Altered ECM deposition by diabetic foot ulcer‐derived fibroblasts implicates fibronectin in chronic wound repair

Current chronic wound treatments often fail to promote healing of diabetic foot ulcers (DFU), leading to amputation and increased patient morbidity. A critical mediator of proper wound healing is the production, assembly, and remodeling of the extracellular matrix (ECM) by fibroblasts. However, little is known about how these processes are altered in fibroblasts within the DFU microenvironment. Thus, we investigated the capacity of multiple, primary DFU‐derived fibroblast strains to express, produce, and assemble ECM proteins compared to diabetic patient‐derived fibroblasts and healthy donor‐derived fibroblasts. Gene expression microarray analysis showed differential expression of ECM and ECM‐regulatory genes by DFU‐derived fibroblasts which translated to functional differences in a 3D in vitro ECM tissue model. DFU‐derived fibroblasts produced thin, fibronectin‐rich matrices, and responded abnormally when challenged with transforming growth factor‐beta, a key regulator of matrix production during healing. These results provide novel evidence that DFU‐derived fibroblasts contribute to the defective matrices of DFUs and chronic wound pathogenesis.     

Inhibition of miR‐92a Enhances Fracture Healing via Promoting Angiogenesis in a Model of Stabilized Fracture in Young Mice

MicroRNAs (miRNAs) are endogenous small noncoding RNAs regulating the activities of target mRNAs and cellular processes. Although no miRNA has been reported to play an important role in the regulation of fracture healing, several miRNAs control key elements in tissue repair processes such as inflammation, hypoxia response, angiogenesis, stem cell differentiation, osteogenesis, and chondrogenesis. We compared the plasma concentrations of 134 miRNAs in 4 patients with trochanteric fractures and 4 healthy controls (HCs), and the levels of six miRNAs were dysregulated. Among these miRNAs, miR‐92a levels were significantly decreased 24 hours after fracture, compared to HCs. In patients with a trochanteric fracture or a lumbar compression fracture, the plasma concentrations of miR‐92a were lower on days 7 and 14, but had recovered on day 21 after the surgery or injury. To determine whether systemic downregulation of miR‐92a can modulate fracture healing, we administered antimir‐92a, designed using locked nucleic acid technology to inhibit miR‐92a, to mice with a femoral fracture. Systemic administration of antimir‐92a twice a week increased the callus volume and enhanced fracture healing. Enhancement of fracture healing was also observed after local administration of antimir‐92a. Neovascularization was increased in mice treated with antimir‐92a. These results suggest that plasma miR‐92a plays a crucial role in bone fracture healing in human and that inhibition of miR‐92a enhances fracture healing through angiogenesis and has therapeutic potential for bone repair. © 2014 American Society for Bone and Mineral Research.     

Five Freely Circulating miRNAs and Bone Tissue miRNAs Are Associated With Osteoporotic Fractures

Osteoporosis as a systemic skeletal disorder is characterized by increased bone fragility and the risk of fractures. According to the World Health Organization, osteoporosis is one of the 10 most common diseases and affects approximately 75 million people in Europe, the United States, and Japan. In this context, the identification of specific microRNA (miRNA) signatures is an important step for new diagnostic and therapeutic approaches. The focus of interest on miRNAs as biomarkers came with new publications identifying free circulating extracellular miRNAs associated with various types of cancer. This study aimed to identify specific miRNAs in patients with osteoporotic fractures compared with nonosteoporotic fractures. For the array analysis, miRNAs were isolated from the serum of 20 patients with hip fractures, transcribed, and the samples were pooled into 10 osteoporotic and 10 nonosteoporotic specimens. With each pool of samples, human serum and plasma miRNA PCR arrays were performed, which are able to identify 83 different miRNAs. Subsequently, a separate validation analysis of each miRNA found to be regulated in the array followed with miRNA samples isolated from the serum of 30 osteoporotic and 30 nonosteoporotic patients and miRNA samples isolated from the bone tissue of 20 osteoporotic and 20 nonosteoporotic patients. With the validation analysis of the regulated miRNAs, we identified 9 miRNAs, namely miR‐21, miR‐23a, miR‐24, miR‐93, miR‐100, miR‐122a, miR‐124a, miR‐125b, and miR‐148a, that were significantly upregulated in the serum of patients with osteoporosis. In the bone tissue of osteoporotic patients, we identified that miR‐21, miR‐23a, miR‐24, miR‐25, miR‐100, and miR‐125b displayed a significantly higher expression. A total of 5 miRNAs display an upregulation both in serum and bone tissue. This study reveals an important role for several miRNAs in osteoporotic patients and suggested that they may be used as biomarkers for diagnostic purposes and may be a target for treating bone loss and optimizing fracture healing in osteoporotic patients. © 2014 American Society for Bone and Mineral Research.     

