Bioinspired nanofibers support chondrogenesis for articular cartilage repair

Articular cartilage repair remains a significant and growing clinical challenge with the aging population. The native extracellular matrix (ECM) of articular cartilage is a 3D structure composed of proteinaceous fibers and a hydrogel ground substance that together provide the physical and biological cues to instruct cell behavior. Here we present fibrous scaffolds composed of poly(vinyl alcohol) and the biological cue chondroitin sulfate with fiber dimensions on the nanoscale for application to articular cartilage repair. The unique, low-density nature of the described nanofiber scaffolds allows for immediate cell infiltration for optimal tissue repair. The capacity for the scaffolds to facilitate cartilage-like tissue formation was evaluated in vitro. Compared with pellet cultures, the nanofiber scaffolds enhance chondrogenic differentiation of mesenchymal stems cells as indicated by increased ECM production and cartilage specific gene expression while also permitting cell proliferation. When implanted into rat osteochondral defects, acellular nanofiber scaffolds supported enhanced chondrogenesis marked by proteoglycan production minimally apparent in defects left empty. Furthermore, inclusion of chondroitin sulfate into the fibers enhanced cartilage-specific type II collagen synthesis in vitro and in vivo. By mimicking physical and biological cues of native ECM, the nanofiber scaffolds enhanced cartilaginous tissue formation, suggesting their potential utility for articular cartilage repair.     

Extensive neurite outgrowth and active synapse formation on self-assembling peptide scaffolds

A new type of self-assembling peptide (sapeptide) scaffolds that serve as substrates for neurite outgrowth and synapse formation is described. These peptide-based scaffolds are amenable to molecular design by using chemical or biotechnological syntheses. They can be tailored to a variety of applications. The sapeptide scaffolds are formed through the spontaneous assembly of ionic self-complementary beta-sheet oligopeptides under physiological conditions, producing a hydrogel material. The scaffolds can support neuronal cell attachment and differentiation as well as extensive neurite outgrowth. Furthermore, they are permissive substrates for functional synapse formation between the attached neurons. That primary rat neurons form active synapses on such scaffold surfaces in situ suggests these scaffolds could be useful for tissue engineering applications. The buoyant sapeptide scaffolds with attached cells in culture can be transported readily from one environment to another. Furthermore, these peptides did not elicit a measurable immune response or tissue inflammation when introduced into animals. These biological materials created through molecular design and self assembly may be developed as a biologically compatible scaffold for tissue repair and tissue engineering.     

Cartilage Tissue Engineering with Silk Fibroin Scaffolds Fabricated by Indirect Additive Manufacturing Technology

Advanced tissue engineering (TE) technology based on additive manufacturing (AM) can fabricate scaffolds with a three-dimensional (3D) environment suitable for cartilage regeneration. Specifically, AM technology may allow the incorporation of complex architectural features. The present study involves the fabrication of 3D TE scaffolds by an indirect AM approach using silk fibroin (SF). From scanning electron microscopic observations, the presence of micro-pores and interconnected channels within the scaffold could be verified, resulting in a TE scaffold with both micro- and macro-structural features. The intrinsic properties, such as the chemical structure and thermal characteristics of SF, were preserved after the indirect AM manufacturing process. In vitro cell culture within the SF scaffold using porcine articular chondrocytes showed a steady increase in cell numbers up to Day 14. The specific production (per cell basis) of the cartilage-specific extracellular matrix component (collagen Type II) was enhanced with culture time up to 12 weeks, indicating the re-differentiation of chondrocytes within the scaffold. Subcutaneous implantation of the scaffold-chondrocyte constructs in nude mice also confirmed the formation of ectopic cartilage by histological examination and immunostaining.     

