Nitric oxide enhances cyclooxygenase activity in articular cartilage

Nitric oxide (NO) is a small messenger molecule synthesized by a family of enzymes, the nitric oxide synthases. Cyclooxygenases are a group of proinflammatory enzymes that release prostaglandins including prostaglandin E₂ (PGE₂). Both nitric oxide synthase and cyclooxygenase are involved in the inflammatory cascade of arthritis. However, the relationship between these two enzymes and their products has not been explored in articular cartilage. Here we show that in cultured bovine chondrocytes and explants of human osteoarthritic cartilage both nitric oxide synthase and cyclooxygenase activities were induced by the inflammatory mediators, lipopolysaccharide, and interleukin-1 or tumor necrosis factor-. When nitric oxide synthase activity was inhibited, PGE₂, synthesis was inhibited. NO donors also induced PGE₂ synthesis and NO scavengers inhibited cyclooxygenase activity. Taken together, these results support the concept that PGE₂ synthesis is directly related to NO formation and that NO may modulate cyclooxygenase activity in articular cartilage.accepted by W. B. van den BergFinalist in the 1995 Westinghouse Science Talent Search, the 1995 Otto Burgdorf Competition, and the 1995 St. Johns New York Symposium.     

Observations on the senescence of cells derived from articular cartilage

In the experiments described here, we have sought to determine whether primary cultures of cells derived from articular cartilage will, upon subsequent subculture, undergo in vitro senescence in a manner analogous to that described for several other types of diploid cell. Using cells from the articular cartilage of rabbits, dogs and man, we have established that the population doubling capacity of cultures of these cells is directly related to the specific lifespan of the donor organism. Furthermore, the doubling capacity of the initial cultures of lapine articular chondrocytes is inversely related to the age of the donor rabbit. By these criteria, serially passaged primary cultures of cells derived from articular cartilage appear, a priori, to be a valid system for studies of cellular ageing. Monolayer cultures of lapine chondrocytes appear to "dedifferentiate" after several passages. However, the same cells can be grown as clones, under which conditions they appear to retain better their differentiated properties. Even under these circumstances, lapine articular chondrocytes have a limited capacity for growth, which can be calculated to approximate to the same average number of cell divisions as undergone by monolayer cultures. Lapine chondrocytes frequently transform into established lines of fibroblastic cells. Transformation of canine chondrocytes was more rare, while human chondrocytes have not been observed to transform. This suggests that resistance to transformation is somehow related to lifespan. In addition to furthering our understanding of cellular ageing, studies of the senescence of articular chondrocytes could provide new insights into the aetiology of primary osteoarthritis.     

Chondrocyte and mesenchymal stem cell-based therapies for cartilage repair in osteoarthritis and related orthopaedic conditions

Osteoarthritis (OA) represents a final and common pathway for all major traumatic insults to synovial joints. OA is the most common form of degenerative joint disease and a major cause of pain and disability. Despite the global increase in the incidence of OA, there are no effective pharmacotherapies capable of restoring the original structure and function of damaged articular cartilage. Consequently cell-based and biological therapies for osteoarthritis (OA) and related orthopaedic disorders have become thriving areas of research and development. Autologous chondrocyte implantation (ACI) has been used for treatment of osteoarticular lesions for over two decades. Although chondrocyte-based therapy has the capacity to slow down the progression of OA and delay partial or total joint replacement surgery, currently used procedures are associated with the risk of serious adverse events. Complications of ACI include hypertrophy, disturbed fusion, delamination, and graft failure. Therefore there is significant interest in improving the success rate of ACI by improving surgical techniques and preserving the phenotype of the primary chondrocytes used in the procedure. Future tissue-engineering approaches for cartilage repair will also benefit from advances in chondrocyte-based repair strategies. This review article focuses on the structure and function of articular cartilage and the pathogenesis of OA in the context of the rising global burden of musculoskeletal disease. We explore the challenges associated with cartilage repair and regeneration using cell-based therapies that use chondrocytes and mesenchymal stem cells (MSCs). This paper also explores common misconceptions associated with cell-based therapy and highlights a few areas for future investigation.     

