BACKGROUND:The repair of large-scale bone defects is still facing serious challenges.It is of great significance to develop personalized,low-cost,and osteogenic-inducing tissue engineering scaffolds for bone repair. OBJECTIVE:To explore the process of 3D printing bone tissue engineering scaffold containing pearl composite material by low-temperature condensation deposition method,and further test the physicochemical properties and in vitro biological functions of the composite scaffold. METHODS:Pearl powder was prepared by grinding and sieving.The pearl powder of different qualities was added into the poly-L-lactic acid ink,so that the mass ratio of pearl powder to poly-L-lactic acid was 0,0.1,0.2,0.3,and 0.5,respectively.The 3D-printed poly-L-lactic acid/pearl powder scaffolds were prepared using the low-temperature condensation deposition method.The microstructure,compressive properties,water contact angle,cytocompatibility,and in vitro bone differentiation ability of the printed poly-L-lactic acid/pearl powder composite scaffolds were detected. RESULTS AND CONCLUSION:(1)Scanning electron microscopy showed that the five groups of scaffolds all had micropores with a diameter of 2 μm or even smaller,irregular shapes and interconnectivity.(2)All the five groups had good compressive properties.The compressive strength of the pearl powder 0.5 group was higher than that of the other four groups(P<0.05).The water contact angle of the pearl powder 0.2 group and the pearl powder 0.5 group was smaller than that of the pearl powder 0 group(P<0.01,P<0.001).(3)Bone marrow mesenchymal stem cells were co-cultured with five groups of scaffolds for 1,3,and 5 days,respectively.The cell proliferation in pearl powder 0.1,0.2,0.3,and 0.5 groups cultured for 3 and 5 days was faster than that in pearl powder 0 group(P<0.05).After 1 day of culture,live-dead staining exhibited that the number of cells on the scaffold was small,but all of them were living cells.(4)Bone marrow mesenchymal stem cells were inoculated on the scaffold surface of the pearl powder 0 group and pearl powder 0.1 group respectively for osteogenic differentiation.The alkaline phosphatase activity induced for 4 and 6 days in the pearl powder 0.1 group was higher than that in the pearl powder 0 group(P<0.05).(5)The results showed that the poly-L-lactic acid/pearl powder composite scaffold had good compressive strength,hydrophilicity,cytocompatibility,and osteogenic properties.

Objective:To explore the effect of behavioral habits on the recovery of spinal cord function in patients with cervical spondylotic myelopathy after expansive open-door laminoplasty(ELAP).Methods:Retrospective analysis of clinical data of 183 patients with cervical spondylotic myelopathy who underwent ELAP at the Spinal Surgery Department of Jining Medical University Affiliated Hospital, from February 2019 to October 2022, with complete follow-up information. General clinical data of patients were collected. The patients were followed up at 3 months, 6 months and 12 months after surgery with the modified standard MacNab.The JOA score was used to evaluate the recovery of motor and sensory functions in patients before and 12 months after surgery. The recovery rate of spinal cord function was calculated based on the JOA score, and patients were divided into two groups: the group with good therapeutic effect ( n=143, recovery rate ≥ 50%) and the group with poor therapeutic effect ( n=40, recovery rate<50%). Data statistics were conducted using SPSS 20.0 software for chi-square test, rank sum test, t-test, and Logistic regression analysis. Results:There were significant differences in age ( t=-3.252, P<0.01), smoking ( χ2=21.503, P<0.01), body mass index(BMI) ( t=-5.885, P<0.01), hypertension ( χ2=20.263, P<0.01), coronary heart disease ( χ2=13.272, P<0.01), hospitalization time ( t=-2.278, P=0.02), desk and screen time ( t=-6.589, P<0.01), and frequency of rehabilitation exercise ( χ2=10.927, P<0.01) between the group with good therapeutic effect and the group with poor therapeutic effect. Further multivariate Logistic regression analysis showed that smoking ( B=2.402, OR=11.046, 95% CI=2.334-52.285, P<0.05), high BMI ( B=0.341, OR=1.406, 95% CI=1.076-1.837, P<0.05), hypertension ( B=2.238, OR=9.370, 95% CI=2.153-40.790, P<0.05), long desk and screen time ( B=0.961, OR=2.613, 95% CI=1.540-4.435, P<0.05) and low frequency of rehabilitation exercise ( B=-1.039, OR=0.354, 95% CI=0.201-0.623, P<0.05) were risk factors for spinal cord function recovery in patients with cervical spondylotic myelopathy after ELAP( P<0.05). Conclusion:Smoking, high BMI, hypertension, long desk and screen time, and low frequency of rehabilitation exercise are not adverse to the recovery of neurological function in patients with cervical spondylotic myelopathy after ELAP.

