MSTN modification in goat mediated by TALENs and performance analysis.

Myostatin (MSTN) is a negative regulator of skeletal muscle growth and development. It can inhibit the proliferation of myoblasts and serve as an important candidate gene for animal breed improvement. Mutations of the MSTN gene can cause extensive skeletal muscle hyperplasia and hypertrophy, resulting in "double muscle" symptoms. This leads to reduction of animal fat differentiation and increase of muscle content, thereby meeting the demand for quality consumption of animal meat in the market. In order to obtain a double-muscle phenotype using mutant MSTN gene in cloned goat, the goat MSTN gene was target-modified by TALENs. In this study, the TALENs expression vector was designed and constructed in the first exon sequence of the goat MSTN gene, which was then transfected into the goat fetal fibroblasts. The resistant cell lines were obtained by puromycin selection, and the cell lines with the MSTN gene mutations were analyzed by PCR and gene sequencing, thereby identifying the mutation type(s). The MSTN gene mutant cell lines were used as the nuclear donor cells in somatic cell nuclear transfer procedures in goats, and The morphological structure of the muscle tissue of the goats with MSTN gene mutations was analyzed by tissue section. The body weight of the cloned goats were monitored at different months of age, which provided the growth trend of their weight at different developmental stages. The results show that a total of 109 MSTN gene mutant cell lines were obtained. The mutation efficiency was 79.0% (109/138), of which 46 were biallelic mutations, accounting for 33.3% (46/138) of the total cell lines. Four MSTN gene mutant cell lines (1 biallelic homozygous mutation, 3 non-homozygous mutations) with good growth status were selected for somatic cell nuclear transfer in 12 recipients, of which 4 were pregnant by B-ultrasound at 30 days, indicating the a 33.3% (4/12) pregnancy rate. Two cloned goats were born at the end of the pregnancy. Sequencing analysis showed that there was no mutation in one allele of the M-1 cloned lamb, and the other allele harbored a 3 bp-deletion. The M-2 cloned lamb harbored a 1 bp base insertion in one allele of the MSTN gene, and a deletion of 13 bp in the other allele, resulting in mutations in both alleles and the loss of the protein-coding sequence of MSTN after the mutation site. In addition, the muscle fibers of cloned M-1 goats are tightly arranged and thick, and their monthly body weight is higher than that of normal wild-type goats. However, it is still consistent with the growth trend of normal wild-type goats and the M-1 goats can develop into healthy adults. In summary, this study showed that goat fetal fibroblasts with the multiple MSTN gene mutations were successfully obtained by TALENs technology, and cloned goats with mutant MSTN genes could be generated by somatic cell nuclear transfer method, thereby providing a technical foundation for the cultivation of the "double muscle" phenotype goats, and serving as a reference method for the preparation of other transgenic animals in the future.

Cytobiological effect of micro-arc oxidation coating on 3D-printed titanium alloy scaffold / 中国组织工程研究

BACKGROUND: Micro-arc oxidation (MAO) treatment can improve the biological properties of titanium alloy implants. Previous studies mostly focused on the evaluation of titanium alloy plate, while the effects of the MAO-modified 3D titanium scaffold on the cell growth and differentiation were rarely reported. OBJECTIVE: To investigate the effect of MAO coating on the biological performance of cells seeded onto the 3D-printed porous titanium alloy scaffold. METHODS: Rat bone marrow mesenchymal stem cells were seeded onto the MAO-modified Ti6Al4V alloy scaffolds (experimental group) and unmodified scaffolds (control group). After 4 and 7 days of culture, cell/scaffold constructs were retrieved and processed for the assessment of cell morphology by using scanning electron microscopy, cytoskeletal staining analysis and cell viability assay were also evaluated. At 4, 7 and 11 days of culture, the levels of alkaline phosphatase and osteocalcin in the cell supernatant were detected. At 1, 4, 7 and 11 days of culture, the cell proliferation rate was measured using the MTT assay. RESULTS AND CONCLUSION: (1) At 4 and 7 days of culture, live/dead staining showed that the bone marrow mesenchymal stem cells grew well on the two kinds of scaffolds. The analysis of cytoskeleton staining showed that the cytoskeleton of the experimental group was stereo and polygonal, while the cells on the scaffold surface in the control group were flat and spindle-shaped, spreading along the macro structure of the scaffolds. Under the scanning electron microscopy, the cells in the experimental group arranged closely and spread in a good condition, with interconnected lamellipodia and filopodia that firmly adhered to the scaffold surface in an anchor-shaped structure; in the control group, less filopodia interconnected, less extracellular matrix, and flat and sheet-like cells were observed. (2) With the time increase, the levels of alkaline phosphatase and osteocalcin increased gradually in both groups. The alkaline phosphatase level in the experimental group was significantly higher than that in the control group at 7 and 11 days of culture (P < 0.05), while the osteocalcin level was higher in the experimental group than the control group at 11 days of culture (P < 0.05). (3) With the prolongation of culture time, the number of cells in the two groups increased gradually. The number of cells cultured in the experimental group was significantly higher than that in the control group at 7 and 11 days of culture (P < 0.05). To conclude, the MAO-modified titanium alloy scaffold is favorable for cell adhesion, proliferation and differentiation.

