Application value of bone cement containing rhbFGF and RHBMP-2 in PKP treatment of osteoporotic lumbar compression fracture / 中国骨伤

OBJECTIVE@#To investigate the effect of bone cement containing recombinant human basic fibroblast growth factor (rhbFGF) and recombinant human bone morphogenetic protein-2 (rhBMP-2) in percutaneous kyphoplasty(PKP)treatment of osteoporotic vertebral compression fracture(OVCF).@*METHODS@#A total of 103 OVCF patients who underwent PKP from January 2018 to January 2021 were retrospectively analyzed, including 40 males and 63 females, aged from 61 to 78 years old with an average of (65.72±3.29) years old. The injury mechanism included slipping 33 patients, falling 42 patients, and lifting injury 28 patients. The patients were divided into three groups according to the filling of bone cement. Calcium phosphate consisted of 34 patients, aged(65.1±3.3) years old, 14 males and 20 females, who were filled with calcium phosphate bone cement. rhBMP-2 consisted of 34 patients, aged (64.8±3.2) years old, 12 males and 22 females, who were filled with bone cement containing rhBMP-2. And rhbFGF+rhBMP-2 consisted of 35 patients, aged (65.1±3.6) years old, 14 males and 21 females, who were filled with bone cement containing rhbFGF and rhBMP-2. Oswestry disability index (ODI), bone mineral density, anterior edge loss height, anterior edge compression rate of injured vertebra, visual analog scale (VAS) of pain, and the incidence of refracture were compared between groups.@*RESULTS@#All patients were followed for 12 months. Postoperative ODI and VAS score of the three groups decreased (P<0.001), while bone mineral density increased (P<0.001), anterior edge loss height, anterior edge compression rate of injured vertebra decreased first and then slowly increased (P<0.001). ODI and VAS of group calcium phosphate after 1 months, 6 months, 12 months were lower than that of rhBMP-2 and group rhbFGF+rhBMP-2(P<0.05), bone mineral density after 6 months, 12 months was higher than that of rhBMP-2 and group calcium phosphate(P<0.05), and anterior edge loss height, anterior edge compression rate of injured vertebra of group rhbFGF+rhBMP-2 after 6 months and 12 months were lower than that of group rhBMP-2 and group calcium phosphate(P<0.05). There was no statistical difference in the incidence of re-fracture among the three groups (P>0.05).@*CONCLUSION@#Bone cement containing rhbFGF and rhBMP-2 could more effectively increase bone mineral density in patients with OVCF, obtain satisfactory clinical and radiological effects after operation, and significantly improve clinical symptoms.

Effect of Human Bone Marrow Mesenchymal Stem Cells with Ectopic High OCT4 Expression on T Lymphocyte Function / 中国实验血液学杂志

OBJECTIVE@#To explore the effect of human bone marrow mesenchymal stem cells (MSCs) with ectopic high OCT4 expression on T-cell proliferation, activation and secretion in vitro.@*METHODS@#Peripheral blood mononuclear cells were isolated from healthy children. Anti-CD3 and anti-CD28 monoclonal antibodies were used to activate T lymphocytes, which were stimulated by interleukin (IL)-2 for one week in vitro. Then MSCs with ectopic high OCT4 expression (MSC-OCT4) were co-cultured with activated T lymphocytes. After one week of co-culture, the supernatant was collected and the levels of Th1/Th2 cytokines [IL-2, IL-4, IL-6, IL-10, tumor necrosis factor (TNF)-α and interferon (IFN)-γ] were determined by flow cytometry. The lymphocytes after one week of co-culture were collected and counted by Countstar software. After the proportions of activated/inactivated T cell subsets were determined by flow cytometry, the absolute lymphocyte counts were calculated and expressed as mean ± standard deviation.@*RESULTS@#Compared with control T cell alone culture group, the proliferation of CD3+ T cells, CD3+CD4+ T cells, and CD3+CD8+ T cells were significantly inhibited in MSC group and MSC-OCT4 group. Compared with MSC, MSC-OCT4 could inhibit CD3+CD8+ T cell proliferation better (P =0.049), and mainly inhibited early T cell activation. Compared with control T cell alone culture group, the levels of IL-2 and INF-γ were significantly down-regulated both in MSC group and MSC-OCT4 group.After co-culture with T cells for one week, the level of IL-6 significantly increased in MSC group and MSC-OCT4 group compared with that before co-culture. Compared with control MSC group, MSC-OCT4 group had higher viable cell numbers after 1 week of co-culture (P =0.019), and could resist the inhibition of proliferation by higher concentration of mitomycin C.@*CONCLUSION@#Both MSC and MSC-OCT4 can inhibit the proliferation and activation of IL-2-stimulated T cells in vitro. After overexpression of OCT4, MSC has better proliferation ability in vitro and can inhibit the proliferation of CD3+CD8+ T cells more effectively, which may have a better and more lasting immunosuppressive ability to regulate the balance of Th1/Th2.

