[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.

[Preliminary study on treatment of mandibular osteoradionecrosis by autologous bone marrow mesenchymal stem cells in miniature pigs].

OBJECTIVE: To investigate the treatment effect of mandibular osteoradionecrosis (ORN) by autologous bone marrow mesenchymal stem cells (BMMSC) in miniature pigs. METHODS: Six miniature pigs with mandibular ORN (25 Gy) were used. BMMSC were separated and cultured in vitro and then implanted on to the premolded hydroxyapatite-tricalcium phosphate (HA-TCP). The BMMSC-HA-TCP complexes were implanted into the defective area of mandibular ORN in 4 animals. Implantation of only HA-TCP in 2 animals served as control. Gross observation, spiral CT, coronary CT and histopathologic examination were carried out. RESULTS: Fistula disappeared in animals with BMMSC-HA-TCP complexes 3 months after implantation, while fistula was found in controls. Spiral CT analysis showed that cortical bone repair were found 4 months after BMMSC-HA-TCP complexes implantation, while bone damage (cortical and cancellous bone fracture) increased in controls. Histopathologic examination revealed that 4 months later after BMMSC-HA-TCP complexes implantation, new bone formation and bone cells could be observed, but there was a large number of fibrous tissue and no new bone in controls. CONCLUSIONS: Transplantation of autologous BMMSC with HA-TCP may have therapeutic effect in the treatment of mandibular ORN.

[Expression and clinical significance of Cox-2 and XIAP in malignant tumors of the salivary gland].

PURPOSE: To detect the expression of Cox-2 and XIAP in adenoid cystic carcinoma (ACC) and mucoepidermoid carcinoma (MEC) of salivary glands. To study the role and correlation between the expression of Cox-2 and XIAP in malignant tumors of salivary gland. METHODS: Immunohistochemical SP staining method was used to quantify the protein expression levels of Cox-2 and XIAP in 95 patients with malignant tumors of the salivary gland (50 ACCs,45 MECs),10 patients with pleomorphic adenoma and 10 persons with normal salivary gland tissues. SPSS17.0 software package was used to analyze the data. RESULTS: Cox-2 and XIAP were expressed strongly in malignant tumors of the salivary gland, while they not expressed in pleomorphic adenoma and normal salivary gland tissues. The expression of Cox-2 and XIAP in malignant tumors of salivary gland was correlated with clinical stage and lymphnode metastasis, while they not correlated with primary sites (P>0.05). CONCLUSIONS: The high expression of Cox-2 and XIAP in malignant tumors of salivary gland may play an important role in the development of malignant tumors of the salivary gland.

[The isolation and identification of bone marrow mesenchymal stem cells derived from rabbit mandible].

PURPOSE: To investigate the methods of isolation, culture and identification of BMMSCs derived from rabbit mandible. METHODS: BMMSCs were collected from rabbit mandible and isolated by density gradient centrifugation. Cells were adherently cultured in vitro, and P2 or P3 BMMSCs populations were collected and examined. Cell growth was observed by inverted microscopy; the propagation of BMMSCs were tested by MTT and a growth curve was drawn after statistical analysis; colony-forming unit-fibroblast(CFU-F) was detected by examination of colony formation; the potential of multi-directional differentiation into osteoblasts, adipocytes and skeletal muscle cells was estimated by pertinent methods; the surface marks of BMMSCs were detected by flow cytometry, the data was analysed using SPSS16.0 software package. RESULTS: The majority of adherent cells were long fusiform, and few were small triangle; the growth curve of BMMSCs showed that every passage experienced incubation period, log phase and platform period; the rate of colony formation was 37%. Growth of BMMSCs represented the appearance of CFU-F; BMMSCs after inducted differentiation showed osteogenic and adipogenic potential. The staining of mineralized nodules was positive by alizarin red S and the positive staining of oil red O appeared in lipid drops around cell nucleus. The staining of skeletal muscle cells was positive by desmin immunofluorescence; the cell surface marks assessed with flow cytometry indicated that these BMMSCs expressed CD90 and CD146 in high percentage (about 98.7% and 98.1%, respectively). CONCLUSIONS: The highly uniformed BMMSCs derived from rabbit mandible can be collected. These BMMSCs have the ability of self-replication and propagation, as well as potential of multi-directional differentiation in vitro.

[Research on the adipose-derived stem cells combined with the extract of Eucommiol scaffold material to repair the rabbit mandible defect].

