The successful design and implementation of constructs that mimic the properties of host tissue represents a fundamental goal of tissue engineering and regenerative medicine. Along with the types of cells present, the ECM is a defining component of each tissue in the body and therefore should be considered in the design of culture surfaces and tissue engineered constructs. Common approaches toward ECM incorporation in biomaterial design include (1) the coating of material surfaces with recombinant ECM proteins and peptides1
and (2) the use of decellularized allogenic or xenogenic tissue scaffolding to bridge the tissue defect and influence cellular behavior.2
However, the presentation of individual matrix proteins fails to capture the complex composition and architecture found within native ECM, and decellularized tissues suffer from immunological concerns while also lacking the reproducibility, availability, and tailorability available with synthetic materials.3
Cell-secreted matrix coatings, such as those studied herein, offer the complexity of a biologically relevant ECM, while enabling the potential synergism of an underlying, highly tunable synthetic backbone.
hMSCs represent a valuable tool for testing our hypothesis due to their multipotent nature and secretion of large quantities of matrix in culture, presumably depositing transitional matrices associated with the induced lineages along the way. As the complexity of environmental variables influencing the composition of the hMSC-secreted matrix is not fully understood, recent studies analyzing the ability of DMs to influence naïve MSC differentiation have focused on determining the relevance of various parameters including culture duration and the presence of soluble osteogenic cues.26
In order to minimize the number of experiments necessary to correlate such conditions with DM efficacy to modulate cell phenotype while maximizing experimental returns, we employed a DOE approach to analyze four conditions associated with hMSC-matrix deposition. This strategy is invaluable when a full knowledge of input variables and their effect on the system as a whole is poorly understood. A response surface method approach was employed to observe the curvature of our system response as a result of variable changes, and a Box–Behnken 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. In this instance, a DOE-based approach allowed for more than a 2-fold reduction in the number of experimental groups and 5-fold reduction in the number of experimental samples necessary to perform a complementary, full-factorial designed experiment (n
= 3). In the case of 5 or more variable systems, the reduction in the number of experimental groups becomes even more pronounced, allowing for faster optimization of engineered systems, and in turn, the isolation of those system variables which have the greatest impact and merit further study.
The DOE-based approach in our study revealed unanticipated correlations between conditions under which hMSC-deposited decellularized matrix coatings are created and their resulting capacity to direct the osteogenic differentiation of naïve hMSCs. Previous studies have examined links between the stage of hMSC differentiation prior to decellularization and the ability of the resulting DMs to drive osteogenesis. For example, Hoshiba et al.26
demonstrated that an early stage transitional osteogenic matrix deposited by hMSCs in 2D was most efficient, while Liao et al.32
observed that a more mature mineralized matrix coating was superior in a 3D murine cell model. Our data indicate that DMs deposited by hMSCs cultured in media supplemented with A2P only, and therefore in a comparably undifferentiated state, are more effective at driving hMSC osteogenesis than those DMs deposited by cells exposed to standard osteogenic media.
Mineralization of the DM also appeared unnecessary to drive naïve cell differentiation, as both DM1 and DM2 did not contain detectable levels of calcium prior to decellularization yet enhanced osteogenic differentiation compared to TCP. However, we did observe that culture duration of hMSCs for up to 15 days facilitated greater matrix deposition and more effective osteogenic DMs. Matrix deposition beyond 15 days generally resulted in curling at the edges of the cell layers, producing DMs that failed to completely adhere to the culture dish and did not fully cover the culture area. While no significant two-factor interactions were detected in our study, a trend was noted between culture duration and media type in driving osterix expression in naïve hMSCs (p = 0.089). The capacity of culture duration to drive osterix expression was more pronounced in supplemented media (Fig. a) than osteogenic media (Fig. b).
While the results of our study revealed several novel correlations between DM design and resultant hMSC phenotype, these findings should not be taken as rigid guidelines by which all osteogenic DMs should be engineered. Instead, these findings serve as a proof-of-principle that a DOE-based approach is a useful tool in optimizing unique biologically engineered systems. Differences in our results from previously published studies such as those pertaining to matrix mineralization may be due in part to disparities in the material surface, dimensionality, cell source, cell passage number, decellularization technique, and media type utilized in each study, all of which could affect DM composition and the outcome of a DOE-based optimization study.
To further probe the results from our DOE model, we characterized the osteogenic potential of the DOE-determined optimal matrix (DM1), as well as a second matrix (DM2) whose osteogenic potential was predicted to fall between that of DM1 and our control substrate TCP. Fibronectin-coated TCP also served as a single ECM protein-coated control, as fibronectin has been repeatedly described to influence adhesion, proliferation, and differentiation of numerous cultured cell populations.25
Although both DMs were more effective osteogenic substrates when compared to control surfaces, DM1 significantly outperformed DM2 as indicated by increases in the expression of multiple osteogenic genes, accelerated calcium deposition, and enhanced proliferative potential. These data show the power and accuracy of the DOE-based experimental approach to quickly isolate effective conditions that optimize multivariable input systems.
The assays and methods chosen in our study were selected in an attempt to effectively determine the impact of our substrates on naïve hMSC phenotype. To quantify osteogenic differentiation, we utilized qPCR in addition to ALP and calcium quantification. ALP and calcium are commonly sequestered within decellularized matrices, thereby making it difficult to discern whether measurements are indicative of the newly seeded cells alone or a combination of the cells and matrix. To address this challenge, we maintained unseeded control DMs at each time point and subtracted ALP quantities detected in those matrices from hMSC-seeded DMs. While our qPCR and calcium data confirmed the impressive osteogenic potential of our DMs, ALP activity was only slightly higher in hMSCs cultured on DM1 and DM2 compared to controls, possibly due to feedback inhibition from extracellular ALP present in the scaffolds. This problem was rendered mute in our calcium quantification assays, as the DMs studied contained no appreciable calcium prior to seeding with naïve hMSCs.
qPCR results from our study and previous work38
demonstrate that cell-secreted matrix coatings have the capacity to alter gene expression within progenitor cell populations. However, the method or methods by which this occurs are still unclear. Grunert et al.
demonstrated that glycosaminoglycans present in DMs bind and modulate the efficacy of endogenous inductive factors such as bone morphogenetic protein-2 to direct hMSC osteogenic fate.22
Growth factors deposited by cells within the matrix prior to decellularization may therefore play a major role in determining DM efficacy, with naïve hMSCs likely secreting a unique array of trophic factors in comparison to cells found in a transitionally differentiated state.6
hMSC integrin binding ECM protein motifs may also contribute to the DM capacity to instruct cell phenotype. Previous reports confirm that the decellularization techniques utilized in our study retain key osteogenic ECM components upon cell removal,8
and MSC–ECM protein interactions have been significantly linked to both MSC potential for in vitro
and the pathway by which differentiation occurs.29
The capacity of osteogenic supplements to influence naïve hMSC differentiation may also be modulated by DM cell–substrate interactions, as dexamethasone may not be necessary for the osteogenic differentiation of MSCs cultured in mineralized matrix-coated constructs.32
To date, the mechanism of action for cell-secreted ECMs to direct cell fate, particularly with regard to ECM composition, is unclear. The presence of other components such as residual cell debris or intracellular proteins that may elicit an eventual immune response should be investigated more fully.21
While further characterization of osteogenic DMs and their interactions with progenitor cell populations is undoubtedly needed to fully unlock their mechanism of impact, our novel approach toward eliciting optimal cell-secreted DM design may help to better identify the specific DMs which merit such further study.