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.     

组织工程化软骨修复兔膝关节软骨缺损的实验研究

目的 评估同种异体组织工程软骨修复兔膝关节全层软骨缺损的有效性。方法分离收集成年新西兰大白兔软骨细胞进行体外培养。建立双侧兔膝关节软骨缺损模型，用去端肽胶原（atelocollagen）凝胶与所培养的异体兔关节软骨细胞共同植入兔膝关节软骨缺损处，并设对照组。分别于手术后4周、8周观察大体标本以及组织学修复结果，并进行Wakitan的评分，评估此方法的有效性。结果大体观察结果表明，与对照组相比，实验组缺损处由软骨组织修复而对照组缺损处由纤维样组织填充。组织学观察可以见到实验组关节软骨缺损处有密集的软骨细胞而对照组关节缺损处只有纤维细胞无软骨细胞。结论通过短期观察表明以同种异体软骨细胞-去端肽胶原复合物修复全层软骨缺损的方法是有效可行的，为其进一步临床应用提供了参考。     

应用旋转生物反应器开展软骨组织工程研究

目的 应用旋转生物反应器(RCCS)在体外条件下培育具有一定形状的组织工程化人工软骨,以便为软骨组织工程产品产业化生产奠定基础。方法 将兔关节软骨细胞接种到圆形或方形可降解聚合物材料上,然后在RCCS内进行培养,并以在培养瓶内培养复合体为对照组。在体外培养期间,对复合体培育产物氨基糖胺聚糖(GAG)及DNA含量进行定量测量。经体外培养后,将复合体植入到裸鼠背部皮下,术后不同时间取材,进行大体、组织学等检查。结果 经RCCS培养的复合体在体外培养期间以及体内植入后均有明显软骨形成,而对照组则仅见少量软骨形成。复合体体外培育产物检测结果表明,在RCCS内培育的复合体GAG和DNA含量明显高于对照组。结论 RCCS与以单层生长方式为主的传统细胞培养装置—培养瓶相比,能够提供更适宜的外部环境,从而有利于软骨细胞在支架内形成人工软骨组织。     

同种异体软骨细胞与自体骨髓基质干细胞混合共培养构建软骨皮下移植的可行性研究

背景：软骨组织工程的种子细胞问题是目前研究的热点和难点,如何找到一种既能够避免对自体软骨进行取材又能够达到稳定软骨构建目的的方法呢？本研究尝试利用少量同种异体羊软骨细胞作为软骨诱导微环境提供者,与扩增后的羊自体BMSC混合共培养并植入皮下环境,探讨利用同种异体软骨细胞共培养构建软骨皮下移植的可行性。方法：本实验对山羊软骨细胞和BMSC分别进行取材和分离培养扩增,并将以上细胞分为以下四组进行混合并接种在PGA支架材料上：A组：100%自体软骨细胞;B组：30%自体软骨细胞＋70%自体BMSCs;C组：30%同种异体软骨细胞＋70%自体BMSCs;D组：100%同种异体软骨细胞。经过体外构建6周后植入羊皮下进行体内构建12周,对所形成的组织块进行大体观察和组织学染色等评价。结果：自体软骨细胞组和自体软骨细胞混合自体BMSC组皮下移植后可见成熟软骨组织形成,但同种异体软骨细胞参与的两组（包括同种异体软骨细胞混合自体BMSC的实验组和单纯异体软骨细胞组）在皮下环境中都因为较强的免疫反应未能形成软骨组织。结论：同种异体软骨细胞以及PGA支架材料的存在对于组织工程软骨在羊皮下环境的构建有负面影响。     

利用组织工程技术再生软骨组织的实验研究

目的 在有免疫力的动物体兔体内探索组织工程化软骨生成的影响因素。 方法 经不同物质修猸的聚羟基乙酸支架与软骨细胞体外培养,观察基质产生情况,并将细胞支架复合物体内回植,观察软骨的生成,并进行组织学及超微结构评价。结果 以孵磷脂、多聚赖氨酸及聚乳酸共同修饰的聚羟基乙酸与软骨细胞体外培养,结果基质产生旺盛,体内回植后软骨生成良好。 结论 细胞支架复合物体外培养期间有基质产生,是组织工程化软骨生成的前提     