OBJECTIVE: To investigate the effect of Eucommiol on osteogenic differentiation of adipose-derived stem cells (ADSCs), and the feasibility of applying in mandibular defects repair using ADSCs combined with the extract of Eucommiol scaffold material. METHODS: Forty-eight New Zealand rabbits were randomly divided into four groups and bilateral mandibular defect was prepared. Group A: Implanted the ADSCs combined with the extract of Eucommiol scaffold, group B: Implanted the ADSCs combined with hydroxyapatite materials, group C: Implanted hydroxyapatite materials, group D: The control group. All the experimental animals were sacrificed after 2, 4, 8, 12 weeks. Tissue samples were observed by gross observation, radiographic analysis, hematoxylin-eosin (HE) staining, scanning electron microscope (SEM). The value of imaging analysis and osteogenesis were evaluated. The results were analyzed by SPSS 17.0 statistical software. RESULTS: In vivo imaging and histological staining showed that the healing of bone defect and bone quality in group A was significantly better than those in the other groups. SEM showed well biocompatibility between composite material and tissue without inflammation reaction. By measuring and analyzing the dental CT data and new bone area, the bone in group A was obviously better than those in other groups (P < 0.05). CONCLUSION: The extract of Eucommiol can promote ADSCs into bone cells. The role of ADSCs combined with extract of Eucommiol scaffold materials has significant bone induction. It is expected that this material could become a new composite material and be used to fix the jaw bone defects.

Mesenchymal stromal cell-based treatment of jaw osteoradionecrosis in Swine.

Jaw osteoradionecrosis (ORN) is a common and serious complication of radiation therapy for head and neck cancers. Bone marrow mesenchymal stromal cells (BMMSCs) are multipotent postnatal stem cells and have been widely used in clinical therapies. In the present study, we generated the mandibular ORN model in swine using a combination of single-dose 25-Gy irradiation and tooth extraction. A typical ORN phenotype, including loss of bone regeneration capacity and collagen collapse with the obliteration of vessels, gradually appeared after irradiation. After autologous BMMSC transplantation, new bone and vessels were regenerated, and the advanced mandibular ORN was treated successfully. In summary, we developed a swine model of jaw ORN, and our results indicate that autologous BMMSC transplantation may be a promising therapeutic approach for ORN.

Periodontal ligament stem cell-mediated treatment for periodontitis in miniature swine.

Periodontitis is a periodontal tissue infectious disease and the most common cause for tooth loss in adults. It has been linked to many systemic disorders, such as coronary artery disease, stroke, and diabetes. At present, there is no ideal therapeutic approach to cure periodontitis and achieve optimal periodontal tissue regeneration. In this study, we explored the potential of using autologous periodontal ligament stem cells (PDLSCs) to treat periodontal defects in a porcine model of periodontitis. The periodontal lesion was generated in the first molars area of miniature pigs by the surgical removal of bone and subsequent silk ligament suture around the cervical portion of the tooth. Autologous PDLSCs were obtained from extracted teeth of the miniature pigs and then expanded ex vivo to enrich PDLSC numbers. When transplanted into the surgically created periodontal defect areas, PDLSCs were capable of regenerating periodontal tissues, leading to a favorable treatment for periodontitis. This study demonstrates the feasibility of using stem cell-mediated tissue engineering to treat periodontal diseases.

Transplantation of mesenchymal stem cells is an optimal approach for plastic surgery.

Mesenchymal stem cells (MSCs) are able to differentiate into a variety of cell types, offering promising approaches for stem cell-mediated tissue regeneration. Here, we explored the potential of utilizing MSCs to reconstruct orofacial tissue, thereby altering the orofacial appearance. We demonstrated that bone marrow MSCs were capable of generating bone structures and bone-associated marrow elements on the surfaces of the orofacial bone. This resulted in significant recontouring of the facial appearance in mouse and swine. Notably, the newly formed bone and associated marrow tissues integrated with the surfaces of the recipient bones and re-established a functional bone marrow organ-like system. These data suggested that MSC-mediated tissue regeneration led to a body structure extension, with the re-establishment of all functional components necessary for maintaining the bone and associated marrow organ. In addition, we found that the subcutaneous transplantation of another population of MSCs, the human periodontal ligament stem cells (PDLSCs), could form substantial amounts of collagen fibers and improve facial wrinkles in mouse. By contrast, bone marrow MSCs failed to survive at 8 weeks post-transplantation under the conditions used for the PDLSC transplantation. This study suggested that the mutual interactions between donor MSCs and recipient microenvironment determine long-term outcome of the functional tissue regeneration. Disclosure of potential conflicts of interest is found at the end of this article.

Mesenchymal stem cell-mediated functional tooth regeneration in swine.

Mesenchymal stem cell-mediated tissue regeneration is a promising approach for regenerative medicine for a wide range of applications. Here we report a new population of stem cells isolated from the root apical papilla of human teeth (SCAP, stem cells from apical papilla). Using a minipig model, we transplanted both human SCAP and periodontal ligament stem cells (PDLSCs) to generate a root/periodontal complex capable of supporting a porcelain crown, resulting in normal tooth function. This work integrates a stem cell-mediated tissue regeneration strategy, engineered materials for structure, and current dental crown technologies. This hybridized tissue engineering approach led to recovery of tooth strength and appearance.