A three-dimensional osteochondral composite scaffold for articular cartilage repair

There is a recognized and urgent need for improved treatment of articular cartilage defects. Tissue engineering of cartilage using a cell-scaffold approach has demonstrated potential to offer an alternative and effective method for treating articular defects. We have developed a unique, heterogeneous, osteochondral scaffold using the TheriForm three-dimensional printing process. The material composition, porosity, macroarchitecture, and mechanical properties varied throughout the scaffold structure. The upper, cartilage region was 90% porous and composed of D,L-PLGA/L-PLA, with macroscopic staggered channels to facilitate homogenous cell seeding. The lower, cloverleaf-shaped bone portion was 55% porous and consisted of a L-PLGA/TCP composite, designed to maximize bone ingrowth while maintaining critical mechanical properties. The transition region between these two sections contained a gradient of materials and porosity to prevent delamination. Chondrocytes preferentially attached to the cartilage portion of the device, and biochemical and histological analyses showed that cartilage formed during a 6-week in vitro culture period. The tensile strength of the bone region was similar in magnitude to fresh cancellous human bone, suggesting that these scaffolds have desirable mechanical properties for in vivo applications, including full joint replacement.     

Hydrogels for the repair of articular cartilage defects

The repair of articular cartilage defects remains a significant challenge in orthopedic medicine. Hydrogels, three-dimensional polymer networks swollen in water, offer a unique opportunity to generate a functional cartilage substitute. Hydrogels can exhibit similar mechanical, swelling, and lubricating behavior to articular cartilage, and promote the chondrogenic phenotype by encapsulated cells. Hydrogels have been prepared from naturally derived and synthetic polymers, as cell-free implants and as tissue engineering scaffolds, and with controlled degradation profiles and release of stimulatory growth factors. Using hydrogels, cartilage tissue has been engineered in vitro that has similar mechanical properties to native cartilage. This review summarizes the advancements that have been made in determining the potential of hydrogels to replace damaged cartilage or support new tissue formation as a function of specific design parameters, such as the type of polymer, degradation profile, mechanical properties and loading regimen, source of cells, cell-seeding density, controlled release of growth factors, and strategies to cause integration with surrounding tissue. Some key challenges for clinical translation remain, including limited information on the mechanical properties of hydrogel implants or engineered tissue that are necessary to restore joint function, and the lack of emphasis on the ability of an implant to integrate in a stable way with the surrounding tissue. Future studies should address the factors that affect these issues, while using clinically relevant cell sources and rigorous models of repair.     

The pathology of the end-stage osteoarthritic lesion of the knee: potential role in cartilage repair

The purpose was to explore whether there were any pathological characteristics of the end-stage osteoarthritic sclerotic lesion that have potential to participate in cartilage repair.Specimens harvested following total knee surgery were examined for gross pathology including staining with Safranin O. Multiple small sections of the lesion were placed in tissue culture for 6 weeks. Gross examination and photographic documentation was made at 3 and 6 weeks. At 6 weeks the specimens from culture were subject to histological examination. The pathology of the end-stage osteoarthritic lesion showed sclerotic bone, dead osteons, hypervascularity and scattered cartilaginous aggregates. Additional observations showed multiple pitting on the sclerotic surface, which histologically was related to three events; fragmentation of dead bone, ruptured blood vessels, and eroded aggregates. There were no pathological or biological changes in the specimens following the time in tissue culture.The in-depth pathological evaluation showed the end-stage osteoarthritic lesion to have certain features with potential to facilitate cartilage repair. The cartilaginous aggregates may be a participant in cartilage repair following surgery. The cartilaginous aggregates remained unchanged in the tissue culture absent the normal synovial joint chemical and physical environment and therefore further testing with a different experimental model would be necessary to establish these aggregates as a source of cartilage regeneration. The multiple small depressions in this lesion may have potential to be a “home” for therapeutics.     

Regeneration of ovine articular cartilage defects by cell-free polymer-based implants

The aim of our study was the evaluation of a cell-free cartilage implant that allows the recruitment of mesenchymal stem and progenitor cells by chemo-attractants and subsequent guidance of the progenitors to form cartilage repair tissue after microfracture. Chemotactic activity of human serum on human mesenchymal progenitors was tested in 96-well chemotaxis assays and chondrogenic differentiation was assessed by gene expression profiling after stimulating progenitors with hyaluronan in high-density cultures. Autologous serum and hyaluronan were combined with polyglycolic acid (PGA) scaffolds and were implanted into full-thickness articular cartilage defects of the sheep pre-treated with microfracture. Defects treated with microfracture served as controls. Human serum was a potent chemo-attractant and efficiently recruited mesenchymal progenitors. Chondrogenic differentiation of progenitors upon stimulation with hyaluronan was shown by the induction of typical chondrogenic marker genes like type II collagen and aggrecan. Three months after implantation of the cell-free implant, histological analysis documented the formation of a cartilaginous repair tissue. Controls treated with microfracture showed no formation of repair tissue. The cell-free cartilage implant consisting of autologous serum, hyaluronan and PGA utilizes the migration and differentiation potential of mesenchymal progenitors for cartilage regeneration and is well suited for the treatment of cartilage defects after microfracture.     