High‐dose folic acid and its effect on early stage diabetic foot ulcer wound healing

High‐dose folic acid (HDFA; vitamin B9)—5 mg, given daily, has not been evaluated as a treatment to improve early stage‐diabetic foot ulcer (ES‐DFU) wound healing. However, HDFA has been demonstrated to correct: (a) endothelial dysfunction and decreased nitric oxide (NO) bioavailability, associated with type‐2 diabetes mellitus (T2DM); and (b) hyperhomocysteinemia (HHcy) that may promote impaired DFU‐wound healing. Measures of wound area (cm²) reduction (wound closure; WC), over a 4‐week period (4 W‐WC), greater than 50% of the wound area, have been reported as a robust indicator of the potential for DFU‐wound healing. By using this model, we examined the effectiveness of a wound treatment in promoting progressive healing and complete wound closure for the chronic, nonhealing DFU‐wound. To investigate this possible relationship between HDFA and ES‐DFU wound healing, a retrospective cohort study of medical records, between November 2018 and April 2019, was performed for Veterans with T2DM and ES‐DFUs following treatment with HDFA. During the study period 29 (n = 29) Veterans with ES‐DFU wounds who received HDFA treatment were identified. Medical record reviews of this retrospective cohort of ES‐DFU Veterans receiving HDFA report 90% (26/29) experiencing complete DFU‐wound closure during the study period. Of the 29 Veterans with ES‐DFUs receiving HDFA, the medical records of nine (30%), with healed wounds, provided documentation suitable for 4 W‐WC, pre‐ and post‐HDFA treatment study comparisons. This study documents significant (P < .05) improvements comparing 4 W‐WC values for standard treatment for Veterans with poorly progressing, worsening or stagnating ES‐DFU‐wounds to those for the same subjects following HDFA treatment. These observations suggest that chronic ES‐DFUs treated with HDFA may experience significantly improved wound closure and complete healing (re‐epithelialization) when compared with standard treatments without HDFA. With validation from RCTs, HDFA may be established as an effective treatment to promote wound healing and closure for nonhealing ES‐DFUs.     

结肠癌组织中微小RNA分子组学分析

目的 检测结肠癌组织与癌旁组织中微小RNA（miRNA）的差异表达.方法 采用实时荧光定量PCR法检测20例结肠癌组织与癌旁组织miRNA分子的差异表达,筛选具有显著差异表达（变化倍数大于2.4且P＜0.01）的miRNA,进一步通过聚类分析不同miRNA之间的聚集性.并分析miRNA与其他结肠癌相关蛋白表达的相关性.结果 17个miRNA分子在结肠癌组织中显著下调.聚类分析显示,其中miR763-3、miR451和miR99a表达相近.血浆CK20水平与miR100（r=-0.948）、miR125a-5p（r=-0.948）、miR125b（r=-0.949）、miR145 （r=-0.949）和miR145＊ （r=-0.949）均呈高度负相关（均P＜0.05）.结论 miR145等17种miRNA可作为结直肠诊断的分子标记物;miR100、miR125a-5p、miR125b、miR145和miR145＊可能成为提示结肠癌淋巴转移的分子标志物。     

