Lesions within the articular cartilage layer of synovial joints do not heal spontaneously. Some repair cells may appear, but their failure to become established may be related to problems of adhesion to proteoglycan-rich surfaces. We therefore investigated whether controlled enzymatic degradation of surface proteoglycan molecules to a depth of about 1 μm, using chondroitinase ABC, would improve coverage by repair cells. We created superficial lesions (1.0 × 0.2 × 5 mm) in the articular cartilage of mature rabbit knees and treated the surfaces with 1 U/ml of chondroitinase ABC for four minutes. The defects were studied by histomorphometry and electron microscopy at one, three and six months. At one month, untreated lesions were covered to a mean extent of 28% by repair cells; this was enhanced to a mean of 53% after enzyme treatment. By three months, the mean coverage of both control and chondroitinase-ABC-treated defects had diminished dramatically to 0.2% and 13%, respectively, but at six months both untreated and treated lesions had a similar coverage of about 30%, not significantly different from that achieved in untreated knees at one month. These findings suggest that, with time, chondrocytes near the surface of the defect may compensate for the loss of proteoglycans produced by enzyme treatment, thereby restoring the inhibitory properties of the matrix as regards cell adhesion. This supposition was confirmed by electron microscopy. Our results have an important bearing on attempts made to induce healing responses by transplanting chondrogenic cells or by applying growth factors

We stably transfected early passage chondrocytes with an anti-apoptotic Bcl-2 gene in vitro using a retrovirus vector. Samples of articular cartilage were obtained from 11 patients with a mean age of 69 years (61 to 75) who were undergoing total knee replacement for osteoarthritis. The Bcl-2-gene-transfected chondrocytes were compared with non-transfected and lac-Z-gene-transfected chondrocytes, both of which were used as controls. All three groups of cultured chondrocytes were incubated with nitric oxide (NO) for ten days. Using the Trypan Blue exclusion assay, an enzyme-linked immunosorbent assay and flow cytometric analysis, we found that the number of apoptotic chondrocytes was significantly higher in the non-transfected and lac-Z-transfected groups than in the Bcl-2-transfected group (p < 0.05). The Bcl-2-transfected chondrocytes were protected from NO-induced impairment of proteoglycan synthesis. We conclude that NO-induced chondrocyte death involves a mechanism which appears to be subject to regulation by an anti-apoptotic Bcl-2 gene. Therefore, Bcl-2 gene therapy may prove to be of therapeutic value in protecting human articular chondrocytes

Gene therapy with insulin-like growth factor-1 (IGF-1) increases matrix production and enhances chondrocyte proliferation and survival in vitro. The purpose of this study was to determine whether arthroscopically-grafted chondrocytes genetically modified by an adenovirus vector encoding equine IGF-1 (AdIGF-1) would have a beneficial effect on cartilage healing in an equine femoropatellar joint model. A total of 16 horses underwent arthroscopic repair of a single 15 mm cartilage defect in each femoropatellar joint. One joint received 2 × 10. 7. AdIGF-1 modified chondrocytes and the contralateral joint received 2 × 10. 7. naive (unmodified) chondrocytes. Repairs were analysed at four weeks, nine weeks and eight months after surgery. Morphological and histological appearance, IGF-1 and collagen type II gene expression (polymerase chain reaction, in situ hybridisation and immunohistochemistry), collagen type II content (cyanogen bromide and sodium dodecyl sulphate-polyacrylamide gel electrophoresis), proteoglycan content (dimethylmethylene blue assay), and gene expression for collagen type I, matrix metalloproteinase (MMP)-1, MMP-3, MMP-13, aggrecanase-1, tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) and TIMP-3 were evaluated. Genetic modification of chondrocytes significantly increased IGF-1 mRNA and ligand production in repair tissue for up to nine weeks following transplantation. The gross and histological appearance of IGF-1 modified repair tissue was improved over control defects. Gross filling of defects was significantly improved at four weeks, and a more hyaline-like tissue covered the lesions at eight months. Histological outcome at four and nine weeks post-transplantation revealed greater tissue filling of defects transplanted with genetically modified chondrocytes, whereas repair tissue in control defects was thin and irregular and more fibrous. Collagen type II expression in IGF-1 gene-transduced defects was increased 100-fold at four weeks and correlated with increased collagen type II immunoreaction up to eight months. Genetic modification of chondrocytes with AdIGF-1 prior to transplantation improved early (four to nine weeks), and to a lesser degree long-term, cartilage healing in the equine model. The equine model of cartilage healing closely resembles human clinical cartilage repair. The results of this study suggest that cartilage healing can be enhanced through genetic modification of chondrocytes prior to transplantation

