Intra-articular administration of hyaluronic acid increases the volume of the hyaline cartilage regenerated in a large osteochondral defect by implantation of a double-network gel

Implantation of PAMPS/PDMAAm double-network (DN) gel can induce hyaline cartilage regeneration in the osteochondral defect. However, it is a problem that the volume of the regenerated cartilage tissue is gradually reduced at 12 weeks. This study investigated whether intra-articular administration of hyaluronic acid (HA) increases the volume of the cartilage regenerated with the DN gel at 12 weeks. A total of 48 rabbits were used in this study. A cylindrical osteochondral defect created in the bilateral femoral trochlea was treated with DN gel (Group DN) or left without any implantation (Group C). In both Groups, we injected 1.0 mL of HA in the left knee, and 1.0 mL of saline solution in the right knee. Quantitative histological evaluations were performed at 2, 4, and 12 weeks, and PCR analysis was performed at 2 and 4 weeks after surgery. In Group DN, the proteoglycan-rich area was significantly greater in the HA-injected knees than in the saline-injected knees at 12 weeks (P = 0.0247), and expression of type 2 collagen, aggrecan, and Sox9 mRNAs was significantly greater in the HA-injected knees than in the saline-injected knees at 2 weeks (P = 0.0475, P = 0.0257, P = 0.0222, respectively). The intra-articular administration of HA significantly enhanced these gene expression at 2 weeks and significantly increased the volume of the hyaline cartilage regenerated by implantation of a DN gel at 12 weeks. This information is important to develop an additional method to increase the volume of the hyaline cartilage tissue in a potential cartilage regeneration strategy using the DN gel.     

A hydrophylic polymer system enhanced articular cartilage regeneration in vivo

This study describes a new method for the repair of large articular cartilage defects in the knee joint and compares the effect of two polymer systems on the quality of the repair tissue. The two systems are a newly developed hydrophylic system, based on poly-ethyl-methacrylate (PEMA) polymer and tetra-hydro-furfuryl-methacrylate (THFMA) monomer and the conventional bone cement polymer system, based on poly-methyl-methacrylate (PMMA) polymer and methyl-methacrylate (MMA) monomer. Thirty adult Sandy-lop rabbits were used. Both knees were operated on in each animal, the one defect received either PEMA/THFMA or conventional bone cement and the contralateral defect received no biomaterial (control group). Femora were retrieved at six weeks and the repair tissue was studied by histology, histochemistry and immuno-histochemistry. PEMA/THFMA enhanced the quality of the repair significantly (p<0.0001). By six weeks hyaline-like articular cartilage was the predominant tissue covering the defects and it was fully integrated with the surrounding normal articular cartilage. Immuno-localization showed cartilage components, including collagen type II, distributed evenly throughout its matrix. PMMA/MMA on the other hand did not improve significantly the repair tissue, which was predominately fibro-cartilaginous, poorly bonded to the adjacent normal articular cartilage. The method of implantation is simple and easily reproducible and the new polymer has been well-accepted by the rabbits.     

Biological responses of novel high-toughness double network hydrogels in muscle and the subcutaneous tissues

The study evaluated biological reaction of four types of novel double network gels in muscle and subcutaneous tissues, using implantation tests according to the international guideline. The implantation tests demonstrated that, although poly (2-acrylamide-2-metyl-propane sulfonic acid)/poly (N,N′-dimetyl acrylamide) (PAMPS/PDMAAm) gel induced a mild inflammation at 1 week, the degree of the inflammation significantly decreased into the same degree as that of the negative control at 4 and 6 weeks. This gel has a potential to be applied as artificial cartilage. In addition, Cellulose/Gelatin gel showed the same degree of inflammation as that of the negative control at 1 week, and then, showed a gradually absorbable property at 4 and 6 weeks. This gel has a potential to be applied as an absorbable implant. The PAMPS/polyacrylamide and Cellulose/PDMAAm gels induced a significant inflammation at each week. These DN gels are difficult to be applied as clinical implants in the current situation.     

