Ionic solutes impact collagen scaffold bioactivity.

The structure of ice-templated collagen scaffolds is sensitive to many factors. By adding 0.5 wt% of sodium chloride or sucrose to collagen slurries, scaffold structure could be tuned through changes in ice growth kinetics and interactions of the solute and collagen. With ionic solutes (sodium chloride) the entanglements of the collagen molecule decreased, leading to fibrous scaffolds with increased pore size and decreased attachment of chondrocytes. With non-ionic solutes (sucrose) ice growth was slowed, leading to significantly reduced pore size and up-regulated cell attachment. This highlights the large changes in structure and biological function stimulated by solutes in ice-templating systems.

The effects of scaffold architecture and fibrin gel addition on tendon cell phenotype.

Development of tissue engineering scaffolds relies on careful selection of pore architecture and chemistry of the cellular environment. Repair of skeletal soft tissue, such as tendon, is particularly challenging, since these tissues have a relatively poor healing response. When removed from their native environment, tendon cells (tenocytes) lose their characteristic morphology and the expression of phenotypic markers. To stimulate tendon cells to recreate a healthy extracellular matrix, both architectural cues and fibrin gels have been used in the past, however, their relative effects have not been studied systematically. Within this study, a combination of collagen scaffold architecture, axial and isotropic, and fibrin gel addition was assessed, using ovine tendon-derived cells to determine the optimal strategy for controlling the proliferation and protein expression. Scaffold architecture and fibrin gel addition influenced tendon cell behavior independently in vitro. Addition of fibrin gel within a scaffold doubled cell number and increased matrix production for all architectures studied. However, scaffold architecture dictated the type of matrix produced by cells, regardless of fibrin addition. Axial scaffolds, mimicking native tendon, promoted a mature matrix, with increased tenomodulin, a marker for mature tendon cells, and decreased scleraxis, an early transcription factor for connective tissue. This study demonstrated that both architectural cues and fibrin gel addition alter cell behavior and that the combination of these signals could improve clinical performance of current tissue engineering constructs.

Type II collagen deposition in cruciate ligament precedes osteoarthritis in the guinea pig knee.

OBJECTIVE: To examine the collagens in cruciate ligaments of young Dunkin-Hartley guinea pigs, to determine whether a change in specific collagen types is an early feature of the spontaneous osteoarthritis (OA), which consistently develops in the medial compartment of the knee in this strain. DESIGN: Collagen types I, II, III, IX, and XI were detected by immunofluorescence microscopy in the anterior and posterior cruciate ligaments of animals at 3, 4-5 and 12 weeks of age. Type II collagen in PCL was further analysed by confocal microscopy or biochemical assay after cyanogen bromide digestion, SDS-PAGE and immunoblotting. Interfibrillar proteoglycans were visualized by transmission electron microscopy. RESULTS: Collagen types I and III formed the bulk of fibrous mid-ligament tissue in all animals. Typical cartilage collagens, types II, IX and XI, were identified by immunolabeling where ligaments attached to tibial bone. Type II collagen, normally restricted to the fibrocartilage attachment sites, was also found at separate foci in anterior fiber bundles of the posterior cruciate ligament in 12-week-old animals. Biochemical data confirmed these observations which, together with electron microscopy showing large atypical proteoglycan structures, suggested the deposition of fibrocartilage within the fibrous mid-ligament. CONCLUSIONS: Cruciate ligaments, especially posterior cruciate ligament in Dunkin-Hartley guinea pigs synthesize cartilage-like matrix in mid-ligament prior to the appearance of classical signs of OA.

Up-regulation of matrix metalloproteinase expression and activation following cyclical compressive loading of articular cartilage in vitro.

Osteoarthritis (OA) results in articular cartilage degeneration and subchondral bone remodeling. Excessive or abnormal loading of the joint may contribute to matrix destruction by creating an imbalance between proteinases and their inhibitors. This study investigates whether cyclical loading regulates expression and/or activation of metalloproteinases 2 and 9 (MMPs) in articular cartilage explants. Gelatin zymography, reverse zymography, and MMP activity assays of mechanically loaded bovine cartilage explants (0.5 MPa, 1 Hz, 3 h) showed increased expression and activation of MMPs 2 and 9, whereas expression of the tissue inhibitors of MMPs was unaffected. This shows, for the first time that mechanical loading can influence tissue homeostasis generating an imbalance of proteinases and their inhibitors inducing turnover and/or catabolic events in cartilage.

