S-PE/Wnt3a Loaded Deformable Hydrogel For In Situ Recruitment Of Stem Cells To Efficiently Repair Bone Defects Via The Asymmetric Division

ObjectiveBone defect diseases caused by various reasons are increasing year by year,which affected the living quality of human seriously.Autologous bone grafting is still considered the gold standard in clinical practice,but the puzzle of insufficient donor resources is difficult to address.Moreover,the invasive operations in the transplantation process usually bring irreparable damage to patients.With this regard,tissue engineering based on biomaterials has become a hot topic in the field of bone repair,because the intelligent platform can be built through the reasonable combination of functional materials so as to achieve efficient bone defect repair.Taking the aforementioned pivotal issues into account,it is of great application potential to provide a central platform acts as "lodestone" for the active recruitment and immobilization of bone-related stem cells in the defect area.This strategy will bring new opportunities for clinical bone defect treatment,since it can avoid the damage of invasive surgery depend on endogenous stem cell therapy.In this study,we intend to prepare a novel 3D hydrogel with good biocompatibility,in situ recruitment of stem cells and maintenance of cell proliferation and differentiation balance,thus verify the functional characteristics of hydrogel in this case.We hope this work can open a new avenue for clinical rapid repair of bone defects.Methods1.The SDF-1 analog of small peptide(S-PE)has been screened and synthesized previously,and the cytotoxicity of S-PE against rat BMSCs was detected using the CCK-8 method.The proliferation effect of S-PE on rat BMSCs was determined by EDU staining and the cystain DNA,respectively,the effect of S-PE on rat BMSCs was detected using Annexin V-FITC as well.Western blot was used to evaluate the effect of S-PE on CXCR4 expression and its role in the Wnt/β-catenin signaling pathway of BMSCs,so the expression level of osteogenic differentiation markers OPG and OSX was examined.Furthermore,Alizarin red staining was used to evaluate the effect of SPE on the osteogenic differentiation of rat BMSCs,and Transwell array was performed to assess the effect of S-PE on cell migration of rat BMSCs.2.S-PE was functionalized grafted onto chitosan(CS)using amidation reaction,and the synthesized products were characterized using Fourier infrared transform spectroscopy(FT-IR),ultraviolet-visible(UV-vis),and nuclear magnetic resonance hydrogen(1H NMR)spectroscopy.The effective dosage concentrations of CS and CSPE were identified through the CCK-8 method.The Live/Dead staining was employed to detect the effect of CS-PE on BMSCs proliferation,and the effect of CS-PE on the induction of rat BMSCs migration was verified using Transwell experiment.3.CS was mixed with different concentrations of glycerophosphate(GP)in proportion,and the optimal GP concentration was determined according to the gelation time and pH value.CS,CS-PE,GP,type Ⅰ collagen,and Wnt3a were mixed in various proportions to prepare the CS/GP hydrogel,CS-PE/GP hydrogel,and CS-PE/GP/Wnt3a hydrogel,respectively.The gelation time and pH value were recorded,hydrogel morphological characterization was performed using scanning electron microscopy(SEM).4.Live/Dead staining was carried out to evaluate the effect of CS-PE/GP/Wnt3a hydrogels on the toxicity and proliferation of rat BMSCs,and Transwell was used to verify the profile of CS-PE/GP/Wnt3a hydrogels on the induction of rat BMSC migration.Rat BMSCs were incubated into the Wnt3a-containing platform with type Ⅰcollagen derived from rat tail,CS-PE/GP hydrogels,and CS-PE/GP/Wnt3a hydrogels,respectively,then the samples were underwent an process of osteogenic differentiation for 5-7 days.Immunofluorescence was used to detect aPKCζ,Numb,and OPN expression so as to assess asymmetric cell division(ACD)in the hydrogels.5.The hydrogels were subcutaneously injected into mice,and the materials were obtained after 7 days prior to prepared into single-cell suspensions.The single-cell mouse suspensions were labeled with CD45,F4/80,and CD206,and macrophage infiltration and polarization in the hydrogels were analyzed using flow cytometry.The expression of M1 macrophage marker Nos2 and M2 macrophage marker Arg1 in tissues were detected using immunofluorescence staining.6.As mentioned above,the hydrogels were injected subcutaneously into rats and mice,respectively,then the single-cell suspensions were prepared after 7 days.The samples were labeled with CD44,CD45,CD29,and CD90 for rat cells and CD45,CD54,and CD90 for mouse cells.The proportion of BMSCs recruited in the hydrogels was analyzed using flow cytometry,and tissue stem cell markers were determined using immunofluorescence of CD54 expression.7.A rat critical cranial defect model was established to evaluate the ability of hydrogels to recruit endogenous BMSCs and accelerate bone regeneration in vivo,and the expression of ALT,AST,TBIL,yGTT,ALP,BUN,and CREA in serum was measured using a biochemical analyzer 8 weeks after surgery.HE staining pathology was performed on major tissues and organs(heart,liver,spleen,lung,and kidney);and the effect of bone repair was evaluated using micro-CT.The expression level of the M2 macrophage marker Argl and the new bone marker SOST in the cranial