Melatonin promotes osteogenesis of bone marrow mesenchymal stem cells by improving the inflammatory state in ovariectomized rats / 中国修复重建外科杂志

OBJECTIVE@#To investigate the effects of melatonin (MT) on bone mass and serum inflammatory factors in rats received ovariectomy (OVX) and to investigate the effects of MT on the levels of inflammatory factors in culture medium and osteogenic ability of bone marrow mesenchymal stem cells (BMSCs) stimulated by lipopolysaccharide.@*METHODS@#Fifteen 12-week-old Sprague Dawley (SD) rats were randomly divided into 3 groups. The rats in Sham group only received bilateral lateral abdominal incision and suture, the rats in OVX group received bilateral OVX, and the rats in OVX+MT group received 100 mg/(kg·d) MT oral intervention after bilateral OVX. After 8 weeks, the levels of serum inflammatory factors [interleukin-1β (IL-1β), IL-6, and tumor necrosis factor α (TNF-α)] were detected using ELISA assay. Besides, the distal femurs were detected by Micro-CT to observe changes in bone mass and microstructure, and quantitatively measured bone volume fraction, trabecular thickness, and trabecular number. The BMSCs were extracted from the femurs of three 3-week-old SD rats using whole bone marrow culture method and passaged. The 3rd-5th passage BMSCs were cultured with different concentrations of MT (0, 1, 10, 100, 1 000 µmol/L), and the cell viability was then detected using cell counting kit 8 (CCK-8) to select the optimal concentration of MT for subsequent experiments. Cells were devided into osteogenic induction group (group A) and osteogenic induction+1/5/10 μg/mL lipopolysaccharide group (group B-D). The levels of inflammatory factors (IL-1β, IL-6 and TNF-α) in cell culture medium were detected using ELISA assay after corresponding intervention. According to the results of CCK-8 method and ELISA detection, the cells were intervened with the most significant concentration of lipopolysaccharide for stimulating inflammation and the optimal concentration of MT with osteogenic induction, defining as group E, and the cell culture medium was collected to detect the levels of inflammatory factors by ELISA assay. After that, alkaline phosphatase (ALP) staining and alizarin red staining were performed respectively in groups A, D, and E, and the expression levels of osteogenic related genes [collagen type Ⅰ alpha 1 chain (Col1a1) and RUNX family transcription factor 2 (Runx2)] were also detected by real time fluorescence quantitative PCR (RT-qPCR).@*RESULTS@#ELISA and Micro-CT assays showed that compared with Sham group, the bone mass of the rats in the OVX group significantly decreased, and the expression levels of serum inflammatory factors (IL-1β, IL-6, and TNF-α) in OVX group significantly increased (P<0.05). Significantly, the above indicators in OVX+MT group were all improved (P<0.05). Rat BMSCs were successfully extracted, and CCK-8 assay showed that 100 µmol/L was the maximum concentration of MT that did not cause a decrease in cell viability, and it was used in subsequent experiments. ELISA assays showed that compared with group A, the expression levels of inflammatory factors (IL-1β, IL-6, and TNF-α) in the cell culture medium of groups B-D were significantly increased after lipopolysaccharide stimulation (P<0.05), and in a concentration-dependent manner. Moreover, the expression levels of inflammatory factors in group D were significantly higher than those in groups B and C (P<0.05). After MT intervention, the expression levels of inflammatory factors in group E were significantly lower than those in group D (P<0.05). ALP staining, alizarin red staining, and RT-qPCR assays showed that compared with group A, the percentage of positive area of ALP and alizarin red and the relative mRNA expressions of Col1a1 and Runx2 in group D significantly decreased, while the above indicators in group E significantly improved after MT intervention (P<0.05).@*CONCLUSION@#MT may affect the bone mass of postmenopausal osteoporosis by reducing inflammation in rats; MT can reduce the inflammation of BMSCs stimulated by lipopolysaccharide and weaken its inhibition of osteogenic differentiation of BMSCs.

Isolating culture of adipose mesenchymal stem cells in psoriasis vulgaris patients and differentiation into immune regulation function / 基础医学与临床

Objeetive To study the differences of the biological characteristics and immune regulation function of adipose mesenchymal stem cells (AMSCs)from psoriasis patients and healthy people.Methods AMSCs were isolated and cultured from human psoriatic and healthy adipose tissue,the phenotypes and cell cycle of AMSCs taken from three generation were detected by flow eytometry.Alkaline phosphate enzyme staining and oil red o staining were used respectively to identify their adipogenic and osteogenic capacity.Next,the levels of inflammation antimicrobial proinflammatory factor were detected by PCR and ELISA.Then gene expression profile of AMSCs were screen by gene expression profile chip,as so to bolting the the gene array related with immunology gene.Results There was no significant change in cell morphology,and cell surface markers were expressed high for CD29,CD44,CD73,while lower for CD31,CD45 and HLA-DR.AMSCs of psoriasis patients and healthy people both had the ability of adipogenic and osteogenic differentiation.But the cell cycle showed the third generation AMSCs proliferation rates were slower than that of normal control,as compared with healthy controls,adipogenic differentiation ability was stronger.What'more,the level of inflammatory cytokines in psoriasis group was lower than that in controls such asIL-10,IDO,TGF-β,on the contrary the levels of proinflammatory factor in psoriasis group were higher than that in controls,such as TNF-α,IFN-γ.In addition,gene chip results suggested that psoriasis group AMSCs had obvious expression differences on JAK-STAT pathway with healthy controls.Conclusions Compared with the control,there are significant differences in patients AMSCs proliferation and adipogenic differentiation ability,immune inflammation suppression control ability is weaken,this phenomenon may be associated with JAK-STAT immune pathways related to downgrade.