Effect of Wnt/β-catenin signaling pathway in neural differentiation of human bone marrow mesenchymal stem cells / 中国修复重建外科杂志

OBJECTIVE@#To explore the effect of basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), and the combination of bFGF and EGF in the neural differentiation of human bone marrow mesenchymal stem cells (hBMSCs), and the role of Wnt/β-catenin signaling pathway in this process.@*METHODS@#The identified 4th-generation hBMSCs were divided into five groups according to different induction conditions, namely control group (group A), EGF induction group (group B), bFGF induction group (group C), EGF and bFGF combined induction group (group D), and EGF, bFGF, and Dickkopf-related protein 1 (DKK-1) combined induction group (group E). After 7 days of continuous induction, the cell morphology was observed by inverted fluorescence phase contrast microscopy, levels of genes that were related to neural cells [Nestin, neuron-specific enolase (NSE), microtubule-associated protein 2 (MAP-2), and glial fibrillary acidic protein (GFAP)] and key components of the Wnt/β-catenin signaling pathway (β-catenin and Cyclin D1) were detected by RT-PCR, and the levels of proteins that were related to neural cells (Nestin and GFAP) as well as genes that were involved in Wnt/β-catenin signaling pathway [β-catenin, phosphorylation β-catenin (P-β-catenin), Cytoplasmic β-catenin, and Nuclear β-catenin] were explored by cellular immunofluorescence staining and Western blot.@*RESULTS@#When compared to groups A and B, the typical neuro-like cell changes were observed in groups C-E, and most obviously in group D. RT-PCR showed that the relative expressions of Nestin, NSE, and MAP-2 genes in groups C-E, the relative expressions of GFAP gene in groups D and E, the relative expression of NSE gene in group B, the relative expressions of β-catenin gene in groups C and D, and the relative expressions of Cyclin D1 gene in groups B-D significantly increased when compared with group A ( P<0.05). Compared with group E, the relative expressions of Nestin, NSE, MAP-2, GFAP, β-catenin, and CyclinD1 genes significantly increased in group D ( P<0.05); compared with group C, the relative expression of Nestin gene in group D significantly decreased ( P<0.05), while NSE, MAP-2, and GFAP genes significantly increased ( P<0.05). The cellular immunofluorescence staining showed that the ratio of NSE- and GFAP-positive cells significantly increased in groups C-E than in group A, in group D than in groups C and E ( P<0.05). Western blot assay showed that the relative expression of NSE protein was significantly higher in groups C and D than in group A and in group D than in groups C and E ( P<0.05). In addition, the relative expression of GFAP protein was significantly higher in groups C-E than in group A and in group D than in group E ( P<0.05). Besides, the relative expressions of β-catenin, Cytoplasmic β-catenin, Nuclear β-catenin, and the ratio of Nuclear β-catenin to Cytoplasmic β-catenin were significantly higher in groups C and D than in group A and in group D than in group E ( P<0.05), whereas the relative expression of P-β-catenin protein was significantly lower in groups C and D than in group A and in group D than in group E ( P<0.05).@*CONCLUSION@#Different from EGF, bFGF can induce neural differentiation of hBMSCs. In addition, EGF can enhance the hBMSCs neural differentiation of bFGF, while the Wnt/β-catenin signaling pathway may play a positive regulatory role in these processes.

Exosome-derived circ_0009362 mediates osteogenic differentiation of human bone marrow mesenchymal stem cells by regulating miR-29b-3p / 中华内分泌外科杂志

Objective:To investigate the effect and potential mechanism of serum exosome-derived cirC_0009362 on the osteogenic differentiation of human bone marrow mesenchymalstem cells (hBMSCs) .Methods:Serum samples from patients with osteoporosis (OP) were collected and exosomes were isolated. The expression level of circ_0009362 in exosomes was detected by qRT-PCR. hBMSCs osteogenesis was induced and the expression of circ_0009362 and miR-29b-3p was detected. The interaction between circ_0009362 and miR-29b-3p was detected by dual luciferase reporter assay. Alkaline phosphatase (ALP) kit was used to detect ALP activity and alizarin red (ARS) staining was used to detect calcium deposition.Results:Compared with healthy control group, the expression of circ_0009362 in serum exosomes of OP patients was increased, and the expression of circ_0009362 was decreased after inducing hBMSCs osteogenesis (all P<0.05) . The ALP activity and the percentage of calcium deposition in hBMSCs were decreased by exosomes, and this effect was achieved by secreting circ_0009362. The effect of exosomes was partially offset by circ_0009362 expression in knockdown exosomes (all P<0.05) . The expression of miR-29b-3p was increased after inducing hBMSCs osteogenesis ( P<0.05) . Circ_0009362 had a targeting relationship with miR-29b-3p, and exosomes inhibited the expression of miR-29b-3p by secreting circ_0009362. The ALP activity and the percentage of calcium deposition in hBMSCs were promoted by overexpression of miR-29b-3p, which was partially offset by exosomes (all P<0.05) . Conclusion:Serum exosomes of OP patients inhibit the osteogenic differentiation of hBMSCs by secreting circ_0009362 to down-regulate the expression of miR-29b-3p.

