| Objective:Many therapies,such as autografting and artificial bone substitutes have been used for the improvement and treatment of bone defects during the past years[1,2].However,complications,such as pain,morbidity,and functional disturbance,have hindered their clinical applications[3].Therefore,biotechnological and bone tissue engineering strategies have developed as potential and effective alternatives[4].Periosteum-derived cells?PDCs?have a strong potential for proliferation and osteogenic differentiation,and they are essential for the healing and repair of bone fractures and defects[5-7].Previous studies have shown that PDCs can repair a critical size long-bone defect in mice.In combination with porous scaffold,PDCs were able to treat a long bone defect in rabbits.PDCs possess a positive effect on increasing the bone volume of the rabbit model of skull defects.PDCs and its separated exocrine can enhance the proliferation,migration and osteogenic differentiation of BMSCs.PDCs embedded cells are used to wrap BMSCs and phosphorus tricalcium citrate can create a biomimetic bone graft substitute to enhance bone regeneration.PDCs can achieve good bone filling and new bone area in the bone defects of implant contact range in animal experiments[8-13].Angiogenesis and timely formation of blood vessels have also played important roles in the survival of implanted cells and successful bone regeneration.Therefore,an appropriate cell source for biotechnological and bone tissue engineering applications should have both osteogenic potential and proangiogenic characteristics[14,15].An experimental study has shown that PDCs can produce vascular endothelial growth factor?VEGF?and exhibit proangiogenic properties[16].As an important angiogenic factor,VEGF enhances endothelial cell proliferation,promotes cell survival under ischemic conditions,and accelerates angiogenesis and formation of the microvascular system[17].Furthermore,PDCs contain a greater number of mesenchymal stem cells compared with bone marrow stromal cells?BMSC?[18].Concentrated growth factor?CGF?is a new generation platelet concentrate,and the production process is completely natural without the addition of any biochemical agents[19].Therefore,CGF avoids immunoreactions,toxicity,cross-contamination,and ethical concerns.Moreover,CGF is composed of various growth factors and cytokines,such as transforming growth factor?TGF?,insulin-like growth factor?IGF?,platelet-derived growth factor?PDGF?,and VEGF[20,21].Cell proliferation and osteogenic differentiation are regulated by a variety of growth factors,including bone morphogenetic protein?BMP?,TGF,PDGF,IGF,core-binding factor?I,and epidermal growth factor?EGF?[22].These factors interact with each other in the process of proliferation and osteogenic differentiation,and a single factor is not sufficient to induce proliferation and osteogenesis.CGF is widely used in oral soft and hard tissue repair,implant,extraction site preservation and other related fields.Many clinical studies have shown that CGF can be used as a bone graft material for hydrodynamic piezoelectric internal sinus elevation,periodontal tissue and bone tissue repair,immediate implant and so on[23,24].Studies have also shown that CGF can increase bone formation[25]and differentiation of multiple cell lines such as apical papillary stem cells,bone marrow mesenchymal stem cells[16,26-28],as well as promoting Schwann cell proliferation and neurotrophic factor secretion[21].CGF and PDCs exhibit superior clinical and biotechnological application potential.Nevertheless,studies regarding the effects of CGF on the proliferation,osteogenic differentiation,and angiogenic potential of PDCs are scarce,and their underlying mechanisms are poorly understood.In this study we investigated the effects of CGF on the proliferation of rabbit PDCs and the expression of osteogenic differentiation markers(alkaline phosphatase?ALP?,BMP-2,type I collagen?Col-1?,osteocalcin?OCN?and angiogenesis markers?VEGF?,basic fibroblast growth factor?bFGF?in these cells in vitro.Methods:1.Rabbit PDCs were isolated and extracted by enzymatic digestion and tissue culture methods.The morphological features of rabbit PDCs were observed by inverted microscope.2.Flow cytometry was used to detect the phenotype of PDCs isolated from rabbits by enzymatic digestion and tissue culture methods.3.The proliferation of PDCs isolated from rabbits by enzymatic digestion and tissue culture methods was detected by CCK-8 method.4.Rabbit PDCs isolated by enzymatic digestion and tissue culture methods were induced to