Ginsenoside Rg1/ADSCs supplemented with hyaluronic acid as the matrix improves rabbit temporomandibular joint osteoarthrosis.

OBJECTIVE: To investigate whether and how ginsenoside Rg1/ADSCs supplemented with hyaluronic acid as the matrix can improve rabbit temporomandibular joint osteoarthrosis. METHOD: Isolate and culture adipose stem cells, measure the activity of differentiated chondrocytes by MTT assay and expression of type II collagen in these cells by immunohistochemistry, in order to evaluate the effect of ginsenoside Rg1 on adipose stem cell proliferation and differentiation into chondrocytes.32 New Zealand white rabbits were randomly divided into four groups: blank group, model group, control group and experimental group, 8 in each group. Osteoarthritis model was established by intra-articular injection of papain. Two weeks after successful model building, medication was given for the rabbits in control group and experimental group. 0.6 mL ginsenoside Rg1/ ADSCs suspension was injected into superior joint space for the rabbits in control group, once a week; 0.6 mL ginsenoside Rg1/ ADSCs complex was injected for the rabbits in experimental group, once a week. RESULTS: Ginsenoside Rg1 can promote ADSCs-derived chondrocytes' activity and expression of type II collagen. Scanning electron microscopy histology images showed cartilage lesions of the experimental group was significantly improved in comparison with control group. CONCLUSION: Ginsenoside Rg1 can promote ADSCs differentiate into chondrocytes, and Ginsenoside Rg1/ADSCs supplemented with hyaluronic acid as the matrix can significantly improve rabbit temporomandibular joint osteoarthrosis.

Impacts of Angelica Polysaccharide on Proliferation and Differentiation of Mesenchymal Stem Cells of Rat Bone Marrow.

In literature, antiosteoporotic effects of Angelica sinensis root have been confirmed, but the impact of Angelica sinensis polysaccharide (ASP) on osteoblastic or adipogenic distinction of BMSCs is limited. This paper aimed to explore the role of ASP on proliferation and differentiation of rat BMSCs. Rat BMSCs were subjected to isolation and identification through flow cytometry. The proliferation of rat BMSCs under ASP was performed by CCK-8 kit. Measures of osteogenesis under different concentrations of ASP were detected by using alizarin red staining for mesenchymal cells differentiation and ALP activity assay to identify ALP activity. Quantitative RT-PCR was selected to identify osteoblastic or adipogenic biomarkers from a genetic perspective. Likewise, we have evaluated measures of indicators of Wnt/ß-catenin signal. ASP significantly promoted the proliferation, increased osteogenesis, and decreased adipogenesis of rat BMSCs within the limit of 20-60 mg/L in a dose-dependent manner but was suppressed at 80 mg/L. The expression of cyclin D1 and ß-catenin showed a considerable rise over the course of ASP induced osteogenesis. Dickkopf 1 (DKK1) suppressed the regulation of rat BMSCs differentiation through the mediation of ASP. We have observed that ASP upregulated the osteogenic but downregulated adipogenic differentiation of BMSCs, and our findings help to contribute to effective solutions for treating bone disorders.

The lncRNA H19/miR-541-3p/Wnt/ß-catenin axis plays a vital role in melatonin-mediated osteogenic differentiation of bone marrow mesenchymal stem cells.

Implant dentures become the first choice for denture restoration in patients with tooth loss. However, oral implants often fail in osteoporosis (OP) patients. Melatonin (MT) induces osteogenic differentiation of bone mesenchymal stem cells (BMSCs), suggesting its therapeutic potential in OP treatment. Long non-coding RNA H19 induces osteogenic differentiation of BMSCs, while its regulatory mechanism in MT-involved osteogenic and adipogenic differentiation of BMSCs remains elusive. Ovariectomized (OVX) rat was used to construct an OP model, and bone quality was assessed. Meanwhile, the expression of H19, miR-541-3p, MT and adiponectin (APN) was examined by quantitative reverse transcription-PCR (qRT-PCR) or ELISA. The adipogenic and osteogenic differentiation of BMSCs were determined by oil red O staining and alizarin red S staining, respectively. The targeting relationships between H19, miR-541-3p and APN mRNA were predicted by bioinformatics and confirmed by RNA immunoprecipitation and dual-luciferase reporter assay. The results showed that MT, H19 and APN were down-regulated, while miR-541-3p was up-regulated in the OVX rat model. At the cellular level, MT reduced adipogenic differentiation, heightened osteogenic differentiation of BMSCs, and activated Wnt/ß-catenin pathway, which were reversed by the MT2 selective inhibitor 4-P-PDOT. Overexpressing H19 facilitated the osteogenic differentiation and inhibited the adipogenic differentiation of BMSCs mediated by MT, while H19 knockdown or overexpressing miR-541-3p had the opposite effect. Moreover, H19 functioned as a competitive endogenous RNA and sponged miR-541-3p, and miR-541-3p targeted APN. Overall, MT modulates the osteogenic and adipogenic differentiation of BMSCs by mediating H19/miR-541-3p/APN axis, providing a new reference for the targeted therapy of OP.

