BACKGROUND:The regulation of M1/M2 polarization direction of macrophages is particularly critical in tissue engineering applications,and timely regulation can minimize proinflammatory,anti-inflammatory,or tissue healing responses. OBJECTIVE:To implant lentivirus-mediated miRNA-378a macrophage strain complex collagen to detect the expression level of immune regulation in the in vivo environment,and further clarify the influence of miRNA-378a in promoting macrophage M2 polarization in immune regulation and tissue repair in the in vivo environment. METHODS:Lentivirus-mediated miRNA-378a overexpressing macrophage cell lines and negative control virus macrophage lines were amplified and screened,and the macrophage lines were recovered and cultured together with collagen sponge to form a composite scaffold,which was divided into the following groups:(1)Positive group:miRNA-378a overexpressing macrophage-collagen sponge composite;(2)negative group:negative control of virus-mediated miRNA-378a macrophage-collagen sponge composite;(3)control group:macrophage-collagen sponge;(4)blank control group:collagen sponge.The cell density,phenotype,and adhesion of each group were observed by immunofluorescence and scanning electron microscopy.The cells were implanted into the subcutaneous model of the back of mice,and the mice were sacrificed 4 and 7 days after modeling.The direction of macrophage polarization in the collagen sponge composite of macrophages with miRNA-378a overexpression mediated by lentivirus and its effect on immune regulation and tissue repair were analyzed by gross observation,hematoxylin-eosin staining,MASSON staining,and immunohistochemistry. RESULTS AND CONCLUSION:(1)Under immunofluorescence microscopy,the macrophage cell lines in each group were observed to form a composite scaffold with the collagen sponge.(2)Under scanning electron microscope,lentivirus-mediated miRNA-378a macrophages in the positive group proliferated in cell density,had spherical,elliptic and polygonal differentiation,and had more pseudofeet than other groups.(3)Under general observation,the overall 7-day healing was better than that at 4 days.Lentivirus-mediated miRNA-378a macrophages in the positive group healed better than other groups regardless of 4 and 7 days.(4)Lentivirus-mediated miRNA-378a macrophages in the positive group under hematoxylin-eosin staining and MASSON staining had more amounts of fibrocytes,capillaries,fibroblasts,and collagen fiber hyperplasia.(5)Immunohistochemistry showed that lentivirus-mediated miRNA-378a macrophages in the positive group were more positive in 4-and 7-day M2 polarized cells than in other groups.The macrophages of the control and negative groups in 4-and 7-day M2 polarized cells were greater than that of the blank control group.There was no statistical difference between the control group and the negative group.The number of stained cells in the positive,negative,and control groups regardless of 4 and 7 days was higher than that in the blank control group,and the positive group>negative group ≈ control group>blank control group.(6)It is concluded that macrophages with miRNA-378a overexpression have a large amount of fibroblasts,capillaries,fibroblasts,and collagen fiber hyperplasia in vivo,which has a positive effect on tissue repair,and can promote the polarization of macrophages towards M2 type and inhibit the polarization of M1 type,thus contributing to reducing the inflammatory response of the body.

