Desktop-stereolithography 3D printing of a radially oriented extracellular matrix/mesenchymal stem cell exosome bioink for osteochondral defect regeneration.

Mitochondrial dysfunction and oxidative stress damage are hallmarks of osteoarthritis (OA). Mesenchymal stem cell (MSC)-derived exosomes are important in intercellular mitochondria communication. However, the use of MSC exosomes for regulating mitochondrial function in OA has not been reported. This study aimed to explore the therapeutic effect of MSC exosomes in a three dimensional (3D) printed scaffold for early OA therapeutics. Methods: We first examined the mitochondria-related proteins in normal and OA human cartilage samples and investigated whether MSC exosomes could enhance mitochondrial biogenesis in vitro. We subsequently designed a bio-scaffold for MSC exosomes delivery and fabricated a 3D printed cartilage extracellular matrix (ECM)/gelatin methacrylate (GelMA)/exosome scaffold with radially oriented channels using desktop-stereolithography technology. Finally, the osteochondral defect repair capacity of the 3D printed scaffold was assessed using a rabbit model. Results: The ECM/GelMA/exosome scaffold effectively restored chondrocyte mitochondrial dysfunction, enhanced chondrocyte migration, and polarized the synovial macrophage response toward an M2 phenotype. The 3D printed scaffold significantly facilitated the cartilage regeneration in the animal model. Conclusion: This study demonstrated that the 3D printed, radially oriented ECM/GelMA/exosome scaffold could be a promising strategy for early OA treatment.

Decrease of miR-195 Promotes Chondrocytes Proliferation and Maintenance of Chondrogenic Phenotype via Targeting FGF-18 Pathway.

Slow growth and rapid loss of chondrogenic phenotypes are the major problems affecting chronic cartilage lesions. The role of microRNA-195 (miR-195) and its detailed working mechanism in the fore-mentioned process remains unknown. Fibroblastic growth factor 18 (FGF-18) plays a key role in cartilage homeostasis; whether miR-195 could regulate FGF-18 and its downstream signal pathway in chondrocyte proliferation and maintenance of chondrogenic phenotypes still remains unclear. The present research shows elevated miR-195 but depressed FGF-18 expressed in joint fluid specimens of 20 patients with chronic cartilage lesions and in CH1M and CH3M chondrocytes when compared with that in joint fluid specimens without cartilage lesions and in CH1W and CH2W chondrocytes, respectively. The following loss of function test revealed that downregulation of miR-195 by transfection of miR-195 inhibitors promoted chondrocyte proliferation and expression of a type II collagen α I chain (Col2a1)/aggrecan. Through the online informatics analysis we theoretically predicted that miR-195 could bind to a FGF-18 3' untranslated region (3'UTR), also, we verified that a miR-195 could regulate the FGF-18 and its downstream pathway. The constructed dual luciferase assay further confirmed that FGF-18 was a direct target of miR-195. The executed anti-sense experiment displayed that miR-195 could regulate chondrocyte proliferation and Col2a1/aggrecan expression via the FGF-18 pathway. Finally, through an in vivo anterior cruciate ligament transection (ACLT) model, downregulation of miR-195 presented a significantly protective effect on chronic cartilage lesions. Evaluating all of the outcomes of the current research revealed that a decrease of miR-195 protected chronic cartilage lesions by promoting chondrocyte proliferation and maintenance of chondrogenic phenotypes via the targeting of the FGF-18 pathway and that the miR-195/FGF-18 axis could be a potential target in the treatment of cartilage lesions.

Inhibition of the p38-MAPK signaling pathway suppresses the apoptosis and expression of proinflammatory cytokines in human osteoarthritis chondrocytes.