Interaction of periosteal explants with articular chondrocytes alters expression profile of matrix metalloproteinases

Periosteal tissue is a source of growth factors and of osteochondral progenitor cells which makes it suitable for implantation in chondral defects as known in autologous chondrocyte implantation. The aim of this study was to determine the interaction between periosteal tissue and articular chondrocytes with respect to catabolic effectors such as matrix metalloproteinases (MMPs) and IL‐6. Human articular chondrocytes were cultured for up to 28 days as micromass pellets in coculture either with physical contact to periosteal explants or allowing paracrine interactions only. Expression, secretion, and activation of MMPs and IL‐6 were analyzed in chondrocytes, periosteum, and culture supernatants. Both coculture conditions influence gene expression levels of MMPs and IL‐6 in a time‐, culture‐, and tissue‐dependent manner. Coculturing of periosteum with chondrocytes promotes gene expression and secretion of IL‐6. In periosteum, physical contact inhibits MMP‐2 and MMP‐13 gene expression while paracrine coculture induces expression of IL‐6, MMP‐2, ‐7, and ‐13. Pro‐MMP‐2, ‐7, and ‐13 were detected in supernatants of all culture regimens whereas pro‐MMP‐9 was secreted from periosteum only. As a balanced amount of MMP activity is likely required to achieve sufficient integration of the regenerate tissue with the surrounding healthy cartilage, an exceeding expression of proteinases might result in degradation, hypertrophy or rejection of the graft. © 2010 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 28:1576–1585, 2010     

体内外环境对组织工程软骨组织结构与力学性能的影响

目的 探讨体外构建组织工程化软骨在体、内外环境中力学性能及组织结构的变化,以及微环境对组织形成的影响,为工程化软骨构建提供适当参数。方法 体外培养扩增人胎儿关节软骨细胞,取第2代细胞以6×107个细胞/ml密度接种到聚羟基乙酸/聚乳酸(Polyglycolic acid/Polylactic acid,PGA/PLA)材料制成的圆柱形三维支架上,常规体外培养4周后,分为体内组(C、D组)和体外组（A、B组）,C、D组植于裸鼠皮下,A、B组继续常规培养液培养,于6、12周后取材,以正常软骨作为对照,行大体观察,以及组织学、组织化学、生物力学、超微结构等检测。结果 A、B、C、D4组均形成大体形态良好的透明软骨样组织。C、D组软骨呈乳白色,表面光滑,超微结构上胶原纤维排列致密而有规则,可形成有横纹的粗大胶原纤维,类似正常成人软骨;A、B组颜色偏黄,表面略粗糙,外观及超微结构近似半透明的胎儿关节软骨。工程化软骨植入体内12周后压缩弹性模量及胶原直径分别为（38.28±3.95）MPa和（41.58 ±2.78）nm,明显优于体外同时期组的(4.12±0.63) MPa和（15.83±1.70）nm(P ＜0.01)。结论 组织工程软骨的结构和功能在体内环境逐步成熟,体内组软骨超微结构上能形成粗大胶原纤维网络,胶原的交联增强可能是其力学性能较体外组明显提高的重要原因。     

Comparison of tracheal and nasal chondrocytes for tissue engineering of the trachea

