Influences of Molecular Weights on Physicochemical and Biological Properties of Collagen-Alginate Scaffolds

For tissue engineering applications, biodegradable scaffolds containing high molecular weights (MW) of collagen and sodium alginate have been developed and characterized. However, the properties of low MW collagen-based scaffolds have not been studied in previous research. This work examined the distinctive properties of low MW collagen-based scaffolds with alginate unmodified and modified by subcritical water. Besides, we developed a facile method to cross-link water-soluble scaffolds using glutaraldehyde in an aqueous ethanol solution. The prepared cross-linked scaffolds showed good structural properties with high porosity (~93%) and high cross-linking degree (50–60%). Compared with collagen (6000 Da)-based scaffolds, collagen (25,000 Da)-based scaffolds exhibited higher stability against collagenase degradation and lower weight loss in phosphate buffer pH 7.4. Collagen (25,000 Da)-based scaffolds with modified alginate tended to improve antioxidant capacity compared with scaffolds containing unmodified alginate. Interestingly, in vitro coagulant activity assay demonstrated that collagen (25,000 Da)-based scaffolds with modified alginate (C25-A63 and C25-A21) significantly reduced the clotting time of human plasma compared with scaffolds consisting of unmodified alginate. Although some further investigations need to be done, collagen (25,000 Da)-based scaffolds with modified alginate should be considered as a potential candidate for tissue engineering applications.     

Ultrasound-mediated preparation and evaluation of a collagen/PVP-PCL micro- and nanofiber scaffold electrospun from chloroform/ethanol mixture

In this study, to improve the cellular biocompatibility of PVP-PCL micro- and nanofiber scaffold, a novel electrospun collagen/PVP-PCL micro- and nanofiber scaffold was sucessfully prepared assisted by ultrasonic irradiation using chloroform/ethanol mixtures as solvent. The micro- and nanofibers of the electrospun PCL-PVP scaffolds still presented compact inter-fiber entanglement and three-dimensional netlike network with some certain range of pore space after introducing collagen. The incorporated collagen phase was dispersed as inclusions within the electrospun fibers, and then could be easily released by immersing the scaffold in Hanks simulated body fluid. Meanwhile, the integral triple helix structure of collagen could be maintained after blending with the PVP-PCL mixture due to the weak intermolecular interactions. Furthermore, the suitable mechanical and degradation properties of the PVP-PCL scaffold were still reserved after introducing collagen, and the introduction of collagen could further promote the thermostability of the PVP-PCL scaffold. Above all, the collagen/PVP-PCL scaffold showed no cytotoxicity, better cell proliferation, and improved viability of primary fibroblasts than the PVP-PCL scaffold. In conclusion, blending collagen with the PVP-PCL mixture in this study has potential for promoting the biocompatibility of PVP-PCL micro- and nanofiber scaffolds for tissue engineering.     

Electrospun PU-PEG and PU-PC hybrid scaffolds for vascular tissue engineering

Produced via electrospinning, polyurethane (PU) scaffolds have always attracted the interest of medical applications due of their unique properties such as good adhesion, biocompatibility and excellent mechanical strength. However, the poor hydrophilicity and hemocompatibility of PU presented a problem during PU’s application in the manufacturing of biomedical materials. We hypothesized that the incorporation of polyethylene glycol (PEG) and phosphatidylcholine (PC) into electrospinning solution of PU could improve the cell affinity and hemocompatibility. This research focused on fabricating hybrid PU-PEG and PU-PC random/aligned scaffolds through electrospinning technique and comparing their properties as a potential biocompatible scaffold for vascular tissue engineering. PC was doped into a PU solution in order to prepare an electrospun scaffold through the electrospinning technology while crosslinked electrospun PUPEG hybrid scaffolds were fabricated by photoinduced polymerization. The contact angle dramatically decreased from 122.3±0.8° to 39.1±0.8° with doping of PC in electrospinning solution while it decreased from 122.3±0.8° to 41.6±0.8° with doping of PEG. Furthermore, the mechanical properties of PU scaffolds were altered significantly by the addition of PC. The hemolysis and cytocompatibility assays demonstrated that these composite scaffolds could potentially be used as a smalldiameter vascular graft.     