Biocompatibility and effect on bone formation of a native acellular porcine pericardium: Results of in vitro and in vivo / 北京大学学报(医学版)

OBJECTIVE@#To examine the morphology and biocompatibility of a native acellular porcine pericardium (APP) in vitro and to evaluate its barrier function and effects on osteogenesis when used in guided bone regeneration (GBR) in vivo.@*METHODS@#First, the morphology of APP (BonanGenⓇ) was detected using a scanning electron microscope (SEM). Next, for biocompatibility test, proliferation of human bone marrow mesenchymal stem cells (hBMSCs) were determined using cell counting kit-8 (CCK-8) after being seeded 1, 3 and 7 days. Meanwhile, the cells stained with phalloidine and 4, 6-diamidino-2-phenylindole (DAPI) were observed using a confocal laser scanning microscopy (CLSM) to view the morphology of cell adhesion and pattern of cell proliferation on day 5. A 3-Beagle dog model with 18 teeth extraction sockets was used for the further research in vivo. These sites were randomly treated by 3 patterns below: filled with Bio-OssⓇand coverd by APP membrane (APP group), filled with Bio-OssⓇand covered by Bio-GideⓇmembrane (BG group) and natural healing (blank group). Micro-CT and hematoxylin-eosin (HE) were performed after 4 and 12 weeks.@*RESULTS@#A bilayer and three-dimensional porous ultrastructure was identified for APP through SEM. In vitro, APP facilitated proliferation and adhesion of hBMSCs, especially after 7 days (P < 0.05). In vivo, for the analysis of the whole socket healing, no distinct difference of new bone ratio was found between all the three groups after 4 weeks (P>0.05), however significantly more new bone regeneration was detected in APP group and BG group in comparison to blank group after 12 weeks (P < 0.05). The radio of bone formation below the membrane was significantly higher in APP group and BG group than blank group after 4 and 12 weeks (P < 0.05), however, the difference between APP group and BG group was merely significant in 12 weeks (P < 0.05). Besides, less resorption of buccal crest after 4 weeks and 12 weeks was observed in APP group of a significant difference compared in blank group (P < 0.05). The resorption in BG group was slightly lower than blank group (P>0.05).@*CONCLUSION@#APP showed considerable biocompatibility and three-dimentional structure. Performing well as a barrier membrane in the dog alveolar ridge preservation model, APP significantly promoted bone regeneration below it and reduced buccal crest resorption. On the basis of this study, APP is a potential osteoconductive and osteoinductive biomaterial.

A novel hydroxyapatite fiber scaffold for three-dimensional cell culture / 中国组织工程研究

BACKGROUND: The dense microstructure of biological scaffolds and the limitation of cell growth microenvironment are the two major difficulties in the application of biological scaffolds in bone tissue repair. OBJECTIVE: To prepare fluffy hydroxyapatite/polylactic acid composite fiber scaffold, so that cells can easily enter into the scaffold and to realize three-dimensional culture of bone marrow mesenchymal stem cells. METHODS: Fluffy hydroxyapatite/polylactic acid composite scaffold was prepared by using modified electrospinning technology combined with biomineralization. The physical and chemical properties of the fiber scaffold were measured and observed. Human bone marrow mesenchymal stem cells were inoculated on the fluffy hydroxyapatite/polylactic acid composite scaffold and traditional hydroxyapatite/polylactic acid composite scaffold. Cell proliferation, adherence and morphological changes were detected. RESULTS AND CONCLUSION: (1) The thickness of hydroxyapatite coating in the fluffy hydroxyapatite/polylactic acid composite scaffold was about 8.3μ m, most of hydroxyapatite fibers were in discrete state with a diameter of 8-14μm. The fibers were connected by pores, and the pore diameter was (65±35) μm. The surface area, porosity and water absorption of the scaffold were significantly higher than those of the traditional scaffold (P<0.01).(2) After 12 hours of culture, the adherence of bone marrow mesenchymal stem cells on the two scaffolds was similar, 83% and 81% cells adhered on the traditional and fluffy scaffolds, respectively. (3) After 7 days of culture, the number of proliferated cells in the fluffy hydroxyapatite/polylactic acid composite scaffold was significantly more than that in the traditional hydroxyapatite/polylactic acid composite scaffold (P<0.01).(4) After 7 days of culture, FDA staining and scanning electric microscopy showed that cell-cell independent shape appeared in the traditional scaffold. A large number of cells appeared in the fluffy scaffold and grew into cell clusters with high cell activity, which formed a cell-fiber construction. These results indicate that this new type hydroxyapatite/polylactic acid composite scaffold is beneficial for cell entry and proliferation.

