松质骨骨基质明胶负载软骨细胞体外培养的实验研究

目的　探索松质骨骨基质明胶 (BMG)作为软骨细胞培养支架的可行性。方法　分离幼兔关节软骨细胞 ,体外单层培养扩增 ,并负载于松质骨BMG上体外培养 ,于不同时间取材进行解剖显微镜、组织学、透射电镜观察及免疫组化检测。结果　培养 6d软骨细胞在松质骨BMG上分裂增殖。 1 2d形成 1 0～ 1 2层细胞的软骨组织 ,Safranin -O染色阳性。 1 8d软骨组织厚度增加 ,胶原染色阳性。培养 2 4～ 42d网眼中细胞数量增多 ,周围形成 2 5～ 30层细胞的软骨组织。培养 42d ,解剖显微镜观察显示形成直径 4mm的“圆盘状软骨”。电镜观察显示软骨细胞外有胶原纤维。免疫组化检测显示形成的软骨组织基质中有Ⅱ型胶原。结论　松质骨BMG可作为软骨细胞培养的较好支架     

松质骨基质复合生物蛋白胶构建组织工程软骨的研究

目的尝试采用松质骨基质与生物蛋白胶复合材料构建组织工程软骨。方法体外培养大鼠软骨细胞,接种于松质骨基质／生物蛋白胶材料上行体外培养。采用HE、甲苯胺蓝染色免疫学检测、扫描电镜观察等方法观察所构建的组织工程软骨的特性。结果松质骨基质／生物蛋白胶组的组织学结构更接近于软骨样组织．其Ⅱ型胶原、蛋白多糖基因表达量及蛋白多糖含量明显高于松质骨基质组。结论松质骨基质／生物蛋白胶复合材料可用于构建组织工程软骨,是一种较理想的支架材料。     

松质骨骨基质明胶负载软骨细胞体外培养的实验研究

目的：探索松质骨骨基质明胶（BMG）作为软骨细胞培养支架的可行性。方法：分离幼兔关节软骨细胞，体外单层培养扩增，并负载于松质骨BMG上体外培养，于不同时间取材进行解剖显微镜，组织学，秀射电镜观察及免疫组化检测。结果：培养6d软骨细胞在松质骨BMG上分裂增殖。12d形成10－12层细胞的软骨组织，Safranin-O染色阳性，18d软骨组织厚度增加，胶原染色阳性，培养24－42d网眼中细胞数量增多，周围形成25－20层细胞的软骨组织，培养42d，解剖显微镜观察显示形成直径4mm的“圆盘状软骨”，电镜观察显示软骨细胞外有胶原纤维，免疫组化检测显示形成的软骨组织基质中有II型胶原，结论：松质骨BMG可作为软骨细胞培养的较好支架。     

模拟微重力环境下层状修复体与软骨细胞复合培养的实验研究

[目的]探讨模拟微重力作为软骨组织工程培养方法的作用和胶原/壳聚糖/β-磷酸三钙(trical ciumphosphate,TCP)层状梯度修复体作为关节软骨组织工程支架的可行性.[方法]体外培养新西兰大白兔关节软骨细胞并扩增,吸附于多孔胶原/壳聚糖/β-磷酸三钙层状梯度修复体上,模拟微重力和普通环境下三维立体分别培养3周,通过生长曲线、倒置相差显微镜、组织学、扫描电镜及免疫组织化学检测微重力对软骨细胞培养的影响和支架在三维立体培养对软骨细胞的表型、增殖及功能的影响.[结果]软骨细胞/修复体体外培养3周,软骨细胞模拟微重力培养组明显比普通培养组在层状修复体上分布均匀,修复体中心软骨细胞数量明显较多,并分泌细胞基质,包裹在软骨细胞周围,Ⅱ型胶原免疫组织化学染色阳性.[结论]模拟微重力环境有利于软骨细胞在三维支架上的均匀增殖,有望成为软骨组织工程中的一种重要培养方法;胶原/壳聚糖/β-磷酸三钙层状梯度修复体,细胞相容性良好,有望成为一种比较理想的关节软骨组织工程支架材料.     