Chondrogenic differentiation of rat mesenchymal stem cells on silk fibroin/chondroitin sulfate/hyaluronic acid ternary scaffolds

Cartilage repair is a challenge in bone tissue reconstruction. In this study, silk fibroin (SF), chondroitin sulfate (CS) and hyaluronic acid (HA) were employed to fabricate scaffolds for tissue engineered cartilage by freeze drying technique. The secondary pores were formed in the main pores of SF/CS/HA scaffold which improved the pore connectivity and equilibrium swelling of the scaffold. Furthermore, rat bone marrow mesenchymal stem cells were seeded on the scaffolds to evaluate the cell adhesion and proliferation. Results of hematoxylin/eosin staining and cell counting kit-8 assay showed that the cells migration and differentiation of SF/CS/HA (80/15/5) scaffold were better than that of SF/CS/HA scaffolds with different ratios after 7 days culture. Moreover, immunohistochemistry and scanning electron microscope demonstrated that large amounts of collagen II and proteoglycans of the cells were expressed in the SF/CS/HA 3D scaffold, while the expression of collagen I was barely visible by immunohistochemistry. Abound of extracellular matrix was formed to morphologically round and distributed uniformly throughout the scaffolds. The 3D ternary scaffold could promote the cells chondrogenic differentiation without using any inductive agent and offer potential for cartilage tissue regeneration.     

Influence of high solid concentrations on enzymatic wheat gluten hydrolysis and resulting functional properties

Enzymatic hydrolysis at increased solid concentrations is beneficial with regard to energy and water consumption. This study examines the influence of the solid concentration on the enzymatic hydrolysis of wheat gluten and the resulting functional properties of the hydrolysate. Wheat gluten was mildly hydrolyzed at a solid concentration varying from 10% to 60% to degrees of hydrolysis (DH%) ranging from 3.2% to 10.2%. The gluten was susceptible to hydrolysis at all solid concentrations but the hydrolysis rate was influenced by increasing solid concentrations. Size-exclusion high-performance liquid chromatography revealed an increase in the ratio of peptides with a molecular mass >25 kDa for solid concentrations of 40% and 60%. The water solubility increased on hydrolysis and was independent of the solid concentration during proteolysis. The foam stability was not influenced by the solid concentration at low DH%. At DH% higher than 8%, high solid concentrations increased the foam stability, which might be related to the presence of more peptides with a molecular mass >25 kDa. In addition, we found increased reactor productivity. The results show the potential of hydrolyzing wheat gluten at high solid concentrations, which could lead to large savings for water and energy when applied industrially.     

Jellyfish Collagen: A Biocompatible Collagen Source for 3D Scaffold Fabrication and Enhanced Chondrogenicity

Osteoarthritis (OA) is a multifactorial disease leading to degeneration of articular cartilage, causing morbidity in approximately 8.5 million of the UK population. As the dense extracellular matrix of articular cartilage is primarily composed of collagen, cartilage repair strategies have exploited the biocompatibility and mechanical strength of bovine and porcine collagen to produce robust scaffolds for procedures such as matrix-induced chondrocyte implantation (MACI). However, mammalian sourced collagens pose safety risks such as bovine spongiform encephalopathy, transmissible spongiform encephalopathy and possible transmission of viral vectors. This study characterised a non-mammalian jellyfish (Rhizostoma pulmo) collagen as an alternative, safer source in scaffold production for clinical use. Jellyfish collagen demonstrated comparable scaffold structural properties and stability when compared to mammalian collagen. Jellyfish collagen also displayed comparable immunogenic responses (platelet and leukocyte activation/cell death) and cytokine release profile in comparison to mammalian collagen in vitro. Further histological analysis of jellyfish collagen revealed bovine chondroprogenitor cell invasion and proliferation in the scaffold structures, where the scaffold supported enhanced chondrogenesis in the presence of TGFβ1. This study highlights the potential of jellyfish collagen as a safe and biocompatible biomaterial for both OA repair and further regenerative medicine applications.     