This study aims to investigate the effects of p38-MAPK signaling pathway on the apoptosis and expression of proinflammatory cytokines in human osteoarthritis (OA) chondrocytes. Human articular cartilage specimens were obtained from 57 OA patients and 31 patients with lower extremity traumatic amputations. The expressions of p38-MAPK pathway-related proteins in cartilage tissue were detected by immunochemistry. Cultured chondrocytes isolated from human OA cartilage were assigned into the blank group, the IL-1ß group, the PD (PD980959, ERK pathway inhibitors)+IL-1ß group, the SB (SB203580, p38 pathway inhibitors)+IL-1ß group, and the SP (SP600125, JNK signaling pathway inhibitors)+IL-1ß group. Cell proliferation and apoptosis were detected by MTT assay and flow cytometry. Western blotting was used to detect the expressions of MAPK pathway-related proteins. The mRNA expressions of IL-1, IL-6, and TNF-α were detected by qRT-PCR. The positive rates of p-p38, p-JNK and p-ERK in OA cartilage were higher than those in normal cartilage. Compared with the blank group, cell proliferation rate was decreased, cell apoptotic rate was increased, the mRNA expressions of IL-1, IL-6, TNF-α and the expressions of p-p38, p-JNK and p-ERK were increased in the IL-1ß group, while opposite trends were observed in the PD+IL-1ß, SB+IL-1ß, and SP+IL-1ß groups. Our study provides evidence that inhibition of the p38-MAPK signaling pathway could suppress the apoptosis and expression of proinflammatory cytokines in human OA chondrocytes.

Should we use cells, biomaterials, or tissue engineering for cartilage regeneration?

For a long time, cartilage has been a major focus of the whole field of tissue engineering, both because of the constantly growing need for more effective options for joint repair and the expectation that this apparently simple tissue will be easy to engineer. After several decades, cartilage regeneration has proven to be anything but easy. With gratifying progress in our understanding of the factors governing cartilage development and function, and cell therapy being successfully used for several decades, there is still a lot to do. We lack reliable methods to generate durable articular cartilage that would resemble the original tissue lost to injury or disease. The question posed here is whether the answer would come from the methods using cells, biomaterials, or tissue engineering. We present a concise review of some of the most meritorious efforts in each area, and propose that the solution will most likely emerge from the ongoing attempts to recapitulate certain aspects of native cartilage development. While an ideal recipe for cartilage regeneration is yet to be formulated, we believe that it will contain cell, biomaterial, and tissue engineering approaches, blended into an effective method for seamless repair of articular cartilage.

Comparison of quantitative and semiquantitative dynamic contrast-enhanced MRI with respect to their correlation to delayed gadolinium-enhanced MRI of the cartilage in patients with early rheumatoid arthritis.

OBJECTIVE: The objective of this study was to investigate the correlation between semiquantitative and quantitative dynamic contrast-enhanced (DCE) parameters with delayed gadolinium-enhanced magnetic resonance imaging (MRI) of the cartilage (dGEMRIC). METHODS: Fifteen patients with early rheumatoid arthritis (RA) from the ArthroMark cohort were investigated at a 3-T MRI scanner. The metacarpophalangeal (MCP) joint of the index finger was examined with DCE-MRI and dGEMRIC. Semiquantitative and quantitative DCE perfusion parameters were calculated. The RA MRI score of the second MCP joint and the joint space width were measured. RESULTS: Significant correlations were noted between both semiquantitative and quantitative DCE parameters and the RA MRI score of the second MCP joint. There was a significant negative correlation between DCE parameters and dGEMRIC. No association between joint space width and DCE parameters was observed. CONCLUSIONS: Semiquantitative and quantitative analyses of perfusion are applicable to show that cartilage damage correlates with the inflammation activity despite the absence of joint space narrowing.

Bacterial lipopolysaccharides form procollagen-endotoxin complexes that trigger cartilage inflammation and degeneration: implications for the development of rheumatoid arthritis.

INTRODUCTION: We have previously reported that bacterial toxins, especially endotoxins such as lipopolysaccharides (LPS), might be important causative agents in the pathogenesis of rheumatoid arthritis (RA) in an in vitro model that simulates the potential effects of residing in damp buildings. Since numerous inflammatory processes are linked with the nuclear factor-κB (NF-κB), we investigated in detail the effects of LPS on the NF-κB pathway and the postulated formation of procollagen-endotoxin complexes. METHODS: An in vitro model of human chondrocytes was used to investigate LPS-mediated inflammatory signaling. RESULTS: Immunoelectron microscopy revealed that LPS physically interact with collagen type II in the extracellular matrix (ECM) and anti-collagen type II significantly reduced this interaction. BMS-345541 (a specific inhibitor of IκB kinase (IKK)) or wortmannin (a specific inhibitor of phosphatidylinositol 3-kinase (PI-3K)) inhibited the LPS-induced degradation of the ECM and apoptosis in chondrocytes. This effect was completely inhibited by combining BMS-345541 and wortmannin. Furthermore, BMS-345541 and/or wortmannin suppressed the LPS-induced upregulation of catabolic enzymes that mediate ECM degradation (matrix metalloproteinases-9, -13), cyclooxygenase-2 and apoptosis (activated caspase-3). These proteins are regulated by NF-κB, suggesting that the NF-κB and PI-3K pathways are involved in LPS-induced cartilage degradation. The induction of NF-κB correlated with activation of IκBα kinase, IκBα phosphorylation, IκBα degradation, p65 phosphorylation and p65 nuclear translocation. Further upstream, LPS induced the expression of Toll-like receptor 4 (TLR4) and bound with TLR4, indicating that LPS acts through TLR4. CONCLUSION: These results suggest that molecular associations between LPS/TLR4/collagen type II in chondrocytes upregulate the NF-κB and PI-3K signaling pathways and activate proinflammatory activity.