Platelet lysate 3D scaffold supports mesenchymal stem cell chondrogenesis: An improved approach in cartilage tissue engineering

Articular lesions are still a major challenge in orthopedics because of cartilage's poor healing properties. A major improvement in therapeutics was the development of autologous chondrocytes implantation (ACI), a biotechnology-derived technique that delivers healthy autologous chondrocytes after in vitro expansion. To obtain cartilage-like tissue, 3D scaffolds are essential to maintain chondrocyte differentiated status. Currently, bioactive 3D scaffolds are promising as they can deliver growth factors, cytokines, and hormones to the cells, giving them a boost to attach, proliferate, induce protein synthesis, and differentiate. Using mesenchymal stem cells (MSCs) differentiated into chondrocytes, one can avoid cartilage harvesting. Thus, we investigated the potential use of a platelet-lysate-based 3D bioactive scaffold to support chondrogenic differentiation and maintenance of MSCs. The MSCs from adult rabbit bone marrow (n?=?5) were cultivated and characterized using three antibodies by flow cytometry. MSCs (1?×?10⁵) were than encapsulated inside 60?µl of a rabbit platelet-lysate clot scaffold and maintained in Dulbecco's Modified Eagle Medium Nutrient Mixture F-12 supplemented with chondrogenic inductors. After 21 days, the MSCs-seeded scaffolds were processed for histological analysis and stained with toluidine blue. This scaffold was able to maintain round-shaped cells, typical chondrocyte metachromatic extracellular matrix deposition, and isogenous group formation. Cells accumulated inside lacunae and cytoplasm lipid droplets were other observed typical chondrocyte features. In conclusion, the usage of a platelet-lysate bioactive scaffold, associated with a suitable chondrogenic culture medium, supports MSCs chondrogenesis. As such, it offers an alternative tool for cartilage engineering research and ACI.     

Tissue engineering applications: cartilage lesions repair by the use of autologous chondrocytes

Promising new therapies based on tissue engineering have been recently developed for cartilage repair. The association of biomaterials with autologous chondrocytes expanded in vitro can represent a useful tool to regenerate this tissue. The scaffolds utilised in such therapeutical applications should provide a pre-formed three-dimensional shape, prevent cells from floating out of the defect, have sufficient mechanical strength, facilitate uniform spread of cells and stimulate the phenotype of transplanted cells. Hyaff-11 is a hyaluronic-acid based biodegradable polymer, that has been shown to provide successful cell carrier for tissue-engineered repair. From our findings we can state that human chondrocytes seeded on Hyaff-11 are able to maintain in vitro the characteristic of differentiated cells, expressing and producing collagen type II and aggrecan which are the main markers of cartilage phenotype, down-regulating collagen type I. Moreover, it seems to be a useful scaffold for cartilage repair both in animal models and clinical trials in humans, favouring the formation of a hyaline-like tissue. In the light of these data, we can hypothesise, for the future, the use of autologous chondrocyte transplantation together with gene therapy as a treatment for rheumatic diseases such as osteoarthritis.     

Decellularized Avian Cartilage,a Promising Alternative for Human Cartilage Tissue Regeneration

Articular cartilage defects, and subsequent degeneration, are prevalent and account for the poor quality of life of most elderly persons; they are also one of the main predisposing factors to osteoarthritis. Articular cartilage is an avascular tissue and, thus, has limited capacity for healing and self-repair. Damage to the articular cartilage by trauma or pathological causes is irreversible. Many approaches to repair cartilage have been attempted with some potential; however, there is no consensus on any ideal therapy. Tissue engineering holds promise as an approach to regenerate damaged cartilage. Since cell adhesion is a critical step in tissue engineering, providing a 3D microenvironment that recapitulates the cartilage tissue is vital to inducing cartilage regeneration. Decellularized materials have emerged as promising scaffolds for tissue engineering, since this procedure produces scaffolds from native tissues that possess structural and chemical natures that are mimetic of the extracellular matrix (ECM) of the native tissue. In this work, we present, for the first time, a study of decellularized scaffolds, produced from avian articular cartilage (extracted from Gallus Gallus domesticus), reseeded with human chondrocytes, and we demonstrate for the first time that human chondrocytes survived, proliferated and interacted with the scaffolds. Morphological studies of the decellularized scaffolds revealed an interconnected, porous architecture, ideal for cell growth. Mechanical characterization showed that the decellularized scaffolds registered stiffness comparable to the native cartilage tissues. Cell growth inhibition and immunocytochemical analyses showed that the decellularized scaffolds are suitable for cartilage regeneration.     

