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BACKGROUND@#Implantation of bone marrow mesenchymal stem cells (BMSCs) is a potential alternative for promoting bone defects healing or osseointegration in osteoporosis. However, the reactive oxygen species (ROS) accumulated and excessive inflammation in the osteoporotic microenvironment could weaken the self-replication and multi-directional differentiation of transplanted BMSCs. @*METHODS@#In this study, to improve the hostile microenvironment in osteoporosis, Poloxamer 407 and hyaluronic acid (HA) was crosslinked to synthetize a thermos-responsive and injectable hydrogel to load MnO2 nanoparticles as a protective carrier (MnO2 @Pol/HA hydrogel) for delivering BMSCs. @*RESULTS@#The resulting MnO2 @Pol/HA hydrogel processed excellent biocompatibility and durable retention time, and can eliminate accumulated ROS effectively, thereby protecting BMSCs from ROS-mediated inhibition of cell viability, including survival, proliferation, and osteogenic differentiation. In osteoporotic bone defects, implanting of this BMSCs incorporated MnO2 @Pol/HA hydrogel significantly eliminated ROS level in bone marrow and bone tissue, induced macrophages polarization from M1 to M2 phenotype, decreased the expression of pro-inflammatory cytokines (e.g., TNFa, IL-1b, and IL-6) and osteogenic related factors (e.g., TGF-b and PDGF). @*CONCLUSION@#This hydrogel-based BMSCs protected delivery strategy indicated better bone repair effect than BMSCs delivering or MnO2 @Pol/HA hydrogel implantation singly, which providing a potential alternative strategy for enhancing osteoporotic bone defects healing.
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Objective:To explore the correlations of α-melanocyte stimulating hormone ( α-MSH) levels in serum and synovial fluid with progression of primary knee osteoarthritis (KOA). Methods:A retrospective analysis was conducted of the 96 patients who had been diagnosed as primary KOA at Department of Orthopedics, The First Hospital of Huizhou from October 2018 to October 2019. Radiographic severity of KOA was determined by Kellgren-Lawrence (K-L) grades; α-MSH levels were measured by enzyme-linked immunosorbent assay (ELISA). Levels of pro-inflammatory cytokine interleukin-1 β (IL-1 β) and matrix metalloproteinase-3 (MMP-3) were also detected. Another 64 patients with patellar dislocation, matched in age and gender, were enrolled as controls. The Numeric Pain Scale (NPS) and revised Oxford Knee Score (OKS) were employed to evaluate their symptomatic severity. Receiver operating characteristics (ROC) curve was used to compare α-MSH, IL-1 β and MMP-3 with regard to their diagnostic values in the K-L grading. Results:There were no statistically significant difference in age, gender and body mass index between the 2 groups, showing they were comparable ( P> 0.05). The α-MSH levels in synovial fluid were significantly lower in the KOA patients than in the controls [(16.9±3.8) pg/mL versus (18.8±2.7) pg/mL] ( P<0.001); there were no significant differences between the KOA patients and the controls in the serum α-MSH levels [(24.9±1.8) pg/mL versus (24.8±1.7) pg/mL] ( P>0.05). The α-MSH levels in synovial fluid were negatively correlated with K-L grades ( r=-0.382, P<0.001) and negatively correlated with NPS ( r=-0.382, P<0.001) but positively correlated with OKS ( r=0.339, P<0.001). Moreover, the α-MSH levels in synovial fluid were negatively correlated with the IL-1 β levels in synovial fluid ( r=-0.483, P<0.001) and with the MMP-3 levels in synovial fluid ( r=-0.336, P< 0.001). Conclusions:The level of serum α-MSH may not be correlated with the progression of KOA but the synovial fluid α-MSH is negatively correlated with the progression of KOA. Therefore, the expression level of α-MSH in joint synovial fluid can be used as a potential biomarker for assessment of severity of knee osteoarthritis.