组织工程软骨体外构建的相关实验研究

目的 探索组织工程软骨体外构建技术体系可行性.方法 种子细胞选用胎儿软骨细胞(口服药物流产胎儿,胎龄3～6个月).酶消化法获得第1代细胞,以50×106/ml浓度均匀接种于经聚乳酸(PLA)包埋聚乙醇酸(PGA)高分子聚合物支架,形成细胞-支架复合体,在体外静态培养.分别于2周、4周、8周进行大体观察、扫描电镜及组织学检测.结果 体外构建的组织工程软骨,随培养时间延长,色泽由2周时的乳白色逐渐呈现半透明,8周时接近正常软骨外观.扫描电镜显示软骨细胞与材料具有良好相容性,培养7天PGA纤维之间有基质沉积.HE染色示2周有大量软骨陷窝形成和均匀嗜碱性基质分泌,Safranin'O染色示基质有酸性蛋白多糖分布,Massons's trichome染色示基质有胶原成分,但含量较少,经免疫组织化学检测为特异Ⅱ型胶原.培养4周胶原成分开始明显增多,软骨陷窝形态接近成熟,8周细胞外基质蛋白多糖和Ⅱ型胶原含量丰富且分布均匀.结论 以成熟软骨细胞为种子细胞,运用组织工程技术在体外能构建出具有正常软骨组织结构特征的人组织工程软骨.     

骺板软骨细胞复合三维支架体外构建组织工程软骨的研究

目的探讨将骺板软骨细胞复合三维支架经体外培养,构建组织工程软骨的效果及其生物学特点. 方法将3周龄幼兔第1代骺板软骨细胞与液态的生物凝胶混合,接种于聚磷酸钙纤维/L-聚乳酸(CPPF/PLLA)三维支架材料,构建组织工程软骨组织块,连续培养4周.行大体、倒置显微镜及组织学、Ⅰ型和Ⅱ型胶原免疫组织化学光镜观察,定量检测硫酸糖胺多糖(GAG)含量. 结果构建的组织工程软骨块在培养过程中能保持其初始外形,种子细胞呈稳定的三维均相分布,外观逐渐呈乳白色、半透明,硬度亦不断增加.培养1周有软骨细胞陷窝形成,2周后形成富含Ⅱ型胶原和蛋白聚糖、具有典型软骨组织结构的工程化软骨,且Ⅰ型胶原逐渐转为阴性.4周时构建软骨的组织结构与天然骺板软骨相类似,硫酸GAG含量平均为天然骺板软骨的34%以上. 结论骺板软骨细胞复合三维支架体外培养可生成典型软骨,且可形成类似天然骺板软骨的组织结构,能满足修复骺板缺损的基本要求.体外培养1～2周可能是植入体内修复骺板缺损的较佳时机.     

组织工程软骨的体外实验研究

目的 :研究软骨细胞在纤维胶内体外生成组织工程化软骨的可行性。方法 :将 4 - 10× 10 7cells/mL细胞 /毫升的软骨细胞植入纤维胶 ,在体外培养 2个月后进行组织学、免疫组织化学分析。结果 :细胞在纤维胶内产生大量的软骨特异性Ⅱ型胶原和蛋白多糖。结论 :纤维胶内的软骨细胞可在体外培养的环境下生成大量透明软骨基质。     