BACKGROUND: This study was undertaken to evaluate the feasibility of creating engineered tracheal equivalents grown in the shape of cylindrical cartilaginous structures using sheep nasal cartilage-derived chondrocytes. We also tested sheep tracheal and nasal septum for cell yield and quality of the engineered cartilage each produced. METHODS: Nasal septum and tracheal tissue were harvested from sheep. Chondrocytes from each were separately isolated from the tissues and suspended in culture media. Tracheal and nasal chondrocytes were seeded onto separate polyglycolic acid matrices. Cell-polymer constructs were cultured for 1 week and then wrapped around a 7-mm diameter x 30-mm length silicon tube and implanted subcutaneously on the back of nude mice for 8 weeks (each, n = 6). Both of the tissue-engineered tracheas (TET) were harvested and analyzed for histological, biochemical, and biomechanical properties. These values were compared with native sheep trachea. RESULTS: The morphology and histology of both tracheal-chondrocyte TET and nasal-chondrocyte TET closely resembled that of native sheep trachea. Safranin-O staining showed that tissue-engineered cartilage was organized into lobules with round, angular lacunae, each containing a single chondrocyte. Chondrocytes from the trachea or nasal septum produced tissue with similar mechanical properties and had similar glycosaminoglycan and hydroxyproline content. CONCLUSIONS: This study demonstrates that the property of TET using nasal chondrocytes is similar to that obtained using tracheal chondrocytes. This has the potential benefit of facilitating an autologous approach for repair of segmental tracheal defects using an easily obtained chondrocyte population.     

Variations in matrix composition and GAG fine structure among scaffolds for cartilage tissue engineering

OBJECTIVE: To compare matrix composition and glycosaminoglycan (GAG) fine structure among five scaffolds commonly used for in vitro chondrocyte culture and cartilage tissue engineering. DESIGN: Bovine articular chondrocytes were seeded into agarose, alginate, collagen I, fibrin and polyglycolic acid (PGA) constructs and cultured for 20 or 40 days. In addition to construct DNA and sulfated GAG (sGAG) contents, the delta-disaccharide compositions of the chondroitin/dermatan sulfate GAGs were determined for each scaffold group via fluorophore-assisted carbohydrate electrophoresis (FACE). RESULTS: Significant differences were found in cell proliferation and extracellular matrix accumulation among the five scaffold groups. Significant cell proliferation was observed for all scaffold types but occurred later (20-40 days) in PGA constructs compared to the other groups (0-20 days). By 40 days, agarose constructs had the highest sGAG to DNA ratio, while alginate and collagen I had the lowest levels. Quantitative differences in the Delta-disaccharide composition of the GAGs accumulated in the different scaffolds were also found, with the most striking variations in unsulfated and disulfated delta-disaccharides. Agarose constructs had the highest fraction of disulfated residues and the lowest fraction of unsulfated residues, with a 6-sulfated/4-sulfated disaccharide ratio most similar to that of native articular cartilage. CONCLUSIONS: The similarities and differences among scaffolds in proteoglycan accumulation and GAG composition suggest that the scaffold material directly or indirectly influences chondrocyte proteoglycan metabolism and may have an influence on the quality of tissue engineered cartilage.     

人真皮成纤维细胞体外构建组织工程化软骨的初步探索

目的 利用软骨细胞提供的体外软骨诱导微环境,探讨人真皮成纤维细胞在体外构建软骨的可行性.方法 分别培养猪的软骨细胞与人真皮成纤维细胞,将2种细胞按1:1(软骨细胞:成纤维细胞)比例混匀,以5.0×10~7/ml的终浓度接种于聚羟基乙酸支架(PGA,直径9 mm,高2mm)作为共培养组,相同终浓度的单纯软骨细胞和单纯成纤维细胞分别接种于相同支架作为阳性对照及阴性对照.每组各接种3个标本,每个接种细胞悬液200 μl.全部标本均于体外培养8周后取材,通过大体观察、湿重测定、组织学及免疫组化等相关检测对构建软骨进行评价.结果 软骨细胞组(阳性对照组)基本保持了复合物初始的大小和形状,组织周边和中央均有较均质的软骨陷窝样结构形成,表达软骨特异性细胞外基质;共培养组的组织稍有缩小,组织周边也有软骨陷窝样结构形成,表达软骨特异性细胞外基质,但组织内大部分区域形成了纤维样组织,特别是通过人核抗原免疫组化和对应的Safranin O染色结果,可以看到少量人核抗原阳性的细胞形成了较成熟的陷窝样结构,表达软骨特异性基质.单纯成纤维细胞组(阴性对照组)在体外培养过程中逐渐皱缩变形,未形成软骨样组织.结论 软骨细胞共培养体系可以有效地诱导人真皮成纤维细胞中一定比例的细胞向软骨分化,并能在体外构建软骨样组织.     

