• <tr id="yyy80"></tr>
  • <sup id="yyy80"></sup>
  • <tfoot id="yyy80"><noscript id="yyy80"></noscript></tfoot>
  • 99热精品在线国产_美女午夜性视频免费_国产精品国产高清国产av_av欧美777_自拍偷自拍亚洲精品老妇_亚洲熟女精品中文字幕_www日本黄色视频网_国产精品野战在线观看 ?

    Sequentially releasing self-healing hydrogel fabricated with TGFβ3-microspheres and bFGF to facilitate rat alveolar bone defect repair

    2022-09-06 13:50:18

    aDepartment of Cell Biology,College of Life Science and Technology,Jinan University,Guangzhou 510632,China

    bGuangdong Province Key Laboratory of Bioengineering Medicine,Jinan University,Guangzhou 510632,China

    cBiopharmaceutical R&D Center of Jinan University,Guangzhou 510632,China

    dDepartment of Stomatology,Jinan University Medical College,Guangzhou 510632,China

    Keywords:Self-healing hydrogel TGFβ3 microspheres bFGF Sequential release Alveolar defects

    ABSTRACT Resorption and loss of alveolar bone leads to oral dysfunction and loss of natural or implant teeth.Biomimetic delivery of growth factors based on stem cell recruitment and osteogenic differentiation,as the key steps in natural alveolar bone regenerative process,has been an area of intense research in recent years.A mesoporous self-healing hydrogel (DFH) with basic fibroblast growth factor(bFGF)entrapment and transforming growth factor β3(TGFβ3)-loaded chitosan microspheres (CMs) was developed.The formulation was optimized by multiple tests of self-healing,in-bottle inversion,SEM,rheological,swelling rate and in vitro degradation. In vitro tubule formation assays,cell migration assays,and osteogenic differentiation assays confirmed the ability of DFH to promote blood vessels,recruit stem cells,and promote osteogenic differentiation.The optimum DFH formula is 0.05 ml 4Arm-PEG-DF (20%) added to 1 ml CsGlu (2%) containing bFGF (80 ng) and TGFβ3-microspheres(5 mg).The results of in vitro release studied by Elisa kit,indicated an 95%release of bFGF in 7 d and long-term sustained release of TGFβ3.For alveolar defects rat models,the expression levels of CD29 and CD45,the bone volume fraction,trabecular number,and trabecular thickness of new bone monitored by Micro-CT in DFH treatment groups were significantly higher than others (?P <0.05, vs Model).HE and Masson staining show the same results.In conclusion,DFH is a design of bionic alveolar remodelling microenvironment,that is in early time microvessels formed by bFGF provide nutritious to recruited endogenous stem cells,then TGFβ3 slowly released speed up the process of new bones formation to common facilitate rat alveolar defect repair.The DFH with higher regenerative efficiency dovetails nicely with great demand due to the requirement of complicated biological processes.

    1.Introduction

    Periodontal disease is one of the most common diseases of the oral cavity.It induces plaque accumulation,dysbacteriosis,periodontal pocket formation,gingival recession,tissue destruction,and loss of the alveolar bone,eventually leading to tooth loss.In fact,it is one of the primary reasons behind tooth loss in adults [1,2].Epidemiological studies have revealed that the incidence of periodontal disease is as high as 90% [3].In addition,systemic diseases associated with periodontal disease,e.g.,diabetes,cardiovascular and cerebrovascular diseases,pregnancy,osteoporosis,and Alzheimer’s disease,endanger the physical and mental health of patients[4].Although the progression of periodontal disease can be slowed down by reducing inflammation via mechanical plaque removal based on scaling,lesions resulting from bone loss,particularly that of the alveolar bone,are irreversible [5].Therefore,improving repair and reconstruction methodologies of alveolar bone defects has remained a popular research topic in periodontal disease treatment.

    With recent developments in material and biological sciences,the potential application of tissue engineering to bone regeneration,while also satisfying clinical requirements,has been investigated [6-8].Recently,owing to the detailed study of mobilizable/homing endogenous resident stem cells and growth factors,endogenous tissue regeneration (ERM)has garnered tremendous attention in this field [9,10].In relatively young patients,with active growth and metabolism and excellent function of endogenous stem cells,endogenous regeneration and repair is the preferred alternative [11].Compared to treatments involvingin vitroexpansion of stem cells,ERM circumvents the complex processes involved in tissue engineering and thereby facilitates the development of clinically translatable regenerative approaches[12,13].

    Numerous studies have demonstrated the advantages of cell-and growth factor-encapsulated hydrogel scaffolds in bone regeneration over the past decade [14,15].They have been utilized as a platform to improve regenerative payload delivery owing to their unique physico-chemical properties,e.g.,their porosity and other mechanical properties are tuneable to site-specific tissue types [16,17].Successful bone regeneration requires a complex and coordinated cascade of growth factors (GFs) and cells.Thus,designing a biomaterial capable of improving the stability and longevity of GFs,guiding stem cell homing,and promoting proper cell residency and differentiation simultaneously are crucial steps[18].Further,a single GF cannot meet the requirements of periodontal tissue regeneration during the different stages-a combination of multiple GFs is required [19].Our laboratory has been committed to the research of growth factors and their effects on osteogenic differentiation of human periodontal ligament stem cells (hPDLSCs) [20-22].We have derived the following conclusions.(1) basic fibroblast growth factor (bFGF) can significantly promote the proliferation of mesenchymal stem cells (MSCs) and the formation of micro-vessels during early stages of bone regeneration.(2)transforming growth factorβ3 (TGFβ3) facilitates osteogenic differentiation of hPDLSCsin vitro.Further,synthetically considering our previous studies and clinical requirements,we designed a dual-factor (bFGF and TGFβ3) sequentially releasing system to repair periodontal tissue defects.

    In recent years,multiple biomaterial-based delivery systems have been developed to achieve controlled release of growth factors in bone tissue engineering [23].The microsphere-gel scaffold structure is a widely used candidate owing to the simplicity of its method of preparationthe preparation environment needs to satisfy only a few conditions [15].Further,it enables sequential release of different active ingredients with precise control over the release time and dosage [24].In addition,the injectability and deformability of the microsphere-gel scaffold enables it to adapt to irregular bone defects.It is particularly suited to the narrow oral cavity [25].An injectable hydrogel sulfated chitosan oligosaccharide was prepared in our laboratory,which is capable of effectively enhancing the biological activity of aFGF,enabling neuronal repair[26].This motivated our goal of first encapsulating TGFβ3 in microspheres and then co-loading bFGF and TGFβ3-microspheres into a chitosan hydrogel.As the hydrogel degrades,the bFGF and microspheres in the hydrogel are preferentially released,followed by the delayed release of TGFβ3.This is analogous to the natural healing process,in which bFGF and TGFβ3 are released at different times to aid different stages of healing.

