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。     

细胞载体作为软骨修复中的下一代细胞疗法

背景自从第一位患者接受培养自体软骨细胞移植 （ACI） 以来已过去20多年,新型的软骨修复细胞疗法已经出现.自体培养软骨细胞是第一代细胞疗法,它使用的是混悬培养的自体软骨细胞结合骨膜修补.胶原膜覆盖ACI （CACI）是第二代细胞疗法,它使用的是接种了混悬培养软骨细胞的Ⅰ/Ⅲ型胶原膜上.现今,因关节镜移植术的需求而开发出了第三代细胞疗法,也即采用细胞载体或接种了细胞的支架来移植培养的自体软骨细胞.目的 本文综述了目前基质诱导的自体软骨细胞移植 （MACI） 的情况,它是迄今为止使用最广泛的载体系统.本文还探讨了Ⅰ/Ⅲ型胶原膜的特点、与胶原膜相关的细胞行为、手术技术、康复、临床结果和组织修复效果.研究设计 系统回顾.方法通过搜索 Medline 数据库从1949 年成立以来到 2007年12月的数据,找出相关文献;确认出关于细胞行为、制作过程、手术技术和康复方案的基础和临床研究同行评审的文献.具有原始数据以及使用Ⅰ/Ⅲ型胶原膜的基础和临床研究也被纳入.结果 这些研究获得的数据证实使用MACI治疗的患者在临床结果方面有总体改善.观察到视觉模拟疼痛量表（VAS）评分降低（范围在 1.7～5.32 分）,而改良Cincinnati（范围在3.8～34.2分）评分、Lysholm-Gillquist（范围在23.09～47.6分）评分、Tegner-Lysholm（范围在1.39～3.9分）评分和国际膝关节文献委员会分类量表评分（P〈0.05）均有改善.通过关节镜（包括国际软骨修复学会评分）、MRI 和组织学评估,患者也显示出质量良好的（透明样）组织修复,并且术后并发症发生率较低.结论本综述的结果提示：第三代细胞疗法MACI 程序在治疗有症状的、全层关节软骨缺损修复方面是一种大有前景的方法.     

Minced cartilage without cell culture serves as an effective intraoperative cell source for cartilage repair.

Traumatic articular cartilage injuries heal poorly and may predispose patients to the early onset of osteoarthritis. One current treatment relies on surgical delivery of autologous chondrocytes that are prepared, prior to implantation, through ex vivo cell expansion of cartilage biopsy cells. The requirement for cell expansion, however, is both complex and expensive and has proven to be a major hurdle in achieving a widespread adoption of the treatment. This study presents evidence that autologous chondrocyte implantation can be delivered without requiring ex vivo cell expansion. The proposed improvement relies on mechanical fragmentation of cartilage tissue sufficient to mobilize embedded chondrocytes via increased tissue surface area. Our outgrowth study, which was used to demonstrate chondrocyte migration and growth, indicated that fragmented cartilage tissue is a rich source for chondrocyte redistribution. The chondrocytes outgrown into 3-D scaffolds also formed cartilage-like tissue when implanted in SCID mice. Direct treatment of full-thickness chondral defects in goats using cartilage fragments on a resorbable scaffold produced hyaline-like repair tissue at 6 months. Thus, delivery of chondrocytes in the form of cartilage tissue fragments in conjunction with appropriate polymeric scaffolds provides a novel intraoperative approach for cell-based cartilage repair.     

Autologous chondrocyte implantation and osteochondral cylinder transplantation in cartilage repair of the knee joint. A prospective,comparative trial