Gallotannin causes differentiation and inflammation via ERK­1/­2 and p38 kinase pathways in rabbit articular chondrocytes.

Gallotannin (GT) is a type of tannic acid, derived from plant polyphenols, that is an agonist of plant defense mechanisms. Tannins have two types of structure; condensed tannins are a polymer of flavonoid units, while hydrolysable tannins are carbohydrates. GT is used in medical agents for its anti­viral, anti­bacterial and anti­parasitic effects. The present study investigated the effects of GT on differentiation and inflammation in rabbit articular chondrocytes. GT caused differentiation and inflammatory responses in the rabbit articular chondrocytes. GT treatment induced the expression of type Ⅱ collagen and sulfated proteoglycan, as determined by western blot analysis and alcian blue staining, respectively, in a dose­ and time­dependent manner. Additionally, treatment with GT increased the expression of cyclooxygenase­2 (COX­2) and the production of prostaglandin E2 (PGE2), as determined by western blot analysis and PGE2 assay. GT was confirmed to cause phosphorylation of ERK­1/­2 and p38 kinase. Inhibition of pERK with PD98059 promoted GT­induced type Ⅱ collagen expression. However, the inhibition of p38 with SB203580 suppressed GT­induced COX­2 expression and PGE2 production. In summary, the results demonstrated that GT­induced ERK­1/­2 and p38 kinase have opposite effects on differentiation and inflammation in rabbit articular chondrocytes.

Adipokine actions on cartilage homeostasis.

Epidemiological studies have shown an intriguing correlation between obesity and articular cartilage disease. An increase in mechanical forces across weight-bearing joints has long been considered the primary factor leading to joint degeneration. However, emerging data suggest that additional soluble factors such as the adipocyte-derived molecules "adipokines" may also play an important role in the onset and progression of weight-associated cartilage degradative process. Adipokines are pleiotropic secretory molecules mainly produced by white adipose tissue. Adipokines exert their actions through endocrine, paracrine, autocrine, or juxtacrine cross talk in a wide variety of physiological or pathophysiological processes. In particular, they are mainly involved in the regulation of food intake and energy metabolism, in both health and disease states, and in the inflammatory response. Recent observations have shown that, among adipokines, leptin, adiponectin, resistin, visfatin, and apelin may also participate to the complex mechanisms that regulate skeleton biology, both at bone and cartilage level. Herein, we review the present knowledge about the role of these adipokines in cartilage function as well as in inflammatory and degenerative joint diseases. Moreover, we describe some methodological approaches which can be utilized in the measurement of these adipokines in different biological matrices, like plasma and synovial fluid (SF), and may be helpful to better clarify the involvement of these molecules in cartilage disease.

High throughput proteomic analysis of the secretome in an explant model of articular cartilage inflammation.