[Effect of Angelica sinensis polysaccharide on the osteogenic differentiation of bone marrow mesenchymal stem cells of rats with high glucose levels].

OBJECTIVE: This study aims to evaluate the effect of Angelica sinensis polysaccharide (ASP) on the osteogenic differentiation of the bone marrow mesenchymal stem cells (BMSCs) of rats with high glucose levels. METHODS: Rat BMSCs were isolated and identified by osteogenic and adipogenic differentiation. Then, the BMSCs were divided into three groups as follows: normal control group (5.5 mmol·L⁻¹ glucose), high glucose group (25.5 mmol·L⁻¹ glucose), and ASP+high glucose group (25.5 mmol·L⁻¹ glucose +40 mg·L⁻¹ ASP). The proliferation activities of the BMSCs were detected by CCK8. Alizarin red staining, and alkaline phosphatase activity were used in the examination of osteogenic activity. Quantitative real time-polymerase chain reaction was used to detect the expression levels of the osteogenic genes (Runx2, Osx, OCN, Col-Ⅰ) and the key factors of Wnt/ß-catenin signal pathway (CyclinD1, ß-catenin). In vivo, a type 2 diabetes rat model was established. The rats were divided into three groups, namely, the normal control group (normal rats), diabetes group (diabetic rats), diabetes+ASP group (diabetic rats, ASP feeding). Then, the tibia bone defect was established. The repair of bone defects in each group was observed through histological examination. RESULTS: The proliferation of BMSCs was higher in the high glucose group and ASP+high glucose group than in the normal control group (P<0.05). No significant difference was observed between the high glucose group and ASP+high glucose group (P>0.05). The number of calcium nodules of BMSCs; alkaline phosphatase activity; and the mRNA expression of Runx2, OCN, Osx, Col-Ⅰ, CyclinD1, ß-catenin in the high glucose group were lower than those in the normal control and ASP+high glucose groups (P<0.05). No significant difference was observed between the normal control and ASP+high glucose groups (P>0.05). The bone mass was significantly lower in the bone defect of the diabetes group than in the bone defect of the normal control or diabetes+ASP group (P<0.05). No statistical difference was found between the normal control and diabetes+ASP groups (P>0.05). CONCLUSIONS: ASP can promote the osteogenic differentiation of rat BMSCs under high glucose culture and induce bone regeneration in rats with type 2 diabetes. These features may be related to the activation of the Wnt/ß-catenin signaling pathway.

Effect of adiponectin secreted from adipose-derived stem cells on bone-fat balance and bone defect healing.

The efficacy of adiponectin (APN) in regulating bone metabolism remains controversial. This study aimed to investigate the role of APN secreted from adipose-derived stem cells on adipogenesis and osteogenesis. Human APN gene was transfected via recombinant adenovirus into adipose derived stem cells (ASCs) in vitro and were cocultured with bone marrow mesenchymal stem cells (BMSCs) in using a transwell chamber. Adipogenesis was inhibited in APN-transfected ASCs; in BMSCs, adipogenesis was inhibited, but osteogenesis was promoted in coculture with APN-transfected ASCs. Next, the same adenovirus construct was transfected into the abdominal adipose tissue of a Sprague Dawley rat in vivo, and then a tibia defect was established in the same rat. We confirmed there was higher gene and protein expression of APN in ASCs and the abdominal adipose tissue of these rat models. Development of adipocytes in abdominal adipose tissue was suppressed, and less new bone was formed in the bone defect area. In conclusion, APN secreted from ASCs could directly inhibit adipogenesis in ASCs and BMSCs and promote osteogenesis in the latter. However, APN overexpression in adipose tissue was inversely associated with bone formation in tibia defects potentially due to decreased levels of circulating bone-activating hormones.