脱细胞软骨细胞外基质取向支架复合软骨细胞构建组织工程软骨的实验研究

目的探讨脱细胞软骨细胞外基质(acellular cartilage extracellular matrix,ACECM)取向支架复合软骨细胞构建组织工程软骨的可行性。方法取市售猪关节软骨组织,分离培养关节软骨细胞并传代。取第3代软骨细胞行PKH26荧光标记,MTT检测标记对细胞增殖无影响后,分别取标记及未标记的软骨细胞复合ACECM取向支架并体外培养后,大体观察支架形态,倒置显微镜、荧光显微镜观察软骨细胞在支架中的黏附、生长和分布情况,扫描电镜观察支架中细胞形态,Ⅱ型胶原免疫荧光染色观察软骨细胞外基质分泌情况。将PKH26标记的软骨细胞-支架复合物植入裸鼠背部皮下腔隙,术后观察裸鼠一般情况,4周后分子荧光活体成像系统无创伤性评估细胞-支架复合物生长情况,取材行大体观察以及番红O、甲苯胺蓝、Ⅱ型胶原免疫组织化学染色观察,评价形成软骨组织的能力。结果细胞-支架复合物体外培养7 d,大体观察呈半透明并具有一定硬度;倒置显微镜和荧光显微镜观察软骨细胞在支架上能良好黏附生长,并沿支架管道方向生长,分泌Ⅱ型胶原。细胞-支架复合物植入裸鼠皮下后,分子荧光活体成像系统观察示细胞均存活;术后4周,大体观察见复合物呈类软骨样组织,组织学染色及Ⅱ型胶原免疫组织化学染色示细胞周围软骨细胞外基质分泌,可见"陷窝"样结构形成。结论 ACECM取向支架有利于软骨细胞的黏附、增殖及取向性分布类似于正常软骨结构,并在裸鼠皮下成功异位构建组织工程软骨。     

脱细胞软骨细胞外基质取向支架复合软骨细胞构建组织工程软骨的实验研究北大核心CSCD

目的探讨脱细胞软骨细胞外基质(acellular cartilage extracellular matrix,ACECM)取向支架复合软骨细胞构建组织工程软骨的可行性。方法取市售猪关节软骨组织,分离培养关节软骨细胞并传代。取第3代软骨细胞行PKH26荧光标记,MTT检测标记对细胞增殖无影响后,分别取标记及未标记的软骨细胞复合ACECM取向支架并体外培养后,大体观察支架形态,倒置显微镜、荧光显微镜观察软骨细胞在支架中的黏附、生长和分布情况,扫描电镜观察支架中细胞形态,Ⅱ型胶原免疫荧光染色观察软骨细胞外基质分泌情况。将PKH26标记的软骨细胞-支架复合物植入裸鼠背部皮下腔隙,术后观察裸鼠一般情况,4周后分子荧光活体成像系统无创伤性评估细胞-支架复合物生长情况,取材行大体观察以及番红O、甲苯胺蓝、Ⅱ型胶原免疫组织化学染色观察,评价形成软骨组织的能力。结果细胞-支架复合物体外培养7 d,大体观察呈半透明并具有一定硬度;倒置显微镜和荧光显微镜观察软骨细胞在支架上能良好黏附生长,并沿支架管道方向生长,分泌Ⅱ型胶原。细胞-支架复合物植入裸鼠皮下后,分子荧光活体成像系统观察示细胞均存活;术后4周,大体观察见复合物呈类软骨样组织,组织学染色及Ⅱ型胶原免疫组织化学染色示细胞周围软骨细胞外基质分泌,可见"陷窝"样结构形成。结论 ACECM取向支架有利于软骨细胞的黏附、增殖及取向性分布类似于正常软骨结构,并在裸鼠皮下成功异位构建组织工程软骨。     

胶原凝胶包埋软骨细胞复合聚磷酸钙纤维/左旋聚乳酸支架异体移植修复兔关节软骨缺损

目的探讨胶原凝胶包埋软骨细胞复合聚磷酸钙纤维／左旋聚乳酸(CPPf／PLLA)支架异体移植修复兔关节软骨缺损的有效性和可行性。方法将胶原凝胶包埋的软骨细胞接种CPPf/PLLA支架构建的复合物体外培养3周，行倒置显微镜和扫描电镜观察，并将复合物异体移植入兔关节软骨缺损，术后4、8、12周取材，从大体、组织学和Ⅱ型胶原免疫组织化学对再生软骨组织进行评价。结果复合物体外培养3周，细胞被大量基质包裹，在支架内分布均匀；新形成的组织为透明软骨样组织、表面光滑且与周围组织整合良好、基质内有Ⅱ型胶原分布。结论胶原凝胶包埋软骨细胞接种CPPf／PLLA支架的方法能提高细胞一支架复合物构建质量，胶原凝胶复合CPPf／PLLA支架可作为软骨细胞载体修复关节软骨缺损。     

Tissue-engineered cartilage using fibrin/hyaluronan composite gel and its in vivo implantation

