Dimethyloxalylglycine Promotes Bone Marrow Mesenchymal Stem Cell Osteogenesis via Rho/ROCK Signaling.

BACKGROUND/AIMS: We investigated the role of dimethyloxalylglycine (DMOG) in bone marrow mesenchymal stem cell (BMSC) osteogenesis mediated by RhoA/ROCK. METHODS: BMSCs were cultured with and without DMOG and/or Y-27632 (ROCK1 inhibitor). Cell proliferation, alkaline phosphatase (ALP) levels, and calcium deposits were determined. The expression of Runx2, OSX, p-cofilin, RhoA, and GTP-bound RhoA was determined by real-time RT-PCR and Western blot. Rho-associated coiled-coil-containing protein kinase (ROCK) activity was determined by measuring the phosphorylation of myosin-binding subunit of myosin phosphatase using an ELISA kit. Actin morphology was observed by immunofluorescence. RESULTS: After 24 h, DMOG (0.5 mM) increased the expression of GTP-bound RhoA (+141%, P < 0.001) and enhanced ROCK activity (315%, P < 0.001). DMOG (0.5 mM) enhanced ALP levels after 3, 7, and 21 days of osteogenic induction (all P < 0.001) and strengthened calcium deposition (P < 0.001). In addition, compared with controls, DMOG (0.5 mM) increased the mRNA levels of osteogenesis genes RUNX2 and OSX (all P < 0.001). Furthermore, compared with controls, DMOG increased the expression of p-cofilin (+57%, P < 0.001), which resulted in rearrangement of actin filaments. All these effects were abolished, at least in part, by Y-27632. CONCLUSION: DMOG promotes BMSC osteogenic differentiation via activation of RhoA/ROCK, suggesting clues for future therapies using BMSCs.

[Clinical and radiographic outcomes of navigation-assisted versus conventional total knee arthroplasty].

OBJECTIVE: To assess the early postoperative clinical and radiographic outcomes after navigation-assisted or standard instrumentation total knee arthroplasty (TKA). METHODS: From August 2007 to May 2008, 60 KSS-A type patients underwent 67 primary TKA operations by the same surgical team. Twenty-two operations were performed with the Image-free navigation system with an average age of 64.5 years while the remaining 45 underwent conventional manual procedures with an average age of 66 years. Their preoperative demographic and functional data had no statistical differences (P>0.05). The operative duration, blood loss volume and hospitalization days were compared for two groups. And radiographic data included coronal femoral component angle, coronal tibial component angle, sagittal femoral component angle, sagittal tibial component angle and coronal tibiofemoral angle after one month. And functional assessment scores were evaluated at 1, 3 and 6 months postoperatively. RESULTS: Operative duration was significantly longer for computer navigation (P<0.05). The average blood loss volume was 555.26 ml in computer navigation group and 647.56 ml in conventional manual method group (P<0.05). And hospitalization stay was shorter in computer navigation group than that in conventional method group (7.74 vs 8.68 days) (P=0.04). The alignment deviation was better in computer-assisted group than that in conventional manual method group (P<0.05). The percentage of patients with a coronal tibiofemoral angle within ±3 of ideal value was 95.45% for computer-assisted mini-invasive TKA group and 80% for conventional TKA group (P=0.003). The Knee Society Clinical Rating Score was higher in computer-assisted group than that in conventional manual method group at 1 and 3 montha post-operation. However, no statistical inter-group difference existed at 6 months post-operation. CONCLUSION: Navigation allows a surgeon to precisely implant the components for TKA. And it offers faster functional recovery and shorter hospitalization stay. At 6 months post-operation, there is no statistical inter-group difference in KSS scores.

Overexpression of NMNAT3 improves mitochondrial function and enhances antioxidative stress capacity of bone marrow mesenchymal stem cells via the NAD+-Sirt3 pathway.

Oxidative stress damage is a common problem in bone marrow mesenchymal stem cell (BMSC) transplantation. Under stress conditions, the mitochondrial function of BMSCs is disrupted, which accelerates senescence and apoptosis of BMSCs, ultimately leading to poor efficacy. Therefore, improving mitochondrial function and enhancing the antioxidative stress capacity of BMSCs may be an effective way of improving the survival rate and curative effect of BMSCs. In the present study, we have confirmed that overexpression of nicotinamide mononucleotide adenylyl transferase 3 (NMNAT3) improves mitochondrial function and resistance to stress-induced apoptosis in BMSCs. We further revealed the mechanism of NMNAT3-mediated resistance to stress-induced apoptosis in BMSCs. We increased the level of nicotinamide adenine dinucleotide (NAD+) by overexpressing NMNAT3 in BMSCs and found that it could significantly increase the activity of silent mating type information regulation 2 homolog 3 (Sirt3) and significantly decrease the acetylation levels of Sirt3-dependent deacetylation-related proteins isocitrate dehydrogenase 2 (Idh2) and Forkhead-box protein O3a (FOXO3a). These findings show that NMNAT3 may increase the activity of Sirt3 by increasing NAD+ levels. Our results confirm that the NMNAT3-NAD+-Sirt3 axis is a potential mechanism for improving mitochondrial function and enhancing antioxidative stress capacity of BMSCs. In the present study, we take advantage of the role of NMNAT3 in inhibiting stress-induced apoptosis of BMSCs and provide new methods and ideas for breaking through the bottleneck of transplantation efficacy of BMSCs in the clinic.