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Objective:To investigate the correlation of mRNA and protein levels of Ghrelin in shoulder tissue and synovial fluid with severity of rotator cuff tear(RCT) and frozen shoulder(FS).Methods:Recruited for this study were 66 RCT and 66 FS patients who had been definitely diagnosed at Department of Orthopedics, The First Hospital of Huizhou from January 2018 to September 2019. Another 60 patients with rotator instability were recruited as a control group. After the severity of RCT was evaluated by ultrasonographic images, the RCT group was further divided into 3 subgroups according to the severity of RCT: a massive full thickness tear (MFTT) subgroup, a non massive full thickness tear (MFTT) subgroup, and a partial thickness tear (PTT) subgroup. The FS group was conventionally divided into 31 cases of acute phase and 35 cases of adhesive phase. Samples of subacromial bursa and shoulder joint bursa tissues and shoulder joint synovial fluid were collected. The expression of Ghrelin mRNA was detected by RT-PCR and the expression of Ghrelin protein in joint synovial fluid was detected by ELISA. The symptoms and functions of shoulder joint were evaluated by visual analogue scale (VAS) and Constant-Murley functional score.Results:There were no significant differences in gender or age between the RCT, FS and control groups, showing compatibility ( P>0.05).The expression of Ghrelin mRNA in subacromial synovial capsule and shoulder joint capsule and the expression of Ghrelin protein in shoulder synovial fluid in the RCT and FS groups were significantly lower than those in the control group ( P<0.05).The expression of Ghrelin mRNA in subacromial synovial capsule and shoulder joint capsule and the expression of Ghrelin protein in shoulder synovial fluid in the PTT subgroup were the highest, followed sequentially by NMFTT and MFTT subgroups, with significant differences between subgroups ( P<0.05). For FS patients, the expression levels of Ghrelin mRNA and protein in the acute phase were significantly lower than in the adhesive phase ( P<0.05). The relative expression of Ghrelin mRNA in joint capsule and the expression of Ghrelin protein in shoulder synovial fluid were negatively correlated with VAS scores and IL-6 levels (all P<0.05), and positively correlated with the Constant-Murley scores (all P<0.05). Conclusions:The expression of Ghrelin in shoulder tissue and synovial fluid is negatively correlated with the progress of rotator cuff tear and frozen shoulder.Local supplementation of Ghrelin may be a potential therapy for rotator cuff tear and frozen shoulder.
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ObjectiveTo explore the effects of glycomacropeptide (GMP) in human milk and formula milk on proliferation ofbifidobacterium infantis and their dose-response relationship.Methods Casein was isolated from the milk of 30 healthy postpartum women from Guangzhou Women and Children's Medical Center in September 2014, and hydrolyzed by rennet to obtain GMP, which was then purified by ultrafiltration and ion exchange chromatography. Human milk GMP and cow milk GMP (0, 250, 500, 1 000, 1 500, 2 000 and 3 000 mg/L) were added tobifidobacterium infantis liquid medium, and cultured under anaerobic conditions. Concentration of bacteria was measured by turbidimetric microplate assay (detection of OD600 nmvalue of medium). Difference of proliferative activities ofbiifdobacterium infantis in human milk GMP and cow milk GMP was compared with independent samplest-test.ResultsPurified human milk GMP concentration was 1 712.20 mg/L, with a purity of 80.3%. Increasing the cow milk GMP initial concentration in the culture medium at 250-2 000 mg/L could increase the concentration and proliferation rate ofbiifdobacteria infantis. When cultured at 36 h with GMP of various concentrations, the proliferation ofbiifdobacteria infantis maintained at a logarithmic phase. Therefore, 36 h was chosen as the test time point to compare the proliferation ofbifidobacterium infantis. At 36 h, when GMP in the medium was 1 000, 1 500, 2 000 and 3 000 mg/L, concentrations ofbiifdobacteria infantis in human milk GMP were 2.255±0.036, 2.583±0.088, 2.877±0.080 and 3.219±0.081, respectively, which were significantly higher than those in cow milk GMP (2.115±0.053, 2.312±0.064, 2.542±0.090 and 2.894±0.076;t=4.867, 5.569, 6.192 and 6.516; allP<0.01).Conclusions Both human milk GMP and cow milk GMP can promote the proliferation ofbiifdobacterium infantisin vitro, and the proliferative activity in human milk is greater than in cow milk at the same concentration of GMP.