Culture of chondrocytes in alginate surrounded by fibrin gel: characteristics of the cells over a period of eight weeks

OBJECTIVE: To produce tissue engineered cartilage by human articular chondrocytes in vitro for further use in in vivo manipulations for the treatment of cartilage defects. METHODS: Human articular chondrocytes were cultured in 0.5%, 1.0%, and 2.0% of alginate for up to four weeks. The optimal concentration of an alginate matrix for cell replication and for aggrecan synthesis by chondrocytes was determined. DNA content in the different culture conditions was measured after two and four weeks. Aggrecan synthesis rates and accumulation in the surrounding extracellular matrix were assessed by [(35)S]sulphate incorporation after the same periods of culture. To follow the outgrowth of chondrocytes from the alginate beads, chondrocytes were cultured for four weeks in 0.5 or 1.0% alginate surrounded by 0.25 or 0.5% fibrin gel. DNA content of each culture was measured after different culture periods. Finally, human chondrocytes in 1.0% alginate beads were embedded in 0.5% fibrin gel for eight weeks. Immunohistochemical analysis for aggrecan, type I and II collagen was performed weekly. RESULTS: At two weeks the DNA content in each culture significantly increased in 0.5 and 1.0% alginate cultures in comparison with baseline values. This increase continued until week 4 at the three alginate concentrations. Aggrecan synthesis at two weeks was highest in 0.5 and 1.0% alginate cell cultures. At four weeks aggrecan synthesis rates decreased independently of the alginate concentrations. Aggrecan mainly accumulated in the interterritorial matrix. Proliferation of chondrocytes in alginate and outgrowth of these cells in the surrounding fibrin gel were evident throughout the culture period. The accumulation of aggrecan and type II collagen around the cells, in alginate as well as in fibrin gel, gradually increased over the culture period. Type I collagen appeared after six weeks in alginate and in the surrounding fibrin. CONCLUSION: Human chondrocytes proliferate in this culture system, show an outgrowth into the surrounding fibrin, and synthesise a cartilage-like matrix for up to eight weeks.     

Dynamic reassembly of peptide RADA16 nanofiber scaffold

Nanofiber structures of some peptides and proteins as biological materials have been studied extensively, but their molecular mechanism of self-assembly and reassembly still remains unclear. We report here the reassembly of an ionic self-complementary peptide RADARADARADARADA (RADA16-I) that forms a well defined nanofiber scaffold. The 16-residue peptide forms stable beta-sheet structure and undergoes molecular self-assembly into nanofibers and eventually a scaffold hydrogel consisting of >99.5% water. In this study, the nanofiber scaffold was sonicated into smaller fragments. Circular dichroism, atomic force microscopy, and rheology were used to follow the kinetics of the reassembly. These sonicated fragments not only quickly reassemble into nanofibers that were indistinguishable from the original material, but their reassembly also correlated with the rheological analyses showing an increase of scaffold rigidity as a function of nanofiber length. The disassembly and reassembly processes were repeated four times and, each time, the reassembly reached the original length. We proposed a plausible sliding diffusion model to interpret the reassembly involving complementary nanofiber cohesive ends. This reassembly process is important for fabrication of new scaffolds for 3D cell culture, tissue repair, and regenerative medicine.     

Autologous chondrocytes transplantation

Hunter's observation in 1743 that cartilage "once destroyed, is not repaired" has not essentially changed for two and a half centuries. At present, there is no well-established procedure for the repair of cartilage defect with articular cartilage. 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. Good clinical results suggest that this technique should be a promising procedure for repairing articular cartilage defect.     

Marrow-derived cells populate scaffolds composed of xenogeneic extracellular matrix.