Effect of FGF-21 on implant bone defects through hepatocyte growth factor (HGF)-mediated PI3K/AKT signaling pathway.

Implant bone defects are the most common phenomenon in the processes of bone transplantation. Evidences have identified that fibroblast growth factor-21 (FGF-21) encourages osteogenesis for patients with implant bone defects. The purpose of this study was to investigate the role of FGF-21 and its potential mechanism in bone mesenchymal stem cells (BMSCs). RT-PCR, Western blotting, flow cytometry, immunofluorescence and immunohistochemistry assays were performed to analyze the role of FGF-21 and intracellular signaling pathways involved in BMSCs. It was shown that FGF-21 increased viability of BMSCs. Treatment with FGF-21 decreased the apoptosis of BMSCs by decreasing pro-apoptosis protein Caspase-3. Results indicated that FGF-21 (2 mg/kg) treatment up-regulated HGF, PI3K and AKT expression in BMSCs. In addition, the protective effects of FGF-21 on BMSCs were canceled by PI3K/AKT inhibitor in BMSCs. Results found that knockdown of HGF abolished FGF-21-decreased PI3K/AKT signal pathway. Furthermore, results demonstrated that FGF-21 presented beneficial effects for implant bone defects in rat model. In conclusion, these results indicate that FGF-21 can improve implant bone defects through HGF-mediated PI3K/AKT signaling pathway in BMSCs.

Scutellarin enhances osteoblast proliferation and function via NF-κB-mediated CXCR4 induction.

In the current study, we aimed to examine the function of scutellarin on human osteoblast proliferation and osteogenic function. The results indicated that scutellarin enhanced osteoblast proliferation over a seven day period. This increase in cell proliferation was associated with corresponding increases in osteoblast activity, as measured by alkaline phosphatase (ALP) secretion, intracellular calcium ion influx, and calcium deposition. These anabolic effects were associated with C-X-C chemokine receptor type 4 (CXCR4) mRNA levels and protein induction. Knockdown of CXCR4 reversed the scutellarin-induced increases in cell proliferation, ALP activity, and calcium deposition. Furthermore, scutellarin increased p65 phosphorylation in a dose-dependent manner, which resulted in the increased binding of phosphorylated p65 to the CXCR4 gene promoter region, to increase CXCR4 protein expression. p65 phosphorylation inhibition resulted in a decrease in CXCR4 protein expression. A p65 inhibitor blocked scutellarin-induced increases in osteoblast proliferation and function. Moreover, in a rat model of estrogen-deficient osteoporosis, scutellarin restored ovariectomy-induced bone loss in mice. Taken together, both cellular and animal models support the novel findings that scutellarin increased osteoblast proliferation and function through NF-κB/p65-mediated CXCR4 induction.

[Effect of TGF-ß3 transfected with adipose derived stem cells and OGP-HA-chondroitin sulfate scaffold on repair of condylar cartilage in rabbits].

