OBJECTIVETo investigate the capacity of human endometrial stromal stem cells for differentiation into osteoblasts and chondroblasts and their potential as seeding cells in bone tissue engineering.

METHODSHuman endometrial stromal stem cells were obtained from hysterectomy tissues from 15 women during normal menstrual cycles and induced to differentiate into osteoblasts and chondroblasts. The differentiated cells were examined with cytochemistry.

RESULTSA population of endometrial stromal stem cells was successfully isolated from human endometrial tissue and showed stable proliferation in vitro. After treatment with osteoblast and chondroblast revulsant, the endometrial stromal stem cells differentiated towards osteoblasts were verified by positive staining with alizarin red and towards chondroblasts by positive staining with Alcian blue.

CONCLUSIONEndometrial stromal stem cells obtained from human endometrial tissue with multilineage potential can differentiate into osteoblasts and chondroblasts in vitro, and may serve as candidate autogenous seeding cells for bone tissue engineering.

Adult ; Cell Differentiation ; Cells, Cultured ; Chondrocytes ; cytology ; Endometrium ; cytology ; Female ; Humans ; Middle Aged ; Osteoblasts ; cytology ; Stromal Cells ; cytology

This study was purposed to investigate the influence of inflammatory microenvironment on mesenchymal stem cells (MSCs) and regulatory effect of MSCs on osteoblast formation. The MSCs were isolated from synovial fluid of patients with rheumatoid arthritis (RASF-MSCs) and were cultured, the immunotypes of RASF-MSCs were detected by flow cytometry, the ability to differentiate RASF-MSCs into osteoblasts and adipocytes was determined by means of osteogenic and adipogenic induction, the regulatory effect of RASF-MSCs on osteoblast formation was assayed by co-culturing RASF-MSCs whth CD14(+) monocytes and in situ tartrate-resistant acid phosphatase staining. The results showed that RASF-MSCs highly expressed CD105, CD73, CD29, CD44, CD166 and HLA-ABC. Meanwhile, they lowly expressed CD34, CD45, CD31, HLA-DR, CD80 and CD86. However, RASF-MSCs decreased multi-differentiation capability as compared with BM-MSCs. More interestingly, RASF-MSC significantly promoted osteoclasts formation (p < 0.05) when co-cultured with monocytes. It is concluded that MSCs from rheumatoid arthritis synovial fluid exert typical MSC phenotypes but displayed decline of multi-differentiation capability. RASF-MSCs especially show promoting effect on osteoclastogenesis. The findings of this study may contribute to the understanding biological behavior of MSCs in pathological microenvironment.
Arthritis, Rheumatoid ; Bone Marrow Cells ; cytology ; Cell Differentiation ; Cells, Cultured ; Humans ; Mesenchymal Stromal Cells ; cytology ; Osteoblasts ; cytology ; Synovial Fluid ; cytology

Bone marrow mensenchymal stem cells (BM-MSCs) are capable of supporting the survival, differentiation and migration of hematopoietic stem cell, and have a profound application prospect in transplantation and treatment of graft-versus-host disease (GVHD). This review aims to illustrate the interaction between BM-MSCs and other intra-bone marrow cells, including hematopoietic stem cells, endothelial cells and osteoblasts. The investigation of their regulating mechanism will help better understanding of the BM-MSCs' role in hematopoiesis.
Bone Marrow Cells ; cytology ; Cell Communication ; physiology ; Endothelial Cells ; cytology ; Humans ; Mesenchymal Stromal Cells ; cytology ; Osteoblasts ; cytology

OBJECTIVETo test the capacity of the stem cells derived from human exfoliated deciduous teeth in in vitro differentiation into osteoblasts.

METHODSStem cells were isolated from the exfoliated deciduous teeth of healthy children and sorted into CD34(+)/CD117(+) cells and the remaining mixed cells using flow cytometry. After in vitro cell culture, the differentiation capacity into osteoblasts of the two groups of cells was evaluated by detecting the markers of osteoblasts using immunocytochemical techniques and fluorescent quantitative PCR. Mineralization assay was performed to identify the cell differentiation.

RESULTSThe cells isolated by typsin digestion grew in the manner of fibroblasts. After a 30-day culture of the two groups of cells, immunocytochemistry detected the expressions of osteoblast markers RUNX-2, OC, and BSP. After 40 days of cell culture, the mRNA expressions of RUNX-2, OC and BSP genes were significantly different between the two groups. At day 50 of cell culture, the CD34(+)/CD117(+) cells exhibited positivity for von Kossa's staining and alizarin red staining, but the mixed cells showed negative staining results.

CONCLUSIONThe purified CD34(+)/CD117(+) stem cells derived from exfoliated deciduous teeth of healthy children possess the capacity to differentiate into osteoblasts and form calcium deposits and mineralized nodules in vitro.