INTRODUCTION: The source of cells that participate in wound repair directly affects outcome. The extracellular matrix (ECM) and other acellular biomaterials have been used as therapeutic scaffolds for cell attachment and proliferation and as templates for tissue repair. The ECM consists of structural and functional proteins that influence cell attachment, gene expression patterns, and the differentiation of cells. OBJECTIVE: The objective of this study was to determine if the composition of acellular matrix scaffolds affects the recruitment of bone marrow-derived cellular elements that populate the scaffolds in vivo. METHODS: Scaffolds composed of porcine tissue ECM, purified Type I collagen, poly(L)lactic coglycolic acid (PLGA), or a mixture of porcine ECM and PLGA were implanted into subcutaneous pouches on the dorsum of mice. The origin of cells that populated the matrices was determined by first performing bone marrow transplantation to convert the marrow of glucose phosphate isomerase 1b (Gpi-1(b)) mice to cells expressing glucose phosphate isomerase 1a (Gpi-1(a)). RESULTS: A significant increase in Gpi-1(a) expressing cells was present in sites implanted with the porcine ECM compared to sites implanted with either Type I collagen or PLGA. Use of recipient mice transplanted with marrow cells that expressed beta-galactosidase confirmed that the majority of cells that populated and remodeled the naturally occurring porcine ECM were marrow derived. Addition of porcine ECM to the PLGA scaffold caused a significant increase in the number of marrow-derived cells that became part of the remodeled implant site. CONCLUSION: The composition of bioscaffolds affects the cellular recruitment pattern during tissue repair. ECM scaffolds facilitate the recruitment of marrow-derived cells into sites of remodeling.     

透明质酸保护组织工程软骨拮抗硝普钠抑制作用的机制研究

目的 研究透明质酸对壳聚糖复合支架与再分化软骨细胞构建的组织工程软骨的保护作用.方法 藻酸钠微球包被体外单层扩增培养的去分化软骨细胞2周以恢复其表型,Ⅱ型胶原蛋白表达的免疫组织化学监测分化状态.扫描电镜观察再分化软骨细胞在壳聚糖复合支架上的生长,3周后用硝普钠或(和)透明质酸以及β1整合素特异性阻断抗体作用于该组织工程软骨,反转录聚合酶链反应(RT-PCR)和Westem blot检测软骨特异性的Ⅱ型胶原或聚集蛋白聚糖(aggrecan)的表达.结果 藻酸钠微球包被去分化软骨细胞2周能显著提高Ⅱ型胶原的表达.壳聚糖支架支持再分化软骨细胞的贴附、增殖和迁徙.硝普钠呈剂量依赖性地抑制组织工程软骨上的软骨细胞Ⅱ型胶原和aggrecan mRNA的表达,而透明质酸显著提高Ⅱ型胶原和aggrecan mRNA的表达.当用特异性抗体阻断β1整合素后,透明质酸不能逆转硝普钠对组织工程软骨中Ⅱ型胶原蛋白表达的抑制作用.结论 藻酸钠微球包被去分化的软骨细胞2周能恢复细胞的表型.透明质酸通过β1整合素信号通路拮抗低浓度硝普钠对组织工程软骨的抑制作用,保护软骨组织.     

Patient-to-Patient Variability in Autologous Pericardial Matrix Scaffolds for Cardiac Repair

The pursuit of alternate therapies for end-stage heart failure post-myocardial infarction has led to the development of a variety of in situ gelling materials to be used as cellular or acellular scaffolds for cardiac repair. Previously, a protocol was established to decellularize human and porcine pericardia and process the extracellular matrix (ECM) into an injectable form. The resulting gels were found to retain components of the native extracellular matrix; cell infiltration was facilitated in vivo, and neovascularization was observed by 2 weeks. However, the assertion that an injectable form of human pericardial tissue could be a potentially autologous scaffold for myocardial tissue engineering requires assessment of the patient-to-patient variability. With this work, seven human pericardia from a relevant patient demographic are processed into injectable matrix materials that gel when brought to physiologic conditions. The resulting materials are compared with respect to their protein composition, glycosaminoglycan content, in vitro degradation, in vivo gelation, and microstructure. It is observed that a diminished collagen content in a subset of samples prevents in vitro gelation but not in vivo gelation at lower ECM concentrations. The structure is similarly fibrous and porous across all samples, implying the cell infiltration may be similarly facilitated. The biochemical composition as characterized by tandem mass spectrometry is comparable; basic ECM components are conserved across all samples, and the presence of a wide variety of ECM proteins and glycoproteins demonstrate the retention of biochemical complexity post-processing. It is concluded that the variability within human pericardial tissue specimens does not prevent them from being processed into injectable scaffolds; therefore, pericardial tissue offers a promising source as an autologous, injectable biomaterial scaffold.     