BACKGROUND:There are some problems such as bone fusion failure or peri-implant infection after implantation of pure titanium implants.Therefore,surface improvement of titanium implants has become a hot topic in research.Macrophages are an immune defense line of the body in response to external stimuli,and the relevant immune response of any biomaterials implanted in the body is related to macrophages. OBJECTIVE:The graphene oxide/hydroxyapatite composite coating on titanium alloy surface was prepared by electrochemical deposition method.The surface characteristics of the coating and the growth and polarization of macrophage RAW264.7 on the surface were analyzed. METHODS:The graphene oxide coating and graphene oxide/hydroxyapatite composite coating on titanium alloy surface was prepared by electrochemical deposition method.The physical properties of the coating were characterized.Pure titanium sheets(blank group),titanium sheets deposited with pure GO coating(control group)and titanium sheets deposited with graphene oxide/hydroxyapatite composite coating(experimental group)were co-cultured with macrophages RAW264.7,respectively.Cell proliferation was detected by CCK-8 assay and DAPI staining.Cell adhesion on the surface of titanium was observed by scanning electron microscopy,and cell polarization phenotype was detected by flow cytometry. RESULTS AND CONCLUSION:(1)Under scanning electron microscope,the pure titanium sheet showed directional scratches and a few punctate pits.After the pure graphene oxide coating was deposited,the surface of titanium sheet showed more cracks,gullies and particles of uneven size,and the wrinkle-like structure characterized by graphene oxide.After the composite coating was deposited,the surface of the titanium sheet was smooth,and the pellet size was more uniform.The water contact angle of composite coated titanium sheet was lower than that of pure titanium sheet and pure graphene oxide coated titanium sheet(P<0.05).(2)CCK-8 assay and DAPI staining showed that compared with the blank group and the control group,the cell proliferation in the experimental group was faster.Scanning electron microscopy showed that RAW264.7 cells all adhered to the surface of the three groups of titanium sheets.With the extension of culture time,cell morphology changed from round to spindle shape.After 7 days of culture,cells in the blank group extended short and few pseudopods;cells in the control group extended long and more pseudopods,and cells in the experimental group extended short and more pseudopods,and the overall cell fullness in the experimental group was the best.Flow cytometry results showed that the cells in the experimental group showed a higher proportion of M2 polarization in the anti-inflammatory direction.(3)These findings conclude that graphene oxide/hydroxyapatite composite coating has good physical,chemical,and biological properties.

BACKGROUND:Titanium and titanium-coated materials are widely used in the field of oral implantology,but there are still phenomena such as peri-implantitis,implant loss and loosening.Therefore,the surface modification of pure titanium has become a hot topic in oral medicine research. OBJECTIVE:To investigate the physical and osteogenic properties of hydroxyapatite-graphene oxide composite coating on titanium surface. METHODS:Hydroxyapatite-graphene oxide composite coatings were prepared on the titanium surface by electrodeposition at voltages of 10,30,and 50 V.The micromorphology and hydrophilic properties of the coatings were characterized,and the composite coatings prepared under the optimal voltage conditions were screened for animal experiments.Fifty-four SD rats were selected to prepare defects of 2 mm in diameter and 7 mm in depth on the femoral head of both hind limbs,and were randomly divided into 3 groups with 18 rats in each group:no titanium material was implanted in the blank group;pure titanium material was implanted in the pure titanium group,and coated titanium material loaded with hydroxyapatite-graphene oxide composite coating was implanted in the coated group.The osteogenesis effect was observed by X-ray,Micro-CT scan,and pathological section staining at 4,8,and 12 weeks after implantation. RESULTS AND CONCLUSION:(1)Under scanning electron microscopy,when the voltage was 10 V,there were a lot of cracks and clumps in the coating.When the voltage rose to 30 V,there were still some small clumps in the coating,but the overall uniformity was relatively flat.When the voltage was 50 V,the coating was more evenly distributed and cracks and spots were reduced.The hydrophilicity of the composite coating prepared at 50 V voltage was the best.In summary,the composite coating material prepared at 50 V voltage was selected in animal experiments.(2)The X-ray film showed that the implant position was relatively fixed,and no serious postoperative inflammation occurred.The results of Micro-CT scan showed that the new bone formation rate and bone formation volume on the implant surface of the coated group were better than those of the pure titanium group(P<0.001).The results of Micro-CT scan were further confirmed by hematoxylin-eosin staining and Masson staining in pathological sections.Immunohistochemical staining of pathological sections showed that the expressions of osteopontin and bone morphogenetic protein 2 in the pure titanium group were higher than those in the blank group at week 12 after implantation(P<0.001),and those in the coated group were higher than those in the pure titanium group at week 12 after implantation(P<0.001).(3)The results show that the hydroxyapatite-graphene oxide composite coating material has good physical and osteogenic properties.

