Injectable temperature-sensitive hydrogel system incorporating deferoxamine-loaded microspheres promotes H-type blood vessel-related bone repair of a critical size femoral defect.

Insufficient vascularization is a major challenge in the repair of critical-sized bone defects. Deferoxamine (DFO) has been reported to play a potential role in promoting the formation of H-type blood vessels, a specialized vascular subtype with coupled angiogenesis and osteogenesis. However, whether DFO promotes the expression of H-type vessels in critical femoral defects with complete periosteal damage remains unknown. Moreover, stable drug loading systems need to be designed owing to the short half-life and high-dose toxic effects of DFO. In this study, we developed an injectable DFO-gelatin microspheres (GMs) hydrogel complex as a stable drug loading system for the treatment of critical femoral defects in rats. Our results showed that sustained release of DFO in critical femoral defects stimulated the generation of functional H-type vessels. The DFO-GMs hydrogel complex effectively promoted proliferation, formation, and migration of human umbilical vein endothelial cells in vitro. In vivo, the application of the DFO-GMs hydrogel complex expanded the distribution range and prolonged the expression time of H-type vessels in the defect area and was positively correlated with the number of osterix+ cells and new bone tissue. Topical application of the HIF-1α inhibitor PX-478 partially blocked the stimulation of H-type vessels by DFO, whereas the osteogenic potential of the latter was also weakened. Our results extended the local application of DFO and provided a theoretical basis for targeting H-type vessels to treat large femoral defects. STATEMENT OF SIGNIFICANCE: Abundant functional blood vessels are essential for bone repair. The H-type blood vessel is a functional subtype with angiogenesis and osteogenesis coupling potential. A drug loading system with long-term controlled release was first used to investigate the formation of H-type blood vessels in critical femoral defects and promotion of bone repair. Our results showed that the application of DFO-GMs hydrogel complex expanded the distribution range and expression time of H-type vessels, and was positively correlated with the number of osteoblasts and volume of new bone tissue. These results expanded the local application approach of DFO and provide a theoretical basis for targeting H-type vessels to treat large femoral defects.

Can metformin relieve tibiofemoral cartilage volume loss and knee symptoms in overweight knee osteoarthritis patients? Study protocol for a randomized, double-blind, and placebo-controlled trial.

BACKGROUND: Osteoarthritis (OA) is the most common joint disease, and is most frequently seen in the knees. However, there is no effective therapy to relieve the progression of knee OA. Metformin is a safe, well-tolerated oral medication that is extensively used as first-line therapy for type 2 diabetes. Previous observational studies and basic researches suggested that metformin may have protective effects on knee OA, which needs to be verified by clinical trials. This study, therefore, aims to examine the effects of metformin versus placebo on knee cartilage volume loss and knee symptoms in overweight knee OA patients by a randomized controlled trial over 24 months. METHODS: This protocol describes a multicenter, randomized, double-blind, and placebo-controlled clinical trial aiming to recruit 262 overweight knee OA patients. Participants will be randomly allocated to the two arms of the study, receiving metformin hydrochloride sustained-release tablets or identical inert placebo for 24 months (start from 0.5 g/day for the first 2 weeks, and increase to 1 g/day for the second 2 weeks, and further increase to 2 g/day for the remaining period if tolerated). Primary outcomes will be changes in tibiofemoral cartilage volume and Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) score over 24 months. Secondary outcomes will be changes in visual analogue scale (VAS) knee pain, tibiofemoral cartilage defects, effusion-synovitis volume, and tibiofemoral bone marrow lesions maximum size over 24 months. The primary analyses will be intention-to-treat analyses of primary and secondary outcomes. Per-protocol analyses will be performed as the secondary analyses. DISCUSSION: If metformin is proved to slow knee cartilage volume loss and to relieve knee symptoms among overweight knee OA patients, it will have the potential to become a disease modifying drug for knee OA. Metformin is a convenient intervention with low cost, and its potential effects on slowing down the structural progression and relieving the symptoms of knee OA would effectively reduce the disease burden worldwide. TRIAL REGISTRATION: ClinicalTrials. gov NCT05034029 . Registered on 30 Sept 2021.

Advanced oxidation protein products induce inflammatory responses and invasive behaviour in fibroblast-like synoviocytes via the RAGE-NF-κB pathway.

