Extracellular matrix (ECM) stiffness is closely related to the physiological and pathological states of breast tissue. The current study was aimed to investigate the effect of silk fibroin/collagen composite hydrogels with adjustable matrix stiffness on the growth and phenotype of normal breast epithelial cells. In this study, the enzymatic reaction of horseradish peroxidase (HRP) with hydrogen peroxide (H₂O₂) was used to change the degree of cross-linking of the silk fibroin solution. The rotational rheometer was used to characterize the composite hydrogel's biomechanical properties. Human normal mammary epithelial cell line MCF-10A were inoculated into composite hydrogels with various stiffness (19.10-4 932.36 Pa) to construct a three dimensional (3D) culture system of mammary epithelial cells. The CCK-8 assay was applied to detect the cell proliferation rate and active states in each group. Hematoxylin-Eosin (HE) staining and whole-mount magenta staining were used for histological evaluation of cell morphology and distribution. The results showed that with the increase of matrix stiffness, MCF-10A cells exhibited inhibited proliferation rate, decreased formation of acinus structures and increased branching structures. Meanwhile, with the increase of matrix stiffness, the polarity of MCF-10A cells was impeded. And the increase of matrix stiffness up-regulated the expression levels of mmp-2, mmp-3, and mmp-9 in MCF-10A cells. Among the genes related to epithelial-mesenchymal transition (EMT), the expression level of the epithelial marker gene E-cadherin was significantly down-regulated, while the interstitial cell marker gene Vimentin was up-regulated, and the expression levels of Snail, Wnt5b and Integrin β1 in the Wnt pathway were up-regulated. These results suggest that the silk fibroin/collagen composite hydrogels with adjustable matrix stiffness regulates the proliferation and the phenotype of MCF-10A cells. The effects of increased matrix stiffness may be closely related to the changes of the polar structures and function of MCF-10A cells, as well as the occurrence of ECM-remodeling and EMT.
Collagen/metabolism* ; Epithelial Cells/metabolism* ; Fibroins/pharmacology* ; Humans ; Hydrogels/metabolism* ; Hydrogen Peroxide ; Phenotype

Silk fibroin, a natural fibrin, is a suitable matrix biomaterial for wound repair due to its unique properties such as good biocompatibility, tunable biodegradation and mechanical properties, low host inflammatory response, low cost, ease of fabrication, etc. Silk fibroin can be used alone or in combination with other materials to construct various dressings including scaffolds, hydrogels, films, smart mats, and microneedles, which can meet the needs of different wound repair and regulate the wound repair process. Thus, the application research of silk fibroin in skin tissue engineering has increased dramatically. Compared with other natural materials, silk fibroin promotes tissue regeneration and wound repair by improving cell proliferation, migration, and differentiation behavior at different stages, showing unique advantages in different dimensions. Based on the development of silk fibroin wound repair materials in the recent years, this review focuses on the mechanism and application prospect of silk fibroin and its composite materials in wound repair.
Fibroins/metabolism* ; Biocompatible Materials/therapeutic use* ; Tissue Engineering ; Hydrogels ; Fibrin ; Tissue Scaffolds

A repetitive DNA fragment, named P1, was amplified by PCR with the full-length Minor Ampullate Spidroin gene sequence of Araneus ventricosus as template. P1 was ligated with pPic3.5 and PKT expression vectors and transferred into GS115 and BL21(DE3) competence cells, respectively. SDS-PAGE and Western blot were used to analyze the recombinant his-tag fusion protein. With expressed in different expression systems, soluble P1 induced proteins could be obtained as the same size. Furthermore, the expression level and purification recovery efficiency were also higher in GS115 than that of BL21(DE3). Additionally, the expression level could be improved after optimizing the incubation and induction conditions of GS115. In this research, Pichia pastoris expression system is more suitable for the native repetitive Gly/Ala-rich spider spidroin gene sequence expression than Escherichia coli system. The data can help the native full-length MiSp gene expression and large-scale exploitation of recombinant of spider silk proteins.
Animals ; Escherichia coli ; genetics ; metabolism ; Fibroins ; biosynthesis ; classification ; genetics ; Genetic Vectors ; genetics ; Pichia ; genetics ; metabolism ; Recombinant Fusion Proteins ; biosynthesis ; Spiders ; classification ; genetics ; metabolism

