Cartilage injuries often represent irreversible tissue damage because cartilage has only a low ability to regenerate. Thus, cartilage loss results in permanent damage, which can become the starting point for osteoarthritis. In the past, bioactive glass scaffolds have been developed for bone replacement and some of these variants have also been colonized with chondrocytes. However, the hydroxylapaptite phase that is usually formed in bioglass scaffolds is not very suitable for cartilage formation (chondrogenesis). This interdisciplinary project was undertaken to develop a novel slowly degrading bioactive glass scaffold tailored for cartilage repair by resembling the native extracellular cartilage matrix (ECM) in structure and surface properties. When colonized with articular chondrocytes, the composition and topology of the scaffolds should support cell adherence, proliferation and ECM synthesis as a prerequisite for chondrogenesis in the scaffold.

To study cell growth in the scaffold, the scaffolds were colonized with human mesenchymal stromal cells (hMSCs) and primary porcine articular chondrocytes (pACs) (27,777.8 cells per mm³) for 7 – 35 d in a rotatory device. Cell survival in the scaffold was determined by vitality assay. Scanning electron microscopy (SEM) visualized cell ultramorphology and direct interaction of hMSCs and pACs with the bioglass surface. Cell proliferation was detected by CyQuant assay. Subsequently, the production of sulphated glycosaminoglycans (sGAGs) typical for chondrogenic differentiation was depicted by Alcian blue staining and quantified by dimethylmethylene blue assay assay. Quantitative real-time polymerase chain reaction (QPCR) revealed gene expression of cartilage-specific aggrecan, Sox9, collagen type II and dedifferentiation-associated collagen type I. To demonstrate the ECM-protein synthesis of the cells, the production of collagen type II and type I was determined by immunolabelling.

The bioactive glass scaffold remained stable over the whole observation time and allowed the survival of hMSCs and pACs for 35 days in culture. The SEM analyses revealed an intimate cell-biomaterial interaction for both cell types showing cell spreading, formation of numerous filopodia and ECM deposition. Both cell types revealed initial proliferation, decreasing after 14 days and becoming elevated again after 21 days. hMSCs formed cell clusters, whereas pACs showed an even distribution. Both cell types filled more and more the pores of the scaffold. The relative gene expression of cartilage-specific markers could be proven for hMSCs and pACs. Cell associated sGAGs deposition could be demonstrated by Alcian blue staining and sGAGs were elevated in the beginning and end of the culturing period. While the production of collagen type II could be observed with both cell types, the synthesis of aggrecan could not be detected in scaffolds seeded with hMSCs.

hMSCs and pACs adhered, spread and survived on the novel bioactive glass scaffolds and exhibited a chondrocytic phenotype.

The superficial zone (SFZ) of articular cartilage has unique structural and biomechanical features, and is important for joint long-term function. Previous studies have shown that TGF-β/Alk5 signaling upregulating PRG4 expression maintains articular cartilage homeostasis. However, the exact role and molecular mechanism of TGF-β signaling in SFZ of articular cartilage homeostasis are still lacking. In this study, a combination of in vitro and in vivo approaches were used to elucidate the role of Alk5 signaling in maintaining the SFZ of articular cartilage and preventing osteoarthritis initiation. Mice with inducible cartilage SFZ-specific deletion of Alk5 were generated to assess the role of Alk5 in OA development. Alterations in cartilage structure were evaluated histologically. The chondrocyte apoptosis and cell cycle were detected by TUNEL and Edu staining, respectively. Isolation, culture and treatment of SFZ cells, the expressions of genes associated with articular cartilage homeostasis and TGF-β signaling were analyzed by qRT-PCR. The effects of TGF-β/Alk5 signaling on proliferation and differentiation of SFZ cells were explored by cells count and alcian blue staining. In addition, SFZ cells isolated from C57 mice were cultured in presence of TGF-β1 or SB505124 for 7 days and transplanted subcutaneously in athymic mice. Postnatal cartilage SFZ-specific deletion of Alk5 induced an OA-like phenotype with degradation of articular cartilage, synovial hyperplasia as well as enhanced chondrocyte apoptosis, overproduction of catabolic factors, and decreased expressions of anabolic factors in chondrocytes. qRT-PCR and IHC results confirmed that Alk5 gene was effectively deleted in articular cartilage SFZ cells. Next, the PRG4-positive cells in articular cartilage SFZ were significantly decreased in Alk5 cKO mice compared with those in Cre-negative control mice. The mRNA expression of Aggrecan and Col2 were decreased, meanwhile, expression of Mmp13 and Adamts5 were significantly increased in articular cartilage SFZ cells of Alk5 cKO mice. In addition, Edu and TUNEL staining results revealed that slow-cell cycle cell number and increase the apoptosis positive cell in articular cartilage SFZ of Alk5 cKO mice compared with Cre-negative mice, respectively. Furthermore, all groups of SFZ cells formed ectopic solid tissue masses 1 week after transplantation. Histological examination revealed that the TGF-β1-pretreated tissues was composed of small and round cells and was positive for alcian blue staining, while the SB505124-pretreated tissue contained more hypertrophic cells though it did stain with alcian blue. TGF-β/alk5 signaling is an essential regulator of the superficial layer of articular cartilage by maintaining chondrocyte number, its differentiation properties, and lubrication function. Furthermore, it plays a critical role in protecting cartilage from OA initiation

