨细胞和软骨细胞分化的潜能,并且其在成骨和成软骨诱导培养基中培养2周时,其可表达成骨和成软骨的标志。山羊BMSCs来源丰富、取材容易,创伤小,是理想的组织工程种子细胞,具有广阔的应用前景。
2、BrdU标记山羊BMSCs的最佳时间为48小时;最佳终浓度为15μmol/L;标记阳性率＞90%。BrdU对细胞无毒副作用,安全性高,对细胞的生长增殖及成骨、成软骨分化无明显影响。
3、利用双腔搅拌式生物反应器,以山羊BMSCs为种子细胞,在同一块β-TCP生物陶瓷上可同时构建组织工程化骨和软骨。山羊体内实验证明,在生物反应器中进行体外培养时,力学刺激有利于改善山羊关节软骨的形成质量。
BACKGROUND
Articular cartilage tissue lacks the blood supply to support self-repair and remolding.Because of this limited capacity for spontenous repair,minor articular cartilage injury can lead to irreversible degeneration of the joints.It is particularly prominent when the damage is involved the subchondral bone injury.The Articular subchondral bone cartilage plays an important role in the conduction of stress in the joint,and it acts as a strong support.Once osteochondral defect occurs in the jiont,the abnormal stress distribution and the collapse of articular suiface will be inevitable because the cartilage loses the solid suppot of subchondral bone,which can trigger a vicious circle that will result in the damage of normal articular cartilage.Eventually, it can lead to osteoarthritis or bony ankylosis.
At present,many procedures have been proposed for the treatment of focal articular-cartilage injury,mainly including bone marrow stimulation and transplantation.Bone marrow stimulation includes subchondral drilling and microfracture techniques;the latter includes autogenous chondrocyte implantation (ACI) and mosaicplasty.However,all the technology above have their own limitations.The development of tissue engineering provides a new way for osteochondral defects repairing.Today complex organs and tissues construction are the direction for the development of tissue engineering.Ideal tissue-engineered repair materials should be able to provide a better intergration of the cartilage and subchondral bone of the host tissue,ACI technology can not reach this aim,and Mosaicplasty technology has many limitations.As we have known,bone-bone intergration model is much better than that of cartilage-cartilage and cartilage-bone model.The ideal tissue-engineered cartilage should meet the bone-bone intergration, which can support cartilage restoration and avoid the adverse combination of cartilage and bone problems.Studies have already reported numerous way of tissue engineering of bone and tissue engineering cartilage construct separately successful, and now composite osteochondral construction research have become the focus of attention in this field.
At present,the bioreactor are the hot spots and important direction of development in the field of tissue engineering research,and its aim is to simulate the physiological environment of tissues and organs in vivo,which can promote the regeneration of them.For the normal bone and cartilage tissue,not only physical and chemical factors but also normal stress stimulation of articular cartilage also play an important role in maintain the physiological functions of cartilage.The appropriate mechanical load is a necessary physiological stimulus,which can overcome the deficiencies on the traditional cell culture.Mechanical factors affect the biological behavior of chondrocytes in an extremely complex process,involving cell types, morphology,growth cycle and distribution.Research has proved that external forces can affect the BMSCs differentiation towards osteoblasts and adipocyte,which suggest that stress factors may have multilineage differentiation potential for stem cells.Different forces in different ways of load,size and frequency of the role,can adjust and maintain normal chondrocyte biology through a mechanical signal transduction mechanism.Some studies show that fluid shear stress can increase the deposition of minerals,and enhance osteoblast phenotype expression and osteoblast secretion of extracellular matrix material in the stent with good distribution.Studies have found that a range of shear stress can promote the seed cells and vector-attached, and retain the osteoblast phenotype in vitro.In addition,stress load can adjust chondrocyte proliferation,matrix metabolism and synthesis in vitro.Although this mechanism of mechanical stimulation have not yet been fully clarified,it suggests that cells cultured in vitro,whether single-layer or three-dimensional culture,can improve their biological behavior of cultured cells through being exerted a certain dynamic mechanical stimulation,such as the promotion of cartilage cells extracellular matrix proteoglycan and collagen synthesis.By analoging the mechanical load in vivo, to study the seed cells for matrix synthesis,morphology,mechanical signal transduction mechanism and mechanical properties such as electricity,bioreactor has a great significance in the aspect of repairing osteochondral damage using tissue engineering methods.
Calcium phosphate bioceramics has been widely accepted as an excellent scaffold for bone tissue engineering.The most widely used is hydroxyapatite(HA) andβ-tricalcium phosphate(β-TCP).β-TCP has a good biocompatibility, degradability and bone conduction capacity,moderate mechanical strength, microstructure and the degradation rate in vivo can be manipulated,and so on. Recently,scholars have found that the bioceramic aperture diameter of the inner connecting bone formation has an important impact,and it is necessary that the diameter must be larger than 50μm for the formation of mineralized bone.Calcium phosphate bioceramics has been used to construct tissue-engineered cartilage. Scholars have used the calcium phosphate as a scoffold to construct chondral tissue with chondrocyte and stem cells successful.