Effect of micro RNA-335-5p regulating bone morphogenetic protein 2 on osteogenic differentiation of human bone marrow mesenchymal stem cells / 中国修复重建外科杂志

Objective: To investigate the effect of micro RNA (miR)-335-5p regulating bone morphogenetic protein 2 (BMP-2) on the osteogenic differentiation of human bone marrow mesenchymal stem cells (hBMSCs). Methods: hBMSCs were cultured in vitro and randomly divided into control group (group A), miR-335-5p mimics group (group B), miR-335-5p mimics negative control group (group C), miR-335-5p inhibitor group (group D), and miR-335-5p inhibitor negative control group (group E). After grouping treatment and induction of osteogenic differentiation, the osteogenic differentiation of cells in each group was detected by alkaline phosphatase (ALP) and alizarin red staining; the expressions of miR-335-5p and BMP-2, Runt-related transcription factor 2 (Runx2), osteopontin (OPN), and osteocalcin (OCN) mRNAs were detected by real-time fluorescence quantitative PCR analysis; the expressions of Runx2, OPN, OCN, and BMP-2 proteins were detected by Western blot. Results: Compared with group A, the relative proportion of ALP positive cells and the relative content of mineralized nodules, the relative expressions of BMP-2, miR-335-5p, OPN, OCN, Runx2 mRNAs, the relative expressions of Runx2, OPN, OCN, and BMP-2 proteins in group B were significantly increased ( P0.05). Conclusion: miR-335-5p can up-regulate BMP-2 expression and promote osteogenic differentiation of hBMSCs.

A novel hydroxyapatite fiber scaffold for three-dimensional cell culture / 中国组织工程研究

BACKGROUND: The dense microstructure of biological scaffolds and the limitation of cell growth microenvironment are the two major difficulties in the application of biological scaffolds in bone tissue repair. OBJECTIVE: To prepare fluffy hydroxyapatite/polylactic acid composite fiber scaffold, so that cells can easily enter into the scaffold and to realize three-dimensional culture of bone marrow mesenchymal stem cells. METHODS: Fluffy hydroxyapatite/polylactic acid composite scaffold was prepared by using modified electrospinning technology combined with biomineralization. The physical and chemical properties of the fiber scaffold were measured and observed. Human bone marrow mesenchymal stem cells were inoculated on the fluffy hydroxyapatite/polylactic acid composite scaffold and traditional hydroxyapatite/polylactic acid composite scaffold. Cell proliferation, adherence and morphological changes were detected. RESULTS AND CONCLUSION: (1) The thickness of hydroxyapatite coating in the fluffy hydroxyapatite/polylactic acid composite scaffold was about 8.3 µm, most of hydroxyapatite fibers were in discrete state with a diameter of 8-14 µm. The fibers were connected by pores, and the pore diameter was (65±35) µm. The surface area, porosity and water absorption of the scaffold were significantly higher than those of the traditional scaffold (P < 0.01). (2) After 12 hours of culture, the adherence of bone marrow mesenchymal stem cells on the two scaffolds was similar, 83% and 81% cells adhered on the traditional and fluffy scaffolds, respectively. (3) After 7 days of culture, the number of proliferated cells in the fluffy hydroxyapatite/polylactic acid composite scaffold was significantly more than that in the traditional hydroxyapatite/polylactic acid composite scaffold (P < 0.01). (4) After 7 days of culture, FDA staining and scanning electric microscopy showed that cell-cell independent shape appeared in the traditional scaffold. A large number of cells appeared in the fluffy scaffold and grew into cell clusters with high cell activity, which formed a cell-fiber construction. These results indicate that this new type hydroxyapatite/polylactic acid composite scaffold is beneficial for cell entry and proliferation.

In vitro hematopoietic support of human umbilical cord blood cd34+ cells by human skeletal muscle-derived pericytes/perivascular cells / 中国组织工程研究

BACKGROUND: Perivascular cells have been shown to be the precursor cells of mesenchymal stem cells, which regulate the behavior of hematopoietic stem cells and support hematopoiesis through cell-to-cell contact or paracrine effects. Hematopoietic support of human skeletal muscle-derived pericytes/perivascular cells (hMD-PCs) remains to be studied. OBJECTIVE: To identify the biological characteristics of hMD-PCs isolated from human skeletal muscle and to study their supporting effect on umbilical cord blood CD34+ cells in vitro. METHODS: (1) hMD-PCs with phenotype CD146+ CD56-CD34-CD144-CD45- were sorted from human skeletal muscle by enzymatic digestion and multiparameter fluorescence-activated cell sorting, and their biological characteristics were identified. (2) The in vitro culture system of umbilical cord blood CD34+ cells co-cultured with human CD146+ hMD-PCs (experimental group) or with human bone marrow mesenchymal stem cells (positive control group) was established. After 1, 2 and 4 weeks of co-culture, the number of cells, the colony formation ability and immunophenotype were measured and statistically analyzed. RESULTS AND CONCLUSION: (1) CD146+ hMD-PCs were sorted by multiparameter fluorescence-activated cell sorting and the purity was (91.5±1.85)% (n=5). CD146+ hMD-PCs expressed mesenchymal surface markers CD73, CD90, CD105, CD44, and did not express hematopoietic cell and endothelial cell markers CD45, CD34, and CD31. After induced culture, CD146+ hMD-PCs could differentiate into osteoblasts, chondrogenesis, adipocytes and myoblasts. (2) There were no significant differences in the cell number, colony f ormation ability or immunophenotype (CD45+, CD34+ CD33-, CD14+, CD10+/CD19+) between experimental and positive control groups (P > 0.05, n=6). The number of cells in the blank control group without feeder was significantly decreased at 1 week of culture, and there was almost no cell survival at 2 weeks of culture. (3) In summary, CD146+ hMD-PCs, like human bone marrow mesenchymal stem cells, have hematopoietic support capacity in vitro.