AIMS: Rheumatoid arthritis (RA), which mainly results from fibroblast-like synoviocyte (FLS) dysfunction, is related to oxidative stress. Advanced oxidation protein products (AOPPs), which are proinflammatory mediators and a novel biomarker of oxidative stress, have been observed to accumulate significantly in the serum of RA patients. Here, we present the first investigation of the effects of AOPPs on RA-FLSs and the signalling pathway involved in AOPP-induced inflammatory responses and invasive behaviour. METHODS: We used different concentrations of AOPPs (50 to 200 µg/ml) to treat RA-FLSs. Cell migration and invasion and the expression levels of tumour necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), matrix metalloproteinase-3 (MMP-3), and MMP-13 were investigated. Western blot and immunofluorescence were used to analyze nuclear factor-κB (NF-κB) activation. RESULTS: AOPPs promoted RA-FLS migration and invasion in vitro and significantly induced the messenger RNA (mRNA) and protein expression of TNF-α, IL-6, MMP-3, and MMP-13 in dose- and time-dependent manners. Moreover, AOPPs markedly activated the phosphorylation of nuclear factor-κB (NF-κB) p65 protein, which triggered inhibitory kappa B-alpha (IκBα) degradation, NF-κB p65 protein phosphorylation, and NF-κB p65 translocation into the nucleus. Furthermore, treatment with a neutralizing antibody specific to receptor for advanced glycation end products (RAGE) significantly suppressed aggressive behaviour and inflammation, decreased TNF-α, IL-6, MMP-3, and MMP-13 expression, and blocked AOPP-induced NF-κB pathway activation. CONCLUSION: The results indicate that AOPPs can enhance aggressive behaviour and the inflammatory response in RA-FLSs via the RAGE-NF-κB pathway. These results present AOPPs as a new class of potentially important mediators of progressive disease in RA patients. Cite this article: Bone Joint Res 2021;10(4):259-268.

Serum KL-6, CA19-9, CA125 and CEA are Diagnostic Biomarkers for Rheumatoid Arthritis-Associated Interstitial Lung Disease in the Chinese Population.

INTRODUCTION: This study aimed to evaluate the role of tumor marker carbohydrate antigen (CA) 125 (CA125), CA19-9, carcinoembryonic antigen (CEA) and Krebs von den Lungen-6 (KL-6) in the diagnosis and determination of the severity of interstitial lung disease (ILD) in rheumatoid arthritis (RA) patients. METHODS: A retrospective analysis was performed. Fifty RA patients (24 patients with ILD and 26 patients without ILD), 10 healthy subjects and 14 patients with other connective tissue disease-associated interstitial lung disease were included. Serum levels of KL-6 and tumor markers CA19-9, CA125 and CEA were measured. Chest HRCT of patients with ILD was scored quantitatively according to the degree of fibrosis. Data on the C-reactive protein, erythrocyte sedimentation rate, rheumatoid factors and anti-cyclic peptide containing citrulline (anti-CCP) were also collected. RESULTS: Serum levels of KL-6, CA19-9, CA125 and CEA in the RA-ILD group were significantly higher than those in the RA-no-ILD group. The serum KL-6 level was positively correlated with the HRCT fibrosis score (r = 0.63, p = 0.002). The logistic regression analysis showed that CA19-9 and smoking were associated with RA-ILD [OR = 1.118, 95% CI = (1.038, 1.204), p = 0.003 for CA19-9, OR = 14.969, 95% CI = (1.750, 128.043), p = 0.013 for smoking]. CONCLUSIONS: KL-6 level and tumor markers were elevated in RA-ILD, and strongly associated with the severity of ILD, supporting their value as pathogenically relevant biomarkers, which can contribute to noninvasive detection of this extra-articular disease complication.

Interstitial lung disease (ILD) is a common pulmonary manifestation of RA associated with high morbidity and mortality. Our retrospective study was performed to investigate the clinical utility of tumor marker carbohydrate antigen (CA) 125 (CA125), CA19-9, carcinoembryonic antigen (CEA) and Krebs von den Lungen-6 (KL-6) in the diagnosis and determining the severity of RA-ILD. Fifty RA patients (24 patients with ILD and 26 patients without ILD), 10 healthy subjects and 14 patients with other connective tissue disease-associated interstitial lung disease (CTD-ILD) were included. The results showed KL-6 level and tumor markers were elevated in RA-ILD, and strongly associated with the severity of ILD, which meant KL-6 and tumor markers might be useful pathogenically relevant biomarkers and could be predictors for the diagnosis and determination of severity of ILD in RA.