[Investigation on soil - transmitted nematode infections in national surveillance sites in Jiangsu Province from 2006 to 2015].

OBJECTIVE: To understand the status of soil-transmitted nematode infections in rural residents so as to provide the evidence for formulating the guidance for prevention and control of the diseases. METHODS: The national surveillance sites of soil-transmitted nematode infections were established in Shuyang County, Suqian City, Jiangsu Province from 2006 to 2015. At least 1 000 fecal samples of residents aged 3 years or above were collected in every autumn, and the intestinal helminth eggs were detected with the Kato-Katz technique and the Enterubius vermicularis eggs were detected by the cellophane tape method for children aged 3-12 years. The soil samples were collected from vegetable fields, lavatories, courtyards and kitchens to examine Ascaris lumbricoides eggs and larvae of hookworm. RESULTS: The infection rates of soil-transmitted nematodes in residents and E. vermicularis in children reduced from 1.81% (19/1 049) and 4.72% (5/106) in 2006 to 0.25% (3/1 180) and 0 (0/263) in 2015, respectively, in the surveillance sites. The infection intensity was mild in all the infected cases. The soil samples were negative for detecting A. lumbricoides eggs and hookworm larvae. CONCLUSIONS: The infection rates of soil-transmitted nematodes in the residents and E. vermicularis in the children show a decreasing trend and keep at a low level of prevalence in Shuyang County.

Generation of human lactoferrin transgenic cloned goats using donor cells with dual markers and a modified selection procedure.

The objective was to use dual markers to accurately select genetically modified donor cells and ensure that the resulting somatic cell nuclear transfer kids born were transgenic. Fetal fibroblast cells were transfected with dual marking gene vector (pCNLF-ng) that contained the red-shifted variant of the jellyfish green fluorescent protein (LGFP) and neomycin resistance (Neo) markers. Cell clones that were G418-resistant and polymerase chain reaction-positive were subcultured for several passages; individual cells of the clones were examined with fluorescence microscopy to confirm transgenic integration. Clones in which every cell had bright green fluorescence were used as donor cells for nuclear transfer. In total, 86.7% (26/30) cell clones were confirmed to have transgenic integration of the markers by polymerase chain reaction, 76.7% (23/30) exhibited fluorescence, but only 40% (12/30) of these fluorescent cell clones had fluorescence in all cell populations. Moreover, through several cell passages, only 20% (6/30) of the cell clones exhibited stable LGFP expression. Seven transgenic cloned offspring were produced from these cells by nuclear transfer. Overall, the reconstructed embryo fusion rate was 76.6%, pregnancy rates at 35 and 60 days were 39.1% and 21.7%, respectively, and the offspring birth rate was 1.4%. There were no significant differences between nuclear transfer with dual versus a single (Neo) marker (overall, 73.8% embryo fusion rate, 53.8% and 26.9% pregnancy rates, and 1.9% birth rate with five offspring). In conclusion, the use of LGFP/Neo dual markers and an optimized selection procedure reliably screened genetically modified donor cells, excluded pseudotransgenic cells, and led to production of human lactoferrin transgenic goats. Furthermore, the LGFP/Neo markers had no adverse effects on the efficiency of somatic cell nuclear transfer.

