Immunomodulatory hybrid micro-nanofiber scaffolds enhance vascular regeneration

The inertness of synthetic polymer materials and the insufficient mechanical strength of reprocessed decellu-larized extracellular matrix(dECM)limited their promotive efforts on tissue regeneration.Here,we prepared a hybrid scaffold composed of PCL microfibers and human placental extracellular matrix(pECM)nanofibers by co-electrospinning,which was grafted with heparin and further absorbed with IL-4.The hybrid scaffold with improved hemocompatibility firstly switched macrophages to anti-inflammatory phenotype(increased by 18.1%)and then promoted migration,NO production,tube formation of endothelial cells(ECs),and migration and maturation of vascular smooth muscle cells(VSMCs),and ECM deposition in vitro and in vivo.ECs coverage rate increased by 8.6%and the thickness of the smooth muscle layer was 1.8 times more than PCL grafts at 12 wks.Our study realized the complementary advantages of synthetic polymer materials and dECM materials,and opened intriguing perspectives for the design and construction of small-diameter vascular grafts(SDVGs)and immune-regulated materials for other tissue regeneration.

Recent advances of natural and bioengineered extracellular vesicles and their application in vascular regeneration

The progression of cardiovascular diseases such as atherosclerosis and myocardial infarction leads to serious vascular injury,highlighting the urgent need for targeted regenerative therapy.Extracellular vesicles(EVs)composed of a lipid bilayer containing nuclear and cytosolic materials are relevant to the progression of cardiovascular diseases.Moreover,EVs can deliver bioactive cargo in pathological cardiovascular and regulate the biological function of recipient cells,such as inflammation,proliferation,angiogenesis and polarization.However,because the targeting and bioactivity of natural EVs are subject to several limitations,bioengineered EVs have achieved wide advancements in biomedicine.Bioengineered EVs involve three main ways to acquire including(i)modification of the EVs after isolation;(ii)modification of producer cells before EVs’isolation;(iii)synthesize EVs using natural or modified cell membranes,and encapsulating drugs or bioactive molecules into EVs.In this review,we first summarize the cardiovascular injury-related disease and describe the role of different cells and EVs in vascular regeneration.We also discuss the application of bioengineered EVs from different producer cells to cardiovascular diseases.Finally,we summarize the surface modification on EVs which can specifically target abnormal cells in injured vascular.

A hierarchical vascularized engineered bone inspired by intramembranous ossification for mandibular regeneration

Mandibular defects caused by injuries,tumors,and infections are common and can severely affect mandibular function and the patient's appearance.However,mandible reconstruction with a mandibular bionic structure remains challenging.Inspired by the process of intramembranous ossification in mandibular development,a hierarchical vascularized engineered bone consisting of angiogenesis and osteogenesis modules has been produced.Moreover,the hierarchical vascular network and bone structure generated by these hierarchical vascularized engineered bone modules match the particular anatomical structure of the mandible.The ultra-tough polyion complex has been used as the basic scaffold for hierarchical vascularized engineered bone for ensuring better reconstruction of mandible function.According to the results of in vivo experiments,the bone regenerated using hierarchical vascularized engineered bone is similar to the natural mandibular bone in terms of morphology and genomics.The sonic hedgehog signaling pathway is specifically activated in hierarchical vascularized engineered bone,indicating that the new bone in hierarchical vascularized engineered bone underwent a process of intramembranous ossification identical to that of mandible development.Thus,hierarchical vascularized engineered bone has a high potential for clinical application in mandibular defect reconstruction.Moreover,the concept based on developmental processes and bionic structures provides an effective strategy for tissue regeneration.

Gibberellin promotes cambium reestablishment during secondary vascular tissue regeneration after girdling in an auxin-dependent manner in Populus

Secondary vascular tissue(SVT)development and regeneration are regulated by phytohormones.In this study,we used an in vitro SVT regeneration system to demonstrate that gibberellin(GA)treatment significantly promotes auxin-induced cambium reestablishment.Altering GA content by overexpressing or knocking down ent-kaurene synthase(KS)affected secondary growth and SVT regeneration in poplar.The poplar DELLA gene GIBBERELLIC ACID INSENSITIVE(PtoGAI)is expressed in a specific pattern during secondary growth and cambium regeneration after girdling.Overexpression of PtoGAI disrupted poplar growth and inhibited cambium regeneration,and the inhibition of cambium regeneration could be partially restored by GA application.Further analysis of the PtaDR5:GUS transgenic plants,the localization of PIN-FORMED 1(PIN1)and the expression of auxin-related genes found that an additional GA treatment could enhance the auxin response as well as the expression of PIN1,which mediates auxin transport during SVT regeneration.Taken together,these findings suggest that GA promotes cambium regeneration by stimulating auxin signal transduction.