[Comparative study between hypoxia and hypoxia mimetic agents on osteogenesis of bone marrow mesenchymal stem cells in mouse].

OBJECTIVE: ?To compare the effects on the osteogenesis of bone marrow mesenchymal stem cells (BMSCs) between hypoxia and hypoxia mimetic agents dimethyloxalylglycine (DMOG) under normal oxygen condition. METHODS: ?BMSCs were isolated and cultured from healthy 3-4 weeks old Kunming mouse. Cell phenotype of CD29, CD44, CD90, and CD34 was assayed with flow cytometry; after osteogenic, adipogenic, and chondrogenic induction, alizarin red staining, oil red O staining, and toluidine blue staining were performed. The passage 3 BMSCs were cultured under normal oxygen in control group (group A), under 1%O2 in hypoxia group (group B), and under normal oxygen and 0.5 mmol/L DMOG in DMOG intervention group (group C). BMSCs proliferation was estimated by methyl thiazolyl tetrazolium assay at 1, 2, 3, and 4 days. Alkaline phophatase (ALP) expression was determined at 7 and 14 days after osteogenic induction. Western blot was employed for detecting hypoxia inducible factor-1α (HIF-1α) at 24 hours. Real time fluorescence quantitative PCR was employed for detecting the mRNA expression of runt-related transcription factor 2 (RUNX2) and Osterix at 3 and 7 days. Alizarin red staining was applied to assess the deposition of calcium tubercle at 21 days. RESULTS: ?The BMSCs presented CD29(+), CD44(+), CD90(+), and CD34(-); and results of the alizarin red staining, oil red O staining, and toluidine blue staining were positive after osteogenic, adipogenic, and chondrogenic induction. No significant difference in BMSCs proliferation was observed among 3 groups at 1 day (P>0.05); compared with group A, BMSCs proliferation was inhibited in group C at 2, 3, and 4 days, but no significant difference was observed (P>0.05); compared with group A, BMSCs proliferation was significantly promoted in group B (P<0.05). At each time point, compared with group A, the ALP expression, HIF-1α protein relative expression, and mRNA relative expressions of RUNX2 and Osterix were significantly up-regulated in groups B and C (P<0.05); compared with group B, the ALP expression, the RUNX2 and Osterix mRNA relative expression were significantly up-regulated in group C (P<0.05); compared with group C, the HIF-1α protein relative expression was significantly up-regulated in group B (P<0.05). The alizarin red staining showed little red staining materials in group A, some red staining materials in group B, and a large number of red staining materials in group C. CONCLUSIONS: ?Hypoxia can promote BMSCs proliferation, DMOG can not influence the BMSCs proliferation; both hypoxia and DMOG can improve osteogenic differentiation of BMSCs, and DMOG is better than hypoxia in improving the BMSCs osteogenesis.

[CEMENTLESS TOTAL HIP ARTHROPLASTY FOR TREATMENT OF Crowe TYPE â ¢ DEVELOPMENTAL DYSPLASIA OF HIP IN ADULTS].

OBJECTIVE: To investigate the value of cementless total hip arthroplasty (THA) for the treatment of Crowe type Ⅲ developmental dysplasia of hip (DDH) in adults. METHODS: Between September 2013 and September 2015, 50 patients (51 hips) with Crowe type Ⅲ DDH were treated. There were 20 males (20 hips) and 30 females (31 hips), with the average age of 39 years (range, 19-55 years). The left side was involved in 34 cases, the right side in 15 cases, and both sides in 1 case. All patients had the symptoms of limp walking and hip pain. The disease duration was 10-47 months (mean, 26 months). The sign of "4" number test and Trendenleburg sign were positive; the Harris score was 38.9±7.1. The bilateral lower extremities discrepancy was 2.5-4.0 cm (mean, 3.3 cm) before operation. All the patients underwent cementless THA, and acetabulum by structural femoral head autograft was performed in 28 cases (28 hips). RESULTS: After operation, the incision healed by first intention. Only 2 patients (2 hips) had femoral nerve palsy, and 7 patients (7 hips) had leg swelling, which were cured after symptomatic treatment. All the patients were followed up 6-18 months (mean, 10 months). The sign of limp walking was improved after operation, hip pain was relieved in 46 patients (46 hips) and only 4 patients (5 hips) still had mild pain. The X-ray films showed bony healing at 3-6 months (mean, 5 months) after operation. At last follow-up, the patients had equal limb length with the discrepancy less than 1 cm (mean, 0.4 cm). At last follow-up, the Harris score was significantly increased to 91.2±2.8 (t=-79.77, P=0.00). CONCLUSIONS: The cementless THA is an effective method to treat Crowe type Ⅲ DDH in adults.