differentiate into osteogenesis,chondrogenesis and adipogenesis,and the corresponding indicators were detected.5.CCK-8 method was used to detect the effect of CGF on the proliferation of rabbit PDCs.6.Scanning electron microscopy?SEM?was used to observe the ultrastructure of CGF membrane and rabbit PDCs cultured with CGF membrane.7.Alkaline phosphatase?ALP?activity assay was used to detect the effect of CGF on the ALP activity of rabbit PDCs.8.Western blotting was used to detect the expression of osteogenic differentiation-related factors?BMP-2,Col-1,OCN?and angiogenesis-related factors?VEGF,bFGF?in rabbit PDCs.9.The expression of BMP-2,Col-1,OCN,VEGF and bFGF in rabbit PDCs was detected by qRT-PCR.10.The secretion of osteogenic differentiation-related factors?BMP-2,Col-1,OCN?and angiogenesis-related factors?VEGF,bFGF?in rabbit PDCs supernatant was detected by ELISA.Results:1.Morphological manifestations and changes of rabbit PDCsRabbit PDCs were irregular,spindle-shaped and triangular at the initial stage of culture under inverted microscope.With the increase of culture time,the cells gradually became wide and spindle-shaped.And then number of cells increased significantly,and the cells grew in a long spindle shape and a whirlpool shape.There was no significant difference in morphological characteristics between PDCs isolated by tissue culture and enzymatic digestion methods.2.Phenotype and surface antigen expression of rabbit PDCs isolated by enzymatic digestion and tissue culture methods were detected by flow cytometryFlow cytometry was used to analyze the cell phenotypes of P3 rabbit PDCs,which were CD34,CD45,CD105 and CD166,respectively.The cell phenotypes of rabbit PDCs obtained by tissue culture method and enzymatic digestion method were CD34 and CD45negative,CD105 and CD 166 positive.There was no significant difference in the phenotype of rabbit PDCs obtained by tissue culture and enzymatic digestion methods?P all<0.05?.3.Proliferation of PDCs isolated from rabbits by enzymatic digestion and tissue culture methodsCCK-8 results showed that the proliferative ability of PDCs extracted by the two methods increased gradually with the increase of culture time,and increased significantly on the day 3 and 5 after culture.At 7 and 9 days,PDCs proliferated slightly slowly and entered the relative plateau stage,and then showed an upward trend of proliferation.At each time point,there was no significant difference in cell proliferation between tissue culture and enzyme digestion methods for isolation and extraction of PDCs?P all>0.05?.4.Isolation and extraction of rabbit PDCs by enzymatic digestion and tissue culture methods for osteogenesis,chondrogenesis and adipogenic differentiation and detection?1?Osteogenic induced differentiation resultsAt 7 days of induction,alizarin red S staining showed that a large number of orange and orange granules with different sizes were distributed in PDCs,indicating that they chelated with calcium salts to form complex?calcium salts existed in PDCs?.The levels of OSX and OCN mRNA in rabbit PDCs increased significantly.There was no significant difference between tissue culture and enzymatic digestion methods?P>0.05?.?2?Chondrogenesis induced differentiation resultsThe results of qRT-PCR showed that the levels of Col2-a1 and Acan mRNA in rabbit PDCs increased significantly.There was no significant difference between tissue culture and enzymatic digestion methods?P>0.05?.?3?Adipogenic induced differentiation resultsAfter 3 days of induction,the morphology of rabbit PDCs cells gradually changed from spindle cells to polygonal and round-like cells,and there were bright,highly refractive and compartmentalized lipid droplets in the cytoplasm.The number and volume of lipid droplets increased with the prolongation of induction time and fused with each other.Oil red O staining at 7 days of induction showed a large amount of red lipid deposition.The levels of Pparg and Fabp4 in rabbit PDCs increased significantly.There was no significant difference between tissue culture and enzymatic digestion methods?P>0.05?.5.Effects of CGF on the proliferation of rabbit PDCsCCK-8 results showed that the proliferative ability of PDCs in control group,1×CGF and 2×CGF group increased with the prolongation of culture time?from the3days to the 21 days?.On day 3-21,the proliferation rate of PDCs in 1×CGF and 2×CGF group was significantly higher than that in control group,and 2×CGF group was significantly higher than 1×CGF group,the difference was statistically significant?P<0.05?.6.The ultrastructure of CGF membrane,PDCs