Insights into the present and future of cartilage regeneration and joint repair.

Knee osteoarthritis is the most common joint disease. It causes pain and suffering for affected patients and is the source of major economic costs for healthcare systems. Despite ongoing research, there is a lack of knowledge regarding disease mechanisms, biomarkers, and possible cures. Current treatments do not fulfill patients' long-term needs, and it often requires invasive surgical procedures with subsequent long periods of rehabilitation. Researchers and companies worldwide are working to find a suitable cell source to engineer or regenerate a functional and healthy articular cartilage tissue to implant in the damaged area. Potential cell sources to accomplish this goal include embryonic stem cells, mesenchymal stem cells, or induced pluripotent stem cells. The differentiation of stem cells into different tissue types is complex, and a suitable concentration range of specific growth factors is vital. The cellular microenvironment during early embryonic development provides crucial information regarding concentrations of signaling molecules and morphogen gradients as these are essential inducers for tissue development. Thus, morphogen gradients implemented in developmental protocols aimed to engineer functional cartilage tissue can potentially generate cells comparable to those within native cartilage. In this review, we have summarized the problems with current treatments, potential cell sources for cell therapy, reviewed the progress of new treatments within the regenerative cartilage field, and highlighted the importance of cell quality, characterization assays, and chemically defined protocols.

GDF5 reduces MMP13 expression in human chondrocytes via DKK1 mediated canonical Wnt signaling inhibition.

OBJECTIVE: Growth differentiation factor 5 (GDF5) is important for joint formation and associated with osteoarthritis (OA). Its role for the homeostasis of cartilage extracellular matrix (ECM) is, however, unknown. The canonical Wnt signaling pathway is also implemented in OA and activation of the pathway has detrimental effects on the cartilage ECM. The objective of this study was to investigate the effect of GDF5 stimulation on the Wnt signaling pathway and on the expression of known modulators of cartilage ECM. DESIGN: Human chondrocytes were cultured in the pellet mass system and stimulated with increasing concentrations of GDF5. Expression of matrix modulating enzymes and canonical Wnt inhibitors dickkopf 1 (DKK1) and frizzled related protein (FRZB) were measured with quantitative PCR (qPCR). Protein levels of matrix metalloprotease 13 (MMP13), DKK1 and ß-catenin were measured with enzyme-linked immunosorbent assay (ELISA). Canonical Wnt signaling was stimulated with Wnt3a and small molecule CHIR-99021 and DKK1 was blocked with small molecule WAY-262611. RESULTS: In this study, we show that GDF5 stimulation of human chondrocytes inhibits expression of the cartilage ECM degrading enzymes MMP13 and ADAMTS4 and stimulates the expression of cartilage anabolic genes ACAN and SOX9. We further show that the stimulation inhibits the canonical Wnt signaling pathway through expression of the canonical Wnt inhibitors DKK1 and FRZB. Finally we show that inhibition of MMP13 expression through GDF5 stimulation is mediated by DKK1. CONCLUSION: Herein, we provide evidence of a previously unknown link between GDF5 signaling and canonical Wnt signaling that may contribute to the understanding of the molecular mechanisms of OA.

Cartilage Repair in Football (Soccer) Athletes: What Evidence Leads to Which Treatment? A Critical Review of the Literature.

The prevalence of focal articular cartilage lesions among athletes is higher than in the general population. Treatment goals differ considerably between the professional and recreational athlete. High financial stakes and the short duration of a professional career influence the treatment selection for the professional athlete, while such parameters weigh differently in recreational sports. This article describes our investigation of the relation between sports and a high prevalence of focal cartilage lesions. In addition, we provide a critical review of the best available evidence for cartilage surgery and treatment selection, evaluate specific patient profiles for professional and recreational athletes, and propose a treatment algorithm for the treatment of focal cartilage lesions in football (soccer) players.

Characteristic Markers of the WNT Signaling Pathways Are Differentially Expressed in Osteoarthritic Cartilage.