Effects of PLGA nanofibrous scaffolds structure on nerve cell directional proliferation and morphology

Electrospinning has been recognized as an efficient technique for the fabrication of neural tissue engineering scaffolds. Many approaches have been developed on material optimization, electrospinning techniques, and physical properties of scaffolds to produce a suitable scaffold for tissue engineering aspects. In this study, structural properties of scaffolds were promoted by controlling the speed of fiber collection without any post-processing. PLGA scaffolds, in two significantly different solution concentrations, were fabricated by the electrospinning process to produce scaffolds with the optimum nerve cell growth in a desired direction. The minimum, intermediate and maximum rate of fiber collection (0.4, 2.4, 4.8 m/s) formed Random, Aligned and Drown-aligned fibers, with various porosities and hydrophilicities. The scaffolds were characterized by fiber diameter, porosity, water contact angle and morphology. Human nerve cells were cultured on fiber substrates for seven days to study the effects of different scaffold structures on cell morphology and proliferation, simultaneously. The results of MTT assay, the morphology of cells and scaffold characterization recommend that the best structure to promote cell direction, morphology and proliferation is accessible in an optimized hydrophilicity and porosity of scaffolds, which was obtained at the collector linear speed of 2.4 m/s.     

复合酶法制备澳洲坚果蛋白肽及其抗氧化活性研究

本研究以前期制备的澳洲坚果蛋白为原料,经复合酶（木瓜蛋白酶和中性蛋白酶）酶解制备澳洲坚果蛋白肽,采用水解度为指标,利用单因素试验与正交试验考察各酶解因素对澳洲坚果蛋白水解度的影响,同时通过不同分子量（3、10、30 kDa）的超滤离心管对制备的蛋白肽进行初步的分离,并基于DPPH自由基清除能力对不同分子量的澳洲坚果蛋白肽的抗氧化活性进行评价。结果表明：各酶解因素对复合酶酶解制备澳洲坚果蛋白水解度的影响依次为酶解初始pH>复合酶配比>酶添加量>酶解时间;最佳复合酶酶解条件为复合酶配比1∶5、酶添加量12 000 U/g、酶解液初始pH 9.0、酶解时间360 min,在此条件下澳洲坚果蛋白的水解度为21.88%;同时,通过分析不同分子量的澳洲坚果蛋白肽组分发现,不同分子量的澳洲坚果蛋白肽均具有抗氧化活性,分子量在3~10 kDa的肽段组分具有较强的抗氧化能力,其DPPH自由基清除能力达到80.97%,且随着蛋白肽组分浓度的增加,其抗氧化能力也逐渐增强。     

Potential of Thermolysin-like Protease A69 in Preparation of Bovine Collagen Peptides with Moisture-Retention Ability and Antioxidative Activity

Bovine bone is rich in collagen and is a good material for collagen peptide preparation. Although thermolysin-like proteases (TLPs) have been applied in different fields, the potential of TLPs in preparing bioactive collagen peptides has rarely been evaluated. Here, we characterized a thermophilic TLP, A69, from a hydrothermal bacterium Anoxybacillus caldiproteolyticus 1A02591, and evaluated its potential in preparing bioactive collagen peptides. A69 showed the highest activity at 60 °C and pH 7.0. We optimized the conditions for bovine bone collagen hydrolysis and set up a process with high hydrolysis efficiency (99.4%) to prepare bovine bone collagen peptides, in which bovine bone collagen was hydrolyzed at 60 °C for 2 h with an enzyme–substrate ratio of 25 U/g. The hydrolysate contained 96.5% peptides that have a broad molecular weight distribution below 10000 Da. The hydrolysate showed good moisture-retention ability and a high hydroxyl radical (•OH) scavenging ratio of 73.2%, suggesting that the prepared collagen peptides have good antioxidative activity. Altogether, these results indicate that the thermophilic TLP A69 has promising potential in the preparation of bioactive collagen peptides, which may have potentials in cosmetics, food and pharmaceutical industries. This study lays a foundation for the high-valued utilization of bovine bone collagen.     