Cell Differentiation ; physiology ; Cells, Cultured ; Child ; Dental Pulp ; cytology ; Humans ; Osteoblasts ; cytology ; Osteogenesis ; physiology ; Stem Cells ; cytology ; Tooth, Deciduous ; cytology

Osteoclasts and osteoblasts are not exist alone,while communicating with each other through direct contact, diffusible paracrine factors and cell-bone matrix interaction. Co-culture system of osteoblast with osteoclast,including direct co-culture and indirect co-culture. It should be according to the ratio of osteoclasts and osteoblasts under the pathology, choosing the same species. Compared with lonely culture of osteoblasts or osteoclasts,co-culture system is much closer to the microenvironment in vivo. It benefits to explain the interactions between osteoblasts and osteoclasts, exploring molecular communication in bone diseases. It was mainly used to investigate the pharmacological mechanism of herbal and western medicine in bone remodeling. Some osteoporosis drugs (such as epimedium,sanchi, fructus psoraleae, ranelate strontium) not only promoted osteoblastic bone formation, but also inhibited osteoclastic bone resorption in the system,so as to balance bone homeostasis. At the same time,it has been used to study medical physics and assess biomedical materials in recent years. Considerably,the co-cultrue system will be used to study the subchondral bone remodeling and its pharmacological mechanism of herbal and western medicine in osteoarthritis.
Animals ; Bone Remodeling ; Cell Communication ; Coculture Techniques ; Humans ; Osteoblasts ; cytology ; Osteoclasts ; cytology

OBJECTIVETo determine whether human alveolar bone osteoblasts (HABO) attachment to commercial available guided tissue regeneration (GTR) membranes can be enhanced by coating with freshly prepared human platelet-rich plasma (PRP) and platelet poor plasma (PPP).

METHODSHuman osteoblasts established from tissue explants were used at 4 th passage in culture. Human whole blood from healthy subjects was collected and centrifuged twice to produce the PRP fraction and PPP fraction. Double-sided adhesive tape was used to fix 3 mm discs of each membrane and cover-slides to the bottom of a 24-well tissue culture plate. A (GoreTex-ePTFE), B (GoreTex-Resolut) and C (Inion-GTR) membranes were studied. Cover-slides were positive control. Membranes or cover-slides were exposed to PRP, PPP or PBS respectively for 2 hours. Membranes and cover-slides were seeded with osteoblasts (5 x 10(7) cells/L) and allowed to attach for 24 hours. After staining with hematoxylin, the number of attached cells per mm(2) was counted using a light microscope with graticule. The the ultrastructure of osteoblasts attachment to the membranes was observed by scanning electronic microscopy.

RESULTSPRP and PPP-treated membranes significantly enhanced osteoblasts attachment compared to PBS-treated membranes (P < 0.05). There was more osteoblasts attachment in the PRP-treated membranes than in the PPP-treated membranes (P < 0.05). Cover-slides showed more osteoblasts attachment than the three membranes (P < 0.05). B and C membranes showed higher cell attachment than A membranes (P < 0.05). SEM showed that osteoblasts attached to the membranes treated by PRP were spindle and stretched well, and there were platelets, fibrins in a interlaced mesh on the membranes, which appeared to grow in a multiplayer style. The osteoblasts attached to the membranes treated by PPP or PBS were round and partially attached.

CONCLUSIONSPRP and PPP could improve attachment of osteoblasts in the three membranes, and PRP altered and enhanced the way of the attachment to the membranes.

Cell Adhesion ; Cells, Cultured ; Culture Media ; Humans ; Osteoblasts ; cytology ; Platelet-Rich Plasma ; Tooth Socket ; cytology

OBJECTIVEThe aim of this study was to investigate the circadian rhythm of the proliferative index of mandibular osteoblasts of goats in a normal situation.

METHODSTwenty four healthy goats were selected and randomly divided into six groups. They were killed, and a piece of bone tissue with 2 cm x 2 cm x 0.5 cm was obtained from right mandibular mental foramen region at 0:00, 4:00, 8:00, 12:00, 16:00 and 20:00 time points, after they had been fed for one week. The primary cell culture method was used to cultivate osteoblasts from the bone. The method of anti-5-bromodeoxyuridine monoclonal antibody and the flow cytometric analysis were used to detect the proliferative index of the mandibular osteoblasts at different time. The data were analyzed using the method of single cosine rhythmometry.

RESULTSThe proliferative indexes of the mandibular osteoblasts of goats were higher at the nighttime and lower in the daytime. The undulation of the proliferative indexes displayed a significant circadian rhythm (P < 0.01). The peak value was obtained at about 19:44, and the lowest value at about 7:44.

CONCLUSIONThe proliferative indexes of the mandibular osteoblasts of goats are not stable within 24 hours in a normal situation, but with significant circadian rhythm. There are different proliferative indexes at different time points.