Neuroprotection against vascular dementia after acupuncture combined with donepezil hydrochloride：P300 event related potential

Acupuncture can be used to treat various nervous system diseases.Here,168 vascular dementia patients were orally administered donepezil hydrochloride alone（5 mg/day,once a day for 56 days）,or combined with acupuncture at Shenting（DU24）,Tianzhu（BL10）,Sishencong（Extra）,Yintang（Extra）,Renzhong（DU26）,Neiguan（PC6）,Shenmen（HT7）,Fengchi（GB20）,Wangu（GB12） and Baihui（DU20）（once a day for 56 days）.Compared with donepezil hydrochloride alone,P300 event related potential latency was shorter with an increased amplitude in patients treated with donepezil hydrochloride and acupuncture.Mini-Mental State Examination score was also higher.Moreover,these differences in P300 latency were identified within different infarcted regions in patients treated with donepezil hydrochloride and acupuncture.These findings indicate that acupuncture combined with donepezil hydrochloride noticeably improves cognitive function in patients with vascular dementia,and exerts neuroprotective effects against vascular dementia.

Evidence for novel age-dependent network structures as a putative primo vascular network in the dura mater of the rat brain

With chromium-hematoxylin staining, we found evidence for the existence of novel age-dependent network structures in the dura mater of rat brains. Under stereomicroscopy, we noticed that chromium-hematoxylin-stained threadlike structures, which were barely observable in 1-weekold rats, were networked in specific areas of the brain, for example, the lateral lobes and the cerebella, in 4-week-old rats. In 7-week-old rats, those structures were found to have become larger and better networked. With phase contrast microscopy, we found that in 1-week-old rats, chromium-hematoxylin-stained granules were scattered in the same areas of the brain in which the network structures would later be observed in the 4- and 7-week-old rats. Such age-dependent network structures were examined by using optical and transmission electron microscopy, and the following results were obtained. The scattered granules fused into networks with increasing age. Cross-sections of the age-dependent network structures demonstrated heavily-stained basophilic substructures. Transmission electron microscopy revealed the basophilic substructures to be clusters with high electron densities consisting of nanosized particles. We report these data as evidence for the existence of age-dependent network structures in the dura mater, we discuss their putative functions of age-dependent network structures beyond the general concept of the dura mater as a supporting matrix.

Cartilage oligomeric matrix protein enhances the vascularization of acellular nerves

Vascularization of acellular nerves has been shown to contribute to nerve bridging.In this study,we used a 10-mm sciatic nerve defect model in rats to determine whether cartilage oligomeric matrix protein enhances the vascularization of injured acellular nerves.The rat nerve defects were treated with acellular nerve grafting（control group） alone or acellular nerve grafting combined with intraperitoneal injection of cartilage oligomeric matrix protein（experimental group）.As shown through two-dimensional imaging,the vessels began to invade into the acellular nerve graft from both anastomotic ends at day 7 post-operation,and gradually covered the entire graft at day 21.The vascular density,vascular area,and the velocity of revascularization in the experimental group were all higher than those in the control group.These results indicate that cartilage oligomeric matrix protein enhances the vascularization of acellular nerves.

Neuroprotective effects of tetrandrine against vascular dementia

Tetrandrine is one of the major active ingredients in Menispermaceae Stephania tetrandra S.Moore,and has specific therapeutic effects in ischemic cerebrovascular disease.Its use in vascular dementia has not been studied fully.Here,we investigated whether tetrandrine would improve behavioral and cellular impairments in a two-vessel occlusion rat model of chronic vascular dementia.Eight weeks after model establishment,rats were injected intraperitoneally with 10 or 30 mg/kg tetrandrine every other day for 4 weeks.Behavioral assessment in the Morris water maze showed that model rats had longer escape latencies in training trials,and spent less time swimming in the target quadrant in probe trials,than sham-operated rats.However,rats that had received tetrandrine showed shorter escape latencies and longer target quadrant swimming time than untreated model rats.Hematoxylin-eosin and Nissl staining revealed less neuronal necrosis and pathological damage,and more living cells,in the hippocampus of rats treated with tetrandrine than in untreated model rats.Western blot assay showed that interleukin-1β expression,and phosphorylation of the N-methyl-D-aspartate 2B receptor at tyrosine 1472,were lower in model rats that received tetrandrine than in those that did not.The present findings suggest that tetrandrine may be neuroprotective in chronic vascular dementia by reducing interleukin-1β expression,N-methyl-D-aspartate receptor 2B phosphorylation at tyrosine 1472,and neuronal necrosis.

