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Development of an Accurate and Proactive Immunomodulatory Strategy to Improve Bone Substitute Material-Mediated Osteogenesis and Angiogenesis.

ABSTRACT: Background: Treatment of large bone defects represents a major clinical problem worldwide. Suitable bone substitute materials are commonly required to achieve successful bone regeneration, and much effort has been spent to optimize their chemical compositions, 3D architecture and mechanical properties. However, material-immune system interactions are increasingly being recognized as a crucial factor influencing regeneration. Here, we envisioned an accurate and proactive immunomodulation strategy via delivery of IL-4 (key regulator of macrophage polarization) to promote bone substitute material-mediated regeneration. Methods: Four different IL-4 doses (0 ng, 10 ng, 50 ng and 100 ng) were delivered into rat large cranial bone defects at day 3 post-operation of decellularized bone matrix (DBM) material implantation, and the osteogenesis, angiogenesis and macrophage polarization were meticulously evaluated. Results: Micro-CT analysis showed that immunomodulation with 10 ng IL-4 significantly outperformed the other groups in terms of new bone formation (1.23-5.05 fold) and vascularization (1.29-6.08 fold), achieving successful defect bridging and good vascularization at 12 weeks. Histological analysis at 7 and 14 days showed that the 10 ng group generated the most preferable M1/M2 macrophage polarization profile, resulting in a pro-healing microenvironment with more IL-10 and less TNF-α secretion, a reduced apoptosis level in tissues around the materials, and enhanced mesenchymal stem cell migration and osteogenic differentiation. Moreover, in vitro studies revealed that M1 macrophages facilitated mesenchymal stem cell migration, while M2 macrophages significantly increased cell survival, proliferation and osteogenic differentiation, explaining the in vivo findings. Conclusions: Accurate immunomodulation via IL4 delivery significantly enhanced DBM-mediated osteogenesis and angiogenesis via the coordinated involvement of M1 and M2 macrophages, revealing the promise of this accurate and proactive immunomodulatory strategy for developing new bone substitute materials.

SUBMITTER: Zheng ZW

PROVIDER: S-EPMC6276091 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BACKGROUND/AIM:The present study investigates the in vivo tissue reaction and the integration behavior of an injectable bone substitute material (IBS) composed of a water-based gel combined with nano hydroxyapatite particles and biphasic calcium phosphate granules. The results of the IBS were compared to biphasic bone substitute granules (BBSM) of the same chemical composition. MATERIALS AND METHODS:The subcutaneous implantation model in 40 female 5-week-old CD-1 mice up to 60 days after implantation was used for conduction of the in vivo experiments. Moreover, established histological, histopathological and histomorphometrical methods were applied. RESULTS:The results showed that the IBS was gradually invaded by cells and complex tissue elements. Thus, the implant bed could be distinguished in two areas, i.e. an outer and inner region. While the outer region started to interact with the peri-implant tissue by evoking multinucleated giant cells and at earlier time points by undergoing a continuous high vascularization, the inner part was free of peri-implant cells for at least 30 days, starting to undergo a similar tissue reaction at a later time point. The bone substitute granules allowed for a fast tissue influx between the interspaces of the granules starting at day 10. While the vessel density did not differ in both groups up to the end of the study, the amount of vascularization was significantly higher over the entire observation period in the BBSM group. Moreover, the amount of biomaterial-associated multinucleated giant cells (BMGCs) was significantly higher in the IBS group in the period of between 15 to 30 days after implantation, while comparable BMGC numbers were found in both groups towards the end of the study. CONCLUSION:IBS can build a barrier-like structure that is able to control the soft tissue influx into the central regions of the implantation bed, which could not be observed in other bone substitute granules of the same chemical composition. This directed integration behavior is assumed to be in accordance with the concept of Guided Bone Regeneration (GBR). Furthermore, BMGCs can significantly influence the process of angiogenesis within an implant bed of a biomaterial but not the maturity of blood vessels.

Project description:Rationale: Angiogenesis and osteogenesis are highly coupled processes which are indispensable to bone repair. However, the underlying mechanism(s) remain elusive. To bridge the gap in understanding the coupling process is crucial to develop corresponding solutions to abnormal bone healing. Epidermal growth factor-like protein 6 (EGFL6) is an angiogenic factor specifically and distinctively up-regulated during osteoblast differentiation. In contrast with most currently known osteoblast-derived coupling factors, EGFL6 is highlighted with little or low expression in other cells and tissues. Methods: In this study, primary bone marrow mesenchymal stem cells (MSCs) and osteoblastic cell line (MC3T3-E1) were transduced with lentiviral silencing or overexpression constructs targeting EGFL6. Cells were induced by osteogenic medium, followed by the evaluation of mineralization as well as related gene and protein expression. Global and conditional knockout mice were established to examine the bone phenotype under physiological condition. Furthermore, bone defect models were created to investigate the outcome of bone repair in mice lacking EGFL6 expression. Results: We show that overexpression of EGFL6 markedly enhances osteogenic capacity in vitro by augmenting bone morphogenic protein (BMP)-Smad and MAPK signaling, whereas downregulation of EGFL6 diminishes osteoblastic mineralization. Interestingly, osteoblast differentiation was not affected by the exogenous addition of EGFL6 protein, thereby indicating that EGFL6 may regulate osteoblastic function in an intracrine manner. Mice with osteoblast-specific and global knockout of EGFL6 surprisingly exhibit a normal bone phenotype under physiological conditions. However, EGFL6-deficiency leads to compromised bone repair in a bone defect model which is characterized by decreased formation of type H vessels as well as osteoblast lineage cells. Conclusions: Together, these data demonstrate that EGFL6 serves as an essential regulator to couple osteogenesis to angiogenesis during bone repair.

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.

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Development of an Accurate and Proactive Immunomodulatory Strategy to Improve Bone Substitute Material-Mediated Osteogenesis and Angiogenesis.

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