In this study,the goat BMSCs was acted as seed cells,β-TCP bioceramics was acted as osteochondral scoffold for tissue engineering construction osteochondral graft. Firstly,the goat BMSCs were aspirated and differentiated into osteoblasts and chondrocyte induced by different induction medium and flow cytometry,which can identify its capacity to differentiation;then the goat BMSCs were labeled with bromodeoxyuridine,and explored the optimal concentration,time and cytotoxicity of BrdU labeling of goat BMSCs in vitro,and confirm its feasibility for stem cells labelling and tracer means for goat;Finally,we used the method of negative pressure suction in order to get the combination of goat BMSCs attached toβ-TCP ceramics, and we designed and producted the double-chamber stirring bioreactor,in which BMSCs could inducte and differentiate to osteoblasts and chondrocyte respectively; and the goats knee joint osteochondral defects model were created,and transplanted the engineered graft into the osteochondral defect site.Then,we observed the treatment effect of this tissue engineering mothod for repairing the osteochondral defect,which can provide a new method for clinical treatment of osteochondral defect using autologous BMSCs.
MATERIAL AND METHODS
1.Isolation,culture,osteogenic,identification,osteogenic and chondrogenic differentiation of goat BMSCs
10-month-old goat was used in this experiment(The Southern Medical University institutional ethics committee approved all experiments in advance). 10ml bone marrow was aspirated;BMSCs were isolated and cultured using the whole bone marrow culture method in vitro.The 4rd passages of BMSCs(P4) were identified by flow cytometry.Add osteogenic and chondrogenic induction medium to the goat BMSCs.Osteogenic induction medium composition as follows:final concentration of 100nmol/L dexamethasone,10mmol/lβ- glycerophosphate,50ug/ml ascorbic acid of 10%FBS high glucose DMEM;chondrogenesis induction medium composition as follows:final concentration of 6.25μg/ml insulin,6.25μg/ml transferrin,50ug/ml ascorbic acid,100nmol/L dexamethasone and 10 ng/ml TGF-β1 of 10%FBS high glucose DMEM.Cytochemical staining, immunocytochemical staining and reverse transcription-polymerase chain reaction (RT-PCR) and Western blot were performed to detect of the collagenⅠ,collagenⅡand Aggrecan expression at 0,1,2,4 weeks to identify the success of differentiation. The data was analyzed by SPSS 13.0 statistical software,p value＜0.05 was regarded significant difference.The doubling time of P4 and p8 goat BMSCs was analyzed with two-sample t test;the relative expression of mRNA and protein were analysised with One-way ANOVA,multiple comparisons between groups using LSD method.
2.In vitro bromodeoxyuridine labeling of goat bone marrow mesenchymal stem cells.
The 4~(rd) passage of goat BMSCs was incubated with BrdU at different concentrations(0,5,10,15 and 20μmol/L) for incubating time(12,24,48 and 72 h),to identify the optimal BrdU concentration and incubating time for goat BMSCs labeling.Immunofluorescence and trypanblau strain were performed respectively to calculate the labeling positive rate and the cells' survival rate for different time after BrdU labeling in vitro.Then we compared the difference of osteogencic and chondrogenic potentiality before and after BrdU labeling.MTT analysis was used to detect of the rate of cell proliferation and cytotoxicity of BrdU.Labeling rate and the survival rate before and after BrdU labeling was analyzed by factorial analysis of variance;P＜0.05 was regarded significant difference.The doubling time of P4 goat BMSCs of before and after BrdU labeling were analyzed with two-sample t test; P＜0.05 was regarded significant difference.
3.The manufacture of double-chamber stirring bioreactor and tissue-engineered osteochondral graft repairing goat knee osteochondral defects in vivo.
The double-chamber stirring bioreactor was designed and manufactured.Collect the 4rd-passage goat BMSCs and inoculate them to to theβ-TCP bioceramic composite at the density of 5×10~7/ml.After induction in the double-chamber stirring bioreactor respectively for two weeks,the BMSCs-β-TCP bioceramic composites were transplanted into the defect area of goat knee joints.A defect of 6mm in diameter and 12mm in depth was created in the femoral medial condyle weight-bearing areas of both posterior limbs.
Drilling at weight-bearing cartilage with the external diameter of 6mm trepan, bone and cartilage debris were removed.Autogenous goat BMSCs-β-TCP bioceramic composite was implanted into the defect area by press-fit method.After the implantation,the BMSCs-gel formed by 1.2%in algal and 102mmol/l sodium chloride crosslinked were covered on the surface of defect.According to whether the graft experienced mechanical stimulation during cultured and inducted in bioreactor, the goats were divided into three groups:group A:Mechanical stimulation+ two-direction induction in double-chamber bioreactor;group B:two-direction induction without mechanical stimulation;groups C:control group.Each group has four goats,totally 24 knees,12 goats.The goats were sacrificed at 12 weeks and 24 weeks after operation.Then detected the following items:general observation; histology and histochemical detection:HE staining,Toluidine blue staining,Masson staining,collagenⅡimmunohistochemistry,Brdu immunofluorescence detection in the time point of 12 weeks.O'Driscoll,Keeley and Salter Histomorphology score. The data was showed as(?)±s,and two-way ANOVA and LSD was used for stastistcs analysis,P＜0.05 was regarded significant difference
RESULTS
1.The goat BMSCs of primary culture were cambiform or spindle-like,and it has the character of good refractive and adherent growth.With the extension of culture time,the growth of BMSCs was colony-like and typical spindle-like of cells can be observed in the colony.The cells would reach 80%of fusion after 7～8d cultivation,90%of fusion after 9～10d cultivation.Most of the cells were long spindle-like;cell growth rate was faster than the primary cells.After cultured for 2～3d,the cells began to enter the logarithmic-like growth phase,and 7～8d into the plateau phase.The result of flow cytometry showed:cell surface antigen expression of MSCs markers CD29 can reach 86.4%,and the expression of hemopoietic stem cell marker CD34 was only 0.4%.Under the induction of osteogenic induction medium and the chondrogenic induction medium,the goat BMSCs could be differentiated toward osteoblasts and chondrocytes respectively;Identified osteoblast formed from BMSCs with alkaline phosphatase stain and alizarin red staining,the result showed positive in 2 weeks.Identified chondrocyte formed from BMSCs with toluidine blue and Alican blue staining,the result showed positive in 2 weeks.RT-PCR,Western blot and immunohistochemical staining results were confirmed osteoblastic and chondrogenic cell differentiation and,after the BMSCs were induced towards osteogenesis and chondrogenesis for 2 weeks,The expression CollagenⅠ,Ⅱand Aggrecan mRNA and protein had no significance stastistic difference with that of induction 4 weeks.and it has significance stastistic difference with induction 1 week and non-induction.
