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Expression of bone morphogenetic protein and its receptors in different stages of hypertrophic scar

Xin HUANG ; Haiming WEI ; Minhua LIANG ; Hui MAI ; Gefei ZHU ; Xiaopin TENG

Chinese Journal of Tissue Engineering Research.2013;(50):8691-8696. doi:10.3969/j.issn.2095-4344.2013.50.012

BACKGROUND:Bone morphogenetic proteins are associated with the formation and development of scars. Fibroblasts are closely related to the proliferation and maturation of scars. However, the expression of bone morphogenetic proteins in fibroblasts in different stage of hypertrophic scars remains poorly understood.
OBJECTIVE:To observe the expression of bone morphogenetic protein 2/4, bone morphogenetic protein 7, bone morphogenetic protein receptor IA in fibroblasts in different stage of hypertrophic scars.
METHODS:The immunohistochemical SP method was employed to detect the expression of bone morphogenetic protein 2/4, bone morphogenetic protein 7, bone morphogenetic protein receptor IA in fibroblasts in 20 cases of proliferative stage hypertrophic scar and 20 cases of maturation stage hypertrophic scar. Al samples were obtained from the Department of Plastic and Aesthetic Surgery, the People’s Hospital of Guangxi Zhuang Autonomous Region, China.
RESULTS AND CONCLUSION:The expression of bone morphogenetic protein 2/4 in fibroblasts in proliferative hypertrophic scars significantly elevated compared with mature hypertrophic scars (P<0.05). In various stages of hypertrophic scars, the expression of bone morphogenetic protein 7 and bone morphogenetic protein receptor IA in fibroblasts was not obviously different (P>0.05). Results demonstrated that bone morphogenetic protein 2/4 expression down-regulated during the development from proliferative stage to maturation stages in fibroblasts in hypertrophic scars. However, there was no change in the expression of bone morphogenetic protein 7 and bone morphogenetic protein receptor IA.

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Culture of rabbit’s articular chondrocytes using type Ⅱ collagenase enzyme digestion method

Hu YAN ; Youxin SU ; Xueyi LIN ; Baojun CHEN ; Bihong ZHOU ; Qing ZHANG

Chinese Journal of Tissue Engineering Research.2013;(50):8647-8653. doi:10.3969/j.issn.2095-4344.2013.50.005

BACKGROUND:At present, the separation and culture technique of chondrocytes has been mature, but the chondrocytes grow slowly which are prone to degenerate using the present technique. It is not conducive to the fol ow-up test.
OBJECTIVE:To investigate and improve the separation and culture method of articular chondrocytes of New Zealand rats at 4 weeks of age.
METHODS:New Zealand rats aged 4 weeks were selected to take cartilage tissues from the bilateral knees that were resected under aseptic condition. Chondrocytes were isolated by type Ⅱ col agenase enzyme digestion and mechanical isolation method. The cells were cultured and passaged, and then identified by morphologic observation, toluidine blue staining and type Ⅱ col agen enzyme immunohistochemical methods. Growth curve was pictured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide method.
RESULTS AND CONCLUSION:Inverted microscope observation showed that the primary cultured chondrocytes adhered at 6 hours after cultivation. The monolayer formation occurred at 72 hours after cultivation, and the cells were ready to be passaged at 96 hours after cultivation. In the fourth generation, some cells represented a spindle-like appearance. In the fifth generation, most cells turned into irregular shape appearance, and cellproliferation capacity diminished. Toluidine blue staining showed that the nuclei of cultured chondrocytes were blue and cytoplasm was pale blue. Immunofluorescent staining showed that cultured chondrocytes had a positive expression of col agen type Ⅱ and the color was tawny. Proliferative rate of chondrocytes in the first to third generations had no differences (P<0.05), while differences were found compared with the fourth generation in 4-7 days (P<0.05) and the fifth generation in 1-7 days (P<0.05). The results indicate that type Ⅱ col agenase enzyme digestion and mechanical isolation method is successful for isolating, cultivating New Zealand rat articular chondrocytes in vitro, and the first to third generations can be the best choice for the experiments of knee osteoarthritis.