This study employed a targeted high-throughput proteomic approach to identify the major proteins present in the secretome of articular cartilage. Explants from equine metacarpophalangeal joints were incubated alone or with interleukin-1beta (IL-1ß, 10ng/ml), with or without carprofen, a non-steroidal anti-inflammatory drug, for six days. After tryptic digestion of culture medium supernatants, resulting peptides were separated by HPLC and detected in a Bruker amaZon ion trap instrument. The five most abundant peptides in each MS scan were fragmented and the fragmentation patterns compared to mammalian entries in the Swiss-Prot database, using the Mascot search engine. Tryptic peptides originating from aggrecan core protein, cartilage oligomeric matrix protein (COMP), fibronectin, fibromodulin, thrombospondin-1 (TSP-1), clusterin (CLU), cartilage intermediate layer protein-1 (CILP-1), chondroadherin (CHAD) and matrix metalloproteinases MMP-1 and MMP-3 were detected. Quantitative western blotting confirmed the presence of CILP-1, CLU, MMP-1, MMP-3 and TSP-1. Treatment with IL-1ß increased MMP-1, MMP-3 and TSP-1 and decreased the CLU precursor but did not affect CILP-1 and CLU levels. Many of the proteins identified have well-established extracellular matrix functions and are involved in early repair/stress responses in cartilage. This high throughput approach may be used to study the changes that occur in the early stages of osteoarthritis.

Associations of horse age, joint type, and osteochondral injury with serum and synovial fluid concentrations of type II collagen biomarkers in Thoroughbreds.

OBJECTIVE: To determine the effects of horse age, osteochondral injury, and joint type on a synthesis biomarker and 3 degradative biomarkers of type II collagen in Thoroughbreds. ANIMALS: Healthy rested adult (3- to 12-year-old) Thoroughbreds (n = 19), yearling (1- to 2-year-old) Thoroughbreds (40), and Thoroughbred racehorses (2 to 7 years old) undergoing arthroscopic surgery for removal of osteochondral fragments that resulted from training or racing (41). PROCEDURES: Samples of blood and metacarpophalangeal, metatarsophalangeal, or carpal joint synovial fluid (SF) were collected from all horses. Commercially available assays were used to analyze SF and serum concentrations of type II collagen biomarkers of synthesis (carboxy propeptide of type II collagen [CPII]) and degradation (cross-linked C-telopeptide fragments of type II collagen [CTX II], neoepitope generated by collagenase cleavage of type I and II collagen [C1,2C], and neoepitope generated by collagenase cleavage of type II collagen [C2C]). RESULTS: Osteochondral injury affected concentrations of CPII, CTX II, C1,2C, and C2C in SF, serum, or both, compared with concentrations in healthy adult horses. Compared with adult horses, yearling horses had increased SF or serum concentrations of degradative biomarkers (CTX II, C1,2C, and C2C). Concentrations were higher in carpal than metacarpophalangeal or metatarsophalangeal joints for all biomarkers in osteochondral-injured horses. Variable differences in SF concentrations between joint types were detected in healthy adult and yearling horses. CONCLUSIONS AND CLINICAL RELEVANCE: Horse age, osteochondral injury, and joint type all significantly affected type II collagen biomarker concentrations in SF and serum of Thoroughbreds.

IL-3 inhibits TNF-alpha-induced bone resorption and prevents inflammatory arthritis.

IL-3, a cytokine secreted by activated T cells is well known to regulate the proliferation, differentiation, and survival of pluripotent hematopoietic stem cells. IL-3 functions as a link between the immune and the hematopoietic system. In this study, we suggest an important new role of IL-3 in inhibition of TNF-alpha-induced bone resorption in vitro and prevention of inflammatory arthritis in mice. We show here that IL-3 potently and irreversibly inhibits TNF-alpha-induced bone resorption in hematopoietic precursors of monocyte/macrophage lineage. IL-3 showed an inhibitory effect on TNF-alpha-induced bone resorption even in the presence of proinflammatory cytokines such as IL-1alpha, TGF-beta(1), TGF-beta(3), IL-6, and PGE(2). We found that IL-3 prevented TNF-alpha-induced c-fos nuclear translocation and AP-1 DNA-binding activity. Interestingly, IL-3 pretreatment prevented the development of inflammatory arthritis in mice induced by a mixture of anti-type II collagen mAbs and LPS. Furthermore, IL-3 prevented cartilage and bone loss in the joints indirectly through inhibition of inflammation. Thus, we provide the first evidence that IL-3, a strong regulator of hematopoiesis, also plays an important role in inhibition of TNF-alpha-induced bone resorption and prevention of inflammatory arthritis in mice.

Interleukin-6 and high mobility group box protein-1 in synovial membranes and osteochondral fragments in equine osteoarthritis.