Engineered cartilage generated by nasal chondrocytes is responsive to physical forces resembling joint loading

OBJECTIVE: To determine whether engineered cartilage generated by nasal chondrocytes (ECN) is responsive to different regimens of loading associated with joint kinematics and previously shown to be stimulatory of engineered cartilage generated by articular chondrocytes (ECA). METHODS: Human nasal and articular chondrocytes, harvested from 5 individuals, were expanded and cultured for 2 weeks into porous polymeric scaffolds. The resulting ECN and ECA were then maintained under static conditions or exposed to the following loading regimens: regimen 1, single application of cyclic deformation for 30 minutes; regimen 2, intermittent application of cyclic deformation for a total of 10 days, followed by static culture for 2 weeks; regimen 3, application of surface motion for a total of 10 days. RESULTS: Prior to loading, ECN constructs contained significantly higher amounts of glycosaminoglycan (GAG) and type II collagen compared with ECA constructs. ECN responded to regimen 1 by increasing collagen and proteoglycan synthesis, to regimen 2 by increasing the accumulation of GAG and type II collagen as well as the dynamic modulus, and to regimen 3 by increasing the expression of superficial zone protein, at the messenger RNA level and the protein level, as well as the release of hyaluronan. ECA constructs were overall less responsive to all loading regimens, likely due to the lower extracellular matrix content. CONCLUSION: Human ECN is responsive to physical forces resembling joint loading and can up-regulate molecules typically involved in joint lubrication. These findings should prompt future in vivo studies exploring the possibility of using nasal chondrocytes as a cell source for articular cartilage repair.     

The pathophysiology of osteoarthritis

Osteoarthritis (OA) is a complex disease whose pathogenesis includes the contribution of biomechanical and metabolic factors which, altering the tissue homeostasis of articular cartilage and subchondral bone, determine the predominance of destructive over productive processes. A key role in the pathophysiology of articular cartilage is played by cell/extra-cellular matrix (ECM) interactions, which are mediated by cell surface integrins. In a physiologic setting, integrins modulate cell/ECM signaling, essential for regulating growth and differentiation and maintaining cartilage homeostasis. During OA, abnormal integrin expression alters cell/ECM signaling and modifies chondrocyte synthesis, with the following imbalance of destructive cytokines over regulatory factors. IL-1, TNF-alpha and other pro-catabolic cytokines activate the enzymatic degradation of cartilage matrix and are not counterbalanced by adequate synthesis of inhibitors. The main enzymes involved in ECM breakdown are metalloproteinases (MMPs), which are sequentially activated by an amplifying cascade. MMP activity is partially inhibited by the tissue inhibitors of MMPs (TIMPs), whose synthesis is low compared with MMP production in OA cartilage. Intriguing is the role of growth factors such as TGF-beta, IFG, BMP, NGF, and others, which do not simply repair the tissue damage induced by catabolic factors, but play an important role in OA pathogenesis.     

