Static and dynamic compression modulate matrix metabolism in tissue engineered cartilage.

Static and dynamic compression are known to modulate the metabolism of articular cartilage. The present study focused on determining the effects of compressive loading on the metabolism of sulfated glycosaminoglycans (S-GAG) and protein in tissue engineered cartilage constructs. Cartilage constructs were subjected to static or dynamic compression for 24 h and radiolabeled with 35SO4 and 3H-proline to assess the total synthesis and percentage retention of S-GAG and total protein, respectively. Static compression at an amplitude of 50% suppressed the synthesis of both S-GAG and protein by 35% and 57%, respectively. Dynamic compression at an amplitude of 5% had stimulatory effects on synthesis that were dependent on the static offset compression amplitude (10% or 50%) and dynamic compression frequency (0.001 or 0.1 Hz). Thus, tissue engineered cartilage demonstrated the ability to respond to mechanical loading in a manner similar to that observed with articular cartilage. Mechanical loading may therefore potentially be used to modulate the growth of cartilaginous tissues in vitrd, potentially facilitating the culture of functional cartilage tissues suitable for implantation.     

软骨组织工程支架材料研究进展

各种原因引起的关节软骨损伤在临床工作中非常常见,但临床治疗手段有限,组织工程的发展为关节软骨损伤的修复提供了新的途径。在软骨组织工程中支架材料起着重要作用,选择合适的载体是一个首先要解决的问题。本文对目前软骨组织工程支架材料的现状做一综述,指出了当前软骨组织工程所面临的问题,并针对此问题对未来软骨组织工程材料的研究作出了展望。     

采用自体成熟关节软骨细胞的软骨组织工程修复

目的探讨使成熟软骨细胞转化成代谢活跃、增殖迅速的再生软骨祖细胞，而后利用这种细胞构建自体源性工程组织，修复成熟关节软骨的缺损。方法成熟兔关节软骨细胞进行普通单层培养和转化生长因子(TGF)-β1、碱性成纤维细胞生长因子(bFGF)联合诱导培养。培养细胞进行细胞计数、Ⅱ型胶原免疫组织化学染色和逆转录一聚合酶链反应(RT-PCR)检测。将诱导培养的再生软骨祖细胞与聚乳酸载体(PDLLA)一道构建自体源性工程软骨，修复成熟关节软骨缺损。修复组织进行组织形态学和免疫组织化学研究。结果研究发现成熟关节软骨细胞在体外单层培养中增殖缓慢。而联合TGF-β1、bFGF诱导培养可促进成熟软骨细胞的增殖，10d内细胞增殖189倍。标准单层培养4～5代细胞经密集培养不表达Ⅱ型胶原。而联合细胞因子诱导培养6代的细胞在体外密集培养下，可很快恢复Ⅱ型胶原的表达。利用再生软骨祖细胞构建的自体源性工程软骨可修复软骨缺损。修复组织表达Ⅱ型胶原。结论成熟关节软骨细胞经TGF-β和bFGF联合诱导培养可形成再生软骨祖细胞，此细胞可用于构建自体源性工程组织，修复成熟关节软骨的缺损。     

细胞移植及软骨组织工程技术在关节软骨损伤临床应用进展

细胞移植和软骨组织工程技术是治疗关节软骨损伤的新方法,也是当今国内外关节骨科的研究热点。本文综述了国际上各种细胞移植和组织工程技术在关节软骨损伤临床的应用,包括软骨细胞移植技术、联合组织工程支架的软骨细胞移植技术和干细胞移植技术,着重介绍了各种技术的特点和临床应用现状及进展,以期为国内软骨损伤治疗技术选择提供思路。     

Current concepts in tissue engineering technique for repair of cartilage defect

Hunter's observation in 1743 that cartilage "once destroyed, is not repaired," has not essentially changed for 250 years. At present, there is no well-established procedure for the repair of cartilage defect with articular cartilage, which has the same biochemical and biomechanical properties as the surrounding normal intact cartilage. In 1994, transplantation of human autologous chondrocytes in suspension, as reported by Brittberg et al., provided a potential procedure for articular cartilage repair. We have improved their procedure and developed a new technique which creates new cartilage-like tissue by cultivating autologous chondrocytes embedded in Atelocollagen gel for 3 weeks before transplantation. These improvements maintained the chondrocyte phenotype, evenly distributed chondrocytes throughout the osteochondral defects, and decreased the risk of leakage of grafted chondrocytes into the defects. Good clinical results suggest that this technique should be a promising procedure for repairing articular cartilage defect.     