Transforming growth factor ß1 promotes fibroblast-like synoviocytes migration and invasion via TGF-ß1/Smad signaling in rheumatoid arthritis.

Migration and invasion are important characteristics of rheumatoid arthritis fibroblast-like synoviocytes (RA-FLSs), which are involved in joint damage and contribute to rheumatoid arthritis (RA) pathology. However, the underlying mechanisms remain unclear. Because epithelial-mesenchymal transition (EMT) is a key mechanism related to migration and invasion in cancer cells, we investigated the relationship between EMT and RA-FLSs and explored whether the transforming growth factor ß1 (TGF-ß1)/Smad signaling pathway is involved. In vivo, fibroblast-like synoviocytes (FLSs) were isolated from the synovium of RA or osteoarthritis (OA) patients and cultured for 4-8 passages. EMT markers were detected by immunofluorescence and Western blotting. RA-FLSs were treated with TGF-ß1 or Smad2/3 small interfering RNA (siRNA), EMT markers were detected, and migration and invasion were assessed by Transwell assays. EMT markers could be detected in FLSs; when compared with osteoarthritis fibroblast-like synoviocytes (OA-FLSs), E-cadherin and vimentin decreased, while N-cadherin and α-smooth muscle actin (α-SMA) increased in RA-FLSs. Furthermore, TGF-ß1 enhanced migration and invasion by inducing EMT via activating Smad2/3 in RA-FLSs. Phosphorylation of Smad2/3 was accompanied by degradation of Smad3. Silencing Smad2/3 blocked EMT and inhibited the migration and invasion induced by TGF-ß1. Matrix metalloproteinase 9 (MMP9) and vimentin were not affected when cells were treated with TGF-ß1 or Smad2/3 siRNA. The TGF-ß1/Smad signaling pathway is involved in EMT and contributes to migration and invasion in RA-FLSs. Interestingly, vimentin decreased in RA-FLSs, but there is no correlation between vimentin and TGF-ß1/Smad signaling pathway. Thus, further research on vimentin should be conducted.

Development of a centrally vascularized tissue engineering bone graft with the unique core-shell composite structure for large femoral bone defect treatment.

Great effort has been spent to promote the vascularization of tissue engineering bone grafts (TEBG) for improved therapeutic outcome. However, the thorough vascularization especially in the central region still remained as a major challenge for the clinical translation of TEBG. Here, we developed a new strategy to construct a centrally vascularized TEBG (CV-TEBG) with unique core-shell composite structure, which is consisted of an angiogenic core and an osteogenic shell. The in vivo evaluation in rabbit critical sized femoral defect was conducted to meticulously compare CV-TEBG to other TEBG designs (TEBG with osteogenic shell alone, or angiogenic core alone or angiogenic core+shell). Microfil-enhanced micro-CT analysis has been shown that CV-TEBG could outperform TEBG with pure osteogenic or angiogenic component for neo-vascularization. CV-TEBG achieved a much higher and more homogenous vascularization throughout the whole scaffold (1.52-38.91 folds, p < 0.01), and generated a unique burrito-like vascular network structure to perfuse both the central and peripheral regions of TEBG, indicating a potential synergistic effect between the osteogenic shell and angiogenic core in CV-TEBG to enhance neo-vascularization. Moreover, CV-TEBG has generated more new bone tissue than other groups (1.99-83.50 folds, p < 0.01), achieved successful bridging defect with the formation of both cortical bone like tissue externally and cancellous bone like tissue internally, and restored approximately 80% of the stiffness of the defected femur (benchmarked to the intact femur). It has been further observed that different bone regeneration patterns occurred in different TEBG implants and closely related to their vascularization patterns, revealing the potential profound influence of vascularization patterns on the osteogenesis pattern during defect healing.

Microarray Expression Profile of Circular RNAs in Peripheral Blood Mononuclear Cells from Rheumatoid Arthritis Patients.