Collagen expression in chicken tibial dyschondroplasia.

Collagen expression in growth plate cartilage derived from broiler chickens with tibial dyschondroplasia was studied and compared with samples from unaffected birds. Normal growth plate contains 12% collagen (dry weight) and dyschondroplastic growth plate 19% collagen compared with articular cartilage, which contains 55%. Dyschondroplastic growth plate collagens were more resistant to extraction by pepsin treatment than were those from unaffected growth plate. Normal and dyschondroplastic growth plate cartilages contain similar amounts of type I collagen (5% of the total collagen) but dyschondroplastic growth plate cartilage contains slightly less type II and type XI collagens, and significantly more type X collagen (25% as compared to 11%) than in normal growth plate. The levels of the mature collagen cross-link, hydroxylysyl-pyridinoline, are very low in normal growth plate but are six times higher in dyschondroplastic lesions. Immunolocalisation studies show that there is little change to the normal patterns of collagen organisation in dyschondroplastic growth plate. Investigation of metalloproteinase activity showed there to be a reduction in MMP-2 levels in dyschondroplastic growth plate compared to normal growth plate. In vitro studies on articular, normal growth plate and dyschondroplastic growth plate chondrocytes cultured in alginate or on plastic revealed differences between the cell types. When plated on plastic, articular chondrocytes rapidly assume a fibroblastic morphology. In contrast, normal growth plate chondrocytes retain their polygonal morphology whereas chondrocytes derived from dyschondroplastic cartilage initially exhibit both fibroblastic and polygonal phenotypes but gradually change to totally fibroblastic. These morphological changes are reflected by the collagen synthesis in vitro. Chondrocytes derived from normal articular cartilage synthesised collagen types I, II and X when cultured in alginate but type X synthesis was lost when cultured on plastic. Chondrocytes derived from normal growth plate cartilage synthesised predominantly type X collagen when cultured in either system. Chondrocytes derived from dyschondroplastic growth plate exhibited a similar phenotype to normal growth plate chondrocytes when cultured in alginate beads, but showed signs of dedifferentiation with reduced type X collagen and increased type I collagen when plated on plastic. These results suggest that the chondrocytes in dyschondroplastic growth plate cartilage are at a different stage of maturity than normal resulting in a cartilage that is failing to turn over at a normal rate.

Differential scanning calorimetric studies of superficial digital flexor tendon degeneration in the horse.

Differential scanning calorimetry (DSC) of equine superficial digital flexor tendons revealed the presence of a small exothermic peak at 23 degrees C of unknown origin, and a large endothermic peak at 70 degrees C due to denaturation of cross-linked collagen fibres. In the central degenerated core of damaged tendons the denaturation temperature remained at 70 degrees C but the enthalpy decreased in relation to the extent of degeneration of the tendon. We suggest that this reduction in enthalpy is due to depolymerisation and denaturation of the collagen fibres. This contention is supported by the observed increased activity of the degradative enzyme cathepsin B secreted by the fibroblasts. DSC analysis of cultured porcine tendon fibroblasts revealed a multicomponent endotherm, denaturation beginning at 46 degrees C, a temperature capable of being achieved within the tendon during intensive exercise. DSC clearly has considerable potential in complementing morphological and biochemical studies to determine the aetiology and progress of equine tendon degeneration.

Characterisation of articular and growth plate cartilage collagens in porcine osteochondrosis.

