Hydroxypatite Nanowire/Polylactic Acid Composite Membranes Loaded With SDF-1 Promote Periodontal Bone Tissue Regeneration

Background and Objective:Periodontitis is an infectious and destructive disease of the supporting tissues around the teeth,which can cause gingival inflammation,formation of periodontal pockets,alveolar bone resorption,if we do not intervene eventually cause tooth loosening and loss,seriously affect the quality of life of patients.Periodontitis is the leading cause of tooth loss in adults.Obtaining complete regeneration of supporting periodontal tissues such as periodontal ligament,alveolar bone,and cementum has always been an ideal goal of periodontitis treatment.At present,clinically used periodontal treatment modalities such as supragingival scaling,subgingival curettage and flap surgery can effectively control the progression of inflammation but cannot achieve the repair and regeneration of periodontal tissues.The development of regenerative surgery has offered promising prospects for treating periodontitis.Periodontal regenerative surgery mainly includes barrier membrane-mediated guided tissue regeneration(GTR)and bone grafting.GTR utilizes membrane materials as barriers,preventing gingival epithelium and connective tissue from contacting with root surface,providing space and allowing regenerative periodontal ligament stem cells(PDLSCs)to preferentially colonize the root surface,leading to the formation of new cementum on exposed root surfaces in periodontal pockets,allowing the integration of periodontal ligament fibers,and thereby facilitating periodontal tissue regeneration.Currently,collagen membranes and polylactic acid(PLA)membranes are the most common medical barrier membranes.Although the current membranes can function as barrier and provide space for regeneration,they lack osteogenesis ability;thus,clinically,it is necessary to put the membrane and bone graft materials into the periodontal defect area separately to achieve a good restoration effect.However,complex procedures are difficult to perform and time-consuming,and may increase the risk of bleeding and infection.To improve the current deficiencies of medical barrier membranes,our research group developed hydroxyapatite(HAp)/PLA(HP)composite membranes with dual functions,osteogenic induction,and barrier protection,to promote periodontal tissue regeneration.However,it is a complex procedure that involves stem cell recruitment,angiogenesis,osteogenic differentiation for bone tissue regeneration.Although HAp has a certain osteoinductive capacity,it lacks the ability to recruit stem cells and promote angiogenesis,which leads to a relatively low efficiency and quality of bone regeneration.A promising solution to enhance the biological function of HP composite membranes is to endow them with the capacity to facilitate stem cell recruitment,cell proliferation,angiogenesis,and osteogenic differentiation simultaneously.According to in situ tissue engineering principles,multifunctional effects can be achieved by loading chemokines onto the composite membranes.Stromal cell-derived factor-1(SDF-1),a chemokine that activates C-X-C chemokine receptor type 4(CXCR4),is crucial in various stem cell activities including migration,homing,and angiogenesis.A previous study carried out by our group indicated that SDF-1 enhanced the in vitro proliferation and migration of PDLSCs,and its local application in rat periodontal bone defects significantly improved the in vivo proliferation and migration of mesenchymal stem cells(MSCs),thereby promoting the angiogenesis and periodontal bone defect repair.Therefore,loading SDF-1 may endow HP composite membranes with the ability to recruit stem cells and stimulate angiogenesis.In this study,SDF-1 was loaded by a freeze-drying method onto the HAp surface of an HP composite membrane to construct a multifunctional SDF-1-HAp/PLA(S-HP)composite membranes,and the physicochemical characterization and SDF-1 release pattern were detected.Furthermore,the effect of S-HP composite membranes on the proliferation,migration,and adhesion of PDLSCs were designed in vitro,to clarify the optimal concentration of SDF-1 in vitro,and to detect the vascularization and osteogenic differentiation effect of S-HP composite membranes.To further clarify the function of S-HP composite membranes in periodontal bone repair,a rat periodontal bone defect model was constructed to investigate the effects of S-HP composite membranes on MSCs recruitment,angiogenesis and osteogenic differentiation in the process of bone repair.Finally,the synergistic effect of S-HP composite membranes in promoting periodontal bone regeneration was assessed,aiming at providing a novel therapeutic strategy for periodontal tissue repair and regeneration.Materials and Methods:1.Preparation,physicochemical characterization and SDF-1 release detection of S-HP