BACKGROUND: Current methods used to restore the joint surface in patients with localized articular cartilage defects include transplantation of an autologous osteochondral cylinder and implantation of autologous chondrocytes. The purpose of this study was to evaluate the clinical and histological outcomes of these two techniques. METHODS: We performed a prospective clinical study to investigate the two-year outcomes in forty patients with an articular cartilage lesion of the femoral condyle who had been randomly treated with either transplantation of an autologous osteochondral cylinder or implantation of autologous chondrocytes. Biopsy specimens from representative patients of both groups were evaluated with histological staining, immunohistochemistry, and scanning electron microscopy. RESULTS: According to the postoperative Lysholm score, the recovery after autologous chondrocyte implantation was slower than that after osteochondral transplantation at six months (p < or = 0.015), twelve months (p < or = 0.001), and twenty-four months (p < or = 0.012). On the basis of the Meyers score and the Tegner activity score, the results were equally good with the two methods two years after treatment. Histomorphological evaluation of biopsy specimens within two years after autologous chondrocyte implantation demonstrated a complete, mechanically stable resurfacing of the defect in all patients. The tissue consisted mainly of fibrocartilage, while localized areas of hyaline-like regenerative cartilage could be detected close to the subchondral bone. Although a gap remained at the site of the transplantation in all five biopsy specimens examined as long as two years after osteochondral cylinder transplantation, histomorphological analysis and scanning electron microscopy revealed no differences between the osteochondral transplants and the surrounding original cartilage. CONCLUSIONS: Both treatments resulted in a decrease in symptoms. However, the improvement provided by the autologous chondrocyte implantation lagged behind that provided by the osteochondral cylinder transplantation. Histologically, the defects treated with autologous chondrocyte implantation were primarily filled with fibrocartilage, whereas the osteochondral cylinder transplants retained their hyaline character, although there was a persistent interface between the transplant and the surrounding original cartilage. Limitations of our study included the small number of patients, the relatively short (two-year) follow-up, and the absence of a control group.     

Stem cells and debrided waste: two alternative sources of cells for transplantation of cartilage

Implantation of autologous chondrocytes and matrix autologous chondrocytes are techniques of cartilage repair used in the young adult knee which require harvesting of healthy cartilage and which may cause iatrogenic damage to the joint. This study explores alternative sources of autologous cells. Chondrocytes obtained from autologous bone-marrow-derived cells and those from the damaged cartilage within the lesion itself are shown to be viable alternatives to harvest-derived cells. A sufficient number and quality of cells were obtained by the new techniques and may be suitable for autologous chondrocyte and matrix autologous chondrocyte implantation.     

Evaluation of articular cartilage with 3D-SPGR MRI after autologous chondrocyte implantation

To assess the maturation process of the cartilage after autologous chondrocyte implantation (ACI), we performed a longitudinal study with three-dimensional spoiled gradient-recalled (3D-SPGR) magnetic resonance imaging (MRI). Five knees of five patients on which ACI of the femoral condyle was performed were studied. The signal intensity of reparative tissue approached that of normal articular cartilage with time. The volume of reparative tissue remained at an almost constant level after implantation. During second-look arthroscopy, the areas on which ACI was performed were covered with hyaline-like cartilage, and the reparative tissue removed by biopsy consisted of normal chondrocytes and extracellular matrix. The increased signal intensity of the reparative tissue represents maturation of implanted autologous chondrocytes. 3D-SPGR MRI is thought to be useful for evaluating reparative tissue after autologous chondrocyte implantation.     

New Technique for Cell-Seeded Collagen Matrix-Supported Autologous Chondrocyte Transplantation

Autologous chondrocyte transplantation/implantation (ACT/ACI) is becoming increasingly common for the treatment of large cartilage defects in the knee joint. The traditional ACT technique involves injection of a suspension of cells into the cartilage defect, which is covered with a periosteal flap or collagen membrane. The technique requires extensive suturing to create an effective seal; however, cell leakage remains a potential problem. Matrix-induced autologous chondrocyte implantation (MACI/MACT) avoids this potential problem by using a membrane on which chondrocytes are seeded and cultured for several days, before the membrane is cut to the correct size and shape of the defect. Time-consuming extensive suturing is unnecessary. However, cutting and repeated manipulation of the seeded membrane may result in the loss of critical chondrocytes. A modified technique termed ACT-collagen membrane seeding (ACT-Cs) has been developed in which expanded chondrocytes are applied to the collagen membrane after it has been cut to size, substantially reducing the risk of viable cell loss while retaining the ease and speed of the MACI/MACT procedure. In addition, the seeding of mitotically active chondrocytes onto the membrane after expansion and immediately before transplantation allows direct application of high cell concentrations.     