Bone morphogenetic protein (BMP) has a crucial role in osteochondrogenesis of bone formation as well as in the repair of fractures. The interaction between hedgehog protein and BMPs is inferred from recent molecular studies. Hedgehog genes encode secreted proteins which mediate patterning and growth during skeletal development. We have shown that Indian hedgehog gene (Ihh) is expressed in cartilage anlage and later in mature and hypertrophic chondrocytes. This finding suggests that Ihh may regulate the development of chondrocytes. Our results in this study have shown that Ihh transcripts were expressed in hypertrophic chondrocytes in mice at three days but not at three weeks, although a similar expression pattern of α1 (X) collagen could be observed in both types of cartilage. To investigate the possibility that there are direct and age-dependent functions of Ihh in chondrocytes, cultured chondrocytes were treated with the amino-terminal fragment of Sonic hedgehog protein (Shh-N) which can functionally substitute for Ihh protein. Shh-N did not affect the proliferation and differentiation of chondrocytes from three-week-old mice but had a significant effect on three-day-old mice. It enhanced proliferation up to 128% of the control culture in a dose-dependent manner. Although there was no effect in Shh-N-treated cultures, Shh-N enhanced the stimulatory effect of parathyroid hormone (PTH) on the synthesis of proteoglycans. Because the effects of Shh-N on chondrocyte differentiation in this culture system differed from those of bone morphogenetic protein-2 (BMP2) and PTH, in terms of proteoglycan synthesis and ALPase activity, it is unlikely that BMP2 or PTH/PTH-related protein mediates the direct effects of Ihh in chondrocytes. Our study shows that Ihh can function in chondrocytes in a direct and age-dependent fashion

The discrepancy between successful experimental studies of cartilage repair and the clinical results is unexplained. We have evaluated the effect of metabolic alterations in joint homeostasis owing to an articular defect on the outcome of cartilage repair using tissue engineering methods. We used 21 adolescent Dutch goats divided into three groups. The control knees were left untreated while the contralateral knee was randomised to receive either no treatment (N), early treatment (E) or late treatment (L). The metabolism of proteoglycans in the surrounding joint surface was determined and correlated with the O’Driscoll score used to quantify the histological aspect of the repair of the defect. Synthesis of proteoglycan (PG) was increased in all groups. The release of glucosaminoglycan (GAG) was significantly higher in the untreated but not after early transplantation (1.3 v 1.8 NS). The cartilage repair scores in the early treatment group were not as good as those of the normal control group, but were significantly better when compared with both the untreated defects and the late treated defects. Defects which had been treated late showed a significantly decreased score when compared with those which had had early treatment or the normal control group and did not differ (p = 0.12) from those with no treatment. The histological and biochemical scores closely resembled the macroscopic and functional parameters which showed a significant deterioration for the late treated group and those without treatment compared with animals treated early. Thus, tissue-engineered cartilage repair is negatively influenced by altered matrix metabolism. Early treatment showed significantly better results for repair of cartilage than late or no treatment, with a concurrent decrease in the detrimental disturbance of cartilage metabolism which constituted a protective effect on the articulation

