Study On The In Situ Engineered Hydrogel For Loading PGRN To Promote Regeneration Of Inflammatory Periodontal Bone Defect

The aim of in situ periodontal tissue engineering is creating a suitable microenvironment for proliferation and differentiation of seed cells in vivo by using biological materials or their combining with growth factors,stimulate the body’s own periodontal repair potential,thus to promote periodontal tissue reconstruction.It is a novel research direction of periodontal tissue regeneration.Periodontal ligament stem cells(PDLSCs)are the main functional cells for periodontal regeneration and target cells for induction of in situ tissue engineering.However,the osteogenic property of PDLSCs is affected by the inflammatory microenvironment.Immune factors,especially the imbalance of macrophage polarization and T cell differentiation,are important factors affecting the stability of alveolar bone.Therefore,the ideal periodontal regeneration strategy should have multiple functions such as antiinflammatory,immune regulation,and promoting the osteogenic differentiation.Progranulin(PGRN)has been attracted much attention due to their diverse biological activities.Our previous studies have also demonstrated that exogenous PGRN shows immune regulation and bone generation in the treatment of periodontitis,suggesting that it may be an important factor in promoting periodontal regeneration.However,like other bioactive factors,PGRN has a short half-life and is easily degraded by a variety of enzymes.Exploring suitable scaffold materials as a controlled release system of PGRN is an urgent problem to realize its application in periodontal tissue regeneration.The ideal scaffold material requires good biocompatibility and proper biodegradability,and it could also enhance osteogenic activity and immune regulation.Hydrogels have a highly three-dimensional network of highly hydrates,and the structure of their macromolecules is very similar to that of extracellular matrix(ECM)in vivo,which mimic ECMs of natural tissues so as to be more imitative than other forms.In periodontal tissue engineering,injectable hydrogels can not only be highly adapted to the irregular periodontal bone defect morphology but also provide the possibility for regular injection in periodontal pocket for minimally invasive treatment during periodontal regeneration.Natural biological macromolecules hyaluronic acid(HA)and carboxymethyl chitosan(CMCTS)are often used for preparation of bioengineering scaffolds,drug carriers and biological dressings because of their excellent biomedical properties.Introducing aldehyde groups into part of HA is the most simple and effective way to self-crosslink CMCTS-HA to form injectable hydrogels.However,this composite hydrogel based on dynamic covalent chemical crosslinking has not been applied to treat periodontal bone defects.In this study,the role of the scaffold in periodontitis bone defects and its use as a controlled release carrier of PGRN were studied.Based on the conformed bioactivity of CMCTS,aldehyde-modified HA was prepared and covalent reacted with the amino group of CMCTS to prepare injectable in situ self-crosslinked CMCTS-HA composite hydrogel(CA).The effect of PGRN-loading CA on osteogenesis and immunomodulation were systematically studied in order to provide meaningful reference for the selection of scaffold materials and growth factors in periodontal in situ tissue engineering.Materials and Methods:1.Effects of CMCTS or Carboxymethy Chitin on the proliferation and differentiation of PDLSCsThe human periodontal ligament stem cells(hPDLSCs)were treated with CMCTS or carboxymethyl chitin(CMCT),respectively.Cell viability,pluripotent differentiation ability and mineralization ability,as well as the gene and protein expression of classic bone forming molecules alkaline phosphatase(ALP),osteopontin(OPN),runt related transcription factor 2(Runx2)and osteocalcin(OCN)were determined to analyze the effects of CMCTS and CMCT on proliferation and osteogenic differentiation of hPDLSCs.2.Preparation,characterization and osteogenic activity of injectable in situ dynamic hydrogel CAThe hyaluronic acid aldehyde(HAD)was prepared by oxidation of hyaluronic acid(HA)with sodium periodate,and the formation of aldehyde group was characterized by reaction with Fehling’s solution and Fourier Transform infrared spectroscopy(FTIR).In situ hydrogel(CA)was prepared by mixed HAD and CMCTS with extrusion from double syringes.The chemical structure,microscopic morphology,rheology,self-healing ability,cytocompatibility,histocompatibility and osteogenic properties of the hydrogels with different oxidation degrees HAD and CMCTS were systematically characterized to screen the suitable injectable hydrogel for subsequent loading of bioactive factors.3.The role of CA combined with or without PGRN in the regeneration of inflammatory periodontal bone defect and immune regulationIn situ CA hydrogel was prepared by the mixture of CMCTS and HAD2,and the CA membrane was prepared by freeze-dried compaction method.The release of PGRN from PGRN-loading CA hydrogel and membrane was detected with BSA as the control.The osteogenic effect of CA combined with PGRN on hPDLSCs was investigated in vitro.In addition,CA membrane combined with PGRN was implanted into the rat skull defect model to detect osteogenic ability in a non-inflammatory environment by MicroCT and histology.The polarization of mouse macrophage cell line RAW264.7 was induced by LPS and IL-4,respectively in vitro.The expression levels of M1 polarization factors(iNOS,TNF-α and IL-1β)and M2 polarization factors(TGF-β,Arg1 and IL-10)were detected by qRT-PCR to analyze the effects of CA+PGRN on the polarization state of macrophages.And PGRN-loading CA hydrogel was applied to the periodontitis related bone defect model.HE staining,Masson staining and immunohistochemistry were