IADR Abstract Archives
Towards Regeneration of the Cranial Suture: Pore Curvature Regulates Mesenchymal Stem Cell Fate in Macroporous Tissue Engineering Scaffolds
Objectives: Craniosynostosis is a debilitating disease characterized by premature fusion of cranial bones, recently suggested as a stem-cell disease involving depletion of the suture mesenchyme. We aim to use three-dimensional tissue engineering scaffolds to design an optimized microenvironment which recapitulates the cranial suture as a stem-cell niche. Our central hypothesis is that principle curvature of scaffold macropores is a critical design criteria to guide cell differentiation.
Methods: Nanofibrous, macroporous tissue engineering scaffolds are fabricated from poly(L-lactic acid) by a sugar-sphere porogen method and thermally-induced phase separation. Sugar-spheres are selectively isolated in the ranges: 65-120um diameter (small) and 250-425um diameter (large). We isolated bone marrow stromal cells and cranial suture mesenchymal stem-cells from wild-type mice and cultured on scaffolds for up to 3 weeks (growth media, n > 4). Gene expression was assessed by qPCR; protein expression and distribution by IHC. Cytochalasin-D (CytD) was used to inhibit actin polymerization, verteporfin (VP) was used to inhibit YAP signaling (5 uM) and lysophosphatidicacid (LPA) was used to induce YAP signaling (25 uM) in vitro. Image analysis performed with Fiji.
Results: We demonstrate sufficiently steep curvature (small-pores, diameter <125um) caused significantly decreased nuclear aspect ratio (NAR) and increased cell-clustering while shallow curvature (large-pores, diameter >250um) caused significantly increased NAR and increased distance between cells. Alterations in curvature-mediated cell organization correlate to differential modulation of YAP/TAZ signaling, by increased gene expression of YAP-related genes in large-pores: YAP1, CTGF, Cy61, peaking at 48 hours and maintained through 3-weeks culture in vitro, and increased osteogenesis (RUNX2, COL1A1, OSX expression, p<0.01). In contrast, small-pores maintain stem-cell marker expression (Gli1, CD44, CD146, p<0.01) and minimal YAP activation. By immunofluorescence we demonstrate YAP activation in large-pores by quantitative increases in YAP nuclear translocation and decreased phosphorylation, from 48 hours in vitro (p < 0.05); small-pores maintain YAP phosphorylation and cytosolic localization. Cytoskeletal disruption by CytD decreases YAP activity and osteogenic differentiation in large-pore constructs, where cytoskeletal tension may regulate nuclear activity. Additionally, we demonstrate that direct inhibition of YAP signaling by VP in large-pores, and activation of YAP signaling by LPA in small-pores dysregulates curvature-mediated gene expression(p<0.05).
Conclusions: Our evidence suggests that pore curvature of synthetic scaffolds influences osteogenic differentiation potential of mesenchymal stem-cells (MSCs) because of cells sensing and responding to unique microenvironments imparted by spherical curvature of macropores by YAP/TAZ modulation.
Methods: Nanofibrous, macroporous tissue engineering scaffolds are fabricated from poly(L-lactic acid) by a sugar-sphere porogen method and thermally-induced phase separation. Sugar-spheres are selectively isolated in the ranges: 65-120um diameter (small) and 250-425um diameter (large). We isolated bone marrow stromal cells and cranial suture mesenchymal stem-cells from wild-type mice and cultured on scaffolds for up to 3 weeks (growth media, n > 4). Gene expression was assessed by qPCR; protein expression and distribution by IHC. Cytochalasin-D (CytD) was used to inhibit actin polymerization, verteporfin (VP) was used to inhibit YAP signaling (5 uM) and lysophosphatidicacid (LPA) was used to induce YAP signaling (25 uM) in vitro. Image analysis performed with Fiji.
Results: We demonstrate sufficiently steep curvature (small-pores, diameter <125um) caused significantly decreased nuclear aspect ratio (NAR) and increased cell-clustering while shallow curvature (large-pores, diameter >250um) caused significantly increased NAR and increased distance between cells. Alterations in curvature-mediated cell organization correlate to differential modulation of YAP/TAZ signaling, by increased gene expression of YAP-related genes in large-pores: YAP1, CTGF, Cy61, peaking at 48 hours and maintained through 3-weeks culture in vitro, and increased osteogenesis (RUNX2, COL1A1, OSX expression, p<0.01). In contrast, small-pores maintain stem-cell marker expression (Gli1, CD44, CD146, p<0.01) and minimal YAP activation. By immunofluorescence we demonstrate YAP activation in large-pores by quantitative increases in YAP nuclear translocation and decreased phosphorylation, from 48 hours in vitro (p < 0.05); small-pores maintain YAP phosphorylation and cytosolic localization. Cytoskeletal disruption by CytD decreases YAP activity and osteogenic differentiation in large-pore constructs, where cytoskeletal tension may regulate nuclear activity. Additionally, we demonstrate that direct inhibition of YAP signaling by VP in large-pores, and activation of YAP signaling by LPA in small-pores dysregulates curvature-mediated gene expression(p<0.05).
Conclusions: Our evidence suggests that pore curvature of synthetic scaffolds influences osteogenic differentiation potential of mesenchymal stem-cells (MSCs) because of cells sensing and responding to unique microenvironments imparted by spherical curvature of macropores by YAP/TAZ modulation.