Our study establishes a rabbit model of disc degeneration which requires neither a chemical nor physical injury to the disc. Disc degeneration similar to that seen in man was created at levels proximal (L4-L5) and caudal (L7-S1) to a simulated lumbar fusion and was studied for up to nine months after arthrodesis. Loss of the normal parallel arrangement of collagen bundles within the annular lamellae was observed in intervertebral discs adjacent to the fusion at three months. By six months there was further disorganisation as well as loss of distinction between the lamellae themselves. By nine months the structure of the disc had been replaced by disorganised fibrous tissue, and annular tears were seen. There was an initial cellular proliferative response followed by loss of chondrocytes and notochordal cells in the nucleus pulposus. Degeneration was accompanied by a decrease in the monomer size of proteoglycans. Narrowing of the disc space, endplate sclerosis and the formation of osteophytes at adjacent disc spaces were observed radiologically

We studied the presence of anabolic growth factors in human herniated intervertebral discs (IVD) using a reverse transcriptase-polymerase chain reaction (RT-PCR) and immunohistochemistry. Messenger RNA (mRNA) was isolated from the nucleus pulposus using oligo (dT). 25. superparamagnetic beads and probing with gene-specific primers in RT-PCR. mRNA coding for TGF-α (3/10), EGF (0/10), TGF-β1 (0/10) and TGF-β3 (2/10) or the EGF receptor (EGF-R; 0/10) and TGF-β type-II receptor (0/10) was found only occasionally. Beta-actin was always present and positive sample controls confirmed the validity of the RT-PCR assay. These RT-PCR findings were confirmed using immunohistochemical staining of EGF and TFG-β, whereas TGF-α protein was always found associated with discocytes. We conclude that the nucleus pulposus of the herniated IVD is vulnerable to proteolytic degradation and depletion of proteoglycans due to the lack and/or low production of anabolic growth factors/receptors which could increase the local synthesis of the extracellular matrix

Composites of chondrocytes and polymerised fibrin were supplemented with insulin-like growth factor-I (IGF-I) during the arthroscopic repair of full-thickness cartilage defects in a model of extensive loss of cartilage in horses. Repairs facilitated with IGF-I and chondrocyte-fibrin composites, or control defects treated with chondrocyte-fibrin composites alone, were compared before death by the clinical appearance and repeated analysis of synovial fluid, and at termination eight months after surgery by tissue morphology, collagen typing, and biochemical assays. The structure of cartilage was evaluated histologically by Toluidine Blue reaction and collagen type-I and type-II in situ hybridisation and immunohistochemistry. Repair tissue was biochemically evaluated by DNA assay, proteoglycan quantitation and characterisation, assessment of collagen by reverse-phase high-performance liquid chromatography, and collagen typing using cyanogen bromide digestion and peptide separation by polyacrylamide gel electrophoresis. The results at eight months showed that the addition of IGF-I to chondrocyte grafts enhanced chondrogenesis in cartilage defects, including incorporation into surrounding cartilage. Gross filling of defects was improved, and the tissue contained a higher proportion of cells producing type-II collagen. Measurements of collagen type II showed improved levels in IGF-I-treated defects, supporting in situ hybridisation and immunohistochemical assessments of the defects. IGF-I improves the repair capabilities of chondrocyte-fibrin grafts in large full-thickness repair models

Although success has been achieved with implantation of bone marrow mesenchymal stem cells (bMSCs) in degenerative discs, its full potential may not be achieved if the harsh environment of the degenerative disc remains. Axial distraction has been shown to increase hydration and nutrition. Combining both therapies may have a synergistic effect in reversing degenerative disc disease. In order to evaluate the effect of bMSC implantation, axial distraction and combination therapy in stimulating regeneration and retarding degeneration in degenerative discs, we first induced disc degeneration by axial loading in a rabbit model.

The rabbits in the intervention groups performed better with respect to disc height, morphological grading, histological scoring and average dead cell count. The groups with distraction performed better than those without on all criteria except the average dead cell count.

