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In the U.S., 30% of adults suffer joint pain, most commonly in the knee, which severely limits mobility and is often attributed to injury of cartilage and underlying bone in the joint. Current treatment methods such as microfracture result in less resilient fibrocartilage with eventual failure; autografting can cause donor site morbidity and poor integration. To overcome drawbacks in treatment, tissue engineers can design cell-instructive biomimetic scaffolds using biocompatible materials as alternate therapies for osteochondral defects. Nanofibrous poly (L-lactic acid) (PLLA) scaffolds of uniform, spherical, interconnected and well-defined pore sizes that are fabricated using a thermally-induced phase separation and sugar porogen template method create an extracellular matrix-like environment which facilitates cell adhesion and proliferation. Herein we report that chondrogenesis and endochondral ossification of rabbit and human bone marrow stromal cells (BMSCs) can be controlled by scaffold pore architecture, particularly pore size. Small-pore scaffolds support enhanced chondrogenic differentiation in vitro and cartilage formation in vivo compared to large-pore scaffolds. Endochondral ossification is prevented in scaffolds with very small pore sizes; pore interconnectivity is critical to promote capillary ingrowth for mature bone formation. These results provide a novel strategy to control tissue regenerative processes by tunable architecture of macroporous nanofibrous scaffolds.

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To mimic the bone matrix of mineralized collagen and to impart microporous structure to facilitate cell migration and bone regeneration, we developed a nanofibrous (NF) polymer scaffold with highly interconnected pores and three-dimensional calcium phosphate coating utilizing an electrodeposition technique. The mineral content, morphology, crystal structure, and chemical composition could be tailored by adjusting the deposition temperature, voltage, and duration. A higher voltage and a higher temperature led to a greater rate of mineralization. Furthermore, nearly linear calcium releasing kinetics was achieved from the mineralized 3D scaffolds. The releasing rate was controlled by varying the initial electrodeposition conditions. A higher deposition voltage and temperature led to slower calcium release, which was associated with the highly crystalline and stoichiometric hydroxyapatite content. This premineralized NF scaffold enhanced bone regeneration over the control scaffold in a subcutaneous implantation model, which was associated with released calcium ions in facilitating osteogenic cell proliferation.

Craniosynostosis is defined as congenital premature fusion of one or more cranial sutures. While the genetic basis for about 30% of cases is known, the causative genes for the diverse presentations of the remainder of cases are unknown. The recently discovered cranial suture stem cell population affords an opportunity to identify early signaling pathways that contribute to craniosynostosis. We previously demonstrated that enhanced BMP signaling in neural crest cells (caA3 mutants) leads to premature cranial suture fusion resulting in midline craniosynostosis. Since enhanced mTOR signaling in neural crest cells leads to craniofacial bone lesions, we investigated the extent to which mTOR signaling is involved in the pathogenesis of BMP-mediated craniosynostosis by affecting the suture stem cell population. Our results demonstrate a loss of suture stem cells in the caA3 mutant mice by the newborn stage. We have found increased activation of mTOR signaling in caA3 mutant mice during embryonic stages, but not at the newborn stage. Our study demonstrated that inhibition of mTOR signaling via rapamycin in a time specific manner partially rescued the loss of the suture stem cell population. This study provides insight into how enhanced BMP signaling regulates suture stem cells via mTOR activation.

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.