OBJECTIVE: It is well known that expression of markers for WNT signaling is dysregulated in osteoarthritic (OA) bone. However, it is still not fully known if the expression of these markers also is affected in OA cartilage. The aim of this study was therefore to examine this issue. METHODS: Human cartilage biopsies from OA and control donors were subjected to genome-wide oligonucleotide microarrays. Genes involved in WNT signaling were selected using the BioRetis database, KEGG pathway analysis was searched using DAVID software tools, and cluster analysis was performed using Genesis software. Results from the microarray analysis were verified using quantitative real-time PCR and immunohistochemistry. In order to study the impact of cytokines for the dysregulated WNT signaling, OA and control chondrocytes were stimulated with interleukin-1 and analyzed with real-time PCR for their expression of WNT-related genes. RESULTS: Several WNT markers displayed a significantly altered expression in OA compared to normal cartilage. Interestingly, inhibitors of the canonical and planar cell polarity WNT signaling pathways displayed significantly increased expression in OA cartilage, while the Ca(2+)/WNT signaling pathway was activated. Both real-time PCR and immunohistochemistry verified the microarray results. Real-time PCR analysis demonstrated that interleukin-1 upregulated expression of important WNT markers. CONCLUSIONS: WNT signaling is significantly affected in OA cartilage. The result suggests that both the canonical and planar cell polarity WNT signaling pathways were partly inhibited while the Ca(2+)/WNT pathway was activated in OA cartilage.

Influence of pore size on the redifferentiation potential of human articular chondrocytes in poly(urethane urea) scaffolds.

The chemical and physical properties of scaffolds affect cellular behaviour, which ultimately determines the performance and outcome of tissue-engineered cartilage constructs. The objective of this study was to assess whether a degradable porous poly(urethane urea) scaffold could be a suitable material for cartilage tissue engineering. We also investigated whether the post-expansion redifferentiation and cartilage tissue formation of in vitro expanded adult human chondrocytes could be regulated by controlled modifications of the scaffold architecture. Scaffolds with different pore sizes, < 150 µm, 150-300 µm and 300-500 µm, were seeded with chondrocytes and subjected to chondrogenic and osteogenic induction in vitro. The poly(urethane urea) scaffold with the smaller pore size enhanced the hyaline-like extracellular matrix and thus neocartilage formation. Conversely, the chondrocytes differentiated to a greater extent into the osteogenic pathway in the scaffold with the larger pore size. In conclusion, our results demonstrate that poly(urethane urea) may be useful as a scaffold material in cartilage tissue engineering. Furthermore, the chondrogenic and the osteogenic differentiation capacity of in vitro expanded human articular chondrocytes can be influenced by the scaffold architecture. By tailoring the pore sizes, the performance of the tissue-engineered cartilage constructs might be influenced and thus also the clinical outcome in the long run.

Debridement of cartilage lesions before autologous chondrocyte implantation by open or transarthroscopic techniques: a comparative study using post-mortem materials.

We compared the quality of debridement of chondral lesions performed by four arthroscopic (SH, shaver; CU, curette; SHCU, shaver and curette; BP, bipolar electrodes) and one open technique (OPEN, scalpel and curette) which are used prior to autologous chondrocyte implantation (ACI). The ex vivo simulation of all five techniques was carried out on six juvenile equine stifle joints. The OPEN, SH and SHCU techniques were tested on knees harvested from six adult human cadavers. The most vertical walls with the least adjacent damage to cartilage were obtained with the OPEN technique. The CU and SHCU methods gave inferior, but still acceptable results whereas the SH technique alone resulted in a crater-like defect and the BP method undermined the cartilage wall. The subchondral bone was severely violated in all the equine samples which might have been peculiar to this model. The predominant depth of the debridement in the adult human samples was at the level of the calcified cartilage. Some minor penetrations of the subchondral end-plate were induced regardless of the instrumentation used. Our study suggests that not all routine arthroscopic instruments are suitable for the preparation of a defect for ACI. We have shown that the preferred debridement technique is either open or arthroscopically-assisted manual curettage. The use of juvenile equine stifles was not appropriate for the study of the cartilage-subchondral bone interface.

Genome-wide expression profiling reveals new candidate genes associated with osteoarthritis.

