Bone tissue engineering from marrow stromal cells: effects of growth factors and biomaterials

The tissue engineering of bone is a promising new field of research to address the problems arising from the loss of bony tissue occurring through infection, loss of blood supply or disease, such as osteoporosis, or as a complication of a fracture or genetic disorders. However, due to the complex composition of bone and many critical parameters in the tissue engineering (osteogenic factors, scaffold material, mechanical forces applied on cell-polymer constructs, cell source) influencing bone formation, there are still many obstacles that need to be surmounted to obtain in vitro engineered bone-like tissue. The goal of this thesis was to optimize two of the key factors that influence tissue engineering, the osteogenic agents and the scaffold material, in order to improve the formation of bone-like tissue. The strategy that we applied to engineer bone-like tissue involved aspiration of rat marrow stromal cells (rMSCs) from the bone marrow, cell expansion in two-dimensional cell culture to obtain a high cell number, dynamic cell seeding on poly(L-lactic-co-glycolic acid) (PLLGA) fiber meshes in spinner flasks, and finally in vitro cultivation of the cell-polymer constructs under treatment with osteogenic agents. At first, the optimization of the culture conditions for the bone cell culture of rMSCs was a necessary prerequisite for a successful approach to bone tissue engineering. To this end, we investigated the optimum cell seeding density at rMSC isolation, the influence of basal media with different nutrient concentrations and the time point, at which the osteogenic supplements may most advantageously be added. To improve bone tissue engineering, it is essential to overcome the problem of limited extracellular matrix formation in cell-polymer constructs of rMSCs when cultured with the standard osteogenic agents (dexamethasone, s-glycerophosphate and ascorbic acid). Thus, we included transforming growth factor-s1 (TGF-s1), which was considered to be a promising growth factor to improve the matrix formation in our three-dimensional cell culture system, in order to solve the outlined problem. It was shown that the application of TGF-s1 in addition to the standard osteogenic agents is indispensible to obtain a coherent bone-like extracellular matrix. We established culture conditions and dosing regimens, such that TGF-s1 preserved or even increased osteoblastic cell differentiation. Furthermore, the combined application of the growth factors TGF-s1 and bone morphogenetic protein-2 (BMP-2) offers a chance to enhance the osteoblastic differentiation of rMSCs in three-dimensional cell culture. Concerning the key factor the scaffold material, the examination of various biomaterials revealed that poly(D,L-lactic acid)-poly(ethylene glycol)-monomethyl ether (Me.PEG-PLA) diblock copolymers, especially Me.PEG2-PLA40 (PEG block of 2 kDa, PLA block of 40 kDa) are promising candidates for the use as scaffold materials in bone tissue engineering, because they provide controlled protein adsorption and positively influenced osteoblastic differentiation. At last, the successful grafting of an amine-reactive derivative [ST-NH-PEG2-PLA20] [1] of the low-adhesive Me.PEG-PLA diblock copolymers with an RGD (Arg-Gly-Asp)-peptide � the alphavbeta3/alphavbeta5 integrin subtype specific cyclic RGD-peptide [c(-RGDfK-)] [2] � was demonstrated by a receptor-ligand mediated cell adhesion. These results bear a great potential to achieve selective cell adhesion in implant technology and tissue engineering. References 1. Tessmar, J. K. , Mikos, A. G. , and Gopferich, A. (2002). Amine-reactive biodegradable diblock copolymers. Biomacromolecules. 3, 194-200. 2. Kantlehner, M. , Schaffner, P., Finsinger, D. , Meyer, J., Jonczyk, A. , Diefenbach, B. , Nies, B. , Holzemann, G. , Goodman, S. L. , and Kessler, H. (2000). Surface Coating with Cyclic RGD Peptides Stimulates Osteoblast Adhesion and Proliferation as well as Bone Formation. Chembiochem.Europ. J.Chem.Biol. 1, 107-114.