传代对残耳软骨细胞体内软骨形成能力的影响

目的：研究传代对残耳软骨细胞体内软骨形成能力的影响。方法分离培养人残耳软骨细胞,将第3-8代细胞分别复合聚羟基乙酸/聚乳酸支架,构建组织工程化软骨;体外培养4周后植入裸鼠体内观察8周。采用组织学染色观察各组标本的软骨形成情况;Real-time PCR检测软骨分化相关基因的表达;生物力学分析新生软骨的弹性模量。结果各代复合物体外培养4周时均不能形成软骨组织,但第3-5代残耳软骨细胞COL 2A1、第3-4代的SOX 9和第3代的DLK 1仍可维持较高的表达水平（P〈0.05）;体内植入8周后,第3-6代复合物均有不同程度的弹性软骨结构形成,并随代次增高而减少,第3-6代复合物的弹性模量明显高于第7、8代。结论残耳软骨细胞传至第4代仍能保持良好的体内软骨形成能力,但扩增传代对残耳软骨细胞软骨表型去分化的影响在第7代后已无法逆转。     

组织工程骨软骨复合物的构建与形态学观察

目的探讨采用组织工程技术构建骨软骨复合物的可行性。方法将骨髓基质细胞(BMSCs)成诱导软骨后接种于快速成形的三维支架材料聚乳酸／聚羟乙酸共聚物(PLGA)构建组织工程软骨，经成骨诱导的BMSCs接种于聚乳酸／聚羟乙酸共聚物／磷酸三钙(PLGA／TCP)构建组织工程骨，在体外分别培养2周后，将两种工程化组织及两者以无损伤线缝合形成的组织工程骨软复合体分别植入自体股部肌袋，术后8周取材，行组织学观察。结果术后组织学观察表明。组织工程软骨在体内可形成软骨组织组织工程骨在体内可形成骨组织，两者的复合体在体内可形成骨软骨复合物。结论以骨髓基质细胞为种子细胞、以快速成形的生物降解材料为支架体外构建的组织工程骨软骨复合物，可在体内形成骨软骨组织，有望用于骨软骨缺损的修复。     

Differences in cartilage-forming capacity of expanded human chondrocytes from ear and nose and their gene expression profiles

The aim of this study was to evaluate the potential of culture-expanded human auricular and nasoseptal chondrocytes as cell source for regeneration of stable cartilage and to analyze the differences in gene expression profile of expanded chondrocytes from these specific locations. Auricular chondrocytes in monolayer proliferated less and more slowly (two passages took 26.7 ± 2.1 days and were reached in 4.37 ± 0.30 population doublings) than nasoseptal chondrocytes (19.3 ± 2.5 days; 5.45 ± 0.20 population doublings). However, auricular chondrocytes produced larger pellets with more cartilage-like matrix than nasoseptal chondrocytes (2.2 ± 0.71 vs. 1.7 ± 0.13 mm in diameter after 35 days of culture). Although the matrix formed by auricular and nasoseptal chondrocytes contained collagen X, it did not mineralize in an in vitro model or after in vivo subcutaneous implantation. A DNA microarray study on expanded auricular and nasoseptal chondrocytes from the same donors revealed 1,090 differentially expressed genes. No difference was observed in the expression of known markers of chondrogenic capacity (e.g., collagen II, FGFR3, BMP2, and ALK1). The most striking differences were that the auricular chondrocytes had a higher expression of anabolic growth factors BMP5 and IGF1, while matrix-degrading enzymes MMP13 and ADAMTS5 were higher expressed in nasoseptal chondrocytes. This might offer a possible explanation for the observed higher matrix production by auricular chondrocytes. Moreover, chondrocytes isolated from auricular or nasoseptal cartilage had specific gene expression profiles even after expansion. These differently expressed genes were not restricted to known characterization of donor site subtype (e.g., elastic), but were also related to developmental processes.     