Recombinant human bone morphogenetic protein-2 combined with autologous bone grafting and fusion in the treatment of thoracolumbar tuberculosis / 中国组织工程研究

BACKGROUND: The efficacy and safety of recombinant human bone morphogenetic protein-2 (rhBMP-2) combined with autologous bone grafting for the treatment of spinal degenerative diseases such as lumbar spondylolisthesis, spinal canal stenosis and intervertebral disc herniation have been recognized, but few clinical studies have been conducted on the efficacy and safety in the treatment of spinal infectious diseases such as spinal tuberculosis. OBJECTIVE: To evaluate the clinical efficacy and safety of rhBMP-2 combined with autologous bone grafting for spinal tuberculosis. METHODS: Clinical data of thoracolumbar tuberculosis admitted in the First Affiliated Hospital of Guangzhou University of Chinese Medicine from November 2010 to May 2018 were retrospectively analyzed. All patients underwent posterior pedicle screw fixation plus bone graft for spinal fusion, with (experimental group) or without (control group) the use of rhBMP-2. In the experimental group, 33 patients were treated with posterior pedicle fixation and autologous bone graft for spinal fusion combined with rhBMP-2 (1 mg). In the control group, 35 patients underwent posterior pedicle fixation and autologous bone graft. Visual analogue scale, the American Spinal Injury Association (ASIA), perioperative complications and fusion rate were statistically analyzed. The study protocol was approved by the Ethics Committee of the First Affiliated Hospital, Guangzhou University of Chinese Medicine. Informed consent was obtained from each patient. RESULTS AND CONCLUSION: All patients were followed up for more than 1 year. During the follow-up period, no fracture or movement of the internal fixation or distinct collapse of the vertebral body were found. There was no significant difference between the two groups in terms of operative time, intraoperative blood loss, length of stay, and proportion of perioperative complications (P > 0.05). There was a significant improvement in visual analogue scale scores and ASIA grades in the two groups at 1 week and 1 year after operation (P 0.05). The fusion rate in the experimental group was significantly higher than that in the control group at 6 months after operation (P 0.05). These findings indicate that rhBMP-2 combined with autologous bone for the treatment of thoracolumbar tuberculosis can accelerate bone fusion with favorable efficacy and safety in a short time.

In vivo study on the repairment of distal femur defects in rabbit with nano-pearl powder bone substitute / 中南大学学报(医学版)

OBJECTIVES@#To evaluate the repairing ability of nano-pearl powder bone substitute in rabbit with defect of distal femur bone.@*METHODS@#Thirty-two New Zealand rabbits were randomly divided into four groups: a nano-pearl powder/recombinant human bone morphogenetic protein 2 (rhBMP-2)/hyaluronic acid group, a nano-pearl powder/hyaluronic acid group, a nano-pearl powder group and a blank control group (=8 in each group). A defect with the diameter of 7 mm and height of 10 mm was prepared at the distal femoral metaphysis line of the rabbit.Different bone substitutes were planted, and the effect of repair was evaluated by macroscopic observation, imaging examination, and histopathological examination.@*RESULTS@#The results of imageology showed that: the bone repairing effect in the nano-pearl powder/rhBMP-2/hyaluronic acid group was better than that in the pure pearl powder group and the nano-pearl powder/hyaluronic acid group, and which in the 3 experimental groups was better than that in the blank control group; The results of histology showed that: at the 4th, 8th and 12th weeks after the modeling operation, the speed of bone repair in the nano-pearl powder/rhBMP-2/hyaluronic acid group was faster than that in the pure pearl powder group and the nano-pearl powder/hyaluronic acid group, and which in the blank control group was far slower than that in the 3 experimental groups. The results of immunohistochemistry staining for osteocalcin antibody showed that: the osteogenic effect in the nano-pearl powder/rhBMP-2/hyaluronic acid group was better than that in the pure pearl powder group and the nano-pearl powder/hyaluronic acid group (both 0.05); however, there was significant difference between the pure pearl powder group and the blank control group (0.05), but the osteogenic effect in the nano-pearl powder/hyaluronic acid group was better than that in the pure pearl powder group and the blank control group (both <0.05).@*CONCLUSIONS@#Nano-pearl powder and its bone substitute can promote the repair of bone defect, and the nano-pearl powder which contains rhBMP-2 has better osteogenic and repairing effect on defect.