Cytokine production in synovial membranes (SM) and osteochondral fragments (OCF) may influence the development of equine osteoarthritis (OA). In this study, the presence of interleukin (IL)-6 and cytoplasmic and extracellular high mobility group box protein (HMGB)-1 in SM and osteochondral tissue from healthy and diseased equine joints was investigated by immunohistochemistry. Additionally, microscopic synovitis was graded. IL-6 was commonly found in SM cells and in chondrocytes in uncalcified cartilage of OCF, whereas little staining was detected in healthy cartilage. Cytoplasmic and/or extracellular HMGB-1 was widespread only in SM from diseased joints, and also detected in OCF in areas of cartilage damage, fibrous repair tissue, and tidemark reduplication. Joints with OCF and cartilage lesions (without OCF) showed significantly higher median synovitis scores than healthy joints (p=0.013 and p=0.042, respectively). The study identifies OCF as a source of inflammatory mediators in equine OA, as shown by the presence of IL-6 and extracellular HMGB-1 in the fragment. Based upon HMGB-1 release in SM and OCF, further studies to investigate possible involvement of HMGB-1 in the pathogenesis of OA are warranted.

Effect of dietary nutrients on osteochondrosis lesions and cartilage properties in pigs.

OBJECTIVE: To evaluate dietary ingredients involved in cartilage and bone metabolism and their influence on osteochondrosis lesions in swine. ANIMALS: 80 crossbred gilts (mean initial weight, 39 kg). PROCEDURES: Pigs (10 pigs/treatment) were fed a corn-soybean meal basal (control) diet or the basal diet supplemented with additional minerals (copper and manganese or silicon), amino acids (proline and glycine; a combination of leucine, isoleucine, and valine; or methionine and threonine), or fatty acids (provided by fish oil) for 84 days. Pigs were then slaughtered and the distal portion of the left femur was collected for determination of osteochondrosis lesions at the femoral condyle. After evaluation of external joint surfaces, the distal portion of the femur was sectioned to evaluate lesions in the growth plate and articular cartilage. Additionally, a cartilage specimen was obtained from the patella for analysis. RESULTS: Pigs fed diets containing high amounts of methionine and threonine or the diet containing all additional ingredients had significantly lower total severity scores, compared with scores for pigs fed the control diet or a diet supplemented with fish oil. Pigs fed diets containing additional proline and glycine, copper and manganese, methionine and threonine, or all additional ingredients had significantly lower overall scores, compared with scores for pigs fed the control diet or a diet supplemented with fish oil. CONCLUSIONS AND CLINICAL RELEVANCE: Dietary manipulation decreased the severity of osteochondrosis lesions, compared with results for pigs fed a control diet. However, additional research on optimal concentrations and combinations of dietary components is needed.

Synthesis and release of human cartilage matrix proteoglycans are differently regulated by nitric oxide and prostaglandin-E2.

OBJECTIVES: Recent studies showed beneficial effects of COX-2 inhibition on proteoglycan turnover of both IL-1beta/tumour necrosis factor alpha (TNFalpha) damaged cartilage and of osteoarthritic cartilage. Although proteoglycan release and content were normalised, proteoglycan synthesis was only partially influenced. Prostaglandin-E2 is the main product formed by COX-2. We therefore evaluate the role of prostaglandin-E2 in relation to nitric oxide in disturbing cartilage proteoglycan turnover. METHODS: Human healthy cartilage, alone or in the presence of IL-1beta+TNFalpha, was cultured for 7 days with or without prostaglandin-E2 or the selective COX-2 inhibitor (celecoxib 10 microM). Changes in cartilage matrix proteoglycan turnover, levels of prostaglandin-E2 and nitric oxide were determined. RESULTS: Proteoglycan synthesis and release of the cartilage were not affected by prostaglandin-E2 alone. Addition of IL-1beta+TNFalpha to healthy cartilage resulted in inhibition of proteoglycan synthesis and increase in proteoglycan release. When prostaglandin-E2 was added, in addition to IL-1beta+TNFalpha, proteoglycan release increased further, but proteoglycan synthesis was not influenced further. Addition of a selective COX-2 inhibitor to the IL-1beta+TNFalpha treated cartilage inhibited the enhanced prostaglandin-E2 production and almost completely normalised proteoglycan release, whereas synthesis remained unaffected. Also, the enhanced NO-levels remained elevated. Prostaglandin-E2 levels correlated significantly with proteoglycan release, whereas NO levels correlated significantly with proteoglycan synthesis. CONCLUSION: The present results suggest involvement of prostaglandin-E2 in enhanced cartilage proteoglycan release but not synthesis, although healthy cartilage has to be sensitised by IL-1beta+tumour necrosis factor alpha (TNFalpha). IL-1beta+TNFalpha induced NO seems to be involved in inhibition of proteoglycan synthesis, independent of prostaglandin-E2, and thus seems insensitive to regulation by (selective) COX-2 inhibitors.