    In summary,we fabricated a self-healing hydrogel system (DFH) consisting of TGFβ3-microspheres and bFGF to biologically mimic the micro-environment of alveolar bone defects.The effectiveness of DFH was evaluated bothin vitroandin vivo.Based on the results,we expect it to be applied in the field of periodontal tissue engineering in the future.

    2.Materials and methods

    2.1.Materials

    Chitosan was purchased from Zhejiang Jinke Pharmaceutical Co.Ltd.(Hangzhou,China).Chitosan-glutamate (CsGlu) was obtained from Wuhan Yuancheng Technology Development Co.Ltd.(Wuhan,China).4-arm polyethylene glycolbenzaldehyde (4Arm-PEG-DF) was purchased from Wuhu Ponsure Biological Technology Co.Ltd.(Wuhu,China).bFGF and TGFβ3 were provided by Jinan University Biopharmaceutical R&D Centre (Guangzhou,China).The enzyme-linked immunosorbent assay (ELISA) kits were purchased from Wuhan Huamei Biotechnology Co.Ltd.(Wuhan,China).hPDLSCs were obtained from our laboratory.ECV304-eGFP cells were purchased from the Chinese Academy of Sciences (Shanghai,China).All cell culture plates and bottles were obtained from Corning Company(Corning,NY).

    2.2.The role of bFGF and TGFβ3 during the proliferation and differentiation of hPDLSCs

    Sequential administration of bFGF and TGFβ3 was carried out following the revision presented in [27].In brief,cells were seeded into 12-well plates and cultured in the following media for 12 or 21 d:basic medium(NC),osteogenic induction medium (OM),OM with 40 ng/ml bFGF,OM with 1000 ng/ml TGFβ3,OM with simultaneous application of bFGF and TGFβ3,and OM with 40 ng/ml bFGF pre-treated during the first 3/6/9 d and TGFβ3 during the remaining period.The experimental categories are depicted in Fig.1A and 1C.After culturing for 12 or 21 d,the osteogenic differentiation of hPDLSCs was observed using ALP staining and Alizarin Red staining.

    Fig.1-Sequential application of bFGF and TGFβ3 significantly increased ALP activity and mineral deposition in hPDLSCs.(A).Schematic diagram of sequential administration of bFGF and TGFβ3(12 d).(B).After 12 d of culture,ALP staining and Alizarin red staining were performed.(C).Schematic diagram of sequential administration of bFGF and TGFβ3(21d).(D).After 21 d of culture,Alizarin red staining was performed.(n=3,?P <0.05,??P <0.01,vs.NC).

    2.3.Preparation and characterization of DFH

    DFH comprised two constituents-TGFβ3 chitosan microspheres (CMs) and bFGF self-healing hydrogel.The self-healing hydrogel was prepared by forming a Schiff base bond between the amine group on CsGlu and the active carbonyl group on 4Arm-PEG-DF.First,CMs were preparedvia emulsification and cross-linking (Fig.2A).Next,CMs were incubated in TGFβ3 solution at 4°C for 48 h to produce TGFβ3-loaded CMs.Then,TGFβ3-loaded CMs were added into the bFGF-CsGlu solution and mixed with the 4Arm-PEG-DF solution to obtain DFH.

    2.4.Optimization and characterization of DFH

    In order to optimize the composition of DFH,its gel time,scanning electron microscopy (SEM),rheological properties,and compression properties were recorded.Vial inversion method was used to determine its gelation times (Table 1).SEM(XL30,Philips,Germany)was used to observe its structure.A rotational rheometer (Kinexus Pro,Malvern) was used to measure the rheological properties of DFH.The reformation of DFH fragments into a whole was observed macroscopically to investigate the self-healing properties of DFH.Finally,the dynamic universal testing machine (ELF3200,Bose,America) was used to evaluate the mechanical properties of DFH.

    Table 1-The gelation time of CsGlu and 4Arm-PEG-DF in different proportions.

    Table 2-The group of in vivo recruitment of stem cells.

    Fig.2-Preparation and characterization of DFH.(A).Flow chart of TBFH preparation.(B).The morphology of CM was observed via SEM.(C).SEM observation of the structure of DFH and the microspheres in it.(D).Dyes of different colours were added to the DFH,chopped,and then combined to observe the self-healing process of the hydrogel.(E).Changes in mechanical properties of the DFH before and after self-healing.

    2.5.In vitro degradation and release characteristics of DFH

    The weight loss method was applied to evaluate degradation of DFHin vitro.The weighed (W0) DFH was immersed in Phosphate Buffer Saline (PBS,pH 7.4) containing 20 μg/ml lysozyme,and continuously shaken in a constant temperature shaker at 37°C.At predetermined times,portions of DFH were retrieved,its surface moisture was removed,and the DFH was weighed (Wt).The degradation rate (DR) of the gel was calculated using the following formula(n=3):

    To ascertain the release profile,the release profile of bFGF and TGFβ3 from the DFHin vitrowas evaluated by ELISA.The DFH were placed in the 12-well plates containing PBS(pH=7.4) under the sustained oscillation.At predetermined intervals,100 μl the supernatant was collected and evaluated with bFGF ELISA and TGFβ3 ELISA,followed by adding an equal volume of fresh PBS in well plates.Calculate the protein release rate and draw the cumulative release curve.

    2.6.Proliferation and osteogenic differentiation of cells in DFH

    DFH and hPDLSCs(3×105cells/well)were mixed and added to a 24-well plate.After culturing for 1,3 and 7 d,the proportions of living and dead cells were recorded via staining using Calcein-AM/PI living cell/dead cell double staining kit (Zeye Biotechnology Co.Ltd.Shanghai,China).To evaluate hPDLSCs osteogenesis differentiation,ALP staining was executed after culturing in the inducing medium for 7 d and 14 d.

    2.7.Promotion of tubule formation and cell migration by DFH in transwell

    DFH (300 μl/well) was added to a 24-well plate to observe the tube-formation behaviour of human umbilical vein endothelial cells (HUVECs) (Shanghai,China) on DFH.Following the gelation of DFH,HUVECs with red fluorescence(1×105cells/well) were added to the surface of the gel.After culturing for 24 h,the formation of the lumen was observed.Transwell migration assay was used to evaluate the ability of DFH to recruit MSCs.MSCs (5×104cells/well) were seeded into the upper chamber of a Transwell (24-well) plate.DFH containing different concentrations of TGFβ3 was added to the lower chamber.After culturing for 48 h,it was stained with a crystal violet solution to measure the number of migrated cells.

    2.8.Animals

    Specific-pathogen free male Sprague-Dawley (SD) rats(250±20 g)with certificate no.44007200069979 were supplied by the Guangdong Medical Laboratory Animal centre(Guangdong,China).They were kept in separate animal rooms at constant temperature (25±2°C) and humidity (55%± 10%) on a 12-h light/dark cycle with free access to water and food.The experimental protocol was approved by the Ethics Review Committee for Animal Experimentation ofJinan University (ethical review no.20200826-11),and all the experiments were conducted following the National Institutes of Health Guide for the Care and Use of Laboratory Animals(NIH Publications No.8023,revised 1996).