Biological characteristics of human bone marrow mesenchymal stem cells in the microenvironment of rabbit bladder acellular matrix graft / 西安交通大学学报(医学版)

Objective: To explore the effects of the microenvironment of rabbit bladder acellular matrix graft (BAMG) on proliferation, cell surface markers, and molecular protein level of human bone marrow mesenchymal stem cells (hBMSCs). Methods: We prepared BAMG immersion fluid medium and detected its effect on the proliferation of hBMSCs by MTT method. The expressions of CD44, CD45, CD73 and PDGFRβ were detected by flow cytometry. The expressions of PPAR, OCN and α-SMA were detected by RT-PCR, and the expression of OCT was detected by Western blot. Results: hBMSCs had good compatibility with BAMG. The MTT method showed that BAMG and BAMG immersion medium did not affect the proliferation capacity of hBMSCs. The surface of hBMSCs cells cultured with immersion fluid still expressed CD44, CD73 and PDGFRβ, but not CD45. RT-PCR showed that OCN, PPAR, and α-SMA were all expressed. Western blot test also showed the positive expression of OCT-4. Conclusion: hBMSCs can still keep their original biological characteristics in the microenvironment of rabbit BAMG. It can be the seed cells and combined substrate materials for urinary system tissue engineering.

Inhibitory Effect of Lnc-1708 on Osteogenic Differentiation of Human Bone Marrow Mesenchymal Stem Cells in vitro / 中山大学学报(医学科学版)

@#【Abstract】 【Objective】To investigate the differential expression of long non-coding RNA-1708(lncRNA-1708)in osteogenic differentiation of human bone marrow mesenchymal stem cells(hBMSC)and its effect on osteogenic differentiation of hBMSC.【Methods】Purchased 3 groups of hBMSC from different sources and cultured in vitro. qRT-PCR was used to detect the expression of lncRNA-1708 after osteogenic differentiation of three groups of hBMSC,and the relationship between lncRNA-1708 and hBMSC osteogenic differentiation was analyzed. LncRNA-1708 overexpressing lentivirus and lncRNA-1708 interferenced plasmid were transfected respectively,to obtain stable hBMSC cell line. After 14-day osteogenic differentiation on transfected hBMSC,RT-PCR was used to detect the expressions of runt-related transcription factor 2(RUNX2)and alkaline phosphatase(ALP)mRNA,and alkaline phosphatase staining was made.【Results】The expression of lnc-1708 decreased after osteogenic differentiation of hBMSC for 7 d(P < 0. 001).Two cell lines,which respectively express high lncRNA-1708 and low lncRNA-1708,were constructed successfully. In lnc-1708-overexpressed BMSC,the mRNA levels of osteogenic markers RUNX2 and ALP were both significantly down-regulated(0.46±0.03 vs.1.00±0.02,0.15±0.07 vs. 1.02±0.28,P < 0.01). On the contrary,in the lnc-1708-silencing BMSC,the expressions of RUNX2 and ALP mRNA level were significantly up-regulated(1.62±0.18 vs. 1.00±0.04,1.58±0.11 vs. 1.01±0.18,P < 0.01).【Conclusion】LncRNA-1708 may have an inhibitory effect on osteogenic differentiation of hBMSC.

The role of mir-124-3p in inhibiting osteogenic differentiation of bone marrow mesenchymal stem cell and reducing bone quality in senile osteoporosis / 中华内分泌代谢杂志

Objective To investigate different expression levels between young and old bone marrow mesenchymal stem cells in microRNAs (miRNAs) that are significantly conserved between humans and mice.Additional studies have been conducted to discover changes in miRNA expression in old mice relative to that in young adults and discussed the roles of miRNAs in primary osteoporosis.Methods MiRNAs that are highly conserved between human and mice,and are expressed at significantly different levels in the bone marrow mesenchymal stem cells of young and old people were identified by searching the Gene Expression Omnibus (GEO) database.Human bone mesenchymal stem cells (hBMSCs) were transfected with miRNA mimics,and their relative alkaline phosphatase (ALP) activity levels were then determined.Micro-CT scanning was employed to quantitatively characterize cortical and cancellous bones of young and old mice,and to confirm that these mice accurately modeled natural aging osteoporosis.Simultaneously,we investigated differences in expression levels of miRNAs that influence ALP activity in hBMSCs in the two groups of mice.Correlations between miRNA expression levels,and parameters of bone mass and bone strength were studied.Results 28 miRNAs were found to be more than 2 fold up-regulated (down-regulated) with statistical significance (P＜0.05) in the GEO database.We also found that ALP activity was lower in hBMSCs transfected with 4 miRNAs (mir-124-3p,mir-126-3p,mir-128-3p,mir-424-5p,P＜0.05 or P＜ 0.01).The micro-CT scans indicated that the mice are accurately modeled natural aging osteoporosis.Expression of mir-124-3p increased significantly in older mice.This upregulation correlated positively with trabecular separation,and negatively with trabecular pattern factor in trabecular bone.However,in cortical bone,its expression correlated positively with trabecular separation,and negatively with bone volume fraction,trabecular number,and bone mineral density (P＜ 0.05).Conclusion Hsa-mir-124-3p,which is expressed differently in young and old bone marrow stromal cells,inhibited the osteogenic differentiation of hBMSCs.Upregulation of this miRNA in the bone tissue of aged mice may be related to the development of osteoporosis.