Black phosphorus nanosheets-enabled DNA hydrogel integrating 3D-printed scaffold for promoting vascularized bone regeneration

The classical 3D-printed scaffolds have attracted enormous interests in bone regeneration due to the customized structural and mechanical adaptability to bone defects.However,the pristine scaffolds still suffer from the absence of dynamic and bioactive microenvironment that is analogous to natural extracellular matrix(ECM)to regulate cell behaviour and promote tissue regeneration.To address this challenge,we develop a black phosphorus nanosheets-enabled dynamic DNA hydrogel to integrate with 3D-printed scaffold to build a bioactive gel-scaffold construct to achieve enhanced angiogenesis and bone regeneration.The black phosphorus nanosheets reinforce the mechanical strength of dynamic self-healable hydrogel and endow the gel-scaffold construct with preserved protein binding to achieve sustainable delivery of growth factor.We further explore the effects of this activated construct on both human umbilical vein endothelial cells(HUVECs)and mesenchymal stem cells(MSCs)as well as in a critical-sized rat cranial defect model.The results confirm that the gel-scaffold construct is able to promote the growth of mature blood vessels as well as induce osteogenesis to promote new bone formation,indicating that the strategy of nano-enabled dynamic hydrogel integrated with 3D-printed scaffold holds great promise for bone tissue engineering.

Construction of Oriented Structure in Inner Surface of Small-Diameter Artificial Blood Vessels:A Review

There is an urgent need for small-diameter artificial blood vessels in clinic.Physical,chemical and biological factors should be integrated to avoid thrombosis and intimal hyperplasia after implantation and to promote successful fabrication of small-diameter artificial blood vessels.From a physical perspective,the internal oriented structures of natural blood vessels plays an important role in guiding the directional growth of cells,improving the blood flow environment,and promoting the regeneration of vascular tissue.In this review,the effects of the oriented structures on cells,including endothelial cells(ECs),smooth muscle cells(SMCs)and stem cells,as well as the effect of the oriented structures on hemodynamics and vascular tissue remodeling and regeneration are introduced.Various forms of oriented structures(fibers,grooves,channels,etc.)and their construction methods are also reviewed.Conclusions and future perspectives are given.It is expected to give some references to relevant researches.

Are bone marrow regenerative cells ideal seed cells for the treatment of cerebral ischemia?

Bone marrow cells for the treatment of ischemic brain injury may depend on the secretion of a large number of neurotrophic factors. Bone marrow regenerative cells are capable of increasing the secretion of neurotrophic factors. In this study, after tail vein injection of 5-fluorouracil for 7 days, bone marrow cells and bone marrow regenerative cells were isolated from the tibias and femurs of rats, and then administered intravenously via the tail vein after focal cerebral ischemia. Immunohistological staining and reverse transcription-PCR detection showed that transplanted bone marrow cells and bone marrow regenerative cells could migrate and survive in the ischemic regions, such as the cortical and striatal infarction zone. These cells promote vascular endothelial cell growth factor mRNA expression in the ischemic marginal zone surrounding the ischemic penumbra of the cortical and striatal infarction zone, and have great advantages in promoting the recovery of neurological function, reducing infarct size and promoting angiogenesis. Bone marrow regenerative cells exhibited stronger neuroprotective effects than bone marrow cells. Our experimental findings indicate that bone marrow regenerative cells are preferable over bone marrow cells for cell therapy for neural regeneration after cerebral ischemia. Their neuroprotective effect is largely due to their ability to induce the secretion of factors that promote vascular regeneration, such as vascular endothelial growth factor.

MicroRNAs as Modulators of Endothelial Differentiation of Stem Cells

MicroRNAs (miRs) are a class of small (~22 nucleotides), widely distributed, and highly conserved non-coding RNA molecules and play an important post-transcriptional regulatory role by targeting mRNA. Embryonic and induced pluripotent stem cells (ESCs and iPSC, respectively) hold great promise for vascular regenerative therapies. However, several limitations currently prohibit their therapeutic use. The importance of miRs in controlling the gene expression profile of a particular cell type is emerging and a multitude of miRs have been identified that play key roles in vascular development and regeneration. A combination of pluripotency transcription factors and different miRs not only enhances the pluripotency of stem cells but also has been reported to enhance their endothelial differentiation. This review will summarize the findings that focus different miR clusters in the induction, maintenance, and directed endothelial differentiation of ESCs and iPSCs.