    2.9.DFH-induced recruitment of MSCs in the muscle pocket model of SD rats

    As indicated by the categories presented in Table 2,DFH was implanted into the muscle pocket of SD rats to observe DFH-induced recruitment of MSCsin vivo.Each rat was injected with 100 μl DFH.The rats were euthanized seven d after the operation,and the gel and surrounding tissues were fixed with 4% paraformaldehyde and embedded in paraffin for histological sectioning.Following dewaxing and hydration,immunohistochemical analysis was performed on the sections to evaluate the expression of CD29 and CD45.Finally,the sections were dehydrated and sealed for microscopy.

    2.10.DFH-induced repair of alveolar bone injury in SD rats

    The experimental process of alveolar bone injury repair is depicted in Fig.5A.After the rats were anesthetized(using 3%sodium pentobarbital),the left gingiva of the maxillary incisor of each rat was lacerated with a pointed scalpel to expose the surface of the alveolar bone.Then,a 1.5 mm diameter dental drill was used to drill a hole and create a spherical defect with a diameter of 2 mm and a depth of 1 mm.

    One week after the operation,the periodontal defect was observed using Micro-CT and rats exhibiting unqualified alveolar bone defects were discarded.24 animals were categorized in the following classes-Model group,Blank hydrogel group,DFH-L (DFH with 80 ng/ml bFGF and 1000 ng/ml TGFβ3),and DFH-H (DFH with 80 ng/ml bFGF and 4000 ng/ml TGFβ3).Each rat was injected with 100 μl DFH.The state of repair of the alveolar bones of these rats was observed after 12 weeks.

    2.11.Quantitative real-time PCR

    DFH scaffolds were retrieved after seven d.The total RNA of the tissue was extracted using the TRIzol reagent and reverse transcribed using the PrimeScriptTM-RT reagent kit (TaKaRa) by following the manufacturer’s instructions.Real-time polymerase chain reaction (PCR) was performed using 2×SYBR Green PCR Master Mix on a Real-Time PCR System.All the primer sequences were designed using the primer 5.0 software.The following primer sets were used: CD29-forward 5′-CTACTGGTCCCGACATCATC-3′and reverse 5′-TGTCACGGCACTCTTGTAAA-3′;CD45-forward 5′-ACCACATATCTTCC AGGTGCC-3′and reverse 5′-CCATTGGAGAGAGTGACGTTT-3′;and GAPDH-forward 5′-CTCTGCTCCTCCCTGTTCTA-3′and reverse 5′-TCGTTGATGGCA ACAATGTC-3′.The relative expressions of CD29 and CD45 were calculated via the 2-ΔΔCt method using GAPDH as a reference gene.

    2.12.Histochemical staining

    Briefly,alveolar bone tissue was fixed with 4%paraformaldehyde and embedded in paraffin for histological sectioning.Following dewaxing and hydration,the sections were examined using HE staining to analyse the formation of new bone.Masson’s trichrome staining was utilised to further analyse the formation of collagen.The sections were analysed,and images were captured with a microscope(Olympus IX71;Tokyo,Japan).

    2.13.Statistical analysis

    All data are expressed as the mean ± standard deviation(n=3).Statistical analyses were performed using the GraphPad Prism 6 software (GraphPad Software Inc.,La Jolla,CA,USA).Differences between more than two groups were analysed using one-way ANOVA followed by Tukey’s HSD comparison test.The threshold for statistical significance was set to beP <0.05.

    3.Results and discussion

    3.1.Significant increase of ALP activity and mineral deposition in hPDLSCs by the sequential application of bFGF and TGFβ3

    The sequential application of multiple GFs found in real-world biological processes is essential to periodontal regeneration.To validate the different functions of bFGF and TGFβ3 during the osteogenic differentiation of hPDLSCs,we designed anin vitrocell experiment by slightly altering the method of sequential administration [27].As depicted in Fig.1B and 1D,compared to the NC group,the combination of bFGF and TGFβ3 significantly enhanced the expression of ALP,with the performance of the 40b(6d)/1000T(6d) group being significantly better than those of others (??P < 0.01,vsNC).Pre-treatment with 40 ng/ml bFGF for 6 d,followed by that with 1000 ng/ml TGFβ3 for 6 to 18 d,produced the best mineralization promotion ability amongst all groups(??P <0.01,vsNC).This indicates that bFGF pre-treatment for approximately 6 d,followed by TGFβ3 administration,significantly enhanced the osteogenic differentiation ability of hPDLSCs.Some studies have concluded that bFGF can activate endothelial cell proliferation and migration to promote angiogenesis through multiple signalling pathways[28].However,although the establishment of the new vascular network is attributed to bFGF,subsequent creeping replacement of endogenous stem cells and new bone formation are attributed to TGFβ3 [29,30].Ge et al.found that bFGF exerts a time-dependant antagonistic effect on ALP activity induced by OM and BMP-2,while the sequential treatment of low-dose bFGF and BMP-2 can promote ALP activity in hPDLSCs [27].In this study,we employed TGFβ3 instead of BMP2.TGFβ3 and BMP2 belong to the same family and are two classical growth factors involved in bone regeneration.Although TGFβ3 exhibits obvious advantages over BMP2 in terms of recruiting endogenous stem cells[31],TGFβ3 is still in the laboratory research stage due to the limitation of its industrialized amount.Our laboratory has overcome the difficulty of refolding recombinant human TGFβ3 and achieved large-scale production [32],but finer details of the plan are yet to be finalized.

    3.2.Preparation,optimization,and characterization of DFH

    Exogenous growth factors in periodontal tissues cannot fully exert their biological activities owing to their short halflife and fast diffusion rate.Therefore,it is necessary to design an appropriate system capable of controlling growth factors for therapeutic purposes.The proposed DFH system,composed of TGFβ3-microspheres and bFGF,is a promising candidate.The CM preparation process is depicted in Fig.2A.The microspheres adsorbed TGFβ3 proteins owing to the combined effects of electrostatic properties and the internal pore structure.SEM revealed that the CMs were completely spherical with a particle size primarily distributed between 20 and 30 μm(Fig.2B).Then,TGFβ3-microspheres were added into the bFGF-CsGlu solution,followed by the addition of 4Arm-PEG-DF.The mixed solution was gelled via Schiff-based bond formation within 5 min (Table 1).The optimized DFH composition was ascertained to be 2%CsGlu:20%4Arm-PEGDF=1:0.05(v/v),with the concentration of microspheres being 5 mg/ml(Fig.S1).

    DFH exhibits a layered porous structure with a porosity of approximately 84.3%,which lies within the porosity range of 50%-95% of natural alveolar bones,and a pore size between 100 and 300 μm,as observed via SEM.Numerous studies have established the requirement of good porosity and interconnected porous structures in engineered scaffolds that allow cells to attach and proliferate,thereby promoting angiogenesis,to mimic natural scaffold structures.The optimal range for pore size is 100-500 μm [15].In this study,the prepared DFH exhibited a porosity of 84.3%and a pore size distribution between 100 and 300 μm.Thus,its structure is similar to that of healthy dense bone and favourable for bone regeneration(Fig.2C).

