Skeletal stem cells: phenotype, biology and environmental niches informing tissue regeneration.

Advances in our knowledge of the biology of skeletal stem cells, together with an increased understanding of the regeneration of normal tissue offer exciting new therapeutic approaches in musculoskeletal repair. Skeletal stem cells from various adult tissues such as bone marrow can be identified and isolated based on their expression of a panel of markers associated with smooth muscle cells, pericytes and endothelial cells. Thus, skeletal stem cell-like populations within bone marrow may share a common perivascular stem cell niche within the microvascular network. To date, the environmental niche that nurtures and maintains the stromal stem cell at different anatomical sites remains poorly understood. However, an understanding of the osteogenic and perivascular niches will inform identification of the key growth factors, matrix constituents and physiological conditions that will enhance the ex vivo amplification and differentiation of osteogenic stem cells to mimic native tissue critical for tissue repair. This review will examine skeletal stem cell biology, the advances in our understanding of the skeletal and perivascular niche and interactions therein and the opportunities to harness that knowledge for musculoskeletal regeneration.

Genetic manipulation of human mesenchymal progenitors to promote chondrogenesis using "bead-in-bead" polysaccharide capsules.

Articular cartilage defects arising from trauma or degenerative diseases fail to repair spontaneously. We have adopted a non-viral gene delivery and tissue engineering strategy, in which Sox-9 transfected human mesenchymal progenitors have been encapsulated within alginate/chitosan polysaccharide capsules to promote chondrogenesis. Human bone marrow stromal cells and articular chondrocytes were transfected with flag-tagged Sox-9 plasmid and after 7 days in static culture, large regions of cell-generated matrix containing cartilage proteoglycans were observed as confirmed by positive Alcian blue staining and Sox-9 immunohistochemistry. Further, after 28 days, in vitro and in vivo, samples encapsulated with Sox-9 transfected cells demonstrated large regions of cartilaginous matrix as confirmed by positive Alcian blue staining, Sox-9 and type-II collagen immunohistochemistry, absent in samples encapsulated with untransfected cells. Extracted protein from in vivo constructs was further assessed by western blot analysis and positive expression of Sox-9 and type-II collagen was observed in Sox-9 transfected constructs which was absent in untransfected cells. Regions of cartilage-like matrix were significantly increased in Sox-9 constructs in comparison with untransfected constructs, confirming Sox-9 gene delivery enhances chondrogenesis in targeted cell populations, outlining the potential to promote cartilaginous construct formation with therapeutic implications for regeneration of human articular cartilage tissue defects.

Formation of a human-derived fat tissue layer in P(DL)LGA hollow fibre scaffolds for adipocyte tissue engineering.

Development of adipose tissue-engineering strategies, where human bone marrow stromal cells (HBMSC) are combined with three-dimensional scaffolds, is likely to prove valuable for soft tissue restoration. In this study, we assessed the function of poly(DL-lactide-co-glycolide) (P(DL)LGA) hollow fibres in facilitating the development of HBMSC-derived adipocytes for advancement of an associated adipocyte layer. The large surface area of 75:25 P(DL)LGA fibres facilitated the rapid generation of extensive adipocyte aggregates from an undifferentiated HBMSC monolayer, where the fat-laden cells stained positive with Oil Red O and expressed the adipocyte marker, fatty acid binding protein 3 (FABP3). Following implantation subcutaneously in severely compromised immunodeficient mice, the adipogenic phenotype of the PLGA-adipocyte graft was maintained for up to 56 days. Confocal microscopy showed associated LipidTOX Deep Red neutral lipid staining in an (FL)P(DL)LGA fibre-adipocyte graft after 56 days, critical evidence demonstrating maintenance of the adipocyte phenotype in the subcutaneous graft. To support adipose tissue advancement in a defined volume, the P(DL)LGA-adipocyte scaffold was encapsulated within alginate/chitosan hydrogel capsules (typical diameters, 4.0 mm). In a 28-day in vivo trial in immunodeficient mice, clusters of the capsules were maintained at the subcutaneous site. An adipocyte tissue layer advancing within the surrounding hydrogel was demonstrated.

The effect of pre-coating human bone marrow stromal cells with hydroxyapatite/amino acid nanoconjugates on osteogenesis.

Aqueous colloidal suspensions of positively charged, amino acid-functionalized hydroxyapatite (HAp) nanoparticles (HAp/alanine and HAp/arginine) were added to a HBMSC suspension to effect non-specific cell surface deposition due to favourable attractive electrostatic interactions. Subsequent maintenance of these hybrid precursors under in vitro basal (non-osteogenic) culture conditions for up to 21 days, either as a monolayer or as a 3D pellet culture system, resulted in significantly increased levels of markers of osteoblast differentiation in comparison with uncoated cells. In monolayer culture, osteogenic activity could be further enhanced in a dose-dependent manner by surface derivatization of the amino acid-stabilized nanoparticles with the cell surface-specific binding peptide arginine-glycine-aspartic acid (RGD). Significantly, in 3D pellet culture conditions all HAp nanoconjugates promoted osteoblast differentiation, whereas for uncoated cells even soluble osteogenic culture additives were ineffectual. We therefore tested these constructs for in vivo activity by subcutaneous implantation in immunocompromised mice. New osteoid formation was observed in samples recovered after 21 days, comparable to the extensive areas of mineralized extracellular matrix produced in vitro. Overall, these studies outline the potential of biomolecular/hydroxyapatite nanoconjugates to promote osteogenic cell differentiation in vitro and hence provide new models to examine skeletal cell differentiation and function. Moreover, the pre-coating of HBMSCs enables the formation of viable hybrid multicellular 3D constructs with demonstrable activity both in vitro and in vivo.