Hydrogen attenuates the inhibition of P.g-LPS on osteogenic capacity of huma periodental ligament cells / 实用口腔医学杂志

Objective:To observe the effects of hydrogen on osteogenic capacity of human periodontal ligament cells(hPDLCs)stim-ulated with P.g-LPS.Methods:hPDLCs were cultured and divided into 4 groups:control(C)group,osteogenic induction(OI) group,OI +1 00 ng/ml LPS(OILPS)group and OIPLS +3%H2 (H2 OIPLS)group,and treated respectively.Alizarin red staining (ARS)was carried out 3 weeks after treatment.ALP and OC mRNA expression of the cells was examined by RT-PCR after 7-d treat-ment.Results:LPS decreased A value of ARS(P <0.01 ),ALP mRNA expression(P <0.001 )and OC mRNA expression(P <0.001 )of the cells.H2 increased the A value(P <0.05),ALP mRNA expression(P <0.01 )and OC mRNA(P <0.01 )of the cells treated by LPS.Conclusion:High concentration of P.g-LPS can inhibit osteogenic capacity of hPDLCs,while hydrogen can impair the P.g-LPS induced suppression of hPDLC's osteogenesis.

Ossificação do pedículo vascular de um retalho livre de fíbula: relato de caso / Ossification of vascular pedicle in fibula free flap: a case report

Introdução: Várias são as vantagens da utilização de retalhos fibulares para as reconstruções de defeitos craniomaxilofaciais, incluindo a baixa morbidade da área doadora, boa qualidade óssea possibilitando a realização de implantes osteointegrados quando indicados, além da possibilidade de inclusão de uma ilha de pele quando indicado. Durante a dissecção do retalho, próximo à região do pedículo vascular, normalmente inclui-se um cuff muscular e uma faixa de periósteo. O potencial osteogênico do periósteo transplantado tem sido objeto de estudo. Relato de caso: paciente de 15 anos, submetido à reconstrução microcirúrgica com um retalho fibular para um defeito mandibular pós-ressecção de um sarcoma ósseo. Evoluiu com aumento de volume, de consistência óssea na região cervical próximo à cervicotomia realizada para anastomose vascular. Exames de imagem mostravam características ósseas da massa. Foi então submetido à nova cervicotomia e exploração da massa, sendo observada uma formação de tecido ósseo no local da anastomose vascular. Exame anatomopatológico da peça mostrava formação de tecido ósseo adjacente ao retalho periostal. Discussão: Durante a dissecção do retalho fibular, a osteotomia é realizada a alguns centímetros da articulação do joelho, isto a fim de facilitar a dissecção do pedículo vascular na região do oco poplíteo. O pedículo vascular fica então envolto por uma cuff muscular e por uma tira de periósteo. Este mantém sua capacidade osteogênica, que pode ser ativada de acordo com o estímulo do local. A ossificação do periósteo do pedículo vascular de retalhos livres de fíbula permanece um evento raro, porém relatado por centros diferentes.

Introduction: The use of fibula flaps for the reconstruction of craniomaxillofacial defects has many advantages, including the low morbidity of the donor area, good bone quality for use of osseointegrated implants, and the possibility to include a skin island, when indicated. During the dissection of the flap, a muscle "cuff" and a periosteal strip are usually included near the region of the vascular pedicle. The osteogenic potential of the transplanted periosteum has been the object of studies. Case report: A 15-year-old male patient underwent microsurgical reconstruction using a fibula flap for a mandibular defect caused by the resection of a bone sarcoma. He developed increased volume and bone consistency in the cervical region next to the area where a cervicotomy was performed for vascular anastomosis. Imaging examinations showed the characteristics of the bone mass. He then underwent a new cervicotomy and mass exploratory surgery because bone tissue formation was observed at the site of vascular anastomosis. Anatomopathological examination of the specimen showed bone tissue formation next to the periosteal flap. Discussion: During fibula flap dissection, osteotomy is performed a few centimeters from the knee joint to facilitate the dissection of the vascular pedicle in the region of the popliteal fossa. Then, the vascular pedicle is surrounded by a muscle cuff and periosteal strip. This maintains its osteogenic capacity, which can be activated according to the stimulus of the area. Although periosteal ossification of the vascular pedicle in fibula free flaps is a rare event, it has been reported in different centers.