A Unique Marine-Derived Collagen: Its Characterization towards Biocompatibility Applications for Tissue Regeneration

Biomedical engineering combines engineering and materials methods to restore, maintain, improve, or replace different types of biological tissues. In tissue engineering, following major injury, a scaffold is designed to support the local growth of cells, enabling the development of new viable tissue. To provide the conditions for the mechanical and structural properties needed for the restored tissue and its appropriate functioning, the scaffold requires specific biochemical properties in order to ensure a correct healing process. The scaffold creates a support system and requires a suitable material that will transduce the appropriate signals for the regenerative process to take place. A scaffold composed of material that mimics natural tissue, rather than a synthetic material, will achieve better results. Here, we provide an overview of natural components of marine-derived origin, the collagen fibers characterization schematic is summarized in the graphical abstract. The use of collagen fibers for biomedical applications and their performances in cell support are demonstrated in an in vitro system and in tissue regeneration in vivo.     

Preparation, structure, and in vitro degradation behavior of the electrospun poly(lactide-co-glycolide) ultrafine fibrous vascular scaffold

The PLGA ultrafine fibrous scaffold was successfully fabricated by electrospinning. The morphology and properties of the PLGA vascular scaffolds were examined. In particular, the in vitro degradation behavior of the electrospun PLGA vascular scaffolds was investigated by means of morphology, microstructure, mass loss, M_w, and breaking strength characterization. The results showed that electrospun scaffold possessed ultrafine fibrous and porous structure, and had adequate mechanical properties to be developed as a substitute for native blood vessels. In vitro degradation study showed that the PLGA ultrafine fibrous scaffold could biodegrade in the PBS solution, and the mass loss, M_w, and breaking strength studies indicated that degradation rate of the electrospun PLGA nanofibers was greater in the first 2 weeks. After the degradation of 2 weeks, the degradation slowed down. Furthermore, with the extension of the degradation time, the thermal decomposition temperature of the PLGA scaffold decreased gradually. The results indicated that the electrospun PLGA vascular scaffold could be considered as an ideal candidate for tissue-engineered blood vessel.     

Interaction of antibiotic with PAN and cationic-dyeable PET fibers in development of infection resistant biomedical materials

Interaction of a representative antibiotic, doxycycline (Doxy), with commercial poly(acrylonitrile) (PAN) and cationic-dyeable poly(ethylene terephthalate) (PET) fiber was studied in development of infection resistant biomedical materials. Regular PET was also employed for a comparison purpose. Their interactions were investigated at different treatment temperatures, times, and pHs. Fibers were also hydrolyzed by 1 % NaOH for 1 or 2 hours at 85°C and 100°C to study effect of hydrolysis on antibiotic sorption. Infection-resistant characteristics of the substrates were evaluated by zone of inhibition (ZOI) test. Results revealed that a significant chemical change occurred in PAN and cationic-dyeable PET due to hydrolysis. Additional functional groups obtained by hydrolysis not only enhanced sorption of the antibiotics but also provided greater ZOI values, indicating substantial improvement in sustained infection resistance properties.     

Assessment of protein entrapment in hydroxyapatite scaffolds by size exclusion chromatography

Although it is well known that the textural properties of scaffolds play an important role in the process of tissue regeneration, the investigation of such effects remain difficult especially at the micro/nano level. Texture confers the material the additional ability to entrap/concentrate molecules circulating in the body fluid regardless of their binding affinity to the material. The goal of the present work is to isolate protein entrapment from protein adsorption phenomena in two macroporous hydroxyapatite scaffolds with identical chemical structure, similar macroporosity but different micro/nanoporosity using proteins of different sizes. This was achieved implementing size exclusion chromatography and using the scaffolds as chromatographic columns. The results showed that the larger the crystal size and the lower the packing density of the crystals composing the scaffold increased protein retention but decreased the protein dwelling time in the column. Differences in the amount of protein retained depended on the protein type.     