BACKGROUND:M2 macrophages have the function of reducing inflammatory factors and promoting tissue healing.Therefore,how to regulate M2 polarization of macrophages has been a hot research topic in recent years,and some miRNAs have been found to have this function. OBJECTIVE:To investigate the effects of miR-378a on the polarization of the Raw264.7 macrophage cell line. METHODS:The M1 polarization of macrophages was induced by lipopolysaccharide and interferon-γ.Interleukin-4 induced M2 polarization and the expression of endogenous miR-378a in each cell type was detected using qRT-PCR to verify whether miR-378a was involved in the polarization of macrophages.By transfection with lentivirus as the vector of overexpression of miR-378a,the stable expression of miR-378a cell lines was screened.Macrophage M1 polarization was induced synergically by lipopolysaccharide and interferon-γ.Macrophage M2 polarization was induced by interleukin-4.The levels of M1/M2 polarization-related cytokines in the supernatant of the macrophage culture medium were determined by enzyme-linked immunosorbent assay.qRT-PCR was used to detect the polarization characteristics of M1/M2-type macrophages and the mRNA expression levels of related cytokines. RESULTS AND CONCLUSION:(1)The expression level of endogenous miR-378a in Raw264.7 cells of each group increased after macrophage polarization.(2)Compared with the non-transfected group,the expressions of proinflammatory cytokine-induced nitric oxide synthase,tumor necrosis factor-α,interleukin-6 and interleukin-1β in macrophage M1 induced polarization were significantly decreased in the miR-378a transfection group(P<0.05);the levels of inducible nitric oxide synthase,tumor necrosis factor-α and interleukin-6 in cell supernatant were also significantly decreased(P<0.05).(3)Compared with the non-transfected group,the expressions of CD206,interleukin-10 and arginase-I in macrophage M2 induced polarization were significantly increased(P<0.05);the levels of CD206 and interleukin-10 in cell supernatant were also significantly increased(P<0.05)in the miR-378a transfection group.(4)It is indicated that overexpression of miR-378a promotes the M2 polarization of macrophages and inhibits the M1 polarization of macrophages.

Objective: To investigate the effects of over expression and low expression of antisense transcripts of circular RNA cerebellar degeneration associated protein 1 (CDR1as) in Balb/C mouse bone marrow mesenchymal stem cells (BMSCs) on factors related to osteogenesis and angiogenesis. Methods: BMSCs were cultured and identified in vitro. The lentiviral (LV) vector containing the overexpressed and silenced circRNA CDR1as genes and the control lentivirus were respectively transfected into mouse BMSCs, and stable cell lines were screened. The cells were divided into the circRNACDR1as over expression group and the over expression control group, and the CircRNACDR1as low expression group and the low expression control group. The components were stained with Alizarin Red S and alkaline phosphatase after 14 and 21 days of osteoinduction; qRT-PCR was used to detect the target genes circRNA CDR1as, osteogenic differentiation markers alkaline phosphatase (ALP), runt- related transcription factor 2 (RUNX2), osteocalcin (OCN), osteopontin (OPN), osterix(Osx), collagen I (COL-1), and the mRNA expression levels of vascular endothelial grown factor (VEGF) and angiogenin-1 (Ang-1). Results: The results of alizarin red staining and alkaline phosphatase staining showed that the extracellular matrix calcium precipitation and ALP staining area of the over expression experimental group was greater than its control group, and those of the low expression experimental group was less than its control group. As the number of days of osteogenic induction increased, the calcium precipitation and ALP staining in each group also increased. RT-PCR results showed that the mRNA expression levels of circRNA CDR1as, ALP, RUNX2, OCN, OPN, OSX, COL-1, VEGF and Ang-1 in the over expression experimental group BMSCs were significantly increased (P<0.001). In the low expression experimental group, the mRNA expression levels of circRNA CDR1as, ALP, RUNX2, OCN, OPN, OSX, COL-1, VEGF and Ang-1 in BMSCs were significantly reduced (P<0.001). Conclusion Over expression of the circRNA CDR1as gene promotes the osteogenic differentiation and angiogenesis of BMSCs. Low expression of the circRNA CDR1as gene inhibits the osteogenic differentiation and angiogenesis of BMSCs.