Recent Progress in Cartilage Tissue Engineering—Our Experience and Future Directions

Given the limited spontaneous repair that follows cartilage injury, demand is growing for tissue engineering approaches for cartilage regeneration. There are two major applications for tissue-engineered cartilage. One is in orthopedic surgery, in which the engineered cartilage is usually used to repair cartilage defects or loss in an articular joint or meniscus in order to restore the joint function. The other is for head and neck reconstruction, in which the engineered cartilage is usually applied to repair cartilage defects or loss in an auricle, trachea, nose, larynx, or eyelid. The challenges faced by the engineered cartilage for one application are quite different from those faced by the engineered cartilage for the other application. As a result, the emphases of the engineering strategies to generate cartilage are usually quite different for each application. The statuses of preclinical animal investigations and of the clinical translation of engineered cartilage are also at different levels for each application. The aim of this review is to provide an opinion piece on the challenges, current developments, and future directions for cartilage engineering for both applications.     

The growth of chondrocytes using Gelfoam® as a biodegradable scaffold

Successful articular cartilage resurfacing must overcome several problems: the implant must easily fit the defect, it must be stable within the defect before full incorporation of repair tissue has occurred, and the reparative tissue must closely approximate the structure of normal hyaline cartilage. To this end, several natural and synthetic components have been used, both in vivo and in vitro, to provide a scaffold. These include isolated chondrocyte allografts, intact cartilage allografts, periossteal grafts, reconstructed collagen sponges, hydrogels and carbon fibres. However, promising results have been reported using three dimensional scaffolds in culture with isolated chondrocytes with subsequent implantation. This preliminary in vitro study utilizes Gelfoam^® (a purified gelatin sponge) as such a scaffold. The biocompatibility of Gelfoam with both chondrocytes and osteoblast cells was first confirmed. The ability of chondrocytes to replicate and differentiate within Gelfoam scaffolds was assessed biochemically by measurement of the DNA content and glycosaminoglycans (GAG) production over 25 days in culture. The distribution of the cartilagenous matrix produced was observed by light microscopy, and the constituents of this matrix were assessed using specific antibodies and immunolocalization.     

In vivo effects of isolated implantation of salmon-derived crosslinked atelocollagen sponge into an osteochondral defect

We have developed crosslinked salmon-derived atelocollagen (SC) sponge, which has a denaturation temperature of 47°C. Sixty-four knees of 32 mature rabbits were randomly divided into 4 groups after creating an osteochondral defect in the femoral trochlea. Defects in Groups I, II, and III were filled with the crosslinked SC sponge, the crosslinked porcine collagen (PC) sponge, and the non-crosslinked PC sponge, respectively. In Group IV, defects were left untreated as the control. At 12?weeks after implantation, the histological score showed that Group I was significantly greater than Groups III (P?=?0.0196) and IV (P?=?0.0021). In addition, gene expression of type-2 collagen, aggrecan, and SOX9 was the greatest in Group I at 12?weeks. The fundamental in vivo properties of the crosslinked SC sponge showed that this is a promising biomaterial, specifically as a scaffold for cartilage tissue engineering.     

Enhanced mechanical properties of thermosensitive chitosan hydrogel by silk fibers for cartilage tissue engineering

Articular cartilage has limited repair capability following traumatic injuries and current methods of treatment remain inefficient. Reconstructing cartilage provides a new way for cartilage repair and natural polymers are often used as scaffold because of their biocompatibility and biofunctionality. In this study, we added degummed chopped silk fibers and electrospun silk fibers to the thermosensitive chitosan/glycerophosphate hydrogels to reinforce two hydrogel constructs which were used as scaffold for hyaline cartilage regeneration. The gelation temperature and gelation time of hydrogel were analyzed by the rheometer and vial tilting method. Mechanical characterization was measured by uniaxial compression, indentation and dynamic mechanical analysis assay. Chondrocytes were then harvested from the knee joint of the New Zealand white rabbits and cultured in constructs. The cell proliferation, viability, production of glycosaminoglycans and collagen type II were assessed. The results showed that mechanical properties of the hydrogel were significantly enhanced when a hybrid with two layers of electrospun silk fibers was made. The results of GAG and collagen type II in cell-seeded scaffolds indicate support of the chondrogenic phenotype for chondrocytes with a significant increase in degummed silk fiber–hydrogel composite for GAG content and in two-layer electrospun fiber–hydrogel composite for Col II. It was concluded that these two modified scaffolds could be employed for cartilage tissue engineering.     

