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COLLAGEN BIOMIMETIC SCAFFOLDS FOR BONE AND TISSUE ENGINEERING

University Orthopaedics, Southampton General Hospital, Southampton SO16 6YD.

The formation of biomimetic environments using scaffolds containing cell recognition sequence and osteo-inductive factors in combination with bone cells offers tremendous potential for bone and cartilage regeneration. In tissues, collagen forms the scaffold by mediating the flux of chemical and mechanical stimuli. Recently, a synthetic 15-residue peptide P-15, related biologically to the active domain of type I collagen, has been found to promote attachment and the osteoblast phenotype of human dermal fibroblasts and periodontal ligament fibroblasts on particulate anorganic bone mineral (ABM). The aim of this study was to exam the ability of the collagen peptide, P-15, to promote human osteoprogenitor attachment, proliferation and differentiation on cell culture surfaces and 3-D scaffolds.

Selected human bone marrow cells were cultured on particulate microporous anorganic bone mineral (‘pure ‘ hydroxyapatite based on x-ray diffraction standard JCPDS9-432) phase and polygalactin vicryl mesh adsorbed with or without P-15 in basal or osteogenic conditions. Cell adhesion, spreading and patterning were examined by light and confocal microscopy following incorporation of cell tracker green and ethidium homodimer fluorescent labels. Osteoprogenitor proliferation and differentiation was assessed by DNA content and alkaline phosphatase specific activity. Growth and differentiation on 3-D ABM structures were examined by confocal and scanning electron microscopy (SEM).

P-15 promoted human osteoprogenitor cell attachment and patterning on particulate bovine anorganic bone mineral phase and polygalactin vicryl mesh over 5–24 hours compared to culture on ABM and vicryl mesh alone as observed by photomicroscopy. Increased alkaline phosphatase specific activity was enhanced following culture on P-15 adsorbed matrices as recognized by enhanced expression of alkaline phosphatase, type I collagen, osteocalcin and cfba-1. The presence of mineralised bone matrix and extensive cell ingrowth and cellular bridging between 3-D ABM matrices and polygalactin vicryl mesh adsorbed with P-15 was observed by confocal microscopy and alizarin red staining. SEM confirmed the 3-D structure of newly formed cell constructs and cellular ingrowth on and between the P-15 modified inorganic bone mineral materials. Negligible cell growth was observed on ABM alone or polygalactin vicryl mesh alone.

These observations demonstrate that the synthetic 15-residue collagen peptide, P-15, when adsorbed to ABM or polygalactin vicryl mesh, can stimulate human osteoprogenitor attachment and spreading. They also demonstrated that P-15 coupled 3-D matrices stimulate human osteoprogenitor differentiation and materialisation. The studies indicate that a synthetic analogue of collagen provides a biomimetic environment supportive for cell differentiation and tissue regeneration and indicate a potential for the use of extracellular matrix cue in the development of biomimetic environments for bone tissue engineering.

Abstracts prepared by Dr P E Watkins, Hodgkin Building, Guys Campus, King’s College London.