Our findings suggest that bMSC implantation and distraction stimulate regenerative changes in degenerative discs in a rabbit model.

The weight-bearing status of articular cartilage has been shown to affect its biochemical composition. We have investigated the topographical variation of sulphated glycosaminoglycan (GAG) relative to the DNA content of the chondrocyte in human distal femoral articular cartilage.

Paired specimens of distal femoral articular cartilage, from weight-bearing and non-weight-bearing regions, were obtained from 13 patients undergoing above-knee amputation. After papain enzyme digestion, spectrophotometric GAG and fluorometric DNA assays assessed the biochemical composition of the samples. The results were analysed using a paired t-test.

Although there were no significant differences in cell density between the regions, the weight-bearing areas showed a significantly higher concentration of GAG relative to DNA when compared with non-weight-bearing areas (p = 0.02).

We conclude that chondrocytes are sensitive to their mechanical environment, and that local loading conditions influence the metabolism of the cells and hence the biochemical structure of the tissue.

In this study a combination of autologous chondrocyte implantation (ACI) and the osteochondral autograft transfer system (OATS) was used and evaluated as a treatment option for the repair of large areas of degenerative articular cartilage. We present the results at three years post-operatively. Osteochondral cores were used to restore the contour of articular cartilage in 13 patients with large lesions of the lateral femoral condyle (n = 5), medial femoral condyle (n = 7) and patella (n = 1). Autologous cultured chondrocytes were injected underneath a periosteal patch covering the cores. After one year, the patients had a significant improvement in their symptoms and after three years this level of improvement was maintained in ten of the 13 patients. Arthroscopic examination revealed that the osteochondral cores became well integrated with the surrounding cartilage. We conclude that the hybrid ACI/OATS technique provides a promising surgical approach for the treatment of patients with large degenerative osteochondral defects.

Acetabular dysplasia was produced in 24 immature white rabbits. A rotational acetabular osteotomy was then carried out and radiological and histological studies of the articular cartilage were made.

In the hips which did not undergo osteotomy, radiographs at 26 weeks showed that residual subluxation remained and arthritic changes such as narrowing of the joint space or dislocation were still seen. However, in the operated group there was a remarkable increase in cover, but arthritic changes were not observed. After 24 weeks, the Mankin grading score in the operated group was significantly lower than that in the non-operated group. The latter hips showed an irregular surface of the cartilage, exfoliation and proliferation of synovial tissue. In those undergoing osteotomy, primary cloning of chondrocytes or hypercellularity was seen and at 24 weeks after operation and metaplasia of the cartilage in the fibrous tissue was observed in the boundary between the medial area of the acetabulum and the acetabular fossa.

Perilesional changes of chronic focal osteochondral defects were assessed in the knees of 23 sheep. An osteochondral defect was created in the main load-bearing region of the medial condyle of the knees in a controlled, standardised manner. The perilesional cartilage was evaluated macroscopically and biopsies were taken at the time of production of the defect (T0), during a second operation one month later (T1), and after killing animals at three (T3; n = 8), four (T4; n = 8), and seven (T7; n = 8) months. All the samples were histologically assessed by the International Cartilage Repair Society grading system and Mankin histological scores. Biopsies were taken from human patients (n = 10) with chronic articular cartilage lesions and compared with the ovine specimens. The ovine perilesional cartilage presented with macroscopic and histological signs of degeneration. At T1 the International Cartilage Repair Society ‘Subchondral Bone’ score decreased from a mean of 3.0 (sd 0) to a mean of 1.9 (sd 0.3) and the ‘Matrix’ score from a mean of 3.0 (sd 0) to a mean of 2.5 (sd 0.5). This progressed further at T3, with the International Cartilage Repair Society ‘Surface’ grading, the ‘Matrix’ grading, ‘Cell Distribution’ and ‘Cell Viability’ grading further decreasing and the Mankin score rising from a mean of 1.3 (sd 1.4) to a mean of 5.1 (sd 1.6). Human biopsies achieved Mankin grading of a mean of 4.2 (sd 1.6) and were comparable with the ovine histology at T1 and T3.