performed to analyze the repair of periodontal bone defect in inflammatory environment.The polarization of macrophage and Th17/Treg cell balance were determined by qRT-PCR and flow cytometry to study the immunomodulatory effects on the microenvironment of inflammatory periodontal bone defect.Results:1.The effect of CMCTS or CMCT on the proliferation and differentiation of hPDLSCsIn this study,100 μg/mL of CMCTS and CMCT significantly promoted the proliferation and clonal formation of hPDLSCs.The mineral synthesis ability of the CMCTS group was significantly higher than that of the other groups.Through further analysis of the osteogenic inducers,it was showed that CMCTS combined with conditioned medium for osteogenic induction could promote osteogenic differentiation of hPDLSCs from 7 to 21 d.The gene expression levels of osteoblast differentiation markers ALP,RUNX2,OPN and OCN were significantly increased in group of CMCTS and the protein expression levels of ALP and OPN were consistent with the gene expression data.However,the osteogenic effect of CMCT was lower than that of CMCTS because it mainly induced the increase of ALP expression.2.The characterization and osteogenic differentiation activity of in situ selfcrosslinked network dynamic hydrogel CAHAD was prepared by sodium periodate oxidation in ethanol-water mixed phase,which is simple and easy to operate.The presence of aldehyde group was proved by FTIR and Fehling reagent.HAD reacted with CMCTS by Schiff base and trigger gelation,which was proved by infrared spectroscopy.Through detecting the characterization of gelatinization,water absorption,swelling degree,fluidity and selfhealing ability,it was found that hydrogel could be formed quickly when extruded from the double syringe.However,the hydrogels with different crosslinking degrees were related to their gel formation time,viscosity,porous structure,self-healing ability and cytotoxicity.Therefore,the HAD/CMCTS hydrogels(CA)with the best crosslinking degree were selected.In vivo degradation experiment found that the material implanted into the subcutaneous tissue and muscle of rats did not cause obvious fibrotic envelope.HE staining showed that the inflammation gradually subsided after 10 days,and the material basically degraded and the inflammation subsided after 20 days,indicating that CA has good histocompatibility.The results of in vitro osteogenesis experiments showed that the addition of CA compounds to the osteogenic induction culture system could significantly promote the expressions of ALP,OPN and Collagen I.It also promoted mineral deposition during bone generation.3.The effect of CA combined with PGRN on the regeneration of inflammatory periodontal bone defect and immune regulationModerately crosslinked CA hydrogel and freeze-dried membrane had a good slowrelease effect on PGRN,and release active factors.The results of in vitro osteogenic experiments showed that CA and the combined effect of PGRN and CA significantly induced the mineralization and expression of osteogenic factors ALP,Runx2 and OPN.In the rat skull defect model,the new bone volume fraction(BV/TV)detected by MicroCT and the proportion of new bone in the defect area through histological detection after the PGRN-CA treatment were significantly higher than those of the control and CA groups.The expression level of M1/M2 polarization-related genes in macrophages was detected in vitro and it was found that CA+PGRN significantly downregulated the expression levels of LPS-induced M1 polarization-related genes iNOS,IL-1β TNF-α and significantly up-regulated the expression of IL-4-induced M2 polarization related genes IL-10 and TGF-β.CA also inhibited the polarization of M1,but the effect on M2 was not obvious.CA hydrogel loaded with PGRN was injected into the defect area of inflammatory periodontal bone defect in rat model and the repair effect was observed regularly.Histological results showed that the CA had a slight osteogenic effect,while there were much more new bone trabeculae in the CA combined with PGRN group.Histologically,the proportion of new bone in the CA combined with PGRN group was significantly higher than that in the control and CA groups.qRT-PCR for gingival tissue showed that the expression of M1 macrophage-related factors iNOS,IL-1β and TNF-α was significantly down-regulated in the PGRN-CA group comparing with the control,while the M2 macrophage and Treg cells related factor IL-10 was significantly up-regulated.CA also inhibited the expression of M1-related factors.The flow cytometry was used to detect the proportion of Th17 and Treg cells in submaxillary lymph nodes of rats.Compared with the control group,the proportion of Th17 in CA and CA+PGRN groups was significantly decreased,the proportion of Treg was increased,and the ratio of Th17/Treg was significantly decreased.The proportion of The Th17 ratio and Th17/Treg ratio in CA+PGRN group were significantly lower than those in CA group.Conclusion:1.Both CMCTS and CMCT can promote osteogenic differentiation of periodontal stem cells in vitro,and CMCTS has the stronger osteogenic activity.2.The aldehyde-based HAD prepared by the oxidation of HA in water-ethanol phase can react with the amino group on CMCTS molecules to form imine chain to obtain injectable hydrogel(CA).Moderately crosslinked CA gels have the properties of fast gel formation,high viscosity,strong water absorption,moderate swelling rate,uniform porous structure,rapid self-healing,low cytotoxicity,and good tissue compatibility.It can promote osteogenic differentiation of periodontal stem cells in vitro and regulate immune function in vivo and in vitro.3.CA hydrogel loaded with PGRN can slowly release PGRN and maintain its high biological activity.It provides a potential feasible way for regulating the immune microenvironment of periodontitis and promoting the regeneration of bone defect.