Hybrid expression cassettes consisting of a milk protein promoter and a cytomegalovirus enhancer significantly increase mammary-specific expression of human lactoferrin in transgenic mice.

It is very important to develop an effective, specific, and robust expression cassette that ensures a high level of expression in the mammary glands. In this study, we designed and constructed a series of mammary gland-specific vectors containing a complex hybrid promoter/enhancer by utilizing promoter sequences from milk proteins (i.e., goat ß-casein, bovine αs1-casein, or goat ß-lactoglobulin) and cytomegalovirus enhancer sequences; vectors containing a single milk protein promoter served as controls. Chicken ß-globin insulator sequences were also included in some of these vectors. The expression of constructs was analyzed through the generation of transgenic mice. Enzyme-linked immunosorbent assay (ELISA) analysis revealed that the hybrid promoter/enhancer could drive the expression of recombinant human lactoferrin (rhLF) cDNA at high levels (1.17-8.10 mg/ml) in the milk of transgenic mice, whereas control promoters achieved a very low rhLF expression (7-40 ng/ml). Moreover, the expression of rhLF was not detected in the serum or saliva of any transgenic animal. This result shows that all constructs, driven by the hybrid promoter/enhancer, had high mammary gland-specific expression pattern. Together, our results suggest that the use of a hybrid promoter/enhancer is a valuable alternative approach for increasing mammary-specific expression of recombinant hLF in a transgenic mouse model.

Reconstruction of critical sized calvarial defects by porous nano-hydroxyapatite/polyamide 6 composite with bone marrow mesenchymal stem cells in rat / 华西口腔医学杂志

<p><b>OBJECTIVE</b>To evaluate the effects of the nano-hydroxyapatite/polyamide 6 (n-HA/PA6) on the proliferation and osteoblastic differentiation of rat bone marrow mesenchymal stem cells (BMSCs), and the feasibility of using both for constructing tissue engineered bone in the calvarias of rats with critical sized defects.</p><p><b>METHODS</b>The third passage of BMSCs were cultured in osteoblastic medium and seeded on the scaffolds of n-HA/PA6, the proliferation of the BMSCs was tested by MTT (3-{4,5-dimethylthiazol-2yl}-2,5-diphenyl-2H-tetrazolium-bromide) on scheduled dates, and the osteoblastic differentiation of the BMSCs were measured by alkaline phosphatase (ALP) staining. Furthermore, the scaffolds with or without BMSCs in rat calvarial defects, after 4 weeks, 8 weeks, and 16 weeks have been implanted. Histology and scanning electron microscope were used to test the bone healing in the different groups.</p><p><b>RESULTS</b>The BMSCs seeded on the n-HA/PA6 grew well, the proliferation of cells was not affected by the scaffold, and the staining of ALP was also positive. At 4 week and 8 week after implantation, the n-HA/PA6 with BMSCs showed more new bone formation on the surface of scaffolds, with a better osseointegration of implant and host bone when compared with the group of n-HA/PA6 without BMSCs. However, there was no significant difference between these two groups at 16 week.</p><p><b>CONCLUSION</b>The porous n-HA/PA6 has no negative effects on the proliferation and osteoblastic differentiation of rat BMSCs, and using BMSCs as seed cells and n-HA/PA6 as scaffolds is a good choice for constructing tissue engineered bone due to the enhanced new bone formation and osseointegration.</p>

Comparative study on inorganic composition and crystallographic properties of cortical and cancellous bone / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>To comparatively investigate the inorganic composition and crystallographic properties of cortical and cancellous bone via thermal treatment under 700 °C.</p><p><b>METHODS</b>Thermogravimetric measurement, infrared spectrometer, X-ray diffraction, chemical analysis and X-ray photo-electron spectrometer were used to test the physical and chemical properties of cortical and cancellous bone at room temperature 250 °C, 450 °C, and 650 °C, respectively.</p><p><b>RESULTS</b>The process of heat treatment induced an extension in the a-lattice parameter and changes of the c-lattice parameter, and an increase in the crystallinity reflecting lattice rearrangement after release of lattice carbonate and possible lattice water. The mineral content in cortical and cancellous bone was 73.2wt% and 71.5wt%, respectively. For cortical bone, the weight loss was 6.7% at the temperature from 60 °C to 250 °C, 17.4% from 250 °C to 450 °C, and 2.7% from 450 °C to 700 °C. While the weight loss for the cancellous bone was 5.8%, 19.9%, and 2.8 % at each temperature range, the Ca/P ratio of cortical bone was 1.69 which is higher than the 1.67 of stoichiometric HA due to the B-type CO₃²⁻ substitution in apatite lattice. The Ca/P ratio of cancellous bone was lower than 1.67, suggesting the presence of more calcium deficient apatite.</p><p><b>CONCLUSION</b>The collagen fibers of cortical bone were arrayed more orderly than those of cancellous bone, while their mineralized fibers ollkded similar. The minerals in both cortical and cancellous bone are composed of poorly crystallized nano-size apatite crystals with lattice carbonate and possible lattice water. The process of heat treatment induces a change of the lattice parameter, resulting in lattice rearrangement after the release of lattice carbonate and lattice water and causing an increase in crystal size and crystallinity. This finding is helpful for future biomaterial design, preparation and application.</p>

