Project description:Collagen and chitosan are frequently used natural biomaterials in tissue engineering. However, most collagen is derived from animal tissue, with inconsistent quality and pathogen transmittance risks. In this context, we aimed to use a reliable Type-III recombinant human collagen (RHC) as an alternative biomaterial together with chitosan to develop novel photo-responsive bioinks for three-dimensional (3D) bioprinting. RHC was modified with methacrylic anhydride to obtain the RHC methacryloyl (RHCMA) and mixed with acidified chitosan (CS) to form composites CS-RHCMA. The characterizations demonstrated that the mechanical properties and the degradation of the bioinks were tunable by introducing the CS. The printabilities improved by adding CS to RHCMA, and various structures were constructed via extrusion-based 3D printing successfully. Moreover, in vitro tests confirmed that these CS-RHCMA bioinks were biocompatible as human umbilical vein endothelial cells (HUVECs) were sustained within the constructs post-printing. The results from the current study illustrated a well-established bioinks system with the potential to construct different tissues through 3D bioprinting.
Project description:Injectable recombinant collagen hydrogels (RCHs) are crucial in biomedical applications. Culture conditions play an important role in the preparation of hydrogels. However, determining the characteristics of hydrogels under certain conditions and determining the optimal conditions swiftly still remain challenging tasks. In this study, a machine learning approach was introduced to explore the correlation between hydrogel characteristics and culture conditions and determine the optimal culture conditions. The study focused on four key factors as independent variables: initial substrate concentration, reaction temperature, pH level, and reaction time, while the dependent variable was the elastic modulus of the hydrogels. To analyze the impact of these factors on the elastic modulus, four mathematical models were employed, including multiple linear regression (ML), decision tree (DT), support vector machine (SVM), and neural network (NN). The theoretical outputs of NN were closest to the actual values. Therefore, NN proved to be the most suitable model. Subsequently, the optimal culture conditions were identified as a substrate concentration of 15% (W/V), a reaction temperature of 4 °C, a pH of 7.0, and a reaction time of 12 h. The hydrogels prepared under these specific conditions exhibited a predicted elastic modulus of 15,340 Pa, approaching that of natural elastic cartilage.
Project description:To investigate the virological properties of a SARS-CoV-2 variant, Omicron BA.2, we generated chimeric recombinant viruses that express GFP and encodes the S gene of B.1.1 (ancestral D614G-bearing virus), Delta, BA.1 and BA.2. To verify the genome sequence of the working viruses, we performed viral RNA-sequencing of the viral stock.
Project description:At present, the clinical treatment of osteomyelitis and osteomyelitis-induced bone defects is challenging, easy to recur, drug toxic side effects, secondary or multiple surgeries, etc. The design of biodegradable composite biomaterials to improve antibiotics in the local precise anti-infection at the same time to complete the repair of bone defects is the current research hot spot. Herein, a composite hydrogel with a double bond at the end (FA-MA) was prepared by affinity addition reaction between fish collagen (FA) and methacrylic anhydride (MA) under photoinitiator initiation conditions, then, FA-MA was amino-activated by EDC/NHC, and vancomycin was attached to FA-MA via amide bonding to prepare FA-MA-Van hydrogels, and finally, the composite hydrogel microspheres were prepared by microfluidic technology. The structure of the hydrogel was confirmed by SEM (elemental analysis), optical microscopy, FTIR, and XPS to confirm the successful preparation. The composite hydrogel microspheres showed the better antimicrobial effect of hydrogel microspheres by bacterial coated plate experiments and SEM morphology results, with the antimicrobial class reaching 99.8%. The results of immunofluorescence staining and X-ray experiments showed that the hydrogel microspheres had a better effect on promoting bone repair. This engineered design of hydrogel microspheres provides clinical significance for treating osteomyelitis at a later stage.
Project description:Existing treatment methods encounter difficulties in effectively promoting skin wound healing, making this a serious challenge for clinical treatment. Extracellular vesicles (EVs) secreted by stem cells have been proven to contribute to the regeneration and repair of wound tissue, but they cannot be targeted and sustained, which seriously limits their current therapeutic potential. The recombinant human collagen III protein (rhCol III) has the advantages of good water solubility, an absence of hidden viral dangers, a low rejection rate and a stable production process. In order to achieve a site-specific sustained release of EVs, we prepared a rhCol III hydrogel by cross-linking with transglutaminase (TGase) from Streptomyces mobaraensis, which has a uniform pore size and good biocompatibility. The release profile of the rhCol III-EVs hydrogel confirmed that the rhCol III hydrogel could slowly release EVs into the external environment. Herein, the rhCol III-EVs hydrogel effectively promoted macrophage changing from type M1 to type M2, the migration ability of L929 cells and the angiogenesis of human umbilical vein endothelial cells (HUVECs). Furthermore, the rhCol III-EVs hydrogel is shown to promote wound healing by inhibiting the inflammatory response and promoting cell proliferation and angiogenesis in a diabetic rat skin injury model. The reported results indicate that the rhCol III-EVs hydrogel could be used as a new biological material for EV delivery, and has a significant application value in skin wound healing.