Resurfacing potential of heterologous chondrocytes suspended in fibrin glue in large full-thickness defects of femoral articular cartilage: an experimental study in the goat.

A large full-thickness articular-cartilage defect was created in the medial femoral condyle of 32 adult goats. The defects were xenografted with isolated rabbit chondrocytes suspended in fibrin glue. Sham operated goats, where only a standardized defect was created, were used as controls. Results of cartilage repair were assessed after 3, 8, 13, 26 and 52 weeks. The repair tissue was evaluated macroscopically, histologically and biochemically. Results indicated that xenografted rabbit chondrocytes survived the transplantation and maintained their potential to produce matrix in fibrin glue, particularly if they were located in a non-weight-bearing area. In terms of an immunological reaction to xenografted chondrocytes, only mild signs of synovitis were observed in both groups and rejection of transplanted cells did not occur. From 3 weeks gradually progressive resolvement of the fibrin glue was observed with subsequent replacement by fibrous tissue. Initially xenografted defects histologically showed better tendency for cartilage regeneration, however, 52 weeks after surgery no significant differences could be detected in the repair tissue of both groups macroscopically, histologically and on biochemical scoring. The amount of collagen type II in the newly synthesized matrix was 75% 1 year after surgery. This study shows that isolated heterologous chondrocytes can be used for transplantation in articular cartilage defects, however, fibrin glue does not offer enough biomechanical support to the cells to maintain its function as a three-dimensional scaffold.     

Modulation of chondrocyte functions and stiffness-dependent cartilage repair using an injectable enzymatically crosslinked hydrogel with tunable mechanical properties

We developed an injectable hydrogel system to evaluate the effect of hydrogel stiffness on chondrocyte cellular functions in a three-dimensional (3D) environment and its subsequent influence on ectopic cartilage formation and early-stage osteochondral defect repair in a rabbit model. The hydrogels, composed of gelatin-hydroxyphenylpropionic acid (Gtn-HPA) conjugate, were formed using oxidative coupling of HPA moieties catalyzed by hydrogen peroxide (H₂O₂) and horseradish peroxidase (HRP). The storage modulus (G′) of the hydrogels, which was tunable by changing the H₂O₂ and Gtn-HPA concentrations, ranged from 570 Pa to 2750 Pa. It was found that the cellular functions of chondrocytes encapsulated in hydrogels, including cell proliferation, biosynthesis of collagen and sulfated glycosaminoglycans (sGAG), as well as gene expression of type I (Col-I) and type II collagen (Col-II), were strongly affected by the stiffness of the hydrogels. Of note, chondrocytes cultured within the Gtn-HPA hydrogel of medium stiffness (G′ = 1000 Pa) produced highest level of sGAG production, as well as highest ratio of Col-II to Col-I gene expression among the Gtn-HPA hydrogels of different stiffness. Consistent with the results from in vitro and in vivo ectopic cartilage formation, osteochondral defect repair in a rabbit model showed stiffness-dependent tissue repair, with defects implanted with chondrocytes in hydrogels of medium stiffness having markedly more hyaline cartilage formation, smoother surface and better integration with adjacent cartilage, compared to defects treated with hydrogels of low or high stiffness. These results suggest that the tunable stiffness of Gtn-HPA hydrogels modulates chondrocyte cellular functions, and has a dramatic impact on cartilage tissue histogenesis and repair.     

DNA repair by articular chondrocytes. I. Unscheduled DNA synthesis following ultraviolet irradiation in monolayer culture

The hypothesis that aging of articular chondrocytes at a cellular level results from loss of DNA repair capability was studied by measuring unscheduled DNA synthesis (UDS). Cultured rabbit and human articular chondrocytes were irradiated with 254 nm ultraviolet light (20 J/m²) following treatment with 10 mM hydroxyurea. Neither the “in vitro senescence” nor spontaneous transformation that developed during serial passage of rabbit chondrocytes was accompanied by diminution of UDS. Synthesis of sulfated glycosaminoglycans declined more rapidly than the ability of the cells to divide. Levels of UDS by chondrocytes from old donors, rabbit or human, were comparable to those of younger individuals. UDS was greater in human than rabbit chondrocytes. Similar data have been reported previously for dermal fibroblasts but do not necessarily indicate that there is a direct or causative relationship between UDS capability and the longevity of mammalian species. X-Irradiation of rabbit chondrocytes or cartilage explants, in doses up to 40 000 rads, yielded no measurable UDS.     

