BACKGROUND: Both hydroxyapatite (HA) and large diameter TiO2 nanotubes have excellent biocompatibility, but bone-forming ability of nano-HA (nHA) deposited large diameter TiO2 nanotubes is rarely reported.OBJECTIVE: To evaluate the bone-forming ability of nHA/large-diameter TiO2 nanotube composite coating.METHODS: Large-diameter TiO2 nanotubes were prepared by anodic oxidation method, and then nHA was electrochemically deposited on the surface of TiO2 nanotubes. Preosteoblasts MC3T3-E1 were co-cultured with the nHA/large diameter TiO2 nanotube composite, pure titanium and TiO2 nanotube coatings, respectively. At 0.5, 1, 2 hours after culture, the initial cell adhesion was observed. At 1, 3, 5 day after culture, cell proliferation was assessed. At 2 days after culture, cell morphology was observed. At 3 and 7 days after osteogenic induction, intracellular alkaline phosphatase activity was detected. At 14 days after osteogenic induction, mineralization of extracellular matrix was detected.RESULTS AND CONCLUSION: (1) After 2 hours of culture, the number of adherent cells on the composite coating was significantly lower than that on the TiO2 nanotube coating (P < 0.05), but slightly higher than that on the pure titanium coating with no statistical difference. (2) After 1, 3, 5 days of culture, the cell proliferation on the composite coating was significantly lower than that on the TiO2 nanotube coating (P < 0.05), but slightly higher than that on the pure titanium with no statistical difference. (3) The cells on the pure titanium showed a spindle-shape, while those on the TiO2 nanotube coating processed filopodia. The cells on the composite coating showed polygonal shape with a larger number of filopodia. (4) The intracellular alkaline phosphatase activity of the composite coating group was significantly higher than that of the pure titanium group and TiO2 nanotube group. The trend of mineralization of extracellular matrix was ranked from high to low: the composite coating group > TiO2 nanotube group > pure titanium group. To conclude, the nHA/large diameter TiO2 nanotube composite coating not only has good biocompatibility, but also has the ideal ability to promote bone formation.

Objective:To investigate the expression of microRNA-296 (miR-296) in rabbit hypertrophic scars and its role in human fibroblasts (HFbs).Methods:The experimental method was used. Twelve healthy adult New Zealand long-eared rabbits regardless gender were randomly divided into normal control group and scar group, with 6 rabbits in each group. The rabbit ear hypertrophic scar model was created in scar group according to the literature, and the rabbits in normal control group did not receive any treatment. On 60 days after setting up the models in scar group, hematoxylin-eosin staining was performed to observe the growth and arrangement of fibroblasts (Fbs) in the ear scars and skin tissue of rabbits in the two groups. The mRNA expressions of miR-296 and transforming growth factor-β 1 (TGF-β 1) in ear scars and skin tissue of rabbits in the two groups were detected by real-time fluorescent quantitative reverse transcription polymerase chain reaction, and the correlation of mRNA between miR-296 and TGF-β 1 was performed with Pearson regression analysis. Two batches of HFbs were used and transfected respectively with corresponding sequences, with the 1st batch being divided into TGF-β 1 wild type+miR-296 negative control group and TGF-β 1 wild type+miR-296 mimic group and the 2nd batch being divided into TGF-β 1 mutant type+miR-296 negative control group and TGF-β 1 mutant type+miR-296 mimic group. At 48 h after transfection, luciferase reporter gene detection kit was used to detect the luciferase and renal luciferase expression of TGF-β 1 in the cells of each group, with their ratio being used to reflect the gene expression level. Two batches of HFbs were used, and each batch of cells were divided into miR-296 negative control group and miR-296 mimic group, being transfected with the corresponding sequences. At 0 (immediately), 12, 24, 36, and 48 h after transfecting the first batch of cells, the cell proliferation was detected by thiazolyl blue method. At 24 h after transfecting the second batch of cells, the expression of TGF-β 1 and collagen type Ⅰ was detected by Western blotting. The number of samples in cell experiments was 3. Data were statistically analyzed with analysis of variance for factorial design, independent sample t test. Results:On 60 days after setting up the models in scar group, the Fbs of rabbit ear scar tissue in scar group proliferated and arranged disorderly, while the growth and arrangement of Fbs in rabbit ear skin tissue in normal control group were normal. The mRNA expression of miR-296 of rabbit scar tissue in scar group (0.65±0.11) was significantly lower than 1.19±0.12 of rabbit ear skin tissue in normal control group ( t=5.175, P<0.01). The mRNA expression of TGF-β 1 of rabbit ear scar tissue in scar group (1.47±0.06) was significantly higher than 1.10±0.03 of rabbit ear skin tissue in normal control group ( t=12.410, P<0.01). Pearson regression analysis showed that there was a negative correlation between the mRNA expression of miR-296 and TGF-β 1 in the ear scars and skin tissue of 12 rabbits ( F=7.278, P<0.05). At 48 h after transfection, the gene expression of TGF-β 1 of cells in TGF-β 1 wild type+miR-296 mimic group was significantly lower than that in TGF-β 1 wild type+miR-296 negative control group ( t=35.190, P<0.01), while the gene expression of TGF-β 1 of cells in the two TGF-β 1 mutant type groups were close ( P>0.05). The HFbs proliferation ability in miR-296 mimic group was significantly lower than that in miR-296 negative control group at 12, 24, 36, and 48 h after transfection( t=3.275, 11.980, 10.460, 17.260, P<0.05 or P<0.01). At 24 h after transfection, the protein expressions of TGF-β 1 and type Ⅰ collagen of cells in miR-296 negative control group were significantly higher than those in miR-296 mimic group ( t=3.758, 29.390, P<0.05 or P<0.01). Conclusions:The miR-296 expression in rabbit hypertrophic scars is down-regulated; miR-296 can inhibit the proliferation of HFbs and the expression of type Ⅰ collagen by down regulating the expression of TGF-β 1.