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Secondary fracture risk assessment:Bone mineral density and bone metabolism of elderly women within 6-12 months after hip fractures

Zijun ZHANG ; Wen ZHAO ; Xi ZHAO ; Haizhou PENG

Chinese Journal of Tissue Engineering Research.2013;(50):8635-8640. doi:10.3969/j.issn.2095-4344.2013.50.003

BACKGROUND: Prospective studies concerning bone metabolism and bone mineral density variation after fractures have been reported from the 1960s, but these studies are mainly focused on tibia and fibula and ankle fractures in patients with low sample size.
OBJECTIVE:To observe the changes in bone mineral density and bone metabolism indexes in elderly women within 6 to 12 months after hip fractures, and to analyze the correlation.
METHODS:We selected 48 elderly women with hip fractures admitted in the Department of Orthopedics, Beijing Aerospace General Hospital in China from May 2011 to July 2013. Standards for fol ow-up were developed to measure the bone mineral density and bone metabolism indexes in the L 1-4 spinal segments and both sides of the hip. The bone metabolism indexes included bone alkaline phosphatase, osteocalcin, cross-linked C-terminal peptide of col agen I, and serum tartrate-resistant acid phosphatase 5b levels. Multiple linear regression analysis was performed based on measurements of bone mineral density and bone metabolism indexes after fracture healing.
RESULTS AND CONCLUSION:After fracture healing, bone mineral density of the fractured hip and lumbar vertebra was significantly lower than the baseline value. There was no statistical difference in bone mineral density between the healthy hip and the baseline value. At 6 months after fractures, bone alkaline phosphatase, osteocalcin, cross-linked C-terminal peptide of col agen I, and serum tartrate-resistant acid phosphatase 5b levels were significantly higher than the baseline values. At 12 months after fractures, osteocalcin level was significantly higher than the baseline value, while other indexes of bone metabolism measurements showed no statistical difference from the baseline values. When healing of hip fractures met the clinical and radiographic standards, the partial regression coefficient of delta-Z score reached peak in the changes of serum osteocalcin and bone mineral density of the fractured hip. Under clinical healing of fractures, serum osteocalcin level exhibits a higher value for the assessment of recovery speed of bone mineral density. Monitoring corresponding bone metabolism indexes after fracture healing can improve the accuracy of judging bone mineral density changes to reduce the risk of secondary fractures.

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Neurogenin2 gene-regulated Schwann cells differentiate into neurons

Xinlong XU ; Qingsong XIE ; Hongsong PAN ; Xiaojie WEI ; Zaifeng CHEN

Chinese Journal of Tissue Engineering Research.2013;(49):8590-8595. doi:10.3969/j.issn.2095-4344.2013.49.020

BACKGROUND:It is confirmed that astrocytes can differentiate into neurons by Neurogenin2 gene regulation, suggesting that Schwann cells may also differentiate into neurons by gene regulation.
OBJECTIVE:To evaluate the feasibility of Schwann cells differentiating into neurons by Neurogenin2 gene regulation.
METHODS:Rats Schwann cells were isolated, purified and identified. Then the Schwann cells were transfected with Neurogenin2 via green fluorescent protein gene-plentivirus. To induce neuronal differentiation, the Schwann cells were cultured in serum-free Dulbecco’s modified Eagle’s medium containing epidermal growth factor, basic fibroblast growth factor and brain-derived neurotrophic factor for 2 weeks. The morphology of induced cells was observed by microscope, and myelin basic protein and neuron-specific enolase were detected by immunocytochemistry.
RESULTS AND CONCLUSION:After transfection with Neurogenin2 via green fluorescent protein gene-plentivirus and induced differentiation, immunofluorescence assay demonstrated that 12.56%of the induced cellexpressed neuron-specific enolase, but the control group did not express neuron-specific enolase. Neurogenin2 gene-transfected Schwann cells can express neuron-specific enolase, suggesting Neurogenin2 gene may regulate transdifferentiation of Schwann cells into neurons.