用组织工程方法桥接周围神经缺损的实验研究

目的 寻找修复周围神经缺损的新的有效替代材料。方法 将体外培养扩增的许旺细胞接种在生物可吸收材料聚羟基乙酸纤维支架上培养２周,形成一种新的神经桥接物,并对其进行了观察和动物移植试验。结果 发现接种的许旺细胞可在聚羟基乙酸纤维上良好吸附、迁移形成一种新型的、非管状的、具有三维纵向细胞排列和导层粘蛋白通道的组织工程化神经桥接物,用其修复近交的Ｗｉｓｔａｒ大鼠１５ｍｍ坐骨神经缺损时,取得了接近自体神经移     

藻酸钙复合材料在软骨组织工程中的应用前景

软骨缺乏血运,仅靠自身修复能力有限,近年来随着组织工程的兴起,构建出人工的组织工程化软骨来替代自身缺损的软骨组织已广为研究,研究重点也集中在寻找适合软骨细胞生长,且能满足自身内部环境并能降解的支架材料上。藻酸钙支架构建方式简单;具有水凝胶及三维多孔结构2种形式;在改变物理及化学因素的条件下能改变自身材料的力学及生物特性;且能与其他因素(如高分子聚合物、生长因子)构建出新型的复合材料,因此在软骨组织工程上具有一定的应用前景。本文主要概括了藻酸钙支架材料的特点和优势,就复合其他成分(生长因子、基因片段)的藻酸钙水凝胶和三维结构2种不同形式,联合整复软骨缺损甚至软骨及软骨下骨的应用前景作出展望。     

人组织工程软骨的实验研究

目的　了解组织工程研究的基本原理 ,完善制作组织工程化人软骨的技术方法 ,探讨天然可吸收材料组织 ,引导再生胶原膜作为人软骨细胞体外培养支架的可行性 ,为广泛开展组织工程的研究与应用开辟新的途径。方法　利用组织工程技术制作人组织工程化软骨 ,并应用组织学方法 ,对获得的人组织工程化组织形态和功能进行表征。结果　培养的软骨细胞均匀沉降于医用组织引导再生胶原膜上 ,1周后形成一层乳白色软骨样组织 ;植入 8周后从裸鼠体内获得软骨样组织 ,组织学检测证实软骨细胞的存在 ,软骨细胞可以分泌硫酸软骨素。结论　医用组织引导再生胶原膜以其特有的三维结构和细胞网结功能 ,可以作为组织工程技术应用研究中软骨细胞培养的支架 ;利用组织工程技术 ,能够完成人组织工程化软骨的制作。     

软骨组织工程种子细胞及其培养方法

关节软骨损伤是临床常见疾病，软骨组织自身修复能力低下，治疗缺乏有效手段，容易导致创伤性关节炎和功能障碍。随着对种子细胞培养方法研究的深入、生物材料学上的进展，组织工程学为关节软骨损伤修复提供了新的方法，有望解决这一难题。本对软骨组织工程中用到的几种种子细胞及其培养方法进行了综述。     

组织工程技术修复皮肤缺损的动物实验

目的　探索应用组织工程技术修复包括皮下组织的皮肤缺损的方法。方法　长枫杂交仔猪 2 0只 ,取腹部 2cm× 2cm全厚皮肤 ,酶消化法获取表皮细胞与成纤维细胞。经原代培养 ,将处于对数生长期的表皮细胞、成纤维细胞分别与 30 %氧化异丙烯F 1 2 7(pluronicF 1 2 7)混匀成细胞悬液后 ,种植于聚羟基乙酸 (polyglycolicacid ,PGA)形成细胞 生物材料复合物 ,用于修复自体背部直径 4cm皮肤缺损 ,以单纯生物材料 (PGA 30 %氧化异丙烯F 1 2 7)修复作为对照组。术后 1、2、4、8周取材 ,通过组织学方法评价新生组织。结果　实验组 :第 1周 ,形成含表皮与真皮两层结构的组织工程化皮肤 ;第 2周 ,表皮与真皮均较前增厚 ;第 8周 ,组织工程化皮肤的结构与正常皮肤相似 ,仅缺乏毛发、毛囊和汗腺等附属器。对照组 :则无皮肤形成 ,仅见大量肉芽组织。结论　以原代培养的表皮细胞与成纤维细胞作为种子细胞 ,PGA 30 %氧化异丙烯F 1 2 7为细胞载体的方法可修复皮肤缺损。     