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Objective To detect the effects of bifidobacterium or bifidobacterium cultured supernatant on the mRNA expression of tumor necrosis factor receptor-associated factor 6 (TRAF6),glycogen synthase kinase-3β (GSK-3β) and the miRNA-146a in rat small intestinal epithelial cell(IEC-6) induced by lipopolysaccharide (LPS).Methods IEC-6 in logarithmic phase were randomly divided into LPS group,cultured supernatant group and inactivated bacteria group.All the 3 groups were exposed to 5 mg/L LPS for 5 hours,and then 1 mL sterile saline was added in LPS group and culturing continued for 24 hours ; 1 mL bifidobacterium cultured supernatant was added in cultured supernatant group and culturing continued for 24 hours;1 mL inactivated bifidobacterium 1 x 1010 CFU/L added in inactivated bacteria group and continued culturing for 24 hours.The mRNA expressions of TRAF6,GSK-3 β and miRNA-146a were detected by reverse transcription-polymerase chain reaction (RT-PCR).Results The level of TRAF6,GSK-3 β of culture supematant group (0.72 ± 0.05,0.46 ± 0.14) were all lower than LPS group (1.01 ± 0.14,1.02 ± 0.25),but the level of miRNA-146a(3.05 ± 0.40) was higher than that in LPS group(1.01 ± 0.12),and there were significant differences between them (t =5.278,6.316,13.218,P =0.000).The level of GSK-3 β of inactivated bacteria group(0.59 ±0.13) was significantly lower than that in LPS group(t =4.837,P =0.000).The levels of TRAF6 and miRNA-146a of inactivated bacteria group(1.05 ±0.11,0.78 ±0.22) had no significant differences with LPS group (t =0.732,1.463,P ＞ 0.05).The level of TRAF6 of cultured supernatant group was lower than that in inactivated bacteria group,and the level of miRNA-146a was higher than that in inactivated bacteria group,and there were significant differences between 2 groups (t =6.009,14.687,P =0.000).Conclusions Bifidobacterium cultured supernatant and inactivated bacteria both have certain protective effect on the IEC-6 induced by LPS.One of the protective mechanisms of bifidobacterium cultured supernatant may be achieved by elevating the expression of miRNA-146a,and decreasing the levels of inflammation related factor TRAF6 and damage related factor GSK-3β.The protective effects of inactivated bifidobacterium may be achieved by decreasing the level of damage related factor GSK-3β.
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Objective To investigate the effects of lipopolysaccharides (LPS) in different concentrations on the proliferation and interleukin(IL)-6,IL-1 β and tumor necrosis factor-α(TNF-o) secretion of intestinal epithelial cells (IEC-6) of rats in vitro.Methods IEC-6 of rats were divided into normal group (0 mg/L,group A),0.1 mg/L group (group B),0.5 mg/L group (group C),1.0 mg/L group (group D),5.0 mg/L group (group E) and 10.0 mg/L group(group F).Different groups cells were exposed to LPS with different concentrations for 3 h,5 h and 7 h.Thiazolyl blue(MTT) was performed to investigate the proliferation of IEC-6.The concentrations of IL-6,IL-1 β and TNF-α in culture supernatant were detected by enzyme linked immunosorbent assay(ELISA).Results The proliferation rate of IEC-6 was gradually lower while the concentration of LPS increased.After co-culture with LPS 3h and 5 h,the proliferation rates of group B,group C,group D,group E and group F had no significant difference with those of group A (all P ＞ 0.05);after co-culture with LPS 7 h,the proliferation rates of group B,group C,and group D had no significant difference with those of group A (all P ＞ 0.05);the proliferation rates of group E and group F had significant difference with those of group A(t =4.216,P =0.014;t =14.991,P =0.000).The proliferation rates of group E and group F were lower after co-culture with LPS 5 h than 7 h,and there were significant differences (t =2.576,P =0.033;t =2.975,P =0.018);but there was no significant differences between group E and group A after co-culture with LPS 5 h (P ＞ 0.05).Group B,group C,group D,group E and group F all had a significant higher level of IL-6 than group A after co-culture with LPS 3 h,5 h and 7 h(all P ＜0.01).In addition,group E had the highest level of IL-6 at all time points.And the peak level of IL-6 rose after co-culture with LPS 5 h.The TNF-α level and IL-1 β level of group B,group C,group D,group E and group F all had no significant differences than that of group A after co-culture with LPS 3 h,5 h and 7 h (all P ＞ 0.05).Conclusions In a certain concentration,incubation time range,the proliferation rates of IEC-6 cells were gradually lower while the concentration of LPS increased.Co-cultured IEC-6 cells with LPS(0-10.0 mg/L) can stimulate them secrete to IL-6.The highest level of IL-6 was of group E after 5 h co-culture.LPS had no effects on TNF-α and IL-1 β level of IEC-6 cells cultural supernatant.So 5.0 mg/L concentration of LPS stimulating IEC-6 cells for 5 h can be chosen to build the IEC-6 inflammatory models.