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Application of VEGFA and FGF-9 enhances angiogenesis, osteogenesis and bone remodeling in type 2 diabetic long bone regeneration.

ABSTRACT: Although bone regeneration is typically a reliable process, type 2 diabetes is associated with impaired or delayed healing processes. In addition, angiogenesis, a crucial step in bone regeneration, is often altered in the diabetic state. In this study, different stages of bone regeneration were characterized in an unicortical bone defect model comparing transgenic type 2 diabetic (db-/db-) and wild type (WT) mice in vivo. We investigated angiogenesis, callus formation and bone remodeling at early, intermediate and late time points by means of histomorphometry as well as protein level analyses. In order to enhance bone regeneration, defects were locally treated with recombinant FGF-9 or VEGFA. Histomorphometry of aniline blue stained sections indicated that bone regeneration is significantly decreased in db-/db- as opposed to WT mice at intermediate (5 days post operation) and late stages (7 days post operation) of bone regeneration. Moreover, immunohistochemical analysis revealed significantly decreased levels of RUNX-2, PCNA, Osteocalcin and PECAM-1 in db-/db- defects. In addition, osteoclastogenesis is impaired in db-/db- indicating altered bone remodeling. These results indicate significant impairments in angiogenesis and osteogenesis in type 2 diabetic bones. Importantly, angiogenesis, osteogenesis and bone remodeling could be reconstituted by application of recombinant FGF-9 and, in part, by VEGFA application. In conclusion, our study demonstrates that type 2 diabetes affects angiogenesis, osteogenesis and subsequently bone remodeling, which in turn leads to decreased bone regeneration. These effects could be reversed by local application of FGF-9 and to a lesser degree VEGFA. These data could serve as a basis for future therapeutic applications aiming at improving bone regeneration in the type 2 diabetic patient population.

SUBMITTER: Wallner C

PROVIDER: S-EPMC4350939 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Objective: The purpose of this study was to observe the effect of the accordion technique (AT) during the distraction phase on chondrogenesis and bone regeneration in a rat femoral distraction osteogenesis (DO) model, and investigate its potential mechanism for reducing the total treatment time of DO. Methods: Fifty-four male Sprague-Dawley (SD) rats that were specific-pathogen-free (SPF) were subjected to DO surgery on the right femur. The distraction rate was 0.5 mm/day for 10 days, following a latency period of 5 days. Rats were randomly divided into Control (no AT, n = 18), Group LA (low amplitude with AT, n = 18), and Group HA (high amplitude with AT, n = 18) according to different AT protocols in the distraction phase. Rats were respectively euthanized by anesthesia overdose at 2, 4 and 6 weeks of the consolidation phase, and the femurs were harvested. Digital radiography, micro-computed tomography (micro-CT), biomechanical tests, and histomorphological analysis were used to assess the quality of regenerated bone in the distraction area. Results: Digital radiographic, micro-CT, biomechanical tests, and histological analysis revealed an increase in early-stage callus formation (p < 0.05) and improved blood supply to the callus tissue in Group LA, as compared to both the Control and Group HA. The enhanced differentiation of fibrous and cartilaginous tissue into bone tissue was also observed in Group LA, leading to improved strength and stiffness (p < 0.05) of the regenerated bone at 6 weeks of the consolidation phase. The angiogenic (hypoxia-inducible factor-1α (HIF-1α) and vascular endothelial growth factor (VEGF), p < 0.05) and osteogenic (runt-related transcription factor 2 (RUNX2), osteocalcin (OCN) and osteopontin (OPN), p < 0.05) biomarkers were higher expressed in Group LA at 2 and 4 weeks of consolidation phase, whereas decreased at 6 weeks of consolidation phase. Conclusion: The application of AT with low amplitude during the distraction phase can enhance chondrogenesis and bone regeneration by activating the angiogenesis factor pathway and upregulating the expression of osteogenic-related biomarkers such as HIF-1α, VEGF, RUNX2, OCN, and OPN.