The articular and growth plate cartilages of osteochondrotic pigs were examined and compared with those from clinically normal animals. Both types of osteochondrotic cartilage showed considerable localised thickening apparently due to a lack of ossification. Histological examination of cartilage lesions demonstrated a breakdown in the normal pattern of chondrocyte maturation. Articular cartilage lesions lacked mature clones of chondrocytes in the calcifying region. Growth plate cartilage showed an accumulation of disorganised hypertrophic chondrocytes rather than the well-defined columns seen in normal tissue. The overall percentages of collagen in osteochondrotic lesions from both articular and growth plate cartilage were significantly reduced compared with levels in unaffected cartilage. There were substantial increases in the proportion of type I collagen in lesions from both osteochondrotic articular and growth plate cartilages and a reduction in the proportion of type II collagen. Type X collagen was detected in osteochondrotic but not normal articular cartilage. The proportion of type X collagen was unchanged in osteochondrotic growth plate cartilage. The levels of the collagen cross-links, hydroxylysylpyridinoline, hydroxylysyl-ketonorleucine and dehydrohydroxylysinonorleucine were radically reduced in samples from osteochondrotic growth-plate cartilage lesions when compared with normal tissue. Less dramatic changes were observed in articular cartilage although there was a significant decrease in the level of hydroxylysylketonorleucine in osteochondrotic lesions. Immunofluorescence examination of osteochondrotic lesions showed a considerable disruption of the organisation of the collagenous components within both articular and growth-plate cartilages. Normal patterns of staining of types I and VI collagen seen at the articular surface in unaffected tissue were replaced by a disorganised, uneven stain in osteochondrotic articular cartilage lesions. Incomplete removal of cartilage at the ossification front of osteochondrotic growth plate was demonstrated by immunofluorescence staining of type IX collagen. Type X collagen was produced in the matrix of the calcifying region of osteochondrotic articular cartilage by small groups of hypertrophic chondrocytes, but was not detected in normal articular cartilage. The distribution of type X collagen was unchanged in osteochondrotic growth plate cartilage.

Quantification and immunolocalisation of porcine articular and growth plate cartilage collagens.

The collagens of growth plate and articular cartilage from 5-6 month old commercial pigs were characterised. Growth plate cartilage was found to contain less total collagen than articular cartilage as a proportion of the dry weight. Collagen types I, II, VI, IX and XI are present in both growth plate and articular cartilage whereas type X is found exclusively in growth plate cartilage. Types III and V collagen could not be detected in either cartilage. Type I collagen makes up at least 10% of the collagenous component of both cartilages. There are significant differences in the ratios of the quantifiable collagen types between growth plate and articular cartilage. Collagen types I, II, and XI were less readily extracted from growth plate than from articular cartilage following pepsin treatment, although growth plate cartilage contains less of the mature collagen cross-links, hydroxylysyl-pyridinoline and lysyl-pyridinoline. Both cartilages contain significant amounts of the divalent reducible collagen cross-links, hydroxylysyl-ketonorleucine and dehydro-hydroxylysinonorleucine. Immunofluorescent localisation indicated that type I collagen is located predominantly at the surface of articular cartilage but is distributed throughout the matrix in growth plate. Types II and XI are located in the matrix of both cartilages whereas type IX is predominantly pericellular in the calcifying region of articular cartilage and the hypertrophic region of the growth plate. Collagen type VI is located primarily as a diffuse area at the articular surface.

The application of scanning confocal microscopy in cartilage research.

Scanning confocal microscopy has been used in conjunction with immunofluorescent localization to address two areas of debate in cartilage research. With the enhanced resolution and optical sectioning capability of this new technique, we have demonstrated that type IX collagen is preferentially located in an area around the chondrocyte, even in young cartilage. We have also shown that cathepsin B production is not confined to de-differentiated chondrocytes. The advantages and versatility of scanning confocal microscopy have thus clearly been demonstrated.

Monoclonal antibody sandwich ELISA for the potential detection of chicken meat in mixtures of raw beef and pork.

A sandwich ELISA (enzyme-linked immunosorbent assay) has been developed successfully for the detection of defined amounts of chicken meat (1-100%) in beef and pork meat mixtures. The assay uses a monoclonal antibody (BC9) specific to a chicken muscle soluble protein to capture this protein from complex meat mixtures. Further immunorecognition of the captured protein was attained with rabbit polyclonal antibodies against chicken muscle proteins (anti-CHSP). A commercial goat anti-rabbit immunoglobulin conjugated to peroxidase was used to detect the anti-CHSP antibodies bound to the chicken protein. Subsequent enzymic conversion of substrate gave clear optical density differences when assaying mixtures of beef and pork meats containing variable amounts of chicken meat.

Immunodetection of cathepsins B and L present in and secreted from human pre-malignant and malignant colorectal tumour cell lines.