Differentiation of preosteoblasts using a delivery system with BMPs and bioactive glass microspheres

Bone morphogenetic proteins (BMPs) and 45S5 Bioglass microspheres (bioactive GM) can increase the differentiation of osteoblasts. Recombinant human BMP-2 (rhBMP-2) is presently the BMP most frequently used in delivery systems and it has already been used in clinical bone healing studies. We have developed a delivery system that combines a collagen Type I gel, BMP and bioactive GM. Since BMP-9 seems to be more osteogenic than BMP-2, we compared the differentiation of MC3T3-E1 preosteoblasts induced by our delivery system containing either a peptide derived from BMP-9 (pBMP-9), or rhBMP-2, both at 100 ng/mL. After 5 days, alkaline phosphatase staining showed that pBMP-9 induced more differentiation than rhBMP-2 in all experimental conditions. Also, bioactive GM increased this BMP effect. Since preosteoblasts secreted matrix metalloproteinases (MMPs) that can degrade collagen, we then studied the influence of the delivery system on MMPs production. We observed that MMP-2 was the major MMP involved in all experimental conditions. In addition, pBMP-9 with bioactive GM generated less MMP-2 than did rhBMP-2 on days 3 and 5. Thus, a delivery system using collagen Type I gel with pBMP-9 and bioactive GM seems to be a promising system for bone regeneration.     

The evaluation of collagen gel with various connection states by using MRI

To noninvasively evaluate the connection states of collagen fiber, a characterizing factor of the physical property, is considered to be helpful in the evaluation of cartilage functions. The purpose of this study was to examine how the connection states of collagen influence the MRI parameters by evaluating the collagen gel with various connection states using MRI. MRI was performed to six type I collagen gel samples with various connection status and a water sample. The evaluation parameters included T1 relaxation time, T2 relaxation time, and diffusion coefficient. With regard to gel samples with cross-links, the T2 relaxation time was shortened in proportion to the dose of glutaraldehyde. It is considered that as the glutaraldehyde concentration increases, the distance between protons in water molecules decreases; this is followed by a stronger bipole–bipole interaction, resulting in a shorter T2 relaxation time. The diffusion coefficient for gel samples with cross-links also decreased with increasing glutaraldehyde concentrations. However, gel samples without glutaraldehyde were almost the same as that of the water. This result suggested that the degree of entrapment of water inside the gel samples without cross-links, even when it converted into gel, was found to be nearly equal to that of the free water.     

Comparison of various mixtures of β-chitin and chitosan as a scaffold for three-dimensional culture of rabbit chondrocytes

With the use of a recently created chitosan neutral hydrogel, we have been able to create various mixtures of chitin and chitosan without changing their characteristics even at room temperature. The aim of this study was the initial comparison of various mixtures of β-chitin and chitosan as a scaffold for rabbit chondrocyte culture. We created five types of sponges: pure β-chitin, pure chitosan, 3:1, 1:1, and 1:3 β-chitin-chitosan. The absorption efficiencies of chondrocytes in all five types of sponges were found to be around 98%. The mean concentrations of chondroitin sulfate were statistically different neither at week 2 nor at week 4 postculture between the types of sponges. The content of hydroxyproline in the β-chitin sponge was significantly greater than in other sponges at week 4 postculture. From the histochemical and immunohistochemical findings, the cartilage-like layer in the chondrocytes-sponge composites of all five types of sponges was similar to hyaline cartilage. However, only immunohistochemical staining of type II collagen in the pure β-chitin sponge was closer to normal rabbit cartilage than other types of sponges. The pure β-chitin sponge was superior to other sponges concerning the content of extracellular matrices of collagen.     

Behavior of tissue-engineered human cartilage after transplantation into nude mice

