ObjectiveTo review the research progress of endogenous repair strategy (ERS) in intervertebral disc (IVD).MethodsThe domestic and foreign literature related to ERS in IVD in recent years was reviewed, and its characteristics, status, and prospect in the future were summarized.ResultsThe key of ERS in IVD is to improve the vitality of stem/progenitor cells in IVD or promote its migration from stem cell Niche to the tissue that need to repair. These stem/progenitor cells in IVD are derived from nucleus pulposus, annulus fibrosus, and cartilaginous endplate, showing similar biological characteristics to mesenchymal stem cells including the expression of the specific stem/progenitor cell surface markers and gene, and also the capacity of multiple differentiations potential. However, the development, senescence, and degeneration of IVD have consumed these stem/progenitor cells, and the harsh internal microenvironment further impair their biological characteristics, which leads to the failure of endogenous repair in IVD. At present, relevant research mainly focuses on improving the biological characteristics of endogenous stem/progenitor cells, directly supplementing endogenous stem/progenitor cells, biomaterials and small molecule compounds to stimulate the endogenous repair in IVD, so as to improve the effect of endogenous repair.ConclusionAt present, ERS has gotten some achievements in the treatment of IVD degeneration, but its related studies are still in the pre-clinical stage. So further studies regarding ERS should be carried out in the future, especially in vivo experiments and clinical transformation.
ObjectiveTo review the research progress of the co-culture system for constructing vascularized tissue engineered bone. MethodsThe recent literature concerning the co-culture system for constructing vascularized tissue engineered bone was reviewed, including the selection of osteogenic and endothelial lineages, the design and surface modification of scaffolds, the models and dimensions of the co-culture system, the mechanism, the culture conditions, and their application progress. ResultsThe construction of vascularized tissue engineered bone is the prerequisite for their survival and further clinical application in vivo. Mesenchymal stem cells (owning the excellent osteogenic potential) and endothelial progenitor cells (capable of directional differentiation into endothelial cell) are considered as attractive cell types for the co-culture system to construct vascularized tissue engineered bone. The culture conditions need to be further optimized. Furthermore, how to achieve the clinical goals of minimal invasion and autologous transplantation also need to be further studied. ConclusionThe strategy of the co-culture system for constructing vascularized tissue engineered bone would have a very broad prospects for clinical application in future.
Abstract： Objective To investigate and improve the method of isolation, induction and culture, amplification in vitro of human endothelial progenitor cells (EPCs) in human bone marrow, thus establish a foundation for EPCs to participate in basic research and clinical application. Methods WHuman bone marrow mononuclear cells (hBMMNCs) were isolated by density gradient centrifugation and cultured in the 6plateds coating human fibronections(HFN group), coating gelatinum (coating gelatinum group) and coating nothing (coating nothing group) respectively. After culturing for 4-7d endothelial cell basal medium-2(EBM-2) cell colonyforming units (CFUs) appeared, then select EPCsliked CFUs for cultivation which was named the pick method, CD34+ KDR+ and CD133+ KDR+ double positive cells were detected in flow cytometry, and CD133, CD34, CD31, vWF and KDR expression were detected with cell immunochemical test. Results hBMMNCs were isolated from human bone marrow more effectively with OptiprepTM cell separating medium, and induced and obtained more EPCs, and cultured and amplificated in vitro. Flow cytometer showed CD133+ KDR+ double positive cells reaching up to 70.4%±5.4%, CD34+ KDR+ double positive cells reaching up to 69.1%±8.7%. EPCs grew vigorously in coating HFN group and coating gelatinum group, both HFN and gelatinum promote EPCs adherence and growth, but there were no statistically difference in two groups (Pgt;0.05).Surface mark of adherent cells cultured 7d such as CD133, CD31, vWF and KDR showed positive, and cells cultured 14d such as CD34 showed positive. Conclusion The method of picking CFUs can obtain more EPCs from human bone marrow with success and can amplificate EPCs in vitro, thus introducing another simple and effective method to purify EPCs, further widening range and increasing method to purify EPCs.
