Polydimethylsiloxane (PDMS) and hydroxyapatite (HA) were combined in our laboratory to fabricate an elastic porous cell scaffold with pore-forming agent, and then the scaffold was used as culture media for rat bone marrow derived mesenchymal stem cells (rBMSCs). Different porous materials (square and circular in shape) were prepared by different pore-forming agents (NaCl or paraffin spheres) with adjustable porosity (62%-76%). The HA crystals grew on the wall of hole when the material was exposed to SBF solutions, showing its biocompatibility and ability to support the cells to attach on the materials.
Animals ; Biocompatible Materials ; chemistry ; Dimethylpolysiloxanes ; chemistry ; Durapatite ; chemistry ; Mesenchymal Stromal Cells ; cytology ; Porosity ; Rats ; Tissue Scaffolds

In order to set up a base for stem cells to be widely used in clinical medicine, we tried to optimize, in this study, the technique that induces human mesenchymal stem cells (hMSCs) to differentiate into neural stem cells by using cerebrospinal fluid (CSF) from the different groups. After the induction, presence of neural stem cells was confirmed with microscope observation, flow cytometry analysis, immunohistochemistry and fluorescent immunohistochemistry. At the same time, we also compared and analysed the data of the number of stem cells when it totally met the requirements for clinical treatment and the days required. At last, we confirmed that hMSCs could be induced to differentiate into neural stem cells, and that the number of cells totally met the requirements for clinical treatment. But there were some differences both in the number of cells and the days required. Among the groups, the group that marrow mesenchymal stem cells from patients own induced by CSF from healthy volunteers used the shortest time and the quantity of the cells was significantly higher than those of the others.
Cell Differentiation ; Cerebrospinal Fluid ; chemistry ; Culture Media ; chemistry ; Flow Cytometry ; Humans ; Immunohistochemistry ; Mesenchymal Stromal Cells ; cytology ; Neural Stem Cells ; cytology

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

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 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

To prepare poly(lactic acid/glycolic acid/ asparagic acid-co- polyethylene glycol) (PLGA-[ASP-PEG]) and examine the cellular biocompatibility. PLGA-[ASP-PEG] was obtained by bulk ring-opening copolymerization method, examined by infrared spectrometry (IR) and 1H nuclear magnetic resonance spectroscopy (1H NMR). Bone marrow stromal cells(BMSCs) were cultured with PLGA-[ASP-PEG] (experiment gruop) and PLGA (control group) in vitro respectively, and were observed by phase-contrast microscopy and scanning electron microscopy. The resuls showed that PLGA-[ASP-PEG] was obtained and proved by IR and 1H NMR. The BMSCs of the experiment group could well attach to and extend on the surface of the PLGA-[ASP-PEG], and could proliferate and secrete better extracellular matrix, compared with control. The PLGA-[ASP-PEG] has good cellular a biocompatibility. It can be used as a biomaterial for bone tissue engineering.
Aspartic Acid ; chemistry ; Biocompatible Materials ; Bone Matrix ; cytology ; Lactic Acid ; chemistry ; Mesenchymal Stromal Cells ; cytology ; Polyesters ; Polyethylene Glycols ; chemistry ; Polyglycolic Acid ; chemistry ; Polymers ; 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

BACKGROUNDRepair of large bone defects remains a challenge for clinicians. The present study investigated the ability of mesenchymal stem cells (MSCs) and/or periosteum-loaded poly (lactic-co-glycolic acid) (PLGA) to promote new bone formation within rabbit ulnar segmental bone defects.

METHODSRabbit bone marrow-derived MSCs (passage 3) were seeded onto porous PLGA scaffolds. Forty segmental bone defects, each 15 mm in length, were created in the rabbit ulna, from which periosteum was obtained. Bone defects were treated with either PLGA alone (group A), PLGA + MSCs (group B), periosteum-wrapped PLGA (group C) or periosteum-wrapped PLGA/MSCs (group D). At 6 and 12 weeks post-surgery, samples were detected by gross observation, radiological examination (X-ray and micro-CT) and histological analyses.

RESULTSGroup D, comprising both periosteum and MSCs, showed better bone quality, higher X-ray scores and a greater amount of bone volume compared with the other three groups at each time point (P < 0.05). No significant differences in radiological scores and amount of bone volume were found between groups B and C (P > 0.05), both of which were significantly higher than group A (P < 0.05).