Cartilage lacks the ability to regenerate structural defects. Therefore, autologous grafting has been used routinely to replace cartilaginous lesions. Because tissue engineering of human cartilage with the help of bioresorbable polymer scaffolds is possible in experimental models, the demand for the clinical application grows. In this study we present an analysis of the behavior of transplants made of human chondrocyte pools, agarose and the resorbable polymer scaffold Ethisorb and a preliminary comparison with transplants made of single patients' cells and Ethisorb but without the additional ingredient agarose. Chondrocytes were isolated from the matrix of human septal cartilage by enzymatic digestion. The pool cells were kept in monolayer culture for 2 weeks, the single patients' cells for 3–4 weeks. Chondrocyte pools were suspended in agarose and seeded into the resorbable polymer scaffold Ethisorb. Single patients' cells were seeded without agarose. All cell–polymer constructs were kept in perfusion culture for 10–14 days and transplanted subcutaneously into thymusaplastic nude mice. Additionally we implanted Ethisorb fleeces embedded in agarose without chondrocytes. After 6, 12 and 24 weeks the animals were sacrificed and the specimens were explanted and analyzed histochemically and immunohistochemically. Polymer scaffolds not seeded with chondrocytes did not show cartilage formation. Resorption was complete after 12 weeks in vivo. Transplants from cell pools remained mechanically stable over 24 weeks apart from four transplants that were resorbed completely. Cartilage formation was observed in all pool-specimens with the presence of chondronic structures and a homogeneous matrix containing hyaline cartilage-specific matrix molecules such as collagen type II. Single patients' transplants showed hyaline cartilage matrix synthesis and mechanical stability as well. Chondrocyte pools are a suitable method to study cartilage engineering of human cells in vitro and in vivo in experimental models. Under clinical conditions it is, however, necessary to study the generation of cartilage from single patients' cells. We showed that it is possible without additional ingredients such as agarose. However, variations in the preliminary results show that the clinical application with human cells is more difficult than one would expect when using human chondrocyte pools. Further studies need to be performed to find out which individual factors influence the in vitro engineered cartilage's fate in vivo. © 1999 Kluwer Academic Publishers     

Hydroxyapatite-ceramic for juxta-articular implantation

The histocompatibility of hydroxyapatite-ceramic (HAC) has been proven extensively. For the reconstruction of juxta-articular cancellous bone defects with this synthetic material, the mechanical properties of the HAC-bone regeneration complex needed to be investigated. In order not to alter the specific ability of the articular structures to distribute and absorb loading stress, the physiological force-transmitting performance of the subchondral zone must be achieved by filling the defect within HAC. This study deals with the influence of a physiological load on the remodelling within HAC-filled subchondral bone defects. As orientation is the important factor affecting the physical properties of hard tissue, we show the morphological aspect of functional adaptation of the hydroxyapatite-bone compound determined by the orientation of the bone collagen fibres. By biomechanical methods, the elastic properties of the resulting ceramo-osseous regeneration complex were tested. Reproducible subchondral bone defects were prepared in medial femoral condyles of rabbits, leaving a 0.5 mm coplanar layer of bone and cartilage. The defects were filled with granules of HAC. Polarizing microscopy revealed the dynamical aspect of the bony integration of the material and the remodelling process under physiological locomotion. It showed a rapid ongrowth of collagen fibres on the ceramic surface. By its increasing orientation to domains from woven texture to economical trabecular architecture, the load-bearing facility is documented. Indenting the articular surface on an impressive force testing machine 18 months after HAC implantation proved the equal elastic response of the ceramo-osseous regeneration complex with the overlying structures in comparison with the integrity of not-operated femoral condyles. When integrated by bone, HAC fulfils in our dynamic animal model physiological demands even in large bone defects close to articular surfaces.On the occasion of his 60th birthday, we dedicate this study to Professor K. H. Jungbluth, Head of Trauma and Reconstructive Surgery Department, University Hospital of Hamburg.     

Engineering cartilage tissue interfaces using a natural glycosaminoglycan hydrogel matrix — An in vitro study

Hydrogels are suitable matrices for cartilage tissue engineering on account of their resemblance to native extracellular matrix of articular cartilage and also considering its ease of application, they can be delivered to the defect site in a minimally invasive manner. In this study, we evaluate the suitability of a fast gelling natural biopolymer hydrogel matrix for articular cartilage tissue engineering. A hydrogel based on two natural polymers, chitosan and hyaluronic acid derivative was prepared and physicochemically characterized. Chondrocytes were then encapsulated within the hydrogel and cultured over a period of one month. Cartilage regeneration was assessed by histological, biochemical and gene expression studies. Chondrocytes maintained typical round morphology throughout the course of this investigation, indicating preservation of their phenotype with sufficient production of extracellular matrix and expression of typical chondrogenic markers Collagen type 2 and aggrecan. The results suggest that the natural polymer hydrogel matrix can be used as an efficient matrix for articular cartilage tissue engineering.     