Objective:A new type of bio-ink and polycaprolactone (PCL) were used to construct an integrated osteochon-dral composite tissue block by multi-nozzle 3D bioprinter. And the repair results to osteochondral defects were evaluated.Methods:In freeze-drying group: Freeze-dried composite scaffold made by silk fibroin (SF) and β-tricalcium phosphate was used to repair osteochondral defects, as control. In the 3D printing group: PCL was used to printed a hollow multi-layer cylinder frame by 3D biological printer. Extracellular matrix, SF and bone marrow mesenchymal stem cells were used as chondral bio-ink. Then, chon-dral bio-ink was used to print tissue-engineered cartilage on top of PCL frame. Before implantation of cartilage defect, autogenous cancellous bone was filled in PCL frame, then the tissue-engineered osteochondral composite was used to repair osteochondral defects. In mosaic group: Autologous osteochondral transplantation was performed. The repair results of the above three groups were compared by histological score, biochemical analysis and biomechanical test to evaluate the effect of repairing rabbit cartilage defects.Results:The compression modulus of neo-cartilage in the 3D print group 2.56±0.30 MPa was close to that of the mosaic group 2.51±0.13 MPa ( P>0.05), and significantly higher than that of freeze-dried group 1.37±0.14 MPa ( F=11.058, P<0.05). The sGAG contents in the 3D print group 14.49±0.7 μg/mg was close to that of the mosaic group 14.98±0.81 μg/mg ( P>0.05), and significantly higher than that of freeze-dried group 8.72±0.73 μg/mg ( F=20.973, P<0.05). However, there was no significant difference in collagen content between the three groups ( P>0.05). The results of ICRS cartilage repair histology score showed that the scores of the 3D print group were close to those of the mosaic group in the matrix, cell distribution, cell viability and subchondral bone ( P>0.05), and were significantly higher than those of freeze-dried group in the surface and cartilage mineralization scores ( F=19.544, P<0.05). Conclusion:Using the new bio-ink to make bone cartilage composite scaffold by 3D bio printing can simplify the construction of tissue-engineered bone cartilage composite tissue in vitro, and can repair cartilage defects in vivo.

Objective:To investigate the relationship between the changes of inflammatory cytokine levels and prognosis of patients with critical coronavirus disease 2019 (COVID-19) undergoing invasive mechanical ventilation (IMV).Methods:A retrospective study was conducted. The clinical date of critical COVID-19 patients undergoing IMV who were hospitalized in Wuhan Union Hospital, Tongji Medical College of Huazhong University of Science and Technology from February 4th to March 25th in 2020 were collected. At the same time, the inflammatory cytokine levels including interleukins (IL-2, IL-4, IL-6, IL-10) and tumor necrosis factor-α (TNF-α) at 48 hours before IMV and 48 hours after IMV of all the patients, as well as the 48 hours after weaning or right before death were recorded. Multivariate unconditional Logistic regression analysis was used to screen the independent risk factors of death during hospitalization.Results:Among the 43 patients, 13 patients improved and 30 died. Compared with the survival group, the patients in the non-survival group were older (years old: 67.6±7.3 vs. 58.5±11.9, P < 0.05), with higher rates of hypertension, diabetes and coronary heart disease (53.3% vs. 15.4%, 63.3% vs. 23.1%, 26.7% vs. 0%, all P < 0.05), and the time from onset to admission to hospital, admission to ICU and IMV were longer (days: it was 9.17±5.00 vs. 5.07±2.49, 17.10±7.11 vs. 12.23±5.05, and 17.90±7.46 vs. 12.61±5.60, respectively, all P < 0.05). The IL-6 and TNF-α levels on 48 hours after IMV in the non-survival patients increased significantly as compared with those before 48 hours and the surviving patients. Especially, the IL-6 levels increased significantly as compared with those at 48 hours after IMV and 48 hours after weaning in the surviving patients [ng/L: 800.00 (194.25, 2 000.00) vs. 22.03 (6.66, 28.21), 3 204.00 (1 264.88, 5 000.00) vs. 5.00 (3.98, 12.27), both P < 0.01]. The IL-10 level before death in the non-survival patients increased significantly as compared with that at 48 hours after weaning in the surviving patients [ng/L: 55.89 (26.07, 100.14) vs. 3.53 (2.76, 12.36), P < 0.05]. There were no significant differences in the levels of IL-2 and IL-4 between the two groups at every time point. The variables of age, basic diseases, the IL-6 level after IMV were included in the multivariate unconditional Logistic regression analysis, which showed that age [odds ratio ( OR) = 0.821, 95% confidence interval (95% CI) was 0.695-0.968], hypertension ( OR = 0.027, 95% CI was 0.002-0.378), diabetes mellitus ( OR = 0.054, 95% CI was 0.005-0.611), coronary heart disease ( OR = 0.042, 95% CI was 0.002-0.968) and the IL-6 level after IMV ( OR = 0.902, 95% CI was 0.819-0.994) were independent risk factors for death during hospitalization in patients with critical COVID-19 undergoing IMV (all P < 0.05). Conclusions:The levels of inflammatory cytokine including IL-6, IL-10, and TNF-α increased significantly with aggravation in critical COVID-19 patients undergoing IMV, especially IL-6. IL-6 was an independent risk factor for death of critical COVID-19 patients undergoing IMV.