As a biomaterial to be used for reparation in the case of trauma, the silk fibroin, particularly its effect on the transcription and expression of VEGF gene, is a concern. In this study, the ECV304 cell's growth shape and growth curve on the regenerated silk fibroin film were observed, and its VEGF secretion level was measured by ELISA test. It was found that the regenerated silk fibroin film did not interfere with ECV304 cell's growth and function. The L929 cell transfected with human VEGF gene grew on the regenerated silk fibroin film; the real-time quantitative RT-PCR method and ELISA test were used for detecting the transcription and expression of VEGF gene. The results showed the regenerated silk fibroin film did not interfere with the transcription and expression of VEGF gene. Therefore, the regenerated silk fibroin film is a safe biomaterial for inducing vascularization with no untoward effect on the reparation of trauma.
Animals ; Biocompatible Materials ; pharmacology ; Cell Line ; Endothelial Cells ; cytology ; metabolism ; Fibroins ; pharmacology ; Humans ; Silk ; pharmacology ; Transcription, Genetic ; Vascular Endothelial Growth Factor A ; genetics ; metabolism

Spider silk is a natural protein fibroin with excellent character as it is light and tenacious. It has a wild potential applications in the biomedical field due to its good biocompatibility and degradation. Arginine-glycine-aspartic acid (RGD) is a highly conserved amino acid sequence of many adhesion protein. Biological materials binding with RGD peptide in the surface can promote cells adhesion, migration and proliferation. Our lab had constructed the 16 muhimers with the introduced RGD peptide codons which involve cell adhesion for the first time. It was found that the mechanical capability of the 16 mulimer protein was very limited because of the big gap in molecular weight with nature spider proteins when it was used to made biomaterial scaffold.In this paper,based on the 16 multimers of the highly, repetitive sequence of spider dragline silk and with RGD peptide condons which has been constructed by our lab forestall, it was used to construct the 32 and 64 multimers sequence of spider dragline silk by the strategy of "head to tail". The 32 and 64 multimers were ligated into prokaryotic expression vector pET-30a, and then the B121 (DE3) pLysS. The fragments were in agreement with the desired through digestion, agarose gel electrophoresis respectively. By registration into the GenBank data-base, the serial numbers of DQ469929 and DQ837297 were gained respectively. The expression of recombinant protein was introduced by the addition of IPTG. SDS-PAGE analysis shows that the molecular weight of products expressed here are 102 kD and 196.6kD in agreement with the desired respectively. It was the first time for the high polymer spider dragline silk protein expressed in prokaryotic biology. Furthermore, a larger quantity of synthetical proteins with high density fermentation were searched after, and a suit of high efficient purification methods for 32 multimers protein were established.
Animals ; Escherichia coli ; genetics ; metabolism ; Fermentation ; Fibroins ; biosynthesis ; genetics ; Molecular Sequence Data ; Oligopeptides ; biosynthesis ; chemistry ; genetics ; Polymers ; metabolism ; Protein Engineering ; Recombinant Proteins ; biosynthesis ; genetics ; isolation & purification ; Transformation, Bacterial

OBJECTIVETo evaluate the feasibility of the tissue engineered submandibular gland constructed in vivo based on submandibular gland cells and silk fibroin-chitosan (SFCS).

METHODSSubmandibular gland cells were obtained and purified. The second generation cells labeled by 5'-BrdU were seeded on SFCS(5 mm × 5 mm × 5 mm). Submandibular gland cells seeded on SFCS was implanted beneath the skin on the back. At 3, 7, 14 d post-implantation, implant sites were examined local and systemic responses. After paraffin embedding, serial sections 6 mm thick were cut and stained with either hematoxylin and eosin or brdu tissue stain for immunohistochemical studies and examined the responses of tissue. Scanning electron microscope was used to observe the growth behavior submandibular gland cells on SFCS scaffolds.