ObjectiveTo observe the effects of aquaporin 1 (AQP1) on the proliferation and migration of endothelial progenitor-endothelial progenitor cells (EPC).MethodsBone marrow cells of AQP1 wild-type (WT) (n=6) and knockout-type (KO) mice (n=6) were isolated and differentiated into EPC in vitro. Immunofluorescence was used to detect cell surface antigens to identify EPC. Live cell kinetic imaging and quantification technology, transwell migration assays, as well as scratch test were used to compare the function of EPC between AQP1 WT and KO mice.ResultsEPC culture showed that cells were initially suspended and gradually adhered to typical mesenchymal stem cells within 7 days. After cultured on special medium for endothelial cells they were adhered and differentiated, and fusiform or polygonal, paving stone-like EPC were observed around 14 days. When cultured by special medium of EPC, CD133 and CD31 were positively detected after 7 days, and CD34 and Flk-1 were positively detected after 14 days. Positive expression of AQP1 was only detected in EPC of AQP1 WT mice. Functional studies of EPC revealed there was no significant difference in the proliferation of EPC between AQP1 WT and KO group mice. Transwell assay showed that EPC migration ability of AQP1 KO mice was significantly weaker than that of WT mice. The scratch healing ability of EPC in AQP1 KO mice was significantly lower than that of WT mice.ConclusionsEPC initially shows the characteristics of stem cells and with the prolongation of culture time, EPC gradually shows the characteristics of endothelial cells. AQP1 affects the EPC migration rather than proliferation.
Objective To observe endothelial progenitor cells (EPCs) participating in the formation of neovascularization in lung adenocarcinoma. Methods EPCs were transfected by recombinant adenovirus carrying LacZ gene in optimal transfection concentration, and then EPCs were injected into animal models of lung adenocarcinoma through the tail vein; afterwards, lung tissues were taken out for pathological examination in the 6th, 7th, 8th week respectively. EPCs were observed to take part in the angiogenesis in the lung adenocarcinoma through X-gal chromogenic dye. Results The optimal multiplicity of infection (MOI) of AD5F35LacZ transfected EPCs was 400. When MOI was 400, maximum transfection efficiency was 97.13±2.08. After 2 weeks, LacZ gene-transfected EPCs began to proliferate in vitro culture, then the EPCs were transplanted into animal models of lung cancer to be involved in the neovascularization formation in the 8th week after transplantation. Conclusion EPCs are involved in the formation of tumor neovascularization after transplantation.
Endometriosis (EM) is a common benign gynecological disease with complex pathogenesis and lack of unified understanding. In recent years, the theory of stem/progenitor cells has gradually been recognized by scholars. The presence of stem/progenitor cells in the endometrium and researchers’ understanding of stem/progenitor cell specific markers has been further developed, which is of great significance for sorting stem/progenitor cells and further elucidating their roles in the pathogenesis of EM. At present, more endometrial stem cell signaling pathways have been studied including Wnt, Hedgehog, Notch, phosphatidylinositol-3-kinase/protein kinase B, Smad/connective tissue growth factor, CXCL12/CXCR4, etc. These signaling pathways can regulate stem cell involvement in the pathogenesis of EM. Exploring how signaling pathways to regulate stem cell involvement in the pathogenesis of EM can help elucidate the specific pathogenesis of EM and provide new directions for its treatment. This paper will summarize them.