Human articular cartilage samples were retrieved from the resected material of patients undergoing total knee replacement. Samples underwent automated controlled freezing at various stages of preparation: as intact articular cartilage discs, as minced articular cartilage, and as chondrocytes immediately after enzymatic isolation from fresh articular cartilage. Cell viability was examined using a LIVE/DEAD assay which provided fluorescent staining. Isolated chondrocytes were then cultured and Alamar blue assay was used for estimation of cell proliferation at days zero, four, seven, 14, 21 and 28 after seeding. The mean percentage viabilities of chondrocytes isolated from group A (fresh, intact articular cartilage disc samples), group B (following cryopreservation and then thawing, after initial isolation from articular cartilage), group C (from minced cryopreserved articular cartilage samples), and group D (from cryopreserved intact articular cartilage disc samples) were 74.7% (95% confidence interval (CI) 73.1 to 76.3), 47.0% (95% CI 43 to 51), 32.0% (95% CI 30.3 to 33.7) and 23.3% (95% CI 22.1 to 24.5), respectively. Isolated chondrocytes from all groups were expanded by the following mean proportions after 28 days of culturing: group A ten times, group B 18 times, group C 106 times, and group D 154 times. This experiment demonstrated that it is possible to isolate viable chondrocytes from cryopreserved intact human articular cartilage which can then be successfully cultured

Introduction: Osteoarthritis is the most common joint disease in the world. Biochemical and genetic factors as well as mechanical stress contribute to lesions in the cartilage. The present study analyses the effect of b-Endorphin on the cells of articular cartilage. Materials and methods: We used rat articular cartilage for the study. After tripsinizing the cartilage and isolating the chondrocytes the cells were cultured in a culture medium. B-Endorphin was dissolved in the culture medium at concentrations of 1 and 10 mM. Only the culture medium was added to the control wells. Naloxone 1 mM was added for co-treatment with b-Endorphin and naloxone. Thirty minutes later, b-Endorphin was added, thus blocking its receptors. Results: We studied the effect of this procedure on chondrocytes’ proliferating activity and on the proteoglycan synthesis of the extracellular matrix. An increase was observed in the incorporation of 3H-Thymidine, which in turn reflected an increase in the chondrocytes’ proliferating activity. In addition, 35S incorporation analyses were made of cultures which assessed proteoglycan synthesis which showed an increase in the extracellular-matrix forming activity. Differences between the groups with b-endorphin, b-endorphin + naloxone and the control group were found to be highly significant (p< 0.01). Conclusions: B-endorphin has a stimulating effect upon chondrocytes and proteoglycans present in the extracellular matrix in culture. These stimulating effects are mediated by the interaction with a specific opioid receptor, present in the articular cartilage cells. It may be conceived that trophic stimulation of cartilage cells in the early stages of the disease might partly mitigate the loss of joint surface

