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。     

关节软骨缺损修复研究进展

关节软骨缺损是临床常见疑难病症之一。滑膜关节表面缺损后难以修复。现就关节软骨缺损的自发修复、自体或异体移植修复、软骨膜或骨膜移植修复、软骨细胞移植修复，以及三维立体细胞培养及组织工程技术修复等五个方面，综述了滑膜关节软骨缺损修复重建的方法学进展     

组织细胞移植修复关节软骨缺损的研究现状

关节软骨组织的自身修复能力很差,临床治疗中要使修复或再生的软骨能恢复到滑膜关节正常的无痛运动,修复的新组织须在结构、组成、机械性能和持久耐用方面与正常关节软骨相似。目前关节软骨修复损伤的方法主要有两大类：一类为刺激关节软骨自身修复的方法,另一类为组织细胞移植方法。前者包括,清创术和灌洗法、软骨下骨钻孔术、微骨折、截骨术等,后者包括软骨移植、骨膜／软骨膜移植、软骨细胞及间充质细胞移植等［１］。本文就组织细胞移植修复关节软骨缺损的研究进展进行了综述。一、关节软骨移植关节软骨移植是用完整的正常关节软骨…     

筋膜软骨细胞修复关节软骨大面积缺损的实验研究

目的　探讨在筋膜上培养软骨细胞修复关节软骨大面积缺损的能力和生物特性。方法　将幼兔关节软骨消化、分离和传代后的软骨细胞在兔筋膜上培养 ,分别用培养后的新鲜、冻存筋膜软骨细胞和游离软骨细胞移植修复关节软骨大面积缺损。术后 6、12及 2 4周取材 ,通过大体标本、光学显微镜、扫描、透射电镜、放射自显影以及一氧化氮含量测定等方法进行观察。结果　分离后的关节软骨细胞在兔筋膜上生长代谢良好 ,冻存后的筋膜软骨细胞生物活性无损伤 ,移植后修复的关节软骨缺损在细胞形态、生物特性与正常关节软骨组织相同。结论　筋膜可以作为软骨细胞移植较为理想的载体 ,用其修复关节软骨大面积缺损是一种有效可行的方法。     

膝关节软骨损伤的外科治疗进展

关节软骨损伤后,软骨缺损通常缺乏自行修复能力,要求外科修复。传统外科治疗软骨损伤包括关节镜下冲洗清理术、微骨折术、自体骨软骨移植术、异体骨软骨移植术和自体软骨细胞移植等方法。关节冲洗清理术去除了关节内致痛因素,操作简单,应用广泛,早期疗效确切。微骨折术及自体骨软骨移植对小面积的软骨缺损修复较为理想,然而远期临床观察发现钻孔渗透修复的纤维软骨会降低微骨折术后疗效,相对于重建负重区关节面完整性自体骨软骨移植更具有优势。自体软骨细胞移植及异体骨软骨移植适用于更大面积的软骨缺损,异体骨软骨移植术后存活率受到局部排斥反应影响,从而降低了远期疗效。软骨组织工程技术可最大限度地提高自体软骨细胞移植的修复质量,实现修复组织接近透明软骨,但对于累及软骨下骨板、反应性骨水肿、严重骨量丢失或下肢轴线不良具有局限性。近年来许多新技术陆续应用于软骨损伤治疗领域,创伤小、操作简便、恢复快、疗效好、花费低、多技术联合应用的外科修复技术将会成为未来的治疗软骨损伤的重要手段。目前如何提高软骨修复质量,更具抗压、耐磨性,仍亟待解决。     

