Various members of the insulin-like growth factor (IGF) family of growth factors are highly expressed in bone tissue and are vitally important for the normal development and function of bone. Recent studies have shown that IGF-I can associate with the extra-cellular matrix proteins vitronectin (VN) and fibronectin (FN) via IGF binding protein-5 (IGFBP-5). Furthermore, when these complexes are pre-bound to a tissue culture surface they can stimulate enhanced responses in epithelial cell types in vitro. More recently, transforming growth factor-beta 1 (TGF-β1), epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) have also been shown to interact with VN and to elicit functional responses in various cell types. Taken together, these findings indicate that exploitation of the adhesive properties of these ECM proteins might allow immobilisation of various growth factors at the culture surface. This may provide a novel means of coating engineered biomaterial constructs with agents which can elicit specific functional effects in therapeutically important cells, such as those used in cell-based therapeutics for the replacement and / or regeneration of damaged bone tissue. Since both VN and FN are also important matrix components of bone, this study sought to investigate the hypothesis that select pre-bound combinations of these matrix proteins and growth factors could also stimulate functional responses in bone cells and the therapeutically important so called mesenchymal stem cells. Thus it is reported here that pre-bound combinations of VN, IGFBP-5 and IGF-I or FN IGFBP-5 and IGF-I significantly stimulate cell migration in the osteoblast-like SaOS-2 cells. While, VN, IGFBP-5 and IGF-I stimulated cell proliferation over 72 hr, FN, IGFBP-5 and IGF-I did not. Moreover, I found that VN, IGFBP-5 and IGF-I could facilitate alkaline phosphatase (ALP) expression in SaOS-2 cells. VN, FN and EGF on the other hand could sustain SaOS-2 cells for up to 12 days in culture, but could not sustain ALP expression; hence it is possible that these cells may have entered a state of quiescence in response to this treatment. Extending these studies to cells derived from clinical samples, pre-bound combinations of VN / IGFBP-5 / IGF-I were not able to support initiation of human mesenchymal stem cell (hMSC) cultures. Nevertheless, VN alone in serum free media stimulated substantial metabolic activity and protein synthesis in hMSCs once the cultures were established. Moreover, the addition of IGFBP-3 or -5 together with IGF-I can enhance the response to levels equivalent to that observed with 10% FCS. I also report that the responses to VN and TGF-β1 are synergistic and stimulate greater hMSC metabolic activity than 10% FCS. Interestingly, hMSCs cultured in IGF-I or TGF-β1 and low concentrations of VN aggregated, an effect that was not observed when higher concentrations of VN were used. I hypothesise that this aggregation effect was due to endogenous protease activity, and therefore examined MMP-2 and 9 activity in hMSC conditioned media. Both pro-MMP-2 and pro-MMP-9 were constitutively expressed by hMSCs but there was no evidence of the active forms in the conditioned media, indicating that neither IGF-I nor TGF-β1 affect MMP-2 or -9 expression or activation in serum-free media. However, hMSC conditioned media could degrade IGFBP-5, suggesting that there is proteolytic activity within the conditioned media which may impact on the function of ECM / growth factor components in serum-free media settings. Thus, while ECM and growth factors may stimulate desirable responses in therapeutically important cells in serum-free culture, the role that endogenously expressed proteases have on the efficacy of such media supplements needs to be examined closely. Taken together, the studies reported in this thesis provide proof of principle data indicating that select combinations of ECM proteins and growth factors could be utilised in bone tissue engineering applications. This may be achieved for example, as a biomaterial coating, or could form the basis of a viable alternative media supplement for the serum-free culture of hMSCs.
| Identifer | oai:union.ndltd.org:ADTP/265641 |
| Date | January 2007 |
| Creators | Parker, Anthony James |
| Publisher | Queensland University of Technology |
| Source Sets | Australiasian Digital Theses Program |
| Detected Language | English |
| Rights | Copyright Anthony James Parker |
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