RESULTSGeneral observation: at the 3, 7, 14 d after in vivo implantation, capsule formed in the surface of insert. Histological observation: in experimental group, submandibular gland cells proliferate on the SFCS scaffold fused to form unit 14 d after implantation. Brdu immunohistochemical observation: the results of labelled cells were positive by immunohistochemical method at each time point. Cytokeratin-8 (CK-8) immunohistochemical observation: the results of labelled cells were positive by immunohistochemical method at each time point. With time, the positive cells gradually increased. Scanning electron microscope: the shape of the SFCS scaffold was mesh. At earlier, submandibular gland cells presence in disorder at attach to the SFCS. At the 14 d submandibular gland cells proliferate on the SFCS scaffold and form functional unit.

CONCLUSIONSSuch constructed tissue engineered submandibular gland based on submandibular gland cells and SFCS is promising.

Animals ; Cell Proliferation ; Cells, Cultured ; Chitosan ; chemistry ; Female ; Fibroins ; chemistry ; Keratin-8 ; metabolism ; Male ; Rats ; Rats, Sprague-Dawley ; Submandibular Gland ; cytology ; metabolism ; Tissue Engineering ; methods ; Tissue Scaffolds ; chemistry

To develop the stable transformants of the silkworm (Bombyx mori) BmN cells that could continuously express the exogenous gene based on a non-transposon vector, an expression cassette containing human granucyto-macrophage colony-stimulating factor (hGM-CSF) gene driven by ie-1 promoter from B. mori nucleopolyhedrovirus was inserted into pIZT-V5-His to form a recombinant vector pIZT-IE-hGM-CSF, followed by transfecting the constructant into BmN cells, the stable ie-hGM-CSF cell lines were obtained after being selected with Zeocin. PCR result using the genomic DNA of the transformed BmN cells as template illustrated a specific fragment of ie-hGM-CSF, and Western blotting analysis using an antibody against hGM-CSF demonstrated a specific band with a molecular weight of 22 kDa in the transformed cells, meanwhile, the expression level of hGM-CSF determined by ELISA was about 2 814.7 pg in 10(6) transformed BmN cells.
Animals ; Animals, Genetically Modified ; Bombyx ; cytology ; genetics ; metabolism ; Cell Line ; Fibroins ; genetics ; Genetic Vectors ; genetics ; Granulocyte-Macrophage Colony-Stimulating Factor ; biosynthesis ; genetics ; Humans ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; Transformation, Genetic

Based on the character of strong promoter of the fibroin gene and high level secretion of fibroin of Bombyx mori, we amplified the promoter of heavy chain gene (Fib-H) and its downstream signal peptide sequence (FibHS) by PCR. After that, we cloned the PCR product in pBluescriptII SK (+) to form the vector pSK-FibHS and analyzed its sequence. The sequence identity was 99% comparable to that of the reported sequence by Blast on line. Then we digested pSk-Ser-DsRed-PolyA with Sal IKpn I to get DsRed-PolyA DNA fragment and subcloned it into vector pSK-FibHS to generate a transitorily secretory expression vector pSK-FibHS-DsRed-PolyA. After identified the recombinant plasmid by restriction enzyme digestion, we transfected pSK-FibHS-DsRed-PolyA into BmN cells by liposome. From the cells transfected with the recombinant vector, what the red fluorescence could be detected verified that the recombinant vector could express DsRed in BmN cells transiently. Furthermore, when silkworm had been injected with the recombinant vector pSK-FibHS-DsRed-PolyA, red fluorescence could be observed in the lumen of silk gland of silkworm. The result indicated that DsRed expressed transiently and was secreted into lumen of the silk gland. Therefore, we supposed that the cloned sequence (FibHS) possessed signal peptide bio-function. Moreover, this study would lay a foundation for the research on secretory expression of exogenous gene by silk gland bioreactor.
Amino Acid Sequence ; Animals ; Base Sequence ; Bombyx ; genetics ; metabolism ; Cloning, Molecular ; Fibroins ; biosynthesis ; genetics ; Insect Proteins ; biosynthesis ; genetics ; Luminescent Proteins ; biosynthesis ; genetics ; Molecular Sequence Data ; Promoter Regions, Genetic ; genetics

OBJECTIVETo investigate the effect of bioactivity glass 45S5- silk fibroin(BG45S5- SF) membrane on growth, proliferation and differentiation of human dental pulp stem cells(hDPSC), and to provide new ideas and method for the regeneration of pulp-dentine complex.

