Delivery of Mechanical and Chemical Stimuli to Advance Regeneration

Innovations are needed to improve outcomes in the treatment of heart muscle disorders. Regenerative medicine is rapidly showing promise for treating cardiac injury and disease with combinations of biomaterials and stem cells to restore the physiologic function that has been lost. One of the biomedical engineering approaches to mimic the natural niche of stem cells is microfabrication of scaffolds with or without adding a chemical reagent. In this thesis, I have shown human mesenchymal stem cell (hMSC) function depends not only on chemical factors but also on the physical cues of the microenvironmental niche for tissue regeneration. In the first part of the thesis, the physical environment is recapitulated with controlled modes of mechanical strain applied to substrata containing three-dimensional features in order to analyze the effects on cell morphology, focal adhesion distribution, cell proliferation, and gene expression. 10% strain at 1 Hz is delivered for 48h to hMSCs cultured on flat surfaces, or on substrata with microtopographic posts 15 μm high spaced 75 μm apart. Introducing strain to microtopography produced stable semicircular focal adhesions, with anchored actin preferentially spanning post to post. Anisotropic strain caused a two-fold increase in the proliferation of hMSCs over equibiaxial strain with or without the posts. Strain dominated microtopography for expression of genes coding proteins related to muscle function, cell adhesion, extracellular matrix remodeling, and cell differentiation (p<0.05). In the second part of the thesis, local release of drugs is mimicked because chemicals have many advantages for tissue repair. A microrod delivery device was fabricated of poly (ethylene glycol) dimethacrylate (PEGDMA) hydrogel loaded with mechano growth factor (MGF). The elution profile and bioactivity of the peptide was determined. MGF is a member of the IGF-1 family with an E domain that is both anti-apoptotic agent and a stem cell homing factor. The injectable microrods are 30 kPa stiffness and 15 μm width by 100 μm length, chosen to match heart stiffness and myocyte size. Successful encapsulation of native MGF peptide within microrods was achieved with delivery of MGF for two weeks, as measured by high-performance liquid chromatography (HPLC). Migration of hMSCs increased with MGF microrod treatment (1.72 ± 0.23, p<0.05). Inhibition of the apoptotic pathway in neonatal rat ventricular myocytes was induced by 8 hours of hypoxia (1% O2). Protection from apoptosis by MGF microrod treatment was shown by the TUNEL assay and increased Bcl2 expression (2 ± 0.19, p<0.05). Microrods without MGF regulated the cytoskeleton, adhesion, and proliferation of hMSCs, and MGF had no effect on these properties. This thesis has yielded new information about how cells respond to local physical and chemical cues. Altogether, results suggest that it is possible to fabricate a stable and well-understood polymer system into microdevice platform to serve as both a mechanical stimulus as well as provide highly-localized, long-term delivery of bioactive peptides. This basic understanding of the microenvironment may be important to improve tissue regeneration

Cyclic strain dominates over microtopography in regulating cytoskeletal and focal adhesion remodeling of human mesenchymal stem cells

Human bone marrow-derived mesenchymal stem cell (hMSCs) function depends on chemical factors and also on the physical cues of the microenvironmental niche. Here, this physical microenvironment is recapitulated with controlled modes of mechanical strain applied to substrata containing three-dimensional features in order to analyze the effects on cell morphology, focal adhesion distribution, and gene expression. Ten percentage of strain at 1 Hz is delivered for 48 h to hMSCs cultured on flat surfaces, or on substrata with 15 μm-high microtopographic posts spaced 75 μm apart. Adding strain to microtopography produced stable semicircular focal adhesions, and actin spanning from post to post. Strain dominated over microtopography for expression of genes for the cytoskeleton (caldesmon-1 and calponin 3), cell adhesion (integrin-α2, vinculin, and paxillin), and extracellular matrix remodeling (MMP13) (p < 0.05). Overall, attention to external mechanical stimuli is necessary for optimizing the stem cell niche for regenerative medicine