2.We performed immunofluorescence staining after labeled by the BrdU.The goat BMSCs' nucleus show green fluor under fluorescence microscope.The labeling positive rate increased gradually with the increase of incubating time and concentration of BrdU.More than 90%goat BMSCs could be labeled when the cells were incubated for 48h and the concentration of BrdU maintaining 15μmol/L at the same time,and the survival rate of labeled cells were more than 98%.but the labeling rate did not increase with prolong of time and the increase of concentration of BrdU.It indicated that the optimal time for labeling goat BMSCs is 48h and the optimal concentration is 15μmol/L of BrdU.The morphous,growth,proliferation and differentiation of the labeled BMSCs were not influenced compare to the unlabelled BMSCs.
3.After 12 weeks,the result showed the operation area of group A articular surface was smooth,with the surrounding normal cartilage naturally straight flush, transparent form new cartilage tissue;Group B restoration surgery the basic integrity of the cartilage tissue,but the center of many not fully integrated,there is slight depression,surface wear,good subchondral bone formation;control group, depression joint operation areas,non-articular cartilage formation,such as lack of bone joints.BrdU immunofluorescence confirmed that the new cartilage cells from some of the goat implanted autologous BMSCs.Histological examination showed thatβ-TCP bioceramic degradation has been absorbed.After 24 weeks,the operation area of group A was more smooth,and the surrounding normal cartilage naturally straight flush,transparent form new cartilage,subchondral bone formation in good condition;Group B restoration surgery the basic integrity of the cartilage tissue, center is not yet fully integrated,there was slight depression;CollagenⅡimmunohistochemistry of cartilage that was new brown area.Group C has no formation of articular cartilage.The growth and the intergration of subchondral bone of group A and B were better.
The result of histological test showed that:the quality of goat cartilage formed in group A in vivo is superior to that of group B.O'Driscoll Keeley and Salter histomorphology score:group A of 12 weeks(n＝4) 16.00±0.82,24 weeks(n＝4) 18.75±0.96;group B(n＝4) 12 weeks 11.00±0.82,24 weeks(n＝4) 14.75±0.96; group C did not form cartilage,12 weeks and 24 weeks in each group,the difference was statistically significant(P＜0.05).
CONCLUSIONS
1、We isolated the goat BMSCs from iliac of adult Chinese goats.And we established a method about isolation,culture and identification for the goat BMSCs.The 4rd passage BMSCs cultured by the whole bone marrow culture method was of higher purity and thriving.This experiment confirmed that the goat BMSCs had a great potentiality of proliferation and osteogenic and chondrogenic differentiation in vitro.When cultured in osteogenic and chondrogenic induction medium for 2 weeks,the cells could express the characteristic of osteocyte and chondrocyte.Goat BMSCs are an ideal seed cell for tissue engineering and it has broad application prospects,which provide an experimental foundation for goat BMSCs being used in bone and cartilage tissue engineering in vivo.
2、BrdU can be used as a labeling marker for goat BMSCs.When the concentration is 15μmol/L and the incubation time is 48 hours,the optimal labeling effect can be gained.Goat BMSCs labeled with BrdU is of high efficiency.BrdU has no obvious cytotoxicity and influence on the growth, proliferation and differentiation to the cells.
3、We can construct tissue engineering osteochondral graft simultaneously at the same piece ofβ-TCP bioceramics with goat BMSCs act as seed cells and cultured and inducted in double-chamber stirring bioreactor.The experiment in vivo of goat proved that mechanical stimulation could improve the quality of goat cartilage formation.