Chondrozyten – ein Zelltyp, verschiedene Subpopulationen

Chondrocytes represent the most important cell source for engineering of cartilaginous tissues. Depending on the tissue type and the localization within the tissue, these cells may behave differently. Numerous studies have been done to compare articular, nasal, auricular, and costal chondrocytes in order to evaluate differences between knee and ankle joint cartilage and to investigate topographical variations within an articular joint. Moreover, the zonal structure of articular cartilage needs to be considered because it leads to phenotypical differences between chondrocytes of the superficial and the deeper zones. Several studies indicate, however, that even differentiated chondrocytes demonstrate a certain plasticity and strive to adapt their phenotypes to a new mechanical and biochemical environment. The aim of this review is to report on similarities and differences of chondrocytes from different tissues, zones, and topographical locations. In particular, an overview of recent results from comparative studies is presented, and possible consequences for the design of tissue engineering models are discussed.     

Three-dimensional seeding of chondrocytes encapsulated in collagen gel into PLLA scaffolds

Tissue engineering approaches have been clinically tried to repair damaged articular cartilages. It is an essential step to uniformly seed chondrocytes into 3D scaffolds in order to reconstruct tissue-engineered cartilages in vitro, but the tissue engineering could not have been provided with efficient cell seeding methods. Type I collagen is clinically used and known as a cytocompatible material, having recognition sites for integrins. Collagen gel encapsulating chondrocytes has been tried for making regenerated cartilages, but it is found difficult to have the gel keep its original shape after long-term culture, because of shrinking. On the other hand, 3D scaffolds, either of a nonwoven structure or a sponge-like structure, involve difficulty in that chondrocytes could not be uniformly seeded, although they have adequate initial mechanical properties. In this study, by combining collagen gelation with a nonwoven PLLA scaffold, we achieved uniform cell seeding into the 3D scaffold. Bovine articular chondrocytes were mixed with type I collagen solution, and the solution was poured into the nonwoven PLLA scaffold (1.5 mm thick, diameter 15 mm). The collagen-chondrocyte mixture was made into gel at 37 degrees C for 1 h. The 0.39% collagen mixture was viscous enough to prevent cells from precipitating during gelation. Almost all chondrocytes were able to be incorporated into the PLLA scaffolds by mixing with collagen solution and subsequently making into gel, while 30-40% of the chondrocytes seeded as a cell suspension were not trapped into the PLLA scaffolds. The method presented, where chondrocytes were mixed with collagen solution, and the mixture was incorporated into a 3D scaffold, then made into gel in the scaffold, could serve as an alternative for in vitro cartilage regeneration, also simultaneously having the advantages of both materials.     

软骨细胞和骨髓间质干细胞混合培养构建组织工程软骨的实验研究

目的 探讨软骨细胞和骨髓间质干细胞(bone mesenchymal stem cells,BMSCs)混合培养对构建组织工程软骨的影响,并确定两者的最佳比例.方法 体外分离培养兔(1月龄)关节软骨细胞和BMSCs,按不同比例(软骨细胞和BMSCs的浓度比为:4:1、2:1、1:1、1:2、1:4及单纯软骨细胞)混合培养一代.以4×107/ml的细胞终浓度接种40μl于聚乳酸-羟基乙酸共聚物[poly(lactic-co-glycolic acid),PLGA:4mm×4mm×2mm),静态培养2 d,然后移至周期性压力场(压力0～200 kPa,频率:0.1 Hz,时间:8 h/d)培养3周.收获组织工程软骨行大体观察,组织切片,定量检测糖胺聚糖(glyeosaminoglycans,GAGs)含量、DNA含量及Ⅱ型胶原染色面积.结果 软骨细胞和BMSCs混合培养组与单纯软骨细胞组相比,体积较大,表面光滑,有弹性,有光泽.组织学检查显示混合培养组结构致密,细胞外基质分布更均匀,其中软骨细胞和BMSCs的浓度比为2:1组可见软骨陷窝.混合培养组的Ⅱ型胶原染色面积、GAGs含量、DNA含量高于单纯软骨细胞组,其中软骨细胞和BMSCs的浓度比为2:1组含量最高,与其他比例混合组比较,差异有统计学意义.结论 软骨细胞和BMSCs混合培养能提高组织工程软骨的质量,其中以软骨细胞和BMSCs的浓度比为2:1最佳.     

