Bone marrow-derived stromal cells and cartilage tissue engineering

Articular cartilage repair is one of the most challenging issues which remains to be resolved in clinic work. Discovering of bone marrow stromal cells (BMSC) and its application in tissue engineering provide new methods for the treatment of cartilage defects. High seeding density, appropriate cytokines and three-dimensional culture play important roles in the process of inducing BMSC differentiating into chondrocytes, suitable scaffold is also essential in reconstructing cartilage in vitro by methods of tissue engineering.     

Detection,cloning and expression of bone morphogenetic protein-1 from human osteosarcoma cell lines

AIM:To manufacture recombinant protein of the highly conserved domain in human bone morphogenetic protein-1(BMP-1) using gene engineering methods as antigen for making wide spectrum antibody to BMP-1.METHODS:We analyzed the gene sequences and protein structures of BMP-1 and its related proteins, and chose a highly conserved fragment as target gene. Total RNA was prepared from human osteosarcoma cell line Saos-2, then the target gene was amplified with RT-PCR. The PCR product was cloned into prokaryotic expression vector pMAL c2 to get recombinant vector BMP-1(322-588aa)-pMAL c2. After transforming the recombinant plasmid into DH5-alpha and screening, several prositive clones were got for sequencing. Finally the transformed cells was induced with IPTG to get fusion protein.RESULTS:The BMP-1 gene fragment was successfully cloned into vector pMAL c2, and was able to express efficiently with IPTG inducement. The amount of expressed fusion protein is about 66%-72% in total volume of bacterial proteins.CONCLUSIONS:The recombinant protein contains several key domains(2 CUB domains and 1 EGF domain), which are shared by BMP-1 and its related proteins. Specific wide spectrum antibody to human BMP-1 and its related proteins may be generated with this recombinant protein antigen.     

Selection and amplification of the liver stem cell subset from rat bone marrow cells with a medium containing cholestatic serum in vitro

AIM: To explore the feasibility of direct separat and selective enlargement of the bone marrow-derived liver stem cells (BDLSC) from bone marrow cells with a culture system containing cholestatic serum in vitro. METHODS: Bone marrow cells of rats were cultured with selective media containing 2%, 5%, 7% and 10% cholestatic rat serum, respectively. The BDLSC were then induced to proliferate with the addition of hepatocyte growth factor (HGF) on the firth day. BDLSC were characterized using immunocytochemistry and RT-PCR for lineage markers, glycogen staining and urea synthetic assay for functions 2 weeks later. RESULTS: Bone marrow cells were unble to form colony in the presence of 2% cholestatic serum and apopotosis appeared gradually in 7% or 10% cholestatic serum. The BDLSC survived in the medium containing 5% cholestatic serum while the other types of cells did not. The survival cells proliferated with a high speed during the second week and then formed hepatocyte-like colony-forming units (H-CFU). Cells in the H-CFU expressed the characteristic proteins of fetal hepatocytes. Furthermore, they had glycogen storage and urea synthesis functions, two of the critical features of hepatocytes. CONCLUSION: The selective micro-environment effectively selected BDLSC from the bone marrow cell, and will be a new way to provide an abundant source of donor hepatocytes for clinical cell therapy.     

Biological characteristics of long passaging rat mesenchymal stem cells

AIM:To investigate multi-potential of rat bone marrow mesenchymal stem cells (rBMMSC) and mutation inclination, the rBMMSC were long passaged in vitro. METHODS:Cellular cycles of different passages were assayed by FACSan flow cytometry and karyotypes of passage 6, passage 25 and passage 45 were compared by G-binding analysis. RESULTS:The early passages and long-term passages all showed strong proliferation; passage 6, passage 25 and passage 45 all showed normal karyotype. CONCLUSION:Long-term culture and passage of rBMMSC still remains strong proliferation. With this capability, the mutation inclination is not enhanced.     

Adult rat and human bone marrow mesenchymal stem cells differentiate into neurons with Musk's polypeptide

AIM: To investigate the differentiation from adult rat and human bone marrow mesenchymal stem cells (BMMSCs) into neuron with musk polypeptide (Mu-P).METHODS: Adult rat and human BMMSCs were induced with Mu-P.Neuron-specific enolase (NSE),neurofilament (NF),Nestin,glial fibrillary acidic protein (GFAP) were detected by immunohistochemistry.RESULTS: Simple methods with Mu-P induced adult rat and human BMMSCs exhibiting a neuronal phenotype,expressing Nestin at 3 hours to 5 hours,and expressing NE and NF at 5 hours to 7 days.But the neuron-like cells didn't express the glial astrocyte marker GFAP.CONCLUSION: Adult rat and human BMMSCs can be induced to differentiate into neurons with Mu-P.     

Effects of PD98059 on the differentiation from mesenchymal stem cells to osteoblasts

AIM: To investigate the effects of PD98059 on the differentiation from mesenchymal stem cells to osteoblasts.METHODS: hMSC were separated from human marrow and expanded in cuture medium. hMSC were induced with dexamethasone, β-glycerophosphate, vitamin C which acted as osteoblast differentiation inducer. PD98059 was added into the osteoblasts induction medium. The cells were assayed with cell morphology, alkaline phosphatase (AP) activity and calcium deposition. RESULTS: The isolated cultured MSC comprised a single phenotypic population and displayed a fibroblast-like morphology. After induced with osteoblasts induction medium, the cells showed changes in cell morphology from spindle-shape to cuboidal and polygonal. The AP activity increased gradually and reached the peak in 12 days, then decreased. Many scattered tangerice calcium nodes were observed. PD 98059 significantly inhibited AP activity and calcium deposition in a dose-dependent manner. A striking observation of the present study was that a few adipocytes appeared in cultures that were treated with PD 98059 and osteogenic differentiation medium. CONCLUSION: These results indicated that osteogenic diferentiation from the hMSCs was related to the activation of the ERK.     