关节软骨缺乏血供,软骨自身修复能力低下,一旦发生缺损,往往导致不可逆性的关节退变。而这种自身修复能力的先天不足,在合并有软骨下骨的复合型缺损时,则显得尤为突出。作为关节软骨的坚强支撑,软骨下骨对关节内的应力传导发挥着重要作用。骨软骨缺损一旦发生,由于失去了坚强的软骨下骨支撑,软骨面局部的应力异常分布及塌陷则难以避免,由此引发的恶性循环将造成正常关节软骨的连锁破坏,最终导致骨性关节炎甚至骨性强直。
目前临床上较为成熟的软骨修复技术包括骨髓刺激和移植两大类:前者如软骨下钻孔术和微骨折技术;后者有自体骨软骨镶嵌成型术(Mosaicplasty)和自体软骨细胞移植(ACI)术。但上述技术本身均有其各自的局限性。组织工程学的发展为骨软骨缺损的修复提供了新的思路,复合器官及组织构建是当今组织工程学发展的方向。理想的组织工程化修复应提供软骨及软骨下骨宿主组织的良好结合,已知骨—骨结合远远优于软骨—软骨以及软骨—骨的结合,而理想的组织工程化复合骨软骨,则能够首先满足骨—骨的结合,一期恢复对软骨的力学支撑,并且避免软骨与骨结合不良的问题。组织工程骨和组织工程软骨分别构建成功的报道早已不胜枚举,这也使得骨软骨复合组织构建成为研究者们关注的热点。
目前,生物反应器是组织工程领域的研究热点及重要发展方向,其目的在于体外模拟动物机体组织、器官的生理环境,以促进组织的再生。体内正常的骨及软骨组织,其所处生理环境不仅仅限于物理及化学因素,正常的应力刺激是维持关节软骨生理功能的关键。在组织工程学研究中,适当的力学载荷作为一个必要的生理性刺激因素,可以在一定程度上克服传统细胞培养条件的不足。力学因素对于软骨细胞生物学行为的影响过程极其复杂,涉及到细胞类型、形态、生长周期及分布部位等诸多方面。有学者研究表明:流体剪切力可增加矿物质的沉积,增强成骨细胞表型的表达,并使成骨细胞分泌的细胞外基质在支架材料内获得良好的分布。还有研究发现,一定范围的剪切力可促进种子细胞与载体的贴附,并维持成骨细胞在体外的表型。另外,压力负荷可以调节体外构建的组织工程化软骨中细胞的增殖、基质代谢及合成。由生物反应器模拟体内的力学载荷进行组织构建,研究其对于种子细胞的基质合成、形态、力学信号传导机制及电机械性能等,对于以组织工程学方法进行骨软骨损伤的修复具有重要意义。
磷酸钙生物陶瓷作为一种优秀的骨组织工程支架材料已被广泛接受,其中应用最多的是羟基磷灰石(HA)和β-磷酸三钙(β-TCP)。β-TCP具有良好生物相容性和降解性、骨传导能力佳、机械强度适中、微结构和体内降解速率可操控等优点。磷酸钙生物陶瓷在被用作骨支架材料的同时,也已被用于组织工程软骨的构建,已有学者使用β-TCP复合软骨细胞或成软骨诱导的干细胞成功地进行了软骨构建。
本研究以山羊BMSCs作为种子细胞,β-TCP生物陶瓷作为组织工程骨及软骨的构建材料。首先进行山羊BMSCs的成骨成软骨方面的诱导,以鉴定其多向诱导分化的能力;进而采用5-溴脱氧尿嘧啶核苷(BrdU)标记山羊BMSCs,研究其对山羊BMSCs标记的可行性及其最佳标记浓度和时间,以及对标记对细胞的增殖的影响,为确认下一步山羊体内实验形成的新生软骨组织来源提供研究基础;最后,利用负压抽吸的方法,使山羊BMSCs结合于β-TCP生物陶瓷,设计并制作双腔生物反应器,在其中分别进行成骨诱导分化和成软骨诱导分化;并制作山羊膝关节骨软骨缺损,将在双腔生物反应器中分别诱导2周的β-TCP-细胞复合物移植于山羊膝关节骨软骨缺损部位,观察其对骨软骨缺损的治疗效果,为临床利用自体BMSCs进行组织工程修复骨软骨缺损提供寻找一种新的方法。
材料与方法
1.山羊BMSCs的分离、培养和鉴定及向成骨细胞、成软骨细胞诱导分化
抽取10月龄健康中国青山羊骨髓,全骨髓培养法培养BMSCs,体外培养至第4代(P4)时行流式细胞术鉴定,并加入成骨诱导培养基和成软骨诱导培养基。成骨诱导培养基成分为:终浓度为100nmol/l地塞米松、10mmol/lβ-磷酸甘油、50ug/ml抗坏血酸的10%FBS高糖DMEM;成软骨诱导培养基成分:终浓度为6.25μg/ml胰岛素、6.25μg/ml转铁蛋白、50ug/ml抗坏血酸、100nmol/L地塞米松及10 ng/ml TGF-β1的10%FBS高糖DMEM。诱导两周后分别行细胞化学染色、免疫细胞化学染色,并采用逆转录-聚合酶链反应(RT-PCR)和蛋白印迹试验(Western blot)检测0、1、2、4周的Ⅰ型胶原、Ⅱ型胶原和Aggrecan的表达。采用SPSS 13.0统计软件分析,结果用(?)±s表示,p值＜0.05为有统计学差异。第4代和第8代的细胞倍增时间的比较,采用两独立样本t检验;各时间点mRNA和蛋白的相对表达量的比较,应用多个样本均数比较的方差分析(One-way ANOVA),组间多重比较采用LSD法。
2、BrdU体外标记山羊骨髓间充质干细胞的研究
利用BrdU标记山羊BMSCs,检测其最佳标记时间、标记剂量及毒性作用,探讨其作为山羊BMSCs标记示踪方法的可行性。培养山羊BMSCs,取第4代细胞以浓度分别为5、10、15和20gmol/L的BrdU进行标记,分别记为A、B、C、D组;另1孔不含BrdU,作为空白对照(E组)。分别标记12、24、48和72h后,采用免疫荧光法检测各组细胞阳性率,并用苔盼蓝拒染法测定标记后细胞活率。并观察山羊BMSCs标记后向骨和软骨细胞的分化;使用MTT法检测标记后细胞的增殖能力。细胞阳性标记率和标记前后存活率的均值比较采用析因设计资料的方差分析;细胞标记前、后的细胞倍增时间的比较采用两独立样本的t检验,以P＜0.05为有显著性差异。
3、双腔搅拌式生物反应器的制作及组织工程骨软骨修复山羊膝关节缺损的体内研究
制作双腔搅拌式生物反应器。取第4代山羊BMSCs,以5×10~7/ml的细胞密度与β-TCP生物陶瓷相复合。在双腔生物反应器中诱导2周后,将BMSCs—β-TCP生物陶瓷复合物植入山羊膝关节缺损区域内。于山羊的双后肢股骨内髁负重区制造直径6mm和深度12mm的缺损,使用外径为6mm的环锯,在负重位置的软骨上钻孔,取出骨和软骨碎屑后,压配方式植入直径6mm,长度12mm的BMSCs—β-TCP生物陶瓷复合物,缺损区域表面用复合山羊BMSCs的1.2%藻酸钠与102mM氯化钙交联形成的凝胶覆盖。根据植入物在生物反应器中是否经力学刺激,将山羊分为三组:A组:力学刺激+双向诱导组;B组:单纯双向诱导组;C组:空白对照组。每组山羊各4只,共12只24个膝关节。术后圈养,不限活动,于术后12周、24周分别处死各组2只山羊。进行如下项目检测:大体观察;组织学及组织化学检测:HE染色;甲苯胺蓝染色;Masson染色,Ⅱ型胶原免疫组化,并观察Brdu的表达。进行O'Driscoll,Keeley and Salter组织形态学评分。评分后的数据以(?)±s表示,应用两因素方差分析,组间比较用LSD法,以P＜0.05为有显著性差异。
结果