自体骨膜移植治疗关节软骨损伤的研究现状

关节软骨损伤修复大致分两类，即内源性修复和外源性修复。内源性修复也称骨髓刺激术，外源性修复包括生物移植和组织工程化关节软骨，前者包括骨膜、软骨膜移植，软骨细胞移植和骨-软骨移植；后者是体外构建种子细胞-载体复合物注入缺损区或者利用种子细胞悬液注入缺损区再用骨膜或软骨膜覆盖封闭软骨缺损的开口。两者均涉及到骨膜的成软骨作用。骨膜移植应用于临床治疗软骨损伤已有近20a历史，它有取材方便、对机体损害小等特点，但存在许多影响因素，限制了它的应用。本文就骨膜移植治疗关节软骨损伤的现状作一综述。     

同种异体软骨细胞移植术后关节软骨蛋白多糖的测定

目的 应用Pluronic F-127负载同种异体软骨细胞移植修复兔全厚关节软骨损伤,对于新生的修复组织进行基质蛋白多糖含量测定,以探讨此方法修复全厚关节软骨损伤的可行性.方法 取3个月龄新西兰大白兔关节软骨细胞体外培养扩增,与20%Plurortic F-127凝胶混合.选27只健康同种成年大白兔,人为造成双侧膝关节软骨缺损.实验组软骨缺损处植入培养的软骨细胞/Pluronic F-127混合物,对照组缺损处单纯注入Pluronic F-127凝胶和空白对照.然后,对修复组织进行大体观察及蛋白多糖含量测定.结果 移植的软骨细胞-载体复合物中的软骨细胞能良好地生长,12周时再生组织与周围正常软骨组织外观相似,界限模糊.实验组与对照组各时期蛋白多糖含量均有非常显著性差异,实验组不同时期的蛋白多糖含量之间均有显著性差异,实验组12周时蛋白多糖含量与正常软骨组织无显著性差异.结论 Pluronic F-127负载同种异体软骨细胞移植是治疗关节软骨缺损的有效方法.     

同种异体软骨细胞移植术后关节软骨蛋白多糖的测定

目的应用Pluronic F-127负载同种异体软骨细胞移植修复兔全厚关节软骨损伤，对于新生的修复组织进行基质蛋白多糖含量测定，以探讨此方法修复全厚关节软骨损伤的可行性。方法取3个月龄新西兰大白兔关节软骨细胞体外培养扩增，与20％Pluronic F-127凝胶混合。选27只健康同种成年大白兔，人为造成双侧膝关节软骨缺损。实验组软骨缺损处植入培养的软骨细胞／Pluronic F-127混合物，对照组缺损处单纯注入Pluronic F-127凝胶和空白对照。然后，对修复组织进行大体观察及蛋白多糖含量测定。结果移植的软骨细胞-载体复合物中的软骨细胞能良好地生长，12周时再生组织与周围正常软骨组织外观相似，界限模糊。实验组与对照组各时期蛋白多糖含量均有非常显著性差异，实验组不同时期的蛋白多糖含量之间均有显著性差异，实验组12周时蛋白多糖含量与正常软骨组织无显著性差异。结论Pluronic F-127负载同种异体软骨细胞移植是治疗关节软骨缺损的有效方法。     

天然生物支架材料在软骨修复中的研究进展

关节软骨缺乏再生能力，外伤或疾病引起的软骨缺损需利用软骨或其它材料修复。自体软骨来源有限，且容易造成供区缺损，应用受到限制。异体软骨曾广泛应用，但可引起免疫排斥反应，而导致细胞死亡及功能丧失。骨膜移植曾风行一时，但其存在远期效果不稳定的缺陷，使得人们不断探索更完善的修复方法。体外软骨细胞培养成功，引发人们尝试直接用软骨细胞修复软骨缺损。1968年，Chertman等将软骨细胞悬液注射到关节软骨缺损部位，结果表明：缺损为滑膜成纤维组织修复，镜下仅见少量新生软骨细胞结节。1977年，Green等以脱钙骨作为支架，并接种上软骨细胞，移植到缺损部位。虽未成功，可喜的是作者第一次提出：如能找到一种合适的支架材料，将软骨细胞接种于其上，即有可能形成良好的软骨组织修复。当前，在组织工程中开发为细胞培养支架的生物支架材料主要分为两类，即天然生物支架材料和人工合成的支架材料。天然生物支架材料具有细胞信号识别，促进细胞的黏附、增殖和分化、良好的生物相容性及良好的生物降解性等优点，显示出人工合成支架材料无可比拟的优势。本文就天然生物支架材料在软骨修复中的现状和研究进展做如下综述。     

