BACKGROUNDDendritic cells are professional antigen-presenting cells found in an immature state in epithelia and interstitial space, where they capture antigens such as pathogens or damaged tissue. Matrix metallopeptidase 13 (MMP-13), a member of the collagenase subfamily, is involved in many different cellular processes and is expressed in murine bone marrow-derived dendritic cells (DCs). The function of MMP-13 in DCs is not well understood. Here, we investigated the effect of MMP-13 on DC maturation, apoptosis, and phagocytosis.

METHODSBone marrow-derived dendritic cells were obtained from C57BL/6 mice. One short-interfering RNA specific for MMP-13 was used to transfect DCs. MMP-13-silenced DCs and control DCs were prepared, and apoptosis was measured using real-time polymerase chain reaction and Western blotting. MMP-13-silenced DCs and control DCs were analyzed for surface expression of CD80 and CD86 and phagocytosis capability using flow cytometry.

RESULTSCompared to the control DCs, MMP-13-silenced DCs increased expression of anti-apoptosis-related genes, BAG1 (control group vs. MMP-13-silenced group: 4.08 ± 0.60 vs. 6.11 ± 0.87, P = 0.008), BCL-2 (control group vs. MMP-13-silenced group: 7.54 ± 0.76 vs. 9.54 ± 1.29, P = 0.036), and TP73 (control group vs. MMP-13-silenced group: 4.33 ± 0.29 vs. 5.60 ± 0.32, P = 0.001) and decreased apoptosis-related genes, CASP1 (control group vs. MMP-13-silenced group: 3.79 ± 0.67 vs. 2.54 ± 0.39, P = 0.019), LTBR (control group vs. MMP-13-silenced group: 9.23 ± 1.25 vs. 6.24 ± 1.15, P = 0.012), and CASP4 (control group vs. MMP-13-silenced group: 2.07 ± 0.56 vs. 0.35 ± 0.35, P = 0.002). Protein levels confirmed the same expression pattern. MMP-13-silenced groups decreased expression of CD86 on DCs; however, there was no statistical difference in CD80 surface expression. Furthermore, MMP-13-silenced groups exhibited weaker phagocytosis capability.

CONCLUSIONThese results indicate that MMP-13 inhibition dampens DC maturation, apoptosis, and phagocytosis.

Animals ; Apoptosis ; drug effects ; physiology ; Bone Marrow Cells ; cytology ; Dendritic Cells ; cytology ; drug effects ; metabolism ; Female ; Lipopolysaccharides ; pharmacology ; Matrix Metalloproteinase 13 ; metabolism ; physiology ; Mice ; Mice, Inbred C57BL ; RNA, Small Interfering

OBJECTIVETo downregulate the expression of pituitary tumor transforming gene 1 (PTTG1) in osteosarcoma (OS) cells by siRNA technology and to investigate related biological impact on cell proliferation, cell cycle and cell invasion of OS.

METHODSThree OS cell lines and osteoblast hFOB1.19 cell line were used in this study. Control siRNA and PTTG1 siRNA were employed to transfect OS U2OS cells, and PTTG1 protein level was detected by Western blot after the transfection. Effects of PTTG1 siRNA on cell proliferation, cell cycle and cell invasion were investigated by CCK-8, flow cytometry and Boyden chamber, respectively. Finally, activity of Akt and its downstream target gene expression were analyzed by Western blot in U2OS cells upon various treatments.

RESULTSExpression of PTTG1 protein in 3 OS cells (MG-63, SaOS-2 and U2OS) was significantly higher than that in osteoblast hFOB1.19, among which U2OS cells displayed the highest level. PTTG1 siRNA markedly downregulated the expression of PTTG1 protein in U2OS cells, leading to obvious inhibition of cell proliferation, altered cell cycle distribution and reduced ability of invasion of U2OS cells. Moreover, downregulation of PTTG1 reduced the expression of p-Akt (S473 and T308), MMP-2 and MMP-9 proteins, along with enhanced expression of p21 and E-cadherin proteins.

CONCLUSIONSPTTG1 may be tightly linked to the development of OS and therefore may serve as a novel target for precision therapy of OS.