Evaluation of the influences of exercise, birth date, and osteochondrosis on plasma bone marker concentrations in Hanoverian Warmblood foals.

OBJECTIVE: To determine whether plasma concentrations of bone turnover markers in growing Hanoverian foals are influenced by age, housing conditions, or osteochondrosis. ANIMALS: 165 healthy foals and 119 foals with osteochondrosis. PROCEDURES: Foals were allocated according to birth date and housing management into groups of early-born (born before March 31, 2001; n = 154 foals, 88 of which were healthy and 66 of which had osteochondrosis) and late-born (born after March 31, 2001; 130 foals, 77 of which were healthy and 53 of which had osteochondrosis) foals. Plasma osteocalcin and carboxyterminal propeptide of type I collagen concentrations were analyzed as markers of bone formation, and carboxyterminal telopeptide of type I collagen concentration was analyzed as a marker of bone resorption. Foals underwent radiographic evaluation to screen for osteochondrosis. RESULTS: Plasma concentrations of osteocalcin, carboxyterminal propeptide of type I collagen, and carboxyterminal telopeptide of type I collagen decreased with age, but these changes were more distinct in late-born foals than in early-born foals. Neither sex nor predisposition to develop osteochondrosis affected the pattern of bone marker changes in either group. CONCLUSIONS AND CLINICAL RELEVANCE: An age-related decrease in concentrations of bone markers was seen during the first 200 days of life. Changes in bone marker concentrations were similar for foals with osteochondrosis and healthy foals. The correlation between the decrease in bone marker concentration and date of birth indicates that there are differences in skeletal development between early- and late-born foals.

Contrast agent enhanced pQCT of articular cartilage.

The delayed gadolinium enhanced MRI of cartilage (dGEMRIC) technique is the only non-invasive means to estimate proteoglycan (PG) content in articular cartilage. In dGEMRIC, the anionic paramagnetic contrast agent gadopentetate distributes in inverse relation to negatively charged PGs, leading to a linear relation between T1,Gd and spatial PG content in tissue. In the present study, for the first time, contrast agent enhanced peripheral quantitative computed tomography (pQCT) was applied, analogously to dGEMRIC, for the quantitative detection of spatial PG content in cartilage. The suitability of two anionic radiographic contrast agents, gadopentetate and ioxaglate, to detect enzymatically induced PG depletion in articular cartilage was investigated. First, the interrelationships of x-ray absorption, as measured with pQCT, and the contrast agent solution concentration were investigated. Optimal contrast agent concentrations for the following experiments were selected. Second, diffusion rates for both contrast agents were investigated in intact (n=3) and trypsin-degraded (n=3) bovine patellar cartilage. The contrast agent concentration of the cartilaginous layer was measured prior to and 2-27 h after immersion. Optimal immersion time for the further experiments was selected. Third, the suitability of gadopentetate and ioxaglate enhanced pQCT to detect the enzymatically induced specific PG depletion was investigated by determining the contrast agent concentrations and uronic acid and water contents in digested and intact osteochondral samples (n=16). After trypsin-induced PG loss (-70%, p<0.05) the penetration of gadopentetate and ioxaglate increased (p<0.05) by 34% and 48%, respectively. Gadopentetate and ioxaglate concentrations both showed strong correlation (r=-0.95, r=-0.94, p<0.01, respectively) with the uronic acid content. To conclude, contrast agent enhanced pQCT provides a technique to quantify PG content in normal and experimentally degraded articular cartilage in vitro. As high resolution imaging of e.g. the knee joint is possible with pQCT, the present technique may be further developed for in vivo quantification of PG depletion in osteoarthritic cartilage. However, careful in vitro and in vivo characterization of diffusion mechanics and optimal contrast agent concentrations are needed before diagnostic applications are feasible.