Objective:To explore the characteristics and data related to the degradation and biocompatibility of the domestically procuded polyglycolic acid (PGA) nerve conduit.Methods:This study was conducted in the Department of Burns and Plastic Surgery, the Northern Theater General Hospital between January and August 2022. PGA nerve conduits were immersed in 15 ml normal saline for in vitro experiments of degradation. The experimental period was 12 weeks. The pH of the immersion solution were tested weekly and the rates of mass loss were calculated. The in vitro experiments for biocompatibility were conducted in both of the experimental group and the control group. In the experimental group, a DMEM solution containing 10% of fetal bovine serum was used as the extraction medium, and domestically produced PGA nerve conduit was immersed in the extraction medium in the control group. An extraction medium was firstly prepared for a controlled extraction time of 72 hours±2 hours at 37 ℃±1 ℃. The pH of both experimental and control groups were tested at 24, 48 and 72 hours. The 72-hour extracts of both experimental and control groups were used to prepare the single cell suspension. The single cell suspension containing the cultured RSC 96 were separately seeded into 96-well plates for Methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay, to observe the effect of PGA nerve conduits on the proliferation of RSC 96. Then RSC 96 were incubated for 24 hours with the extracts of both experimental and control groups. The effect of PGA nerve conduits on the migration of RSC 96 was observed with Transwell assay. Twenty-four adult male SD rats were used and divided into experimental group and control group, with 12 rats per group, for the in vivo study of degradation and biocompatibility. Rats in the experimental group were implanted with domestically produced PGA nerve conduits, and Neurotubes, a US made nerve conduit, were implanted in the rats of control group. The PGA nerve conduits were implanted in a bluntly prepared gap between the biceps femoris muscle and the gluteus maximus muscle of the right hind limb of rats for the in vivo degradative experient. The degradation and biocompatibility of the PGA nerve conduit were evaluated by means of gross observation, tests of blood routine, liver and kidney functions and histological examinations at 2, 4, 8 and 12 weeks after implantation. SPSS 19.0 software was used for statistical analysis of the experimental data, and the results were expressed as Mean±SD. T test was used for inter-group comparison and Turkey method was used for intra-group and inter-group comparison. P<0.05 was used to determine whether the difference was statistically significant. Results:The rate of in vitro degradation was found at 1.470%±0.026% in week 4, and thereafter, a gradually accelerated degradation rate was observed and at a 32.180%±0.040% of degradation rate in week 12. The pH of the immersion solution decreased slowly in the first 2 weeks, with the pH at 6.200±0.061 in week 2. The pH then suddenly dropped to 3.930±0.118 in week 3, and then decreased slowly, with a pH 2.560±0.003 in week 12. Both MTT and Transwell experiments showed that the extract of PGA nerve conduits had no effect on the proliferation and migration of RSC 96, nor a significant difference existed in comparison with the corresponding control groups ( P>0.05). The experiments of in vivo degradation of PGA nerve conduits showed that the nerve conduits in both experimental and control groups had good support at week 2 after implantation. At weeks 4-12 after implantation, the nerve conduits in both groups gradually softened and collapsed, but the conduits were in one piece and not broken. The blood routine, liver and kidney functions showed no statistically significant difference between the 2 groups over the same period, and between each time point within the groups and the groups before implantation ( P>0.05). No obvious abnormal appearance of livers and kidneys was found in the rats sacrificed at each observation time point. Also, there was no obvious degeneration and necrosis of the muscle tissue around the conduits in the 2 groups, and no obvious inflammatory cells infiltration was found from the histological examinations. Morphology of the muscle tissue remained normal. Conclusion:Domestically produced PGA nerve conduit is fund good in both of biocompatibility and biodegradability. It is safe and reliable, and it provides a basis for the further experients in repair of nerve defects.