Abstract
Cartilage and bone degeneration are major healthcare problems affecting millions of individuals worldwide. Elucidation of the processes modulating the cell-matrix interactions involved in cartilage or bone formation offer tremendous potential in the development of clinically relevant strategies for cartilage and bone regeneration. We have therefore adopted an ex vivo tissue engineering approach to investigate chondrogenesis and osteogenesis using a mix human mesenchymal progenitor populations encapsulated in biomineralised polysac-charide templates with or without the addition of type-I collagen.
Alginate/chitosan polysaccharide capsules containing 2.5mg/ml type-I collagen and TGF-beta-3 were encapsulated with human bone marrow cells (HBMC), articular chondrocytes or a co-culture at a ratio of 2:1 respectively and placed in a rotating (Synthecon) biore-actor or held in static 2D culture conditions for 28 days, to determine whether the presence of type-I collagen within the alginate could promote the synthesis of an extracellular matrix.
Constructs were stained with alcian blue, sirius red and von Kossa. In bioreactor samples encapsulated with HBMC and type-I collagen, viable cells were present within lacunae, surrounded by a matrix of proteo-glycans and fibrous collagen, which was mineralized. Immunohistochemistry and polarised light microscopy indicated an organised collagenous matrix with extensive expression of type I collagen and bone sialoprotein with small regions of type II collagen. Type X collagen was also expressed indicating the presence of hypertrophic chondrocytes. Within the static HBMC groups, smaller areas of matrix were generated with decreased expression of type-I and type-II collagen. Co-culture bioreactor samples also demonstrated regions of new mineralised bone matrix; however these were less prominent than in the HBMC only groups. No matrix formation was observed in chondrocyte cultures although the cells remained viable as assessed by live/dead staining. Biochemical analysis indicated significantly increased (p< 0.05) DNA in all bioreactor samples in comparison with static constructs and significantly increased protein in HBMC bioreactor constructs in comparison with other cell types.
These studies outline a unique tissue engineering approach, utilizing individual and mixed human mesen-chymal progenitor populations coupled with innovative polysaccharide templates containing type I collagen and bioreactor systems to promote chondrogenic and osteo-genic differentiation.
Correspondence should be addressed to Mr Carlos Wigderowitz, Senior Lecturer, University Department of Orthopaedic and Trauma Surgery, Ninewells Hospital and Medical School, Dundee DD1 9SY.