自体软骨细胞移植治疗关节软骨缺损的进展

关节软骨主要是由少量的软骨细胞和细胞外基质组成的无血管组织软骨损伤后，其自我修复能力很弱。当关节软骨缺损面积〉2cm^2，深度〉4mm时，通常不能自行修复，持续发展会导致骨关节炎。目前，可采用关节镜下清创灌洗术、微骨折术、开放性自体骨膜移植术、自体骨软骨移植术和自体软骨细胞移植（autologous chondrocyt eimplantation，ACI）等方法修复关节软骨。     

The osteochondral dilemma: review of current management and future trends

The management of articular cartilage defects remains challenging and controversial. Hyaline cartilage has limited capacity for self‐repair and post‐injury cartilage is predominantly replaced by fibrocartilage through healing from the subchondral bone. Fibrocartilage lacks the key properties that characterize hyaline cartilage such as capacity for compression, hydrodynamic permeability and smoothness of the articular surface. Many reports relate compromised function associated with repaired cartilage and loss of function of the articular surface. Novel methods have been proposed with the key aim to regenerate hyaline cartilage for repair of osteochondral defects. Over the past decade, with many exciting developments in tissue engineering and regenerative cell‐based technologies, we are now able to consider new combinatorial approaches to overcome the problems associated with osteochondral injuries and damage. In this review, the currently accepted surgical approaches are reviewed and considered; debridement, marrow stimulation, whole tissue transplantation and cellular repair. More recent products, which employ tissue engineering approaches to enhance the traditional methods of repair, are discussed. Future trends must not only focus on recreating the composition of articular cartilage, but more importantly recapitulate the nano‐structure of articular cartilage to improve the functional strength and integration of repair tissue.     

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.     

Repair of articular cartilage defects with cultured chondrocytes on polysulphonic membrane: experimental studies in rabbits

INTRODUCTION: Autologous osteochondral transplantation is one method that can be used to create hyaline or hyaline-like repair in a defect area. The purpose of the present study was to repair full-thickness articular cartilage defects in 9 rabbit knee joints with autologous cultured chondrocytes. METHODS: An articular cartilage defect was created on the patellar groove of the femur. The defect was filled with chondrocytes cultured in vitro and placed into the knee on a polysulphonic membrane. At 8 weeks after the operation, the reparative tissue was analyzed macroscopically and histologically. RESULTS: At 8 weeks after the operation, the surfaces of the reparative tissue were smooth, and the defects were filled with mature hyaline cartilage in 5 cases. In 2 cases, the reparative hyaline cartilage was immature and there was worse integration of grafted tissue into the adjacent normal cartilage. In 2 cases, the surface of the grafted area was irregular, and the reparative tissue was disintegrated and incompletely differentiated. CONCLUSION: The results suggest that transplantation of autologous chondrocytes cultured in vitro and placed into the knee on polysulphonic membrane is effective in repairing an articular cartilage defect.     

Rabbit articular cartilage defects treated by allogenic chondrocyte transplantation

Articular cartilage defects have a poor capacity for repair. Most of the current treatment options result in the formation of fibro-cartilage, which is functionally inferior to normal hyaline articular cartilage. We studied the effectiveness of allogenic chondrocyte transplantation for focal articular cartilage defects in rabbits. Chondrocytes were cultured in vitro from cartilage harvested from the knee joints of a New Zealand White rabbit. A 3 mm defect was created in the articular cartilage of both knees in other rabbits. The cultured allogenic chondrocytes were transplanted into the defect in the right knees and closed with a periosteal flap, while the defects in the left knees served as controls and were closed with a periosteal flap alone, without chondrocytes. Healing of the defects was assessed at 12 weeks by histological studies. Allogenic chondrocyte transplantation significantly increased the amount of newly formed repair tissue (P=0.04) compared with that found in the control knees. The histological quality score of the repair tissue was significantly better (P=0.05), with more hyaline characteristics in the knees treated with allogenic chondrocytes than in the control knees. Articular cartilage defects treated with allogenic chondrocyte transplantation result in better repair tissue formation with hyaline characteristics than those in control knees.     