Changes of MicroRNA Profile and MicroRNA‐mRNA Regulatory Network in Bones of Ovariectomized Mice

Growing evidence shows the possibility of a role of microRNAs (miRNA) in regulating bone mass. We investigated the change of miRNAs and mRNA expression profiles in bone tissue in an ovariectomized mice model and evaluated the regulatory mechanism of bone mass mediated by miRNAs in an estrogen‐deficiency state. Eight‐week‐old female C3H/HeJ mice underwent ovariectomy (OVX) or sham operation (Sham‐op), and their femur and tibia were harvested to extract total bone RNAs after 4 weeks for microarray analysis. Eight miRNAs (miR‐127, ‐133a, ‐133a*, ‐133b, ‐136, ‐206, ‐378, ‐378*) were identified to be upregulated after OVX, whereas one miRNA (miR‐204) was downregulated. Concomitant analysis of mRNA microarray revealed that 658 genes were differentially expressed between OVX and Sham‐op mice. Target prediction of differentially expressed miRNAs identified potential targets, and integrative analysis using the mRNA microarray results showed that PPARγ and CREB pathways are activated in skeletal tissues after ovariectomy. Among the potential candidates of miRNA, we further studied the role of miR‐127 in vitro, which exhibited the greatest changes after OVX. We also studied the effects of miR‐136, which has not been studied in the context of bone mass regulation. Transfection of miR‐127 inhibitor has enhanced osteoblastic differentiation in UAMS‐32 cells as measured by alkaline phosphatase activities and mRNA expression of osteoblast‐specific genes, whereas miR‐136 precursor has inhibited osteoblastic differentiation. Furthermore, transfection of both miR‐127 and miR‐136 inhibitors enhanced the osteocyte‐like morphological changes and survival in MLO‐Y4 cells, whereas precursors of miR‐127 and ‐136 have aggravated dexamethasone‐induced cell death. Both of the precursors enhanced osteoclastic differentiation in bone marrow macrophages, indicating that both miR‐127 and ‐136 are negatively regulating bone mass. Taken together, these results suggest a novel insight into the association between distinct miRNAs expression and their possible role through regulatory network with mRNAs in the pathogenesis of estrogen deficiency–induced osteoporosis. © 2014 American Society for Bone and Mineral Research.     

Statins may be associated with six‐week diabetic foot ulcer healing

Diabetic foot ulcers (DFUs) affect 1.5 million Americans annually, of which only a minority heal with standard care, and they commonly lead to amputation. To improve care, investigations are underway to better understand DFU pathogenesis and develop more effective therapies. Some currently used medications may improve healing. One small, randomized clinical trial found statins improve DFU healing. In this secondary analysis of a large multisite prospective observational cohort of 139 patients with DFUs receiving standard care, we investigated whether there was an association between 6‐week DFU wound size reduction and use of a variety of medications including alpha‐blockers, beta‐blockers, angiotensin converting enzyme inhibitors (ACEi) and statins. We found no significant (p < 0.05) association between six‐week wound reduction and use of any of the evaluated drugs; however, statins did trend toward an association (p = 0.057). This suggests a potential benefit of statins on DFU healing, and larger, targeted studies are warranted.     

Inflammatory cytokines induce specific time‐ and concentration‐dependent MicroRNA release by chondrocytes,synoviocytes, and meniscus cells