Bioinspired porous microspheres for sustained hypoxic exosomes release and vascularized bone regeneration

Exosomes derived from mesenchymal stem cells(MSCs)have demonstrated regenerative potential for cell-free bone tissue engineering,nevertheless,certain challenges,including the confined therapeutic potency of exosomes and ineffective delivery method,are still persisted.Here,we confirmed that hypoxic precondition could induce enhanced secretion of exosomes from stem cells from human exfoliated deciduous teeth(SHEDs)via comprehensive proteomics analysis,and the corresponding hypoxic exosomes(H-Exo)exhibited superior potential in promoting cellular angiogenesis and osteogenesis via the significant up-regulation in focal adhesion,VEGF signaling pathway,and thyroid hormone synthesis.Then,we developed a platform technology enabling the effective delivery of hypoxic exosomes with sustained release kinetics to irregular-shaped bone defects via injection.This platform is based on a simple adsorbing technique,where exosomes are adsorbed onto the surface of injectable porous poly(lactide-co-glycolide)(PLGA)microspheres with bioinspired polydopamine(PDA)coating(PMS-PDA microspheres).The PMS-PDA microspheres could effectively adsorb exosomes,show sustained release of H-Exo for 21 days with high bioactivity,and induce vascularized bone regeneration in 5-mm rat calvarial defect.These findings indicate that the hypoxic precondition and PMS-PDA porous microsphere-based exosome delivery are efficient in inducing tissue regeneration,hence facilitating the clinical translation of exosome-based therapy.

A baicalin-loaded coaxial nanofiber scaffold regulated inflammation and osteoclast differentiation for vascularized bone regeneration

We demonstrate a simple,effective and feasible method to address the shrinkage of Poly(lactic-co-glycolic acid)(PLGA)through a core-shell structure fiber strategy.The results revealed that introducing size-stable poly-caprolactone(PCL)as the core fiber significantly improved the PLGA-based fibrous scaffold’s dimensional maintenance.We further utilized fish collagen to modify the PLGA shell layer(PFC)of coaxial fibers and loaded baicalin(BA)into the PCL core layer(PCL-BA)to endow fibrous scaffold with more functional biological cues.The PFC/PCL-BA fibrous scaffold promoted the osteogenic differentiation of bone mesenchymal stem cells and stimulated the RAW264.7 cells to polarize into a pro-reparative phenotype.Importantly,the in vivo study demonstrated that the PFC/PCL-BA scaffold could regulate inflammation and osteoclast differentiation,favor neovascularization and bone formation.This work tactfully combined PLGA and PCL to establish a drug release platform based on the core-shell fibrous scaffold for vascularized bone regeneration.

Fabrication of a bio-instructive scaffold conferred with a favorable microenvironment allowing for superior implant osseointegration and accelerated in situ vascularized bone regeneration via type H vessel formation

The potential translation of bio-inert polymer scaffolds as bone substitutes is limited by the lack of neovascularization upon implantation and subsequently diminished ingrowth of host bone,most likely resulted from the inability to replicate appropriate endogenous crosstalk between cells.Human umbilical vein endothelial cell-derived decellularized extracellular matrix(HdECM),which contains a collection of angiocrine biomolecules,has recently been demonstrated to mediate endothelial cells(ECs)-osteoprogenitors(OPs)crosstalk.We employed the HdECM to create a PCL(polycaprolactone)/fibrin/HdECM(PFE)hybrid scaffold.We hypothesized PFE scaffold could reconstitute a bio-instructive microenvironment that reintroduces the crosstalk,resulting in vascularized bone regeneration.Following implantation in a rat femoral bone defect,the PFE scaffold demonstrated early vascular infiltration and enhanced bone regeneration by microangiography(μ-AG)and micro-computational tomography(μ-CT).Based on the immunofluorescence studies,PFE mediated the endogenous angiogenesis and osteogenesis with a substantial number of type H vessels and osteoprogenitors.In addition,superior osseointegration was observed by a direct host bone-PCL interface,which was likely attributed to the formation of type H vessels.The bio-instructive microenvironment created by our innovative PFE scaffold made possible superior osseointegration and type H vessel-related bone regeneration.It could become an alternative solution of improving the osseointegration of bone substitutes with the help of induced type H vessels,which could compensate for the inherent biological inertness of synthetic polymers.