Polymer Mechanics as a Model for Short-Term and Flow-Independent Cartilage Viscoelasticity

Articular cartilage is the load bearing soft tissue that covers the contacting surfaces of long bones in articulating joints. Healthy cartilage allows for smooth joint motion, while damaged cartilage prohibits normal function in debilitating joint diseases such as osteoarthritis. Knowledge of cartilage mechanical function through the progression of osteoarthritis, and in response to innovative regeneration treatments, requires a comprehensive understanding of the molecular nature of interacting extracellular matrix constituents and interstitial fluid. The objectives of this study were therefore to (1) examine the timescale of cartilage stress-relaxation using different mechanistic models and (2) develop and apply a novel (termed "sticky") polymer mechanics model to cartilage stress-relaxation based on temporary binding of constituent macromolecules. Using data from calf cartilage samples, we found that different models captured distinct timescales of cartilage stress-relaxation: monodisperse polymer reptation best described the first second of relaxation, sticky polymer mechanics best described data from ～1-100 seconds of relaxation, and a model of inviscid fluid flow through a porous elastic matrix best described data from 100 seconds to equilibrium. Further support for the sticky polymer model was observed using experimental data where cartilage stress-relaxation was measured in either low or high salt concentration. These data suggest that a complete understanding of cartilage mechanics, especially in the short time scales immediately following loading, requires appreciation of both fluid flow and the polymeric behavior of the extracellular matrix.     

Preparation of a biphasic scaffold for osteochondral tissue engineering

Tissue engineering has been developed as a prospective approach for the repair of articular cartilage defects. Engineered osteochondral implants can facilitate the fixation and integration with host tissue, and therefore promote the regeneration of osteochondral defects. A biphasic scaffold with a stratified two-layer structure for osteochondral tissue engineering was developed from biodegradable synthetic and naturally derived polymers. The upper layer of the scaffold for cartilage engineering was collagen sponge; the lower layer for bone engineering was a composite sponge of poly(DL-lactic-co-glycolic acid) (PLGA) and naturally derived collagen. The PLGA–collagen composite sponge layer had a composite structure with collagen microsponge formed in the pores of a skeleton PLGA sponge. The collagen sponge in the two respective layers was connected. Observation of the collagen/PLGA–collagen biphasic scaffold by scanning electron microscopy (SEM) demonstrated the connected stratified structure. The biphasic scaffold was used for culture of canine bone-marrow-derived mesenchymal stem cells. The cell/scaffold construct was implanted in an osteochondral defect in the knee of a one-year old beagle. Osteochondral tissue was regenerated four months after implantation. Cartilage- and bone-like tissues were formed in the respective layers. The collagen/PLGA–collagen biphasic scaffold will be useful for osteochondral tissue engineering.     

Type I Collagen Immobilized Poly(caprolactone) Nanofibers: Characterization of Surface Modification and Growth of Fibroblasts

Poly(caprolactone) (PCL) electrospun nanofibers were modified by aminolysis and collagen was immobilized on the aminolysed PCL nanofibers. Considering low immunogenic response collagen elicits, immobilization of the same is anticipated to enhance the tissue engineering application of the PCL nanofibers. Amino groups were introduced into PCL nanofibers through aminolysis process. Aminolysis of PCL nanofibers was confirmed by electron dispersive X‐ray analysis (EDX). Collagen was immobilized on aminolysed PCL nanofibers using glutaraldehyde as crosslinker. The collagen crosslinking on to PCL nanofibers was established by attenuated total reflectance‐Fourier transform infrared (ATR‐FTIR) spectroscopy. The fiber morphologies of PCL nanofibers at different stages were characterized by scanning electron microscopy (SEM). The change in hydrophobicity of PCL nanofibers due to aminolysis and collagen immobilization was determined by water contact angle measurements. Aminolysis followed by collagen immobilization had reduced the intrinsic hydrophobicity of PCL nanofibers. NIH 3T3 fibroblasts were cultured for 2 days on PCL nanofibers, aminolysed PCL nanofibers, and aminolysed PCL nanofibers crosslinked with collagen. Cell attachment and growth were observed by MTT assay in each case. Collagen immobilization improved the biocompatibility of the PCL nanofibers. Thus the modified PCL nanofibers can be used as suitable broad spectrum scaffold for skin, cartilage, bone, cardiac constructs for efficient tissue engineering applications.     