Effect of rhBMP-2 and Chitosan in Differentiation of Periodontal Ligament Stem Cells into an Osteoblastic Lineage

Aims: To analyze the effect of rhBMP-2 and Chitosan in differentiation of Periodontal Ligament Stem Cells (PDLC) into an osteoblastic lineage. Study Design: This study was designed as in vitro study and osteogenic biomarkers were determined in the culture supernatant. Place and Duration of Study: Laboratory of Oral Biology Faculty of Dentistry Universitas Indonesia. Jakarta 10430 Indonesia, January – September 2016. Methodology: Human periodontal ligament stem cells (PDLC) were isolated from the root of vital teeth, followed by identification of stem cells by antibody anti  STRO-1. Chitosan was used at the concentration of 0.15%.  The culture cells were divided into four groups as follow, the control group (PDLC) and treatment groups with recombinant human Bone Morphogenic protein 2 (rhBMP-2), the combination chitosan-rhBMP-2 and chitosan only. The levels of alkaline phosphatase (ALP) was determined by colorimetry and osteocalcin and collagen type I were measured using ELISA. Results: The results showed that levels of ALP tended to increase is in all groups. At day 14, the highest levels of ALP was in chitosan treated group. The concentration of collagen type 1 managed to raise is in all groups on days 14, and the highest levels Collagen type 1 occurred in RH BMP-2 and chitosan treated cells, after that decrease in all groups until day 21(p < 0.05).  Osteocalcin concentration tended to increase is in all groups, and at days 21, the highest levels in with rhBMP-2 + chitosan.   Conclusion: The rhBMP-2, chitosan, and its combination induce differentiation of periodontal ligament stem cells into the osteoblastic lineage.

Does Recombinant Human Bone Morphogenic Protein 2 Affect Perioperative Blood Loss after Lumbar and Thoracic Spinal Fusion?

STUDY DESIGN: Retrospective cohort design. PURPOSE: This study aimed to determine whether recombinant human bone morphogenic protein 2 (rhBMP-2) reduces total perioperative blood loss during lumbar and thoracic fusion. OVERVIEW OF LITERATURE: Previous studies on rhBMP-2 versus iliac crest bone grafting in thoracic and lumbar fusions have yielded mixed results regarding reductions in blood loss and have largely neglected the postoperative period when analyzing total blood loss. Additionally, these studies have been limited by heterogeneity and sample size. METHODS: We analyzed the blood loss patterns of 617 consecutive adult patients undergoing lumbar and/or thoracic fusions requiring subfascial drain placement at a single institution from January 2009 to December 2016. Patients were divided into BMP and non-BMP cohorts, and a propensity score analysis was conducted to account for the differences between cohorts. RESULTS: At a per-level fused basis, the BMP group exhibited a significant reduction in the intraoperative (66.1 mL per-level fused basis; 95% confidence interval [CI], 127.9 to 4.25 mL; p=0.036) and total perioperative blood loss (100.7 mL per-level fused basis; 95% CI, 200.9 to 0.5 mL; p=0.049). However, no significant differences were observed in an analysis when not controlling for the number of levels or when examining the postoperative drain output. CONCLUSION: RhBMP-2 appears to reduce both intraoperative and total blood loss during lumbar and thoracic fusions on a per-level fused basis. This total reduction in blood loss was achieved via intraoperative effects because RhBMP-2 had no significant effect on the postoperative drain output.

Small peptide KR-12-a5 promotes the osteogenic differentiation of human bone marrow mesenchymalstem cells / 上海交通大学学报（医学版）

Objective: To investigate the effect of KR-12 analog, KR-12-a5, on osteogenic differentiation of human bone marrow mesenchymal stem cells (HBMSCs). Methods: Osteogenic differentiation-related experiments were performed using HBMSCs. KR-12-a5 was used as additional stimulation under osteogenesis inducing environment. By alkaline phosphatase (ALP) staining assay, ALP quantitative assay, alizarin red staining and elution quantitative analysis, and real-time quantitative polymerase chain reaction (RT-qPCR), the effect of KR-12-a5 on osteogenic differentiation of HBMSCs was verified. Results: As the concentration of KR-12-a5 increased, the staining intensity of ALP and alizarin red also increased. The strongest staining effect was exhibited at the highest concentration (50 μg/mL) of KR-12-a5. Quantitative tests showed similar results. After 3 days of stimulation with KR-12-a5, the mRNA level of RUNX2 increased in a dose-dependent manner. On the 7th day, the expression levels of ALP, COL1A1, BSP and BMP2 in the KR-12-a5-treated group showed significant changes. On the 14th day, OSX, OCN and OPN levels increased significantly. Conclusion: KR-12-a5 promoted the osteogenic differentiation of HBMSCs in a dose-dependent manner.