A novel tissue-engineered bone in repairing femoral head defect and necrosis.

OBJECTIVE: To evaluate the therapeutic effects of AACB/BMP/bFGF, a novel tissue-engineered bone, in repairing femoral head defect and necrosis in dog models. METHODS: Dog models of avascular necrosis of femoral head (ANFH) were established by liquid nitrogen freezing method. Group A was untreated; Groups B, C, and D were implanted with AACB, AACB/BMP, and AACB/BMP/bFGF complex, respectively; Group E was grafted with autologous cancellous bone. Samples were collected at 3 w, 6 w, and 12 w after operation. A series of examinations were carried out to investigate the effects of the materials in repairing femoral head defect, including anatomical observation, X-ray examination, histological analysis, and vascular immunohistochemical staining. RESULTS: Our results indicated that, compared with AACB alone and AACB/BMP, AACB/BMP/bFGF complex could exert the most efficient therapeutic effects in dog ANFH models. X-ray examination further confirmed that AACB/BMP/bFGF complex could effectively repair the injuries in dog ANFH models, almost to a comparable level with cancellous bone autografts. Moreover, histological analysis indicated that AACB/BMP/bFGF complex greatly enhanced the new bone formation, which would contribute to the healing of ANFH. Furthermore, vascular immunohistochemical staining revealed that AACB/BMP/bFGF complex could significantly stimulate the revascularization in defect areas, reflecting the post-injury healing process in these models. CONCLUSION: AACB/BMP/bFGF complex has great potential in repairing femoral head defect by enhancing osteogenesis and revascularization. The novel tissue-engineered bone would be widely used in clinical applications for ANFH treatment, especially as an alternative for autografts.

[Total hip arthroplasty for the treatment of developmental dysplasia of the hip in adults].

OBJECTIVE: To summarize techniques of the total hip arthroplasty (THA) in the treatment of developmental dysplasia of the hip (DDH) with severe osteoarthritis in adults. METHODS: From March 2000 to January 2006, 24 patients (27 hips) with DDH were treated by THA with an cementless cup. There were 7 males and 17 females, with the average age of 49.6 years (ranging from 26 years to 63 years). Unilateral DDH occurred in 21 patients and bilateral DDH occurred in 3 patients. Based on the Crowe classification, there were 16 hips in 15 patients of type I, 4 hips in 4 patients of type II, 4 hips in 3 patients of type III, 3 hips in 2 patients of type IV. Except for 3 patients with bilateral DDH, the other patients' ill lower limbs were 2-7 cm shorter than the healthy lower ones. RESULTS: All the patients were followed up from 9 months to 6.5 years and no one had infection, dislocation, femur fracture and so on after the operation. In 18 patients, the pain was completely relieved and the function of the hip joints was good. After the gluteus medius exercise, the claudication of 3 patients after the operation disappeared. In 3 patients, the ill lower limbs were more than 1 cm shorter than the healthy lower ones and the other patients' ill lower limbs were less than 1 cm shorter than the healthy lower ones. Two patients' lower limbs were been lengthened 4-5 cm. All the patients' sciatic nerves were not injured. The Harris scores were 46.5 +/- 7.2 preoperatively and 84.0 +/- 5.7 postoperatively (P < 0.05). CONCLUSION: THA with deepening the medial wall of the acetabulum at the true acetabulum and choosing small cementless cup in adult could obtain favorable results.

[Study on the effect of composite of basic fibroblast growth factor and partially deproteinized bone on the repair of femoral head defects].

OBJECTIVE: To evaluate the effect of composite (bFGF/PDPB) of basic fibroblast growth factor (bFGF) and partially deproteinized bone (PDPB) on the repair of femoral head defect. METHODS: Forty-eight femoral heads with defect derived from 24 New Zealand rabbits were divided into 3 groups at random, which were implanted with bFGF/PDPB (group A), PDPB (group B) and nothing (group C) respectively. The rabbits were sacrificed at 2, 4, and 8 weeks after operation, and then the femoral heads were obtained. The specimens injected with Chinese ink were created. Then X-ray examination, histopathological and morphological examination of blood vessel, and image analysis were made. RESULTS: The bone defects healed completely 8 weeks after operation in group A. The implants in the repaired tissue were not substituted completely in group B. The bone defects did not heal completely in group C. Two weeks after operation, affluent newly formed vessels were seen in repaired areas in group A. No significant difference between group A and group B was observed 8 weeks after operation. In group C, newly formed vessels were scarce 2, 4, and 8 weeks after operation. There were 3 sides rated excellent, 2 good and 1 fair in group A; 1 excellent, 2 good, 2 fair and 1 poor in group B; and 1 fair and 5 poor in group C according to the X-ray evaluation 8 weeks after operation. Eight weeks after operation, the volume fraction of bone trabecula in repaired tissue was higher in group A than that in group B (P < 0.05), and the fraction in group C was the lowest among the 3 groups (P < 0.05). CONCLUSION: The composite of bFGF and PDPB can effectively promote the repair of femoral head defect of rabbit.