自体软骨细胞团块植入修复兔陈旧性关节软骨缺损的实验研究

目的 观察自体软骨细胞团块植入对兔关节软骨缺损的修复作用. 方法 24只成年新西兰大白兔48侧膝关节,随机分为三组(n=16)并制备双膝关节股骨滑车软骨缺损模型.空白对照组无特殊处理,骨膜移植组将骨膜覆盖缺损并缝合于缺损两侧的股骨髁上,实验组将自体软骨细胞团块植入缺损中.术后3、6个月分别取材(n=8),进行大体和组织学观察,修复组织行Wakitani评分并进行比较. 结果实验组共成功取材11个缺损关节,9个为透明软骨修复,2个因植入细胞生长状态差未修复;骨膜移植组修复组织为纤维软骨或纤维组织,修复组织薄,基质异染弱;空白对照组仅有少量纤维组织填充缺损底部.修复组织Wakitani评分:实验组3.82分,骨膜移植组6.71分,空白对照组9.23分,差异有统计学意义(F=5.96,P=0.00). 结论自体软骨细胞团块植入能较好修复关节软骨缺损,修复的质量与植入细胞的质量有关.     

Autologous chondrocyte implantation--technique and long-term follow-up

Cartilage has a limited capacity for self repair after injury. This biological deficiency has led to a variety of surgical attempts to improve the repair of injured articular cartilage surfaces over the past 50 years. The first example of clinical cartilage tissue engineering was performed in 1987 when a knee with an articular cartilage defect on the femoral condyle was treated by implanting the patient's own chondrocytes that had been expanded in vitro into the defect in combination with a covering mechanical membrane-the periosteum. This technology is either termed autologous chondrocyte transplantation (ACT) or autologous chondrocyte implantation (ACI). Today, many modifications of the technique exist, from the first generation to now second and third generations of chondrocyte implantation. This paper describes the basic techniques for the clinical use of chondrocyte implantation and gives an update on the clinical results.     

A Cell-Free Collagen Type I Device for the Treatment of Focal Cartilage Defects

The purpose of this study was to evaluate the potential value of a cell-free collagen type I gel plug for the treatment of focal cartilage defects. Cellular migration and proliferation was addressed in vitro, and the formation of repair tissue in a nude mouse-based defect model. A cell-free plug made of collagen type I was placed in the center of an incubation plate. Surrounding space was filled with a collagen type I gel (Arthro Kinetics, Esslingen, Germany) seeded with 2 × 10⁵ human articular chondrocytes/mL gel. After cultivation for up to 6 weeks in vitro, samples were subject to histological and immunohistochemical staining and gene expression analysis. Subsequently, chondral defects of human osteochondral blocks were treated with the plug, and specimens were cultivated subcutaneously in nude mice for 6 weeks. The repair tissue was evaluated macroscopically, and collagen type II production was investigated immunohistochemically. In vitro, morphology of immigrated cells did not show any differences, as did collagen type II gene expression. After 4 weeks, the plug was homogeneously inhabited. After 6 weeks of cultivation in nude mice, collagen gel plug treatment led to a macroscopically excellent repair tissue. Histological staining revealed a tight bonding, and the collagen gel plug started to be remodeled. We conclude that the novel collagen gel plug device offers an environment favorable for the migration of articular chondrocytes and leads to a good-quality repair tissue in the nude mouse model. The arthroscopic transplantation of a collagen gel plug may be one option in the treatment of focal cartilage defects.     

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.