Combinatorial screen of dynamic mechanical stimuli for predictive control of MSC mechano-responsiveness.

Mechanobiological-based control of mesenchymal stromal cells (MSCs) to facilitate engineering and regeneration of load-bearing tissues requires systematic investigations of specific dynamic mechanical stimulation protocols. Using deformable membrane microdevice arrays paired with combinatorial experimental design and modeling, we probed the individual and integrative effects of mechanical stimulation parameters (strain magnitude, rate at which strain is changed, and duty period) on myofibrogenesis and matrix production of MSCs in three-dimensional hydrogels. These functions were found to be dominantly influenced by a previously unidentified, higher-order interactive effect between strain magnitude and duty period. Empirical models based on our combinatorial cue-response data predicted an optimal loading regime in which strain magnitude and duty period were increased synchronously over time, which was validated to most effectively promote MSC matrix production. These findings inform the design of loading regimes for MSC-based engineered tissues and validate a broadly applicable approach to probe multifactorial regulating effects of mechanobiological cues.

Microdevice arrays with strain sensors for 3D mechanical stimulation and monitoring of engineered tissues.

Native and engineered tissue development are regulated by the integrative effects of multiple microenvironmental stimuli. Microfabricated bioreactor array platforms can efficiently dissect cue-response networks, and have recently integrated critical 2D and 3D mechanical stimulation for greater physiological relevance. However, a limitation of these approaches is that assessment of tissue functional properties is typically limited to end-point analyses. Here we report a new deformable membrane platform with integrated strain sensors that enables mechanical stretching or compression of 3D cell-hydrogel arrays and simultaneous measurement of hydrogel construct stiffness in situ. We tested the ability of the integrated strain sensors to measure the evolution of the stiffness of cell-hydrogel constructs for two cases. First, we demonstrated in situ stiffness monitoring of degradable poly (ethylene glycol)-norbornene (PEG-NB) hydrogels embedded with mesenchymal stromal cells (MSCs) and cultured with or without cyclic tensile stimulation for up to 15 days. Whereas statically-cultured hydrogels degraded and softened throughout the culture period, mechanically-stimulated gels initially softened and then recovered their stiffness corresponding to extensive cell network and collagen production. Second, we demonstrated in situ measurement of compressive stiffening of MSC-seeded PEG-NB gels cultured statically under osteogenic conditions, corresponding to increased mineralization and cellularization. This measurement technique can be generalized to other relevant bioreactor and organ-on-a-chip platforms to facilitate online, non-invasive, and high-throughput functional analysis, and to provide insights into the dynamics of engineered tissue development that are otherwise not available.

Combinatorial screening of 3D biomaterial properties that promote myofibrogenesis for mesenchymal stromal cell-based heart valve tissue engineering.

The physical and chemical properties of a biomaterial integrate with soluble cues in the cell microenvironment to direct cell fate and function. Predictable biomaterial-based control of integrated cell responses has been investigated with two-dimensional (2D) screening platforms, but integrated responses in 3D have largely not been explored systematically. To address this need, we developed a screening platform using polyethylene glycol norbornene (PEG-NB) as a model biomaterial with which the polymer wt% (to control elastic modulus) and adhesion peptide types (RGD, DGEA, YIGSR) and densities could be controlled independently and combinatorially in arrays of 3D hydrogels. We applied this platform and regression modeling to identify combinations of biomaterial and soluble biochemical (TGF-ß1) factors that best promoted myofibrogenesis of human mesenchymal stromal cells (hMSCs) in order to inform our understanding of regenerative processes for heart valve tissue engineering. In contrast to 2D culture, our screens revealed that soft hydrogels (low PEG-NB wt%) best promoted spread myofibroblastic cells that expressed high levels of α-smooth muscle actin (α-SMA) and collagen type I. High concentrations of RGD enhanced α-SMA expression in the presence of TGF-ß1 and cell spreading regardless of whether TGF-ß1 was in the culture medium. Strikingly, combinations of peptides that maximized collagen expression depended on the presence or absence of TGF-ß1, indicating that biomaterial properties can modulate MSC response to soluble signals. This combination of a 3D biomaterial array screening platform with statistical modeling is broadly applicable to systematically identify combinations of biomaterial and microenvironmental conditions that optimally guide cell responses. STATEMENT OF SIGNIFICANCE: We present a novel screening platform and methodology to model and identify how combinations of biomaterial and microenvironmental conditions guide cell phenotypes in 3D. Our approach to systematically identify complex relationships between microenvironmental cues and cell responses enables greater predictive power over cell fate in conditions with interacting material design factors. We demonstrate that this approach not only predicts that mesenchymal stromal cell (MSC) myofibrogenesis is promoted by soft, porous 3D biomaterials, but also generated new insights which demonstrate how biomaterial properties can differentially modulate MSC response to soluble signals. An additional benefit of the process includes utilizing both parametric and non parametric analyses which can demonstrate dominant significant trends as well as subtle interactions between biochemical and biomaterial cues.

Heart valve regeneration: the need for systems approaches.