Objective : To explore the feasibility of antler powder/silk fibroin/polyvinyl alcohol scaffolds as tissue engineering bone scaffolds and the relationship between their degradation performance and the healing speed of bone defects. Methods : Antler powder/silk fibroin/polyvinyl alcohol scaffolds and nano hydroxyapatite/silk fibroin/polyvinyl alcohol scaffolds were prepared by 3D printing. The whole bone marrow culture method was used to prepare blood cell sheets of Altay big tail sheep’s iliac bone marrow. With observation times of 1, 2 and 3 months, the mandibular defects of 4 sheep were established. The experimental group was coated with antler powder/silk fibroin/polyvinyl alcohol scaffolds. The control group was coated with nanohydroxyapatite/silk fibroin/polyvinyl alcohol scaffolds. The negative control group was coated with gel-free sponges. According to the self-control method of the bilateral mandible defect area, scaffolds wrapped with cell membranes or gel sponges wrapped with cell membranes were implanted. At the ends of the first, second and third months after implantation, the experimental animals were killed, cone beam CT was performed, and paraffin sections were taken for HE staining to evaluate the effect of different scaffold materials on bone regeneration and scaffold degradation. Results: Scanning electron microscopy showed that both groups had regular pores and good continuity, and there was no difference in pore size and porosity between the two groups (P > 0.05). The results of CBCT imaging showed that in 3 months after operation, the experimental group had significantly better repair effects on bone defects than the control group, and the degradation rate matched the bone repair rate. The bone mineral density in the center of the defect was higher than that of the control group, which was close to that of normal bone tissue. The central bone mineral density of the experimental group at each time point was higher than those of the control group and the negative control group, and the difference was statistically significant (P < 0.05). HE staining results showed that the bone cells in the experimental group were more active, with more new capillaries and bone trabeculae formed, and the scaffold material absorbed more than the control group. Conclusion The antler powder/silk fibroin/polyvinyl alcohol scaffold can promote the repair of critical bone defects. Its degradability matches its bone tissue healing rate. It is expected to become a promising scaffold material for bone tissue engineering.

Objective: To investigate the level of hypoxia inducible factor-1α (HIF-1α) on osteoblasts and angiogenesis-associated cytokines in bone marrow mesenchymal stem cells (BMSCs) from SD rats. Methods: BMSCs were isolated and cultured and identified by flow cytometry. Plasmid vectors containing upregulated and downregulated HIF-1α gene and a control vector were constructed. The plasmids were transfected into BMSCs by Lipofectamine®LTX transfection reagent, and the cells were divided into an overexpression experimental group, an overexpression control group, a low expression experimental group and a low expression control group. All components were stained with a lizarin red 3 d and 7 d after osteogenesis induction. The mRNA expression levels of the target gene HIF-1α, osteogenic differentiation-specific markers, including Runt-related transcription factor 2 (Runx2) and angiogenic markers, including platelet-derived growth factor-BB (PDGF-BB) and transforming growth factor-β (TGF-β), were detected by RT-PCR. Western blot was used to detect the protein expression of the target proteins HIF-1α, Runx2, and PDGF-BB. Results: The CD29- and CD45-positivity rates of BMSC surface markers identified by flow cytometry were 98.2% and 4.2%, respectively. RT-PCR results showed that the mRNA expression of HIF-1α, Runx2, TGF-β and PDGF-BB was observably increased (P < 0.001). The mRNA expression levels of HIF-1α, Runx2, TGF-β and PDGF-BB in BMSCs from the low expression experimental group were significantly reduced (P < 0.001). Western blot results showed that the expression levels of HIF-1α, Runx2 and PDGF-BB in BMSCs from the overexpression experimental group were all increased (P < 0.001). The expression levels of HIF-1α, Runx2 and PDGF-BB in BMSCs from the low expression experimental group were reduced (P < 0.001). Alizarin red staining results showed that the area of calcium nodules in the low expression experimental group was smaller than that in low expression control group, the area of red calcium nodules in the over expression experimental group was larger than that in over expression control group, and with the increase of osteogenic induction time, the calcification area of each group also increased. Conclusion Upregulation and downregulation of HIF-1α can regulate the osteogenic differentiation and the expression of angiogenesis related factors of BMSCs.

Three dimensionally printed composite porous bone tissue engineering scaffolds have become a research focus. Composite polyvinyl alcohol (PVA) has good biocompatibilityand degradability, but it cannot be prepared indepen⁃dently because it cannot resist highmechanical resistance. This material shows many advantages, such as good biocom⁃patibility, degradability and mechanical properties, when compounded with other materials with good mechanical proper⁃ties and good biocompatibility. Therefore, 3D printed composite PVA scaffold material can optimize the performance of PVA scaffolds. This article reviews 3D printing bone scaffold technology, polyvinyl alcohol (PVA), and composite PVA scaffolds for in vivo and in vitro bone formation.