Response of human chondrocytes prepared for autologous implantation to growth factors

This study investigated metabolism of autologous chondrocytes after initial expansion immediately before implantation. Chondrocytes cultured in either monolayers or alginate beads were treated with insulin-like growth factor-1 (IGF-1), osteogenic protein-1 (OP-1), or a combination. Proteoglycan synthesis and DNA content were tested in both cultures. Alginate beads also were analyzed with live/dead cell assay, safranin O/fast green stain for histology, and immunohistochemistry with antibodies against collagen type II and VI, aggrecan, decorin, and fibronectin. In monolayers, autologous chondrocytes changed their morphologic appearance. In alginate, they maintained chondrocytic phenotype. Growth factors, especially combined, promoted cell survival and induced chondrocyte proliferation. OP-1 stimulated the largest cartilage-specific matrix and the most accumulation of collagen type II and fibronectin, although the overall matrix synthesized by autologous chondrocyte implantation cells was smaller than that produced by normal chondrocytes. The clinical implications of this study suggest a significant promise for anabolic growth factors in cartilage repair as a potential modifying therapy for the enhancement of chondrocytic phenotype of autologous chondrocytes.     

Arthroscopic three dimensional autologous chondrocyte transplantation with navigation-guided cartilage defect size assessment

Introduction

The treatment of large full thickness cartilage defects with matrix guided autologous chondrocyte transplantation shows promising results. However, in many cases an arthrotomy is needed to implant the cell seeded scaffolds. Recently techniques have been developed for arthroscopically guided ACT implantation. Correct defect mapping, to assess size and depth of the chondral lesions, and precise scaffold preparation and fixation are crucial for successful chondrocyte transplantation and remain to be not sufficiently optimized.

Method

In the present study, the geometries of two cartilage defects in cadaver knees were three times assessed, measured and transferred to biodegradable scaffolds with a navigation system by three different executors. The scaffolds were arthroscopically implanted into the cartilage defects.

Results

The cartilage defect assessment was reproducible between all executors for all defect geometries. The implanted scaffolds showed a correct defect filling.

Conclusion

The study showed the feasibility of an arthroscopic implantation of scaffolds for autologous chondrocytes transplantation. Navigation was a useful tool to exactly assess the cartilage defect geometry and allowed a precise transfer of navigated cartilage defect geometries for individualized scaffold preparation. Navigation can help to accomplish and optimize arthroscopically guided chondrocyte transplantations.     

The role of autologous chondrocyte implantation in the treatment of symptomatic chondromalacia patellae

Purpose

Chondromalacia patella is a distinct clinical entity of abnormal softening of the articular cartilage of the patella, which results in chronic retropatellar pain. Its aetiology is still unclear but the process is thought to be a due to trauma to superficial chondrocytes resulting in a proteolytic enzymic breakdown of the matrix. Our aim was to assess the effectiveness of autologous chondrocyte implantation on patients with a proven symptomatic retropatellar lesion who had at least one failed conventional marrow-stimulating therapy.

Methods

We performed chondrocyte implantation on 48 patients: 25 received autologous chondrocyte implantation with a type I/III membrane (ACI-C) method (Geistlich Biomaterials, Wolhusen, Switzerland), and 23 received the Matrix-assisted Chondrocyte Implantation (MACI) technique (Genzyme, Kastrup, Denmark).

Results

Over a mean follow-up period of 40.3 months, there was a statistically significant improvement in subjective pain scoring using the visual analogue scale (VAS) and objective functional scores using the Modified Cincinnati Rating System (MCS) in both groups.

Conclusions

Chondromalacia patellae lesions responded well to chondrocyte implantation. Better results occurred with MACI than with ACI-C. Excellent and good results were achieved in 40% of ACI-C patients and 57% of MACI patients, but success of chondrocyte implantation was greater with medial/odd-facet lesions. Given that the MACI procedure is technically easier and less time consuming, we consider it to be useful for treating patients with symptomatic chondral defects secondary to chondromalacia patellae.     

Atelocollagen-associated autologous chondrocyte implantation for the repair of chondral defects of the knee: a prospective multicenter clinical trial in Japan

Background

New tissue-engineering technology was developed to create a cartilage-like tissue in a three-dimensional culture using atelocollagen gel. The minimum 2-year followup outcome of transplanting autologous chondrocytes cultured in atelocollagen gel for the treatment of full-thickness defects of cartilage in knees was reported from the single institution. The present multicenter study was conducted to determine clinical and arthroscopic outcomes in patients who underwent atelocollagen-associated autologous chondrocyte implantation for the repair of chondral defects of the knees.

