Interactive effects of growth factors and three-dimensional scaffolds on multipotent mesenchymal stromal cells.

The creation of tissue-engineered constructs with autologous cells is a central goal in regenerative medicine. With respect to ligament replacement, we have evaluated the influences of matrix and growth factors on hMSCs (human mesenchymal stromal cells). hMSCs were seeded in two different 3D (three-dimensional) systems consisting of either a collagen type I gel or a synthetic PLA [poly-(L-lactic acid)] scaffold. After cultivation for 14 days with rhTGFbeta1 (recombinant human transforming growth factor beta1), rhPDGF-BB (recombinant human platelet-derived growth factor homodimer of B-chain) or rhBMP13 (recombinant human bone morphogenetic protein 13), we assessed the proliferation potential, mRNA expression and protein expression of various matrix-interacting and matrix-degrading molecules by quantitative real-time RT (reverse transcriptase)-PCR, immunohistochemistry and gelatin zymography in comparison with unstimulated cells. Cellular reactions to the type of scaffold or soluble factors could be found in the expression of tenascin-C as well as integrin subunits alpha1, alpha3 and beta1. Collagen type X expression was induced by 3D culture and stimulated by rhTGFbeta1 on PLA. The expression of MMP-1 (matrix metalloproteinase 1) tended to increase, and MMP-13 was induced in the collagen culture system. The activation of MMP-2 was stimulated by the cultivation of MSCs within the collagenous matrix. These results demonstrated that various interactive effects of growth factors and scaffolds influence the cell-biological behaviour of MSCs. It is important to take these complex interactions, which partly differ from differentiated cells, into account in further tissue-engineering approaches.

Human mesenchymal progenitor cell responses to a novel textured poly(L-lactide) scaffold for ligament tissue engineering.

Biocompatibility and cell seeding capability of a new cell scaffold made of textured polylactic acid (PLA) fibers was investigated as a new material for tissue engineering of anterior cruciate ligaments (ACL). Adhesion and proliferation of human mesenchymal progenitor cells (MPC) was investigated after 15 days by scanning electron microscopy and standard histology. Expression of collagen type I and III, fibronectin, tenascin C, decorin, smooth muscle actin, and the matrix metalloproteinases MMP-1 and MMP-2, as well as their tissue inhibitors TIMP-1 and TIMP-2 was analyzed using real-time PCR. Protein expression of collagen I and III, tenascin C, and proliferating nuclear antigen (PCNA) was determined by immunohistology. Apoptosis was analyzed by detection of p53 expression and TUNEL staining. MPC seeded the scaffold homogeneously and showed good cell growth and no increased rate of apoptosis. After 15 days, the matrix forming genes collagen type I, tenascin C, and decorin were upregulated, indicating the formation of a ligament-like matrix. MMP-1 and TIMP-1 were also significantly increased, suggesting initial matrix remodeling. It was concluded that the new porous PLA scaffold allowed homogeneous cell seeding, a fibroblastic phenotype and the production of a ligament-like matrix and, therefore, might be a suitable cell carrier for ACL tissue engineering.

Mesenchymal progenitor cells communicate via alpha and beta integrins with a three-dimensional collagen type I matrix.

BACKGROUND/AIMS: The aim of our study was to investigate interactions of mesenchymal progenitor cells (MPCs) with collagen matrices. METHODS: Human bone-marrow-derived MPCs were cultivated in collagen type I gels with and without inhibition of beta(1)-integrin by a specific antibody. Collagen gel contraction, cell morphology, expression of integrin subunits and several genes related to matrix synthesis and turnover as well as MPC differentiation were analyzed over 14 days. RESULTS: Human MPCs markedly contracted free-floating collagen gels. Contraction was nearly completely inhibited by blocking beta(1)-integrin. Cellular morphology was elongated in the absence and mostly round in the presence of the antibody. Expression of integrin alpha(1), alpha(2) and beta(1) subunits showed several changes partly dependent on beta(1)-integrin blocking. Expression of matrix metalloproteinase-1 was elevated irrespective of beta(1)-integrin blocking and tenascin-C was subsequently induced during gel contraction. Spontaneous induction of chondrogenic, osteogenic or adipogenic differentiation was observed neither in the presence nor in the absence of the beta(1)-integrin antibody. CONCLUSION: Our results indicate that the interaction of human MPCs with fibrillar collagen type I involves beta(1)- and alpha-integrin subunits and induces changes in gene expression related to extracellular matrix synthesis and turnover but not differentiation to the chondrogenic, osteogenic or adipogenic phenotype.