seeded onto CGF membrane were observed by SEMThe surface ultrastructure of CGF,PDCs seeded onto CGF membranes was analyzed by SEM.Dense porous three-dimensional fibrin network composed of thin and thick fibrin elements was observed in CGF membranes.When PDCs were seeded onto CGF membranes,the CGF membranes were completely covered by PDCs,with slender or polygonal morphological characteristics,and many platelet-like cell components could be observed.7.Effects of CGF on protein,mRNA and secretion of osteogenic differentiation related factors in rabbit PDCs?1?ALP activity resultsIn 1×CGF group,the ALP level on the 21 days was higher than that on the 3,7 and14 days,and the ALP activity on the 14 days was higher than that on the 3 and 7 days.Compared with the control group,ALP activity in 1×CGF and 2×CGF group increased significantly on the 3,7,14 and 21 days?P<005?.2×CGF group significantly higher than1×CGF group on the 7,14 and 21 days?P<0.05?.?2?Western blotting test resultsWestern blotting results showed that the relative protein levels of BMP-2,Col-1 and OCN in 1×CGF and 2×CGF group were significantly higher than those in control group on the 3,7,14 and 21 days,and there was a significant difference between the three groups on the 3,7,14 and 21 days?P<0.05?.?3?Real Time PCR resultsThe results of qRT-PCR showed that the mRNA expression of BMP-2,Col-1 and OCN in 1×CGF and 2×CGF group increased significantly on the 3,7,14 and 21 days compared with the control group,which was consistent with the trend of the changes of Western blotting indices over time.On the 3,7,14 and 21 days,there was a significant difference between the three groups?P<0.05?.?4?ELISA resultsWe further examined the concentration of Col-1,OCN and BMP-2 in the cell supernatant at different time points after CGF membrane treatment.ELISA results showed that the levels of Col-1 and OCN in CGF membrane were not detected during the experiment,while BMP-2 showed a slow release,which was higher on the 14 days.At the same time point,PDCs+2×CGF group higher than PDCs+1×CGF group and PDCs control group?P<0.05?.8.Effects of CGF on vascular related factors protein,mRNA and secretion level of rabbit PDCs?1?CD34 and VEGF immunohistochemical resultsThe positive expression of CD34 was yellow or brown granular or tubular structure.Immunohistochemical staining showed that PDCs were scattered on the surface of CGF membrane and fibrin network,CD34 positive cells and VEGF positive expression were scattered in the network of CGF membrane.?2?Western blotting test resultsWestern blotting results showed that the relative protein levels of VEGF and bFGF in 1×CGF and 2×CGF group were significantly higher than those in control group?P<0.05?.At the 3,7,14 and 21 days,there was a significant difference between the1×CGF and 2×CGF groups?P<0.05?.?3?Real Time PCR resultsThe results of qRT-PCR showed the mRNA expression of VEGF and bFGF in1×CGF and 2×CGF group increased significantly at the 3,7,14 and 21 days compared with the control group?P<0.05?.VEGF mRNA expression in 2×CGF group higher than1×CGF group at 7,14 and 21 days?P<0.05?.bFGF mRNA expression in 2×CGF group higher than 1×CGF group at each time point?P<0.05?.?4?ELISA test resultsELISA results showed that the release of VEGF and bFGF in CGF membrane was slow.2×CGF group was higher than 1×CGF group at each time point?P<0.05?.At same time point,PDCs+2×CGF group and PDCs+1×CGF group higher than that in control group?P<0.05?.Conclusion:1.Rabbit PDCs can be isolated and extracted by tissue culture and enzymatic digestion methods in vitro,which can provide a more stable source of PDCs for tissue engineering research and application related fields.Meanwhile,the phenotype of rabbit PDCs and the ability of three-line differentiation in vitro prove that they have the characteristics of mesenchymal stem cells and the potential of multi-line differentiation of mesenchymal stem cells.2.Three-dimensional fibrin network in CGF is conducive to cell growth and extension.CGF can promote PDCs proliferation,up-regulate ALP activity and promote BMP-2,Col-1,OCN cell expression and secretion of related factors in vitro,thereby promoting PDCs osteogenic differentiation rabbits.3.CGF conditioned medium can promote the expression of VEGF,bFGF protein and mRNA in rabbit PDCs,and CGF membrane fibrin network contains CD34 positive cells and upregulates the secretion of VEGF and bFGF in the culture microenvironment,which provides a new avenue for the bone tissue engineering neovascularization field. |
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