Rabbit bone marrow-derived mesenchymal stem cells(MSCs) are multipotent. We studied the adipogenic and osteogenic differentiation potent using adipogenic supplement (AS) or osteogenic supplement (OS) in vitro. Specific markers of this induced adipogenic and osteogenic lineage were identified. The findings showed that the rabbit MSCs are capable of differentiating into adipogenic and osteogenic lineages spontaneously. On the 21st day, approximately 75% rabbit MSCs were induced to adipogenic or osteogenic cells in medium containing AS or OS, respectively. These results demonstrated that the differentiation of MSCs could be regulated in vitro. The underlying molecular mechanisms of adipogenic or osteogenic differentiation await elucidation.
Adipose Tissue ; cytology ; Animals ; Bone and Bones ; cytology ; Cell Differentiation ; Cell Lineage ; In Vitro Techniques ; Mesoderm ; cytology ; Rabbits ; Stem Cells ; cytology

The research of bone tissue engineering bases on three basic directions of seed cells, scaffold materials and growth information. Stem cells have been widely studied as seed cells. Human urine-derived stem cell (hUSC) is extracted from urine and described to be adhesion growth, cloning, expression of the majority of mesenchymal stem cell markers and peripheral cell markers, multi-potential and no tumor but stable karyotype with passaging many times. Some researches proposed that hUSC might be a new source of seed cells in tissue engineering because of their invasive and convenient obtention, stable culture and multiple differentiation potential.
Bone and Bones ; Cell Differentiation ; Humans ; Mesenchymal Stromal Cells ; Stem Cells ; cytology ; Tissue Engineering ; Urine ; 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

This study was aimed to investigate the distribution of compact bone mesenchymal stem cells(MSC) marked with lentiviral plasmid pGC FU-RFP-LV in lung tissue and bone marrow of mouse. The MSC were infected by lentivirus with infection efficiency 78%, the infected MSC were injected into BALB/c mice via tail veins in concentration of 1×10(6) /mouse. The mice were randomly divided into 4 group according to 4 time points as 1, 2, 5 and 7 days. The lung tissue and bone marrow were taken and made of frozen sections and smears respectively in order to observed the distributions of MSC. The results indicated that the lentiviral infected MSC displayed phenotypes and biological characteristics which conformed to MSC by immunophenotyping analysis and induction differentiation detection. After the MSC were infected with optimal viral titer MOI = 50, the cell growth no significantly changed; the fluorescent microscopy revealed that the distributions of MSC in bone marrow on day 1, 2, 5 and 7 were 0.50 ± 0.20, 0.67 ± 0.23, 0.53 ± 0.14, 0.33 ± 0.16; those in lung tissue were 0.55 ± 0.15, 0.47 ± 0.13, 0.29 ± 0.13, 0.26 ± 0.08. It is concluded that the distribution of MSC in lung tissue reaches a peak on day 1, while distribution of MSC in bone marrow reaches a peak on day 2. The distribution of mouse MSC relates with RFP gene expression and implantation of MSC in lung tissue and bone marrow.
Animals ; Bone Marrow Cells ; cytology ; Bone and Bones ; cytology ; Female ; Lung ; cytology ; Male ; Mesenchymal Stromal Cells ; cytology ; Mice ; Mice, Inbred BALB C ; Mice, Inbred C57BL

OBJECTIVETo study cellular compatibility of improved scaffold material with deproteinized heterogeneous bone and provide experimental basis on choosing the scaffold material in bone tissue engineering.

METHODSBone marrow stromal cells (BMSC) were co-cultured with heterogeneous deproteinized bone in vitro. The contrast phase microscope, scanning electron microscope, MTT assay, flow cytometry were performed and the BGP content and ALP activities were detected in order to observe the cell growth, adhesion in the material, cell cycle and cell viability.

RESULTSThe scaffold material of deproteinized heterogeneous bone had no inhibitory effect on cellular proliferation, differentiation and secretion function of BMSCs.

CONCLUSIONSThe established heterogeneous deproteinized bone has good biocompatibility with BMSCs and is a potentially ideal scaffold material for bone tissue engineering.

Bone and Bones ; cytology ; Cells, Cultured ; Materials Testing ; Stromal Cells ; Tissue Engineering ; methods

Weight-bearing bone is constantly adapting its structure and function to mechanical environments. Loading through routine exercises stimulates bone formation and prevents bone loss, but unloading through bed rest and cast immobilization as well as exposure to weightlessness during spaceflight reduces its mass and strength. In order to elucidate the mechanism underlying unloading-driven bone adaptation, ground-based in vitro and in vivo analyses have been conducted using rotating cell culturing and hindlimb suspension. Focusing on gene expression studies in osteoblasts and hindlimb suspension studies, this minireview introduces our recent understanding on bone homeostasis under weightlessness in space. Most of the existing data indicate that unloading has the opposite effects to loading through common signaling pathways. However, a question remains as to whether any pathway unique to unloading (and not to loading) may exist.
Adaptation, Physiological ; Animals ; Bone and Bones ; cytology ; physiology ; Hindlimb Suspension ; physiology ; Humans ; Osteoblasts ; physiology ; Weightlessness

OBJECTIVETo study the effects of various methods of cryopreservation on the bioactivity of tissue engineered bone.

