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.
Objective To determine the transfection efficiency of recombinant adenovirus to endothelial progenitor cells(EPCs) and provide the base of lung cancer therapy by transfecting human herpes simplex virusthymidine kinase（HSV-TK） gene to EPCs. Methods Admove recombinant adenovirus 5F35(AD5F35) which transfected with βgalactosidase(AD5F35LacZ) to the 24 well plate cultivated with EPCs and transfect the EPCs. Stain the EPCs with LacZ kit and calculate the transfection efficiency. Results The blue stain cells were cells transfected successfully with AD5F35LacZ under the optical microscope. The transfection efficiencies of adenovirus to EPCs were different under the premise of the different multiplicity of infection(MOI). In a certain range, the transfection efficiencies rise with the MOI rise. When MOI was 400,the proportion of blue stain cell is the highest, which was 98.38%±1.25%. Conclusion Recombinant adenovirus can transfect EPCs successfully. The transfection efficiencies rise with the MOI rise. When the MOI is 400,the transfection efficiency is the highest.
【摘要】 目的 比较密度梯度离心法及全骨髓培养法分离培养内皮祖细胞的差异。方法 取4周雄性近交系C57BL/6J小鼠骨髓,分别使用密度梯度离心法及全骨髓培养法培养,观察细胞贴壁情况和细胞形态,并行DiIacLDL及FITCUEAI双染、vWF、eNOS及细胞表面标志检测。结果 密度梯度离心法培养细胞可形成典型铺路石样改变及形成血管样结构;而全骨髓培养法贴壁细胞形态多样,较多呈长梭形铺展生长,部分细胞呈类圆形及纺锤形。比较两种方法培养细胞摄取DiIacLDL、结合FITCUEAI双阳性率以及vWF、eNOS及细胞表面标志表达阳性率,差别均有统计学意义(Plt;005)。应用密度梯度离心法,随着培养时间延长,表达CD34、CD133及FLk1细胞逐渐增多(Plt;005)。结论 密度梯度离心法及全骨髓培养法在EGM2MV培养体系下均可培养出内皮祖细胞,但密度梯度离心法较全骨髓培养法培养的内皮祖细胞纯度高。
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 compare canine decel luarized venous valve stent combining endothel ial progenitor cells (EPC) with native venous valve in terms of venous valve closure mechanism in normal physiological conditions. Methods Thirty-six male hybrid dogs weighing 15-18 kg were used. The left femoral vein with valve from 12 dogs was harvested to prepare decelluarized valved venous stent combined with EPC. The rest 24 dogs were randomly divided into the experimental group and the control group (n=12 per group). In the experimental group, EPC obtained from the bone marrowthrough in vitro ampl ification were cultured, the cells at passage 3 (5 × 106 cells/mL) were seeded on the stent, and the general and HE staining observations were performed before and after the seeding of the cells. In the experimental group, allogenic decelluarized valved venous stent combined with EPC was transplanted to the left femoral vein region, while in the control group, the autogenous vein venous valve was implanted in situ. Color Doppler Ultrasound exam was performed 4 weeks after transplantation to compare the direction and velocity of blood flow in the distal and proximal end of the valve, and the changes of vein diameter in the valve sinus before and after the closure of venous valve when the dogs changed from supine position to reverse trendelenburg position. Results General and HE staining observations before and after cell seeding: the decelluarized valved venous stent maintained its fiber and collagen structure, and the EPC were planted on the decelluarized stent successfully through bioreactor. During the period from the reverse trendelenburg position to the starting point for the closure of the valve, the reverse flow of blood occurred in the experimental group with the velocity of (1.4 ± 0.3) cm/s; while in the control group, there was no reverse flow of blood, but the peak flow rate was decreased from (21.3 ± 2.1) cm/s to (18.2 ± 3.3) cm/s. In the control group, the active period of valve, the starting point for the closure of the valve, and the time between the beginning of closure and the complete closure was (918 ± 46), (712 ± 48), and (154 ± 29) ms, respectively; while in the experimental group, it was (989 ± 53), (785 ± 43), and (223 ± 29) ms, respectively. There was significant difference between two groups (P lt; 0.05).After the complete closure of valve, no reverse flow of blood occurred in two groups. The vein diameter in the valve sinus of the experimental and the control group after the valve closure was increased by 116.8% ± 2.0% and 118.5% ± 2.2%, respectively, when compared with the value before valve closure (P gt; 0.05). Conclusion Canine decelluarized venous valve stent combined with EPC is remarkably different from natural venous valve in terms of the valve closure mechanism in physiological condition. The former rel ies on the reverse flow of blood and the latter is related to the decreased velocity of blood flow and the increased pressure of vein in the venous sinus segment.
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.