Animals ; Cell Division ; Cells, Cultured ; Circadian Rhythm ; Flow Cytometry ; Goats ; Mandible ; cytology ; Osteoblasts ; cytology ; Random Allocation

Samples of healthy and full-term human umbilical cord blood samples were obtained asceptically. Mesenchymal stem cells (MSCs) were isolated by lymphocyte separation medium, and were characterized morphologically by fluorescence-activated cell sorting analysis. Differentiation of chondroblast and osteoblast was induced by 10 ng/ml TGF-beta, 100 ng/ml insulin and 10(-7) mol/L decaesadril, 6.25 microg/ml siderophilin, 10 mmol/L beta-sodium glycerophosphate, 50 microg/ml antiscorbic acid, respectirely; the aim was to investigate the potentiality of differentiation. Umbilical cord blood-derived MSCs were stained positive for MSCs marker CD13, CD90, CD166, CD73, CD44 and HLA-AB, but were negative for hematopoietic stem cell marker CD45, CD34 and HLA-DR. After 21 days induction, Toluidine Blue staining and von-Kossa staining were positive. Immunocytochemistry showed that Collagen II expressed in the induced cells. The results demonstrated that mesenchymal stem cells can be isolated from human umbilical cord blood and differentiated into chondroblasts and osteoblasts in vitro.
Cell Differentiation ; Cell Separation ; Cells, Cultured ; Chondrocytes ; cytology ; Fetal Blood ; cytology ; Humans ; Mesenchymal Stromal Cells ; cytology ; Osteoblasts ; chemistry

OBJECTIVETo compare between MLO-Y4 osteocyte and osteoblast to support osteoclast formation in co-culture system.

METHODSMLO-Y4 cells and murine osteoblast cells were co-cultured with bone marrow cells with or without vitamin D₃ presence.Bone marrow cells were as control group. Tartrat resistant acid phosphatase (TRAP)+ giant cells with three or more nuclei were counted and compared under a microscope at day 9.

RESULTSIn the absence of vitamin D₃, (1963.3 ± 93.1)/plate osteoclasts were observed when MLO-Y4 cells co-cultured with bone marrow cells in 24-well plate.While only (12.7 ± 5.5)/plate osteoclasts were found in the osteoblast group, and (6.0 ± 1.0)/plate in control group. The statistical difference occurs for any two groups (P < 0.05). Vitamin D₃ could significantly increase osteoclast formation in the three groups.

CONCLUSIONSOsteocytes could induce osteoclastogenesis without the presence of vitamin D₃ and vitamin D₃ could enhance the induction effects of MLO-Y4 and osteoblast cells.

Animals ; Cell Line ; Cholecalciferol ; chemistry ; Coculture Techniques ; Culture Media ; chemistry ; Mice ; Osteoblasts ; cytology ; Osteoclasts ; cytology ; Osteocytes ; cytology

OBJECTIVETo investigate a method that constructing a tissue-engineered tendon with a continuous and heterogeneous transition region.

METHODSFibroblasts derived from rabbit epithelial tissue were cultured in vitro and collagen gel was prepared. The experimental groups were scaffold only group, fibroblasts+ chondrocytes group (Fb+ CC group), fibroblasts+ osteoblasts group (Fb+ OB group), fibroblasts+ chondrocytes+ osteoblasts group (Fb+ CC+ OB group). Heterogeneous cell populations(fibroblasts, chondrocytes and osteoblasts) with collagen gel were seeded within three predesigned specific regions (fibrogenesis, chondrogenesis, and osteogenesis) of decellularized rabbit achilles tendons to fabricate a stratified scaffold containing three biofunctional regions supporting fibrogenesis, chondrogenesis, and osteogenesis. The tests of morphology, architecture and cytocompatibility of the scaffolds were performed. Gradient tissue-specific matrix formation was analysed within the predesignated regions via histological staining and immunofluorescence assays.

RESULTSThe HE staining and scanning electron microscopy analysis demonstrated that no major cell fragments or nuclear material was evident, and increased intra-fascicular and inter-fascicular spaces were found, the cytocompatibility of the scaffolds showed that the numbers of viable cells on the scaffold surfaces increase steadily, no significant differences were found between the scaffold only containing ordinary culture medium and scaffold containing gel groups. Histological staining and immunofluorescence assays demonstrated that the cartilage-related markers (GAG, COL2A1) were found only in the chondrogenesis region, but bone-related proteins only in the osteogenesis region of bone tunnel, and fibrosis was remarkable for the fibrogenesis region in the joint cavity. The transitional architecture with ligament-fibrocartilage-bone was constructed in the ligament-bone tunnel interface.

CONCLUSIONSA transitional interface (fiber-fiberocartilage-bone) could be replicated in a decellularized tendon through stratified tissue integration in vitro. The cell-tendon complex offers the advantages of a multi-tissue transition involving controlled cellular interactions and matrix heterogeneity.

Animals ; Bone and Bones ; Cells, Cultured ; Chondrocytes ; cytology ; Collagen ; Fibroblasts ; cytology ; Ligaments ; Osteoblasts ; cytology ; Rabbits ; Tendons ; Tissue Engineering ; methods