    In addition,as the resorption or injury of the alveolar bone in the oral cavity is usually an irregular wound,the ideal repair material should exhibit excellent plasticity and injectability.The self-healing property of DFH is that -NH2 on CsGlu and-CHO on 4Arm-PEG-DF react to form a Schiff base bond.We all know that the Schiff base bond is a reversible covalent bond,so the DFH gel prepared by the Schiff base reaction has the property of self-healing.At the same time,two pieces of gel of different colours were observed to splice together and aggregate into a single entity after 1 h,verifying the viability of the self-healing system(Fig.2D).The mechanical compression test was performed on DFH before and after self-healing(Fig.2E).The results established the constancy of the slope of the gel,which further proved the dynamic/reversible nature of the Schiff-based bonds.Based on the unique physicochemical properties of DFH,its application in tissue engineering is greatly broadened.Also,DFH degradation have a minor effect on the self-healing ability of the gel.In the self-healing ability test of DFH,we found that after the re-healed hydrogel was soaked in PBS buffer for 12 h,only the surface of the gel became less flat due to degradation.

    3.3.In vitro release of bFGF and TGFβ3 in DFH

    In the case of multivariate GFs,the order of release and the quantity introduced at each stage are important parameters to ensure the realization of the desired effect.The sequential release of the two proteins (1 ml DFH containing 80 ng bFGF and 1000 ng TGFβ3) was verified viain vitrorelease kinetic studies.The results (Fig.3A) revealed that,during the initial stage,DFH swelled sufficiently in the PBS solution and started to dissolve rapidly.Alongside the morphologic change of DFH,bFGF was released rapidly,and the released proportion became 20% in 24 h.However,it was ensured via encapsulation that the released amount of TGFβ3 remained lower than the detection limit during this 24 h duration.Over time,the hydrogel gradually swelled sufficiently,and the cross-linked structure decelerated the dissolution of the gel.Then,bFGF was released slowly,with its proportion reaching 95% of the total amount after 168 h (7 d),when it was almost completely released.However,the released amount of TGFβ3 did not exceed the detection threshold until 36 h (4.60% ±2.61%).At the end of the experiment(after 14 d),the released amount of TGFβ3 was observed to be 55% approximately.Thus,the viability of the proposed biomimetic binary delivery system was verified.

    3.4.Microenvironment mimicked by DFH to promote tubule formation,induce cell migration,and aid osteogenic differentiation of hPDLSCs

    The ability of DFH to promote microvascular formation and promote stem cell migration and differentiationin vitrowas evaluated using Calcein-AM/PI staining,tubule formation assay,crystal violet staining,and ALP staining.As illustrated in Fig.3B,hPDLSCs survived in the gel for more than 7 d After 24 h of culture,HUVECs formed tubular structures on DFH via ligation,migration,and differentiation.The tubular structures were particularly numerous and distinct in DFH containing 80 ng/ml bFGF(Fig.4A).Further,in the Transwell cell migration assay Fig.4B,the DFH group loaded with 250 ng/ml TGFβ3 recruited a higher number of cells compared to the other groups,indicating that DFH induced the migration of MSCs.In addition,the ALP staining of empty DFH was lighter,and the incorporation of TGFβ3 enhanced the pro-osteodifferentiation ability of DFH(Fig.3C).

    Fig.3-The release of GFs in vitro and the proliferation and osteogenic differentiation of hPDLSCs in DFH.(A).In vitro degradation and release curve of the DFH.(B).Surviving hPDLSCs in the DFH were observed via calcein-AM/PI staining,and the numbers of living and dead cells were counted using ImageJ(n=3).(C).The DFH loaded with hPDLSCs was cultured in the osteogenic induction medium for 7 and 14 d and then stained with alkaline phosphatase.

    Fig.4-DFH mimics the microenvironment,promotes the formation of tiny blood vessels,and recruits MSCs in vivo and in vitro.(A).HUVECs were cultured in the DFH for 24 h.Tubular structures were observed using an immunofluorescence microscope.(B).HF-MSC migration was induced by the DFH.After 24 h of culture,the number of migrated stem cells was evaluated via crystal violet staining.(C).In vivo cell recruitment by the DFH.Immunohistochemistry of vascular endothelial cell marker CD45 and the MSC marker CD29 on the recruited cells.The black arrow indicates cell clusters with positive staining of CD29 or CD45.DFH-L:80 ng/ml+1000 ng/ml TGFβ3;DFH-H:80 ng/ml+4000 ng/ml TGFβ3.(n=3,?P <0.05,??P <0.01,vs Control).

    Cell-friendly biomaterials incorporate key physicochemical cues that can instruct and govern cell behaviour bothin vitroandin vivo[33].In this study,in order to simulate the spatial and temporal characteristics of a natural cellular environment,we incorporated bFGF and TGFβ3 within the hydrogel system-bFGF was incorporated in the outer gel,which promoted the formation of new blood vessel network.The establishment of a new vascular network provided the requisite amount of oxygen and nutrients,and played a crucial role in removing waste products,laying the environmental foundation for new bone tissue.TGFβ3 was incorporated within the inner layer of microspheres,and its slow release not only guided the homing of stem cells but also guided the osteogenic differentiation of homing stem cells,thereby promoting complete regeneration of bone tissue.

    3.5.DFH-induced promotion of the formation of tiny blood vessels and recruitment of MSCs in vivo

    The repair of natural bone tissue defects can be primarily divided into four stages-vascularization and cell recruitment,cell proliferation and differentiation,cartilage formation and hard bone tissue formation,and bone remodelling.Typically,the vascularization and cell homing process is completed within 5 d approximately [34,35].Interestingly,the sequential dosing results described in Section 3.1 also corroborated better osteogenic differentiation ability of hPDLSCs following pre-treatment with bFGF for approximately 6 d before the administration of TGFβ3.The rat muscle pocket model was used to study the ability of DFH to promote microangiogenesis and recruit MSCsin vivo.Immunohistochemical analyses were performed to evaluate the expression of CD45 and CD29.

    CD45 is a hematopoietic stem cell marker.As a fibronectin receptor,CD45 participates in multiple cell-cell and cellmatrix interactions,and regulates a variety of important biological functions.In this study,DFH groups were observed to exhibit more positive CD45 cells(?P <0.05,vsboth the blank and TGFβ3 group).Similarly,the number of positive cells in the bFGF group exhibited no statistical difference,which indicates that bFGF played an important role during angiogenesis(Figs.4Cand 4D).The expression of CD29 is positively correlated with the migration of MSCs.The number of CD29 positive cells around the materials both in DFH and TGFβ3 groups were significantly higher than in other groups(Figs.4Eand 4F,?P <0.05).As illustrated in Fig.S4,the expression levels of CD29 and CD45 in the DFH-H group were significantly higher than those in the other groups.Yang et al.found that TGFβ3 helps bioscaffolds to recruit IPFSCsin vitroand supports cell settlement and chondrogenic differentiation of migratory cells [36].This study established that TGFβ3 is the crucial component in DFH that aids the recruitment of MSCs bothin vivoandin vitro.

