John F. Tarlton

Repair of meniscal cartilage white zone tears using a stem cell/collagen-scaffold implant

Injuries to the avascular region of knee meniscal cartilage do not heal spontaneously. To address this problem we have developed a new stem cell/collagen-scaffold implant system in which human adult bone marrow mesenchymal stem cells are seeded onto a biodegradable scaffold that allows controlled delivery of actively dividing cells to the meniscus surface. Sandwich constructs of two white zone ovine meniscus discs with stem cell/collagen-scaffold implant in between were cultured in vitro for 40 days. Histomorphometric analysis revealed superior integration in the stem cell/collagen-scaffold groups compared to the cell-free collagen membrane or untreated controls. The addition of TGF-beta1 to differentiate stem cells to chondrocytes inhibited integration. Biomechanical testing demonstrated a significant 2-fold increase in tensile strength in all constructs using the stem cell/collagen-scaffold compared to control groups after 40 days in culture. Integration was significantly higher when collagen membranes were used that had a more open/spongy structure adjacent to both meniscal cartilage surfaces, whereas a collagen scaffold designed for osteoinduction failed to induce any integration of meniscus. In conclusion, the stem cell/collagen-scaffold implant is a potential therapeutic treatment for the repair of white zone meniscal cartilage tears.

The Role of Up-Regulated Serine Proteases and Matrix Metalloproteinases in the Pathogenesis of a Murine Model of Colitis

Proteinases are important at several phases of physiological and pathological inflammation, mediating cellular infiltration, cytokine activation, tissue damage, remodeling, and repair. However, little is known of their role in the pathogenesis of inflammatory bowel disease. The aim of this study was to assess the role of tissue proteases in a mouse model of colitis. Proteolytic activity was analyzed, using gel and in situ zymography, in colonic tissues from severe combined immunodeficient mice with colitis induced by transfer of CD4(+) T lymphocytes. Serine proteinase levels increased in colitic tissue, with major species of 23 kd, 30 kd, and 45 kd. Co-migration and inhibition studies indicated that the 23-kd proteinase was pancreatic trypsin and that the 30-kd species was neutrophil elastase. Matrix metalloproteinase (MMP)-9 expression, and MMP-2 and MMP-9 activation, was elevated in colitic tissues. Proteinase levels followed a decreasing concentration gradient from proximal to distal colon. Proteolysis was localized to infiltrating leukocytes in diseased severe combined immunodeficient mice. Transmural inflammation was associated with serine proteinase and MMP activity in overlying epithelium and with marked subepithelial proteolytic activity. The results demonstrate a clear elevation in the levels and activation of proteases in colitis, potentially contributing to disease progression through loss of epithelial barrier function.

Prognostic value of markers of collagen remodeling in venous ulcers

A 25 patient study was conducted into the relationship between markers of collagen metabolism in venous ulcer exudates and healing status, and their prognostic value in predicting healing performance. Wounds were sampled on at least 5 occasions over 12 months, the frequencies of which were determined by the need for clinic attendance. Specimens were taken from several sites on each ulcer using sterile preweighed filters. Wound margins were traced and sites recorded for each collection. Sample sites were evaluated for severity as improving, static, or deteriorating according to subsequent wound progression. Specimens were analyzed for levels of proenzyme and active forms of matrix metalloproteinases 2 and 9, neutrophil elastase, and type I collagen C propeptide. There was an overall trend of greater expression of all markers with increasing severity of wound site, this being highly significant for pro‐matrix metalloproteinase‐9 (p = 0.006). For samples collected simultaneously from improving and deteriorating regions of the same wound, paired data analysis showed statistically significant differences for pro‐matrix metalloproteinase‐9 (p < 0.001), neutrophil elastase (p < 0.005) and activated matrix metalloproteinase‐9 (p < 0.05). Taken overall, these data show the potential of markers of collagen biochemistry as predictors of repair in venous ulcers; in particular pro‐matrix metalloproteinase‐9 and neutrophil elastase were found to be accurate prognostic indicators of subsequent healing.