composite membranesFirst,ultralong HAp nanowires were prepared and made into HAp paper through filtration.By evaporating the solution of PLA and dichloromethane,a thin membrane on one side of the HAp nanowire paper was prepared.Subsequently,SDF-1 was loaded onto the HAp side of S-HP composite membranes through freeze-drying method.X-ray diffraction(XRD)analysis was used to assess the crystal phase of HAp nanowires.Scanning electron microscopy(SEM)was used to observe the microstructure and appearance of HAp nanowires and PLA.The morphology and crystal phase structure of the materials were further detected by transmission electron microscopy(TEM).Fourier transform infrared(FTIR)spectroscopy was used for qualitative analysis the surface functional groups of HP and S-HP composite membranes.The zeta potential of SDF-1,HAp and HAp loaded with SDF-1(S-H)were detected to further determine whether SDF-1 can be loaded on the HAp surface of HP composite membranes.The contact angle VCA Optima surface analysis system was used to evaluate the hydrophilicity and hydrophobicity of the HAp surface of HP and S-HP composite membranes by measuring the water contact angle(WCA).The concentration of released SDF-1 was measured by an enzyme-linked immunosorbent assay(ELISA)kit,and SDF-1 release curve was obtained.2.The effect of the S-HP composite membranes on the biological activity of PDLSCs in vitroPDLSCs were inoculated on HAp surface of S-HP composite membranes loaded with different concentrations of SDF-1.The effects of S-HP composite membranes on the proliferation and migration of PDLSCs were measured by cell counting kit 8(CCK-8)and Transwell migration assay respectively,and the optimal load concentration of SDF-1 was obtained,which was applied to the subsequent experiments in vitro.Subsequently,confocal laser scanning microscope(CLSM)and SEM were used to observe the attachment,extension,and morphology of PDLSCs on HAp.Quantitative real-time polymerase chain reaction(qRT-PCR)was used to detect S-HP composite membranes angiogenesis and the expression of angiogenesis related genes vascular endothelial growth factor(VEGF),basic fibroblast growth factor(bFGF)and stem cell factor(SCF).After osteogenic induction,osteogenic genes ALP,runt-related transcription factor2(Runx2),osteopontin(OPN),osteocalcin(OCN)and osteoblast-related proteins(ALP,OPN and OCN)were detected by ALP activity,qRTPCR and immunofluorescence staining.3.The effect of S-HP composite membranes on periodontal bone regeneration in vivoThe periodontal bone defect model was established using male Wistar rats and the S-HP composite membranes were implanted into the wound area.Samples were collected at 3 days,1 week,2 weeks,4 weeks,and 8 weeks,and mandibular bone samples were collected.Immunofluorescence staining was used to detect the recruitment of CD90+/CD34-stem cells and the positive expression of CXCR4 in vivo.CD31 and alpha-smooth muscle actin(α-SMA),which are related to angiogenesis,were identified using immunohistochemical staining.Osteogenic/osteoclastic activity in periodontal bone defects was detected by Tartrate-resistant acid phosphatase(TRAP)staining.Microcomputed tomography(micro-CT),hematoxylin and eosin(H&E)staining,Masson’s trichrome staining and immunohistochemical staining were used to assess the bone regeneration effects of the S-HP composite membranes in vivo.Results:1.Preparation,physicochemical characterization and SDF-1 release detection of S-HP composite membraneS-HP composite membranes loaded with SDF-1 with stable physical and chemical properties were successfully prepared.HAp nanowires were pure-phase hydroxyapatite without impurities,as confirmed by XRD analysis.The length of the HAp nanowires is 50-100 μm,and the typical lattice spacing was 0.34 nm(002)detected by SEM and TEM.PLA side had a relatively smooth and flat structure with uniform pore structure.After SDF-1 was loaded on the HAp side of HP membranes,the morphology of HAp side was barely changed,which was similar to the original morphology.FTIR analysis showed that there were vibration peaks belonging to amide I and amide II on HAp loaded with SDF-1(S-H),confirming that SDF-1 was successfully loaded onto HAp nanowires.Zeta potential detection results showed that the potential of S-H was between HAp and SDF-1,which further confirmed that SDF-1 could be efficiently loaded onto the HAp side by physical adsorption and electrostatic attraction.WCA test results showed that HAp side of S-HP composite membranes still had good hydrophilicity compared with that before SDF-1 loading,which was conducive to cell adhesion.The results of the ELISA experiments demonstrated that the loaded SDF-1 on S-HP showed a burst release of approximately 30%within 24 h.Then,the release rate significantly decreased and showed a long-lasting release,which was almost completely released(93%)by 14 days.2.S-HP composite