The importance of scaffold biomaterials has been emphasized for in vitro culture of tissue-engineered cartilage in a three-dimensional (3D) environment. In this study, we examined the feasibility of fibrin glue, mixed with hyaluronic acid (HA) as a composite scaffold. Fibrin glue has been a useful cell delivery matrix for cartilage tissue engineering and HA is a key component of normal articular cartilage. Our hypothesis is that compared to fibrin itself, a fibrin/HA composite can have significantly enhanced properties, due mainly to the added benefits of HA in the matrix. Pieces of cartilage were isolated from rabbit knees and the chondrocytes were harvested through enzymatic digestion. Both fibrin and fibrin/HA composite were prepared and subsequently implanted in nude mice (n = 9, each group) for 1, 2, and 4 weeks, respectively. The retrieved specimens were then analyzed and the results were compared. Cartilage-like tissue formation was detected earlier with fibrin/HA specimens. They produced significantly higher amounts of the extracellular matrix (ECM) molecules, GAG, and collagen at each time point than those in fibrin. Interestingly, the fibrin/HA composite was also competent in maintaining its initial size. Histology--Safranin O/fast green and Alcian blue--of the retrieved specimens found more intense, uniform staining in the fibrin/HA composites. Analysis of the gene expression of the ECM molecules also confirmed the benefits of the composite with added HA in the maintenance of phenotypic stability. The present study suggests that fibrin/HA composite may serve as a dependable cell delivery vehicle as well as a structural basis for tissue-engineered cartilage.     

纤维蛋白凝胶和脱钙骨基质支架材料复合软骨细胞修复兔膝关节软骨缺损的实验研究

目的：探讨纤维蛋白凝胶和脱钙骨基质支架材料复合软骨细胞作为软骨组织工程支架的可行性及有效性,并为后续研究可注射性材料做基础。方法：体外分离培养软骨细胞后接种到纤维蛋白凝胶和脱钙骨基质支架材料体外培养4周,然后植入兔膝关节软骨缺损区继续培养4、8、12周后取材,分别行大体、组织学、Ⅱ型胶原免疫组织化学观察。并进行Wakitani评分,观察其体内修复关节缺损效果。结果：大体观察4周后,实验组软骨缺损区可有乳白色组织修复,12周可修复完全,并无明显凹凸感。光镜下8周可见大量软骨细胞修复,并在TB染色下见Ⅱ型胶原比4周时明显增多。12周时软骨陷窝结构形成,细胞形态排列及Ⅱ型胶原与正常软骨组织相近。结论：纤维蛋白凝胶和脱钙骨基质支架材料复合软骨细胞可以作为软骨组织工程支架材料,能够用于再生修复软骨的缺损。并为构建可注射性修复材料提供途径。     

Injectable tissue-engineered cartilage using a fibrin sealant

OBJECTIVE: To investigate a commercially available fibrin sealant as a vehicle for developing injectable tissue-engineered cartilage. METHODS: Fibrin glue was mixed with autogenous chondrocytes from rabbits (n = 15). This isolate was injected along their nasal dorsa using 1 of 3 different fibrin glue concentrations. The samples were harvested at 8 weeks and compared with elastin and hyaline cartilage controls. RESULTS: Neocartilage was created along a linear injection tract on the dorsa of the nasal bones in 5 of 15 rabbits. Higher thrombin concentrations proved to be directly correlated with successful creation of injectable cartilage. Histologically, the staining patterns of both hematoxylin-eosin and safranin O stains were identical to that of normal auricular control cartilage. The presence of elastin fibers was observed following Verhoeff staining. No foreign body reaction was observed from the host. CONCLUSIONS: This study demonstrated a successful method for percutaneous injection of tissue-engineered cartilage as a mixture of chondrocytes suspended in fibrin glue. The thrombin concentration, along with the concentration of fibrinogen and chondrocytes, must be optimized to succeed consistently in cartilage growth.     