In knee osteoarthritis (OA), concentrations of interleukin (IL)‐1β and tumor necrosis factor (TNF)‐α increase in joint tissues and synovial fluid which incite a catabolic cascade and further the progression of OA. Several microRNAs (miRNA) have been associated with apoptosis (miR‐16), inflammation (miR‐22, miR‐146a), and matrix degradation (miR‐140, miR‐27b) in developed OA or its symptoms. In this study, the time‐ and concentration‐dependent nature of cellular and extracellular miRNAs in synoviocytes, meniscus cells, and chondrocytes as influenced by inflammatory cytokines was investigated. For time‐dependent studies, three cell types were stimulated with 10 ng/ml IL‐1β or 50 ng/ml TNF‐α for 8, 16, and 24 h. For concentration‐dependent studies, chondrocytes were stimulated with a higher level of IL‐1β (20 ng/ml) or TNF‐α (100 ng/ml) for 8 h. Cellular and extracellular expressions of miR‐22, miR‐16, miR‐146a, miR‐27b, and miR‐140 were analyzed by RT‐PCR. Time‐dependent cellular miRNA expressions were similar across the three cell types with miR‐146a significantly up‐regulated and miR‐27b significantly down‐regulated at all time points. However, chondrocytes exhibited a unique extracellular miRNA profile with an increased release rate of miR‐27b at 24 h. Our findings support further research into the characterization of miRNAs in synovial fluid for the development of early detection strategies of OA or cartilage injury. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:779–790, 2016.     

Serial surgical debridement: A retrospective study on clinical outcomes in chronic lower extremity wounds

This investigation was conducted to determine if a correlation exists between wound healing outcomes and serial debridement in chronic venous leg ulcers (VLUs) and diabetic foot ulcers (DFUs). We retrospectively analyzed the results from two controlled, prospective, randomized pivotal trials of topical wound treatments on 366 VLUs and 310 DFUs over 12 weeks. Weekly wound surface area changes following debridement and 12-week wound closure rates between centers and patients were evaluated. VLUs had a significantly higher median wound surface area reduction following clinical visits with surgical debridement as compared with clinical visits with no surgical debridement (34%, p =0.019). Centers where patients were debrided more frequently were associated with higher rates of wound closure in both clinical studies ( p =0.007 VLU, p =0.015 DFU). Debridement frequency per patient was not statistically correlated to higher rates of wound closure; however, there was some minor evidence of a positive benefit of serial debridement in DFUs (odds ratio—2.35, p =0.069). Our results suggest that frequent debridement of DFUs and VLUs may increase wound healing rates and rates of closure, though there is not enough evidence to definitively conclude a significant effect. Future clinical research in wound care should focus on the relationship between serial surgical wound debridement and improved wound healing outcomes as demonstrated in this study.     

Synovium‐Derived MicroRNAs Regulate Bone Pathways in Rheumatoid Arthritis

Articular bone erosion in rheumatoid arthritis (RA) is mediated by the interaction between inflammation and pathways regulating bone metabolism. Inflammation promotes osteoclastogenesis and also inhibits osteoblast function, further contributing to the persistence of erosions. MicroRNAs (miRNAs) are important regulators of skeletal remodeling and play a role in RA pathogenesis. We therefore determined the expression of miRNAs in inflamed synovial tissue and the role they play in pathways regulating osteoblast and osteoclast function. Using the serum transfer mouse model of RA in C57BL/6 mice, we performed Fluidigm high‐throughput qPCR‐based screening of miRNAs from nonarthritic and arthritic mice. Global gene expression profiling was also performed on Affymetrix microarrays from these same synovial samples. miRNA and mRNA expression profiles were subjected to comparative bioinformatics. A total of 536 upregulated genes and 417 downregulated genes were identified that are predicted targets of miRNAs with reciprocal expression changes. Gene ontology analysis of these genes revealed significant enrichment in skeletal pathways. Of the 22 miRNAs whose expression was most significantly changed (p < 0.01) between nonarthritic and arthritic mice, we identified their targets that both inhibit and promote bone formation. These miRNAs are predicted to target Wnt and BMP signaling pathway components. We validated miRNA array findings and demonstrated that secretion of miR‐221‐3p in exosomes was upregulated by synovial fibroblasts treated with the proinflammatory cytokine TNF. Overexpression of miR‐221‐3p suppressed calvarial osteoblast differentiation and mineralization in vitro. These results suggest that miRNAs derived from inflamed synovial tissues may regulate signaling pathways at erosion sites that affect bone loss and potentially also compensatory bone formation. © 2016 American Society for Bone and Mineral Research.     