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Transforming growth factor-beta 3 induces differentiation of adipose-derived mesenchymal stem cells into fibrochondrocytes

Shengnan LEI ; Xing FAN

Chinese Journal of Tissue Engineering Research.2013;(49):8570-8575. doi:10.3969/j.issn.2095-4344.2013.49.017

BACKGROUND:The repair of temporomandibular joint disc is stil a great clinical chal enge. Pluripotent adipose-derived mesenchymal stem cells bring a hope for fibrocartilage tissue repair. Up to now, there are few studies concerning transforming growth factor-β3 to induce differentiation of adipose-derived mesenchymal stem cells into fibrochondrocytes.
OBJECTIVE:To observe the growth and morphology of adipose-derived mesenchymal stem cells under the induction of transforming growth factor-β3.
METHODS:We observed the morphology of fibrochondrocytes differentiated from adipose-derived mesenchymal stem cells under the induction of transforming growth factor-β3. Histological and immunofluorescence staining were employed to detect the expression of extracellular matrix Ⅰ, Ⅱ col agens and proteoglycansthe produced by adipose-derived mesenchymal stem cells. We also evaluated the feasibility of adipose-derived mesenchymal stem cells as seed cells for fibrocartilage tissue engineering.
RESULTS AND CONCLUSION:Under the inverted fluorescence microscope, induced cells showed obvious aggregation growth, polygonal shape, and increased secretion of extracellular matrix. Alcian blue staining results showed that under the induction of transforming growth factor-β3, adipose-derived mesenchymal stem cells exhibited the distinctly deep blue, indicating a large number of glycosaminoglycan was synthesized. Immunostaining results showed that under the induction of transforming growth factorβ3, adipose-derived mesenchymal stem cells synthesized extracellular matrix Ⅰ, Ⅱ col agen. These findings suggest that transforming growth factorβ3 can induce adipose-derived mesenchymal stem cells to differentiate into fibrouscartilage-like cells, and that adipose-derived mesenchymal stem cells can serve as seed cells for fibrocartilage tissue engineering.

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Sequential cytokine induced killer cells therapy for acute myeloid leukemia after autologous peripheral blood stem cell transplantation

Dingming WAN ; Li LI ; Xinsheng XIE ; Ling SUN ; Hui SUN ; Zhongxing JIANG ; Yi ZHANG ; Weijie CAO ; Zhilei BIAN ; Xuefang ZHOU

Chinese Journal of Tissue Engineering Research.2013;(49):8551-8556. doi:10.3969/j.issn.2095-4344.2013.49.014

BACKGROUND:Cytokine induced kil er cells therapy as an effective means of adoptive immunotherapy, becomes a new way to treat acute myeloid leukemia. But, the researches about sequential cytokine induced kil er cells therapy after autologous peripheral blood stem celltransplantation in acute myeloid leukemia patients are stil less, which deserve further research.
OBJECTIVE:To observe the clinical efficiency and safety of sequential cytokine induced kil er cells therapy after autologous peripheral blood stem celltransplantation in acute myeloid leukemia M2 patients.
METHODS:Total y 45 patients with low-or intermediate-risk acute myeloid leukemia M2 were recruited in this study. Among them, 19 patients received sequential cytokine induced kil er cells therapy after autologous peripheral blood stem celltransplantation and 26 patients only received autologous peripheral blood stem celltransplantation. The relapse rate, disease-free survival, and overal survival were compared between two groups, and safety of cytokine induced kil er cells therapy was observed.
RESULTS AND CONCLUSION:(1) Compared with the patients only receiving autologous peripheral blood stem celltransplantation, the relapse rate was lower (21.05%vs. 38.46%;P<0.05), and elevated percentages of the disease-free survival and overal survival were observed in the patients receiving sequential cytokine induced kil er cells therapy after autologous peripheral blood stem celltransplantation (P<0.05). (2) The 19 patients who received sequential cytokine induced kil er cells therapy after autologous peripheral blood stem celltransplantation al completed the treatment scheme successful y. Only four patients appeared to have chil s and fever, and no more side effects were observed. These findings suggested that the sequential cytokine induced kil er cells therapy after autologous peripheral blood stem celltransplantation can improve the disease-free survival and overal survival of low-or intermediate-risk acute myeloid leukemia M2 patients without remarkable side effects, which is a safe, effective and feasible way for the treatment of acute myeloid leukemia M2.