    In short,DFH mimics the microenvironment of damaged tissue.It promotes the formation of tiny blood vessels and effectively recruits endogenous MSCs both in rat muscle pockets implanting expression and alveolar bone injuries in SD rats.The biocompatibility study revealed that DFH exhibits good biocompatibilityin vivo(Fig.S3).

    3.6.Repair of alveolar bone injury by DFH in SD rats

    Currently,research on stomatology tissue engineering is plagued by several problems-the lack of a satisfactory animal model is one of the foremost.In this study,the SD rat alveolar bone injury model was established by slightly revising the method outlined in[37](the modelling method is described in Section 2.10).Micro-CT was utilised to investigate the changes in alveolar bone loss during the testsin vivo.The loss of alveolar bone in the model group increased over time,and it did not heal naturally in 12 weeks(Figs.5Band 5C),which was confirmed by clinical dentists.Then,the proposed method was applied for subsequent experiments.

    Fig.5-Repair of alveolar bone defects in SD rats.(A).Operational flow chart of alveolar bone injury repair.(B).The model of alveolar bone injury was established.One week later,the alveolar bone injury of rats was observed via Micro-CT.In the figure,the yellow dotted line indicates the location of the normal alveolar bone,and the red dotted line indicates the area of the injury.(C).After 12 weeks of administration,the alveolar bone repair states of different groups of DFH were observed via Micro-CT.(D).After 12 weeks of administration,the alveolar bone defect areas of different groups of DFH were calculated.(E-H).The volume fraction,trabecular number,trabecular separation,and trabecular thickness of the newly formed alveolar bone were analysed using the CTAn software(n=3,?P <0.05,??P <0.01,vs Model).

    After 12 weeks of the operation,the alveolar bone defect in the blank gel group did not recover.Compared to the model and blank gel groups,DFH groups regenerated alveolar bone in the defective area,and the amount of new bone tissue in the DFH group were significantly higher than that in other groups(Fig.5D).The bone volume fraction,trabecular number,and trabecular thickness of new bone (analysed using the CTAn software) in DFH groups were significantly higher than those in the model and blank gel groups(?P <0.05).The DFH-H group exhibited the best treatment effect(Figs.5E-5H).

    The regenerative tissue was studied in greater detail using HE and Masson’s trichrome staining histological analyses(Fig.S7).HE staining of alveolar bone specimens at 12 weeks revealed the absence of any new bone formation at the defective sites in the model group and blank gel group.Bone resorption occurs at the bone defect site,with only a small amount of tissue fibre filling.New bone tissue in the DFH-H group was significantly greater than in other groups.The material in the bone defect was completely degraded,and a large amount of fibrous tissue was observed in the defect without any inflammatory cells.Masson staining revealed the low amount of blue fibrous tissue filling in the bone defect in the model and blank gel groups,with no obvious new bone formation.Compared to the model group,the DFH group,bFGF group and TGFβ3 group exhibited a large amount of blue fibrous tissue filling in the defect,with the undegraded material surrounded by blue fibrous tissue.New bone tissue was observed to have grown inwards from the edge of the bone defect.In summary,we successfully constructed a SD rat alveolar bone injury model.The model group were incapable of healing naturally.The DFH groups significantly promoted bone regeneration (?P <0.05).The results verified the operational viability of the DFH hydrogel system.

    For an active biological material,the ability to recruit stem cellsin vivois critical when the body is incapable of healing by itself.The proposed DFH hydrogel was verified to be capable of recruiting MSCs and repairing alveolar bone defects that are difficult to heal.The presence of DFH in alveolar bone defects promoted the formation of vascular networks and the expression of genes involved in the recruitment of MSCs confirms this view.Other studies have established that bFGF binds to FGFR receptors on the cell surface and activates VEGF-A to induce angiogenesis and provide necessary nutrients to injured sites.TGFβ3 plays a crucial role in guiding the migration and differentiation of MSCs.Pang et al.observed that TGFβ3 not only promotes the migration of hBMSCs directly through the TGFβsignalling pathway but also upregulates the secretion of MCP1 in vascular cells in a Smad3-dependant manner,thereby greatly enhancing the migration ability of TGFβ3 to hBMSCs [31].In addition,TGFβ3 promotes the osteogenic differentiation of MSCs by activating the p38-MAPK pathway,thereby initiating bone regeneration [22].However,most of the previous studies implemented a single growth factor or two simultaneous growth factors to promote tissue repair.In this study,a sequential drug delivery biomimetic carrier was constructed to further accelerate the repair of bone tissue by simulating the microenvironment of naturally damaged tissue.Although the sequential release system involving bFGF and BMP2 was reported by Ge et al.,they did not publish any reports on the more complex case of animal experiments[27].Besides,compared to BMP2,TGFβ3 not only promotes endochondral osteogenesis but also recruits endogenous stem cells.Therefore,we feel that this study expands the existing research and expect it to be helpful for future clinical use.

    4.Conclusion

    In this study,we simulated the inherent damage repair process of periodontal tissue and designed a self-repairing hydrogel system that releases bFGF and TGFβ3 sequentially.DFH is a porous (porosity: 84.3% ± 6.5%) hydrogel with good injectability and self-healing properties.Therefore,it is convenient for injection and administration in the narrow oral cavity.In addition,it has been established via gel-loaded cell experiments that DFH not only promotes tube formation by HUVECs and recruitment of MSCs but also promotes the osteogenic differentiation of hPDLSCs.We also successfully established a modified SD rat model of alveolar bone defect.After 12 weeks ofin situinjection of DFH into the alveolar bone defect,it was found that the DFH group promoted the repair of the alveolar bone injury to a much greater extent than the control group.In summary,DFH was verified to exhibit good bone repair capability.As it is convenient for injection and administration in the narrow oral cavity,it can be expected to be used in clinical practice.

    Conflicts of interest

    The authors report no conflicts of interest.The authors are responsible for the content and writing of this paper.

    Acknowledgments

    The authors would like to acknowledge the faculty and staff at the Biopharmaceutical R&D centre of Jinan University,especially Yangfan Li,an excellent postgraduate student of Jinan University.This work was supported by grants from the Guangzhou Science and Technology Program Key Project (Grant No.201803010044),Guangdong Province College Characteristic Innovation Project (2019KTSCX011),Guangdong Province Natural Sciences Fund Project(2021A1515012480),the Key Areas Research and Development Program of Guangzhou (202103030003),and Guangdong Province Special Fund Projects(Yueziranzihe,2021,No.50).

    Supplementary materials

    Supplementary material associated with this article can be found,in the online version,at doi:10.1016/j.ajps.2022.03.003.