1.原代培养的山羊BMSCs形态呈纺锤状或梭状,折光性好,贴壁生长,随着培养时间延长,细胞呈集落状生长,集落中细胞形态为典型的梭形细胞,并逐渐融合成片。培养7-8d约80%融合,9～10d可形成90%融合的细胞单层。传代细胞大部分为长梭形,细胞生长速度较原代细胞明显增快,潜伏期为2～3d后开始进入对数生长期,7～8d进入平台期。流式细胞术检测细胞表面抗原,可见表达BMSCs标志物CD29,而不表达传代造血干细胞标志物CD34。在成骨细胞诱导分化培养基、成软骨细胞诱导分化培养基的作用下,能分化出成熟的成骨细胞和软骨细胞;2周后分别行成骨细胞鉴定可见碱性磷酸酶染色、茜素红染色呈阳性,行成软骨细胞鉴定可见甲苯胺蓝和阿利新蓝染色阳性。RT-PCR、Western blot及免疫细胞染色结果均证实其向成骨和成软骨细胞的分化。第4代和第8代细胞的形态及生长增殖状况无明显差别(t＝0.342,p＝0.741),并且,在成骨及成软骨诱导2周时,CollagenⅠ、Ⅱ和Aggrecan mRNA和蛋白的表达量与诱导后4周无统计学差异(p＞0.05),与未诱导组及诱导1周组有统计学差异(p＜0.05)。
2.第4代山羊BMSCs经BrdU标记后行免疫荧光染色,在荧光显微镜下胞核呈绿色荧光。随着标记时间的延长和BrdU剂量的增加,标记率逐渐增高,BrdU终浓度为15μmol/L且标记48h后细胞标记率＞90%,但标记时间继续延长和BrdU浓度继续增加,细胞标记率无明显增高,表明BrdU标记山羊BMSCs的最佳浓度为15μmol/L,最佳标记时间为48h。与正常未标记细胞比较,标记后细胞的形态及生长增殖状况无明显差别(t＝0.178,p＝0.862),活细胞数＞98%。15gmol/LBrdU标记山羊BMSCs48h后不影响向成骨和成软骨细胞分化的能力。
3.术后12周取材,A组修复区可见部分软骨样组织,关节软骨无磨损,修复的软骨呈白色半透明外观,与周围正常关节软骨有连续性,可见一明显的凹陷,无明显软骨下骨外露;B组修复区也可见部分软骨样组织,关节软骨在修复区可见磨损,软骨呈白色不透明外观,软骨下骨形成较好;空白对照组术区关节凹陷,无关节软骨组织形成,关节下骨缺如。BrdU免疫荧光证实,新生软骨中部分细胞来源于植入的山羊自体BMSCs。组织学检查示,β-TCP生物陶瓷已基本吸收降解。术后24周取材,见A组山羊手术区关节表面较为光滑,与周边正常软骨自然连续平齐,透明的新生软骨组织形成,软骨下骨形成完好;B组山羊手术区修复的软骨组织基本完整,中心部位仍未完全融合,有微小凹陷;Ⅱ型胶原免疫组化示新生软骨组织呈棕黄色。C组术区关节凹陷,无关节软骨组织形成。A组和B组,软骨下骨的生长及与周围组织的结合均较好,无植入物脱落现象的发生。
组织学检测结果显示:A组在山羊体内形成的软骨质量优于B组,O'DriscollKeeley and Salter组织形态学评分为:A组12周(n＝4)16.00±0.816,24周(n＝4)18.75±0.957;B组(n＝4)12周11.00±0.816,24周(n＝4)14.75±0.957;C组未形成软骨,12周及24周时各组比较,差异有统计学意义(P＜0.05)。
结论
1、髂骨骨髓穿刺可分离出BMSCs。全骨髓培养法培养的BMSCs生长至第4代时,纯度较高,活力旺盛。本实验证明,山羊BMSCs具有向成骨细胞和软骨细胞分化的潜能,并且其在成骨和成软骨诱导培养基中培养2周时,其可表达成骨和成软骨的标志。山羊BMSCs来源丰富、取材容易,创伤小,是理想的组织工程种子细胞,具有广阔的应用前景。
2、BrdU标记山羊BMSCs的最佳时间为48小时;最佳终浓度为15μmol/L;标记阳性率＞90%。BrdU对细胞无毒副作用,安全性高,对细胞的生长增殖及成骨、成软骨分化无明显影响。
3、利用双腔搅拌式生物反应器,以山羊BMSCs为种子细胞,在同一块β-TCP生物陶瓷上可同时构建组织工程化骨和软骨。山羊体内实验证明,在生物反应器中进行体外培养时,力学刺激有利于改善山羊关节软骨的形成质量。
BACKGROUND
Articular cartilage tissue lacks the blood supply to support self-repair and remolding.Because of this limited capacity for spontenous repair,minor articular cartilage injury can lead to irreversible degeneration of the joints.It is particularly prominent when the damage is involved the subchondral bone injury.The Articular subchondral bone cartilage plays an important role in the conduction of stress in the joint,and it acts as a strong support.Once osteochondral defect occurs in the jiont,the abnormal stress distribution and the collapse of articular suiface will be inevitable because the cartilage loses the solid suppot of subchondral bone,which can trigger a vicious circle that will result in the damage of normal articular cartilage.Eventually, it can lead to osteoarthritis or bony ankylosis.
At present,many procedures have been proposed for the treatment of focal articular-cartilage injury,mainly including bone marrow stimulation and transplantation.Bone marrow stimulation includes subchondral drilling and microfracture techniques;the latter includes autogenous chondrocyte implantation (ACI) and mosaicplasty.However,all the technology above have their own limitations.The development of tissue engineering provides a new way for osteochondral defects repairing.Today complex organs and tissues construction are the direction for the development of tissue engineering.Ideal tissue-engineered repair materials should