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Human placenta-derived mesenchymal stem cell transplantation promotes tendon graft healing in a bone tunnel

Lifeng YANG ; Wei LIU ; Ye ZHOU ; Zishan FENG ; Li ZHANG ; Wei ZHAO ; Zhi LI

Chinese Journal of Tissue Engineering Research.2013;(49):8539-8544. doi:10.3969/j.issn.2095-4344.2013.49.012

BACKGROUND:Studies have shown that human placenta-derived mesenchymal stem cells with strong proliferative ability have rich sources and can remarkably promote tendon-bone healing after celltransplantation.
OBJECTIVE:To observe the effect of human placenta-derived mesenchymal stem cells on tendon-bone healing in a bone tunnel.
METHODS:Human placenta-derived mesenchymal stem cells were separated using adherent separation screening method. Thirty 8-week-old male Sprague-Dawley rats were randomly divided into two groups, 15 rats as experimental group and 15 rats as control group. Experimental group were subjected to transplantation of human placenta-derived mesenchymal stem cells and the control group were injected with saline solution.
RESULTS AND CONCLUSION:Stem cells, new vessels, and fibrocartilage hyperplasia were observed on the tendon-bone interface with microscope at 2, 4 and 6 weeks after celltransplantation in the experimental group. Biomechanical y, the maximum pul out load in the experimental group was significantly higher than that in the control group at 4 and 6 weeks after celltransplantation (P<0.05). These findings suggest that human placenta-derived mesenchymal stem cells can accelerate early tendon-bone healing in a bone tunnel and strengthen the biomechanical strength.

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Intraperitoneal transplantation of human umbilical cord mesenchymal stem cells in the treatment of acute liver injury in rats

Xuefeng ZHENG ; Xia LIU

Chinese Journal of Tissue Engineering Research.2013;(49):8527-8531. doi:10.3969/j.issn.2095-4344.2013.49.010

BACKGROUND:In vitro experiments have confirmed that human umbilical cord mesenchymal stem cells can be induced to differentiate into hepatocyte-like cells, thus which can be considered to function as liver repair.
OBJECTIVE:To observe the therapeutic effect of human umbilical cord mesenchymal stem celltransplantation on acute liver injuries in rats through in vivo animal experiments.
METHODS:Healthy Sprague-Dawley rats were randomly divided into three groups:normal control group without modeling, celltransplantation group, and PBS group. Rat models of acute liver injury were prepared by 10%CCl4-olive oil solution in the celltransplantation and PBS groups which were fol owed by intraperitoneal injection of 0.5 mL human umbilical cord mesenchymal stem cellsuspension and 0.5 mL PBS, respectively.
RESULTS AND CONCLUSION:Hematoxylin-eosin staining showed that pathological changes related to acute liver injury appeared at 24 hours after intraperitoneal injection of CCl 4 . Then, the liver structure recovered at 7 days after celltransplantation, but it did not recover til the 14th day after PBS injection. Compared with the normal control group, serum alanine aminotransferase and aspartate aminotransferase levels were significantly increased in the other two groups (P<0.05-0.01). In the celltransplantation group, the serum alanine aminotransferase and aspartate aminotransferase levels were significantly lower than those in the PBS group at 3 days after treatment (P<0.05-0.01), and recovered normal y after 7 days. cells positive for anti-human nucleoprotein antibody were found in the portal area of liver tissues in the celltransplantation group after 3 days of transplantation, and then cells positive for anti-human albumin antibody appeared after 7 days. These findings indicate that intraperitoneal transplantation of human umbilical cord mesenchymal stem cells can improve liver function and repair injured liver tissues after acute liver injury in rats to some extent.

