Experimental Study Of MMP-8-responsive Injectable Hydrogel For Intraoral Drug Delivery And Bone Regeneration

Oral cavity is the beginning of human digestive system.Oral health and beauty are of great importance for general health and well-being.Oral diseases,such as caries,trauma,and periodontitis cause worldwide health problems and economic losses.Periodontal disease,with 90%of the world population affected,was deemed to be the leading cause of tooth loss in adult.Periodontitis is an inflammatory disease of teeth supporting tissues caused by initial accumulation of sub-gingival bacteria,released toxins and enzymes,as well as the stimulation of body’s immune response.The progression of periodontitis is characterized by destruction of periodontal ligaments,formation of periodontal pocket and the resorption of alveolar bone.Finally,all the teeth support structures were disrupted.In analogy to periodontitis affecting the periodontal tissue of natural teeth,the tissue inflammation surrounding dental implant is termed as peri-implantitis.With dental implant becoming an increasing popular therapy to replace missing teeth,more evidence raised on the frequent complications of peri-implant inflammations,which could lead to the total failure of implants.Therefore,strategies for prevention and treatment of periodontal and peri-implant diseases are of great significance and urgency.Plaque and its products are considered as the initiating factors of periodontitis and periimplant inflammation.The interaction of plaque accumulation,toxin,enzyme release and immune response promotes the development of periodontal disease.The progression of periodontitis is characterized by periodontal ligament destruction,periodontal pocket formation and alveolar bone resorption.Periodontal tissue regeneration is the ultimate goal of periodontal therapy,but at present the treatment of periodontal disease is mainly engaged in infection control while lack of the ability of tissue regeneration and repair.Recovering the periodontal tissue structure and function is difficult to achieve and periodontal tissue regeneration becames the promising method to achieve the oral health.Thus,it is of great value to develop a treatment scheme that can both inhibit the development of inflammation and promote periodontal regeneration.The particularities of mouth,such as the movement of the tongue,saliva scour,make oral dosing system face with special requirements.Using the materials which can be implemented and self-regulated according to the special microenvironment.Biologically responsive materials showed obvious advantages,which has become a new trend in the design of a new type of biomedical materials.This study focused on the significantly increased MMP-8 during the development of periodontitis.According to this,the aim of this study was to develop an MMP-8 responsive material:an injectable hydrogel formed with MMP-8 sensitive peptide as the crosslink and acrylic acid modified PEG as the frame.The physical and chemical properties,biodegradability and biocompatibility of the hydrogel were evaluated.The drug release characteristics were investigated to measure its use as a drug carrier.Drugs and proteins were loaded to investigate its potential applications as tissue regeneration scaffolds.This material can achieve self-regulation according to the concentration of MMP-8 in the microenvironment and achieve on-demand release of loaded drugs,showing its advantages in the medical applications.Specific works have been carried out as follows:Part 1 Synthesis of the MMP-8 responsive hydrogelObjectives:To synthesize the MMP-responsive hydrogel by using acrylic PEG as skeleton and the MMP-8-specific peptide as crosslinker.To evaluated the morphology,performance and biosafety of the responsive gel system.Experiment 1:Synthesis and characterization of the peptidesMaterials and methods:Fmoc solid phase synthesis method was used to synthesize the MMP-8 sensitive peptide and non-specific peptide.After the successful synthesis of the aimed peptides were confirmed by mass spectrometry,the purity of the synthesized peptide was detected and purified by HPLCResult:The MMP-8 sensitive peptide of CGPQGIWGQC and a non-specific peptide of CGDQGIAGFC were successfully synthesized.The peptides were purified by HPLC and the purity degrees were higher than 95%,providing a basis for the development of the MMP-8 responsive hydrogel.Experiment 2:Construction and characterization of the hydrogel systemMaterials and methods:The acrylic modified PEG was crosslinked with the specific peptide to synthesize the MMP-8 responsive hydrogels,hydrogel produced by nonspecific peptide was control.Vial-tilting method was used to detect gelling time,rheometer was used to measure the gelling process and SEM was used for the gel morphology observations.FTIR,swelling ration test and degradation time experiments were used to characterize the gel and investigate the gel properties in vitro,such swelling,responsive degradation.MTT and the cell adhesion on the gel surface were also evaluated to determine biological safety of the gels.Results:Vial-tilting experiments showed the synthesized MMP-8 responsive hydrogel gelled fast in 5 minutes at 37℃;The rheological performance is good,SEM showed the gel has a high porous and interconnected microstructure,the pore size is about 100 microns;It has good swelling property.The degradation time of the gel was about one week in vitro,and the presence of MMP-8 could accelerate the degradation of the gel.With the increase of MMP-8 concentration,the degradation was further accelerated.MTT and cell adhesion assay confirmed that the gel had good biosafetyConclusions:The MMP-8 responsive hydrogel can be developed with MMP-8 sensitive peptide and PEG-DA.The high porous and interconnected microstructure,good rheological,swelling and MMP-8 responsive degradation properties of MMP-8 sensitive hydrogel make the gel a good material as drug carrier or tissue regeneration scaffold,illustrating its good application prospect in oral drug delivery system and cell and tissue regeneration.Part 2 Application of MMP-8 responsive in-situ injectable gel for intraoral drug deliveryObjectives:To evaluate