Characterization and antibacterial effect of Ag-nHA-nTiO2/polyamide 66 nanocomposite membrane on oral bacteria / 华西口腔医学杂志

<p><b>OBJECTIVE</b>Undried silver-hydroxyapatite-titania (Ag-nHA-nTiO2) nanoparticles slurry was used to make membrane with polyamide 66 (PA66) by co-polymerization method. The purpose of this study is to test the physical and chemical characteristics and antibacterial ability.</p><p><b>METHODS</b>The morphology, chemical components and structures of the membrane were characterized by atomic absorption spectrometer (AAS), X-ray diffraction (XRD), scanning electron microscope (SEM) and energy-dispersive X-ray analysis (EDX). Staphylococcus aureus (S. aureus), Escherichia coli (E. coli), Porphyromonas gingivalis (P. gingivalis), Fusobacterium nucleatum (F. nucleatum) and Streptococcus mutans (S. mutans) were utilized to test the antibacterial effect.</p><p><b>RESULTS</b>XRD results demonstrated that the membrane have characteristic diffraction peaks of pure hydroxyapatite (HA). A homogeneous distribution of the Ca, P, Ti and Ag element in the membrane was confirmed by EDX. Both surface and section showed porous structure which was confirmed by SEM and the average hole size was 20-30 microm. The bacteria assay reflected to the antibacterial effect, 50.10% of S. aureus and 56.31% of E. coli were killed. However, 91.84% of P. gingivalis, 90.64% of F. nucleatum and 90.49% of S. mutans were killed and pictures of SEM showed obviously fewer cells on the surface.</p><p><b>CONCLUSION</b>The nanocomposite membrane could be one of the bioactive materials with antibacterial properties for oral guided bone regeneration technique.</p>

Experimental study on the reconstruction of mandibular defects with a new bioactive artificial bone nano-hydroxyapatite/polyamide-66 in dogs / 中华口腔医学杂志

<p><b>OBJECTIVE</b>This study was designed to investigate reconstruction of segmental defect in the mandible using a new bionic materials of nano-hydroxyapatite -polyainides-66 (n-HA/PA66).</p><p><b>METHODS</b>Two defects (15 mm x 10 mm x 5 mm) were created in the mandibular bodies of dogs. One of defects was reconstructed with n-HA/PA66, another not repaired as a blank control. At 2, 4, 8, 12, 16 weeks after operation. Evaluation of effects of n-HA/PA66 on reconstruction of the mandibular defects was carried out by means of radiography and histology.</p><p><b>RESULTS</b>From 2 to 8 weeks after operation, some fiber tissue grew into the space between n-HA/PA66 and mandibular bone. The ossification was observed at 12 weeks post-operation. At 16 weeks, the n-HA/PA66 was connected directly to the mandibular bone by the newborn bone.</p><p><b>CONCLUSIONS</b>The new artificial bone of n-HA/PA66 has the effects of osteoconduction and osteoinduction, with a good biocompatibility and is an ideal bone substitute material for reconstruction of mandibular defect.</p>

Nano biomedical material and its application / 中国医学科学院学报

Nano biomedical material is a new area that shows promising prospect. In this paper, the researches and applications of nano inorganic biomaterial, nano polymer biomaterial and nano composite biomaterial are reviewed. The developmental tendency of nano biomedical material is also forecasted.