membranes promoted the proliferation,migration,adhesion,angiogenic and osteogenic differentiation of PDLSCsThe results of CCK-8 and Transwell migration experiments indicated that HP composite membranes(S250-HP)loaded with 250 ng/mL SDF-1 had the best effect to promote cell proliferation and migration.Therefore,S-HP composite membranes loaded with 250 ng/mL SDF-1(collectively referred to as S-HP)was selected for the subsequent in vitro experiments.The results of CLSM showed that both HP composite membranes and S-HP composite membranes enhanced the differentiation of PDLSCs into polygonal osteoblast-like cells.Under SEM,PDLSCs extended many pseudopodlike structures and were tightly attached to HAp nanowires.Matrigel tube formation assay showed that SDF-1 and S-HP composite membranes promoted the formation of intertwined capillary-like network structures of PDLSCs.The results of qRT-PCR showed that both SDF-1 and S-HP composite membranes significantly promoted the expression of angiogenesis-related genes(VEGF,bFGF and SCF)of PDLSCs at 1,4 and 7 days.HP composite membranes had no effect on the vasculogenic differentiation of PDLSCs.After osteogenic induction,ALP activity and expression of osteogenic genes(ALP,Runx2,OPN and OCN)detected by qRT-PCR showed that both HP and SHP composite membranes promoted the osteogenic differentiation of PDLSCs.S-HP composite membranes had the optimal effect on osteogenic differentiation,while SDF1 slightly promoted osteogenic differentiation of PDLSCs.OPN and OCN positive cells were observed on both HP and S-HP composite membranes,and the positive expression of PDLSCs on S-HP composite membranes was more significant which detected by immunofluorescence staining.3.S-HP composite membranes promoted the regeneration of periodontal bone tissues by promoting MSCs recruitment,angiogenesis and osteogenesis.The results of immunofluorescence double staining showed that CD90+/CD34stem cells were expressed in each stage of wound healing after S-HP composite membranes were implanted into the periodontal bone defect areas of rats.S-HP composite membranes facilitated the engraftment of MSCs to the periodontal bone defect area most significantly on the 3rd day,and then showed a decreasing trend.In addition,the results of immunofluorescence staining indicated that CXCR4+cells were expressed at all time periods after S-HP composite membranes were implanted,and the maximal expression appeared when S-HP composite membranes were implanted for 7 days,which further confirmed that S-HP composite membranes could promote the recruitment of MSCs in the early stage of periodontal bone defect healing.Immunohistochemical staining was used to detect the expression of angiogenesis related markers CD31 and α-SMA.The results showed that S-HP composite membranes had positive cell expression at week 1 and 2,and the expression was most significant at week 2,which confirmed that S-HP composite membranes enhances vascular regeneration in periodontal bone defect area.TRAP staining was used to detect the activity of osteoclasts in the periodontal bone defect area.The results showed that the number of positive osteoclasts in the S-HP composite membranes was the highest at day 7,indicating that S-HP composite membranes could increase early osteogenic/osteoclastic activity in the periodontal bone defect area.Micro-CT,H&E,Masson and immunohistochemical staining showed that bone volume/tissue volume(BV/TV)and trabecular thickness(Tb.Th)increased in periodontal bone defect area implanted with S-HP composite membranes.trabecular separation(Tb.Sp)decreased,and the newly formed bone tissue became more mature and denser.Conclusions:1.S-HP multifunctional composite membrane was successfully fabricated,featuring outstanding physicochemical properties and enabling the phased release of SDF-1:an initial burst release effect within the early stage(within 24 hours)accounting for approximately 30%of the total release,followed by a sustained release,reaching a cumulative release rate of 93%within 14 days.2.S-HP composite membranes promoted the proliferation and migration of PDLSCs in vitro.When the SDF-1 concentration was 250 ng/mL,the effect was the most significant,and PDLSCs adhered well on S-HP composite membranes,indicating that the composite membrane had optimal biocompatibility and could significantly promote the angiogenesis and formation of PDLSCs resulting in osteogenic differentiation.3.In vivo experiments demonstrated that in the early stages of periodontal defects,S-HP composite membrane released SDF-1 into the defect area,recruited a significant number of MSCs,and provided a sufficient cell source to repair the damaged area.In the early and middle stages of periodontal bone defects,S-HP composite membrane promoted the formation of blood vessels,and new bone in the periodontal defect area and improved the quality of new bone.