Bone Neoplasms ; metabolism ; pathology ; Cadherins ; metabolism ; Cell Cycle ; drug effects ; physiology ; Cell Movement ; Cell Proliferation ; drug effects ; physiology ; Down-Regulation ; Humans ; Matrix Metalloproteinase 2 ; metabolism ; Matrix Metalloproteinase 9 ; metabolism ; Neoplasm Invasiveness ; Osteosarcoma ; metabolism ; pathology ; RNA, Small Interfering ; pharmacology ; Securin ; genetics ; metabolism ; Transfection

The matrix metalloproteinases (MMPs) play a key role in the normal physiology of connective tissue during development, morphogenesis, and wound healing. Dysregulation of their activity has been implicated in numerous diseases including encephalopathy and the process of bone loss. Thus, MMPs may play a role in the encephalopathy and post-transplantation bone disease by immunosuppressive drugs such as cyclosporine (CsA) and tacrolimus. Gelatin zymography of MMP-9 and MMP-2 was performed in the glioma cells and osteoblast after CsA or tacrolimus treatment. Glioma cells or rat osteoblast ROS17/2.8 cells were treated with CsA or tacrolimus to make final concentration from 2 to 250 µM. After incubation, gelatin zymography of MMP-9 and MMP-2 was performed. And the density for the MMP bands were measured using luminescent image analyzer system. Both MMP-9 and MMP-2 activities in the osteoblast cells were decreased depending on the concentration of CsA or tacrolimus. MMP-2 activity was increased after CsA or tacrolimus treatment in the glioma cells. However, MMP-9 activities were decreased after CsA or tacrolimus treatment in the glioma cells. These results indicate that dysregulation of MMPs in the osteoblast and in the glioma cells by immunosuppressive drugs may one of the contributing factors in post-transplantation bone disease and in the encephalopathy by tacrolimus or cyclosporine.
Animals ; Bone Diseases ; Connective Tissue ; Cyclosporine ; Gelatin ; Glioma* ; Matrix Metalloproteinases ; Morphogenesis ; Osteoblasts* ; Physiology ; Rats ; Tacrolimus ; Wound Healing

We compared the bone healing capacity of three different demineralized bone matrix (DBM) products applied using different carrier molecules (hyaluronic acid [HA] vs. carboxymethylcellulose [CMC]) or bone compositions (cortical bone vs. cortical bone and cancellous bone) in a rabbit segmental defect model. Overall, 15-mm segmental defects in the left and right radiuses were created in 36 New Zealand White rabbits and filled with HA-based demineralized cortical bone matrix (DBX), CMC-based demineralized cortical bone matrix (DB) or CMC-based demineralized cortical bone with cancellous bone (NDDB), and the wound area was evaluated at 4, 8, and 12 weeks post-implantation. DBX showed significantly lower radiopacity, bone volume fraction, and bone mineral density than DB and NDDB before implantation. However, bone healing score, bone volume fraction, bone mineral density, and residual bone area at 4, 8, and 12 weeks post-implantation revealed no significant differences in bone healing capacity. Overall, three DBM products with different carrier molecules or bone compositions showed similar bone healing capacity.
Animals ; Bone Matrix/*physiology ; Bone Transplantation ; Carboxymethylcellulose Sodium/*pharmacology ; Histology ; Hyaluronic Acid/*pharmacology ; Rabbits ; *Wound Healing ; X-Ray Microtomography ; X-Rays

BACKGROUND: Demineralized bone matrix (DBM) is used for bone healing due to its osteoinductivity, but it requires a carrier for clinical application. Here, we report the effects on the osteoinductivity of DBM by use of a poloxamer 407-based hydrogel as the carrier, compared to sterile water. METHODS: DBM-W and DBM-H represent 27 wt% of DBM with sterile water and DBM with a poloxamer 407-based hydrogel, respectively. Both of the compositions were applied to human mesenchymal stem cell (MSC) cultures, and monitored for alkaline phosphatase (ALP) staining and ALP activity. Six 10-week-old athymic nude rats were used for abdominal muscle grafting with either DBM-W or DBM-H, and were tested by plane radiography, microfocus X-ray computed tomography (CT), and decalcified histology to evaluate ectopic bone formation. RESULTS: The DBM-W group showed stronger ALP staining at 7, 14, and 21 days of treatment, and significantly higher ALP activity at 7 and 14 days of treatment, compared to the DBM-H group. Plane radiography could not confirm the radio-opaque lesions in the rat ectopic bone formulation model. However, ectopic bone formation was observed in both groups by micro-CT. Compared to the DBM-H group, the DBM-W group showed higher bone volume, percent bone volume and trabecular number, and the difference in percent bone volume was statistically significant. Decalcified histology found bony tissue with lamellation in both groups. CONCLUSIONS: Our results suggest that poloxamer 407-based hydrogel has efficacy as a DBM carrier since it shows ectopic bone formation, but its effects on the quality and quantity of osteoblastic differentiation in rat abdominal ectopic bone and MSC are considered negative.
Animals ; Bone Matrix/*physiology ; Cell Culture Techniques ; Decalcification Technique ; Excipients/*pharmacology ; Hydrogels/pharmacology ; Male ; Mesenchymal Stromal Cells/*drug effects ; Osteogenesis/*drug effects ; Poloxamer/*pharmacology ; Rats ; Rats, Nude