    91麻豆av在线| 日本 av在线| 国产人伦9x9x在线观看| 99国产精品免费福利视频| 日韩中文字幕欧美一区二区| 精品卡一卡二卡四卡免费| 三级毛片av免费| 日本免费一区二区三区高清不卡 | 国产在线观看jvid| 热99国产精品久久久久久7| 色综合欧美亚洲国产小说| 国产精品自产拍在线观看55亚洲| av欧美777| 999久久久国产精品视频| 老司机在亚洲福利影院| 成人影院久久| 最新在线观看一区二区三区| 1024视频免费在线观看| 91成年电影在线观看| 搡老岳熟女国产| 三上悠亚av全集在线观看| 9热在线视频观看99| 多毛熟女@视频| 日本撒尿小便嘘嘘汇集6| 一级,二级,三级黄色视频| 日韩视频一区二区在线观看| 欧美在线黄色| 好男人电影高清在线观看| 亚洲五月色婷婷综合| 午夜日韩欧美国产| 国产精品久久电影中文字幕| 欧美乱妇无乱码| 99精国产麻豆久久婷婷| 丰满的人妻完整版| 亚洲成人精品中文字幕电影 | 国产精品98久久久久久宅男小说| 精品卡一卡二卡四卡免费| 亚洲色图综合在线观看| 最近最新中文字幕大全电影3 | 成熟少妇高潮喷水视频| 久久这里只有精品19| 欧美激情高清一区二区三区| a级片在线免费高清观看视频| 国产三级在线视频| 亚洲欧美激情在线| 9191精品国产免费久久| 搡老乐熟女国产| 一进一出抽搐动态| 欧美日韩乱码在线| 男女床上黄色一级片免费看| 婷婷精品国产亚洲av在线| 国产伦一二天堂av在线观看| 日韩欧美在线二视频| bbb黄色大片| 国产精品偷伦视频观看了| 大陆偷拍与自拍| 国产真人三级小视频在线观看| 天天躁夜夜躁狠狠躁躁| 久久精品亚洲精品国产色婷小说| 在线播放国产精品三级| 69精品国产乱码久久久| 人成视频在线观看免费观看| 亚洲精品粉嫩美女一区| 丁香欧美五月| 日本黄色视频三级网站网址| 国产熟女xx| √禁漫天堂资源中文www| 性少妇av在线| 在线观看免费午夜福利视频| 亚洲国产精品一区二区三区在线| 亚洲第一青青草原| 99re在线观看精品视频| 国产高清视频在线播放一区| 水蜜桃什么品种好| 亚洲午夜理论影院| 精品国内亚洲2022精品成人| 久久伊人香网站| 制服诱惑二区| 性色av乱码一区二区三区2| 99在线人妻在线中文字幕| 日韩精品免费视频一区二区三区| 国产亚洲精品第一综合不卡| 纯流量卡能插随身wifi吗| 亚洲色图av天堂| 搡老乐熟女国产| 久久久国产成人精品二区 | 精品第一国产精品| 最新在线观看一区二区三区| 免费搜索国产男女视频| 琪琪午夜伦伦电影理论片6080| 另类亚洲欧美激情| 操美女的视频在线观看| 俄罗斯特黄特色一大片| 中文字幕人妻丝袜一区二区| 在线观看一区二区三区| 新久久久久国产一级毛片| 亚洲精品成人av观看孕妇| 在线观看66精品国产| 岛国在线观看网站| 黄色毛片三级朝国网站| 搡老乐熟女国产| 精品久久蜜臀av无| 欧美中文综合在线视频| 99久久国产精品久久久| 欧美精品一区二区免费开放| 男女下面插进去视频免费观看| av有码第一页| 在线免费观看的www视频| 日本欧美视频一区| 国产av在哪里看| 乱人伦中国视频| 动漫黄色视频在线观看| 香蕉久久夜色| 一级毛片精品| www.精华液| 老司机午夜十八禁免费视频| 国产精品成人在线| 久久亚洲真实| 免费观看人在逋| 欧美日韩亚洲综合一区二区三区_| 国产蜜桃级精品一区二区三区| 波多野结衣高清无吗| 一边摸一边做爽爽视频免费| 777久久人妻少妇嫩草av网站| 国产精品98久久久久久宅男小说| 国产视频一区二区在线看| 欧美 亚洲 国产 日韩一| 欧美久久黑人一区二区| 久久久久久久久免费视频了| 91大片在线观看| 法律面前人人平等表现在哪些方面| 少妇的丰满在线观看| 很黄的视频免费| 操美女的视频在线观看| 婷婷丁香在线五月| 欧美成人免费av一区二区三区| 最新在线观看一区二区三区| 18禁黄网站禁片午夜丰满| 午夜精品在线福利| av超薄肉色丝袜交足视频| 神马国产精品三级电影在线观看 | 老司机午夜十八禁免费视频| 无人区码免费观看不卡| 一边摸一边抽搐一进一小说| 男人操女人黄网站| 日本一区二区免费在线视频| 99久久精品国产亚洲精品| 非洲黑人性xxxx精品又粗又长| 日本黄色片子视频| 成人鲁丝片一二三区免费| 校园春色视频在线观看| 尤物成人国产欧美一区二区三区| 老女人水多毛片| 淫妇啪啪啪对白视频| 日日摸夜夜添夜夜添av毛片 | 小蜜桃在线观看免费完整版高清| 美女被艹到高潮喷水动态| 人人妻人人看人人澡| 两性午夜刺激爽爽歪歪视频在线观看| 欧美不卡视频在线免费观看| 亚洲人成网站在线播放欧美日韩| 国产爱豆传媒在线观看| 丁香欧美五月| 一进一出好大好爽视频| 麻豆国产97在线/欧美| 我要看日韩黄色一级片| 日韩av在线大香蕉| 一本久久中文字幕| 老司机午夜十八禁免费视频| 人妻久久中文字幕网| 国产综合懂色| 麻豆一二三区av精品| 好看av亚洲va欧美ⅴa在| 黄色配什么色好看| 不卡一级毛片| 波多野结衣高清无吗| 午夜福利欧美成人| 亚洲,欧美精品.