Project description:Regulatory elements contributing to the tissue-specific regulation of the murine alpha 1(I) collagen (Colla1) gene have previously been identified in its promoter region and first intron. Because several lines of evidence indicate that DNA methylation may be involved in the tissue-specific regulation of Colla1 gene expression, we have analyzed the methylation status of the 5' region of the gene by restriction analysis and a methylation-dependent PCR assay. All sites tested were unmethylated in sperm DNA. The region surrounding the start site of transcription was partially or completely methylated in undifferentiated embryonal cell lines, suggesting that it may be marked by de novo methylation during early embryonic development. In differentiated cells and adult tissues, the Colla1 promoter was completely demethylated in collagen-producing and some nonproducing cells, and partially methylated in other nonproducing cells. The first intron was unmethylated in collagen-producing as well as nonproducing cells. Only sites in the first exon showed an inverse correlation with transcriptional activity, i.e., they were unmethylated in cells that express the gene, but predominantly methylated in cells that do not. Our results indicate that the methylation status of a small area (less than 1 kb) downstream of the Colla1 promoter, but not of the promoter itself or the first intron, may be critical for transcriptional activity of the promoter, presumably through an indirect mechanism.
Project description:With the characteristics of low toxicity and biodegradability, recombinant collagen-like proteins have been chemically and genetically engineered as a scaffold for cell adhesion and proliferation. However, most of the existing hydrogels crosslinked with peptides or polymers are not pure collagen, limiting their utility as biomaterials. A major roadblock in the development of biomaterials is the need for high purity collagen that can self-assemble into hydrogels under mild conditions. In this work, we designed a recombinant protein, S-VCL-S, by introducing cysteine residues into the Streptococcus pyogenes collagen-like protein at both the N-and C-termini of the collagen with a trimerization domain (V) and a collagen domain (CL). The S-VCL-S protein was properly folded in complete triple helices and formed self-supporting hydrogels without polymer modifications. In addition, the introduction of cysteines was found to play a key role in the properties of the hydrogels, including their microstructure, pore size, mechanical properties, and drug release capability. Moreover, two/three-dimensional cell-culture assays showed that the hydrogels are noncytotoxic and can promote long-term cell viability. This study explored a crosslinking collagen hydrogel based on disulfide bonds and provides a design strategy for collagen-based biomaterials.
Project description:Collagen possesses distinctive chemical properties and biological functions due to its unique triple helix structure. However, recombinant collagen expressed in Escherichia coli without post-translational modifications such as hydroxylation lacks full function since hydroxylation is considered to be critical to the stability of the collagen triple-helix at body temperature. Here, a proline-deficient E. coli strain was constructed and employed to prepare hydroxylated recombinant collagens by incorporating proline (Pro) and hydroxyproline (Hyp) from the culture medium. By controlling the ratio of Pro to Hyp in the culture medium, collagen with different degrees of hydroxylation (0-88%) can be obtained. When the ratio of Pro and Hyp was adjusted to 12:8 mM, the proline hydroxylation rate of recombinant human collagen (rhCol, 55 kDa) ranged from 40-50%, which was also the degree of natural collagen. After proline hydroxylation, both the thermal stability and cell binding of rhCol were significantly enhanced. Notably, when the hydroxylation rate approached that of native human collagen (40-50%), the improvements were most pronounced. Moreover, the cell binding of rhCol with a hydroxylation rate of 43% increased by 29%, and the melting temperature (Tm) rose by 5 °C compared to the non-hydroxylated rhCol. The system achieved a yield of 1.186 g/L of rhCol by batch-fed in a 7 L fermenter. This innovative technology is expected to drive the development and application of collagen-related biomaterials with significant application value in the fields of tissue engineering, regenerative medicine, and biopharmaceuticals.
Project description:Antibodies, which are important research entities in the field of biopharmaceuticals, hold a key position in the global pharmaceutical market. Nanobodies [a single-domain antibody (VHH)] have gradually shown unique advantages due to their specificity, small molecule size, high affinity, good stability, flexible delivery routes, and fast tissue penetration. The importance of nanobodies in the imaging, diagnosis, and treatment of diseases, especially tumors and autoimmune diseases, is increasing. This review addresses key technological hurdles, such as humanization, immunogenicity, and production scalability, and highlights novel strategies to overcome these challenges, including PEGylation, fusion with long-lived serum proteins, and advanced microbial expression systems. This review summarizes the characteristics, production, and industrialization of nanobodies, including nanobody-derived patents and clinical trials, from 2014 to 2023. Finally, the review explores some challenges associated with nanobody technology in biopharmaceuticals, therapeutic interventions, and diagnostic tools and potential solutions.