Integration of engineered cartilage.

The structure and function of cartilaginous constructs, engineered in vitro using bovine articular chondrocytes, biodegradable scaffolds and bioreactors, can be modulated by the conditions and duration of tissue cultivation. We hypothesized that the integrative properties of engineered cartilage depend on developmental stage of the construct and the extracellular matrix content of adjacent cartilage, and that some aspects of integration can be studied under controlled in vitro conditions. Disc-shaped constructs (cultured for 5+/-1 days or 5+/-1 weeks) or explants (untreated or trypsin treated cartilage) were sutured into ring-shaped explants (untreated or trypsin treated cartilage) to form composites that were cultured for an additional 1-8 weeks in bioreactors and evaluated biochemically, histologically and mechanically (compressive stiffness of the central disk, adhesive strength of the integration interface). Immature constructs had poorer mechanical properties but integrated better than either more mature constructs or cartilage explants. Integration of immature constructs involved cell proliferation and the progressive formation of cartilaginous tissue, in contrast to the integration of more mature constructs or native cartilage which involved only the secretion of extracellular matrix components. Integration patterns correlated with the adhesive strength of the disc-ring interface, which was markedly higher for immature constructs than for either more mature constructs or cartilage explants. Trypsin treatment of the adjacent cartilage further enhanced the integration of immature constructs.     

Degree of differentiation of chondrocytes and their pretreatment with platelet-derived-growth factor. Regulating induction of cartilage formation in resorbable tissue carriers in vivo

Current methods for articular cartilage repair are unpredictable with respect to clinical success. In the present study, we investigated the ability of cells from articular cartilage, perichondrium, and costochondral resting zone to form new cartilage when loaded onto biodegradable scaffolds and implanted into calf muscle pouches of nu/nu mice. Prior in vitro studies showed that platelet derived growth factor-BB (PDGF-BB), but not transforming growth factor beta-1 (TGF-beta 1), basic fibroblast growth factor, or bone morphogenetic protein-2 promoted proliferation and extracellular matrix sulfation of resting zone chondrocytes without causing the cells to exhibit a hypertrophic chondrocyte phenotype. TGF-beta 1 has also been shown to stimulate chondrogenesis by multipotent chondroprogenitor cells like those in the perichondrium. In addition, PDGF-BB has been shown to modulate chondrogensis by resting zone cells implanted in poly(D,L-lactide-co-glycolide) (PLG) scaffolds. In the present study we examined whether the cartilage formation is dependent on state of chondrocyte maturation and whether the pretreatment of chondrocytes with growth factors has an influence on the cartilage formation. Scaffolds were manufactured from 80% PLG with a 75:25 lactide:glycolide ratio and 20% modified PLG with a 50:50 lactide:glycolide ratio (PLG-H scaffolds). For each experimental group, four nude mice received two identical implants, one in each calf muscle resulting in an N = 8 implants: PLG-H scaffolds alone; PLG-H scaffolds with cells derived from either the femoral articular cartilage, costochondral periochondrium, or costochondral resting zone cartilage of 125 g male Sprague-Dawley rats; PLG-H scaffolds with either articular chondrocytes or resting zone chondrocytes that were pretreated with 37.5 ng/ml rhPDGF-BB for 4 h or 24 h before implantation, or with perichondrial cells treated with PDGF-BB plus 0.22 ng/ml rhTGF beta-1 for 4 h and 24 h. At 4 or 8 weeks after implantation, samples were harvested and analyzed histomorphometrically for new cartilage formed, area of residual implant and area of fibrous connective tissue. Only resting zone cells showed the ability to form new cartilage at a heterotopic site in this study. There was no neocartilage found in nude mice with implants loaded with either articular chondrocytes or perichondrial cells. Pretreatment of resting zone chondrocytes for 4 h prior to implantation significantly increased the amount of newly formed cartilage after 8 weeks and suppressed chondrocyte hypertrophy. The amount of fibrous connective tissue around implants containing either articular chondrocytes or perichondrial cells decreased with time, whereas the amount of fibrous connective tissue around implants containing resting zone chondrocytes pretreated with PDGF-BB was increased. The results showed that resting zone cells can be successfully incorporated into biodegradable porous PLG scaffolds and can induce new cartilage formation in a nonweight-bearing site. Articular chondrocytes as well as perichondrial cells did not have the capacity for neochondrogenesis when implanted heterotopically in this model.     