Bone marrow mesenchymal stem cells are induced to differentiate into cardiomyocytes by hepatocyte growth factor in vitro

AIM: To explore a new method of hepatocyte growth factor (HGF) inducing bone marrow mesenchymal stem cells (MSC) to differentiate into cardiomyocytes. METHODS: Bone marrow MSC was cultured with DMEM media (10% fetal calf serum) 4-6 passages, and induced by HGF (10 μg/L) for 30 d. Automatical beating of the differentiated cells was observed daily with transverse microscopy, or under condition of 0.1% isoproterenol or cal-cium-deprived incubation. Specific cardiac myosin in the cells was indentified by immunochemistry. RESULTS: At 14-20 d of differentiation, bone marrow mesenchymal stem cells formed clones, in 10%-50% of which spontaneous beating cell-mass had come to continuously exist. Isoproterenol increased the beating rate and calcium-deprived media inhibited the beating. The cells were identified to be cardiomyocytes by expression of cardiac myosin heavy chain. CONCLUSION: HGF may induce bone marrow mesenchymal stem cells into cardiomyocytes with high efficiency, but the differentiating pathway of stem cells remains to be further studied.     

Are dual-purpose and male layer chickens more resilient against a low-protein-low-soybean diet than slow-growing broilers?

ABSTRACT

1. Although fattening dual-purpose types or male layer hybrid chickens appears more ethical than the common practice of culling day-old male layer chicks, the lower feed efficiency of these birds raises concerns. Replacing feed ingredients that compete with food production by those of lower value for human nutrition would be beneficial.

2. Lohmann Dual (LD), a modern dual-purpose type, Lohmann Brown (LB), a male layer hybrid, and Hubbard JA 957 (HU), a slow-growing broiler type, were fattened for nine weeks on two diets (control or ?20% crude protein; n = 6 × 12 birds). Growth, carcass and meat quality were analysed.

3. Growth performance of HU exceeded that of LD and especially of LB. The growth depression caused by the low-protein diet fed to LD (?7%) was only half of that found in HU (?13%). The LD fed the control diet had the same feed efficiency as the HU fed the low-protein diet. Even the LB had a lower performance and feed efficiency with the low-protein diet in growth. There was a gradient in carcass properties (weight, dressing percentage, breast meat yield, breast proportion and breast angle) from HU to LD to LB, with some additional adverse effects of the low-protein diet especially in HU. There were some breed differences in fatty acid profile in the intramuscular fat.

4. In conclusion, the dual-purpose type used complied with regulations for Swiss organic poultry systems in terms of growth rate and was found to respond less when fed a low-protein diet than the slow-growing broiler type. The LB males were inferior in all growth and carcass quality traits. Future studies need to determine the exact protein and amino acid requirements of dual-purpose and layer hybrid chickens and the economic feasibility of the systems, especially for organic farming.     

Characterization and Film-Forming Properties of Gelatins from Whitemouth Croaker (Micropogonias furnieri) Skin and Bones

The Whitemouth croaker (Micropogonias furnieri), due to its abundance, is considered one of the most traditional and important species fished in the coastal waters and estuaries of South America. The aim of this work was to produce biopolymeric films of gelatins obtained from skin and bones of Whitemouth croaker. The fish gelatins were characterized through the yield, chemical composition, physiochemical, and rheological properties. The films were characterized by tensile strength (TS), elongation at break (EAB), water vapor permeability (WVP), thermogravimetric analysis (TGA), Fourier transform infrared spectroscopy (FT-IR), and optical properties. The values of protein yield, hydroxyproline content, gel strength, melting temperature, gelling temperature, and viscosity of the skin gelatin were higher than the values of the bone gelatin. The films obtained of skin gelatin showed all properties and thermal analysis superior in relation to the films of bone gelatin; this can be justified by the best physicochemical and rheological characteristics of the skin gelatin.     

Neuroprotective effect of fasudil combined with bone marrow-derived neural stem cells on mice with experimental autoimmune encephalomyelitis

AIM: To explore the neuroprotective effect of fasudil combined with bone marrow-derived neural stem cells (BM-NSCs) on the mice with experimental autoimmune encephalomyelitis (EAE). METHODS: Female C57BL/6 mice (8~10 weeks old, n=32) were immunized with myelin oligodendrocyte glycoprotein 35-55 (MOG_35-55) to establish chronic EAE model. The mice were randomly divided into control (ddH₂O) group, fasudil group, BM-NSCs group, and fasudil+BM-NSCs group. The clinical score and body weight were recorded every other day. The expression of neurotrophic factors was determined by immunofluorescence staining. RESULTS: In comparison with ddH₂O group, fasudil combined with BM-NSCs delayed onset and ameliorated severity of EAE. The numbers of brain-derived neurotrophic factor, glial cell-derived neurotrophic factor, nerve growth factor, neurotrophin-3 and ciliary neurotrophic factor positive cells in fasudil group, BM-NSCs group and fasudil+BM-NSCs group were all increased in various extents. In particularly, the expression of these neurotrophic factors in fasudil+BM-NSCs group was significantly higher than that in the mice treated with fasudil or BM-NSCs alone (P<0.01). CONCLUSION: Fasudil combined with BM-NSCs promotes the expression of neurotrophic factors and improves microenvironment of central nervous system, thus playing a positive role in neural restoration and regeneration through a synergistic and superimposed effect.