间充质干细胞向软骨分化及在软骨修复中应用的研究进展

关节软骨是一种负重结缔组织,常因肿瘤、运动、退行性变或老年性疾病造成损伤;然而关节软骨自身修复能力有限,给临床治愈软骨缺损造成了很大困难.近些年出现了多种治疗软骨缺损的方法,包括自体软骨细胞移植、微裂缝和镶嵌成形术,但这些方法各自都有其局限性.近年来,组织工程软骨成为软骨修复研究的新热点,间充质干细胞（MSCs）是其当前最有前景的种子细胞.就MSCs在体外诱导分化为软骨细胞的培养条件及MSCs在软骨修复中应用的研究进展进行综述.     

工程软骨支架的临床应用研究进展

移植工程软骨修复软骨损伤是目前较为理想的治疗方法,构建工程软骨需要种子细胞和支架材料,支架材料的性能对工程软骨的生物特性有重要影响.讨论支架材料的研究进展,比较不同支架材料的工程软骨的临床应用结果,对进一步改善工程软骨的生物学性能有重要意义.结合近年来工程软骨支架的研究和临床应用情况作一综述和展望.     

Porous polymer implants for repair of full-thickness defects of articular cartilage: an experimental study in rabbit and dog.

Full-thickness defects of articular cartilage were repaired by implantation of porous polymer implants in rabbits and dogs. The quality of the repair tissue was determined by collagen typing with antibodies. Implants with varying pore sizes and chemical composition were used. The effect of loading and motion was determined by inserting implants higher than, level with and lower than the surrounding cartilage. It appeared that healing took place by formation of fibrocartilaginous repair tissue containing both type I and type II collagen. Hyaline cartilage was observed in a minority of the rabbits used but not in the dog. Fibrocartilage formation in the dog was simulated by implantation of a porous polymer. Chemical composition of the polymer did not alter the results, neither did loading of the implant. It is concluded that the formation of fibrocartilaginous repair cartilage is stimulated by implantation of a porous polymer. This tissue seemed to function adequately in the dog but did show signs of degeneration in the rabbit.     

Repair of articular cartilage and clinical outcome after osteotomy with microfracture or abrasion arthroplasty for medial gonarthrosis

This study compared the healing of articular cartilage and the clinical outcome after osteotomy with or without marrow stimulation microfracture or abrasion arthroplasty for osteoarthritis of the knee. Patients with osteoarthritis of the medial compartment of the knee were divided into a group undergoing high tibial osteotomy alone (HTO group: 37 knees), a group undergoing osteotomy plus microfracture (MF group: 26 knees), and a group undergoing osteotomy plus abrasion arthroplasty (AA group: 51 knees). The extent of cartilage repair was compared at 1 year after surgery by arthroscopy with reference to Outerbridge's classification, while the clinical outcome was compared at 1, 3, and 5 years postoperatively. Second-look arthroscopy revealed better repair of the femoral condylar cartilage in the AA group than the HTO group (p < 0.0005) or MF group (p < 0.01), with no difference between the HTO and MF groups. Repair of the tibial condylar cartilage was also better in the AA group than the HTO group (p < 0.005), but there was no difference between the AA and MF groups or the MF and HTO groups. There were no differences of the clinical outcome between the three groups. In conclusion, repair of articular cartilage at 1 year postoperatively was accelerated by abrasion arthroplasty, but not by microfracture. However, there was no difference of the clinical outcome within 5 years after surgery, so the clinical utility of marrow stimulation techniques was not apparent in this study.     

大骨节病关节软骨胶原表型表达和软骨细胞异常分化的研究

目的探讨大骨节病关节软骨胶原表型表达的变化特点和软骨细胞异常分化在发病中的意义。方法用单克隆免疫组化法测定５例大骨节病关节软骨Ⅰ、Ⅱ、Ⅲ、Ⅵ、Ⅹ型胶原表型的表达。结果（１）关节软骨表层的Ⅱ型胶原表型表达减少；（２）Ⅰ、Ⅲ和Ⅵ型胶原表型表达见于关节软骨全层，而Ⅹ型胶原仅限于关节软骨钙化层和深层软骨细胞团周围；（３）软骨细胞团有Ⅰ、Ⅱ、Ⅲ、Ⅵ型胶原表型表达，而软骨细胞坏死区内无任何胶原表型表达。结论大骨节病关节软骨胶原表型表达类似于原发性骨关节病的变化，但在关节软骨表层Ⅰ型胶原表型表达增强以及软骨坏死区内无任何胶原表型表达不同于原发性骨关节病。     

