A response surface method approach was employed to observe the curvature of our system response as a result of variable changes, and a BoxBehnken 3-level factorial design was chosen in lieu of a typical 5-level central composite design (CCD), as the more numerous and axial variable levels associated with the CCD are more difficult to carry out with biological systems. mesenchymal stem cells (hMSCs). DOE analysis revealed that matrices created with extended culture period, ascorbate-2-phosphate supplementation, and in ambient oxygen tension exhibited significant correlations with enhanced hMSC differentiation. We validated the DOE model results using DMs predicted to have superior (DM1) Trimebutine maleate or smaller (DM2) osteogenic potential for nave hMSCs. Compared to cells on DM2, hMSCs cultured on DM1 expressed 2-fold higher osterix levels and deposited 3-fold more calcium over 3 weeks. Cells on DM1 coatings also exhibited greater proliferation and viability compared to DM2-coated substrates. This study demonstrates that DOE-based analysis is a powerful tool for optimizing designed systems by identifying significant variables that have the greatest contribution to the target output. Keywords:Extracellular matrix, Mesenchymal stem cells, Multivariable analysis, Osteogenesis == Introduction == The extracellular matrix (ECM) plays a pivotal role in regulating the maintenance and behavior of progenitor cells via physical interactions with cell surface proteins and the modulation of soluble growth factor concentrations within the cellular microenvironment.7,23Thus, capitalizing on the innate ability of the ECM to modulate cell behavior has been of great desire for developing novel biomaterial surfaces that are better able to direct cell phenotype by recapitulating Trimebutine maleate thein vivocellular milieu.41,43The design of cell culture interfaces that maintain cells in their native behavioral state or instruct their phenotypic transformation toward that of a desired tissue would be of great benefit in the advancement of cell-based therapies and tissue engineering. To date, this has been primarily pursued by the deposition of individual ECM proteins on a substrate surface.17,33,36However, this approach fails to accurately mimic the complex protein composition within the endogenous ECM, an environment that can be efficiently generated by a number of cellular populations. Mesenchymal stem cells (MSCs) symbolize a useful tool in the study of cell-secreted matrix coatings, as they produce generous amounts of ECM when cultured on tissue culture plastic (TCP) and other biomaterial surfaces. Upon decellularization of these cultures, the residual ECM covering retains the capacity to alter the phenotype of subsequently seeded cell populations.8,13,22The composition of thein vivoECM associated with each particular tissue has a profound effect on the phenotypic behavior of the engaged neighboring cells. Therefore, it is affordable to expect that cell-secreted matrix coatings of differing composition will manipulate the properties of the adjacent cells during culture to varying degrees. The composition of ECMs depositedin vitroby progenitor cells is dependent upon the culture duration and microenvironmental culture conditions.1,4,20,30Upon decellularization, ECM coatings deposited over different lengths of time and environmental conditions (e.g., oxygen tension, shear causes, soluble factors) will therefore acquire unique compositions that will determine their efficacy in modulating the phenotype of nave stem cell populations.14,26Hence, it is important to determine the ideal conditions under which to culture matrix-depositing MSCs prior to their decellularization for material-based therapeutic strategies. The examination of interactions between large numbers of potentially contributing variables using so-called One Factor At a Time (OFAT) experimental methods is ineffective due to the number of variable permutations and the likelihood of overlooking complex interactions of those variables. Alternatively, multivariable statistical analysis through Trimebutine maleate a design of experiments (DOE) approach addresses these difficulties by markedly reducing the number of variable combinations and repetitions to be examined, while simultaneously determining the Trimebutine maleate significance of single input variables and combinations of such variables toward the ultimate response of the system. DOE represents a powerful tool that has TNFRSF16 previously been applied toward the optimization of developmental protocols from a broad array of scientific fields.9,28,40,42 Substrate-mediated cues directing human MSC (hMSC) fate toward the osteogenic lineage, such as those provided by the native ECM, offer a novel approach toward the development of biomaterial constructs designed to bridge and repair non-healing skeletal defects.5,11,29We hypothesized that a DOE-based approach would provide an efficient means of engineering specific hMSC-secreted matrix coatings optimized to accelerate the osteogenic differentiation of nave hMSCs. We first investigated this hypothesis by analyzing differences in the osteogenic differentiation and proliferation of nave MSCs in the presence of MSC-secreted matrices designed under unique DOE-determined conditions. Upon characterizing cellular responses to these matrices, we validated DOE predictions of matrix efficacy at directing hMSC osteogenic fate with an in-depth analysis of the osteogenic capacity of two designed hMSC-secreted matrix coatings. == Materials and Methods == == Cell Culture == Human bone marrow-derived MSCs (hMSCs, Lonza, Walkersville, MD) were expanded without further characterization in.