胶原蛋白水凝胶在软骨组织工程中的应用

由创伤或骨病所致的关节骨软骨损伤在临床中十分常见,其中软骨缺损者达40.31%。由于关节软骨自身修复能力低下,采用组织工程技术对关节软骨损伤进行修复是目前采用再生医学治疗关节软骨损伤的新方法。组织工程支架按照性状可分为预成型支架材料及水凝胶材料两大类。传统的预成型支架材料移植技术容易给缺损周边软骨带来继发损伤,也存在支架与缺损整合不紧密等问题。如何在避免二次损伤的基础上,应用理想的仿生材料复合种子细胞修复关节不规则软骨损伤将成为未来软骨损伤修复的主要问题。选取微创、仿生并且可以原位塑形的胶原蛋白水凝胶复合种子细胞修复关节软骨损伤为损伤关节软骨的修复带来了希望。本文结合国内外相关文献对目前胶原蛋白水凝胶在软骨组织工程中的应用做一综述。     

Articular cartilage repair using tissue engineering technique--novel approach with minimally invasive procedure

Articular cartilage has very limited potential to spontaneously heal, because it lacks vessels and is isolated from systemic regulation. Although there have been many attempts to treat articular cartilage defects, such as drilling, microfracture techniques, soft tissue grafts or osteochondral grafts, no treatment has managed to repair the defects with long-lasting hyaline cartilage. Recently, a regenerative medicine using a tissue engineering technique for cartilage repair has been given much attention in the orthopedic field. In 1994, Brittberg et al. introduced a new cell technology in which chondrocytes expanded in monolayer culture were transplanted into the cartilage defect of the knee. As a second generation of chondrocyte transplantation, since 1996 we have been performing transplantation of tissue-engineered cartilage made ex vivo for the treatment of osteochondral defects of the joints. This signifies a concept shift from cell transplantation to tissue transplantation made ex vivo using tissue engineering techniques. We have reported good clinical results with this surgical treatment. However, extensive basic research is vital to achieve better clinical results with this tissue engineering technique. This article describes our recent research using a minimally invasive tissue engineering technique to promote cartilage regeneration.     

软骨组织工程中细胞因子功能的研究进展

随着骨关节疾病发病率的增加以及人们对生活质量提出的更高要求,修复软骨损伤成为骨科临床一个重要的问题。软骨组织工程以其不损伤自身组织、可调控、创伤小等优点成为修复软骨组织损伤的突破口。本文就多种细胞因子在促进软骨种子细胞生长起的作用的最新研究做一综述。     

骨-关节软骨复合组织块构建的实验研究

目的探讨采用组织工程技术构建骨．关节软骨复合组织块的可行性。方法将分离、培养的第2代软骨细胞和成骨细胞分别接种于磷酸三钙支架上，培养2周后通过物理方法将两者连接成一个整体，然后接种于裸鼠皮下。术后8周取材，行组织学观察。结果复合组织块在体内分别形成软骨组织和成骨组织，而且两者界面整合良好。结论以软骨细胞和成骨细胞为种子细胞、以磷酸三钙为支架体外构建的组织工程软骨和骨，可在体内形成组织工程骨．关节软骨复合组织块，有望用于关节软骨缺损的修复。     

永生化软骨细胞为基础的工程化软骨修复软骨缺损的实验研究

目的探讨用永生化软骨细胞作为种子细胞修复羊关节软骨缺损的可行性.方法把人端粒酶催化亚基(hTERT)转染羊关节软骨细胞,筛选阳性克隆并通过旋转生物反应器-微载体技术进行大量扩增;将扩增的永生化软骨细胞与β-磷酸三钙复合并在体外培育后,植入羊前肢肱骨头关节面软骨缺损处;3和6个月取材,进行形态学和免疫组织化学评价.结果实验组,术后6个月,缺损区被新生的软骨组织所充填,Ⅱ型胶原染色呈强阳性,与对照组差别有显著性差异(P＜0.01).在材料组,缺损区边缘可见部分新生软骨组织;空白对照组,缺损区新生软骨组织形成.结论永生化软骨细胞在软骨组织工程中具有潜在的应用前景.     