Serum miRNA Signatures Are Indicative of Skeletal Fractures in Postmenopausal Women With and Without Type 2 Diabetes and Influence Osteogenic and Adipogenic Differentiation of Adipose Tissue–Derived Mesenchymal Stem Cells In Vitro

Standard DXA measurements, including Fracture Risk Assessment Tool (FRAX) scores, have shown limitations in assessing fracture risk in Type 2 Diabetes (T2D), underscoring the need for novel biomarkers and suggesting that other pathomechanisms may drive diabetic bone fragility. MicroRNAs (miRNAs) are secreted into the circulation from cells of various tissues proportional to local disease severity and were recently found to be crucial to bone homeostasis and T2D. Here, we studied, if and which circulating miRNAs or combinations of miRNAs can discriminate best fracture status in a well‐characterized study of diabetic bone disease and postmenopausal osteoporosis (n = 80 postmenopausal women). We then tested the most discriminative and most frequent miRNAs in vitro. Using miRNA‐qPCR‐arrays, we showed that 48 miRNAs can differentiate fracture status in T2D women and that several combinations of four miRNAs can discriminate diabetes‐related fractures with high specificity and sensitivity (area under the receiver‐operating characteristic curve values [AUCs], 0.92 to 0.96; 95% CI, 0.88 to 0.98). For the osteoporotic study arm, 23 miRNAs were fracture‐indicative and potential combinations of four miRNAs showed AUCs from 0.97 to 1.00 (95% CI, 0.93 to 1.00). Because a role in bone homeostasis for those miRNAs that were most discriminative and most present among all miRNA combinations had not been described, we performed in vitro functional studies in human adipose tissue–derived mesenchymal stem cells to investigate the effect of miR‐550a‐5p, miR‐188‐3p, and miR‐382‐3p on osteogenesis, adipogenesis, and cell proliferation. We found that miR‐382‐3p significantly enhanced osteogenic differentiation (p < 0.001), whereas miR‐550a‐5p inhibited this process (p < 0.001). Both miRNAs, miR‐382‐3p and miR‐550a‐5p, impaired adipogenic differentiation, whereas miR‐188‐3p did not exert an effect on adipogenesis. None of the miRNAs affected significantly cell proliferation. Our data suggest for the first time that miRNAs are linked to fragility fractures in T2D postmenopausal women and should be further investigated for their diagnostic potential and their detailed function in diabetic bone. © 2016 American Society for Bone and Mineral Research.     

Diabetic foot ulcer,the effect of resource‐poor environments on healing time and direct cost: A cohort study during Syrian crisis

Diabetic foot ulcer (DFU) is one of the slowest healing wounds that hurt the human body. Many studies from developed countries are concerned about materials, procedures, and equipment that accelerate the healing time. In Sweden, the diabetic foot management costs around 24965$/patient. In this review, we would evaluate the healing time of DFUs during what is considered one of the worst humanitarian crisis of the 21st century. 1747 DFUs were studied from the main diabetic foot clinic in Damascus (2014‐2019). We predicted many variables that could prolong the healing time. The cost according to these variables was also reported. The SINBAD Classification was performed to grade the severity of ulcers. We noticed that the median healing time for DFUs was 8 weeks. Almost half of these ulcers healed between 3 and 12 weeks. The time of healing for men was significantly longer than that for women. While the presence of infection doubled the median time of healing, the presence of peripheral artery disease doubled the mean of the direct health care cost. The location of the ulcer acted as another independent risk factor. In conclusion, DFUs face many barriers to heal during a crisis.The environment with resource‐poor settings should be added to the traditional risk factors that delay the healing of DFUs for months or even years. More studies from disaster are as are needed to evaluate low‐cost materials that could be cost effective in applying standard care of the diabetic foot.     