残耳软骨细胞种植于聚羟基丁酸酯-聚羟基己酸酯共聚物形成组织工程软骨

目的探讨以聚羟基丁酸酯-聚羟基己酸酯(PHB-PHH)共聚物为细胞外支架、以残耳软骨作为种子细胞形成组织工程软骨的可能性。方法取先天性小耳畸形8例患者的残耳软骨，以胶原酶消化后种植于PHBPHH支架，体外培养1周后种植于8只裸鼠一侧背部皮下为实验组，另一侧只植入支架材料作为对照组。于4周、8周后取出标本，做大体观察及HE染色、Masson三色染色检查。结果4周时实验组镜下显示有新生软骨形成，但仍有部分支架材料残留；8周时实验组标本大体观察及HE染色、Masson三色染色检查新生软骨与人耳软骨相似，支架材料已完全吸收。对照组无软骨形成。结论以残耳软骨作为种子细胞，以PHB-PHH共聚物为细胞外支架可以形成组织工程软骨，新生软骨大体观察、组织学检查与人耳软骨相似。     

Precultivation of engineered human nasal cartilage enhances the mechanical properties relevant for use in facial reconstructive surgery

OBJECTIVE: To investigate if precultivation of human engineered nasal cartilage grafts of clinically relevant size would increase the suture retention strength at implantation and the tensile and bending stiffness 2 weeks after implantation. SUMMARY BACKGROUND INFORMATION: To be used for reconstruction of nasal cartilage defects, engineered grafts need to be reliably sutured at implantation and resist to bending/tension forces about 2 weeks after surgery, when fixation is typically removed. METHODS: Nasal septum chondrocytes from 4 donors were expanded for 2 passages and statically loaded on 15 x 5 x 2-mm size nonwoven meshes of esterified hyaluronan (Hyaff-11). Constructs were implanted for 2 weeks in nude mice between muscle fascia and subcutaneous tissue either directly after cell seeding or after 2 or 4 weeks of preculture in chondrogenic medium. Engineered tissues and native nasal cartilage were assessed histologically, biochemically, and biomechanically. RESULTS: Engineered constructs reproducibly developed with culture time into cartilaginous tissues with increasing content of glycosaminoglycans and collagen type II. Suture retention strength was significantly higher (3.6 +/- 2.2-fold) in 2-week precultured constructs than in freshly seeded meshes. Following in vivo implantation, tissues further developed and maintained the original scaffold size and shape. The bending stiffness was significantly higher (1.8 +/- 0.8-fold) if constructs were precultured for 2 weeks than if they were directly implanted, whereas tensile stiffness was close to native cartilage in all groups. CONCLUSION: In our experimental setup, preculture for 2 weeks was necessary to engineer nasal cartilage grafts with enhanced mechanical properties relevant for clinical use in facial reconstructive surgery.