be able to provide a better intergration of the cartilage and subchondral bone of the host tissue,ACI technology can not reach this aim,and Mosaicplasty technology has many limitations.As we have known,bone-bone intergration model is much better than that of cartilage-cartilage and cartilage-bone model.The ideal tissue-engineered cartilage should meet the bone-bone intergration, which can support cartilage restoration and avoid the adverse combination of cartilage and bone problems.Studies have already reported numerous way of tissue engineering of bone and tissue engineering cartilage construct separately successful, and now composite osteochondral construction research have become the focus of attention in this field.
At present,the bioreactor are the hot spots and important direction of development in the field of tissue engineering research,and its aim is to simulate the physiological environment of tissues and organs in vivo,which can promote the regeneration of them.For the normal bone and cartilage tissue,not only physical and chemical factors but also normal stress stimulation of articular cartilage also play an important role in maintain the physiological functions of cartilage.The appropriate mechanical load is a necessary physiological stimulus,which can overcome the deficiencies on the traditional cell culture.Mechanical factors affect the biological behavior of chondrocytes in an extremely complex process,involving cell types, morphology,growth cycle and distribution.Research has proved that external forces can affect the BMSCs differentiation towards osteoblasts and adipocyte,which suggest that stress factors may have multilineage differentiation potential for stem cells.Different forces in different ways of load,size and frequency of the role,can adjust and maintain normal chondrocyte biology through a mechanical signal transduction mechanism.Some studies show that fluid shear stress can increase the deposition of minerals,and enhance osteoblast phenotype expression and osteoblast secretion of extracellular matrix material in the stent with good distribution.Studies have found that a range of shear stress can promote the seed cells and vector-attached, and retain the osteoblast phenotype in vitro.In addition,stress load can adjust chondrocyte proliferation,matrix metabolism and synthesis in vitro.Although this mechanism of mechanical stimulation have not yet been fully clarified,it suggests that cells cultured in vitro,whether single-layer or three-dimensional culture,can improve their biological behavior of cultured cells through being exerted a certain dynamic mechanical stimulation,such as the promotion of cartilage cells extracellular matrix proteoglycan and collagen synthesis.By analoging the mechanical load in vivo, to study the seed cells for matrix synthesis,morphology,mechanical signal transduction mechanism and mechanical properties such as electricity,bioreactor has a great significance in the aspect of repairing osteochondral damage using tissue engineering methods.