MicroRNA Profiles in Allograft Tissues and Paired Urines Associate With Chronic Allograft Dysfunction With IF/TA

Despite the advances in immunosuppression, renal allograft attrition over time remains unabated due to chronic allograft dysfunction (CAD) with interstitial fibrosis (IF) and tubular atrophy (TA). We aimed to evaluate microRNA (miRNA) signatures in CAD with IF/TA and appraise correlation with paired urine samples and potential utility in prospective evaluation of graft function. MiRNA signatures were established between CAD with IF/TA versus normal allografts by microarray. Validation of the microarray results and prospective evaluation of urine samples was performed using real‐time quantitative‐PCR (RT‐qPCR). Fifty‐six miRNAs were identified in samples with CAD‐IF/TA. Five miRNAs were selected for further validation based on array fold change, p‐value and in silico predicted mRNA targets. We confirmed the differential expression of these five miRNAs by RT‐qPCR using an independent set of samples. Differential expression was detected for miR‐142‐3p, miR‐204, miR‐107 and miR‐211 (p < 0.001) and miR‐32 (p < 0.05). Furthermore, differential expression of miR‐142‐3p (p < 0.01), miR‐204 (p < 0.01) and miR‐211 (p < 0.05) was also observed between patient groups in urine samples. A characteristic miRNA signature for IF/TA that correlates with paired urine samples was identified. These results support the potential use of miRNAs as noninvasive markers of IF/TA and for monitoring graft function.     

Pulmonary MicroRNA Expression Profiling in an Immature Piglet Model of Cardiopulmonary Bypass‐Induced Acute Lung Injury

After surgery performed under cardiopulmonary bypass (CPB), severe lung injury often occurs in infants. MicroRNAs (miRNAs) are potentially involved in diverse pathophysiological processes via regulation of gene expression. The objective of this study was to investigate differentially expressed miRNAs and their potential target genes in immature piglet lungs in response to CPB. Fourteen piglets aged 18.6 ± 0.5 days were equally divided into two groups that underwent sham sternotomy or CPB. The duration of aortic cross‐clamping was 2 h, followed by 2 h reperfusion. Lung injury was evaluated by lung function indices, levels of cytokines, and histological changes. We applied miRNA microarray and quantitative real‐time polymerase chain reaction (qRT‐PCR) analysis to determine miRNA expression. Meanwhile, qRT‐PCR and enzyme‐linked immunosorbent assay were used for validation of predicted mRNA targets. The deterioration of lung function and histopathological changes revealed the piglets' lungs were greatly impaired due to CPB. The levels of tumor necrosis factor alpha, interleukin 6, and interleukin 10 increased in the lung tissue after CPB. Using miRNA microarray, statistically significant differences were found in the levels of 16 miRNAs in the CPB group. Up‐regulation of miR‐21 was verified by PCR. We also observed down‐regulation in the levels of miR‐127, miR‐145, and miR‐204, which were correlated with increases in the expression of the products of their potential target genes PIK3CG, PTGS2, ACE, and IL6R in the CPB group, suggesting a potential role for miRNA in the regulation of inflammatory response. Our results show that CPB induces severe lung injury and dynamic changes in miRNA expression in piglet lungs. Moreover, the changes in miRNA levels and target gene expression may provide a basis for understanding the pathogenesis of CPB‐induced injury to immature lungs.     

Topical oxygen therapy results in complete wound healing in diabetic foot ulcers

Diabetic foot ulcers (DFUs) are a significant problem in an aging population. Fifteen percent of diabetics develop a DFU over their lifetime, which can lead to potential amputation. The 5‐year survival rate after amputation is 31%, which is greater than the lifetime risk of mortality from cancer. Topical oxygen is a promising technique for the adjunctive therapy of chronic wounds including DFUs, but few controlled studies exist to support its clinical adoption. The aim of this study was to compare a portable topical oxygen delivery system in patients with nonhealing DFUs to standard best practice. Twenty patients were randomized into a topical oxygen group (n = 10), and a nonplacebo control group with regular dressings and standard care (n = 10), and attended the diabetic foot clinic once weekly for 8 weeks. Ulcer surface area over time was analyzed using standardized digital imaging software. DFUs were present without healing for a mean duration of 76 weeks prior to the study. They found a significant difference in healing rate between patients receiving topical oxygen and those receiving standard care. Topical oxygen, therefore, represents a potentially exciting new technology to shorten healing time in patients with nonhealing DFUs. More prospective randomized and powered studies are needed to determine the benefits of topical oxygen, but our current results are very promising.     