CONCLUSIONSImplanted MSCs combined with periosteum have a synergistic effect on segmental bone regeneration and that periosteum plays a critical role in the process. Fabrication of angiogenic and osteogenic cellular constructs or tissue-engineered periosteum will have broad applications in bone tissue engineering.

Animals ; Bone Regeneration ; physiology ; Cells, Cultured ; Lactic Acid ; chemistry ; Mesenchymal Stromal Cells ; cytology ; Periosteum ; cytology ; Polyglycolic Acid ; chemistry ; Rabbits ; Tissue Engineering ; methods ; Tissue Scaffolds ; chemistry

To investigate whether human umbilical cord plasma (HUP) can be used to culture human cord blood mesenchymal stem cells (HUCMSCs), we collected 20 surplus HUP. After being treated with salting out and diasysis, the HUP were used to culture HUCMSCs as 10% volume, and compared with fetal bovine serum (FBS). Morphological characteristics, growth curve and reproductive activity of HUCMSCs cells were observed. The concentration of bFGF and noggin secreted by HUCMSCs cultured with HUP and FBS medium were detected by ELISA. It was found that compared to FBS, the morphology, reproductive activity and characteristic of HUCMSCs cell cultured with HUP were not distinctively different from FBS. The concentration of bFGF in HUP group was significantly higher than that of FBS group, and the concentration of noggin was also different in the two groups. So we concluded that HUP could be used to culture HUCMSCs for a long-time, and the HUP mediumcoild could be more suitable for the culture of human embryonic stem cell (hESC).
Animals ; Cattle ; Cell Culture Techniques ; Cells, Cultured ; Culture Media ; chemistry ; Fetal Blood ; chemistry ; Humans ; Mesenchymal Stromal Cells ; cytology ; Plasma ; chemistry ; Serum ; chemistry

OBJECTIVETo observe and evaluate the effect of transdifferentiation of bone marrow derived stroma cells (BMSCs) into nerve cells by ultrafiltration membrane extract mixture from Angelica sinensis and Hedysarum polybotrys.

METHODSThe BMSCs in vitro cultured after treated by ultrafiltration membrane extract mixture from Angelica sinensis and Hedysarum polybotrys were divided into 5 groups, i.e., the blank group, the low dose group (6 g/L mixture), the high dose group (12 g/L mixture), the combination group (3 g/L mixture + 0.5 mmol/Lbeta-mercaptoethanol), and the positive control group (13-mercaptoethanol). The effects of transdifferentiation of nerve cells were observed using toluidine blue staining in each group. The differences of 5 specific neuroproteins, i.e. neuron-specific enolase (NSE), nestin, neurofilament protein (NFP), microtubule associated protein 2 (MAP2), and glial fibrillary acidic protein (GFAP) were detected using immunohistochemical technique and immunofluorescent technique respectively. The changes of the cell cycle were detected using flow cytometry (FCM).

RESULTSAfter induction BMSCs changed morphologically. The morphological features were weaker in the high and low dose groups than in the combination group and the positive group. Except the blank group, the aforesaid 5 proteins expressed positively in the rest groups. Their expression levels were highest in the positive control group (P <0.05), followed by the combination group (P <0.05). As for the cell proliferation rate detected by FCM, it was the lowest in the positive control group, followed by high dose group, low dose group, and then the combination group (all P <0.05).

CONCLUSIONSThe ultrafiltration membrane extract mixture from Angelica sinensis and Hedysarum polybotrys could effectively induce the transdifferentiation of BMSCs into nerve cells. Its inducing capacities were weaker in the positive control group, but it showed marked proliferation effects on differentiated cells. Therefore, the mixture might be a more ideal medication pathway for effectively inducing BMSCs' transdifferentiation into nerve cells, which might have higher proliferation and be used for clinical research.

Angelica sinensis ; chemistry ; Animals ; Cell Differentiation ; drug effects ; Cells, Cultured ; Drugs, Chinese Herbal ; pharmacology ; Fabaceae ; chemistry ; Mesenchymal Stromal Cells ; cytology ; drug effects ; Mice ; Neurons ; cytology ; Ultrafiltration