BACKGROUND/AIMS: Circular RNAs (circRNAs) compose a large class of RNAs that can be used as biomarkers in clinical blood samples. This study aimed to determine the expression of circRNAs in peripheral blood mononuclear cells (PBMCs) from rheumatoid arthritis (RA) patients to identify novel biomarkers for RA screening. METHODS: We started with a microarray screening of circRNA changes in PBMCs from 5 RA patients and 5 healthy controls. We then confirmed the selected circRNA changes in PBMCs from 30 RA patients and 25 age- and sex-matched controls using the real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Spearman correlation test was performed to assess the correlation of circRNAs and clinical variables. Receiver operating characteristic (ROC) curve was calculated to evaluate the diagnostic value. RESULTS: We identified and verified five circRNAs (092516, 003524, 103047, 104871, 101873) that were significantly elevated in PBMCs from RA patients. Among these RA patients, we detected no significant correlation between the five circRNAs and the disease severity, including disease activity score (DAS28), erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), and health assessment questionnaire (HAQ). Yet, ROC curve analysis suggested that circRNA_104871 has significant value of RA diagnosis (AUC=0.833, P<0.001), followed by circRNA_003524 (AUC = 0.683, P = 0.020), circRNA_101873 (AUC = 0.676, P = 0.026), and circRNA_103047 (AUC = 0.671, P = 0.030). CONCLUSIONS: This study suggests that increased expression of circRNAs circRNA_104871, circRNA_003524, circRNA_101873 and circRNA_103047 in PBMC from RA patients may serve as potential biomarkers for RA patient diagnosis.

Advanced oxidation protein products promote NADPH oxidase-dependent ß-cell destruction and dysfunction through the Bcl-2/Bax apoptotic pathway.

The accumulation of plasma advanced oxidation protein products (AOPPs) has been linked with diverse disorders, including diabetes, chronic kidney disease, obesity, and metabolic syndrome. The aim of the present study was to evaluate the pathophysiological relevance of AOPPs in ß-cell destruction and dysfunction. Exposure of cultured rat ß-cells (INS-1) to AOPPs induced an increase in Bax expression, caspase-3 activity, and apoptosis as well as a decrease in Bcl-2 expression in a dose- and time-dependent manner. AOPP challenge rapidly increased the production of intracellular superoxide by activation of NADPH oxidases, demonstrated by p47phox translocation and interaction with p22phox and gp91phox, and this in turn led to apoptosis. AOPPs treatment resulted in ß-cell apoptosis, AOPPs accumulation, and decreased insulin content in pancreas and plasma in unilateral nephrectomized rats. Chronic inhibition of NADPH oxidase by apocynin prevented ß-cell apoptosis and ameliorated insulin deficiency in AOPP-challenged rats. This study demonstrates for the first time that accumulation of AOPPs promotes NADPH oxidase-dependent ß-cell destruction and dysfunction by the Bcl-2/Bax-caspase apoptotic pathway. This finding may provide a mechanistic explanation for ß-cell destruction and dysfunction in patients with diverse disorders.

Effect of the co-culture of human bone marrow mesenchymal stromal cells with human umbilical vein endothelial cells in vitro.

Mesenchymal stem cells (MSCs) give origin to the marrow tromal environment that supports hematopoiesis. These cells present a wide range of differentiation potentials and a complex relationship with hematopoietic stem cells (HSCs) and endothelial cells. In addition to bone marrow (BM), MSCs can be obtained from other sites in the adult or the fetus. Recent studies have shown that cocultured endothelial cells and osteoblasts are mutually promotive in bone tissues repair. In this study, we observed the effects of coculture of endothelial cells and osteoblasts at different ratios on vasculogenesis and bone formation, and we found that angiogenic effect is more effective when endothelial cells are cocultured with osteoblasts at the ratio of 4:1, and osteogenic effect is more effective at the ratio of 1:4. It is concluded that the co-culture of human bone marrow mesenchymal stromal cells with human umbilical vein endothelial cells could be a promising culture system for bone tissue engineering applications.

[Clinical significance of PCT, CRP, ESR, WBC count as predictors in postoperative early infectious complications with fever after posterior lumbar internal fixation].

OBJECTIVE: To evaluate the role of serum level of procalcitonin (PCT), C-reactive protein (CRP), erythrocyte sedimentation rate (ESR), and white blood cell count (WBC) as predictors in postoperative early infectious complications with fever after posterior lumbar internal fixation (PLIF). METHODS: A retrospective study was conducted from January 2012 to January 2014. Fifty-two patients with fever in the early stage(within 10 days) after the PLIF were collected in the study. They were divided into infection group and non-infection group (group A and group B) according to the results of postoperative blood culture. There were 26 patients in group A and 32 patients in group B. The values of PCT, CRP, ESR, and WBC count were compared and analyzed between two groups. RESULTS: The values of PCT, CRP, and ESR in group A were higher than those of group B. Meanwhile, CRP and ESR in group B were still higher than the normal range. Among the 26 patients with infections (group A), PCT was superior to CRP and ESR, had a good ability in discriminating different kinds of postoperative infections. The area under the ROC curve of serum PCT levels was the largest (CI 95% was 0.81 to 0.98) in the indexs; and ROC curve of WBC count was no statistically significant. When the cut off points of each predictors were evaluated, the higher sensitive was CRP and reached at 90.27% and the higher specific was ESR and reached at 88.50%. CONCLUSION: For the patients with fever at the early stage after the PLIF should be paid attention and reasonable choosing predictors are helpful to identify postoperative infection in the early stage. The CRP and ESR may be influenced by the surgery, and the PCT level is helpful to differentiate infection type.