Pre-malignant and malignant human colorectal tumour epithelial cell lines both secreted precursor forms of the 2 cysteine proteinases, cathepsins B and L. The amount of proteinases secreted by these cell lines varied according to the cell density. Comparison at similar cell densities showed that the pre-malignant, adenoma-derived cell line (PC/AA) secreted as much, or more, of both cathepsin B and L precursors as did the malignant, carcinoma-derived cell line (PC/JW/FI). However, mature forms of cathepsins B and L were detected in the culture media of only the carcinoma-derived cell line, thus indicating that the invasive potential of a tumour may be related to its ability to process extracellularly the secreted precursor enzyme to a mature and consequently active enzyme, rather than to the amount of proteinase synthesized and/or secreted. Similar results were obtained using 2 other epithelium-derived tumour cell lines, HT/29 (carcinoma) and SP/AN (adenoma). Immunolocation studies showed that cathepsin B was lysosomal while cathepsin L appeared to have a distribution more consistent with a plasma membrane association. Purified human cathepsins B and L (mature form) were capable of solubilizing an isolated basement membrane matrix (bovine anterior lens capsule) in vitro, thus indicating that the secreted mature enzymes and the membrane-associated cathepsin L could potentially degrade basal laminae or sub-endothelial basement membranes in vivo.

Production and characterization of monoclonal antibodies specific to chicken muscle soluble proteins.

Three stable hybridoma cell lines (AH4, BC9 and CF2) have been produced which secrete monoclonal antibodies specific for chicken and turkey muscle proteins. Partial characterization by ELISA and SDS-PAGE immunoblotting indicated that the antibodies failed to cross-react with similar extracts of pork, beef, lamb, horse or rabbit. One of the cell lines (AH4) secreted a monoclonal antibody that was also capable of distinguishing between chicken and turkey by indirect ELISA.

Monoclonal antibodies to rabbit liver cathepsin B.

Three stable hybridoma cell lines (AF8, BC11, CE2) have been produced that secrete antibodies specific for cathepsin B. These have been characterized by ELISA, SDS-PAGE immunostaining, immunoprecipitation and immunofluorescent staining. CE2 immunoprecipitated native cathepsin B with retention of enzymic activity, but failed to cross-react with the alkali-denatured enzyme. BC11 bound only to the denatured form of cathepsin B and AF8 cross-reacted with both native and denatured cathepsin B. However, unlike CE2-immunoprecipitated enzyme, activity could be detected only after dissociation of the antigen-AF8 antibody complex. No cross reaction was found with any lysosomal protein including the cysteine proteinases, cathepsins H and L.

Production of a monospecific antiserum to cathepsin L: the histochemical location of enzyme in rabbit fibroblasts.

A polyclonal antibody against rabbit cathepsin L was raised in goats and shown to be specific for both active and inactive enzyme. Using this antibody we have examined the distribution of cathepsin L in primary rabbit skin fibroblasts by immunohistochemistry and found that all the enzyme is located within lysosomal granules. At confluence many cathepsin-L-containing lysosomes were seen in each cell. A new but nonspecific histochemical substrate for cathepsin L was tested and a similar distribution was obtained. Our results indicate that the immunohistochemical technique can be reliably employed for the specific location of cathepsin L in cells and tissues.

The mononuclear cell population in rat leg muscle: its contribution to the lysosomal enzyme activities of whole muscle extracts.

The mononuclear cell fraction of rat hind-limb muscle was obtained by digestion with clostridial collagenase in the presence of calcium ions, filtration through nylon screens and washing to remove the enzyme. Final traces of contaminant myofibrillar debris were separated by isopycnic centrifugation in a Percoll density gradient. Whole muscle, washed cells and Percoll-fractionated cells were extracted in the presence of non-ionic detergent and the supernatants assayed for the lysosomal enzymes cathepsins B + L, N-acetyl-beta-glucosaminidase, beta-glucuronidase and protein. The enzyme levels were highest in the muscle from young rats, but the percentage of recovered activity in the mononuclear cell fraction was little altered by the age of the animal. The values obtained were: cathepsin B + L, 2.4-4.0%; N-acetyl-beta-glucuronidase, 4.3-7.6%; and beta-glucuronidase, 6.3-10.3%. Because of unavoidable losses in preparation these are minimal values and the actual levels of activity from the mononuclear cell fraction in muscle would be higher. The specific activity values of the cell lysates were raised after isopycnic centrifugation and were nearly constant over the age range 65-180 days. Substantially higher specific activity values were obtained for the cells from rats of 38 days. When grown in culture the mononuclear cell fractions were seen to contain mainly fibroblasts and myoblasts with only few leucocytes. The cultures reached confluence by the second week, at which time numerous myotubes had formed. In addition there were groups of large, circular cells with a prominent centrosphere. The origin of these latter cells is uncertain. It was concluded that although total lysosomal enzyme activity was higher in young rats there was little effect of age on the distribution of activity between muscle fibres and mononuclear cells in the muscle.