Objective:To repair the articular cartilage defects of animal models with cartilage tissue block made by multi-nozzle three-dimensional bio-printer and observe its effect.Methods:Bio-ink was made by adding silk fibroin, polyethylene glycol and bone mesenchymal stem cells into extracellular matrix (ECM) solution. Rheological properties of biological ink were evaluated by rheometer, the protein secondary structure of biological ink was identified by Fourier transform infrared spectroscopy, and a tissue engineering scaffold with thickness of 2mm and diameter of 6mm was printed by using a pressure sprinkler loaded with cartilage biological ink. The compression modulus of tissue engineering scaffold was measured by tension machine. The degradation rate of each scaffold was evaluated by dry weight loss method, and the viability and proliferation of cells on the scaffold were evaluated by CCK-8 and live&dead cell staining. The differentiation of cellular cartilage on the scaffold was evaluated by real-time fluorescence quantitative PCR. The scaffold was embedded into the defect area of animal articular cartilage to repair articular cartilage defect according to the principle of autogenous cartilage transplantation. The effect of cartilage repair after 3 months was evaluated by histological staining and biochemical detection.Results:We found that all biological inks showed the flow characteristics of shear thinning. The absorption peak of biological ink amide I region containing silk fibroin moved to 1 623 cm -1. With the increase of silk fibroin content, the mechanical strength and degradability of biological ink were improved, and the compression modulus of 10% and 15% printing stand reached 19.96±5.66 kpa and 26.87±10.68 kpa, respectively. All biological inks had no obvious cytotoxicity. Real-time quantitative PCR showed that when the content of silk fibroin reached 10%-15%, the bone marrow mesenchymal stem cells in the tissue mass had stronger ability to differentiate into cartilage. In vivo studies showed that after 3 months, the sGAG/DNA content of 10% and 15% silk fibroin scaffolds reached 0.25±0.01 μg/ng and 0.24±0.02 μg/ng, respectively, and the collagen/DNA content reached 17.71±0.83 ng/ng and 16.69±2.39 ng/ng, respectively. Tissue engineered cartilage printed with high concentration silk fibroin can better repair articular cartilage defects. Conclusion:TThe chondrogenic differentiation and extracellular matrix (collagen and glycosaminoglycan) secretion of BMSCs were superior to those of the other two scaffolds when the content of silk fibroin reaches 10%-15%. The changes of chondrogenic differentiation ability and extracellular matrix secretion of stem cells from different scaffolds, as well as the repair effect on articular cartilage defects are caused by the differences of mechanical properties of scaffolds, which can be produced by the changes of silk fibroin concentration.