Cartilage Tissue Engineering With Demineralized Bone Matrix Gelatin and Fibrin Glue Hybrid Scaffold: An In Vitro Study

To develop a cartilage‐like tissue with hybrid scaffolds of demineralized bone matrix gelatin (BMG) and fibrin, rabbit chondrocytes were cultured on hybrid fibrin/BMG scaffolds in vitro. BMG scaffolds were carefully soaked in a chondrocyte–fibrin suspension, which was polymerized by submerging the constructs into thrombin–calcium chloride solution. Engineered cartilage‐like tissue grown on the scaffolds was characterized by histology, immunolocalization, scanning electron microscopy, biochemical assays, and analysis of gene expression at different time points of the in vitro culture. The presence of proteoglycan in the fibrin/BMG hybrid constructs was confirmed by positive toluidine blue and alcian blue staining. Collagen type II exhibited intense immunopositivity at the pericellular matrices. Chondrogenic properties were further demonstrated by the expression of gene‐encoded cartilage‐specific markers, collagen type II, and aggrecan core protein. The glycosaminoglycan production and hydroxyproline content of tissue grown on the fibrin/BMG hybrid scaffolds were higher than that of the BMG group. In conclusion, the fibrin/BMG hybrid scaffolds may serve as a potential cell delivery vehicle and a structural basis for cartilage tissue engineering.     

膨体聚四氟乙烯（ePTFE）内支撑组织工程软骨的构建实验研究

目的：探讨骨髓间充质干细胞和软骨细胞混合培养体外构建ePTFE软骨复合体的可能性。方法：实验组：分离、获取、扩增兔骨髓间充质干细胞和软骨细胞,二者按7：3比例混和,接种到以膨体聚四氟乙烯（ePTFE）为内支撑外裹聚羟基乙酸（PGA）支架上,体外培养8周后,行大体、组织学、Ⅱ型胶原免疫组织化学和生物化学检测。对照组：利用实验组支架单纯接种骨髓间充质干细胞行体外培养。结果：实验组：体外培养8周后形成形态良好的软骨样组织复合体,组织学可见成熟软骨陷窝、异染基质、Ⅱ型胶原表达阳性。对照组：无软骨形成。结论：兔骨髓间充质干细胞和软骨细胞混合培养,可以在体外构建出特定形状、结构组织学良好的ePTFE软骨复合体。     

可注射型生物蛋白胶包埋骨髓基质细胞工程化组织的体外实验

目的构建可注射型生物蛋白胶包埋骨髓基质细胞的工程化组织,体外培养并研究其生物学特性,探讨将可注射型生物蛋白胶作为组织工程支架用于临床的实验基础。方法体外培养浇铸有骨髓基质细胞的生物蛋白胶,通过倒置相差显微镜、激光共聚焦显微镜观察载体内细胞生长及载体降解情况,5-溴脱氧尿苷(5-Bromodeocyuridine,BrdU)掺入标记后免疫组化等方法研究可注射型载体内包埋细胞的增殖情况。结果骨髓基质细胞包埋于生物蛋白胶内能很好地存活并增殖,2d后细胞呈典型的成纤维细胞形态;6d后生物蛋白胶边缘部分开始降解,细胞脱落至培养板;体外培养14d,细胞生长良好,大部分生物胶降解,脱落的细胞增多,贴壁生长的细胞形态正常;3周后生物蛋白胶完全降解。结论生物蛋白胶聚合后包埋的种子细胞能够正常增殖,生物蛋白胶是一种理想的适用于微创方法修复组织的可注射型组织工程培养和移植的支架。     

Potential of Fortified Fibrin/Hyaluronic Acid Composite Gel as a Cell Delivery Vehicle for Chondrocytes