PURPOSE: To investigate the feasibility of repairing damaged cartilage of rabbit condylar with TGF-ß3-transfected adipose stem cells combined with OGP (osteogenesis peptide)-HA (hyaluronic acid)-ChS (chondroitin sulfate) scaffold. METHODS: Rabbit ADSCs were isolated and cultured. The expression vector carrying TGF-ß3 gene was constructed using recombinant adenovirus and transfected into rabbit ADSCs. The fluorescence expression of the cells was observed after 14 days and the efficiency of virus transfection was calculated. Western blot was used to detect the expression of TGF-ß3 protein. Fifty rabbits were randomly divided into 5 groups: group A as blank group, group B as TGF-ß3 transfected ADSCs group, group C as OGP-HA-ChS scaffold group, group D as ADSCs complex OGP-HA-ChS group, E group was TGF-ß3 transfected ADSCs with OGP-HA-ChS. After the rabbit temporomandibular joint osteoarthrosis model was established, the animals were transplanted according to the experimental design. The animals in each group were sacrificed after the 3rd and 9th weeks of transplantation. Scanning electron microscopy, histological observation and real-time fluorescent quantitative PCR were performed accordingly. SPSS17.0 software package was used for analysis of variance and t test. RESULTS: Scanning electron microscopy and histological observation showed that the repair of cartilage lesions in group D and E was better than those in group B and C. Compared with group B, the results of real-time quantitative PCR showed that the expression of MMP-3 in group E was similar to that in group A, but significantly lower than that in group C and D (P<0.05). The expression of TIMP-1 in group E was similar to that of group A, but significantly higher than group C and D (P<0.05). CONCLUSIONS: TGF-ß3 transfected ADSCs composite OGP-HA-ChS scaffold has repairing effect on rabbit condylar damaged cartilage.

[RECOMBINANT ADENOVIRUS-MEDIATED BONE MORPHOGENETIC PROTEIN 9 AND ERYTHROPOIETIN GENES CO-TRANSFECTION IN PROMOTING OSTEOGENIC DIFFERENTIATION OF ADIPOSE-DERIVED STEM CELLS IN VITRO].

OBJECTIVE: To investigate the effect of recombinant adenovirus-mediated bone morphogenetic protein 9 (BMP-9) and erythropoietin (EPO) genes co-transfection on osteogenic differentiation of adipose-derived stem cells (ADSCs) in vitro. METHODS: The inguinal adipose tissue was harvested from 4-month-old New Zealand rabbits, ADSCs were isolated with enzyme digestion and adherence method, and multipotent differentiation capacity was identified. The 3rd generation ADSCs were divided into 5 groups: normal cells (group A), empty plasmid control group (group B), BMP-9 or EPO recombinant adenovirus transfected cells (groups C and D), BMP-9 and EPO recombinant adenovirus co-transfected cells (group E). The inverted phase contrast microscope was used to observe the cell growth at 7 days; the expression of cell fluorescence was observed under a fluorescence microscope at 14 days, and viral transfection efficiency was calculated at 48 hours; Western blot was used to detect the expressions of BMP-9 and EPO proteins at 14 days. The expression of alkaline phosphatase (ALP) activity was detected at 3, 7, and 14 days after osteogenic induction, and alizarin red staining was used to detect calcium nodules formation and real-time fluorescence quantitative PCR to detect the expressions of osteopontin (OPN) and osteocalcin (OCN) at 3 weeks. RESULTS: At 7 days after transfected, some cells showed oval, round, and irregular shape under the inverted phase contrast microscope in groups A and B; a few fusiform cells were observed in groups C and D; oval cells increased obviously, and there were only few round cells in group E. The fluorescence microscope observation showed that BMP-9 and EPO, BMP-9/EPO recombinant adenovirus could stably transfected ADSCs, with transfection efficiency of 80%-93%. The expressions of BMP-9 and EPO proteins significantly higher in group E than the other groups by Western blot (P < 0.05). The ALP activity significantly increased in group E when compared with that in the other groups at 3, 7, and 14 days after osteogenic induction (P < 0.05); the number of calcium nodules in group E was significantly more than that in the other groups (P < 0.05). Real-time fluorescence quantitative PCR showed that OPN and OCN genes expressions were significantly higher in group E than other groups (P < 0.05), and in groups C and D than groups A and B (P < 0.05). CONCLUSION: Recombinant adenovirus-mediated BMP-9 and EPO genes can transfect ADSCs, which can stably express in ADSCs, BMP-9/EPO genes co-transfection can more promote the expressions of osteoblast-related genes and protein than non-transfected and single gene transfection.

Bimaxillary Orthognathic Approach to Correct Skeletal Facial Asymmetry of Hemifacial Microsomia in Adults.