Outcome predictors for wound healing in patients with a diabetic foot ulcer

The aim of this study was to identify diabetic foot ulcer (DFU) patients at risk for the development of a hard‐to‐heal wound. This is a post‐hoc analysis of a prospective cohort study including a total of 208 patients with a DFU. The primary endpoints were time to healing and the development of a hard‐to‐heal‐wound. Univariable and multivariable logistic and Cox regression analysis were used to study the associations of patient characteristics with the primary endpoints. The number of previous DFUs [odds ratio (OR): 1.42, 95% confidence interval (CI): 1.01‐1.99, P = .04], University of Texas (UT) classification grade 2 (OR: 2.93, 95% CI: 1.27‐6.72, P = .01), UT classification grade 3 (OR: 2.80, 95% CI: 1.17‐6.71, P = .02), and a diagnosis of foot stand deformation (OR: 1.54, 95% CI: 0.77‐3.08, P = .05) were significantly associated with the development of a hard‐to‐heal wound. Only UT classification grade 3 (HR: 0.61, 95% CI: 0.41‐0.90, P = .01) was associated with time to healing. The number of previous DFUs, UT classification grade, and a diagnosis of foot deformation are significantly associated with development of a hard‐to‐heal wound in patients with a DFU. The only predictor significantly associated with time to healing was UT classification grade 3. These patient characteristics can be used to identify patients at risk for the development of hard‐to‐heal wounds, who might need an early intervention to prevent wound problems.     

MicroRNA‐199a‐3p,microRNA‐193b,and microRNA‐320c are correlated to aging and regulate human cartilage metabolism

MicroRNAs (miRNAs) are small RNAs of ～22 base pairs that regulate gene expression. We harvested cartilage tissue from patients with polydactylism, anterior cruciate ligament injury, and osteoarthritis undergoing total knee arthroplasty and used microarrays to identify miRNAs whose expression is upregulated or downregulated with age. The results were assessed by real‐time PCR and MTT assay in a mimic group, in which synthetic double‐stranded RNA from the isolated miRNA was transfected to upregulate expression, and in an inhibitor group, in which the miRNA was bound specifically to downregulate expression. The expression of two miRNAs (miR‐199a‐3p and miR‐193b) was upregulated with age and that of one miRNA (miR‐320c) was downregulated with age. A real‐time PCR assay showed that type 2 collagen, aggrecan, and SOX9 expression were downregulated in the miR‐199a‐3p mimic group but was upregulated in the inhibitor group. Similar results were observed for miR‐193b. By contrast, ADAMTS5 expression was downregulated in the miR‐320c mimic group and upregulated in the inhibitor group. Cell proliferative activity was upregulated significantly in the miR‐193b inhibitor group compared with the control group. We believe that miR‐199a‐3p and miR‐193b are involved in the senescence of chondrocytes, and miR‐320c is involved in the juvenile properties of chondrocytes. © 2012 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 30:1915–1922, 2012     

Donor Graft MicroRNAs: A Newly Identified Player in the Development of New‐onset Diabetes After Liver Transplantation