INTRODUCTION: Although the extracellular matrix (ECM) is the functional element in articular cartilage and its degradation is central in the pathogenetic process in osteoarthritis (OA), increasing the knowledge about the cellular OA phenotype is essential. The aim of this study is therefore to provide a more complete picture of the cellular and molecular alterations detected in OA cartilage. MATERIAL AND METHODS: Human articular cartilage biopsies were collected from donors with macroscopical and microscopical signs of OA as well as donors with no previous history of OA and with microscopically intact cartilage. RNA was isolated from the biopsies and subjected to whole genome microarray analysis. Important results from the microarray analysis were verified using real-time PCR and immunohistochemistry. RESULTS: Our results reveal several new candidate genes not previously associated with OA to display significantly higher expression in OA cartilage than in normal donor cartilage, including genes involved in bone formation (CLEC3B, CDH11, GPNMB, CLEC3A, CHST11, MSX1, MSX2) and genes encoding collagens (COL13A1, COL14A1, COL15A1, COL8A2). DISCUSSION: This study is the first to report a comprehensive gene expression analysis of human OA cartilage compared to control cartilage from donors lacking macroscopical and microscopical signs of OA using recently developed microarrays containing the whole human genome. Our results could broadly confirm previously published data on many characteristic features of OA as well as adding a panel of genes to the list of genes known to be differentially expressed in OA. Elucidation of the phenotypical alterations occurring in OA chondrocytes is important for the development of effective treatments for OA.

Clonal populations of chondrocytes with progenitor properties identified within human articular cartilage.

The aim of the present study was to identify and characterize progenitor properties of human articular chondrocytes selected by using agarose suspension culture. In this chondrogenic selective culture condition, about 3.6% of seeded surplus chondrocytes from patients undergoing articular chondrocyte transplantation proliferated and formed cell clusters after 6 weeks. Phase-contrast microscopy and transmission electron microscopy revealed four different types of cell clusters differing in cellular content and matrix production. Based on their morphological features, they were named the homogenous (H), the homogenous matrix (HM), the differentiated matrix (DM) and the differentiated (D) cell clusters. All cell clusters showed positive safranin O staining, and matrix was positive for antibodies detecting type II collagen and aggrecan. The clusters were further demonstrated to express the genes for fibroblast growth factor receptor 3, type IIA collagen and type IIB collagen, while type X collagen was not expressed. After subcloning, the H and HM clusters demonstrated the best proliferative capacity. Chondrocytes from these two cell clusters also showed phenotypic plasticity in chondrogenic, adipogenic as well as osteogenic assays. This study demonstrates that existing subpopulations of cells with chondroprogenitor properties can be isolated from human adult articular cartilage using agarose suspension cultures.

Clonal growth of human articular cartilage and the functional role of the periosteum in chondrogenesis.

OBJECTIVE: Clinical cartilage repair with transplantation of cultured chondrocytes, the first described technique introduced in 1994, includes a periosteal membrane but today cells are also implanted without the periosteal combination. The aim of this study was to see if the periosteum had more than a biomechanical function and if the periosteum had a biological effect on the seeded cells tested in an agarose system in which the clonal growth in agarose and the external growth stimulation could be analysed. METHODS: Four different experiments were used to study the growth of human chondrocytes in agarose and the periosteal influence. Human chondrocytes were isolated and transferred to either primary or secondary agarose culture. After 4 weeks, the total number of clones >50 microm was counted. Cocultures of chondrocytes and periosteal tissue, cultures of chondrocytes with conditioned medium from chondrocytes, periosteal cells and fibroblast were used to study a potential stimulatory effect on growth and different cytokines and growth factors were analysed. RESULTS: It was found that the human chondrocytes had different growth properties in agarose with the formation of four different types of clones: a homogenous clone without matrix production, a homogenous clone with matrix production, a differentiated clone with matrix production and finally a differentiated clone without matrix production. The periosteum exerted a paracrine effect on cultured chondrocytes in agarose resulting in a higher degree of cloning. The chondrocytes produced significant amounts of interleukin (IL)-6, IL-8, granulocyte-macrophage colony-stimulating factor (GM-CSF) and transforming growth factor (TGF)-beta. The periosteum produced significant amounts of IL-6, IL-8 and TGF-beta. Cocultures of chondrocytes and periosteum demonstrated a potentiation of IL-6 and IL-8 release but not of TGF-beta and GM-CSF. CONCLUSION: Articular chondrocytes are able to form clones of different properties in agarose and the periosteum has a capacity of stimulating chondrocyte clonal growth and differentiation and secretes significant amounts of IL-6, IL-8, GM-CSF and TGF-beta. It may be that the repair of cartilage defects with seeded chondrocytes could benefit from the combination with a periosteal graft. The production of TGF-beta by implanted chondrocytes could influence the chondrogenic cells in the periosteum to start a periosteal chondrogenesis and together with the matrix from implanted chondrocyte production, a repair of cartilaginous appearance may develop; a dual chondrogenic response is possible.

