OBJECTIVETo construct an injectable tissue-engineered bone graft with fibrin glue (FG), autologous platelet-rich plasma (PRP) and bone marrow stromal cells (BMSCs) cultured in vitro and study its biological characteristics and microscopic structures.

METHODSBMSCs isolated from rabbit iliac bone marrow were culture-expanded in vitro. The injectable tissue-engineered bone constructed from autologous PRP, FG, and BMSCs was cultured in vitro, and its biological characteristics were observed including the time of gel formation, histological features, seed cell survival and microscopic structures.

RESULTSThe constructed injectable tissue-engineered bone began gel formation within 20 to 30 s, and after a week-long culture, the gelatine began to degrade, and numerous well viable fusiform cells could be seen to adhere to the bottom of the Petri dish. Scanning electron microscopy identified globular and olivary cells embedded in the fibrin glue, and numerous small particles could be seen around of the cells.

CONCLUSIONConstruction of an injectable tissue-engineered bone graft with FG, BMSCs and PRP does not require sophisticated techniques and ensures good biological property of the bone graft that can be easily shaped and allow good growth of the seed cells, suggesting great potential of this technique for clinical use.

Bone Marrow Cells ; cytology ; Bone Substitutes ; chemistry ; Cells, Cultured ; Coculture Techniques ; Fibrin Tissue Adhesive ; chemistry ; Platelet-Rich Plasma ; chemistry ; Stromal Cells ; cytology ; Tissue Engineering ; methods

We have established a bone marrow endothelial cell line. This review focuses on the elucidation and analysis of the effects of this bone marrow endothelial cell-conditioned medium (BMEC-CM) on the differentiation and proliferation of hematopoietic and endothelial progenitors as well as embryonic stem cells (ESCs). We will review that (1) BMEC-CM promotes proliferation and differentiation of hematopoietic lineage; (2) BMEC-CM promotes proliferation and differentiation of endothelial lineage; (3) BMEC-CM induces differentiation of hematopoietic stem cells/progenitors into endothelial progenitors; and (4) BMEC-CM induces differentiation of ESCs into hematopoietic cells and endothelial cells. We conclude that the soluble factors secreted by BMECs are able to support the proliferation and differentiation of hematopoietic and endothelium lineages. Moreover, these soluble factors induce hematopoietic cells to differentiate to endothelial cells, and induce ESCs to differentiate towards both endothelial cells and hematopoietic cells. Therefore, this work provides evidence that a close relationship involved in the development of hematopoietic and endothelial lineage. This disclosure will be beneficial for therapy strategy in the treatment of ischemic and tumor diseases, and improve our understanding of the relationship between hematopoietic and endothelial lineages.
Bone Marrow Cells ; chemistry ; Cell Differentiation ; Cell Proliferation ; Culture Media, Conditioned ; chemistry ; Embryonic Stem Cells ; cytology ; Endothelial Cells ; chemistry ; Hematopoietic Stem Cells ; cytology ; Humans

The aim of this study was to establish and optimize two-dimensional electrophoresis method for human bone marrow leukemia cells in order to obtain the profiles with high resolution and reproducibility. The total protein was extracted and separated by isoelectric focusing (IEF) and SDS polyacrylamide gel electrophoresis (SDS-PAGE). The gels were stained with silver nitrate or Coomassie brilliant blue, and then scanned and analyzed with PDQuest 7.4 analysis software. The effects of different protein preparation methods and electrophoresis conditions on the profiles were compared. The results indicated that by optimizing preparation of protein sample and electrophoresis protocols, clear profiles with 780 +/- 73 well separated protein spots on an average were obtained and the match rate was 82 +/- 5% between reproducible gels from leukemia cells of different sub-type. It is concluded that the two-dimensional electrophoresis method of proteome from human bone marrow leukemia cells is established successfully and is suitable for the further comparative proteomic research between leukemia of different types.
Bone Marrow Cells ; chemistry ; Electrophoresis, Gel, Two-Dimensional ; methods ; Humans ; Leukemia ; metabolism ; Proteome ; analysis

OBJECTIVETo study the changes in the biological behavior of bone marrow mesenchymal stem cells (BMSCs) transfected with red fluorescent protein by lentivirus (RFP-BMSCs) seeded on in poly-D, L-lactide acid (PDLLA) scaffolds with bioactive modification by ammonia plasma and Gly-Arg-Gly-Asp-Ser (GRGDS) in vitro.