Calcium phosphate bioceramics has been widely accepted as an excellent scaffold for bone tissue engineering.The most widely used is hydroxyapatite(HA) andβ-tricalcium phosphate(β-TCP).β-TCP has a good biocompatibility, degradability and bone conduction capacity,moderate mechanical strength, microstructure and the degradation rate in vivo can be manipulated,and so on. Recently,scholars have found that the bioceramic aperture diameter of the inner connecting bone formation has an important impact,and it is necessary that the diameter must be larger than 50μm for the formation of mineralized bone.Calcium phosphate bioceramics has been used to construct tissue-engineered cartilage. Scholars have used the calcium phosphate as a scoffold to construct chondral tissue with chondrocyte and stem cells successful.
In this study,the goat BMSCs was acted as seed cells,β-TCP bioceramics was acted as osteochondral scoffold for tissue engineering construction osteochondral graft. Firstly,the goat BMSCs were aspirated and differentiated into osteoblasts and chondrocyte induced by different induction medium and flow cytometry,which can identify its capacity to differentiation;then the goat BMSCs were labeled with bromodeoxyuridine,and explored the optimal concentration,time and cytotoxicity of BrdU labeling of goat BMSCs in vitro,and confirm its feasibility for stem cells labelling and tracer means for goat;Finally,we used the method of negative pressure suction in order to get the combination of goat BMSCs attached toβ-TCP ceramics, and we designed and producted the double-chamber stirring bioreactor,in which BMSCs could inducte and differentiate to osteoblasts and chondrocyte respectively; and the goats knee joint osteochondral defects model were created,and transplanted the engineered graft into the osteochondral defect site.Then,we observed the treatment effect of this tissue engineering mothod for repairing the osteochondral defect,which can provide a new method for clinical treatment of osteochondral defect using autologous BMSCs.
MATERIAL AND METHODS
1.Isolation,culture,osteogenic,identification,osteogenic and chondrogenic differentiation of goat BMSCs
10-month-old goat was used in this experiment(The Southern Medical University institutional ethics committee approved all experiments in advance). 10ml bone marrow was aspirated;BMSCs were isolated and cultured using the whole bone marrow culture method in vitro.The 4rd passages of BMSCs(P4) were identified by flow cytometry.Add osteogenic and chondrogenic induction medium to the goat BMSCs.Osteogenic induction medium composition as follows:final concentration of 100nmol/L dexamethasone,10mmol/lβ- glycerophosphate,50ug/ml ascorbic acid of 10%FBS high glucose DMEM;chondrogenesis induction medium composition as follows:final concentration of 6.25μg/ml insulin,6.25μg/ml transferrin,50ug/ml ascorbic acid,100nmol/L dexamethasone and 10 ng/ml TGF-β1 of 10%FBS high glucose DMEM.Cytochemical staining, immunocytochemical staining and reverse transcription-polymerase chain reaction (RT-PCR) and Western blot were performed to detect of the collagenⅠ,collagenⅡand Aggrecan expression at 0,1,2,4 weeks to identify the success of differentiation. The data was analyzed by SPSS 13.0 statistical software,p value＜0.05 was regarded significant difference.The doubling time of P4 and p8 goat BMSCs was analyzed with two-sample t test;the relative expression of mRNA and protein were analysised with One-way ANOVA,multiple comparisons between groups using LSD method.
2.In vitro bromodeoxyuridine labeling of goat bone marrow mesenchymal stem cells.
The 4~(rd) passage of goat BMSCs was incubated with BrdU at different concentrations(0,5,10,15 and 20μmol/L) for incubating time(12,24,48 and 72 h),to identify the optimal BrdU concentration and incubating time for goat BMSCs labeling.Immunofluorescence and trypanblau strain were performed respectively to calculate the labeling positive rate and the cells' survival rate for different time after BrdU labeling in vitro.Then we compared the difference of osteogencic and chondrogenic potentiality before and after BrdU labeling.MTT analysis was used to detect of the rate of cell proliferation and cytotoxicity of BrdU.Labeling rate and the survival rate before and after BrdU labeling was analyzed by factorial analysis of variance;P＜0.05 was regarded significant difference.The doubling time of P4 goat BMSCs of before and after BrdU labeling were analyzed with two-sample t test; P＜0.05 was regarded significant difference.
3.The manufacture of double-chamber stirring bioreactor and tissue-engineered osteochondral graft repairing goat knee osteochondral defects in vivo.
The double-chamber stirring bioreactor was designed and manufactured.Collect the 4rd-passage goat BMSCs and inoculate them to to theβ-TCP bioceramic composite at the density of 5×10~7/ml.After induction in the double-chamber stirring bioreactor respectively for two weeks,the BMSCs-β-TCP bioceramic composites were transplanted into the defect area of goat knee joints.A defect of 6mm in diameter and 12mm in depth was created in the femoral medial condyle weight-bearing areas of both posterior limbs.