CCL3 promotes hBMSC proliferation and inhibits exosome secretion / 中国病理生理杂志

AIM:To explore the regulatory effect of chemokine CCL 3 on exosome secretion from human bone marrow mesenchymal stem cells(hBMSCs).METHODS: hBMSCs were stimulated with chemokine CCL 3 at different concentrations in vitro.The proliferation of hBMSCs was measured by CCK-8 assay and viable cell counting.Exosome se-cretion from hBMSCs was qualitatively analyzed by transmission electron microscope(TEM)and flow cytometry, and the quantitative analysis was carried out by flow cytometry and nanoparticle tracking analysis(NTA).RESULTS:Compared with control group,the viability of the hBMSCs detected by CCK-8 assay was increased when hBMSCs were treated with CCL3(P<0.05).The results of viable cell counting demonstrated that the number of hBMSCs was raised in CCL 3 group in a dose-dependent manner(P<0.05).The results of flow cytometry showed that hBMSCs expressed 3 CCL3-related spe-cific receptors,CCR1,CCR5 and CCR9.Compared with control group,the fluorescence intensity of CCR9 in CCL3 group was obviously enhanced.However,no significant difference of fluorescence intensity for CCR 5 and CCR1 was observed be-tween the 2 groups.The results of NTA demonstrated that the secretion capacity of CCL 3-induced hBMSCs was far less than that in control group(P<0.05).However, the microvesicles larger than 100 nm in CCL3 groups were increased(P<0.05).The above results indicated that the higher concentration of CCL 3 induced the lower secretion of exosomes.In addi-tion,the results of flow cytometry demonstrated that CCL 3-induced hBMSCs showed lower quantity of CD 9 +exosomes than those in control group(P<0.01).CONCLUSION:CCL3 promotes the proliferation of hBMSCs but depresses the secre-tion of exosomes in a dose-dependent manner.CCL3 affects the size distribution of exosomes and increases the number of nonfunctional microvesicles of larger than 100 nm in size.CCL3 induces the expression of CCR9 in hBMSCs.

Angiogenic ability of 3 different tissues-derived mesenchymal stem cells on endothelial progenitor cells / 中南大学学报(医学版)

Objective:To compare the ability between bone marrow-derived mesenchymal stem cells (MSCs) (BM-MSCs) and adipose-derived MSCs (AD-MSCs) or umbilical cord-derived MSCs (UC-MSCs) on promotion of vessels formation and vessds stabilization relevant to the functions of EPCs.Methods:In vitro,co-culture blood vessel test was performed to compare the angiogenic ability between BM-MSCs,AD-MSCs or UC-MSCs.In vivo,angiogenic assay dependent on basement membrane matrix Matrigel and immunohistochemistry were performed to compare the ability of vessels formation functions between BM-MSCs and AD-MSCs or UC-MSCs.Results:The lengths and dots of vascular structures formed by EPCs on AD-MSCs layer are greater than those by EPCs on BM-MSCs layer and UC-MSCs layer in angiogenic assay in vitro.The stability of the capillary-like structures formed by EPCs with AD-MSCs on Matrigel was more stable than that by the BM-MSCs,UC-MSCs or EPCs.AD-MSCs and EPCs could form abundant functional vessels with blood perfusion in Matrigelin vivo;UC-MSCs and EPCs could form a few functional vessels with blood perfusion in Matrigelin vivo;BM-MSCs and EPCs could form broken vessels with hemocytes leakage in Matrigel in vivo.Conclusion:AD-MSCs have the stronger ability to promote the angiogenesis and stabilize the vessels compared with BM-MSCs or UC-MSCs ex vivo and in vivo.

Effect of dasatinib on biological properties of human bone marrow mesenchymal stem cells / 中国病理生理杂志

AIM:To explore the effect of dasatinib on the viability, migration, cell cycle and apoptosis of human bone marrow mesenchymal stem cells (hBMSCs), as well as the underlying signal pathway to evaluate the influence of dasatinib on hematopoietic microenvironment clinically.METHODS:The cell viability was measured by CCK-8 assay.The migration ability was detected by wound healing assay.The cell cycle and apoptosis were analyzed by flow cytometry.Acridine orange/ethidium bromide staining was also used to detected apoptosis.The secretion of transforming growth factor-β1 (TGF-β1) and tumor necrosis factor-α (TNF-α) were measured by ELISA.The protein levels of cleaved caspase-3, protein kinase B (Akt) and phosphorylated Akt were determined by Western blot.RESULTS:Compared with control group, dasatinib at 1~10 nmol/L suppressed the viability and migration ability of hBMSCs, and dasatinib at concentration of 7 nmol/L was adopted in the following assays.Dasatinib promoted apoptosis, and blocked the cell cycle in G1 phase.In addition, the secretion of TGF-β1 and TNF-α was increased markedly.The protein levels of cleaved caspase-3 was increased, but the protein levels of Akt and phosphorylated Akt were decreased.CONCLUSION:Dasatinib inhibits the viability and migration ability of hBMSCs in a dose-dependent manner, promotes the secretion TGF-β1 and TNF-α, and induces cell cycle arrest and apoptosis.Dasatinib might regulate the biological behaviors of hBMSCs observed above by modulating the expression and phosphorylation of Akt.