| 午夜福利免费观看在线| 久久久精品欧美日韩精品| 综合色av麻豆| 成人无遮挡网站| 午夜福利视频1000在线观看| 久久九九热精品免费| 日本一二三区视频观看| 欧美三级亚洲精品| 夜夜看夜夜爽夜夜摸| 一区二区三区高清视频在线| 国产野战对白在线观看| 国产精品久久久久久精品电影| 亚洲国产欧洲综合997久久,| 一个人免费在线观看的高清视频| 天美传媒精品一区二区| 91在线精品国自产拍蜜月| 中国美女看黄片| 国产亚洲精品久久久com| 亚洲人成电影免费在线| 精品日产1卡2卡| 久久伊人香网站| 啦啦啦观看免费观看视频高清| 人人妻人人看人人澡| 亚洲一区二区三区不卡视频| 亚洲色图av天堂| 熟女电影av网| 97碰自拍视频| 色噜噜av男人的天堂激情| 神马国产精品三级电影在线观看| 少妇丰满av| 亚洲av美国av| a级毛片a级免费在线| 成人午夜高清在线视频| 岛国在线免费视频观看| 亚洲最大成人手机在线| 99精品久久久久人妻精品| 久久久国产成人精品二区| 可以在线观看毛片的网站| 久久精品国产亚洲av涩爱 | 麻豆久久精品国产亚洲av| 午夜影院日韩av| 我的女老师完整版在线观看| 极品教师在线免费播放| 禁无遮挡网站| 亚洲美女搞黄在线观看 | 国产一区二区三区视频了| 国产男靠女视频免费网站| 国产精品一区二区三区四区久久| 18禁在线播放成人免费| 日韩高清综合在线| 在线看三级毛片| 亚洲在线自拍视频| 搞女人的毛片| 12—13女人毛片做爰片一| eeuss影院久久| 亚洲熟妇中文字幕五十中出| 欧美日韩乱码在线| 狂野欧美白嫩少妇大欣赏| 亚洲男人的天堂狠狠| 午夜视频国产福利| 国产精品一区二区三区四区免费观看 | 伊人久久精品亚洲午夜| 在线天堂最新版资源| 无遮挡黄片免费观看| 亚洲18禁久久av| 免费人成视频x8x8入口观看| 午夜精品久久久久久毛片777| 内地一区二区视频在线| 欧美高清成人免费视频www| 久久香蕉精品热| 无遮挡黄片免费观看| 90打野战视频偷拍视频| 国产亚洲av嫩草精品影院| 尤物成人国产欧美一区二区三区| 9191精品国产免费久久| 日本黄色视频三级网站网址| 欧美在线黄色| av在线天堂中文字幕| 国产亚洲精品久久久com| 国产精品伦人一区二区| 免费人成在线观看视频色| 欧美最新免费一区二区三区 | 午夜福利免费观看在线| 久久九九热精品免费| 天堂影院成人在线观看| 黄色一级大片看看| 日韩欧美 国产精品| 国产伦精品一区二区三区视频9| 亚洲av日韩精品久久久久久密| 在线观看午夜福利视频| 亚洲精品456在线播放app | 久久伊人香网站| 啦啦啦韩国在线观看视频| 日韩精品中文字幕看吧| 最好的美女福利视频网| 十八禁网站免费在线| 亚洲五月天丁香| 桃色一区二区三区在线观看| 啦啦啦韩国在线观看视频| 中文亚洲av片在线观看爽| 久久精品国产亚洲av香蕉五月| 亚洲性夜色夜夜综合| 热99re8久久精品国产| 九色国产91popny在线| 少妇熟女aⅴ在线视频| 亚洲专区中文字幕在线| 一进一出抽搐gif免费好疼| 色噜噜av男人的天堂激情| 淫秽高清视频在线观看| 能在线免费观看的黄片| 亚洲av不卡在线观看| 韩国av一区二区三区四区| 国产爱豆传媒在线观看| 国产av不卡久久| 欧美xxxx黑人xx丫x性爽| 亚洲一区高清亚洲精品| 欧美高清成人免费视频www| 亚洲精品在线观看二区| 国产一区二区激情短视频| 亚洲人成电影免费在线| 免费人成在线观看视频色| 日本黄大片高清| 亚洲自偷自拍三级| 97热精品久久久久久| 精品99又大又爽又粗少妇毛片 | 老师上课跳d突然被开到最大视频 久久午夜综合久久蜜桃 | 午夜福利高清视频| 国产精品一及| 桃红色精品国产亚洲av| 老女人水多毛片| 中文亚洲av片在线观看爽| 97人妻精品一区二区三区麻豆| 99国产综合亚洲精品| 国产视频内射| 国产精品亚洲美女久久久| 国产男靠女视频免费网站| 亚洲狠狠婷婷综合久久图片| 日韩 亚洲 欧美在线| 波多野结衣巨乳人妻| 日韩人妻高清精品专区| 国产黄a三级三级三级人| 在现免费观看毛片| 中文字幕久久专区| 亚洲成人久久性| 床上黄色一级片| 欧美色视频一区免费| 欧美一级a爱片免费观看看| 国产视频一区二区在线看| avwww免费| 一个人观看的视频www高清免费观看| 亚洲精品456在线播放app | 国产单亲对白刺激| 国产探花在线观看一区二区| 全区人妻精品视频| 久久精品国产清高在天天线| 一区二区三区四区激情视频 | 一个人免费在线观看的高清视频| 最近最新免费中文字幕在线| 欧洲精品卡2卡3卡4卡5卡区| 成人高潮视频无遮挡免费网站| 国产高清视频在线播放一区| 狂野欧美白嫩少妇大欣赏| 亚洲av一区综合| 亚洲一区高清亚洲精品| 韩国av一区二区三区四区| 亚洲 国产 在线| www.色视频.com| 欧美成狂野欧美在线观看| 给我免费播放毛片高清在线观看| 国产综合懂色| 亚洲av二区三区四区| 欧美bdsm另类| 国产伦人伦偷精品视频| 日日干狠狠操夜夜爽| 嫁个100分男人电影在线观看| 国产69精品久久久久777片| 日日夜夜操网爽| 欧美+亚洲+日韩+国产| 久久精品影院6| 国产精华一区二区三区| 可以在线观看毛片的网站| 久久欧美精品欧美久久欧美| 有码 亚洲区| 少妇人妻精品综合一区二区 | 日本黄大片高清| 亚洲美女视频黄频| 欧美性猛交╳xxx乱大交人| 久久午夜亚洲精品久久| 老司机福利观看| 中文亚洲av片在线观看爽| 婷婷六月久久综合丁香| 啪啪无遮挡十八禁网站| 很黄的视频免费| 成人一区二区视频在线观看| 亚洲电影在线观看av| 精品人妻1区二区| 亚洲av熟女| 乱码一卡2卡4卡精品| 熟妇人妻久久中文字幕3abv| 美女免费视频网站| 色精品久久人妻99蜜桃| 淫妇啪啪啪对白视频| 日韩免费av在线播放| 亚洲熟妇熟女久久| 成人鲁丝片一二三区免费| 欧美+日韩+精品| 动漫黄色视频在线观看| 村上凉子中文字幕在线| 99国产精品一区二区蜜桃av| 蜜桃亚洲精品一区二区三区| 亚洲午夜理论影院| 两个人的视频大全免费| 免费av毛片视频| 99riav亚洲国产免费| 国产精品永久免费网站| 亚洲第一区二区三区不卡| 国产成年人精品一区二区| 午夜福利视频1000在线观看| 免费在线观看日本一区| 欧美区成人在线视频| 亚洲,欧美精品.