METHODShDPSC seed on pure silk fibroin membrane (protein membrane group) and BG45S5-SF membrane with different concentrations(1 000, 5 000 mg/L, composite membrane group A and B, respectively) were prepared, and the materials were incubated in cell culture fluid for 24 h. No material membrane orifice plate was used as blank control group. Contact angle meter was used to measure surface contact angle of protein membrane and composite membrane group(each group had three repeated holes). Cell proliferation was assessed by cell counting kit- 8 on the 4, 7, 14, and 21 days. The state of adhesion and growth of hDPSC on the materials surface was evaluated by scanning electron microscopy and cytoskeleton staining; and alkaline phosphatase (ALP) activity was measured to evaluate the cell differentiation potential. The expression of odontoblastic differentiation-related genes was measured by real-time PCR.

RESULTSSurface contact angle of the protein membrane group and composite membrane group A and group B were 89.51° ± 0.12°, 70.32° ± 0.07° and 71.31° ± 0.09° respectively. hDPSC adhered well on each materials surface on the 7, 14, 21 days, ALP activity and differentiation genes of composite membrane group A and B rised more significantly than the blank control group and protein membrane group did (P<0.05). Dentin matrix protein1(DMP- 1), dentin sialoprotein(DSP), ALP, osteocalcin(OC) mRNA expression reached peak on the 14 days in group A, and in group B on the 21 days. Bone sialoprotein(BSP) mRNA expression in both group A and B reached peak on the 21 days.

CONCLUSIONSBG45S5- SF membrane is able to support the proliferation and showed the potential of odontoblastic differentiation for hDPSC. This finding suggests that BG45S5-SF membrane was a kind of tissue engineering film material with the regeneration potential for pulp-dentine complex.

Cell Adhesion ; Cell Culture Techniques ; Cell Differentiation ; Cell Proliferation ; Ceramics ; therapeutic use ; Dental Pulp ; cytology ; Extracellular Matrix Proteins ; metabolism ; Fibroins ; therapeutic use ; Glass ; Humans ; Membranes, Artificial ; Odontoblasts ; cytology ; Osteocalcin ; metabolism ; Phosphoproteins ; metabolism ; Real-Time Polymerase Chain Reaction ; Sialoglycoproteins ; metabolism ; Stem Cells ; cytology ; Tissue Engineering

RT-PCR was conducted with one degenerate primer designed according to repetitive regions' amino acid sequence of major ampullate spidroin (MaSp) in spiders and adaptor primer in the SMART cDNA Library Construction Kit. By cloning and sequencing of amplified products, one cDNA clone (GenBank Accession No. AY365017) of Argiope amoena MaSp gene was obtained. The deduced amino acid sequence can be distinctly divided into two regions: (1) Repetitive region that consists of an alternating alanine-rich and glycine-rich domain in which many prolines are present; and (2) C-terminal non-repetitive region. The region coding for 272 amino acids of MaSp gene was subcloned into prokaryotic expression vector pET28b(+) and an about 26kD recombinant protein was expressed at high levels in Escherichia coli BL21 (DE3) after induction of IPTG. After being purified with metal-affinity chromatography on Ni(2+) -IDA-Sepharose columns as well as gel filtration chromatography, the recombinant protein was confirmed to be predicted MaSp by means of amino acid composition analysis and N-terminal amino acid sequence analysis. The solubility behavior of recombinant MaSp with C-terminal non-repetitive region in the present study is similar to that of recombinant dragline silk proteins without C-terminal non-repetitive region expressed by bacteria and yeast in the other studies. The result shows that absence or presence of C-terminal non-repetitive region is not a crucial factor affecting the solubility of the recombinant MaSp.
Amino Acid Sequence ; Animals ; Base Sequence ; Chromatography, Affinity ; Cloning, Molecular ; DNA, Complementary ; chemistry ; genetics ; Electrophoresis, Polyacrylamide Gel ; Escherichia coli ; genetics ; Fibroins ; genetics ; metabolism ; Gene Expression ; Molecular Sequence Data ; Molecular Weight ; Plasmids ; genetics ; Recombinant Proteins ; chemistry ; isolation & purification ; metabolism ; Sequence Analysis, DNA ; Sequence Analysis, Protein ; Sequence Homology, Amino Acid ; Spiders ; genetics ; metabolism