The perilesional cartilage in the animal model became chronic at one month and its histological appearance may be considered comparable with that seen in human osteochondral defects after trauma.

The aim of this study was to determine whether subchondral bone influences in situ chondrocyte survival. Bovine explants were cultured in serum-free media over seven days with subchondral bone excised from articular cartilage (group A), subchondral bone left attached to articular cartilage (group B), and subchondral bone excised but co-cultured with articular cartilage (group C). Using confocal laser scanning microscopy, fluorescent probes and biochemical assays, in situ chondrocyte viability and relevant biophysical parameters (cartilage thickness, cell density, culture medium composition) were quantified over time (2.5 hours vs seven days). There was a significant increase in chondrocyte death over seven days, primarily within the superficial zone, for group A, but not for groups B or C (p < 0.05). There was no significant difference in cartilage thickness or cell density between groups A, B and C (p > 0.05). Increases in the protein content of the culture media for groups B and C, but not for group A, suggested that the release of soluble factors from subchondral bone may have influenced chondrocyte survival. In conclusion, subchondral bone significantly influenced chondrocyte survival in articular cartilage during explant culture.

The extrapolation of bone-cartilage interactions in vitro to the clinical situation must be made with caution, but the findings from these experiments suggest that future investigation into in vivo mechanisms of articular cartilage survival and degradation must consider the interactions of cartilage with subchondral bone.

We isolated multilineage mesenchymal progenitor cells from haematomas collected from fracture sites. After the haematoma was manually removed from the fracture site it was cut into strips and cultured. Homogenous fibroblastic adherent cells were obtained. Flow cytometry revealed that the adherent cells were consistently positive for mesenchymal stem-cell-related markers CD29, CD44, CD105 and CD166, and were negative for the haemopoietic markers CD14, CD34, CD45 and CD133 similar to bone-marrow-derived mesenchymal stem cells. In the presence of lineage-specific induction factors the adherent cells could differentiate in vitro into osteogenic, chondrogenic and adipogenic cells.

Our results indicate that haematomas found at a fracture site contain multilineage mesenchymal progenitor cells and play an important role in bone healing. Our findings imply that to enhance healing the haematoma should not be removed from the fracture site during osteosynthesis.

Ovine articular chondrocytes were isolated from cartilage biopsy and culture expanded in vitro. Approximately 30 million cells per ml of cultured chondrocytes were incorporated with autologous plasma-derived fibrin to form a three-dimensional construct. Full-thickness punch hole defects were created in the lateral and medial femoral condyles. The defects were implanted with either an autologous ‘chondrocyte-fibrin’ construct (ACFC), autologous chondrocytes (ACI) or fibrin blanks (AF) as controls. Animals were killed after 12 weeks. The gross appearance of the treated defects was inspected and photographed. The repaired tissues were studied histologically and by scanning electron microscopy analysis.

All defects were assessed using the International Cartilage Repair Society (ICRS) classification. Those treated with ACFC, ACI and AF exhibited median scores which correspond to a nearly-normal appearance. On the basis of the modified O’Driscoll histological scoring scale, ACFC implantation significantly enhanced cartilage repair compared to ACI and AF. Using scanning electron microscopy, ACFC and ACI showed characteristic organisation of chondrocytes and matrices, which were relatively similar to the surrounding adjacent cartilage.

Implantation of ACFC resulted in superior hyaline-like cartilage regeneration when compared with ACI. If this result is applicable to humans, a better outcome would be obtained than by using conventional ACI.

Surgical reconstruction of articular surfaces by transplantation of osteochondral autografts has shown considerable promise in the treatment of focal articular lesions. During mosaicplasty, each cylindrical osteochondral graft is centred over the recipient hole and delivered by impacting the articular surface. Impact loading of articular cartilage has been associated with structural damage, loss of the viability of chondrocytes and subsequent degeneration of the articular cartilage. We have examined the relationship between single-impact loading and chondrocyte death for the specific confined-compression boundary conditions of mosaicplasty and the effect of repetitive impact loading which occurs during implantation of the graft on the resulting viability of the chondrocytes.

