Regenerative medicine techniques are currently being investigated to replace damaged cartilage. Critical to the success of these techniques is the ability to expand the initial population of cells while minimising de-differentiation to allow for hyaline cartilage to form. Three-dimensional culture systems have been shown to enhance the differentiation of chondrocytes in comparison to two-dimensional culture systems. Additionally, bioreactor expansion on microcarriers can provide mechanical stimulation and reduce the amount of cellular manipulation during expansion. The aim of this study was to characterise the expansion of human chondrocytes on microcarriers and to determine their potential to form cartilaginous tissue in vitro.

High-grade human articular cartilage was obtained from leg amputations with ethics approval. Chondrocytes were isolated by collagenase digestion and expanded in either monolayers (104 cells/cm2) or on CultiSpher-G microcarriers (104 cells/mg) for three weeks. Following expansion, monolayer cells were passaged and cells on microcarriers were either left intact or the cells were released with trypsin/EDTA. Pellets from these three groups were formed and cultured for three weeks to establish the chondrogenic differentiation potential of monolayer-expanded and microcarrier-expanded chondrocytes. Cell viability, proliferation, glycosaminoglycan (GAG) accumulation, and collagen synthesis were assessed. Histology and immunohistochemistry were also performed.

Human chondrocytes remained viable and expanded on microcarriers 10.2±2.6 fold in three weeks. GAG content significantly increased with time, with the majority of GAG found in the medium. Collagen production per nanogram DNA increased marginally during expansion. Histology revealed that chondrocytes were randomly distributed on microcarrier surfaces yet most pores remained cell free. Critically, human chondrocytes expanded on microcarriers maintained their ability to redifferentiate in pellet culture, as demonstrated by Safranin-O and collagen II staining. These data confirm the feasibility of microcarriers for passage-free cultivation of human articular chondrocytes. However, cell expansion needs to be improved, perhaps through growth factor supplementation, for clinical utility. Recent data indicate that cell-laden microcarriers can be used to seed fresh microcarriers, thereby increasing the expansion factor while minimising enzymatic passage.

Introduction and Aims: To deliver osteogenic cells into bone defects, the crucial steps are cell attachment and migration in cell-delivery biomaterials. The aim of this study was to examine whether complexes comprised of vitronectin (VN), insulin growth factors (IGFs) and insulin growth factor binding proteins (IGFBPs) could enhance human osteoblasts attachment, especially cell migration in three-dimensional (3-D) culture.

Method: Human osteoblasts derived from alveolar bone chips (passage 4–10) and established human osteoblast cell line SaOS-2 were used. These cells were seeded on scaffolds of type I collagen sponges and poly glycolic acid (PGA) (approx. one millimetre thick, porous structure), which had been coated with VN +/− IGF-I +/− IGFBP-3. Cell attachment and migration were evaluated by cell counting, confocal microscopy, and scanning electron microscopy.

Results: The number of attached human osteoblasts was significantly higher in wells in which pre-bound VN was coated on the polystyrene culture dish or on type I collagen sponges. However, no significant difference of cell attachment was observed when growth factors were bound to these surfaces in the presence of VN. In the two scaffold materials examined, greater cell attachment was found in type I collagen sponges compared to PGA scaffolds. However, coating the scaffolds with complexes comprised of VN + IGF-I or VN + IGFBP-5 + IGF-I enhanced cell attachment on PGA. Moreover, the presence of vitronectin + IGF-I + IGFBP-5 resulted in significantly greater osteoblast migration into deep pore areas as compared to untreated scaffolds or scaffolds treated with different combinations of the VN +/− IGF +/− IGFBP-5.

Conclusion: Complexes of VN + IGFBP-5 + IGF-I enhance the attachment and migration of human osteoblast in three-dimensional culture, which implies that this complex has potential application for use in surface modification of biomaterials for tissue reconstruction.