BACKGROUND:Some studies have indicated that different genes in tooth germ tissue play a role at different time, contributing to tooth germ development.
OBJECTIVE:To observe the expressions of dentin matrix protein 1, enamel protein, col agen I and homeobox gene 1 at different stage of in vitro culture of tooth germ cells.
METHODS:RNA from tooth germ cells was extracted at days 1, 3, 6 of in vitro culture. After reverse transcription, real-time quantitative PCR detection was adopted to measure relative expression of dentin matrix protein 1, enamel protein, col agen I and homeobox gene 1 mRNA.
RESULTS AND CONCLUSION:Dentin matrix protein 1, enamel protein, and col agen I mRNA expressions increased with culture time, and reached the peak at day 3 (P<0.05), whereas homeobox gene 1 mRNA decreased with culture time (P<0.05).

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.

METHODS:Lentiviral vectors carrying bFGF and BMP-2 were constructed to transfect sheep bone marrow mesenchymal stem cells. cells were divided into four groups:bFGF group, BMP-2 group, co-transfection group BACKGROUND:Basic fibroblast growth factor (bFGF) can promote the proliferation of bone marrow stromal cells, and bone morphogenetic protein-2 (BMP-2) has an important significance in the induction of new bone formation.
OBJECTIVE:To analyze the effects of bFGF, BMP-2 and their co-transfection on the proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells and to compare the relative expressions of col agen I, osteocalcin and osteopontin before and after celltransfection, thereby providing theoretical implications for seed cells in the construction of tissue-engineered bone.
and control group. The RNA was extracted using real-time quantitative PCR to detect mRNA levels of col agen I, osteocalcin, and osteopontin.
RESULTS AND CONCLUSION:Significant difference in non-specific osteogenic gene expressions was found among the four groups (P<0.05). bFGF and BMP-2 showed an interaction (P<0.05). Expressions of col agen I and osteocalcin in the co-tranfection group were higher than those in the other three groups (P<0.05), but osteopontin expression exhibited no difference (P>0.05). In vitro experiments showed that the relative expression of col agen I, osteocalcin and osteopontin were higher in the co-transfection group, indicating the cells from the co-transfection group have strongest osteogenic capacity that are suitable for seed cells for bone tissue engineering.

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.

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 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.

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.

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 : 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.