METHODSCircular sheets of PDLLA scaffolds (8 mm in diameter and 1 mm in thickness) were prepared and aminated with PDLLA (group A) or modified with the peptide conjugate A/PDLLA (group PA), with untreated PDLLA as the control (group P). The RFP-BMSCs were seeded on the scaffold materials and their proliferation and metabolic activity were detected using CyQuant NF and Alamar blue staining. The mineralization on the scaffolds was observed using calcein fluorescent dye under a fluorescent microscope. The adhesion and proliferation of RFP-BMSCs were observed by fluorescent microscope, and scanning electron microscope (SEM) was used to confirm the observed adhesion of the seed cells.

RESULTSThe RFP-BMSCs seeded on the 3 scaffolds all showed proliferative activity at different time points after cell seeding, and the cell numbers decreased significantly in the order of PA>A>P (P<0.001). The cell number was significantly greater in group PA than in group A at all the time points except for days 10 (P=0.077) and 12 (P=0.491), and gradually became similar with the passage of time. The metabolic changes of the cells follow a similar pattern of cell proliferation. RFP-BMSCs showed more active proliferation in group A and group PA than in group P. On days 14 and 21, the intensity of green fluorescence decreased in the order of group PA, A and P. The RFP-BMSCs showed better adhesion in group PA than in group A, and the cells in group P appeared more scattered under scanning electron microscope.

CONCLUSIONBioactive modification of PDLLA by ammonia treatment and conjugation with GRGDS peptides may promotes the adhesion, proliferation, metabolism and mineralization of RFP-BMSCs seeded on PDLLA scaffolds.

Bone Marrow Cells ; cytology ; Cell Adhesion ; Cell Proliferation ; Cells, Cultured ; Humans ; Mesenchymal Stromal Cells ; cytology ; physiology ; Oligopeptides ; chemistry ; Osteogenesis ; Polyesters ; chemistry ; Tissue Engineering ; methods ; Tissue Scaffolds ; chemistry

The newly developed approach of tissue engineering has been shown to be great potential on the ligament reconstruction, however, the criterion for the scaffolding material was strict. The scaffold material must have enough strength as well as elasticity, at the same time, it should be biocompatible. As a nature protein, silk is a promising tissue engineering scaffold material for its excellent mechanical property. However, because of the contamination of sericin, the chief problem of silk's medical use is degumming. We compared three degumming reagents to choose the one which has least effect on the mechanical property of silk, and then the best degumming condition was confirmed: 0.4%NazCO3, 90 degrees C, 1 h. Rat bone morrw mesenchymal stem cells (rMSCs) were seeded on the fibroin, and scanning electron microscope (SEM) and fluorescence microscope were used to detect the biocompatibility of it. And the results showed that fibroin had outstanding biocompatibility and cell affinity, which indicated the further use of fibroin in tissue engineering.
Animals ; Biocompatible Materials ; chemistry ; Bone Marrow Cells ; cytology ; Cells, Cultured ; Fibroins ; chemistry ; isolation & purification ; Mesenchymal Stromal Cells ; cytology ; Rats ; Silk ; chemistry ; Tissue Engineering

OBJECTIVETo investigate whether vitamin C can promote the proliferation ability of bone marrow mesenchymal stem cells (BMMSCs) derived from aging mice.

