ABSTRACT
Skeletal stem and progenitor cells (SSPCs) are the multi-potent, self-renewing cell lineages that form the hematopoietic environment and adventitial structures of the skeletal tissues. Skeletal tissues are responsible for a diverse range of physiological functions because of the extensive differentiation potential of SSPCs. The differentiation fates of SSPCs are shaped by the physical properties of their surrounding microenvironment and the mechanical loading forces exerted on them within the skeletal system. In this context, the present review first highlights important biomolecules involved with the mechanobiology of how SSPCs sense and transduce these physical signals. The review then shifts focus towards how the static and dynamic physical properties of microenvironments direct the biological fates of SSPCs, specifically within biomaterial and tissue engineering systems. Biomaterial constructs possess designable, quantifiable physical properties that enable the growth of cells in controlled physical environments both in-vitro and in-vivo. The utilization of biomaterials in tissue engineering systems provides a valuable platform for controllably directing the fates of SSPCs with physical signals as a tool for mechanobiology investigations and as a template for guiding skeletal tissue regeneration. It is paramount to study this mechanobiology and account for these mechanics-mediated behaviors to develop next-generation tissue engineering therapies that synergistically combine physical and chemical signals to direct cell fate. Ultimately, taking advantage of the evolved mechanobiology of SSPCs with customizable biomaterial constructs presents a powerful method to predictably guide bone and skeletal organ regeneration.
ABSTRACT
Biomaterial scaffolds in tissue engineering facilitate tissue regeneration and integration with the host. Poor healing outcomes arise from lack of cell and tissue infiltration, and ill-fitting interfaces between matrices or grafts, resulting in fibrous tissue formation, inflammation, and resorption. Existing tissue engineering scaffolds struggle to recover from deformation to fit irregularly shaped defects encountered in clinical settings without compromising their mechanical properties and favorable internal architecture. This study introduces a synthetic biomaterial scaffold composed of high molecular weight poly (L-lactic acid) (PLLA) and an interpenetrating network of poly (ε-caprolactone) (PCL), in a composition aiming to address the need for conformal fitting synthetic matrices which retain and recover their advantageous morphologies. The scaffold, known as thermosensitive memorized microstructure (TS-MMS), forms nanofibrous materials with memorized microstructures capable of recovery after deformation, including macropores and nanofibers. TS-MMS nanofibers, with 50-500 nm diameters, are formed via thermally induced phase separation (TIPS) of PLLA after in situ polymerization of PCL-diacrylate. A critical partial-melting temperature of TS-MMS at 52°C enables bulk deformation above this temperature, while retaining the nanofibrous and macroporous structures upon cooling to 37°C. Incorporation of drug-loaded poly (lactide-co-glycolide) (PLGA) nanoparticles directly into TS-MMS nanofibers during fabrication allows sustained release of a model drug for up to 40 days. Subcutaneous implantation in vivo using LysM-Cre;td-Tomato; Col1eGFP mice demonstrates successful cellularization and integration of deformed/recovered TS-MMS materials, surpassing the limitations of deformed PLLA scaffolds, to facilitate cell and vasculature infiltration requisite for successful bone regeneration. Additionally we demonstrated a method for embedding controlled release vehicles directly into the scaffold nanofibers; controlled release of simvastatin enhances vascularization and tissue maturation. TS-MMS scaffolds offer promising improvements in clinical handling and performance compared to existing biomaterial scaffolds.
ABSTRACT
Tissue engineering aims to repair, restore, and/or replace tissues in the human body as an alternative to grafts and prostheses. Biomaterial scaffolds can be utilized to provide a three-dimensional microenvironment to facilitate tissue regeneration. Previously, we reported that scaffold pore size influences vascularization and extracellular matrix composition both in vivo and in vitro, to ultimately influence tissue phenotype for regenerating cranial suture and bone tissues, which have markedly different tissue properties despite similar multipotent stem cell populations. To rationally design biomaterials for specific cell and tissue fate specification, it is critical to understand the molecular processes governed by cell-biomaterial interactions, which guide cell fate specification. Building on our previous work, in this report we investigated the hypothesis that scaffold pore curvature, the direct consequence of pore size, modulates the differentiation trajectory of mesenchymal stem cells (MSCs) through alterations in the cytoskeleton. First, we demonstrated that sufficiently small pores facilitate cell clustering in subcutaneous explants cultured in vivo, which we previously reported to demonstrate stem tissue phenotype both in vivo and in vitro. Based on this observation, we cultured cell-scaffold constructs in vitro to assess early time point interactions between cells and the matrix as a function of pore size. We demonstrate that principle curvature directly influences nuclear aspect and cell aggregation in vitro. Scaffold pores with a sufficiently low degree of principle curvature enables cell differentiation; pharmacologic inhibition of actin cytoskeleton polymerization in these scaffolds decreased differentiation, indicating a critical role of the cytoskeleton in transducing cues from the scaffold pore microenvironment to the cell nucleus. We fabricated a macropore model, which allows for three-dimensional confocal imaging and demonstrates that a higher principle curvature facilitates cell aggregation and the formation of a potentially protective niche within scaffold macropores which prevents MSC differentiation and retains their stemness. Sufficiently high principle curvature upregulates yes-associated protein (YAP) phosphorylation while decreased principle curvature downregulates YAP phosphorylation and increases YAP nuclear translocation with subsequent transcriptional activation towards an osteogenic differentiation fate. Finally, we demonstrate that the inhibition of the YAP/TAZ pathway causes a defect in differentiation, while YAP/TAZ activation causes premature differentiation in a curvature-dependent way when modulated by verteporfin (VP) and 1-oleyl-lysophosphatidic acid (LPA), respectively, confirming the critical role of biomaterials-mediated YAP/TAZ signaling in cell differentiation and fate specification. Our data support that the principle curvature of scaffold macropores is a critical design criterion which guides the differentiation trajectory of mesenchymal stem cells' scaffolds. Biomaterial-mediated regulation of YAP/TAZ may significantly contribute to influencing the regenerative outcomes of biomaterials-based tissue engineering strategies through their specific pore design.