Positive effect of oral supplementation with glycosaminoglycans and antioxidants on the regeneration of osteochondral defects in the knee joint.

The effect of oral supplementation with glycosaminoglycans (GAG) and radical scavengers (vitamin E/selenium) on the regeneration of osteochondral defects was investigated in rabbits. After introduction of defined osteochondral defects in the knee joint, groups of ten animals were given a GAG/vitamin E/selenium mixture or a placebo (milk sugar) for 6 weeks. Following sacrifice, histological and histochemical analysis was performed. The amount of synovial fluid was increased in the placebo group, while the viscosity of the synovial fluid was significantly enhanced in the GAG group. The amount of sulfated GAG in the osteochondral regenerates (8.8 +/- 3.6 % vs. 6.0 +/- 5.6 %; p <0.03) was significantly higher in the GAG group. In both groups, the GAG amount in the cartilage of the operated knee was significantly higher than in the non-involved knee (p <0.05). Histological analysis of the regenerates in the GAG group was superior in comparison with the placebo group. For the first time, a biological effect following oral supplementation with GAG was demonstrated in healing of osteochondral defects in vivo. These findings support the known positive clinical results.

Comparison of proteoglycan and collagen in articular cartilage of horses with naturally developing osteochondrosis and healing osteochondral fragments of experimentally induced fractures.

OBJECTIVE: To compare articular cartilage from horses with naturally developing osteochondrosis (OC) with normal articular cartilage and healing cartilage obtained from horses with experimentally induced osteochondral fractures. SAMPLE POPULATION: 109 specimens of articular cartilage from 78 horses. PROCEDURE: Morphologic characteristics, proteoglycan (PG), and type II collagen were analyzed in articular cartilage of OC specimens (group 1), matched healing cartilage obtained 40 days after experimentally induced osteochondral fractures (group 2), and matched normal cartilage from the same sites (group 3). RESULTS: 79 specimens of OC cartilage were obtained from horses. Ex vivo PG synthesis was significantly greater in the femoral cartilage, compared with synthesis in the tibial cartilage, and significantly greater for groups 1 and 2, compared with group 3. For groups 1 and 2, femoral fragments had significantly greater PG content, compared with PG content in tibial fragments. Keratan sulfate content was significantly less in group 3, compared with groups 1 and 2. Cartilage from the OC specimens had loss of structural architecture. The OC tissue bed stained positive for chondroitin sulfate and type II collagen, but the fracture bed did not. CONCLUSIONS AND CLINICAL RELEVANCE: Our analyses could not distinguish articular cartilage from horses with OC and a healing fracture. Both resembled an anabolic, reparative process. Immunohistochemical analysis suggested a chondromyxoid tissue in the OC bed that was morphologically similar to fibrous tissue but phenotypically resembled hyaline cartilage. Thus, tissue in the OC bed may be degenerative cartilage, whereas tissue in the fracture bed may be reparative fibrous callus.

Differentiation of transplanted mesenchymal stem cells in a large osteochondral defect in rabbit.

BACKGROUND: Although accumulating evidence shows that mesenchymal stem cells (MSC) are a promising cell source for articular cartilage repair, the fate of transplanted MSC has not been extensively studied. METHODS: To monitor their persistence and differentiation, we labeled uninduced MSC with a fluorescent dye, PKH26, and transplanted them, in a poly-glycolic-acid scaffold, to full-thickness defects made in the weight-bearing area of rabbit femoral trochleae with hyaluronate sheets. The fate of the labeled cells was monitored for up to 8 weeks. RESULTS: Two weeks after transplantation, immature cartilage containing collagen type II had formed. By 8 weeks, this cartilage had thinned and immunolabeling for collagen type II gradually disappeared from the basal region, which became positive for collagen type I. Most chondrocytes within the regenerated cartilage were PKH26-positive and, therefore, derived from transplanted MSC, whereas osteoblasts within the regenerated bone were a mixture of donor- and host-derived cells. The thickness of the cartilage became thinner up to 8 weeks and then remained stable up to 42 weeks after surgery. DISCUSSION: These results showed that uninduced MSC were able to survive osteochondral defects and differentiated according to the environment, making a major contribution to initial cartilage formation and a partial contribution to bone regeneration.