Zinc-wharton’s Jelly Scafford Regulates Macrophages To Promote Cartilage Regeneration And Differentiation In Rats Under Inflammatory Microenvironment

BackgroundInflammatory factors released by immune cells are considered to be important factors affecting cartilage regeneration.Many diseases accompanying cartilage injury,such as osteoarthritis,are accompanied by inflammation,which makes it more difficult to repair the cartilage tissue that lacks blood vessels and nerves.In the past,cartilage tissue engineering scaffolds focused on properties,mechanical properties and histocompatibility,but ignored anti-inflammatory properties.Zinc,as an important trace metal element in life metabolism,is an important complement of various enzymes,and has definite anti-inflammatory,antioxidation and anti-bacterial effects.ObjectiveIn this study,a zinc ion scaffold with anti-inflammatory properties was constructed by loading zinc with Wharton’s Jelly from the human umbilical cord.To explore the regulatory effects of zinc scaffold extract on macrophage polarization and macrophage-induced inflammatory response in the presence or absence of lipopolysaccharide(LPS),and further elucidate the effects of these regulatory effects on cartilage regeneration.Methods1.Preparation of Zinc-Wharton’s Jelly ScaffoldWharton’s Jelly(WJ)separated from human umbilical cord was decellular by mechanical comminution,hypoosmotic method,enzymatic digestion,repeated freeze-thaw and differential centrifugation technology,then formed by freeze dryer to prepare a Wharton’s Jelly Scaffold(WJS).Zinc-Wharton’s Jelly Scaffold(Zn-WJS)scaffolds were constr-ucted by loading 0.01M(Zn1)and 0.1M(Zn2)zinc acetate in simulated body fluid induced deposition.The surface structure of the scaffold material was observed by scanning electron microscopy(SEM).X-ray photoelectron spectroscopy(XPS)was used to characterize the composition and chemical state of the elements on the scaffold surface.Inductively coupled plasma mass spectrometry(ICP-MS)was used to detect the release characteristics of zinc ion from zinc ion scaffolds.The appropriate concentration of zinc scaffolds was selected according to the release curve for subsequent experiments.The extract of Wharton’s Jelly scaffold was prepared by DMEM medium according to the standard method.CCK8 kit was used to detect the cytotoxicity of scaffold extract and rhodamine-phalloidin staining was used to observe the cell adhesion on the scaffold.2.Regulation of Zinc-Wharton’s Jelly extract on macrophage polarization and macrophage-induced inflammatory responseWith or without LPS induction,the experiment was divided into six groups: blank group(Blank group),WJS extract complete medium macrophage group(WJS group),zincWJS extract complete medium macrophage group(Zn-WJS group),LPS induction group(LPS group),LPS plus WJS extract complete medium macrophage group(LPS + WJS group)and LPS was added to complete culture medium of zinc-WJS extract to culture macrophages(LPS + zinc-WJS group).Three days after culture,the expression of macrophage surface markers CCR7(representing M1 phenotype)and CD206(representing M2 phenotype)were detected by flow cytometry and immunofluorescence double staining.Enzyme-linked immunosorbent assay(ELLISA)kit was used to detect the release of inflammatory factors interleukin-4(IL-4),interleukin-6(IL-6),tumor necrosis factor-alpha(TNF-α)and interleukin-10(IL-10)in the supernatant of macrophage culture.3.Chondrogenesis of Zinc-Wharton’s Jelly in vitroIn order to detect the ability of zinc ion scaffolds to regulate cartilage regeneration in inflammatory microenvironment,conditioned medium(conditioned medium)was prepared to activate macrophages.The experiment was divided into four groups: Rat Bone Marrow Mesenchymal Stem Cells(rBMSCs)+ WJS,zinc scaffolds loaded rBMSCs + conditioned medium(Zn-WJS),rBMSCs + conditioned cartilage culture medium(Conditioned + WJS),zinc ion scaffolds loaded rBMSCs + conditioned cartilage culture medium(Conditioned +WJS),and zinc ion scaffolds loaded rBMSCs + conditioned culture.Conditioned + ZnWJS group,after 21 days of induction of cartilage formation,the cartilage globules were histologically assessed by HE staining,toluidine blue staining and GAG assay.The expression of type II,SOX9 and aggrecan were detected by real-time fluorescence quantitative PCR.Results1.Preparation of Zinc-Wharton’s Jelly Scafford(1)Under scanning electron microscopy,the three-dimensional oriented porous structure,composite tissue engineering scaffolds and the requirements of this experiment were observed before and after loading zinc ions on the Wharton’s Jelly scaffolds.X-ray photoelectron spectroscopy showed that WJS did not contain zinc elements,but zinc ions were loaded on Zn-WJS.Inductively Coupled Plasma Mass Spectrometry(ICP-MS)was used to detect the rapid release of zinc ions from zinc ion scaffolds in 1-4 days.After 7 days,zinc ions entered the plateau phase.The plateau phase zinc ions concentration was Zn1＿1.01mg/L(15u M),Zn2＿7.26 mg L＿1(111u M),respectively.Zn-WJS with Zn1 concentration were selected for subsequent experiments.(2)CCK8 kit showed that the cells cultured with Zn1 and Zn2 scaffolds did not show cytotoxicity in 1,3 and 5 days.The adherence of cells to scaffolds was well demonstrated by GNP staining.2.Regulation of Zinc-Wharton’s Jelly Scafford extract on macrophage polarization and macrophage-induced inflammatory response(1)Immunofluorescence and flow cytometry showed that in the absence of LPS,ZnWJS had no significant effect on inducing macrophage polarization.But in the LPS activated macrophage microenvironment,Zn-WJS could induce macrophage polarization to anti-inflammatory phenotype(M2)(P < 0.05),and inhibit macrophage differe-ntiation to M1 type(P < 0.05).(2)Both WJS group and Zn-WJS group could inhibit the release of IL-6 and TNF-αby macrophages activated by LPS and promote the release of IL-4 and IL-10(P < 0.05),but the effect of Zn-WJS group was more obvious(P < 0.05).3.Chondrogenesis of Zinc-Wharton’s Jelly Scafford in vitro After 21 days of induction culture,rBMSCs loaded with Zn-WJS were successfully differentiated into cartilage.HE staining and toluidine blue staining showed that Cond-itioned+Zn-WJS was superior to Conditioned+WJS group in cartilage formation,close to WJS group and Zn-WJS group.GAG quantitative detection showed that Conditioned +Zn-WJS group>Conditioned+WJS group(P<0.05),type II,SOX9 and aggrecan all supported the relative expression levels of Conditioned+Zn-WJS group.Itioned + Zn-WJS group > Conditioned + WJS group(P < 0.05).Conclusions1.Zinc can be successfully loaded on the Wharton’s Jelly scaffold to become a new type of tissue engineering scaffold,which can control the release concentration of zinc to a certain extent.2.Zn-WJS can play an anti-inflammatory role by inducing macrophage polarization.3.rBMSCs loaded with Zn-WJS can successfully induce chondrocyte and play an anti-inflammatory role in inflammatory microenvironment to promote cartilage repair.It can be used as a new type of tissue engineering scaffold for cartilage repair.SignificanceIn this study,the feasibility of a new type of Zn-WJS as a scaffold material for cartilage tissue engineering was explored,and a new idea for repairing cartilage injury in inflammatory microenvironment was provided.