Osteoclast, a huge coenocytes,originates from mononuclear macrophages or monocytic series hematopoietic precursor cell, plays an important role in the progree of bone resorption. Formation and abnormal activity of osteoclast may cause osteoprosis, rheumatoid arthritis and aseptic loosening after arthroplasty. Therefore, osteoclast is the target for treating these disease. At present, a lot of study on formation of osteoclast were reported, but the study on how to identify and degradation of bone tissue is not yet reported. Bone mineral are seen as important component of identifing osteoclast, and the research suggested that bone matrix is not the essential ingredients of activiting osteoclast, petri dish covered by vitronectin also can make osteoclast occure certain form of bone resorption, vitronectin plays an significant role in activiting osteoclast. Otherwise, the research found that swallowing and secretion of bone matrix degradation products is benefit for differentiation of osteoclast and maintain of function, and this may be therapeutic target for treatment of musculoskeletal disorders.
Animals ; Bone Matrix ; metabolism ; Bone Resorption ; Humans ; Osteoclasts ; physiology

Alveolar Process ; physiology ; Animals ; Biocompatible Materials ; therapeutic use ; Bone Matrix ; transplantation ; Bone Regeneration ; physiology ; Bone Transplantation ; methods ; Dental Implantation ; methods ; Esthetics, Dental ; Guided Tissue Regeneration, Periodontal ; methods ; Humans ; Prosthodontics ; methods ; Tooth Extraction ; methods

To evaluate the biological safety of collagen-based bone repairing material, we implanted the sample or reference substance into rats, and observed relative signs, including the specific inspection targets in animals, blood examination, analysis of immune organ, the pathological examination of organs and tissues, NK cell killing activity assay, lymphocyte group analysis, serum IL-1, IL-6, TNF-alpha detection, detection of immune globulin. Meanwhile, we set control group, sham group, and immunosuppression group. The final results showed that there was no abnormal mental state before and after the experiment. Compared with the control group, the tested group indicated no significant difference in blood test, immune organ analysis, the pathological examination of organs and tissues, NK cell killing activity assay, lymphocyte subset analysis serum IL-1, IL-6, TNF-alphadetection, and detection of immune globulin. Collagen-based bone repairing material produced a slight and transient stimulation on the rats, but created no significant inflammatory responses.
Absorbable Implants ; Animals ; Bone Matrix ; physiology ; transplantation ; Bone Regeneration ; Bone Substitutes ; Collagen ; chemistry ; Implants, Experimental ; Materials Testing ; Prostheses and Implants ; Rats

Matrix metalloproteinases (MMPs) are endocellular proteolytic enzymes. They are so named because they need Ca2+, Zn2+ and other metal ions as their cofactors. MMPs play an important biological role in regulating the formation, remodeling and degradation of extracellular matrix and participate in various physiological and pathological processes of cells. Bone marrow-derived mesenchymal stem cells (BMSCs) are a kind of pluripotent stem cell which has the ability to self-renew and differentiate into functional cells. Meanwhile, they can respond to the damage signals and migrate to injured site for tissue repair and regeneration. MMPs and their inhibitors TIMPs affect the differentiation and migration of BMSCs. This article reviews the roles of MMPs in differentiation and migration of BMSCs.
Bone Marrow Cells ; cytology ; Cell Differentiation ; physiology ; Cell Movement ; physiology ; Humans ; Matrix Metalloproteinases ; physiology ; Mesenchymal Stromal Cells ; cytology

Extracellular matrix (ECM) keeps cell's shape, protects and nourishes cells; it plays a great role in cell proliferation and differentiation. Therefore, ECM is very important in cell and tissue engineering. In this study, after primary mouse osteoblasts and fibroblasts maintained at confluence in vitro were removed, their ECM coated on cell culture plate was prepared, and bone morphogenetic proteins 2 (BMP-2) was detected in the osteoblasts ECM. MC3T3-E1 preosteoblasts cells were seeded on cell culture plates covered with fibroblasts ECM and osteoblasts ECM respectively. The proliferative activity of the cells cultured on fibroblasts ECM was higher than that on osteoblasts ECM and the control group. The alkaline phosphatase activity, relative protein levels of BMP-2 and osteopontin, secreted calcium of the cells cultured on osteoblasts ECM were all the highest. The results indicate that the two different ECMs produced in vitro had different bioactivities, the fibroblasts ECM coated on cell culture plates could accelerate MC3T3-E1 cells proliferation, and the osteoblasts ECM could promote cells osteogenic differentiation.
3T3 Cells ; Alkaline Phosphatase ; metabolism ; Animals ; Animals, Newborn ; Bone Morphogenetic Protein 2 ; metabolism ; Calcium ; metabolism ; Cell Differentiation ; physiology ; Cell Proliferation ; drug effects ; Cells, Cultured ; Extracellular Matrix ; metabolism ; physiology ; Mice ; Osteoblasts ; cytology ; Osteopontin ; metabolism