The Role Of Cucurbitacin In Vascularization Of Bone Tissue Engineering

Bone regeneration is a complex process requiring highly orchestrated interactions between different cells and signals to form new mineralized tissue.Blood vessels serve as a structural template for bone formation and play crucial roles in the maintenance of bone homeostasis by providing key elements into the microenvironment including oxygen,nutrients,hormones,neurotransmitters and growth factors.Bone formation and regeneration depend on the osteogenesis-angiogenesis interplay,which is controlled by a variety of growth factors and several cell types.However,defective angiogenesis at the fracture site has been considered as one of the primary risk factors for unsuccessful bone healing.Over the last decades,attempts have been made to develop effective strategies that can notably promote and improve angiogenesis.Drug delivery-based bone tissue engineering holds great promise for inducing angiogenesis thereby improving bone regeneration.The delivery of a combination of angiogenic molecules?growth factors?and genetic factors?e.g.DNAs,and RNAs?within biomaterial carriers has shown great therapeutic potential in preclinical testing,but a few have been successfully translated to clinical phases due to the limitations,including poor stability and high-dose induced cytotoxicity.Nature product molecules could lead to undesired utility in angiogenesis caused its stable ability and high efficiency.Cucurbitacins?Cus?are tetracyclic terpenes with steroidal structures that are mainly found in the Cucurbitaceae family,e.g.squash,pumpkin,cucumber and melons.As traditional Chinese medicines,the pharmacological properties of cucurbitacin tablets have been extensively studied for decades particularly for the treatment of jaundice.Cucurbitacin B?CuB?and Cucurbitacin E?CuE?are the main bioactive compounds of cucurbitacin tablets.Most of the study focused on Cus'toxicity and anti-cancer effect.These studies have revealed that high-dose Cus can cause apoptosis,and growth suppression of cancer cells.In this study,we found a low dose?no cytotoxicity?of CuB&CuE could significantly promote the tube formation of vascular endothelial cells in vitro for the first time.Then,we developed a novel biomaterial strategy to promote bone tissue engineering vascularization by incorporating CuB/CuE in poly?lactide-co-glycolide??PLGA?and?-tricalcium phosphate??-TCP?biodegradable scaffold by 3D low-temperature rapid prototyping?LT-RP?technology.The bioactivity of drug-loaded scaffold was assessed in a rat critical size calvarial defect model after implantation.The main research contents as follows:1.CuB and CuE were found to promote tube formation of human vascular endothelial cells at low dose in vitro for the first time,with specifically activated VEGFR2^Tyr996 and its key downstream mediators.Cytotoxicity assay was performed to determine the safe dose range of the CuB and CuE in human EA.hy926 endothelial cells?ECs?.Then we found a low dose?<30 nM?of CuB and CuE could significantly promote the tube formation of EA.hy926.The CuB and CuE were determined to impair the zebrafish vascular injury under the safe concentration.Further results showed that low dose CuB and CuE may be an agonist of VEGFR2 and regulating VEGFR2 downstream signaling pathway,which affected ECs'migration and polarization to lead the angiogenesis.Bioinformatics analysis results suggested that the potential protein targets of the Cus may be related the cell metabolism and innate immunity.2.Porous composite scaffolds were prepared by 3D low temperature rapid prototyping technology with well physical and chemical properties and controlled drug release.PLGA and?-TCP were the elements to formulate a porous PT biodegradable scaffold.CuB and CuE were incorporated to PT scaffold respectively?PT/CuB,PT/CuE?.Physical and chemical properties of PT,PT/CuB,and PT/CuE have been characterized by scanning electron microscopy,mechanical testing and micro-CT.The drug release rate will be detected by high performance liquid chromatography.Our results showed that the scaffolds possessed well-designed bio-mimic structure and improved mechanical properties.CuB/CuE were released with linear manner from the biodegradable drug delivery devices PT/CuB and PT/CuE.Alizarin red staining image data demonstrated CuB and CuE composite scaffold increased mineralization of hBMSCs significantly.3.PT/CuB and PT/CuE could promote the neovascularization and bone regeneration in rat critical size calvarial defect model.We investigated the effects of PT/CuB and PT/CuE in rat critical size calvarial defect model.This platform allowed us to explore the potential of CuB and CuE for the treatment of localized bone defects.MRI imaging revealed that blood flow signal significantly increased in PT/CuB and PT/CuE post-surgery compared to sham and PT groups.Micro-CT based angiography also confirmed that PT/CuB composite scaffold promoted angiogenesis with significantly increased vessel volume and density in the cranial bone defect.Bone mineral density and bone volume/tissue volume rate significantly increased in PT/CuB and PT/CuE groups compared with PT and sham groups.VEGF and HIF-1?expression were upregulated in PT/CuB and PT/CuE groups significantly by immunohistochemistry analysis.These results indicated that the bioactive scaffolds containing CuB or CuE could promote bone regeneration by promoting angiogenesis in the local microenvironment of bone defects.In conclusion,this study demonstrated that natural products CuB and CuE promoted tube formation of ea.hy 926 in vitro.Thus,we found that the controlled release of CuB or CuE can be utilized to enhance new bone and microvessel formation in rat critical size calvarial defect model.Taken together,these experiments support CuB and CuE as an agonist for VEGFR2 play in angiogenesis role and highlight its potential use as a therapeutic agent for tissue-engineered bone repair,wound healing,or ischemia.This therapy provides a strategy to improve bone defect repair by integrating a biomimetic scaffold.