Bone defects larger than a critical size are major challenges in orthopedic medicine. We combined tissue-engineered bone and gene therapy to provide osteoprogenitor cells, osteoinductive factors, and osteo-conductive carrier for ideal bone regeneration in critical-sized bone defects. Goat diaphyseal bone defects were repaired with tissue and genetically engineered bone implants, composed of biphasic calcined bone (BCB) and autologous bone marrow derived mesenchymal stem cells (BMSC) transduced with human bone morphogenetic protein-2 (hBMP-2). Twenty six goats with tibial bone defects were divided into groups receiving implants by using a combination of BCB and BMSCs with or without the hBMP-2 gene. In eight goats that were treated with BCB that contained hBMP-2 transduced BMSC, five had complete healing and three showed partial healing. Goats in other experimental groups had only slight or no healing. Furthermore, the area and biochemical strength of the callus in the bone defects were significantly better in animals treated with genetically engineered implants. We concluded that the combination of genetic and tissue engineering provides an innovative way for treating critical-sized bone defects.
Although interaction between BMP and Notch signaling has been demonstrated to be crucial for osteogenic differentiation of mesenchymal stem cells (MSCs), the precise molecular mechanism remains unknown. Here, we show that Notch intracellular domain (NICD) overexpression inhibits BMP9-induced C3H10T1/2 cell osteogenesis in vivo and in vitro. Our results show that activated Notch signaling results in down-regulation of Runx2 and early osteogenesis differentiation factors, without affecting p-Smad1/5/8 expression, and that blocking Notch signaling with DAPT (N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester) significantly increases p-Smad1/5/8 expression. Interestingly, Notch signaling also regulates the cell cycle by increasing PCNA (proliferation cell nuclear antigen) and CyclinD1 expression. Furthermore, similar results were obtained by ectopic bone formation and histological analyses, indicating that Notch signaling activation significantly inhibits BMP9-induced MSC osteogenic, cartilage and adipogenic differentiation. Moreover, we are the first to show that Notch regulates by suppressing JunB synthesis and that the negative effect of Notch is partially reversed by treatment with the JunB activator TPA (12-O-tetradeca-noylphorbol-13-acetate). Our findings demonstrate that Notch signaling significantly enhances cell proliferation but inhibits MSC osteogenic differentiation induced by BMP9 via JunB protein suppression rather than by BMP/Smad signaling regulation.
Age-related decline in the number of mesenchymal stem cells (MSCs) and their reduced capability to differentiate osteogenically, along with diminished availability of growth factors, may be major factors accounting for reduced bone formation in the aging mammalian body. In the first part of the study, we compared the number of MSCs in bone marrow (BM) and the content of bone morphogenetic protein 2 (BMP2) in cortical bone tissue in juvenile, adult, and aged (1, 9, and 24 months, respectively) male rats. To assay the influence of aging on osteogenic differentiation ability, MSCs from the three age groups were transduced with the BMP2 gene. Following gene transduction, the production of BMP2 in culture media, expression of osteogenic proteins (e.g., alkaline phosphatase, type Ialpha1 collagen, osteopontin, and bone sialoprotein), as well as ectopic bone formation in athymic mice were compared. Results showed that the number of MSCs in BM as well as the content of BMP2 in cortical bone tissue decreased with age, but no significant differences between the three age groups were found with regard to production of BMP2 or capability of BMP2 gene-modified MSCs to differentiate osteogenically. The second part of the study applied BMP2 gene-modified autologous MSCs/beta-tricalcium phosphate for repair of bone defects in aged rats with positive results. Our data indicate that the osteogenic potential of MSCs of aged rats can be restored following BMP2 gene transduction and that this technique may be a useful approach in the future planning of gene therapy for age-related osteoporotic fractures.
Background Interleukin (IL)-6/signal transducers and activators of transcription 3 (STAT3) signaling plays an important role in the development of colitis-associated colorectal cancer (CAC). The mechanism of CAC formation remains unclear, and the relationship between miRNAs and the IL-6/STAT3 signaling pathway in the development of CAC is not well understood. In this study, we investigated the relationship between miR-29a-5p and the IL-6/STAT3 signaling pathway in the development of CAC and alterations in 10-11 translocations (TETs) regulated by this network. Methods miR-29a-5p was screened in a CAC mouse model by high-throughput microarray analysis and investigated in human colorectal cancer tissue samples and colon cell lines by quantitative reverse transcription polymerase chain reaction (Q-RTPCR). The expression of miR-29a and TETs was detected by Q-RTPCR, and the expression of STAT3/P-STAT3 and TET3 was detected via Western blot assay. The expression of TET1 and 5-hydroxymethylcytosine (5hmC) was detected through immunofluorescence. Results Our results showed that miR-29a-5p was significantly upregulated and was accompanied by STAT3 activation in the colon tissues of CAC mouse and human colorectal cancer tissues, as compared with normal colon tissues. In contrast, the levels of TETs and 5hmC were decreased. In vitro, overexpression of miR-29a-5p in colonic cell lines (HCT-116 and IEC-6) and RAW264.7 cells increased STAT3 expression, but decreased that of TET3, TET1, and 5hmC. miR-29a-5p downregulation in HCT-116 and IEC-6 cell lines could rescue the expression of STAT3 and TET3. Notably, STAT3 activation induced by IL-6 upregulated miR-29a-5p expression and reduced TET expression in vitro, although STAT3 inhibitor treatment downregulated miR-29a-5p expression, which was induced by IL-6. Conclusions Our studies showed that tumor development occurred with inflammation. The miR-29a-5p/STAT3 signaling axis could play an important role in the development of CAC, and the miR-29a-5p/STAT3 positive feedback loop may amplify the effects of inflammation, lead to decreased levels of TET and 5hmC, and eventually lead to the development of CAC.
OBJECTIVE: To review the clinical and endoscopic features, and outcome of ischemic colitis. METHODS: Sixty cases with the diagnosis of ischemic colitis were retrospectively analyzed. All the patients were under observation in hospital and most of them underwent colonoscopy at least twice: once for diagnosis and then follow‐up after treatment. The demographic data, presenting symptoms, endoscopic findings, laboratory tests, and treatment were reviewed. RESULTS: Fifty‐two of the 60 cases were over 50 years old (87%; mean age, 59.9 years): 40 female, 20 male (2 : 1); 76.0% of these patients had a coexistent disease such as a cardio‐cerebrovascular disorder, diabetes, hematologic diseases or a previous history of abdominal surgery. Abdominal pain (57/60, 95%), hematochezia (55/60, 91.7%), and diarrhea (26/60, 43.3%) were the main complaints. Lesions seen on colonoscopy were more commonly located in the left colon (46/60, 79.3%) and rectum (5/60, 8.6%), and were characteristically segment‐distributed, including hemorrhagic edematous mucosa, erosions, ulcerations, pseudopolyps, and stricture. Ultrasonography revealed colonic wall thickening in 13 cases (13/55, 21.7%), and small to moderate ascites was detected in 4 cases (4/55, 7.3%). In this cohort, most of the patients recovered (49/60, 81.7%) or improved (10/60, 16.7%) after conservative treatment. Only one patient who had a myocardial infarction prior to the onset of the ischemic colitis, died from peritonitis complicated with septic shock. Progress and outcome were associated with the patient's age, severity of the lesions, clinical course, underlying diseases and the complications. CONCLUSION: Colonoscopy is safe and helpful in the early diagnosis of ischemic colitis. Nongangrenous colonic ischemia usually requires only medical management and is associated with a good prognosis.
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