Purpose: Intraarticular use of anaesthetic agents is common for postoperative pain relief after arthroscopic knee surgery. In this study, we have evaluated and compared the effects of Bupivacaine, Levobupivacaine and Tramadol both invivo and invitro experimental rat models on articular cartilage and chondrocytes. Materials and Methods: Invivo Experiment: 1. Injections: Thirty mature Sprague-Dawley rats weighing 230 – 300 g were randomized into 3 groups. Bupivacaine (Group 1), Levobupivacaine (Group 2) and Tramadol (Group 3) were injected into the right knee joints and physiological 0.9% saline into the left. 2. Histopathologic Analysis: The specimens were fixed, decalcified and stained with Hematoxylen and Eosin (H& E) and Toluidin Blue. All slides were examined by the same pathologist, who was blinded to the injectate used in each joint. All samples were evaluated histopathologically according to the recommendation of International Cartilage Repair Society’s osteoarthritis and cartilage histopathology grading and staging system. Invitro Experiment: Articular cartilage cells of the rats were cultured and seeded. Cartilage cell seeded samples (104 cells/mL) were incubated in three different anesthetic agents (0,5%); Bupivacaine, Levobupivacaine, and Tramadol respectively. Cell Titer 96TM Nonradioactivity Cell Proliferation (MTS) assay was used to determine the cell density on the samples. Results:. Invivo: There were pathologic changes like cartilage hypertrophy, active chronic inflammation with abscess formation, cellular proliferation, focal vertical fissures and focal discontunity on cartilage matrix at superficial zone in all three groups on the drug injected sides. Although those histopathologic findings were not found statistically significant when compared the OARSI grade, OA stage and OA score with the control groups (p> 0.05), statistically significant higher OARSI grade, OA stage and OA scores were detected when compared the Levobupivacaine injected group after 10 days with the Levobupivacaine injected group after 48 hours (p< 0.01 [ p=0.008]). Invitro: MTS results show that 0.5% Tramadol is cytotoxic to rat chondrocyte in vitro after 30 min of exposure. Also the cell number in both Bupivacaine and Levobupivacaine treated wells showed decrease throughout 15, 30 and 60 min exposures. Conclusion: No report has been appointed comparing the effects of the mentioned three drugs both invivo and invitro. Although chondrotoxicity of Bupivacaine was less harmful than Levobupivacaine and Tramadol, these findings suggest that local anesthetics negatively affect articular cartilage and chondrocytes. Given that chondrocyte loss has been implicated in the development of chondrosis and osteoarthritis, orthopaedic surgeons should be careful in their preference for pain control with intraarticular drug injections after arthroscopic procedures

Background: To date, conventional freezing and cryopreservation of articular cartilage has had limited success due to the mechanical injury of cells resulting from uncontrolled ice crystal propagation. Frozen then thawed grafts show a total lack of viable articular cartilage cells and weakened matrix. Directional freezing using a precise velocity offers a new approach to the process of freezing, enabling cryopreservation of articular cartilage for long term storage and implantation. Hypothesis: Cryopreservation of articular cartilage using directional freezing maintains significant chondrocyte viability and extra cellular matrix quality. Study Design: Controlled Laboratory Study. Methods: Articular Cartilage, collected from 20 porcine hind legs harvested immediately after slaughter, was transferred to the processing laboratory for cryopreservation and analysis. Cryopreservation was performed using a directional freezing system (MTG 1315). During preparation for freezing cryoprotectants were injected into the matrix using an array of 20 micron needles. Thirty 15mm cylindrical grafts were examined for cell viability and cell density using fluorescent and confocal microscopy and proteoglycan synthesis via . 35. SO. 4. uptake. Biomechanical assessment was performed on a second set of 9 grafts to determine the matrix instantaneous dynamic modulus of elasticity. Results: Chondrocyte viability (53%±9%), viable cell density (18900 ± 4100 cell/mm. 3. , 68%±5.7% viability) and . 35. SO. 4. uptake (59% compared to fresh control) were achieved. Biomechanical measures were mildly impaired (62%±5.2%) compared to fresh control due to the injection of cryoprotectants. In addition, chondrocyte viability in the cryopreserved allograft was preferentially maintained in the superficial zone. Similar results were obtained in human in-vitro studies. Conclusion: Cryopreservation using directional freezing enables the preservation of viable cells within the collagen matrix. These cells are embedded in the supporting hyaline cartilage matrix with good mechanical stability. The behavior of cryopreserved cartilage after transplantation as indicated in sheep transplantations favors the generation of new, healthy hyaline cartilage during one year follow-up. The high percentage of viable cartilage cells, the quality of the matrix following freezing and thawing, and the ability to store these grafts in a hospital facility, are encouraging to meet the growing demand of such allografts in human cartilage repair procedures