Modulation of fibroblast-mediated cartilage degradation by articular chondrocytes in rheumatoid arthritis

OBJECTIVE: To determine the role of chondrocytes and factors released from chondrocytes in cartilage destruction by fibroblast-like synoviocytes (FLS) derived from patients with rheumatoid arthritis (RA). METHODS: RA FLS from 2 patients were implanted into SCID mice, together with fresh articular cartilage or with cartilage that had been stored for 24 hours at 4 degrees C or at 37 degrees C. The invasion of the same RA FLS into the fresh and stored cartilage was compared histologically using a semiquantitative scoring system. In addition, we investigated whether protein synthesis in chondrocytes affects the invasion of RA FLS in vitro. A 3-dimensional cartilage-like matrix formed by cultured chondrocytes was labeled with 35S. After formation of the cartilage-like matrix, protein synthesis was blocked with cycloheximide. The invasion of RA FLS from 6 patients into cycloheximide-treated and untreated matrix was assessed by measuring the released radioactivity in coculture with and without interleukin-1beta (IL-1beta) and tumor necrosis factor alpha (TNFalpha). RESULTS: The SCID mouse experiments showed a significant invasion of RA FLS into the cartilage (overall mean score 3.2) but revealed significant differences when the invasion of the same RA FLS into fresh and stored cartilage was compared. RA FLS that were implanted with fresh articular cartilage showed a significantly higher invasiveness than those implanted with pieces of cartilage that had been stored for 24 hours (overall mean score 2.3). Storage at 37 degrees C and 4 degrees C resulted in the same reduction of invasion (35% and 37%, respectively). In the in vitro experiments, RA FLS rapidly destroyed the cartilage-like matrix. Blocking of chondrocyte protein biosynthesis significantly decreased the invasion of RA FLS, as shown by a decreased release of radioactivity. Addition of IL-1beta, but not TNFalpha, to the cocultures partially restored the invasiveness of RA FLS. CONCLUSION: These data underline the value of the SCID mouse in vivo model of rheumatoid cartilage destruction and demonstrate that chondrocytes contribute significantly to the degradation of cartilage by releasing factors that stimulate RA FLS. Among those, IL-1beta-mediated mechanisms might be of particular importance.     

Technology Insight: adult stem cells in cartilage regeneration and tissue engineering

Articular cartilage, the load-bearing tissue of the joint, has limited repair and regeneration potential. The scarcity of treatment modalities for large chondral defects has motivated attempts to engineer cartilage tissue constructs that can meet the functional demands of this tissue in vivo. Cartilage tissue engineering requires three components: cells, scaffold, and environment. Adult stem cells, specifically multipotent mesenchymal stem cells, are considered the cell type of choice for tissue engineering, because of the ease with which they can be isolated and expanded and their multilineage differentiation capabilities. Successful outcome of cell-based cartilage tissue engineering ultimately depends on the proper differentiation of stem cells into chondrocytes and the assembly of the appropriate cartilaginous matrix to achieve the load-bearing capabilities of the natural articular cartilage. Multiple requirements, including growth factors, signaling molecules, and physical influences, need to be met. Adult mesenchymal stem-cell-based tissue engineering is a promising technology for the development of a transplantable cartilage replacement to improve joint function.     

Joint cartilage regeneration by tissue engineering

The research field of tissue engineering combines cells biology, biomaterial science, and surgery. Major long-term goals are tissue and organ replacement therapies using the patients‘ own cells. Our work is focused on the treatment of severe joint defects and on plastic surgery using in vitro engineered cartilage tissues. The practical approaches in cartilage engineering face problems with three-dimensional cell distribution or cell immobilization raising biocompatibility problems. The tissue engineering of cartilage is based on combining biocompatible cell embedding substances such as fibrin, agarose, alginate, hyaluronic acid and fiber fleece scaffolds of poly α-hydroxy acids (PLLA/PGLA). Different technical approaches were established: a) three-dimensional in vitro cultures of chondrocytes for the development of vital tissue transplants and b) interacting three-dimensional cultures consisting of different cell populations, such as BMP-transfected mesenchymal cells. The preshaped artificial tissue constructs were cultured in perfusion chambers to maintain a stable diffusion of nutrients during the in vitro pre-formation step. Subsequently, pre-formed tissues were implanted into nude mice and into 4mm articular joint defects of rabbits. Transplants were found to produce cartilage typic morphological patterns and matrix. 80% of the transplants remained stable in vivo. However, 20% of the tissues are resorbed or replaced by a fibrous tissue. These results demonstrate that current artificial cartilage transplants are already feasible for plastic reconstruction. The treatment of severe joint defects, however, faces additional problems which are addressed in ongoing studies: (a) the fixation of engineered cartilage in joints, (b) the protection against chronic inflammatory degradation, and (c) the required enormous mechanical stability     