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Improved platelet-rich plasma effects on the proliferation and immunogenicity of human bone marrow mesenchymal stem cells

Sihan LI ; Jianmin DUAN ; Hongtao LI ; Jun WEN

Chinese Journal of Tissue Engineering Research.2013;(49):8505-8511. doi:10.3969/j.issn.2095-4344.2013.49.007

BACKGROUND:Previous experiments have shown that improved platelet-rich plasma can promote the proliferation of human dental pulp cells in a concentration-dependent manner, particularly when the concentration is 10%.
OBJECTIVE:To investigate the effect of platelet-rich plasma at different concentrations on the proliferation and immunogenicity of human bone marrow mesenchymal stem cells.
METHODS:Human bone marrow mesenchymal stem cells from healthy donors were cultured and passaged for 3-4 passages, identified by flow cytometry and differentiation inductions. Platelet-rich plasma samples which were manufactured from the venous blood of the same donor were used for culturing human bone marrow mesenchymal stem cells. The proliferation of human bone marrow mesenchymal stem cells was measured by cellcounting kit-8 method and the growth curves were drawn. The most suitable concentration of platelet-rich plasma was selected to culture human bone marrow mesenchymal stem cells for three generations and the Stro-1 expression rate on the surface of human bone marrow mesenchymal stem cells was determined through flow cytometry.
RESULTS AND CONCLUSION:Platelet-rich plasma at the concentration of 5%-10%evidently promoted the proliferation of human bone marrow mesenchymal stem cells on the 6th and 8th days. The most effective concentration to promote the proliferation was 10%. Platelet-rich plasma at the concentration of 10%stil promoted the proliferation of human bone marrow mesenchymal stem cells on the 10th day, and maintained a better immunogenicity of human bone marrow mesenchymal stem cells compared to the control group. These findings indicate that platelet-rich plasma can promote the proliferation of human bone marrow mesenchymal stem cells in a concentration-dependent manner, and 10%platelet-rich plasma is better to maintain the immunogenicity of human bone marrow mesenchymal stem cells.

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Bone marrow-derived mesenchymal stem cells for high-metastatic potential hepatocellular carcinoma

Tianran LI ; Xiangke DU ; Bin SONG ; Zhengmao WEI ; Tianlong HUO

Chinese Journal of Tissue Engineering Research.2013;(49):8498-8504. doi:10.3969/j.issn.2095-4344.2013.49.006

BACKGROUND:Bone marrow mesenchymal stem cells have the chemotaxis and homing role that promotes immune system reconstruction, eliminate residual lesions and prevent recurrence in patients.
OBJECTIVE:To observe therapeutic effect of human bone marrow mesenchymal stem cells transplantation into high-metastatic potential hepatocellular carcinoma animal models on metastatic potential of high-metastatic potential hepatocellular carcinoma.
METHODS:Nude mouse models of high-metastatic potential hepatocellular carcinoma were established in vivo. In the experimental group, 5×105 cells were injected via the tail vein on day 7 after tumor inoculation, twice a week. In the control group, cellculture medium, 0.2 mL per mouse, was injected by the tail vein. After the start of the experiment, tumor volume was measured every 4 days. After tumor inoculation for 14 days, 21 days, 28 days, 35 days, 42 days, animal models were sacrificed, and then tumor mass and body mass were recorded to calculate the inhibition rate. PCR was employed to detect osteopontin, bone sialoprotein, and integrinα Ⅴ mRNA expression, as wel as bcl-2, bax, caspase3 mRNA expression.
RESULTS AND CONCLUSION:The inhibition rate of tumor mass showed the best results in week 3. As time went on, the tumor inhibition rate was gradual y decreased. Metastasis-related biological factors showed a gradual down-regulated trend, indicating the polarization of tumor apoptotic indexes, that is, anti-apoptotic factor, bcl-2, showed a decreasing trend, while apoptotic factors, bax and caspase3, appeared to have a gradual y increased trend. These findings suggest that human bone marrow mesenchymal stem cells effects to inhibit high-metastatic potential hepatocellular carcinoma animal models appear to vary with time. After human bone marrow mesenchymal stem cells transplantation for 3 weeks, the inhibition performance on high-metastatic potential hepatocellular carcinoma is the best, and then it weakens with time. Human bone marrow mesenchymal stem cells are found to inhibit the metastatic potential of hepatocellular carcinoma.

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