Numerous treatment methods have been applied for use in cartilage repair, including abrasion, drilling, and microfracture. Although chondrocyte transplantation is the preferred treatment, it has some shortcomings, such as difficulty of application (large and posterior condylar regions), poor chondrocyte distribution, and potential cell leakage from the defect region. The cell delivery system of the tissue engineering technique can be used to overcome these shortcomings. We chose fibrin/hyaluronan (HA) composite gel as an effective cell delivery system to resolve these issues. Both components are derived from natural extracellular matrix. In the first trial, fortified fibrin/HA composite gels with rabbit chondrocytes were tested by implantation in nude mice. At 4 weeks, glycosaminoglycan contents in the fibrin/HA composite (0.186 ±  0.006 mg/mg) were significantly higher than those in the presence of fibrin alone (0.153 ± 0.017 mg/mg). As a next step, we applied the fibrin/HA composite gel to animal cartilage defects using full thickness cartilage defect rabbit models. The fibrin/HA composite gel with rabbit chondrocytes (allogenic) was implanted into the experimental group, and the control group was implanted with the fibrin/HA composite gel alone. Implanted chondrocytes with the fibrin/HA composite showed improved cartilage formation. In conclusion, the key to successful regeneration of cartilage is to provide the repair site with a sufficient supply of chondrogenic cells with a suitable delivery vehicle to ensure maximal differentiation and deposition of the proper extracellular matrix. This study suggests the feasibility of tissue-engineered cartilage formation using fibrin/HA composite gel.     

软骨微粒脱细胞基质和纤维蛋白凝胶支架皮下构建组织工程软骨

目的观察皮下植入异体软骨细胞复合异种软骨微粒脱细胞基质(Cartilage microparticle acellular matrix,CMACM)和纤维蛋白胶(Fibrin glue,FG)为支架形成组织工程软骨的可能性。方法制备猪耳廓CMACM,体外培养成年兔的耳软骨细胞,将不同的混合物植人5只成年兔背部皮下。A组:异体软骨细胞复合CMACM和FG;B组:自体软骨细胞复合CMACM和FG;C组:CMACM和FG。将每只兔子背部皮肤均分为6个区,分别植入不同混合物各两个点,以备两次取材。观察并记录皮下植入体的形态变化,分别于植入后8周和12周取材,行组织学检测。结果A组和C组未能形成软骨样组织。B组8周可以形成软骨样组织,周围炎症细胞数量较多;12周时形成的软骨组织成分单一,周围没有炎症反应,类似于正常软骨,且新生的软骨组织中均长有许多小血管,新生软骨的厚度不超过1 mm。结论将同种异体软骨细胞复合CMACM和FG植入皮下,不能形成软骨样组织:而以自体软骨细胞为种子细胞则可以得到软骨样组织,但新生软骨的体积和厚度有限,并且新生的软骨组织中长有许多小血管,可能更有利于新生软骨组织的长期存活。     

Repair of porcine articular cartilage defect with autologous chondrocyte transplantation.

Articular cartilage is known to have poor healing capacity after injury. Autologous chondral grafting remains the mainstay to treat well-defined, full-thickness, symptomatic cartilage defects. We demonstrated the utilization of gelatin microbeads to deliver autologous chondrocytes for in vivo cartilage generation. Chondrocytes were harvested from the left forelimbs of 12 Lee-Sung pigs. The cells were expanded in monolayer culture and then seeded onto gelatin microbeads or left in monolayer. Shortly before implantation, the cell-laden beads were mixed with collagen type I gel, while the cells in monolayer culture were collected and re-suspended in culture medium. Full-thickness cartilage defects were surgically created in the weight-bearing surface of the femoral condyles of both knees, covered by periosteal patches taken from proximal tibia, and sealed with a porcine fibrin glue. In total, 48 condyles were equally allotted to experimental, control, and null groups that were filled beneath the patch with chondrocyte-laden beads in gel, chondrocytes in plain medium solution, or nothing, respectively. The repair was examined 6 months post-surgery on the basis of macroscopic appearance, histological scores based on the International Cartilage Repair Society Scale, and the proportion of characteristic chondrocytes. Tensile stress-relaxation behavior was determined from uniaxial indentation tests. The experimental group scored higher than the control group in the categories of matrix nature, cell distribution pattern, and absence of mineralization, with similar surface smoothness. Both the experimental and control groups were superior to the null group in the above-mentioned categories. Viable cell populations were equal in all groups, but the proportion of characteristic chondrocytes was highest in the experimental group. Matrix stiffness was ranked as null > native cartilage > control > experimental group. Transplanted autologous chondrocytes survive and could yield hyaline-like cartilage. The application of beads and gel for transplantation helped to retain the transferred cells in situ and maintain a better chondrocyte phenotype.