defect area were measured using immunofluorescence staining.Results1.Primary BMSCs were successfully obtained and identified.CCK-8 results identified their maximum cell viability at 100 ng/mL for SDF-1 and 0.4 mM for S-PE,and the EDU results showed that SDF-1 and S-PE promoted cell proliferation(P<0.05).Cell cycle and apoptosis results showed that SDF-1 and S-PE did not block the cell cycle or promote apoptosis.Compared with SDF-1,S-PE incurred BMSCs to express high levels of CXCR4,p-GSK3β,and β-catenin;Immunofluorescence results showed that S-PE promoted β-catenin transfer into the nucleus,and increased expression levels of OPG and OSX(P<0.05)was observed from western blot.Alizarin red staining showed that more mineralized nodules were observed within cells in the group with SPE,which revealed a significant migration-inducing effect of S-PE on rat BMSCs(P<0.05).2.S-PE was grafted onto CS via an amidation approach,and the successful synthesis of CS-PE was confirmed using FT-IR,UV-vis,and 1H NMR characterizations.Furthermore,CCK-8 results indicated the optimal BMSCs activity at 2.5%CS-PE,and Live/Dead staining results showed that CS-PE was non-toxic and cytocompatible with BMSCs.3.According to the examinations of gelation time and pH value of CS and GP,a 60%GP concentration was selected to synthesize the hydrogel.CS/GP-Gel,CS-PE/GP-Gel,and CS-PE/GP/Wnt3a-Gel were prepared from CS and CS-PE with GP,type I collagen,and Wnt3a,in certain proportion to each other,and hydrogels could be formed at 37℃.As expected,a 3D network structure was confirmed in the synthesized hydrogels.4.Live/Dead staining results showed that the three kinds of hydrogels were all nontoxic to BMSCs and had good cytocompatibility.Moreover,the CS-PE/GP/Wnt3a-Gel showed the best migration-inducing effect on rat BMSCs(P<0.001).5.Compared with the control group,more BMSCs were observed to undergo ACD on the collagen functionalized platform containing Wnt3a,and more BMSCs with aPKCζ enriched at one end of the cytosol were observed on the CS-PE/GP/Wnt3a-Gel in comparison to the CS-PE/GP/Wnt3a-Gel group.OPN was also enriched at one end of the cytosol,while Numb distribution did not differ during cytokinesis.6.As the results showed,granulocytes infiltrated into the CS/GP-Gel,CS-PE/GPGel,and CS-PE/GP/Wnt3a-Gel groups,and 22.02%of infiltrated granulocytes in the CS-PE/GP/Wnt3a-Gel group were macrophages,the number was statistically high compared to the CS/GP-Gel(9.8%)and CS-PE/GP-Gel groups.Among the infiltrating macrophages in each group,13.77%,27.95%,and 34.03%were M2 type macrophages,respectively.And the result of CS-PE/GP/Wnt3a-Gel group was statistically significant compared to the other two groups.Immunofluorescence results also confirmed that the expression level of CD54 in CS-PE/GP/Wnt3a-Gel group was higher than that in CSPE/GP-Gel and CS-PE/GP/Wnt3a-Gel.7.Seven days after subcutaneous injection of hydrogels in each group,the CSPE/GP-Gel and CS-PE/GP/Wnt3a-Gel groups could recruit more BMSCs than those in the CS/GP-Gel group(P<0.05,P<0.001,respectively),and the CS-PE/GP/Wnt3a-Gel group also showed better ability to actively recruit stem cells than the CS-PE/GP-Gel group(P<0.01).The CS-PE/GP/Wnt3a-Gel group showed the strongest ability to recruit stem cells(P<0.01),and the immunofluorescence results confirmed that CD54 expression in the CS-PE/GP/Wnt3a-Gel group was higher than in the CS/GP-Gel group and CS-PE/GP-Gel groups.8.After the successful establishment of rat critical cranial defect model,in vivo experiments showed no difference in the expression of ALT,AST,TBIL,yGTT,ALP,BUN,and CREA in rat serum 8 weeks after operation,and histopathological examination of the main organs(heart,liver,spleen,lung,and kidney)exhibited no abnormalities.Micro-CT results at 8 weeks postoperatively displayed optimal cranial bone repair effect in rats treated with CS-PE/GP/Wnt3a-Gel.Besides,quantitative analysis of the remaining defect area revealed that,compared with the NC and CS/GPGel groups,the CS-PE/GP/Wnt3a-Gel group showed more bone formation.On the other hand,the results of HE staining revealed that the mineralization in the new bone area exhibited the histological characteristics of healthy bone,and more new bone formation was observed in the defect area of CS-PE/GP-Gel and CS-PE/GP/Wnt3aGel groups compared with the control group.Masson trichrome staining displayed that the defect edges treated with CS-PE/GP/Wnt3a hydrogel possessed substantial osteoid formation,and immunofluorescence staining collaborated more Arg-1 and SOSTpositive cells in the CS-PE/GP/Wnt3a-Gel group than in the other three groups.Conclusions1.S-PE can play a role simulating the functional domain of SDF-1 to recruit BMSCs.With the low cytotoxicity,it can promote BMSC proliferation and osteogenic differentiation,and be used further as a new functional unit loaded in biomaterials.2.CS-PE possesses good biocompatibility and can recruit stem cells.CSPE/GP/Wnt3a deformable hydrogel exhibited temperature sensitivity,suitable niche environment,the ability to recruit BMSCs in situ,relatively low inflammatory response,and initiate the preliminary tissue repair.Thermal responsive phase transition behavior enables the hydrogels to fill irregular defect areas,maintain dynamic replenishment of stem cell pool,and induce ACD by Wnt3a to achieve rapid bone repair,which holds great application potential.