Bacterial cellulose as a potential scaffold for tissue engineering of cartilage.

Tissue constructs for cartilage with native mechanical properties have not been described to date. To address this need the bacterial cellulose (BC) secreted by Gluconacetobacter xylinus (= Acetobacter xylinum) was explored as a novel scaffold material due to its unusual material properties and degradability. Native and chemically modified BC materials were evaluated using bovine chondrocytes. The results indicate that unmodified BC supports chondrocyte proliferation at levels of approximately 50% of the collagen type II substrate while providing significant advantages in terms of mechanical properties. Compared to tissue culture plastic and calcium alginate, unmodified BC showed significantly higher levels of chondrocyte growth. Chemical sulfation and phosphorylation of the BC, performed to mimic the glucosaminoglycans of native cartilage, did not enhance chondrocyte growth while the porosity of the material did affect chondrocyte viability. The BC did not induce significant activation of proinflammatory cytokine production during in vitro macrophage screening. Hence, unmodified BC was further explored using human chondrocytes. TEM analysis and RNA expression of the collagen II from human chondrocytes indicated that unmodified BC supports proliferation of chondrocytes. In addition, ingrowth of chondrocytes into the scaffold was verified by TEM. The results suggest the potential for this biomaterial as a scaffold for tissue engineering of cartilage.

Health economics benefits following autologous chondrocyte transplantation for patients with focal chondral lesions of the knee.

Autologous chondrocyte transplantation (ACT) for the treatment of cartilage injuries has been in clinical use for several years. Since this new technique is potentially more costly and invasive than traditional conservative therapies, we evaluated the effect of ACT on clinical outcome, absenteeism, disability status, and total direct economic burden in 57 patients with full-thickness chondral lesions of the knee treated between 1987 and 1996. Patients graded good or excellent following ACT in the treatments groups were: femoral condyles (28/33), femoral condyles with anterior cruciate ligament (ACL) repair (5/5), osteochondritis dissecans (7/8), and patellar lesions (9/11). Pre-ACT, 57/57 patients were disabled and post-ACT (mean follow-up 7.3 years) 44/57 had no sickness, 10/57 had minor disability, and 1/57 was disabled. Two of the 57 patients suffered re-injury during the follow-up time. In the 10-year period prior to ACT, the average cost of absenteeism and surgery was SEK 982,457 ($ 122,807) and SEK 47,000 ($ 5,875), respectively, compared to the post-ACT period where both absenteeism and medical costs were dramatically reduced: SEK 9,508 ($ 1,189) and SEK 7,050 ($ 881), respectively. In conclusion, 49 of the 57 patients improved clinically as a result of the ACT treatment. A dramatic cost-saving effect was demonstrated over a projected 10-year period due to reduced absenteeism and disability.

Autologous chondrocytes used for articular cartilage repair: an update.

Articular cartilage in adults has a poor ability to self-repair after a substantial injury; however, it is not known whether there is a cartilage resurfacing technique superior to the existing techniques. It is not satisfactory that at the beginning of the new millennium, there still is a lack of randomized studies comparing different cartilage repair techniques and there still is little knowledge of the natural course of a cartilaginous lesion. To date, various articular cartilage resurfacing techniques have the potential to improve the repair of cartilage defects and reduce the patient's disability. One such cartilage repair technique is autologous chondrocyte transplantation combined with a periosteal graft. Since the first patient was operated on in 1987, much interest in cartilage repair and cell engineering has emerged. The experience with autologous chondrocyte transplantation during the past 13 years with in vitro chondrocyte expansion, cartilage harvest, and postoperative biopsy technique is discussed, and the latest followup of 213 consecutive patients in different subgroups with 2 to 10 years followup is presented. The technique gives stable long-term results with a high percentage of good to excellent results (84%-90%) in patients with different types of single femoral condyle lesions, whereas patients with other types of lesions have a lower degree of success (mean, 74%).

Two- to 9-year outcome after autologous chondrocyte transplantation of the knee.