Methods

At six medical institutes in Japan, we prospectively evaluated the clinical and arthroscopic outcomes of transplanting autologous chondrocytes cultured in atelocollagen gel for the treatment of full-thickness defects of cartilage in 27 patients (27 knees) with cartilage lesions on a femoral condyle or on a patellar facet over 24 months.

Results

The Lysholm score significantly increased from 60.0 ± 13.7 points to 89.8 ± 9.5 points (P = 0.001). Concerning the ICRS grade for arthroscopic appearance, 6 knees (24%) were assessed as grade I (normal) and 17 knees (68%) as grade II (nearly normal). There were few adverse features, except for detachment of the graft in two cases.

Conclusions

We concluded that transplanting chondrocytes in a newly formed matrix of atelocollagen gel can promote restoration of the articular cartilage of the knee.     

Repair of human articular cartilage after implantation of autologous chondrocytes

Tissue engineering is an increasingly popular method of addressing pathological disorders of cartilage. Recent studies have demonstrated its clinical efficacy, but there is little information on the structural organisation and biochemical composition of the repair tissue and its relation to the adjacent normal tissue. We therefore analysed by polarised light microscopy and immunohistochemistry biopsies of repair tissue which had been taken 12 months after implantation of autologous chondrocytes in two patients with defects of articular cartilage. Our findings showed zonal heterogeneity throughout the repair tissue. The deeper zone resembled hyaline-like articular cartilage whereas the upper zone was more fibrocartilaginous. The results indicate that within 12 months autologous chondrocyte implantation successfully produces replacement cartilage tissue, a major part of which resembles normal hyaline cartilage.     

Treatment of a Focal Articular Cartilage Defect of the Talus with Polymer-Based Autologous Chondrocyte Implantation: A 12-Year Follow-Up Period

Autologous chondrocyte implantation (ACI) is a first-line treatment option for large articular cartilage defects. Although well-established for cartilage defects in the knee, studies of the long-term outcomes of matrix-assisted ACI to treat cartilage defects in the ankle are rare. In the present report, we describe for the first time the long-term clinical and radiologic results 12 years after polymer-based matrix-assisted ACI treat a full-thickness talar cartilage defect in a 25-year-old male patient. The clinical outcome was assessed using the visual analog scale and Freiburg ankle score, magnetic resonance imaging evaluation using the Henderson-Kreuz scoring system and T2 mapping. Clinical assessment revealed improved visual analog scale and Freiburg ankle scores. The radiologic analysis and T2 relaxation time values indicated the formation of hyaline-like repair tissue. Polymer-based autologous chondrocytes has been shown to be a safe and clinically effective long-term treatment of articular cartilage defects in the talus.     

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.     

Cartilage repair: A review of Stanmore experience in the treatment of osteochondral defects in the knee with various surgical techniques

Articular cartilage damage in the young adult knee, if left untreated, it may proceed to degenerative osteoarthritis and is a serious cause of disability and loss of function. Surgical cartilage repair of an osteochondral defect can give the patient significant relief from symptoms and preserve the functional life of the joint. Several techniques including bone marrow stimulation, cartilage tissue based therapy, cartilage cell seeded therapies and osteotomies have been described in the literature with varying results. Established techniques rely mainly on the formation of fibro-cartilage, which has been shown to degenerate over time due to shear forces. The implantation of autologous cultured chondrocytes into an osteochondral defect, may replace damaged cartilage with hyaline or hyaline-like cartilage. This clinical review assesses current surgical techniques and makes recommendations on the most appropriate method of cartilage repair when managing symptomatic osteochondral defects of the knee. We also discuss the experience with the technique of autologous chondrocyte implantation at our institution over the past 11 years.     