Drilling at weight-bearing cartilage with the external diameter of 6mm trepan, bone and cartilage debris were removed.Autogenous goat BMSCs-β-TCP bioceramic composite was implanted into the defect area by press-fit method.After the implantation,the BMSCs-gel formed by 1.2%in algal and 102mmol/l sodium chloride crosslinked were covered on the surface of defect.According to whether the graft experienced mechanical stimulation during cultured and inducted in bioreactor, the goats were divided into three groups:group A:Mechanical stimulation+ two-direction induction in double-chamber bioreactor;group B:two-direction induction without mechanical stimulation;groups C:control group.Each group has four goats,totally 24 knees,12 goats.The goats were sacrificed at 12 weeks and 24 weeks after operation.Then detected the following items:general observation; histology and histochemical detection:HE staining,Toluidine blue staining,Masson staining,collagenⅡimmunohistochemistry,Brdu immunofluorescence detection in the time point of 12 weeks.O'Driscoll,Keeley and Salter Histomorphology score. The data was showed as(?)±s,and two-way ANOVA and LSD was used for stastistcs analysis,P＜0.05 was regarded significant difference
RESULTS
1.The goat BMSCs of primary culture were cambiform or spindle-like,and it has the character of good refractive and adherent growth.With the extension of culture time,the growth of BMSCs was colony-like and typical spindle-like of cells can be observed in the colony.The cells would reach 80%of fusion after 7～8d cultivation,90%of fusion after 9～10d cultivation.Most of the cells were long spindle-like;cell growth rate was faster than the primary cells.After cultured for 2～3d,the cells began to enter the logarithmic-like growth phase,and 7～8d into the plateau phase.The result of flow cytometry showed:cell surface antigen expression of MSCs markers CD29 can reach 86.4%,and the expression of hemopoietic stem cell marker CD34 was only 0.4%.Under the induction of osteogenic induction medium and the chondrogenic induction medium,the goat BMSCs could be differentiated toward osteoblasts and chondrocytes respectively;Identified osteoblast formed from BMSCs with alkaline phosphatase stain and alizarin red staining,the result showed positive in 2 weeks.Identified chondrocyte formed from BMSCs with toluidine blue and Alican blue staining,the result showed positive in 2 weeks.RT-PCR,Western blot and immunohistochemical staining results were confirmed osteoblastic and chondrogenic cell differentiation and,after the BMSCs were induced towards osteogenesis and chondrogenesis for 2 weeks,The expression CollagenⅠ,Ⅱand Aggrecan mRNA and protein had no significance stastistic difference with that of induction 4 weeks.and it has significance stastistic difference with induction 1 week and non-induction.
2.We performed immunofluorescence staining after labeled by the BrdU.The goat BMSCs' nucleus show green fluor under fluorescence microscope.The labeling positive rate increased gradually with the increase of incubating time and concentration of BrdU.More than 90%goat BMSCs could be labeled when the cells were incubated for 48h and the concentration of BrdU maintaining 15μmol/L at the same time,and the survival rate of labeled cells were more than 98%.but the labeling rate did not increase with prolong of time and the increase of concentration of BrdU.It indicated that the optimal time for labeling goat BMSCs is 48h and the optimal concentration is 15μmol/L of BrdU.The morphous,growth,proliferation and differentiation of the labeled BMSCs were not influenced compare to the unlabelled BMSCs.
3.After 12 weeks,the result showed the operation area of group A articular surface was smooth,with the surrounding normal cartilage naturally straight flush, transparent form new cartilage tissue;Group B restoration surgery the basic integrity of the cartilage tissue,but the center of many not fully integrated,there is slight depression,surface wear,good subchondral bone formation;control group, depression joint operation areas,non-articular cartilage formation,such as lack of bone joints.BrdU immunofluorescence confirmed that the new cartilage cells from some of the goat implanted autologous BMSCs.Histological examination showed thatβ-TCP bioceramic degradation has been absorbed.After 24 weeks,the operation area of group A was more smooth,and the surrounding normal cartilage naturally straight flush,transparent form new cartilage,subchondral bone formation in good condition;Group B restoration surgery the basic integrity of the cartilage tissue, center is not yet fully integrated,there was slight depression;CollagenⅡimmunohistochemistry of cartilage that was new brown area.Group C has no formation of articular cartilage.The growth and the intergration of subchondral bone of group A and B were better.
The result of histological test showed that:the quality of goat cartilage formed in group A in vivo is superior to that of group B.O'Driscoll Keeley and Salter histomorphology score:group A of 12 weeks(n＝4) 16.00±0.82,24 weeks(n＝4) 18.75±0.96;group B(n＝4) 12 weeks 11.00±0.82,24 weeks(n＝4) 14.75±0.96; group C did not form cartilage,12 weeks and 24 weeks in each group,the difference was statistically significant(P＜0.05).
CONCLUSIONS
1、We isolated the goat BMSCs from iliac of adult Chinese goats.And we established a method about isolation,culture and identification for the goat BMSCs.The 4rd passage BMSCs cultured by the whole bone marrow culture method was of higher purity and thriving.This experiment confirmed that the goat BMSCs had a great potentiality of proliferation and osteogenic and chondrogenic differentiation in vitro.When cultured in osteogenic and chondrogenic induction medium for 2 weeks,the cells could express the characteristic of osteocyte and chondrocyte.Goat BMSCs are an ideal seed cell for tissue engineering and it has broad application prospects,which provide an experimental foundation for goat BMSCs being used in bone and cartilage tissue engineering in vivo.
2、BrdU can be used as a labeling marker for goat BMSCs.When the concentration is 15μmol/L and the incubation time is 48 hours,the optimal labeling effect can be gained.Goat BMSCs labeled with BrdU is of high efficiency.BrdU has no obvious cytotoxicity and influence on the growth, proliferation and differentiation to the cells.
3、We can construct tissue engineering osteochondral graft simultaneously at the same piece ofβ-TCP bioceramics with goat BMSCs act as seed cells and cultured and inducted in double-chamber stirring bioreactor.The experiment in vivo of goat proved that mechanical stimulation could improve the quality of goat cartilage formation.
引文
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