Tissue-engineered heart valves are promising alternatives to address the limitations of current valve replacements, particularly for growing children. Current heart valve tissue engineering strategies involve the selection of biomaterial scaffolds, cell types, and often in vitro culture conditions aimed at regenerating a valve for implantation and subsequent maturation in vivo. However, identifying optimal combinations of cell sources, biomaterials, and/or bioreactor conditions to produce functional, durable valve tissue remains a challenge. Despite some short-term success in animal models, attempts to recapitulate aspects of the native heart valve environment based on 'best guesses' of a limited number of regulatory factors have not proven effective. Better outcomes for valve tissue regeneration will likely require a systems-level understanding of the relationships between multiple interacting regulatory factors and their effects on cell function and tissue formation. Until recently, conventional culture methods have not allowed for multiple design parameters to be considered at once. Emerging microtechnologies are well suited to systematically probe multiple inputs, in combination, in high throughput and with great precision. When combined with statistical and network systems analyses, these microtechnologies have excellent potential to define multivariate signal-response relationships and reveal key regulatory pathways for robust functional tissue regeneration.

A microfabricated platform with hydrogel arrays for 3D mechanical stimulation of cells.

Cellular microenvironments present cells with multiple stimuli, including not only soluble biochemical and insoluble matrix cues but also mechanical factors. Biomaterial array platforms have been used to combinatorially and efficiently probe and define two-dimensional (2D) and 3D microenvironmental cues to guide cell functions for tissue engineering applications. However, there are few examples of array platforms that include dynamic mechanical forces, particularly to enable stretching of 3D cell-seeded biomaterials, which is relevant to engineering connective and cardiovascular tissues. Here we present a deformable membrane platform that enables 3D dynamic mechanical stretch of arrayed biomaterial constructs. Cell-seeded polyethylene glycol norbornene (PEG-NB) hydrogels were bound to miniaturized deformable membranes via a thiol-ene reaction with off-stoichiometry thiol-ene based polydimethylsiloxane (OSTE-PDMS) as the membrane material. Bonding to OSTE-PDMS enabled the 3D hydrogel microconstructs to be cyclically deformed and stretched by the membrane. As a first demonstration, human mesenchymal stromal cells (MSCs) embedded in PEG-NB were stretched for several days. They were found to be viable, spread in the 3D hydrogels, and exhibited a contractile myofibroblast phenotype when exposed to dynamic 3D mechanical deformation. This platform, which is readily scalable to larger arrays, enables systematic interrogation of the relationships between combinations of 3D mechanobiological cues and cellular responses, and thus has the potential to identify strategies to predictably control the construction of functional engineered tissues. STATEMENT OF SIGNIFICANCE: Current high-throughput biomaterial screening approaches fail to consider the effects of dynamic mechanical stimulation, despite its importance in a wide variety of regenerative medicine applications. To meet this need, we developed a deformable membrane platform that enables 3D dynamic stretch of arrayed biomaterial constructs. Our approach combines microtechnologies fabricated with off-stoichiometry thiol-ene based polydimethylsiloxane membranes that can covalently bond cell-seeded polyethylene glycol norbornene 3D hydrogels, a model biomaterial with tunable adhesive, elastic and degradation characteristics. As a first demonstration, we show that human mesenchymal stromal cells embedded in hydrogels and subjected to dynamic mechanical stimulation undergo myofibroblast differentiation. This system is readily scaled up to larger arrays, and will enable systematic and efficient screening of combinations of 3D mechanobiological and biomaterial cues on cell fate and function.

The Therapeutic Potential of Exogenous Adenosine Triphosphate (ATP) for Cartilage Tissue Engineering.

OBJECTIVE: While mechanical stimuli can be used to enhance the properties of engineered cartilage, a promising alternative may be to directly harness the underlying mechanotransduction pathways responsible. Our initial studies on the adenosine triphosphate (ATP)-purinergic receptor pathway demonstrated that stimulation by exogenous ATP improved tissue growth and properties but elicited matrix turnover under high doses (250 µM) potentially due to the accumulation of extracellular inorganic pyrophosphate (ePPi). Therefore, the purpose of this study was to identify the mechanism of ATP-mediated catabolism and determine a therapeutic dose to maximize the anabolic effect. DESIGN: Isolated bovine articular chondrocytes were seeded in high-density, 3-dimensional culture supplemented with varying doses of ATP for 4 weeks. The effects on biosynthesis, matrix metalloproteinase 13 (MMP-13) protein activity, and PPi accumulation were determined. Separate monolayer experiments were conducted to determine the effect of ePPi on MMP-13 activity. RESULTS: High doses of ATP resulted in an increase in ePPi accumulation (by 54%) and MMP-13 activity (by 39%). Monolayer experiments confirmed a link between increased ePPi accumulation and MMP-13 activity, which appeared to require calcium and was inhibited by the MEK1/2 inhibitor U0126. Cultures supplemented with 62.5 to 125 µM ATP favored an anabolic response, which represented the therapeutic dose range. CONCLUSIONS: A therapeutic dose range of exogenous ATP to improve the properties of engineered cartilage has been identified, and a possible catabolic mechanism involving excess PPi was determined. Future research into PPi signal transduction and pathological crystal formation is necessary to maximize the beneficial effect of exogenous ATP on chondrocyte cultures.