Objective To establish the three diamension-model and to observe the contribution of endothelial progenitor cell (EPC) in the angiogenesis and its biological features. MethodsEPC was obtained from the rats’ peripheral blood. Its cultivation and amplification in vitro were observed, and the function of the cultural EPC in vitro was detected. The three diamension-model was established and analyzed. ResultsEPC was obtained from the peripheral blood successfully. The proliferation of the EPC which induced with VEGF(experimental group) was better than that without VEGF （control group） at every different phase (P＜0.01). It was found that EPC grew into collagen-material from up and down in the three diamension-model, and its pullulation and infiltration into the collagen were seen on day 1 after cultivation. With the time flying, there were branch-like constructions which were vertical to the undersurface of collagen and interlaced to net each other. It showed that in experimental group the EPC grew fast, its infiltration and pullulation also were fast, the branch-like construction was thick. But in control group, the EPC grew slowly, infiltration and pullulation were slow, the branch-like construction was tiny and the depth of infiltration into collagen was superficial. The number of new vessels in experimental group was larger than that in the control group at every different phase (P＜0.01). ConclusionRat tail collagen can induce EPC involved in immigration, proliferation and pullulation in angiogenesis. The three-diamension model of EPC can be used to angiogenesis research. VEGF can mobilize and induce EPC to promote the angiogenesis.
Objective To investigate the effects of titanium modified by ultrasonic acid etching/anodic oxidation (UAT) loaded with endothelial progenitor cells-exosome (EPCs-exo) on proliferation and osteogenic and angiogenic differentiations of adipose-derived stem cells (ADSCs). Methods The adipose tissue and bone marrow of 10 Sprague Dawley rats were harvested. Then the ADSCs and EPCs were isolated and cultured by collagenase digestion method and density gradient centrifugation method, respectively, and identified by flow cytometry. Exo was extracted from the 3rd to 5th generation EPCs using extraction kit, and CD9 and CD81 were detected by Western blot for identification. The three-dimensional printed titanium was modified by ultrasonic acid etching and anodic oxidation to prepare the UAT. The surface characteristics of UAT before and after modification was observed by scanning electron microscopy; UAT was placed in EPCs-exo solutions of different concentrations (100, 200 ng/mL), and the in vitro absorption and release capacity of EPCs-exo was detected by BCA method. Then, UAT was placed in DMEM medium containing different concentrations of EPCs-exo (0, 100, 200 ng/mL), and co-cultured with the 3rd generation ADSCs to construct UAT-ADSCs-exo. Cell morphology by laser confocal microscopy, live/dead cell staining, and cell proliferation were observed to evaluate biocompatibility; alkaline phosphatase (ALP) staining and alizarin red staining, RT-PCR detection of osteogenesis-related genes [osteocalcin (OCN), RUNT-related transcription factor 2 (Runx2), ALP, collagen type 1 (COL-1)] and angiogenesis-related gene [vascular endothelial growth factor (VEGF)], immunofluorescence staining for osteogenesis (OCN)- and angiogenesis (VEGF)-related protein expression were detected to evaluate the effect on the osteogenic and angiogenic differentiation ability of ADSCs. Results Scanning electron microscopy showed that micro-nano multilevel composite structures were formed on the surface of UAT. About 77% EPCs-exo was absorbed by UAT within 48 hours, while EPCs-exo absorbed on the surface of UAT showed continuous and stable release within 8 days. The absorption and release amount of 200 ng/mL group were significantly higher than those of 100 ng/mL group (P<0.05). Biocompatibility test showed that the cells in all concentration groups grew well after culture, and the 200 ng/mL group was better than the other groups, with fully spread cells and abundant pseudopodia, and the cell count and cell activity were significantly higher than those in the other groups (P<0.05). Compared with the other groups, 200 ng/mL group showed enhanced ALP activity and mineralization ability, increased expressions of osteogenic and angiogenic genes (OCN, Runx2, COL-1, ALP, and VEGF), as well as increased expressions of OCN and VEGF proteins, with significant differences (P<0.05). Conclusion EPCs-exo can effectively promote the adhesion, proliferation, and osteogenic and angiogenic differentiation of ADSCs on UAT surface, the effect is the most significant when the concentration is 200 ng/mL.