Cell-based therapy options for osteochondral defects. Autologous mesenchymal stem cells compared to autologous chondrocytes

Cartilage defects are multifactorial and site-specific and therefore need a clear analysis of the underlying pathology as well as an individualized therapy so that cartilage repair lacks a one-for-all therapy. The results of comparative clinical studies using cultured chondrocytes in autologous chondrocyte implantation (ACI) have shown some superiority over conventional microfracturing under defined conditions, especially for medium or large defects and in long-term durability. Adult mesenchymal stem cells can be isolated from bone marrow, have the potency to proliferate in culture and are capable of differentiating into the chondrogenic pathway. They represent a promising versatile cell source for cartilage repair but the ideal conditions for cultivation and application in cartilage repair are not yet known or have not yet been characterized. Adding a scaffold offers mechanical stability and advances chondrogenic differentiation for both possible cell sources.     

Zellbasierte Therapieoptionen von Gelenkknorpeldefekten

Cartilage defects are multifactorial and site-specific and therefore need a clear analysis of the underlying pathology as well as an individualized therapy so that cartilage repair lacks a one-for-all therapy. The results of comparative clinical studies using cultured chondrocytes in autologous chondrocyte implantation (ACI) have shown some superiority over conventional microfracturing under defined conditions, especially for medium or large defects and in long-term durability. Adult mesenchymal stem cells can be isolated from bone marrow, have the potency to proliferate in culture and are capable of differentiating into the chondrogenic pathway. They represent a promising versatile cell source for cartilage repair but the ideal conditions for cultivation and application in cartilage repair are not yet known or have not yet been characterized. Adding a scaffold offers mechanical stability and advances chondrogenic differentiation for both possible cell sources.     

Effect of gravity on localization of chondrocytes implanted in cartilage defects.

The transplantation of autologous chondrocytes under a periosteal flap has been used to treat focal cartilage defects. Results have been promising but occasionally involve complications ranging from graft hypertrophy to detachment. The objective of this study was to determine if gravitational forces affect the uniformity of cell distribution within the defect. Using an ex vivo bovine model, the orientation relative to gravity of a repaired full-thickness articular cartilage defect was found to affect the initial distribution of transplanted chondrocytes, prelabeled with 3H-thymidine. After 4 hours, the injected cells (3H-radioactivity) were primarily at the base of the defect (79%) in samples oriented in the up position, primarily at the dependent semicylindrical half of the defect (83%) in samples oriented to the side, and primarily at the periosteal top of the defect (78%) in samples oriented upside-down. Subsequently, the cell distribution remained unchanged after reorientation into other positions. These results indicate that injected chondrocytes localize under the influence of gravity within the initial few hours after injection. Therefore, the defect orientation during this time can be an important factor in the uniformity of cell distribution in the autologous chondrocyte implantation procedure and may be an important determinant of the ultimate clinical outcome.     

Autologous chondrocyte implantation for knee cartilage injuries: moderate functional outcome and performance in patients with high-impact activities

Few studies have assessed the results of autologous chondrocyte implantation in patients with high-impact activities. The purpose of this study was to evaluate the early functional outcome and activity level after 2-stage autologous chondrocyte implantation in professional soldiers and athletes. Nineteen patients with an average age of 32.2 years were treated with autologous chondrocyte implantation and followed up for a minimum of 2 years. All patients except 2 had received previous arthroscopic treatment with debridement and/or microfracture. The mean size of the postdebridement defect was 6.54 cm2. Using Novocart technology (B. Braun-Tetec, Reutlingen, Germany), periosteal patch and matrix-assisted autologous chondrocyte implantation was sequentially performed with no randomization. The average subjective knee evaluation score and Lysholm score improved from 39.16 and 42.42, respectively, preoperatively to 62.4 and 69.4, respectively, at latest follow-up. Median Tegner activity score was 8.8 before injury, 3.8 preoperatively, and 6.15 at latest follow-up. Second-look arthroscopy was performed in 11 patients due to persistent pain, decreased range of movement, and mechanical symptoms. Six of 19 (31.5%) patients with professional or recreational athletic activities returned to preinjury levels of athletic performance.This study shows that mid-term results with autologous chondrocyte implantation in high-performance patients are not as good as have been reported with other similar technologies. Motivational issues during prolonged rehabilitation, multiple surgical interventions before autologous chondrocyte implantation, patient age, and large defects can potentially influence the outcome and overall performance in this selected group of patients.     

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.