Dietary n‐3 polyunsaturated fatty acids reduce disease and colonic proinflammatory cytokines in a mouse model of colitis

Background: n‐3 polyunsaturated fatty acids (PUFAs) reduce the severity of chronic inflammatory bowel disease, probably by means of reduction of immune cell activation or enhancement of the epithelial barrier. Using the severe combined immunodeficient (SCID) mouse model of colitis, this study examined the effect of dietary n‐3 PUFAs on development of colitis and on immunologic, epithelial, and matrix parameters in the intestines of control and colitic animals. Methods: SCID mice were fed n‐3‐enriched or control diet for 3 weeks before colitis induction by transplantation of CD45RBhigh T cells and maintained on the same diet for 4 to 8 weeks. Phenotype of infiltrating cells, epithelial ZO‐1 protein, and mucosal type I collagen were assessed by immunohistology and tissue cytokines by ELISA. Results: Transplanted n‐3‐fed animals had significantly reduced pathology scores, colonic tumor necrosis factor‐&agr;, interleukin‐12, and interleukin‐1&bgr; compared with animals fed standard diet. Proinflammatory cytokines were reduced despite a similar level of immune cell infiltration by T cells, CD11c+ cells, and CD11b+ cells. Neutrophil infiltration was significantly reduced in n‐3‐fed control and colitic mice, and other myeloid populations were reduced in mice on the n‐3 diet. Epithelial ZO‐1 expression was increased, and myofibroblast activation significantly decreased in transplanted n‐3‐fed animals compared with standard diet mice. Submucosal collagen synthesis was enhanced in n‐3‐fed mice. Conclusions: Dietary n‐3 PUFAs reduced clinical colitis and colonic immunopathology in this model of colonic inflammation by decreasing proinflammatory cytokine synthesis, reducing myeloid cell recruitment and activation, and enhancing epithelial barrier function and mucosal wound healing mechanisms.

Influence of housing system and design on bone strength and keel bone fractures in laying hens

The main objectives of the study were to provide an accurate assessment of current levels of old breaks in end-of-lay hens housed in a variety of system designs and identify the important risk factors. Sixty-seven flocks housed in eight broad subcategories were assessed at the end of the production period. Within each flock, the presence of keel fractures was determined and the tibia, humerus and keel bones dissected for measurement of breaking strength. For each house, variations in internal design and perching provision were categorised and the effective heights of the differing structures recorded. All systems were associated with alarmingly high levels of keel damage although variation in mean prevalence between systems was evident with flocks housed in furnished cages having the lowest prevalence (36 per cent) despite also having significantly weaker bones and flocks housed in all systems equipped with multilevel perches showing the highest levels of damage (over 80 per cent) and the highest severity scores.

The relationship between cytokine concentrations and wound healing in chronic venous ulceration