Objective To examine the effect of endothelial progenitor cell (EPC) on lung ischemia-reperfusion injury (LIRI). Methods Twenty-four recipients were randomized into 3 groups including a sham group, a LIRI group, and an EPC group. Rats in the sham group only received anesthesia. Rats in the LIRI and EPC groups received left lung transplantation and received saline or EPC immediately after reperfusion. The partial pressure of oxygen to fraction of inspiratory oxygen (PaO2/FiO2) ratio, wet-to-dry weight ratio and protein levels in the transplanted lung and inflammation-related factors levels in serum were examined. Histological change of transplanted lung were analyzed. The nuclear factor (NF)-κB in the transplanted lung was detected. Results Compared with the LIRI group, the PaO2/FiO2 ratio dramaticly increased, and the wet-to-dry weight ratio and protein level significantly decreased by EPC after reperfusion. The lung histological injury was attenuated by EPC. The pro-inflammatory factors in serum were down-regulated, whereas IL-10 was up-regulated in the EPC group. The expression of NF-κB was decreased by EPC. Conclusion EPC ameliorated LIRI after lung transplantation. The protection of EPC partly associated with anti-inflammation.
ObjectiveTo investigate the ability of autologous peripheral blood endothelial progenitor cells (EPCs) in promoting neovascularization of tissue engineered bone and osteogenesis of bone marrow mesenchymal stem cells (BMSCs). MethodThe peripheral blood EPCs and BMSCs from No. 1-9 New Zealand rabbits were isolated, cultured, and identified. According to the cell types, the third generation of cells were divided into 3 groups:EPCs (group A), BMSCs (group B), and co-cultured cells of EPCs and BMSCs (group C, EPCs:BMSCs=1:2) . Then cells were seeded on the partially deproteinised bone (PDPB) packaged with fibronectin to construct tissue engineered bone. After 4 days, autologous heterotopic transplantation of tissue engineered bone was performed in the rabbit's muscles bag of groups A, B, and C (the right arm, left arm, right lower limb respectively, 2 pieces each part). At 2, 4, and 8 weeks after transplantation, the growth of tissue engineered bone was observed, and the rate of bone ingrowth was calculated by HE staining; the expressions of CD34, CD105, and zonula occludens protein 1(ZO-1) were compared by immunohistochemical staining at each time point in tissue engineered bone among 3 groups. ResultsThe EPCs and BMSCs were isolated and identified successfully; immunofluorescent staining showed that EPCs were positive for CD34, CD133, and von Willebrand factor (vWF), and BMSCs were positive for CD29 and CD90 and were negative for CD34. The tissue engineered bone constructed in 3 groups was transplanted successfully. At 2, 4, and 8 weeks after autologous heterotopic transplantation, the general observations showed that the soft tissue around the tissue engineered bone increased and thickened gradually in each group with time passing; the boundary between bone and soft tissue was not clear; the pore space of tissue engineered bone gradually was filled, especially in group C, the circuitous vascular network could be seen in the tissue engineered bone. HE staining showed capillaries and collagen fibers increased gradually, tissue engineered bone ingrowth rate was significantly higher in group C than groups A and B at 4 and 8 weeks (P<0.05) , and group B was significantly higher than group A (P<0.05) . Immunohistochemical staining showed that the expressions of CD34, CD105, and ZO-1 in tissue engineered bone of 3 groups all increased with the extension of time, showing significant differences between groups at each time point (P<0.05) . At 2 weeks after transplantation, the expression of CD105 in group C was significantly higher than that in groups A and B (P<0.05) ; at 4 and 8 weeks, CD34, CD105, and ZO-1 expressions showed significant differences between 2 groups (P<0.05) ; the expression was the highest in group C, and was the lowest in group B. ConclusionsAutologous peripheral blood EPCs and BMSCs have synergistic effect, and can promote neovascularization and osteogenesis of tissue engineered bone in vivo.