Immuno-SEM characterization of developing bovine cartilage

Collagen is a vital material in the tissues of living organisms. Found almost everywhere in the human body, collagen is important in connective tissues, bone growth, and cartilage. Collagen XI makes up a very small portion of the cartilaginous tissue; however, it plays a key role in cartilaginous tissue. Collagen XI and two collagen XI isoforms, V1b and V2, are critical in the ossification process. The location of collagen XI, V1b, V2, and their specific functions in the ossification process within developing bovine cartilage are not well characterized. In this work, the location of collagens I, II, XI and two collagen XI isoforms, V1b and V2, present in developing bovine cartilage are investigated using the immuno-SEM technique. The results for the locations of collagen I and II indicate a high level of consistency with previous work, thus showing that the technique of immuno-SEM can be used with confidence to determine the location of various collagen types within cartilaginous and mineralized tissue. This work has shown that collagen XI is present in the lower hypertrophic region and also in a pericellular arrangement, within about two microns of cell walls, throughout the cartilaginous tissue. V1b is expressed in the articular surface, mineralized region, resting zone, and the distal edge of the diaphysis. The V2 isoform is most strongly expressed in areas of newly forming cartilage, and disappears with chondrocyte maturation. V2 is present in the distal edge of the epiphysis, as well as in mineralized tissue. Collagen XI and two of its isoforms, V1b and V2, are thought to play a critical role in the ossification process. However, this role is not well understood, and is still being characterized. The detection of collagen XI and two of its isoforms in the osteo-chondral junction as well as at a joint surface further point to collagen XI, V1b, and V2 playing a vital role in the ossification process, and warrants further research as to their specific function within the ossification process.     

Accurate bone registration in knee MR images

Changes of the cartilage morphology over time can tell the progression of osteoarthritis (OA) and show particular promise for evaluating the efficacy of disease-modifying OA drugs. Hence, cartilage matching is required prior to cartilage morphology comparison. An accurate cartilage matching allows one to ensure longitudinal focal and local changes of cartilage morphology due to OA. The method described in this article meets this need. The proposed method consists of three steps. First, the knee femur surfaces are aligned, using the principal axes transformation to correct for different knee joint positions and orientations in the magnetic resonance (MR) scanner. Second, we present a global registration algorithm based on Lipschitz optimization theory for accurately identifying the corresponding points of the knee femur surface. Third, the rigid transformation of the knee femur surface registration is applied to the cartilage surface. Our registration algorithm is efficient and robust, and its performance is evaluated on MR images of pig knees.     

Genipin-crosslinked gelatin scaffolds for articular cartilage tissue engineering with a novel crosslinking method

A novel crosslinking method with directly crosslinking the gelatin gel, being cut to a disc of chosen size beforehand, for the fabrication of porous gelatin scaffold was proposed. This novel method of gel-crosslinking was compared with the traditional methods of mixing-crosslinking and scaffold-crosslinking. The structure of the scaffold fabricated by the gel-crosslinking method shows uniformly distributed and interconnected pores which can be much smaller than those made by the other two methods. All three methods have the last step as freeze-drying; nevertheless, freeze-drying once more will increase the uniformity of the structure and the interconnecting pores. Crosslinking of gelatin was carried out at room temperature with glutaraldehyde (GTA) or genipin (GP). In vitro cell culture of Wistar rat's joint chondrocytes demonstrates that the GTA-crosslinked scaffold is much worse than the GP-crosslinked one; a tissue containing collagen and glycosaminoglycan was produced in the GP-crosslinked scaffold in just 9 days after cell seeding, and a tissue with a cell distribution resembling that of the native cartilage was developed after 30 day cell culture. It was concluded that the novel method is feasible for application in articular cartilage tissue engineering.     

The regeneration of articular cartilage using a new polymer system

A polymer system based on room temperature polymerising poly (ethylmethacrylate) polymer powder and tetrahydrofurfuryl monomer has been investigated as a biomaterial for encouraging articular cartilage repair. This heterocyclic methacrylate polymer system swells slightly in situ and thus provides a good interface with subchondral bone resulting in mechanical stability with favourable uptake kinetics. Another feature of this polymer system is that it exhibits high water uptake which leads to absorption of the surrounding tissue fluid and matrix proteins, including growth factors; this may encourage the formation of new cartilage. Three weeks after implantation the tissue overgrowth contained cartilage components: chondrocytes, collagen type II, chondroitin 4-sulphate and chondroitin 6-sulphate. In addition numerous chondrocyte clones were observed at the edge of the defect and in the newly repaired tissue. By six weeks a superficial articulating surface was continuous with the normal articular cartilage with underlying tissue which showed some evidence of endochondral ossification. By nine weeks the surface covering of new cartilage had a widened and an irregular zone of calcified cartilage with thickened subchondral bone was present. At eight months the resurfaced cartilage remained intact above a remodelled subchondral bone end plate.