Biomedical subjects
P K Bos
Publications and source records attributed to P K Bos.
Specific enzymatic treatment of bovine and human articular cartilage: implications for integrative cartilage repair.
OBJECTIVE: Chondrocyte death in articular cartilage wound edges and the subsequent lack of matrix-producing cells in the interface area are considered to be a major cause of impaired cartilage wound healing and poor integrative cartilage repair. This study was undertaken to investigate whether enzymatic matrix digestion can be used to stimulate integrative cartilage repair via a mechanism of local increase in the amount of vital chondrocytes in cartilage wound edges. METHODS: Full-thickness bovine articular cartilage samples were cultured in vitro for 14 days in standard medium. Samples were either left untreated or treated for 48 hours with 0.3% hyaluronidase or 30 units/ml highly purified collagenase VII. Nuclear and cytoplasmic changes were analyzed to determine cell viability, and the number of vital chondrocytes in wound edges was determined. Subsequently, we investigated whether increased chondrocyte density in the lesion edges resulted in better wound healing. Finally, full-thickness human tibial plateau cartilage explants were tested with similar enzyme treatment protocols to determine the clinical value of our results. RESULTS: In bovine explants a rapid onset of chondrocyte death was observed in wound edges in all treatment groups. This led to low chondrocyte density in a band of 0-150 microm from the lesion edges in untreated and hyaluronidase-treated explants. Treatment with 30 units/ml collagenase resulted in a significant increase in chondrocyte density in this area. The integration experiments demonstrated improved integration of the lesion edges after treatment with collagenase. In human articular cartilage an increase in chondrocyte density at the lesion edges could also be achieved, but only when proteoglycans were depleted from the wound edges prior to collagenase treatment. CONCLUSION: Treatment with highly purified collagenase improves integrative cartilage repair, possibly by increasing the cell density at cartilage wound edges.
Growth factor expression in cartilage wound healing: temporal and spatial immunolocalization in a rabbit auricular cartilage wound model.
OBJECTIVE: The ability of cartilage to regenerate following injury is limited, potentially leading to osteoarthritis. Integrative cartilage repair, necessary for durable restoration of cartilage lesions, can be regarded as a wound healing process. Little is known about the effects of growth factors regulating acute cartilage wound healing in vivo. In this study the temporal expression patterns of growth factors and proteoglycan content in cartilage wound edges in vivo were studied. DESIGN: Cartilage wounds were created in rabbit ear cartilage using a 6 mm biopsy punch. Specimens were subsequently harvested 1, 3, 7, 14 and 28 days after surgery. Paraffin sections were thionin stained to visualize proteoglycan loss and replacement. Immunohistochemical staining of TGFbeta1, TGFbeta3, IGF-1, IGF-II and FGF-2 was used to define growth factor expression at the cartilage wound sites. RESULTS: Almost no effect of cartilage wounding was observed one day after surgery. A decrease of proteoglycan content, with a maximal loss at day 7, and a subsequent restoration was observed at the wound edges. Growth factor expression increased simultaneously. Maximal immunostaining for IGF1, IGFII, FGF2 and TGF-beta3 was observed at day 7, followed by a gradual decrease. Increased expression of TGFbeta1 lasted from day 3 until day 14. CONCLUSION: We have demonstrated the ability of chondrocytes to increase growth factor expression and to restore the rapid decrease in proteoglycan content in the initial phase following acute wounding. A temporal increase in intracellular growth factor expression suggests an autocrine and/or paracrine metabolic stimulation, which can be regarded a sign of chondrocytes repair capacity.
Fixation-dependent immunolocalization shift and immunoreactivity of intracellular growth factors in cartilage.
The effects of fixation on immunolocalization and immunoreactivity in cartilage tissues were studied using monoclonal antibodies against peptides that can effectively stimulate chondrocytes in vitro and have been shown to play a role in musculoskeletal tissue regeneration: transforming growth factor beta1, transforming growth factor beta3, insulin-like growth factor I, insulin-like growth factor II and fibroblast growth factor 2. Paraffin sections fixed in buffered formalin, buffered paraformaldehyde, Carnoy and methacarn, as well as cryosections, were tested. A strong immunoreaction was observed in tissue fixed in formaldehyde-based fixatives, with a resemblance to that in cryopreserved tissues. Immunoreactivity was reduced in alcohol-fixed tissues. Furthermore, a striking intracellular immunolocalization shift from cytoplasm to nucleus was observed using alcohol-based fixatives as compared to cryopreserved or formaldehyde-based fixatives. We concluded that, for the detection and localization of growth factors in cartilage tissues, fixation in buffered formalin or paraformaldehyde is optimal.