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Hybrid Biomaterial with Conjugated Growth Factors and Mesenchymal Stem Cells for Ectopic Bone Formation.

ABSTRACT: Bone is a highly vascularized tissue and efficient bone regeneration requires neovascularization, especially for critical-sized bone defects. We developed a novel hybrid biomaterial comprising nanocalcium sulfate (nCS) and fibrin hydrogel to deliver mesenchymal stem cells (MSCs) and angiogenic factors, vascular endothelial growth factor (VEGF) and fibroblast growth factor 9 (FGF9), to promote neovascularization and bone formation. MSC and growth factor(s)-loaded scaffolds were implanted subcutaneously into mice to examine their angiogenic and osteogenic potential. Micro CT, alkaline phosphatase activity assay, and histological analysis were used to evaluate bone formation, while immunohistochemistry was employed to assess neovessel formation. The presence of fibrin preserved the nCS scaffold structure and promoted de novo bone formation. In addition, the presence of bone morphogenic protein 2-expressing MSC in nCS and fibrin hydrogels improved bone regeneration significantly. While FGF9 alone had no significant effect, the combination FGF9 and VEGF conjugated in fibrin enhanced neovascularization and bone formation more than 4-fold compared to nCS with MSC. Overall, our results suggested that the combination of nCS (to support bone formation) with a fibrin-based VEGF/FGF9 release system (support vascular formation) is an innovative and effective strategy that significantly enhanced ectopic bone formation in vivo.

SUBMITTER: Yuan X

PROVIDER: S-EPMC4948201 | biostudies-literature | 2016 Jul

REPOSITORIES: biostudies-literature

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Hybrid Biomaterial with Conjugated Growth Factors and Mesenchymal Stem Cells for Ectopic Bone Formation.

Yuan Xue X Smith Randall J RJ Guan Huiyan H Ionita Ciprian N CN Khobragade Parag P Dziak Rosemary R Liu Zunpeng Z Pang Manhui M Wang Changdong C Guan Guoqiang G Andreadis Stelios S Yang Shuying S

Tissue engineering. Part A 20160628 13-14

Bone is a highly vascularized tissue and efficient bone regeneration requires neovascularization, especially for critical-sized bone defects. We developed a novel hybrid biomaterial comprising nanocalcium sulfate (nCS) and fibrin hydrogel to deliver mesenchymal stem cells (MSCs) and angiogenic factors, vascular endothelial growth factor (VEGF) and fibroblast growth factor 9 (FGF9), to promote neovascularization and bone formation. MSC and growth factor(s)-loaded scaffolds were implanted subcutan ...[more]

PMID: 27269204

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Project description:BackgroundChemical biology approaches using small molecule inhibitors targeting specific signaling pathways are useful tools to dissect the molecular mechanisms governing stem cell differentiation and for their possible use in therapeutic interventions.MethodsStem cell signaling small molecule library functional screen was performed employing human bone marrow skeletal (mesenchymal) stem cells (hBMSCs). Alkaline phosphatase (ALP) activity and formation of mineralized matrix visualized by Alizarin red staining were employed as markers for osteoblastic differentiation. Global gene expression profiling was conducted using the Agilent microarray platform, and data normalization and bioinformatics were performed using GeneSpring software. Pathway analyses were conducted using the Ingenuity Pathway Analysis (IPA) tool. In vivo ectopic bone formation was performed using hBMSC mixed with hydroxyapatite-tricalcium phosphate granules that were implanted subcutaneously in 8-week-old female nude mice. Hematoxylin and eosin staining and Sirius red staining were performed to identify bone formation in vivo.ResultsAmong the tested molecules, LY411575, a potent ?-secretase and Notch signaling inhibitor, exhibited significant inhibitory effects on osteoblastic differentiation of hBMSCs manifested by reduced ALP activity, mineralized matrix formation, and decreased osteoblast-specific gene expression as well as in vivo ectopic bone formation. Global gene expression profiling of LY411575-treated cells revealed changes in multiple signaling pathways, including focal adhesion, insulin, TGF?, IL6, and Notch signaling, and decreased the expression of genes associated with functional categories of tissue development. Among the affected signaling networks were TGF?1, SPP1, and ERK regulatory networks.ConclusionsWe identified ?-secretase inhibitor (LY411575) as a potent regulator of osteoblastic differentiation of hBMSC that may be useful as a therapeutic option for treating conditions associated with ectopic bone formation.

Project description:BackgroundHedgehog (Hh) signaling is essential for osteoblast differentiation of mesenchymal progenitors during endochondral bone formation. However, the critical role of Hh signaling during adult bone remodeling remains to be elucidated.MethodsA Smoothened (SMO) antagonist/Hedgehog inhibitor, BMS-833923, identified during a functional screening of a stem cell signaling small molecule library, was investigated for its effects on the osteoblast differentiation of human skeletal (mesenchymal) stem cells (hMSC). Alkaline phosphatase (ALP) activity and Alizarin red staining were employed as markers for osteoblast differentiation and in vitro mineralization capacity, respectively. Global gene expression profiling was performed using the Agilent® microarray platform. Effects on in vivo ectopic bone formation were assessed by implanting hMSC mixed with hydroxyapatite-tricalcium phosphate granules subcutaneously in 8-week-old female nude mice, and the amount of bone formed was assessed using quantitative histology.ResultsBMS-833923, a SMO antagonist/Hedgehog inhibitor, exhibited significant inhibitory effects on osteoblast differentiation of hMSCs reflected by decreased ALP activity, in vitro mineralization, and downregulation of osteoblast-related gene expression. Similarly, we observed decreased in vivo ectopic bone formation. Global gene expression profiling of BMS-833923-treated compared to vehicle-treated control cells, identified 348 upregulated and 540 downregulated genes with significant effects on multiple signaling pathways, including GPCR, endochondral ossification, RANK-RANKL, insulin, TNF alpha, IL6, and inflammatory response. Further bioinformatic analysis employing Ingenuity Pathway Analysis revealed significant enrichment in BMS-833923-treated cells for a number of functional categories and networks involved in connective and skeletal tissue development and disorders, e.g., NFκB and STAT signaling.ConclusionsWe identified SMO/Hedgehog antagonist (BMS-833923) as a powerful inhibitor of osteoblastic differentiation of hMSC that may be useful as a therapeutic option for treating conditions associated with high heterotopic bone formation and mineralization.

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Hybrid Biomaterial with Conjugated Growth Factors and Mesenchymal Stem Cells for Ectopic Bone Formation.

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Hybrid Biomaterial with Conjugated Growth Factors and Mesenchymal Stem Cells for Ectopic Bone Formation.

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