BACKGROUND: Hemifacial microsomia (HFM) is the second most common congenital craniofacial deformity after cleft lip and palate. Distraction osteogenesis (DO) is regarded as an alternative and efficient treatment option for patients with HFM. However, DO was not proven effective for all cases, and the results of long-term follow-up were not satisfactory as expected. Compared with DO, the orthognathic surgery approach may offer more stable clinical outcomes for this kind of disease. The purpose of this study is to evaluate the long-term clinical and radiographic outcome of bimaxillary orthognathic surgery in the treatment of adult HFM. METHODS: Eight patients with HFM who had undergone bimaxillary orthognathic surgery between 2008 and 2012 were included in the study. The surgical procedures included Le Fort I osteotomy, inverted-L osteotomy, sagittal split ramus osteotomy, genioplasty, and iliac bone grafting. Pre- and postoperative orthodontic treatments were performed, respectively. Clinical and radiographic examinations were carried out to assess postoperative outcomes. RESULTS: No obvious complications appeared postoperatively and no recurrences occurred during follow-up. All patients obtained satisfactory aesthetic results. Marked improvement in facial contour and occlusion were observed. Plain radiographs showed that the height ratios between the affected and unaffected ramus were ameliorated significantly. CONCLUSION: The bimaxillary orthognathic approach to correct the deformity of adult HFM can obtain stable results in the long-term follow-up, and should be considered as a priority method for the treatment of adult patients with dentofacial deformity. LEVEL OF EVIDENCE V: This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

Overgrowth of costochondral grafts in craniomaxillofacial reconstruction: Rare complication and literature review.

OBJECTIVE: Costochondral grafts (CCGs) have been used for the reconstruction of the craniomaxillofacial defects in various situations. However, there is controversy concerning the growth pattern of CCGs, which is often unpredictable and may manifest as overgrowth or no growth at all. This article summarizes the literature concerning overgrowth of CCGs in craniomaxillofacial reconstruction, and presents an uncommon case of treatment for overgrowth of costal graft in mandibular body reconstruction. MATERIAL AND METHODS: The literature on overgrowth of CCGs in craniomaxillofacial reconstruction was reviewed with a chart. A 25-year-old man received mandibular partial resection because of adamantoblastoma, followed by replacement of costal graft. Two years postoperatively, he began to present with facial asymmetry and malocclusion. Clinical and radiologic image examination showed deviation of the chin to the left side, and overgrowth of the costal graft was diagnosed. Left sagittal split ramus osteotomy (SSRO), genioplasty, and left mandibular angle ostectomy (MAO) were performed. RESULTS: A total of 30 articles containing 68 cases of overgrowth of CCGs in craniomaxillofacial reconstructions have been reported since 1977, including the present case. During a 2-year follow-up, the patient's postoperative facial profile and contour appeared stable clinically and radiographically, and an improved symmetry facial contour and occlusion were achieved. CONCLUSION: The growth of CCGs may be influenced by complex factors such as the function of the mandible, inherent growth capacity, and possibly hormonal factors. Once overgrowth of the costal graft occurs in mandibular body reconstruction, SSRO combined with genioplasty and MAO could be the optimal option to restore a symmetrical face.

[Effects of PRF and released three growth factors on migration of rat adipose tissue-derived stem cells].

PURPOSE: To analyze the effects of PRF and released three growth factors on migration of rat adipose tissue-derived stem cells and to investigate the mechanism of migration. METHODS: The inguinal adipose tissue of rat was excised at aseptic condition to obtain primary ADSCs by enzyme digestion. Multi-directional differentiation was used to identify the ADSCs. PRF membrane was acquired through one time centrifuge. The cell migration was examined by Transwell assay and wound healing assay. The mRNA expression of MMP2 and MT1-MMP was tested by real-time PCR. Statistical analysis was performed using SPSS 13.0 software package. RESULTS: Cell migration test showed that the migration of rat ADSCs in PRF group were significantly higher than those in the negative group（P<0.05） and inhibitor group（P<0.05）. The ADSCs migration effects in three growth factors group at different concentrations showed significant difference（P<0.05）. Real-time PCR showed that gene expressions of MMP2 and MT1-MMP were significantly higher in PRF group than control group (P<0.05). PCR showed that gene expressions of MMP2 and MT1-MMP were significantly higher in three growth factors group than control group (P<0.05). CONCLUTIONS: PRF and three growth factors consistently enhanced the migration of rat ADSCs in a dose-response manner. The migration increase of rat ADSCs may be associated with the up-regulation of MMP2 and MT1-MMP gene expression.