Beta-nerve growth factor promotes neurogenesis and angiogenesis during the repair of bone defects

We previously showed that the repair of bone defects is regulated by neural and vascular signals. In the present study, we examined the effect of topically applied β-nerve growth factor（β-NGF） on neurogenesis and angiogenesis in critical-sized bone defects filled with collagen bone substitute. We created two symmetrical defects, 2.5 mm in diameter, on either side of the parietal bone of the skull, and filled them with bone substitute. Subcutaneously implanted osmotic pumps were used to infuse 10 μgβ-NGF in PBS（β-NGF ＋ PBS） into the right-hand side defect, and PBS into the left（control） defect, over the 7 days following surgery. Immunohistochemical staining and hematoxylin-eosin staining were carried out at 3, 7, 14, 21 and 28 days postoperatively. On day 7, expression of β III-tubulin was lower on the β-NGF ＋ PBS side than on the control side, and that of neurofilament 160 was greater. On day 14, β III-tubulin and protein gene product 9.5 were greater on the β-NGF ＋ PBS side than on the control side. Vascular endothelial growth factor expression was greater on the experimental side than the control side at 7 days, and vascular endothelial growth factor receptor 2 expression was elevated on days 14 and 21, but lower than control levels on day 28. However, no difference in the number of blood vessels was observed between sides. Our results indicate that topical application of β-NGF promoted neurogenesis, and may modulate angiogenesis by promoting nerve regeneration in collagen bone substitute-filled defects.

Buyanghuanwu decoction promotes angiogenesis after cerebral ischemia/reperfusion injury：mechanisms of brain tissue repair

Buyanghuanwu decoction has been shown to protect against cerebral ischemia/reperfusion injury,but the underlying mechanisms remain unclear.In this study,rats were intragastrically given Buyanghuanwu decoction,15 m L/kg,for 3 days.A rat model of cerebral ischemia/reperfusion injury was established by middle cerebral artery occlusion.In rats administered Buyanghuanwu decoction,infarct volume was reduced,serum vascular endothelial growth factor and integrin αvβ3 levels were increased,and brain tissue vascular endothelial growth factor and CD34 expression levels were increased compared with untreated animals.These effects of Buyanghuanwu decoction were partially suppressed by an angiogenesis inhibitor（administered through the lateral ventricle for 7 consecutive days）.These data suggest that Buyanghuanwu decoction promotes angiogenesis,improves cerebral circulation,and enhances brain tissue repair after cerebral ischemia/reperfusion injury.

Effect of Qi-tonifying and Stasis-eliminating Therapy(益气活血法)on Expression of Vascular Endothelial Growth Factor and Its Receptors FIt-l,FIk-1 in the Brain of Intracerebral Hemorrhagic Rats

Objective： To investigate the effects and mechanism of qi-tonifying and stasiseliminating （QTSE） therapy on the expression of vascular endothelial growth factor （VEGF） and its receptors Fit-1 and FIk-1 in the brains of intracerebral hemorrhagic （model） rats. Methods： One hundred and eighty Sprague-Dawley rats were randomly divided into six groups： the normal group （n=5）, the sham-operative （SO） group （n=35）, the model group （n=35）, the QTSE group （n=35）, the QT group （n=35） and the SE group （n=35）. All the rats except those in the normal group and SO group were established into an intracerebral hemorrhage（ICH） model by intracerebral injection of collagenase type Ⅶ and the latter three were orally administered with Buyang Huanwu Decoction （补阳还五汤, a classical recipe for QTSE） or with some of its components for qi-tonification and for stasis-elimination, respectively. To the other three groups, normal saline solutions were given instead. Behavioral tests were carried out in the animals randomly chosen from each group on days 1, 2, 4, 7, 14, 21 and 28 after modeling. The expressions of VEGF, FIk-1 and Fit-1 were determined by immunohistochemistry and the number of vascular segments with positive expression in the injured brain area of the rats was calculated. Results： From day 7 onwards, the asymmetric forelimb use rate in the QTSE group recovered more significantly than that in the other model groups. In the model group, the expressions of VEGF, FIk-1 and Fit-1 appeared on day 1 and reached a peak on day 21, then weakened gradually. In the QTSE group, as compared with the other model groups, a higher level of VEGF expression was shown from day 7 （P〈0.01） and a higher level of Fit-1 expression was shown from the 7th day to the 21st day （P〈0.01）. Conclusion： QTSE therapy can up-regulate the expressions of VEGF and its receptors （FIk-1 and Fit-l） and improve the recovery of kinetic function in the ICH rats, which may be correlated with its action in modulating vascular regeneration to promote the reconstruction of microvascular networks in the damaged areas.