Project description:BackgroundBone tissue engineering is a new concept bringing hope for the repair of large bone defects, which remains a major clinical challenge. The formation of vascularized bone is key for bone tissue engineering. Growth of specialized blood vessels termed type H is associated with bone formation. In vivo and in vitro studies have shown that low level laser therapy (LLLT) promotes angiogenesis, fracture healing, and osteogenic differentiation of stem cells by increasing reactive oxygen species (ROS). However, whether LLLT can couple angiogenesis and osteogenesis, and the underlying mechanisms during bone formation, remains largely unknown.MethodsMouse bone marrow mesenchymal stem cells (BMSCs) combined with biphasic calcium phosphate (BCP) grafts were implanted into C57BL/6 mice to evaluate the effects of LLLT on the specialized vessel subtypes and bone regeneration in vivo. Furthermore, human BMSCs and human umbilical vein endothelial cells (HUVECs) were co-cultured in vitro. The effects of LLLT on cell proliferation, angiogenesis, and osteogenesis were assessed.ResultsLLLT promoted the formation of blood vessels, collagen fibers, and bone tissue and also increased CD31hiEMCNhi-expressing type H vessels in mBMSC/BCP grafts implanted in mice. LLLT significantly increased both osteogenesis and angiogenesis, as well as related gene expression (HIF-1α, VEGF, TGF-β) of grafts in vivo and of co-cultured BMSCs/HUVECs in vitro. An increase or decrease of ROS induced by H2O2 or Vitamin C, respectively, resulted in an increase or decrease of HIF-1α, and a subsequent increase and decrease of VEGF and TGF-β in the co-culture system. The ROS accumulation induced by LLLT in the co-culture system was significantly decreased when HIF-1α was inhibited with DMBPA and was followed by decreased expression of VEGF and TGF-β.ConclusionsLLLT enhanced vascularized bone regeneration by coupling angiogenesis and osteogenesis. ROS/HIF-1α was necessary for these effects of LLLT. LLLT triggered a ROS-dependent increase of HIF-1α, VEGF, and TGF-β and resulted in subsequent formation of type H vessels and osteogenic differentiation of mesenchymal stem cells. As ROS also was a target of HIF-1α, there may be a positive feedback loop between ROS and HIF-1α, which further amplified HIF-1α induction via the LLLT-mediated ROS increase. This study provided new insight into the effects of LLLT on vascularization and bone regeneration in bone tissue engineering.

Project description:BACKGROUND:In bone tissue engineering (BTE), extensive research into vascular endothelial growth factor A (VEGFA)-mediated angiogenesis has yielded inconsistent results. The aim of this study was to investigate the influence on angio- and osteogenesis of adenoviral-mediated delivery of VEGFA alone or in combination with bone morphogenetic protein 2 (BMP2) in bone marrow stromal cells (BMSC) seeded onto a recently developed poly(LLA-co-CL) scaffold. METHODS:Human BMSC were engineered to express VEGFA alone or in combination with BMP2 and seeded onto poly(LLA-co-CL) scaffolds. Changes in angiogenic and osteogenic gene and protein levels were examined by quantitative reverse-transcription polymerase chain reaction (RT-PCR), PCR array, and alkaline phosphatase assay. An in vivo subcutaneous mouse model was used to investigate the effect on angio- and osteogenesis of VEGFA alone or in combination with BMP2, using microcomputed tomography (μCT), histology, immunohistochemistry, and immunofluorescence. RESULTS:Combined delivery of a lower ratio (1:3) of VEGFA and BMP2 (ad-BMP2 + VEGFA) led to upregulation of osteogenic and angiogenic genes in vitro at 3 and 14 days, compared with mono-delivery of VEGFA (ad-VEGFA) and other controls. In vivo, in a subcutaneous mouse model, both ad-VEGFA and ad-BMP2 + VEGFA scaffold explants exhibited increased angiogenesis at 2 weeks. Enhanced angiogenesis was largely related to the recruitment and differentiation of mouse progenitor cells to the endothelial lineage and, to a lesser extent, to endothelial differentiation of the implanted BMSC. μCT and histological analyses revealed enhanced de novo bone formation only in the ad-BMP2 + VEGFA group, corresponding at the molecular level to the upregulation of genes related to osteogenesis, such as ALPL, RUNX2, and SPP1. CONCLUSIONS:Although BMSC expressing VEGFA alone or in combination with BMP2 significantly induced angiogenesis, VEGFA alone failed to demonstrate osteogenic activity both in vitro and in vivo. These results not only call into question the use of VEGFA alone in bone regeneration, but also highlight the importance in BTE of appropriately formulated combined delivery of VEGFA and BMP2.

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Application of VEGFA and FGF-9 enhances angiogenesis, osteogenesis and bone remodeling in type 2 diabetic long bone regeneration.

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