[Tissue engineering vascularized bone repairing segmental femoral bone defects in rabbits].

OBJECTIVE: To investigate the effectiveness and mechanism of tissue engineering vascularized bone in repairing segmental femoral bone defects in rabbits. METHODS: Thirty-two rabbits were randomized into two groups (n = 16 each). A segmental and critical bone defect of 15 mm in length was made at left femur. In experimental group, the tissue engineering bone constructed from autologous bone marrow mesenchymal stem cells plus beta-tricalcium phosphate (beta-TCP) and vascular bundle was implanted into bony defect. In control group, there was no implantation of vascular bundle. Animals were sacrificed at 2, 4, 8 and 12 weeks post-implantation respectively. Histological observation was conducted to determine the process of new bone formation and remodeling. The expression of vascular endothelial growth factor (VEGF) in new bone was measured by immunohistochemistry, real-time PCR and Western blot. RESULTS: As indicated by histological observations over time, new bone formation increased in both groups. It was better in the experimental group than the control group at the beginning of 4 weeks. The expression level of VEGF gradually decreased in each group after an initial rise. And the expression of VEGF was significantly higher than the control group after implantation at all time points and peaked at 4 weeks. CONCLUSION: Tissue engineering vascularized bone accelerates bone repair in critical size defect model of femur in rabbit. Implantation of vascular bundle can promote the secretion of VEGF. And VEGF is an essential mediator of both angiogenesis and ossification.

Osteogenesis and angiogenesis of tissue-engineered bone constructed by prevascularized ß-tricalcium phosphate scaffold and mesenchymal stem cells.

Although vascularized tissue-engineered bone grafts (TEBG) have been generated ectopically in several studies, the use of prevascularized TEBG for segmental bone defect repair are rarely reported. In current study, we investigated the efficacy of prevascularized TEBG for segmental defect repair. The segmental defects of 15 mm in length were created in the femurs of rabbits bilaterally. In treatment group, the osteotomy site of femur was implanted with prevascularized TEBG, which is generated by seeding mesenchymal stem cells (MSCs) into ß-TCP scaffold, and prevascularization with the insertion of femoral vascular bundle into the side groove of scaffold; whereas in the control group, only MSC mediated scaffolds (TEBG) were implanted. The new bone formation and vascularization were investigated and furthermore, the expression of endogenous vascular endothelial growth factor (VEGF) which might express during defect healing was evaluated, as well. At 4, 8, and 12 weeks postoperatively, the treatment of prevascularized TEBG led to significantly higher volume of regenerated bone and larger amount of capillary infiltration compared to non-vascularized TEBG. The expression of VEGF in mRNA and protein levels increased with implantation time and peaked at 4 weeks postoperatively, followed by a slow decrease, however, treatment group expressed a significant higher level of VEGF than control group throughout the whole study. In conclusion, this study demonstrated that prevascularized TEBG by insertion of vascular bundle could significantly promote the new bone regeneration and vascularization compared to non-vascularized TEBG, which could be partially explained by the up-regulated expression of VEGF.

Advanced oxidation protein products decrease expression of nephrin and podocin in podocytes via ROS-dependent activation of p38 MAPK.

Accumulation of plasma advanced oxidation protein products (AOPPs) promotes progression of proteinuria and glomerulosclerosis. To investigate the molecular basis of AOPPs-induced proteinuria, normal Sprague-Dawley rats were treated with AOPPs-modified rat serum albumin. The expression of glomerular podocyte slit diaphragm (PSD)-associated proteins, nephrin and podocin, was significantly decreased coincident with the onset of albuminuria in rats treated with AOPPs. Chronic inhibition of NADPH oxidase by apocynin prevented down-regulation of nephrin and podocin and decreased albuminuria in AOPPs-challenged rats. This suggested that accumulation of AOPPs promotes proteinuria, possibly via down-regulating the expression of PSD-associated proteins.