Joint cartilage regeneration by tissue engineering

Summary The research field of tissue engineering combines cells biology, biomaterial science, and surgery. Major long-term goals are tissue and organ replacement therapies using the patients‘ own cells. Our work is focused on the treatment of severe joint defects and on plastic surgery using in vitro engineered cartilage tissues. The practical approaches in cartilage engineering face problems with three-dimensional cell distribution or cell immobilization raising biocompatibility problems. The tissue engineering of cartilage is based on combining biocompatible cell embedding substances such as fibrin, agarose, alginate, hyaluronic acid and fiber fleece scaffolds of poly α-hydroxy acids (PLLA/PGLA). Different technical approaches were established: a) three-dimensional in vitro cultures of chondrocytes for the development of vital tissue transplants and b) interacting three-dimensional cultures consisting of different cell populations, such as BMP-transfected mesenchymal cells. The preshaped artificial tissue constructs were cultured in perfusion chambers to maintain a stable diffusion of nutrients during the in vitro pre-formation step. Subsequently, pre-formed tissues were implanted into nude mice and into 4mm articular joint defects of rabbits. Transplants were found to produce cartilage typic morphological patterns and matrix. 80% of the transplants remained stable in vivo. However, 20% of the tissues are resorbed or replaced by a fibrous tissue. These results demonstrate that current artificial cartilage transplants are already feasible for plastic reconstruction. The treatment of severe joint defects, however, faces additional problems which are addressed in ongoing studies: (a) the fixation of engineered cartilage in joints, (b) the protection against chronic inflammatory degradation, and (c) the required enormous mechanical stability      

Physiological levels of hydrocortisone maintain an optimal chondrocyte extracellular matrix metabolism

OBJECTIVE: To investigate the effects of physiological doses of hydrocortisone on synthesis and turnover of cell associated matrix (CAM) by human chondrocytes obtained from normal articular cartilage. METHODS: Human articular cartilage cells were obtained from visually intact cartilage of the femoral condyles of five donors and maintained in culture for one week to reach equilibrium in accumulated CAM compounds. 0, 0.05, 0.20, and 1.0 micro g/ml hydrocortisone was added to the nutrient media during the entire culture period. Cells were liberated and levels of CAM aggrecan, type II collagen, and fibronectin, of intracellular IGF-1, IL1alpha and beta, and of their respective plasma membrane bound receptors IGFR1, IL1RI, and the decoy receptor IL1RII, were assayed by flow cytometry. RESULTS: In comparison with controls, hydrocortisone treated chondrocytes, at all concentrations, expressed significantly higher plasma membrane bound IGFR1. Intracellular IGF-1 levels remained unchanged. Together with these changes, reflecting an increased ability to synthesise extracellular matrix (ECM) macromolecules, hydrocortisone treated cells expressed significantly higher amounts of the plasma membrane bound decoy IL1RII. Concurrently, intracellular IL1alpha and beta levels and membrane bound IL1RI were down regulated. Levels of CAM aggrecan, type II collagen, and fibronectin were significantly up regulated in the chondrocytes treated with hydrocortisone. CONCLUSION: 0.05 micro g/ml hydrocortisone treated chondrocytes had decreased catabolic signalling pathways and showed an enhanced ability to synthesise ECM macromolecules. Because IL1 activity was decreased and the expression of IL1RII decoy receptor enhanced, more of the ECM macromolecules produced remained accumulated in the CAM of the chondrocytes. The effects were obtained at doses comparable with physiological plasma levels of hydrocortisone in humans.