METHODSMSCs were cocultured with partialy deproteinised bone to produce tissue engineered bone. The experiment was divided into A, B, C and D group. Group A: Tissue engineered bone was stored in preservation solution with cryopreservation medium. Group B: Tissue engineered bone was stored in preservation solution without cryopreservation medium. Group C: Tissue engineered bone was stored without cryopreservation. Group D: MSCs were cultured without cryopreservation. The tissue engineered bone of group A and B had been cryopreserved at -80 degrees C for three months and thawed three months later. The electronic scanning microscope was used to evaluate the adhesion and distribution of MSCs, cell viability was measured by MTT, ALP activity was detected by p-nitrophosphate, cell cycle was analysed by flow cytometry.

RESULTSMSCs could adhere to the surface of the material and distribute in the hole of material. The cell viability of MSCs adhered to the material was C > A > B group (P < 0.01, P < 0.05). The ALP activity of MSCs adhered to material was C > A > B group (P < 0.01). The cell cycles of different groups did not change significantly; the abnormal cells were not observed.

CONCLUSIONThe choice of proper cryopreservative solution could optimize the bioactivity of tissue engineered bone.

Animals ; Bone and Bones ; cytology ; Cell Adhesion ; Cell Survival ; Cryopreservation ; Flow Cytometry ; Humans ; Mesenchymal Stromal Cells ; cytology ; Rabbits ; Tissue Engineering

In bone tissue engineering, a highly porous artificial extracellular matrix or scaffold is essential to the attachment, proliferation and differentiation of bone cells (osteoblast, osteoclast and osteocytes) and the formation of bone tissue. However, conventional scaffold materials for bone tissue engineering proved less valuable for actual applications because they lack mechanical strength, interconnected channel network, and controllable porosity or channel size. Therefore,to explore the ideal scaffold materials is one of the popular studies on current bone tissue engineering. In this paper, we review, the application and advancement of a newly-developed technology generally known as rapid prototyping (RP) techniques in bone tissue engineering.
Bone Substitutes ; Bone and Bones ; Cell Differentiation ; Cell Division ; Cells, Cultured ; Extracellular Matrix ; Humans ; Osteoblasts ; cytology ; Porosity ; Tissue Engineering

OBJECTIVETo evaluate the therapeutic effect of bone morphogenetic protein 2 (BMP-2) gene modified tissue engineering bone (GMB) combined with vascularized periosteum in the reconstruction of segmental bone defect.

METHODSAdenovirus carrying BMP-2 gene (Ad-BMP-2) was transfected into the isolated and cultured rabbit bone marrow stromal cells (MSCs). The transfected MSCs were seeded on bovine cancellous bone scaffolds (BCB) to construct gene modified tissue engineering bone (GMB). The bilateral rabbits radial defects (2.5 cm long) were created as animal model. The rabbits were divided into five groups to reconstruct the defects with CMB combined with vascularized periosteum (group A); or GMB combined with vascular bundle implantation (group B); or GMB combined with free periosteum (group C); or GMB only (group D); or BCB scaffolds only (group E). Angiogenesis and osteogenesis were observed by X-ray, histological examination, biomechanical analysis and capillary ink infusion.

RESULTSIn group A, the grafted GMB was revascularized rapidly. The defect was completely reconstructed at 8 weeks. The mechanism included both intramemerbrane and endochondral ossification. In group B, the vascular bundle generated new blood vessels into the grafted GMB, but the osteogenesis process was slow in the central zone, which healed completely at 12 weeks. In group C, the free graft of periosteum took at 4 weeks with angiogenesis. The thin extremal callus was formed at 8 weeks and the repairing process almost finished at 12 weeks. Better osteogenesis was found in group D than in group E, due to the present of BMP2 gene-transfected MSCs. The defects in group D were partial repaired at 12 weeks with remaining central malunion zone. The defects in group E should nonunion at 12 weeks with only fibre tissue.

CONCLUSIONSBMP-2 gene modified tissue engineering bone combined with vascularized periosteum which provides periosteum osteoblasts as well as blood supply, has favorable ability of osteogenesis, osteoinduction and osteoconduction. It is an ideal method for the treatment of segmental bone defect.

Animals ; Bone Marrow Cells ; cytology ; Bone Morphogenetic Protein 2 ; genetics ; Bone Regeneration ; Bone Substitutes ; Bone Transplantation ; methods ; Bone and Bones ; pathology ; Cattle ; Mesenchymal Stromal Cells ; cytology ; Periosteum ; blood supply ; transplantation ; Rabbits ; Surgical Flaps ; blood supply ; Tissue Engineering ; methods ; Tissue Scaffolds ; Transfection

OBJECTIVETo study the possibility and mechanism of construction of tissue engineered bone with human bone marrow mesenchymal stem cells (hBMSCs) as seeding cells and partially demineralized bone matrix (pDBM) as scaffold.

METHODShBMSCs are cultured and mutiplified. The 4th grade hBMSCs are seeded on the pDBM, the growth and adhesion of hBMSCs on pDBM are observed under scanning electro microscope. The adhesion efficiency is assessed. The complexes are implanted in the nude mice subcutaneously, the pDBM without cells as control. The grafts are taken out on the 8th and 12th week.

RESULTSThere is new bone formation on the 8th and 12th week in complex group. There is a layer of osteoblast like cells adhered on the surface of most of the new bone, which suggest the possibility of intramembranous ossification. There is no bone formation in control group.

CONCLUSIONSTissue engineered bone can be constructed with hBMScs and pDBM in vivo, and the mechanism of which could be intramembranous ossification.

Animals ; Bone Marrow Cells ; cytology ; Bone and Bones ; Cell Adhesion ; Cell Differentiation ; Humans ; Mesenchymal Stromal Cells ; cytology ; Mice ; Mice, Nude ; Tissue Engineering ; methods