Fresh bovine and porcine femoral condyles were used in this experiment. The percentage of chondrocyte death was found to vary logarithmically with single-impact energy and was predicted more strongly by the mean force of the impact rather than by the number of impacts required during placement of the graft. The significance of these results in regard to the surgical technique and design features of instruments for osteochondral transplantation is discussed.

We used a canine intercalary bone defect model to determine the effects of recombinant human osteogenic protein 1 (rhOP-1) on allograft incorporation. The allograft was treated with an implant made up of rhOP-1 and type I collagen or with type I collagen alone.

Radiographic analysis showed an increased volume of periosteal callus in both test groups compared with the control group at weeks 4, 6, 8 and 10. Mechanical testing after 12 weeks revealed increased maximal torque and stiffness in the rhOP-1 treated groups compared with the control group.

These results indicate a benefit from the use of an rhOP-1 implant in the healing of bone allografts. The effect was independent of the position of the implant. There may be a beneficial clinical application for this treatment.

The purpose of this study was to examine the effects of hyaluronic acid supplementation on chondrocyte metabolism in vitro. The clinical benefits of intra-articular hyaluronic acid injections are thought to occur through improved joint lubrication. Recent findings have shown that exogenous hyaluronic acid is incorporated into articular cartilage where it may have a direct biological effect on chondrocytes through CD44 receptors.

Bovine articular chondrocytes were isolated and seeded into alginate constructs. These were cultured in medium containing hyaluronic acid at varying concentrations. Samples were assayed for biochemical and histological changes.

There was a dose-dependent response to the exposure of hyaluronic acid to bovine articular chondrocytes in vitro. Low concentrations of hyaluronic acid (0.1 mg/mL and 1 mg/mL) significantly increase DNA, sulphated glycosaminoglycan and hydroxyproline synthesis. Immunohistology confirmed the maintenance of cell phenotype with increased matrix deposition of chondroitin-6-sulphate and collagen type II. These findings confirm a stimulatory effect of hyaluronic acid on chondrocyte metabolism.

Peri-tendinous injection of local anaesthetic, both alone and in combination with corticosteroids, is commonly performed in the treatment of tendinopathies. Previous studies have shown that local anaesthetics and corticosteroids are chondrotoxic, but their effect on tenocytes remains unknown. We compared the effects of lidocaine and ropivacaine, alone or combined with dexamethasone, on the viability of cultured bovine tenocytes. Tenocytes were exposed to ten different conditions: 1) normal saline; 2) 1% lidocaine; 3) 2% lidocaine; 4) 0.2% ropivacaine; 5) 0.5% ropivacaine; 6) dexamethasone (dex); 7) 1% lidocaine+dex; 8) 2% lidocaine+dex; 9) 0.2% ropivacaine+dex; and 10) 0.5% ropivacaine+dex, for 30 minutes. After a 24-hour recovery period, the viability of the tenocytes was quantified using the CellTiter-Glo viability assay and fluorescence-activated cell sorting (FACS) for live/dead cell counts. A 30-minute exposure to lidocaine alone was significantly toxic to the tenocytes in a dose-dependent manner, but a 30-minute exposure to ropivacaine or dexamethasone alone was not significantly toxic.

Dexamethasone potentiated ropivacaine tenocyte toxicity at higher doses of ropivacaine, but did not potentiate lidocaine tenocyte toxicity. As seen in other cell types, lidocaine has a dose-dependent toxicity to tenocytes but ropivacaine is not significantly toxic. Although dexamethasone alone is not toxic, its combination with 0.5% ropivacaine significantly increased its toxicity to tenocytes. These findings might be relevant to clinical practice and warrant further investigation.