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 observe the adhesion and prol iferation of late endothel ial progenitor cells (EPCs) planted on nanoporous PLLA scaffold in vitro and to provide a new approach that optimizes tissue engineered material. Methods Male and female New Zealand rabbits (weight 2.5-3.0 kg) were used. Isolated late EPCs from rabbit peri pheral blood were cultured. Electrostatic spinning technique was adopted to prepare misal igned nanofibers, al igned nanofibers and super-al igned nanofibers, and low temperature plasma technique was appl ied to prepare misal igned membrane, al igned membrane and super-al igned membrane. After being divided into group A (cells only), B (misal igned membrane), C (normal membrane), D (al igned membrane) and E (super-al igned membrane), the primary late EPCs (1 × 105/mL) werecultured on scaffolds and MTT method was used to detect cell prol iferation abil ity at 3, 5, 7, 9, 11, 13, 15 and 17 days afterculture. After being divided into group A (misal igned membrane), B (normal membrane), C (al igned membrane) and D (superal igned membrane), precipitation method was appl ied to detect cell adhesion rate at 4, 12 and 24 hours after compound culture, and the morphologic changes of cells were observed at 4, 24 and 72 hours after compound culture. Results Fiber diameters in nanofibrous PLLA scaffolds were 300-400 nm, with a porosity rate of above 90%. At 3, 5, 7, 9, 11, 13, 15 and 17 days after culture, A value of each group was increased with time and the cells in each group grew well, showing there was no significant difference between group A and group B at each time point (P gt; 0.05 ); during the period of 7-15 days after culture, the difference between groups C, D and E and groups A and B was significant (P lt; 0.05). At 4 hours after compound culture, the adhesion rate of group A was superior to that of groups B, C and D (P lt; 0.05); at 12 and 24 hours after compound culture, the adhesion rate of groups B, C and D was remarkably higher than that of group A (P lt; 0.05); significant difference was noted in each group between the time point of 4 hours and the time point of 12 and 24 hours after compound culture (P lt; 0.05), but no significant difference between 12 hours and 24 hours was detected (P gt; 0.05). Morphology observation demonstrated that cells grew well on the scaffolds, the cells in groups A and B grew sporadically and disorderly, while the cells in groups C and D attached and al igned along fiber and prol iferated, with an excretion of ECM. Group D was better at maintaining cell morphology. Conclusion Al igned and superal igned nanofibers of PLLA scaffold can promote the adhesion and prol iferation of seed cells on the scaffold and maintain good cell morphology, which is an appropriate candidate scaffold material for blood vessel tissue engineering. Late EPCs is an ideal cell source for blood vessel tissue engineering.
ObjectiveTo evaluate the biological effect on vascularization during bone repair of prevascularized porous β-tricalcium phosphate (β-TCP) tissue engineered bone (hereinafter referred to as prevascularized tissue engineered bone), which was established by co-culture of endothelial progenitor cells (EPCs) and bone marrow mesenchymal stem cells (BMSCs) based on tissue engineering technology. Methods EPCs and BMSCs were isolated from iliac bone marrow of New Zealand white rabbits by density gradient centrifugation and differential adhesion method. The cells were identified by immunophenotypic detection, BMSCs-induced differentiation, and EPCs phagocytosis. After identification, the third-generation cells were selected for subsequent experiments. First, in vitro tubule formation in EPCs/BMSCs direct contact co-culture (EPCs/BMSCs group) was detected by Matrigel tubule formation assay and single EPCs (EPCs group) as control. Then, the prevascularized tissue engineered bone were established by co-culture of EPCs/BMSCs in porous β-TCP scaffolds for 7 days (EPCs/BMSCs group), taking EPCs in porous β-TCP scaffolds as a control (EPCs group). Scanning electron microscopy and laser scanning confocal microscopy were used to observe the adhesion, proliferation, and tube formation of cells. Femoral condyle defect models of 12 New Zealand white rabbits were used for implantation of prevascularized tissue engineered bone as the experimental group (n=6) and porous β-TCP scaffold as the control group (n=6). The process of vascularization of β-TCP scaffolds were observed. The numbers, diameter, and area fraction of neovascularization were quantitatively evaluated by Microfill perfusion, Micro-CT scanning, and vascular imaging under fluorescence at 4 and 8 weeks. ResultsThe isolated cells were BMSCs and EPCs through identification. EPCs/BMSCs co-culture gradually formed tubular structure. The number of tubules and branches, and the total length of tubules formed in the EPCs/BMSCs group were significantly more than those in the EPCs group on Matrigel (P<0.05) after 6 hours. After implanting and culturing in porous β-TCP scaffold for 7 days, EPCs formed cell membrane structure and attached to the material in EPCs group, and the cells attached more tightly, cell layers were thicker, the number of cells and the formation of tubular structures were significantly more in the EPCs/BMSCs group than in the EPCs group. At 4 weeks after implantation, neovascularization was observed in both groups. At 8 weeks, remodeling of neovascularization occurred in both groups. The number, diameter, and area fraction of neovascularization in the experimental group were higher than those in the control group (P<0.05), except for area fraction at 4 weeks after implantation (P>0.05). ConclusionThe prevascularized tissue engineered bone based on direct contact co-culture of BMSCs and EPCs can significantly promote the early vascularization process during bone defects repair.