METHODSThe senescence-accelerated mouse prone 6 (SAMP6) mice and senescence-accelerated mouse resistant 1 (SAMR1) mice were used as the test group and the control group, respectively, and the SAMP6 mice were examined by micro-CT to verify the senescent phenotype. BMMSCs were harvested from the two mouse lines and cultured in vitro, and the cells from SAMP6 mice were subjected to treatment with different concentrations of vitamin C. The proliferation ability of the cells from the two mouse lines was tested using MTT assay and growth curves, and TeloTAGGG Telomerase PCR ELISA was used to measure the telomerase activity; PCR and Western blotting were performed to detect the expression level of telomerase reverse transcriptase (TERT) in the cells.

RESULTSThe SAMP6 mice displayed a bone senescent phenotype. The proliferation ability of BMMSCs derived from SAMP6 mice and their telomerase activity were significantly lower than those derived from SAMR1 mice (P<0.05). Vitamin C treatment significantly enhanced the proliferation ability of BMMSCs derived from SAMP6 mice in a dose-dependent manner (P<0.05) and increased telomerase activity and TERT expression in the cells (P<0.05). At the concentration of 100 µg/mL, vitamin C produced the strongest effect in promoting the proliferation of BMMSCs from SAMP6 mice, while at the concentration of 1000 µg/ml, growth suppression occurred in the cells.

CONCLUSIONVitamin C can promote the proliferation of BMMSCs from aging mice possibly by increasing the cellular telomerase activity.

Aging ; Animals ; Ascorbic Acid ; chemistry ; Bone Marrow Cells ; cytology ; Cell Proliferation ; Cells, Cultured ; Culture Media ; chemistry ; Hematopoietic Stem Cells ; Mesenchymal Stromal Cells ; cytology ; Mice ; Telomerase ; metabolism

BACKGROUNDWe previously showed that nano-hydroxyapatite/carboxymethyl chitosan (n-Ha/CMCS) displayed excellent mechanical properties, good degradation rates and exceptional biocompatibility, with negligible toxicity. The aim of this study was to determine the effect of the same composite with vascular endothelial growth factor (VEGF)- transfected bone marrow stromal cells (BMSCs) in a rabbit radial defect model.

METHODSThe nano-hydroxyapatite was produced through co-precipitation. The n-HA/CMCS scaffold was produced by particle filtration and lyophilization followed by genipin crosslinking. Total RNA from rabbit bone was reverse-transcribed to synthesize VEGF165-pcDNA3.1 that was transfected into the BMSCs. The composite was implanted into a rabbit radial defect model, and the osteogenic activity examined by gross morphology, X-ray examination and hematoxylin and eosin (HE) staining.

RESULTSThe microstructure and mechanical property of the n-HA/CMCS scaffold resembled natural cancellous bone. Compared with glutaric dialdehyde crosslinked scaffolds, the genipin crosslinked scaffold was less toxic, and displayed a higher capacity to promote cell adhesion and proliferation. Spontaneous fluorescence of the composite permitted visualization of the composite-bone interface and the adhesion behavior of cells on the scaffold under laser scanning confocal microscopy. The scaffold with VEGF-transfected BMSCs bridged the bony defect and promoted healing, with most of the implanted material being replaced by natural bone over time with little residual implant. Using X-ray, we noted obvious callus formation and recanalization of the bone marrow cavity. Furthermore, HE stained sections showed new cortical bone formation.

CONCLUSIONSThe n-HA/CMCS scaffold composite with VEGF-trasnfected BMSCs is biocompatible, nontoxic, promotes the infiltration and formation of the microcirculation, and stimulates bone defect repair. Furthermore, the degradation rate of the composite matched that of growing bone. Overall, this composite material is potentially useful for bone defect repair.