Objective: o study the effect of cleaning treatment with hydrofluoric acid (HF) on the surface and bonding strength of IPS e.max and Vita Mark II ceramic inlays. Methods: Fifty pieces of IPS e.max and Vita Mark II ceramic inlay specimens were made separately using CAD/CAM. After uniformly bonding surfaces using 9% HF etching, they were randomly divided into an untreated control group (group A) and the following experimental groups: neutralizing powder (B group), 37% phosphoric acid (group C), ultrasonic cleaning (group D) and neutralizing powder + 37% phosphoric acid + ultrasonic cleaning (group E). Each set of 8 specimens was bonded to Variolink N resin adhesive under standard conditions. The shear adhesive strength was measured after exposure to a constant-temperature water bath at 37 ℃ for 24 h. The location of the fracture and the type of adhesion failure were recorded. The shear adhesion and the average strength of the connection were analyzed. The remaining 2 specimens were used for scanning electron microscopy (SEM) to observe the surface morphology, including the crystal structure, pore pattern, and residue. Results : The results were similar for the IPS e.max and Vita Mark II inlays. The maximum bond strength was observed in the IPS e.max ceramic inlays in group E, with an average bond strength 11.96 MPa higher than that in group A. Among the Vita Mark II porcelain inlays, the maximum bond strength was observed in group E. The average bond strength was 9.74 MPa higher than that in group A. The results of the statistical analysis were similar for the IPS e.max and Vita Mark II porcelain inlays, with significant differences in the bond strengths between groups C, D, and E and the control group (P < 0.05). There was no significant difference in the adhesive strength between groups B and A. At the same time, there was no significant difference in the bond strength between the treatment groups B, C, D, and E (P > 0.05). SEM revealed that the pores on the surface of ceramics subjected to the acid etching treatment were broadened and uniform, with less residue than observed in the control group. The effects of treatments D and E were the best. Conclusion The HF etching treatment can enhance the bonding strength of IPS e.max and Vita Mark Ⅱ ceramic inlays while leaving little residue, and the joint strength is highest when the joints are treated together.

BACKGROUND:With the promotion of 3D printing technology, 3D printing scaffolds for bone tissue engineering have become the new ideas for jaw bone repair. OBJECTIVE:To compare the physical and biological properties of sheep vertebral bone meal/polyvinyl alcohol (PVA) scaffold, nano-hydroxyapatite (nHA)/PVA scaffold, and sheep vertebral bone meal/PVA nonporous bone plate. METHODS:3D printing technology was used to print sheep vertebral bone meal/PVA scaffold, nHA/PVA scaffold, and sheep vertebral bone meal/PVA nonporous bone plate. Porosity, morphology, water absorption rate and mechanical properties of different scaffolds were detected. Three kinds of scaffolds were al used to culture bone marrow mesenchymal stem cel s, and cel proliferation ability was detected using cel counting kit-8 at 1, 4, 7 days of culture. RESULTS AND CONCLUSION:Under scanning electron microscope, the sheep vertebral bone meal/PVA scaffold and nHA/PVA scaffold exhibited regular and interconnected pores with good continuity and clear network structure;the sheep vertebral bone meal/PVA nonporous bone plate had no obvious pores;however, it had dense and evenly distributed micropores with different sizes on its surface. The porosity of nHA/PVA scaffold was lower than that of the sheep vertebral bone meal/PVA scaffold (P<0.05). The water absorption rate was highest for the nHA/PVA scaffold fol owed by the sheep vertebral bone meal/PVA scaffold and the sheep vertebral bone meal/PVA nonporous bone plate (P<0.05). In contrast, the scaffold toughness was highest for the sheep vertebral bone meal/PVA nonporous bone plate, fol owed by the sheep vertebral bone meal/PVA scaffold and nHA/PVA scaffold. In addition, the cel proliferation activity of cel s cultured on the sheep vertebral bone meal/PVA scaffold was significantly higher than that cultured on the other two kinds of scaffolds. Taken together, the 3D printing sheep vertebral bone/PVA scaffold has good physical and chemical performance.