Objective To investigate the effect of E1A gene on the radiosensitivity of human nasopharyngeal carcinoma cells and its possible mechanism. Methods The E1A gene was transfected into nasopharyngeal carcinoma CNE-2R cells by adenovirus vector. The expression of E1A gene was detected by RT-PCR. Untransfected CNE-2R cells(PBS group)and CNE-2R cells transfected with empty vector Ad-β-gal(Ad-β-gal group)and E1A(Ad-E1A group)were given 0 Gy,2 Gy,4 Gy,6 Gy,8 Gy 6 MV X-ray irradiation. The changes in radiosensitivity of CNE-2R cells were determined by colony-forming assay. Flow cytometry was used to analyze cell apoptosis in each group. The expression of NF-κB, CK2α, Bcl-2, and cleaved caspase-3 was measured by Western blot. Results RT-PCR confirmed that the E1A gene was transfected into CNE-2R cells and stably expressed. The Ad-E1A group had a significantly lower plating efficiency than the PBS group and the Ad-β-gal group(P<0.05). The Ad-E1A group had significantly lower cell survival rate at 2 Gy irradiation than the PBS group and the Ad-β-gal group(0.217 vs. 0.602, P<0.05;0.217 vs. 0.585, P<0.05). The Ad-E1A group had a significantly higher α/β value than the PBS group and the Ad-β-gal group(24.680 vs. 5.268, P<0.05;24.680 vs. 5.132, P<0.05). Flow cytometry results showed that irradiation alone could promote the apoptosis of CNE-2R cells,when combined with E1A gene,the apoptosis rate was significantly increased(P<0.05). Western blot results showed that E1A gene down-regulated the expression of NF-κB/p65,CK2α,and Bcl-2 and up-regulated the expression of cleaved caspase-3. Conclusions E1A gene can enhance the radiosensitivity of nasopharyngeal carcinoma cells by inhibiting the expression of CK2 to block the NF-κB signaling pathway and promoting cell apoptosis.

Objective To investigate the effect of Tiopronin (TIP) on interleukin (IL)-2 immunotherapy of human leukemia KG-1 cells and its possible mechanism.Methods KG-1 ceils in logarithmic growth phase were randomly divided into KG-1 + IL-2 group and KG-1 + IL-2 + TIP group.Methyl thiazolyl tetrazolium (MTI) assay and colony formation assay were used to detect the sensitivity and proliferation of KG-1 cells.The changes of reactive nitric metabolites (RNM) were detected with nitrate reductase method.The production of tumor necrosis factor (TNF)-3 and interferon (IFN)-γ,was detected with enzyme linked immunosorbent assay (ELISA).The expression of CD3ξ was detected with Western blot and real time polymerase chain reaction (RT-PCR).Results IL-2 and IL-2 + TIP could inhibit the growth of KG-1 cells.The inhibitory rate of KG-1 + IL-2 + TIP group was significantly higher than that of KG-1 + IL-2 group,and the sensitivity of KG-1 cells to IL-2 was 6.2 times higher.Both IL-2 and IL-2 + TIP group inhibited the colony formation of KG-1 cells.Compared to KG-1 + IL-2 group,KG-1 + IL-2 + TIP group inhibited the colony formation of KG-1 cells by 3.5 times.The RNM production of KG-1 + IL-2 group was (158.26 ± 3.82) μmol/ml,which was significantly higher than (45.18 ± 4.29) μ mol/ml of KG-1 + IL-2 + TIP group (P ＜ 0.05).The levels of TNF-β and IFN-γin KG-1 + IL-2 + TIP group were (253.28 ± 7.84) pg/ml and (181.25 ±6.41) pg/ml,which was significantly higher than (98.45 ±6.43) pg/ml and (68.74 ±8.26) pg/ml of KG-1 +IL-2 group (P＜0.05).The expression of CD3ξ in KG-1 +IL-2 +TIP group was significantly higher than that in KG-1 + IL-2 group.Conclusions Tiopronin can promote NK/T cell activity and increase the sensitivity of leukemia KG-1 cells to IL-2 by eliminating reactive nitrogen metabolites.