Animals ; Bone Diseases ; surgery ; Bone Marrow Cells ; cytology ; Rabbits ; Stromal Cells ; cytology ; Tissue Engineering ; methods ; Tissue Scaffolds ; chemistry ; Vascular Endothelial Growth Factor A ; chemistry

This study aimed to characterize and magnetic resonance imaging (MRI) track the mesenchymal stem cells labeled with polylysine-coated superparamagnetic iron oxide (PLL-SPIO). Rat bone marrow derived mesenchymal stem cells (rMSCs) were labeled with 25, 50 and 100 microg/mL PLL-SPIO for 24 hours. The labeling efficiency was assessed by iron content, Prussian blue staining, electron microscopy and in vitro MR imaging. The labeled cells were also analyzed for cytotoxicity and differentiation potential. Electron microscopic observations and Prussian blue staining revealed that 75% -100% of cells were labeled with iron particles. PLL-SPIO did not show any cytotoxicity up to 100 microg/mL concentration. Both 25 microg/mL and 50 microg/mL PLL-SPIO labeled stem cells did not exhibit any significant alterations in the adipo/osteo/chondrogenic differentiation potential compared to unlabeled control cells. The lower concentration of 25 microg/mL iron labeled cells emitted an obvious dark signal in T1W, T2WI and T2 * WI MR image. The novel PLL-SPIO enables to label and track rMSCs for in vitro MRI without cellular alteration. Therefore PLL-SPIO may potentially become a better MR contrast agent especially in tracking the transplanted stem cells and other cells without compromising cell functional quality.
Animals ; Bone Marrow Cells ; Cell Differentiation ; Dextrans ; chemistry ; Magnetic Resonance Imaging ; Magnetite Nanoparticles ; chemistry ; Mesenchymal Stromal Cells ; cytology ; Polylysine ; chemistry ; Rats ; Staining and Labeling

In order to improve the bonding strength of the hydroxyapatite (HA) coatings on the metal substrate, we prepared the HA-Ti/HA composite coatings by two-step electrodeposited method, and then we studied the component, microstructure, surface morphologies and the bonding strength of the HA-Ti/HA composite coatings. SBF test and cell culture in vitro were carried out to evaluate the biological properties of the composite coatings. The results showed that the bonding strength of the HA-Ti/HA composite coating (Ti, 51.2wt%) was as high as 21.2 MPa which was 3 times that of pure HA coatings. The coatings' surface was covered by carbonate-apatite layer after being immersed in SBF, and the bone marrow cells attached firmly and proliferated well on the surface of composite coatings. These findings indicate that the composite coatings possess good bioactivity and excellent biocompatibility.
Animals ; Bone Marrow Cells ; cytology ; Cell Adhesion ; Cells, Cultured ; Coated Materials, Biocompatible ; chemistry ; Dogs ; Durapatite ; chemistry ; Electrochemistry ; Surface Properties ; Tissue Engineering ; methods ; Titanium ; chemistry

In order to investigate the effect of Arg-Gly-Asp (RGD) peptide-modified silk biomaterial on the adhesion and proliferation of bone marrow-derived mesenchymal stem cells (MSCs), MSCs of third generation were seeded onto the surface of RGD-decorated silk (silk-RGD group), silk alone (silk group) or tissue culture plate (TCP group). After incubation for 4 or 12 h, MSCs were examined quantitatively by using precipitation method for cell attachment. The cell proliferation, which was defined as cell density, was compared among the three groups after culture for 1, 2, 3, and 4 days. Cell skeleton, which was labeled fluorescently, was observed under laser confocal microscope after 24 h of culture. The results showed that cell adhesion rate in silk-RGD group was higher than in silk group (P<0.05), but similar to that in TCP group after incubation for 4 or 12 h (P>0.05). There were no significant differences in the cell proliferation among the three groups at different time points (P>0.05 for all). Laser confocal microscopy revealed that in silk-RGD group, MSCs, strongly fluorescently stained, spread fully, with stress fibers clearly seen, while in silk group, actin filaments were sparsely aligned and less stress fibers were found. It was concluded that RGD peptide could improve the adhesion of MSCs to the silk scaffold, but had no impact on the proliferation of the cells.
Biocompatible Materials/*chemistry ; Bone Marrow Cells/cytology ; Cell Adhesion/drug effects ; Cell Proliferation/drug effects ; Mesenchymal Stem Cells/*cytology ; Oligopeptides/*chemistry ; *Silk/chemistry ; Tissue Scaffolds