膝关节镜下RAPIDLOC半月板修复患者中短期临床效果分析

目的评价在软骨损伤Ⅳ级以下患者中使用RAPIDLOC进行半月板修复后的短期临床效果。方法通过一组使用RAPIDLOC进行半月板修复前后得到的临床数据进行回顾性研究。2006年7月到2007年5月，共有38位行半月板修复术的患者，将所有病例分成两组，A组包括关节镜下关节软骨正常或者损伤分级为Ⅰ级的患者，B组为关节镜下关节软骨损伤Ⅱ到Ⅲ级的患者。两组病例手术前后均进行Lysholm和Tegner膝关节功能评价，并将两组数据进行统计学比较和分析。结果本次研究中，A组共有22位患者，B组有16位患者；平均随访时间为A组6．5±2．5月，B组6．8±2．3月；A组成功率为90．9％，B组为87．5％：A组患者平均Tegner评分为术前3．5±1．6分，术后7．3±2．5分（P〈0．05），平均Lysholm评分为术前35．1±14．6分，术后88．6±25．5分（P〈0．01）：B组患者平均Tegner评分为术前3．3±2.3分，术后6．8±2．1分（P〈0．05），平均Lysholm评分为术前30．5±17．8分，术后87．8±22．5分（P〈0．01），两组数据进行比较无统计学意义。结论本次研究显示，在软骨损伤Ⅳ级以下患者中，使用RAPIDLOC进行半月板修复均有较高的成功率，两组病例数据之间没有统计学意义。     

Identification of genes required for the spontaneous repair of partial-thickness cartilage defects in immature rats

Purpose: Our previous study showed that partial-thickness articular cartilage defects (PTCDs) created in immature rats spontaneously healed to resemble normal hyaline cartilage, but that of mature rats did not. To identify molecules involved in the spontaneous cartilage repair observed in this model, gene expression was compared between PTCD and sham-operated cartilage of immature and mature rats. Materials and Methods: Six sets of gene comparisons were made at 12, 24, and 48 hours after the creation of PTCDs in immature and mature rats using microarrays. All the genes upregulated in immature cartilage at 12 hours were selected for further analysis if their expression pattern was not irregular such that diminished at 24 hours and re-upregulated at 48 hours. Relationships among genes selected through the above steps were analyzed using Ingenuity Pathway Analysis (IPA) software. After deriving networks, important molecules were further narrowed down by location within a network. Genes were regarded as central if they had relationships with more than 10 molecules in a network. Protein localization in tissues was confirmed by immunohistochemistry. Results: Five networks were identified. Their functional annotations were gene expression, cell cycle, growth and proliferation, and cell signaling. Transforming growth factor-beta (TGF-β) was centrally located in the network with the highest IPA score and mothers against decapentaplegic homolog-3 (Smad3) were centrally located in the second highest ranking network. Phosphorylated Smad3 was detected in the nuclei of chondrocytes in immature cartilage. Conclusions: Our data suggest the possible importance of Smad3 in the TGF-β signaling in the spontaneous healing of PTCDs in immature rats.     

A Paradigm for Functional Tissue Engineering of Articular Cartilage via Applied Physiologic Deformational Loading

Deformational loading represents a primary component of the chondrocyte physical environment in vivo. This review summarizes our experience with physiologic deformational loading of chondrocyte-seeded agarose hydrogels to promote development of cartilage constructs having mechanical properties matching that of the parent calf tissue, which has a Young's modulus E(Y) = 277 kPa and unconfined dynamic modulus at 1 Hz G* = 7 MPa. Over an 8-week culture period, cartilage-like properties have been achieved for 60 x 10(6) cells/ml seeding density agarose constructs, with E(Y) = 186 kPa, G* = 1.64 MPa. For these constructs, the GAG content reached 1.74% ww and collagen content 2.64% ww compared to 2.4% ww and 21.5% ww for the parent tissue, respectively. Issues regarding the deformational loading protocol, cell-seeding density, nutrient supply, growth factor addition, and construct mechanical characterization are discussed. In anticipation of cartilage repair studies, we also describe early efforts to engineer cylindrical and anatomically shaped bilayered constructs of agarose hydrogel and bone (i.e., osteochondral constructs). The presence of a bony substrate may facilitate integration upon implantation. These efforts will provide an underlying framework from which a functional tissue-engineering approach, as described by Butler and coworkers (2000), may be applied to general cell-scaffold systems adopted for cartilage tissue engineering.