Autologous cultured chondrocyte transplantation was introduced in Sweden in 1987 for the treatment of large (1.5-12.0 cm2) full thickness chondral defects of the knee. The clinical, arthroscopic, and histologic results from the first 101 patients treated using this technique are reported in this study. Patients were assessed retrospectively using three types of endpoints: patient and physician derived clinical rating scales (five validated and two new); arthroscopic assessment of cartilage fill, integration, and surface hardness; and standard histochemical techniques. Ninety-four patients with 2- to 9-years followup were evaluable. Good to excellent clinical results were seen in individual groups as follows: isolated femoral condyle (92%), multiple lesions (67%), osteochondritis dissecans (89%), patella (65%), and femoral condyle with anterior cruciate ligament repair (75%). Arthroscopic findings in 53 evaluated patients showed good repair tissue fill, good adherence to underlying bone, seamless integration with adjacent cartilage, and hardness close to that of the adjacent tissue. Hypertrophic response of the periosteum or graft or both was identified in 26 arthroscopies; seven were symptomatic and resolved after arthroscopic trimming. Graft failure occurred in seven (four of the first 23 and three of the next 78) patients. Histologic analysis of 37 biopsy specimens showed a correlation between hyalinelike tissue (hyaline matrix staining positive for Type II collagen and lacking a fibrous component) and good to excellent clinical results. The good clinical outcomes of autologous chondrocyte transplantation in this study are encouraging, and clinical trials are being done to assess the outcomes versus traditional fibrocartilage repair techniques.

Autologous chondrocyte transplantation.

The intrinsic capacity of cartilage to repair chondral injuries is poor. Different techniques to induce cartilage repair with the use of extrinsic chondrogeneic cell sources have been explored in experimental models. Cells can be harvested autologously or as allografts from a healthy part of the donor tissue, isolated, expanded in vitro, and finally implanted into the defect in high densities. Pure chondrocytes, epiphyseal or mature, allogeneic or autologous, and other types of mesenchymal cells have been used. The composition and structure of the extracellular cartilage matrix are maintained through a balance of anabolic and catabolic activities controlled by the unique chondrocytes. They keep the cartilage alive; they alone maintain it and regulate it. It therefore seems important to use true committed chondrocytes to repair a local cartilaginous defect. The rational basis for the use of committed autologous chondrocytes in combination with a covering periosteal membrane in the treatment of deep cartilage defects is presented.

Influence of fibrin sealant (Tisseel) on osteochondral defect repair in the rabbit knee.

Fibrin adhesives have been shown to improve the natural repair of musculoskeletal tissues. Growth hormone (GH) has a chondrogenic effect on immature cartilage. To test if a fibrin adhesive with and without GH could improve the natural repair of a joint surface lesion, we made a 9 x 4 mm2 osteochondral defect in the femoral groove of adult New Zealand rabbits. The defect in one of the knees was filled with the fibrin adhesive Tisseel, while the defect in the other knee was left untreated as a control. Another group of rabbits was treated in both knees with fibrin adhesive with local addition of GH during 1 week on one side. The experiments showed that the fibrin treatment impaired the natural repair of the osteochondral defect and that GH addition had no effect on the healing process. In a second in vitro experiment, chondrocyte migration into the fibrin adhesive Tisseel was compared to migration into rabbit and human blood clots. No cell migration was seen into the fibrin adhesive, while there was migration into the blood clots. We conclude that a fibrin adhesive like Tisseel is not suitable as a scaffold to promote repair of osteochondral defects in the rabbit knee.

Rabbit articular cartilage defects treated with autologous cultured chondrocytes.

Adult New Zealand rabbits were used to transplant autologously harvested and in vitro cultured chondrocytes into patellar chondral lesions that had been made previously and were 3 mm in diameter, extending down to the calcified zone. Healing of the defects was assessed by gross examination, light microscope, and histological-histochemical scoring at 8, 12, and 52 weeks. Chondrocyte transplantation significantly increased the amount of newly formed repair tissue compared to the found in control knees in which the lesion was solely covered by a periosteal flap. In another experiment, carbon fiber pads seeded with chondrocytes were used as scaffolds, and repair significantly increased at both 12 and 52 weeks compared to knees in which scaffolds without chondrocytes were implanted. The histologic quality scores of the repair tissue were significantly better in all knees in which defects were treated with chondrocytes compared to knees treated with periosteum alone and better at 52 weeks compared to knees in which defects were treated with carbon scaffolds seeded with chondrocytes. The repair tissue, however, tended to incomplete the bonding to adjacent cartilage. This study shows that isolated autologous articular chondrocytes that have been expanded for 2 weeks in vitro can stimulate the healing phase of chondral lesions. A gradual maturation of the hyalinelike repair with a more pronounced columnarization was noted as late as 1 year after surgery.