This study investigated confocal laser scanning microscopy (CLSM) as a novel method of imaging of chondrocytes on a collagen membrane used for articular cartilage repair. Cell viability and the effects of surgery on the cells were assessed. Cell images were acquired under four conditions: 1, Pre-operative 2, After handling 3, Heavily grasped with forceps 4, Cut around the edge. Live and dead cell stains were used. Images were obtained for cell counting and morphology. Mean cell density was 1.12–1.68 ± 0.22 × 10. 6. cells/cm. 2. in specimens without significant trauma (n=25 images), this decreased to 0.253 × 10. 6. cells/cm. 2. in the specimens that had been grasped with forceps (p <0.001) (5 images). Cell viability on delivery grade membrane was 86.8±2.1%. The viability dropped to 76.3 ± 1.6% after handling and 35.1 ± 1.7% after crushing with forceps. Where the membrane was cut with scissors, there was a band of cell death where the viability dropped to 17.3 ± 2.0% compared to 73.4 ± 1.9% in the adjacent area (p <0.001). Higher magnification revealed cells did not have the rounded appearance of chondrocytes. CLSM can quantify and image the fine morphology of cells on a MACI membrane. Careful handling of the membrane is essential to minimise chondrocyte death during surgery

Unique progenitor cells have been identified recently and successfully cultured in vitro from human articular cartilage. These cells are able to maintain chondrogenic potential upon extensive expansion. In this study, we have developed a sheep, ex-vivo model of cartilage damage and repair, using these progenitor cells. This study addresses the question can such a model be used to determine factors required for progenitor cell proliferation, differentiation and integration of matrix onto bone. The hypothesis was that sheep allogenic cartilage derived progenitor cells could regenerate artificially damaged sheep articular cartilage in an osteochondral culture model. Progenitor cells were derived from ovine articular cartilage using a differential adhesion assay to fibronectin and expanded clonally. These clonal cells were marked with lentiviral vectors derived from the Human Immunodeficiency Virus-1. When a self-inactivating lentiviral vector encoding a ubiquitous phosphoglycerate kinase promoter, driving a Green Fluorescent Protein (GFP) reporter gene, was used to transduce these cells, up to 80% of these progenitor cells expressed GFP. Normal sheep medial femoral condyles containing about 2mm thick sub-condral bone were obtained and 4mm circular defects created on the cartilage surface using a biopsy punch. Condyles were cultured for two weeks in vitro with GFP labelled progenitor cells within a fibrin glue scaffold (Tisseel Lyo) and matrix production (collagen) as determined by spatially offset Raman spectroscopy and immunohistochemistry was demonstrated. Progenitor cells were able to proliferate and differentiate into collagen producing cells. Such an ex-vivo model system is an effective tool for the analysis of cartilage repair from various sources of stem cells. These ex-vivo experiments and variations on defect type, size, titration of scaffold and progenitor cell numbers requirements can further be used as a basis for screening prior to in vivo experiments

In 1823 J. White excised the head. In 1887 a German surgeon replaced the head with ivory. Interposition arthroplasties were common after WW1. Short-stemmed head replacing prosthesis were developed after WW2. Moores and Thompson designed a more stable intramedullary stem. Acetabular erosion was troublesome—and so replacing both surfaces started in the late 1950s using Teflon cup and metal femur. Unfortunately, these quickly became loose due to wear or sepsis. In 1960, Charnley used a polyethylene cup and stainless-steel femur and fixed both with dental cement. This ‘low friction arthroplast’ became a routine procedure after 1961. In the 1970s there were many ‘Charnley look-alike’ prosthesis with similar problems of poly-wear, granulomas and cysts causing bone loss, loosening, breakages and infection. Resurfacing with two thin shells was developed to reduce the foreign material, the bone resection and the cement used. Unfortunately, neck fractures, avascular necrosis and excessive wear of the poly shell were common. Despite operating theatres with laminar flow of sterile air, space suits and improved cementing techniques, the same problems occurred. To avoid poly and cement, Mittelmayer developed a ceramic screw cup, which did not require cement. Although some screws migrated, they did not wear. Because the un-cemented metal stem remained fixed solid to the femur, un-cemented metal cups and stems were developed. To avoid the poly-wear, ceramic liners became popular. To provide the active patients with a stable joint that requires no restriction in physical activity, a large head in a large cup is desirable. Unfortunately, the large metal-on-metal resurfacing prosthesis produce metal wear ions and nanoparticles which can form hypersensitivities, cysts and pseudotumours. Computer assisted navigation to ensure correct positioning of the prosthetic components is obviously useful for surgeons that use incisions too small to see enough to be certain of the cups position. Presently, articular cartilage research is progressing rapidly and by 2020 most arthritic hip joints will be arthroscopically debrided and resurfaced by an injection of genetically engineered articular cartilage stem cells