OBJECTIVE The importance of wound cytokine function in chronic venous leg ulcers remains poorly understood. This study evaluated the relationship between local and systemic concentrations of wound cytokines and wound healing in patients with chronic venous ulceration. METHODS This prospective observational study was set in a community- and hospital-based leg ulcer clinic. Consecutive patients with chronic leg ulceration and ankle-brachial pressure index >0.85 were prospectively investigated. All patients were treated with multilayer compression bandaging. Wound fluid and venous blood samples were collected at recruitment and 5 weeks later. In the wound fluid and venous blood, cytokines and factors reflecting the processes of inflammation (interleukin 1beta, tumor necrosis factor-alpha), proteolysis (matrix metalloproteinases-2 and -9), angiogenesis (basic fibroblast growth factor [bFGF], vascular endothelial growth factor), and fibrosis (transforming growth factor-beta(1) [TGFbeta(1)]) were measured. Ulcer healing was assessed using digital planimetry at both assessments. RESULTS The study comprised 80 patients (43 men, 37 women). Median (range) ulcer size reduced from 4.4 (0.1-142.4) cm(2) to 2.2 (0-135.5) cm(2) after 5 weeks (P < .001; Wilcoxon signed rank), although 17 of 80 ulcers increased in size. The volume of wound fluid collected strongly correlated with ulcer size (Spearman rank = 0.801, P < .01). Initial wound fluid concentrations of bFGF correlated with ulcer size (Pearson coefficient = 0.641, P < .01), and changes in wound fluid TGFbeta(1) concentrations inversely correlated with changes in ulcer size (Spearman rank = -0.645, P = .032). There were no significant correlations between changes in other factors and ulcer healing. Wound fluid and serum cytokine concentrations correlated poorly. CONCLUSION Wound fluid collection volume correlates with ulcer size. Ulcer healing correlated with increased concentrations of TGFbeta(1), possibly reflecting increased fibrogenesis in the proliferating wound. Aside from this, there was a large variation in wound and serum cytokine levels that largely limits their usefulness as markers of healing.

Regulation of osteoarthritis by omega-3 (n-3) polyunsaturated fatty acids in a naturally occurring model of disease

Summary Objective To examine effects of high omega-3 (n-3) polyunsaturated fatty acid (PUFA) diets on development of osteoarthritis (OA) in a spontaneous guinea pig model, and to further characterise pathogenesis in this model. Modern diets low in n-3 PUFAs have been linked with increases in inflammatory disorders, possibly including OA. However, n-3 is also thought to increases bone density, which is a possible contributing factor in OA. Therefore we aim to determine the net influence of n-3 in disease development. Method OA-prone Dunkin-Hartley (DH) Guinea pigs were compared with OA-resistant Bristol Strain-2s (BS2) each fed a standard or an n-3 diet from 10 to 30 weeks (10/group). We examined cartilage and subchondral bone pathology by histology, and biochemistry, including collagen cross-links, matrix metalloproteinases (MMPs), alkaline phosphatase, glycosaminoglycan (GAG), and denatured type II collagen. Results Dietary n-3 reduced disease in OA-prone animals. Most cartilage parameters were modified by n-3 diet towards those seen in the non-pathological BS2 strain – significantly active MMP-2, lysyl-pyridinoline and total collagen cross-links – the only exception being pro MMP-9 which was lower in the BS2, yet increased with n-3. GAG content was higher and denatured type II lower in the n-3 group. Subchondral bone parameters in the DH n-3 group also changed towards those seen in the non-pathological strain, significantly calcium:phosphate ratios and epiphyseal bone density. Conclusion Dietary n-3 PUFA reduced OA in the prone strain, and most disease markers were modified towards those of the non-OA strain, though not all significantly so. Omega-3 did not increase markers of pathology in either strain.

Induction of cartilage integration by a chondrocyte/collagen-scaffold implant

The integration of implanted cartilage is a major challenge for the success of tissue engineering protocols. We hypothesize that in order for effective cartilage integration to take place, matrix-free chondrocytes must be induced to migrate between the two tissue surfaces. A chondrocyte/collagen-scaffold implant system was developed as a method of delivering dividing cells at the interface between two cartilage surfaces. Chondrocytes were isolated from bovine nasal septum and seeded onto both surfaces of a collagen membrane to create the chondrocyte/collagen-scaffold implant. A model of two cartilage discs and the chondrocyte/collagen-scaffold sandwiched in between was used to effect integration in vitro. The resulting tissue was analysed histologically and biomechanically. The cartilage–implant–cartilage sandwich appeared macroscopically as one continuous piece of tissue at the end of 40 day cultures. Histological analysis showed tissue continuum across the cartilage–scaffold interface. The integration was dependent on both cells and scaffold. Fluorescent labeling of implanted chondrocytes demonstrated that these cells invade the surrounding mature tissue and drive a remodelling of the extracellular matrix. Using cell-free scaffolds we also demonstrated that some chondrocytes migrated from the natural cartilage into the collagen scaffold. Quantification of integration levels using a histomorphometric repair index showed that the chondrocyte/collagen-scaffold implant achieved the highest repair index compared to controls, reflected functionally through increased tensile strength. In conclusion, cartilage integration can be achieved using a chondrocyte/collagen-scaffold implant that permits controlled delivery of chondrocytes to both host and graft mature cartilage tissues. This approach has the potential to be used therapeutically for implantation of engineered tissue.

