Beta 1 integrins in bone formation during development and engineering integrin-specific hydrogels for enhanced bone healing

Healing large bone defects remains a clinical challenge. While autografts are the gold standard treatment for large bone defects, they are limited by availability and donor site pain. Growth factor treatments such as BMP therapy provide a promising alternative but are expensive and present clinical safety concerns, primarily due to delivery of BMPs at supraphysiological doses. Integrins are ECM receptors which mediate crucial cell functions such as adhesion and differentiation. Therefore, understanding the role of integrins in bone formation and directing desired interactions may enable modulation of host cell functions for therapeutic applications. In this work, beta 1 integrins were deleted in osteolineage cells of transgenic mice at three different stages of differentiation to elucidate their role in bone development. We also engineered bioartificial PEG-based matrices which target the pro-osteogenic alpha 2 beta 1 integrin to promote bone healing. Conditional deletion of beta 1 integrins in osteochondroprogenitor cells under the Twist 2 promoter resulted in severe pre-natal skeletal mineralization defects and embryonic lethality. Targeted deletion of beta 1 integrins in osterix-expressing osteoprogenitors resulted in growth abnormalities, reduced calvarial mineralization, impaired femur development, and tooth defects. However, mice lacking beta 1 integrins in osteocalcin-expressing osteoblasts and osteocytes displayed only a mild skeletal phenotype, indicating that beta 1 integrins play an important role in early skeletal development, but are not required for mature osteoblast function. PEG hydrogels functionalized with the integrin-specific GFOGER ligand enhanced bone regeneration, induced defect bridging in combination with low doses of rhBMP-2 and stimulated improved bone healing compared collagen sponges, which are the clinical standard delivery vector for BMP-2 therapy. These results suggest that treatment with bioartificial integrin-specific PEG hydrogels may be a promising clinical strategy for bone regeneration in large bone defects.Ph.D

Additional file 4: Figure S3. of Enhanced in vitro osteogenic differentiation of human fetal MSCs attached to 3D microcarriers versus harvested from 2D monolayers

Kinetics of osteogenic gene expression during osteogenic differentiation of S27 hfMSC in PCL-TCP scaffold cultures. (A) Cultures seeded with 2D monolayer-harvested (2D MNL-harv) and microcarrier-harvested (3D MC-harv) cells, (B) Cultures seeded with microcarrier-harvested (2D MNL-harv) and microcarrier-bound (3D MC-bound) cells. Two-way repeated measures ANOVA with post-hoc Tukey correction was performed between 3D MC-harv, 2D MNL-harv and 3D MC-bound cells using Graphpad, (*p < 0.05 and at least a 2-fold difference in the means). Of the multiple comparisons performed, data representing a single comparison is shown: (A) 3D MC-harv vs 2D MNL-harv, (B) 3D MC-bound vs 2D MNL-harv. (TIFF 544 kb

Additional file 1: Figure S4. of Enhanced in vitro osteogenic differentiation of human fetal MSCs attached to 3D microcarriers versus harvested from 2D monolayers

Gene expression of anti-inflammatory, Wnt inhibition, YAP/TAZ targets and integrins S27 hfMSCs after 7 days of expansion in 2D monolayer-harvested cultures (2D MNL-harv) and harvested and non-harvested 3D MC cultures (3D MC-bound and 3D MC-harv respectively). Results are presented as fold expression levels of genes compared to 2D MNL-harv (*p < 0.05 and at least a 2-fold difference in the means compared to 2D MNL-harv, ($ p < 0.05 and at least a 2-fold difference in the means compared to 3D MC-harv). (TIFF 609 kb

Additional file 5: Table S1. of Enhanced in vitro osteogenic differentiation of human fetal MSCs attached to 3D microcarriers versus harvested from 2D monolayers

List of Taqman Gene Expression Assay IDs for genes investigated by qPCR in this study. (DOCX 13 kb

Distinct biophysical mechanisms of focal adhesion kinase mechanoactivation by different extracellular matrix proteins

Matrix mechanics controls cell fate by modulating the bonds between integrins and extracellular matrix (ECM) proteins. However, it remains unclear how fibronectin (FN), type 1 collagen, and their receptor integrin subtypes distinctly control force transmission to regulate focal adhesion kinase (FAK) activity, a crucial molecular signal governing cell adhesion/migration. Here we showed, using a genetically encoded FAK biosensor based on fluorescence resonance energy transfer, that FN-mediated FAK activation is dependent on the mechanical tension, which may expose its otherwise hidden FN synergy site to integrin α5. In sharp contrast, the ligation between the constitutively exposed binding motif of type 1 collagen and its receptor integrin α2 was surprisingly tension-independent to induce sufficient FAK activation. Although integrin α subunit determines mechanosensitivity, the ligation between α subunit and the ECM proteins converges at the integrin β1 activation to induce FAK activation. We further discovered that the interaction of the N-terminal protein 4.1/ezrin/redixin/moesin basic patch with phosphatidylinositol 4,5-biphosphate is crucial during cell adhesion to maintain the FAK activation from the inhibitory effect of nearby protein 4.1/ezrin/redixin/moesin acidic sites. Therefore, different ECM proteins either can transmit or can shield from mechanical forces to regulate cellular functions, with the accessibility of ECM binding motifs by their specific integrin α subunits determining the biophysical mechanisms of FAK activation during mechanotransduction