New‐onset diabetes after liver transplantation (NODALT) is a frequent complication with an unfavorable outcome. We previously demonstrated a crucial link between donor graft genetics and the risk of NODALT. We selected 15 matched pairs of NODALT and non‐NODALT liver recipients using propensity score matching analysis. The donor liver tissues were tested for the expression of 10 microRNAs (miRNAs) regulating human hepatic glucose homeostasis. The biological functions of potential target genes were predicted using gene ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis. Both miR‐103 and miR‐181a were significantly highly expressed in the NODALT group as compared to the non‐NODALT group. The predicted target genes (e.g. Irs2, Pik3r1, Akt2, and Gsk3b) were involved in glucose import and the insulin signaling pathway. We also observed dysregulation of miRNAs (e.g. let‐7, miR‐26b, miR‐145, and miR‐183) in cultured human hepatocytes treated with tacrolimus or high glucose, the two independent risk factors of NODALT identified in this cohort. The hepatic miRNA profiles altered by tacrolimus or hyperglycemia were associated with insulin resistance and glucose homeostatic imbalance as revealed by enrichment analysis. The disease susceptibility miRNA expressive pattern could be imported directly from the donor and consolidated by the transplant factors.     

Role of anaerobes in polymicrobial communities and biofilms complicating diabetic foot ulcers

Infected tissues in the feet of people with diabetes in the form of a diabetic foot ulcer (DFU) present a complex pathology for clinicians to manage. This is partly attributed to the multi‐factorial nature of the disease, which may include; altered foot architecture leading to excessive plantar pressures and frictional forces peripheral arterial disease and loss of protective sensation. In addition, to the above co‐morbid variables, it is understood that a delayed wound healing state may be perpetuated by the presence of microorganisms residing in the wound tissue. The microbiology of chronic DFUs has often been reported as being polymicrobial. Of growing interest is the presence and potential role of anaerobic microorganisms in the pathology of DFUs and how they may contribute to the infective process or delayed healing. The presence of anaerobes in DFUs has been greatly underestimated, largely due to the limitations of conventional culture methods in identifying them from samples. Advancements in molecular and microscopy techniques have extended our view of the wound microbiome in addition to observing the growth and behaviour (planktonic or biofilm) of microorganisms in situ. This review paper will reflect on the evidence for the role and significance of anaerobes in DFUs and infection. A focus of this review will be to explore recent advancements in molecular genomics and microscopy techniques in order to better assess the roles of anaerobic bacteria in chronic DFUs and in biofilm‐based wound care.     

Evidence of differential microbiomes in healing versus non‐healing diabetic foot ulcers prior to and following foot salvage therapy

Diabetic foot ulcers (DFU) contribute to 80% of lower extremity amputations. Although physicians currently rely on clinical signs along with non‐specific biomarkers of infection, such as erythrocyte sedimentation rate and C‐reactive protein, to diagnose and monitor DFU, there is no specific and sensitive measure available to monitor or prognosticate the success of foot salvage therapy (FST). To address this we performed a prospective, observational microbiome analysis to test the hypotheses that: (i) the initial microbiomes of healed versus non‐healed DFU are distinct; (ii) the microbial load, diversity and presence of pathogenic organism of the DFU change in response to antibiotics treatment; and (iii) the changes in the DFU microbiome during treatment are prognostic of clinical outcome. To test this, microbiome analyses were performed on 23 DFU patients undergoing FST, in which wound samples were collected at zero, four, and eight weeks following wound debridement and antibiotics treatment. Bacterial abundance was determined using quantitative polymerase chain reaction (qPCR). Eleven patients healed their DFU, while FDT failed to heal DFU in the other 12 patients. Microbiome results demonstrated that healing DFUs had a larger abundance Actinomycetales and Staphylococcaceae (p < 0.05), while DFUs that did not heal had a higher abundance of Bacteroidales and Streptococcaceae (p < 0.05). FST marked increases Actinomycetales in DFU, and this increase is significantly greater in patients that healed (p < 0.05). Future studies to confirm the differential microbiomes, and that increasing Actinomycetales is prognostic of successful FST are warranted. Statement of Clinical Significance: Tracking changes in the prevalence of pathogens in diabetic foot ulcers may be a clinical tool for monitoring treatment response to foot salvage therapy and prognosticating the need for further surgical intervention. The initial wound sample microbiome may provide important prognostic information on the eventual clinical outcome of foot salvage therapy. It may serve as an important clinical tool for patient counseling and making surgical decision of pursuing foot salvage versus amputation. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:1596–1603, 2019.