the application of MMP-8 responsive gel as drug controlled release carrier for on-demand drug delivery and the effect of drug loading on its release behavior.Experiment 3:Drug loading,release behavior and activity evaluation Materials and methods:Minocycline hydrochloride(MH),bovine serum albumin(BSA)and antimicrobial polypeptide KSL were loaded as model drugs on MMP-8 responsive gel(M8SH)at a ratio of lmg/100uL,respectively.FTIR,in vitro swelling test,degradation test,MTT and gel surface cell adhesion test were used to investigate the effects of drug loading on gel formation time,gel formation process,gel morphology,FTIR,swelling characteristics,degradation characteristics and biosafety.The controlled release and released drug activity were evaluated by detecting the amount of released drug and antibacterial activity of collected solutions.Results:MMP-8 responsive gels loaded with small molecule model drug MH(M8SH+MH),large molecule model drug BSA(M8SH+BSA)and peptide model drug KSL(ABPM8SH)were successfully developed.The gels loaded with MH and BSA gelled within 5 minutes,and gels loaded with KSL turned into gels within 8 minutes.The loading of MH and BSA didn’t affect its rheological behaviors,while KSL loading had a slight effect on its rheological performance.The water absorption and swelling rations of M8SH,M8SH+MH,M8SH+BSA and ABPM8SH were 28.52,27.56,30.24 and 23.18,respectively.The SEM apertures were 105±10.4um,100±15.7um,103 ±3.4um and 86±10.8um.The degradation time of M8SH,M8SH+MH and M8SH+BSA in PBS were more than 180 h and ABPM8SH was more than 132 h.The presence of MMP-8 can promote the degradation of gels and the release rate of drugs.Conclusions:M8SH can be used as a carrier to deliver and control release of drugs.The simple drug encapsulation does not affect the gel formation time,rheological properties,FTIR,SEM morphology,swelling ration and degradation time of M8SH.The polypeptide as the crosslinker will have an impact on the behaviors of the gels.However,regardless of the loading mode,M8SH maintained good biosafety,drug activity and MMP-8 responsive degradation characteristics.The increase of MMP-8 concentration would promote the degradation of gel and drug release.It is important to note that when M8SH is used as a small molecule drug carrier,drug burst release is obvious,but when it was used as a macromolecular drug or peptide drug carrier,more accurate MMP-8 responsive release can be achieved.As a result,the M8SH could be a good macromolecular drug carrier,and also showed its advantages as tissue engineering scaffold materials.Part 3 Experimental study of MMP-8 responsive hydrogel on bone regenerationObjectives:To mimic extracellular matrix,a dual-release drug delivery system was constructed by drug loading after the MMP-8 responsive gel was modified by adhesive polypeptide RGD.The new smart tissue regeneration scaffold was developed through RGD modification,antimicrobial agents and biological factor loading to evaluate its potential application in tissue regeneration.The bone repair effect was measured by in vitro cell experiments and in vivo animal experiments.Experiment 4:Establishment and characterization of MMP-8 responsive hydrogel tissue regeneration scaffoldMaterials and methods:Minocycline hydrochloride loaded mesoporous silica nanoparticles(MH@MSN)and recombinant bone morphogenesis protein(rhBMP2)were loaded in MMP-8 responsive gel after RGD modification to develop an antimicrobial agents and growth factor double controlled release gel system.Thermogravimetric analysis was used to measure the MH loading.Swelling measurements and SEM were used to evaluate the morphology and physical and chemical characteristics of the gel.The controlled release behaviors of the composite gel were tested by release assay.Results:The composite gel system loaded with MH@MSN and rhBMP-2 was successfully developed.Release assay confirmed that M8SH could be used to develop a double release gel system.The characteristics of rapid gel formation,good hydrophilcity,large pore size,MMP-8 responsive drug release and degradation showed the gel was suitable scaffold for tissue regeneration.Experiment 5:Effects and mechanisms of the composite hydrogel on osteogenesisMaterials and methods:CCK-8 assay was used to evaluate the effects of the composite gel on the proliferation of BMSCs.The cell adhesion on the gel surface was measured to evaluate the biosafety of the gel scaffold.ALP activity assay,ALP staining and Alizarin red staining were used to evaluate the effecct and mechanism of the composite gel on BMSCs osteogenic differentiation to preliminarily explore the application of the MMP-8 responsive hydrogel as tissue regeneration scaffold to repair bone defects.Results:CCK-8 and cell adhesion experiments showed the composite hydrogel had good biocompatibility and could promote BMSCs adhesion and proliferation.ALP activity test,ALP staining and Alizarin red staining showed this hydrogel could promote osteogenic differentiation of BMSCs.Experiment 6:In vivo evaluation of bone tissue regeneration of the composite gel scaffoldMaterials and methods:The experimental skull defect model of SD rats was established.Composite gel scaffolds were developed with the methods used in experiment 4 and 5 and injected into the skull defect of rats.After 8 weeks,the bone defect was evaluated by Micro-CT,HE and MASSON staining to measure the bone tissue repair effect of the composite hydrogel.Results:Micro-CT results showed the volume fraction of new bone in the composite gel scaffold group was higher than that in the control group(p<0.05),which proved that it could promote the formation of new bone,but the new bone did not fill the whole defect area.The MASSON staining and HE staining showed the implantation of composite gel(the experimental group)could promote the healing of the defect,showing a trend of bone regeneration and repair.Conclusion:The developed stimulus-responsive composite gel scaffold has the effect of promoting bone tissue regeneration,showing its good prospect in tissue regeneration,but more studies should be conducted in the future to optimize its physicochemical properties to improve its bone regeneration efficiency.