IL-13 promotes collagen accumulation in Crohn's disease fibrosis by down-regulation of fibroblast MMP synthesis: a role for innate lymphoid cells?

Background Fibrosis is a serious consequence of Crohn’s disease (CD), often necessitating surgical resection. We examined the hypothesis that IL-13 may promote collagen accumulation within the CD muscle microenvironment. Methods Factors potentially modulating collagen deposition were examined in intestinal tissue samples from fibrotic (f) CD and compared with cancer control (C), ulcerative colitis (UC) and uninvolved (u) CD. Mechanisms attributable to IL-13 were analysed using cell lines derived from uninvolved muscle tissue and tissue explants. Results In fCD muscle extracts, collagen synthesis was significantly increased compared to other groups, but MMP-2 was not co-ordinately increased. IL-13 transcripts were highest in fCD muscle compared to muscle from other groups. IL-13 receptor (R) α1 was expressed by intestinal muscle smooth muscle, nerve and KIR+ cells. Fibroblasts from intestinal muscle expressed Rα1, phosphorylated STAT6 in response to IL-13, and subsequently down-regulated MMP-2 and TNF-α-induced MMP-1 and MMP-9 synthesis. Cells with the phenotype KIR+CD45+CD56+/−CD3− were significantly increased in fCD muscle compared to all other groups, expressed Rα1 and membrane IL-13, and transcribed high levels of IL-13. In explanted CD muscle, these cells did not phosphorylate STAT6 in response to exogenous IL-13. Conclusions The data indicate that in fibrotic intestinal muscle of Crohn’s patients, the IL-13 pathway is stimulated, involving a novel population of infiltrating IL-13Rα1+, KIR+ innate lymphoid cells, producing IL-13 which inhibits fibroblast MMP synthesis. Consequently, matrix degradation is down-regulated and this leads to excessive collagen deposition.

Increased expression of promatrix metalloproteinase-9 and neutrophil elastase in canine dilated cardiomyopathy

OBJECTIVE Canine dilated cardiomyopathy, commonly affecting Doberman pinschers, results in extracellular matrix remodelling within the myocardium. The aim of this study was to examine the proteolytic activity in myocardium from Doberman pinschers with dilated cardiomyopathy. METHODS Samples of myocardium, obtained rapidly post mortem from the left ventricular free wall of Dobermans with dilated cardiomyopathy, clinically normal Dobermans and control dogs (non-Dobermans), were examined for proteolytic activity using substrate gel zymography. Gels were analysed by scanning densitometry. RESULTS Promatrix metalloproteinase-9 activity was significantly increased in all Doberman myocardium when compared to controls. A significant increase in an enzyme, identified to be neutrophil elastase by inhibition of its activity by Elastatinal and Western blotting, was also detected in all Dobermans when compared to controls. CONCLUSIONS The results indicate that promatrix metalloproteinase-9 and neutrophil elastase, both of which are implicated in inflammatory responses, are present in significantly elevated levels in Doberman dilated cardiomyopathy and are raised in clinically normal Dobermans. Both proteolytic enzymes degrade a wide variety of connective tissue components and thus the increased levels found may play an important role in the structural remodelling seen in the myocardium and subsequent heart failure. Increased proteolytic enzyme levels in clinically normal Dobermans may be indicative of the predisposition of the breed to dilated cardiomyopathy.