Objective To investigate the effects and its possible mechanisms of tiopronin (TIP) on interleukin-2 (IL-2) immunotherapy of human leukemia KG-1 cells transplanted in nude mice.Methods KG-1 cells (1 x 107/ml) in logarithmic growth phase were injected subcutaneously into the groin of the left hind leg of the 45 5-week-old nude mice.When the subcutaneous tumor diameter was about 8 mm,nude mice were randomly divided into three groups (n =15):Control group (intraperitoneal injection of phosphate buffer),IL-2 group (hypodermic injection of IL-2),IL-2 + TIP group (hypodermic injection of IL-2 and intraperitoneal injection of TIP).The therapeutic effect of TIP combined with IL-2 on human leukemia KG-1 cells transplanted in nude mice was observed.The number of nature killer (NK) cells in peripheral blood of nude mice was detected by flow cytometry.Nitrate reductase assay was used to detect reactive nitric metabolite (RNM) levels in peripheral blood of nude mice.Enzyme linked immunosorbent assay (ELISA) was used to detect the levels of tumor necrosis factor-β (TNF-β) and interferon-γ (IFN-γ) in peripheral blood of nude mice.Terminal-deoxynucleoitidyl transferase mediated nick end labeling (TUNEL) assay was used to analyze apoptosis.Results Both IL-2 and IL-2 + TIP could inhibit the growth of transplanted tumor.Compared with IL-2 group [(54.32 ± 4.32) ％],the tumor inhibition rate of IL-2 + TIP group was (90.15 ± 3.75)％,and its inhibition of tumor growth was more obvious (t =11.893,P ＜ 0.001).The tumor weights of Control group,IL-2 group and IL-2 + TIP group were (0.95 ± 0.05)g,(0.58 ± 0.03)g and (0.27 ± 0.07)g,and there was statistically significant difference among the three groups (F =52.716,P ＜ 0.001).Compared with IL-2 group,the tumor weight of IL-2 + TIP group was significantly reduced (P =0.008).The number of NK cells in IL-2 + TIP group was (0.658 ±0.157)/L,which was significantly higher than (0.452 ±0.124)/L of IL-2 group (P =0.021).The concentration of RNM in IL-2 + TIP group was (42.92 ± 4.68)μmol/ml,which was significantly lower than (163.38 ± 5.49)μmol/ml in IL-2 group (P =0.007).The concentrations of TNF-β and IFN-γin IL-2 + TIP group were (247.68 ± 8.24) pg/ml and (185.61 ±7.58) pg/ml,which were significantly higher than (97.48 ± 7.28)pg/ml (P =0.021) and (70.62 ± 8.47)pg/ml (P =0.015) in IL-2 group.The apoptotic rate of tumor cells in IL-2 + TIP group was (47.38±4.25)％,which was significantly higher than (21.41 ±2.79)％ in IL-2 group (P ＜0.001).Conclusion TIP can increase the sensitivity of leukemia cells to IL-2 immunothe-rapy by removing RNM,promoting NK cells activity and increasing NK cells-induced tumor cell apoptosis.

Aim To investigate the effect of sphingo-sine kinase 1 (SphK1 )on unilateral ureteral obstruc-tion(UUO)-induced tubulointerstitial fibrosis and ex-plore the possible mechanism.Methods The CD-1 mice were randomly divided into four groups:sham-op-eration group(Sham),PF-543 treatment control group (Sham +PF-543),model group(UUO)and PF-543 treatment group(UUO +PF-543).On 1 ,3,7 and 1 4 d after operation,eight mice were selected randomly from each group and sacrificed.The protein expressions of SphK1 ,mature TGF-β1 ,FN,ColⅠ,LC3,Beclin1 ,Atg5 and Atg1 2 were observed by Western blot.The histo-logical changes were examined by Masson′s trichrome stain.Immunhistochemistry was performed to measure the levels of expression of SphK1 ,FN and Col Ⅰ. Transmission electron microscope was used to observe the autophagic body.Results SphK1 expression and autophagy were both upregulated in a mouse model of kidney fibrosis induced by UUO. Meanwhile, in-creased mature TGF-β1 and deposition of extracellular matrix(ECM)were observed in tubulointerstitial areas compared with sham-operated mice.After intraperito-neal injection with the SphK1 specific inhibitor PF-543 in UUO mice,enhanced expression of SphK1 and acti-vated autophagy were significantly abrogated.Howev-er,aggravation of renal fibrosis was detected when SphK1 inhibitor PF-543 was applied to suppress SphK1 expression in UUO mice.Conclusion SphK1 activa-tion is renoprotective through the induction of autoph-agy in the pathogenesis of kidney fibrosis.

Aim To investigate the effects of high glu-cose on cholesterol metabolism in renal tubular cells and the intervention of the anthocyanins. Methods HK-2 cells were grown in the DMEM medium supple-mented with 10% FBS and were divided into 5 groups:normal glucose group, high glucose group, mannitol group, C3G group and Cy group. Effect of anthocya-nins on cell viability was detected with MTT, and cho-lesterol accumulation was detected with Amplex Red Cholesterol Assay kit and Filipin staining. Expression of ABCA1 was detected with RT-qPCR and Western Blot. Results In compared with control groups, HG significantly promoted cholesterol mass inside the cell and decreased the cholesterol concentration in the me-dium after treatment for 24 h or 48 h. The levels of mRNA and protein of ABCA1 were detected with RT-qPCR and Western blot, and both were decreased in the presence of HG. Whereas treatment with C3G and Cy markedly attenuated HG-induced cholesterol mass inside the cell by up-regulating the expression of AB-CA1. Conclusions High glucose can reduce the ex-pressions of the ABCA1, and then decrease cholesterol efflux and increase the cholesterol accumulation in HK-2 cells. Anthocyanins can decrease cholesterol accu-mulation by up-regulating the expression of ABCA1.