| 国产成人aa在线观看| 久久精品国产99精品国产亚洲性色| 精品久久久久久久人妻蜜臀av| 亚洲中文字幕日韩| 亚洲片人在线观看| 夜夜爽天天搞| 欧洲精品卡2卡3卡4卡5卡区| 日本黄大片高清| 日本精品一区二区三区蜜桃| 国产欧美日韩一区二区精品| 精品一区二区三区av网在线观看| 黄色配什么色好看| 国产不卡一卡二| 亚洲成av人片免费观看| av在线老鸭窝| 日本黄大片高清| 成年女人永久免费观看视频| 最新在线观看一区二区三区| 一本综合久久免费| 亚洲片人在线观看| 色精品久久人妻99蜜桃| 日本一本二区三区精品| 成人毛片a级毛片在线播放| 亚洲内射少妇av| 国产日本99.免费观看| 91狼人影院| 久久人人精品亚洲av| 国内毛片毛片毛片毛片毛片| www.www免费av| 国产精品美女特级片免费视频播放器| 国产伦精品一区二区三区四那| 国内少妇人妻偷人精品xxx网站| 观看美女的网站| 国产精品永久免费网站| 三级男女做爰猛烈吃奶摸视频| 中文字幕人成人乱码亚洲影| 亚洲精品久久国产高清桃花| 国产精品永久免费网站| 国语自产精品视频在线第100页| 久久人人爽人人爽人人片va | 久久人人爽人人爽人人片va | 午夜福利视频1000在线观看| 人妻久久中文字幕网| 三级男女做爰猛烈吃奶摸视频| 亚洲无线在线观看| 成年女人毛片免费观看观看9| 欧美日韩国产亚洲二区| 在线观看免费视频日本深夜| 五月伊人婷婷丁香| 熟妇人妻久久中文字幕3abv| 无遮挡黄片免费观看| 长腿黑丝高跟| 麻豆国产97在线/欧美| 一级黄片播放器| 色综合婷婷激情| 97人妻精品一区二区三区麻豆| 中亚洲国语对白在线视频| 国产久久久一区二区三区| 在线十欧美十亚洲十日本专区| 在线观看av片永久免费下载| 国产成年人精品一区二区| 美女cb高潮喷水在线观看| 久久国产精品人妻蜜桃| 久久香蕉精品热| 久久这里只有精品中国| 麻豆国产97在线/欧美| or卡值多少钱| 午夜福利在线观看吧| 久久久久性生活片| 三级毛片av免费| 免费在线观看亚洲国产| 久久久精品大字幕| 欧美日韩综合久久久久久 | 国产亚洲av嫩草精品影院| 中文字幕久久专区| 日本五十路高清| 欧美成人一区二区免费高清观看| 观看免费一级毛片| 亚洲精品一区av在线观看| 亚洲欧美日韩卡通动漫| 可以在线观看的亚洲视频| 欧美最新免费一区二区三区 | 热99在线观看视频| 欧美日韩乱码在线| 国产在线精品亚洲第一网站| 亚洲av二区三区四区| 黄片小视频在线播放| 午夜福利视频1000在线观看| 国产欧美日韩精品一区二区| 嫩草影视91久久| 超碰av人人做人人爽久久| 亚洲国产精品久久男人天堂| 麻豆成人av在线观看| 精品一区二区免费观看| 禁无遮挡网站| 亚洲人成网站高清观看| av在线蜜桃| 免费观看人在逋| av黄色大香蕉| 国产高清视频在线播放一区| 亚洲av第一区精品v没综合| 内地一区二区视频在线| 成人特级黄色片久久久久久久| 久久精品综合一区二区三区| 欧美3d第一页| 亚洲人成网站在线播放欧美日韩| 禁无遮挡网站| 亚洲av日韩精品久久久久久密| 三级毛片av免费| 国产精品99久久久久久久久| avwww免费| 在线观看美女被高潮喷水网站 | 中文字幕精品亚洲无线码一区| 在线观看舔阴道视频| 少妇被粗大猛烈的视频| 中国美女看黄片| 免费av观看视频| 国产精品一区二区三区四区免费观看 | 麻豆成人av在线观看| 深爱激情五月婷婷| 级片在线观看| 婷婷色综合大香蕉| 一本一本综合久久| 黄色配什么色好看| 深夜精品福利| 首页视频小说图片口味搜索| av福利片在线观看| 亚洲激情在线av| 欧美色欧美亚洲另类二区| 精品一区二区三区人妻视频| 午夜亚洲福利在线播放| 亚洲人成网站高清观看| 日韩成人在线观看一区二区三区| 丝袜美腿在线中文| 亚洲三级黄色毛片| 久久久久久久久久黄片| xxxwww97欧美| 三级男女做爰猛烈吃奶摸视频| 精品午夜福利在线看| 桃色一区二区三区在线观看| 国产免费一级a男人的天堂| 乱人视频在线观看| 国内久久婷婷六月综合欲色啪| 韩国av一区二区三区四区| 色综合亚洲欧美另类图片| 亚洲精品456在线播放app | 特大巨黑吊av在线直播| 国产视频一区二区在线看| 黄色视频,在线免费观看| 99热这里只有是精品50| 免费av不卡在线播放| 丝袜美腿在线中文| 好看av亚洲va欧美ⅴa在| 97超视频在线观看视频| 色视频www国产| 久久热精品热| 天堂动漫精品| 最近最新中文字幕大全电影3| 精品久久久久久,| 深爱激情五月婷婷| 亚洲美女搞黄在线观看 | 国产精品人妻久久久久久| 婷婷色综合大香蕉| 久久99热6这里只有精品| 亚洲欧美日韩东京热| 亚洲精品日韩av片在线观看| 午夜免费激情av| 亚洲av二区三区四区| 我要搜黄色片| 成人精品一区二区免费| 一进一出好大好爽视频| 亚洲avbb在线观看| 日韩精品青青久久久久久| 国产人妻一区二区三区在| 亚洲国产精品合色在线| 九九热线精品视视频播放| 级片在线观看| 男女做爰动态图高潮gif福利片| 如何舔出高潮| 亚洲 国产 在线| 嫩草影院入口| 亚洲va日本ⅴa欧美va伊人久久| 我要搜黄色片| 国产午夜精品论理片| 国内毛片毛片毛片毛片毛片| 午夜日韩欧美国产| 少妇裸体淫交视频免费看高清| 欧美乱色亚洲激情| 亚洲七黄色美女视频| 搞女人的毛片| 51午夜福利影视在线观看| 亚洲欧美日韩东京热| 国产av不卡久久| 午夜精品一区二区三区免费看| 黄色女人牲交| 欧美高清性xxxxhd video| 国产一区二区在线观看日韩| 日本撒尿小便嘘嘘汇集6| 久久国产精品影院| 久久午夜福利片| 亚洲色图av天堂| 嫩草影院入口| 黄色配什么色好看| 精品久久久久久,| 97热精品久久久久久| 99国产精品一区二区三区| 欧美性感艳星| 人妻丰满熟妇av一区二区三区| 亚洲精品成人久久久久久| 欧美极品一区二区三区四区| 精品不卡国产一区二区三区| 综合色av麻豆| 免费在线观看亚洲国产| 深夜a级毛片| 俺也久久电影网| 白带黄色成豆腐渣|