Osteochondritis dissecans of the talus treated by the transplantation of tissue-engineered cartilage

Management of osteochondral lesions of the joint has been difficult, because articular cartilage has a poor healing capacity as a result of its lack of vessels, nerve supply, and its isolation of systemic regulation. Although a lot of basic research and surgical treatments for cartilage repair have focused on osteochondral lesions in the knee joint, orthopedic surgeons have recently diverted their attention to osteochondral lesions in the ankle joint, partly because of the widespread introduction of arthroscopy in ankle surgery. There have been many attempts to treat articular cartilage defects in the ankle joint as well as in the knee joint. However, no treatment has achieved efficient healing with hyaline cartilage. Recently, tissue engineering technique for cartilage repair has been gaining much attention in the orthopedic field. In this study, we reported on a patient with osteochondritis dissecans of the talar dome, successfully treated by transplantation of tissue-engineered cartilage made ex vivo using atelocollagen gel and low tibial osteotomy.     

软骨细胞外基质与壳聚糖复合制备组织工程软骨支架及其性能研究

 目的 探讨采用壳聚糖与脱细胞软骨基质复合制备组织工程软骨支架的可行性,检测其理化性能和细胞相容性。方法 取天然人软骨粉碎.取 100 nm~5μm 软骨微丝,脱细胞处理后制备为质量浓度 1%悬液.与质量浓度 2%壳聚糖醋酸溶液按 1颐1(重量比)充分搅拌混合,冷冻干燥制备复合支架。对支架交联,并进行组织学、扫描电镜、孔隙率及吸水性测定、生物力学评估, MTT法分析支架浸提液毒性。分离培养犬软骨细胞.种植到支架上.倒置显微镜、电镜观察细胞在支架的生长、分化情况。结果 组织学显示支架中无细胞碎片残留.II型胶原免疫组化染色阳性。扫描电镜显示支架内孔洞相互连通似海绵状.孔径为(136.2±34.9)μm.孔隙率为 81.4%±3.5%.吸水性约为 1525.7±129.3%。支架纵向弹性模量为(1.940±0.335)MPa。 MTT法显示不同浓度支架浸提液与对照培养液吸光度值比较.差异无统计学意义(P>0.05)。倒置显微镜观察.细胞在支架上粘附良好.扫描电镜下细胞在支架上均匀分布.呈圆形或椭圆形.有基质分泌。结论 软骨细胞外基质和壳聚糖复合制备的仿生三维多孔双相支架.具有较高的孔隙率和吸水性.良好的生物力学特性.无毒.生物相容性良好.是组织工程软骨的良好支架载体。     

Cartilage tissue engineering by expanded goat articular chondrocytes.

In this study we investigated whether expanded goat chondrocytes have the capacity to generate cartilaginous tissues with biochemical and biomechanical properties improving with time in culture. Goat chondrocytes were expanded in monolayer with or without combinations of FGF-2, TGF-beta1, and PDGFbb, and the postexpansion chondrogenic capacity assessed in pellet cultures. Expanded chondrocytes were also cultured for up to 6 weeks in HYAFF-M nonwoven meshes or Polyactive foams, and the resulting cartilaginous tissues were assessed histologically, biochemically, and biomechanically. Supplementation of the expansion medium with FGF-2 increased the proliferation rate of goat chondrocytes and enhanced their postexpansion chondrogenic capacity. FGF-2-expanded chondrocytes seeded in HYAFF-M or Polyactive scaffolds formed cartilaginous tissues with wet weight, glycosaminoglycan, and collagen content, increasing from 2 days to 6 weeks culture (up to respectively 2-, 8-, and 41-fold). Equilibrium and dynamic stiffness measured in HYAFF M-based constructs also increased with time, up to, respectively, 1.3- and 16-fold. This study demonstrates the feasibility to engineer goat cartilaginous tissues at different stages of development by varying culture time, and thus opens the possibility to test the effect of maturation stage of engineered cartilage on the outcome of cartilage repair in orthotopic goat models.