Chitosan-Alginate Biocomposite Containing Fucoidan for Bone Tissue Engineering

Over the last few years, significant research has been conducted in the construction of artificial bone scaffolds. In the present study, different types of polymer scaffolds, such as chitosan-alginate (Chi-Alg) and chitosan-alginate with fucoidan (Chi-Alg-fucoidan), were developed by a freeze-drying method, and each was characterized as a bone graft substitute. The porosity, water uptake and retention ability of the prepared scaffolds showed similar efficacy. The pore size of the Chi-Alg and Chi-Alg-fucoidan scaffolds were measured from scanning electron microscopy and found to be 62–490 and 56–437 µm, respectively. In vitro studies using the MG-63 cell line revealed profound cytocompatibility, increased cell proliferation and enhanced alkaline phosphatase secretion in the Chi-Alg-fucoidan scaffold compared to the Chi-Alg scaffold. Further, protein adsorption and mineralization were about two times greater in the Chi-Alg-fucoidan scaffold than the Chi-Alg scaffold. Hence, we suggest that Chi-Alg-fucoidan will be a promising biomaterial for bone tissue regeneration.     

Cell-interactive polymers for tissue engineering

Tissue engineering is one exciting approach to treat patients who need a new organ or tissue. A critical element in this approach is the polymer scaffold, as it provides a space for new tissue formation and mimics many roles of natural extracellular matrices. In this review, we describe several design parameters of polymer matrices that can significantly affect cellular behavior, as well as various polymers which are frequently used to date or potentially useful in many tissue engineering applications. Interactions between cells and polymer scaffolds, including specific receptor-ligand interactions, physical and degradation feature of the scaffolds, and delivery of soluble factors, should be considered in the design and tailoring of appropriate polymer matrices to be used in tissue engineering applications, as these interactions control the function and structure of engineered tissues.     

Preparation and characterization of blended <Emphasis Type="Italic">Bombyx mori</Emphasis> silk fibroin scaffolds

The aim of this study was to compare physical, mechanical and biological properties of 3-dimensional scaffolds prepared from Bombyx mori silk fibroin (SF), fibroin blended with collagen (SF/C), and fibroin blended with gelatin (SF/G) using a freeze-drying technique. The prepared scaffolds were sponge-like structure that exhibited homogeneous porosity with highly interconnected pores. Average pore size of these scaffolds ranged from 65–147 μm. All biodegradable scaffolds were capable of water absorption of 90 %. The degradation behavior of these scaffolds could be controlled by varying the amount of blended polymer. The SF/C and SF/G scaffolds showed higher compressive modulus than that of SF scaffolds which could be attributed to the thicker pore wall observed in the blended constructs. The less crystalline SF structure was observed in SF/G scaffolds as compared to SF/C scaffolds. Thus, the highest compressive modulus was observed on SF/C matrix. To investigate the feasibility of the scaffolds for cartilage tissue engineering application, rat articular chondrocytes were seeded onto the scaffolds. The MTT assay demonstrated that blending collagen or gelatin into SF sponge facilitated cell attachment and proliferation better than SF scaffolds. The blended SF scaffolds possessed superior physical, mechanical and biological properties in comparison to SF scaffolds and showed high potential for application in cartilage tissue engineering.     

Conductive 3D structure nanofibrous scaffolds for spinal cord regeneration

The complex nature of spinal cord injuries has provided much inspiration for the design of novel biomaterials and scaffolds which are capable of stimulating neural tissue repair strategies. Recently, conductive polymers have gained much attention for improving the nerve regeneration. In our previous study, a three-dimensional (3D) structure with reliable performance was achieved for electrospun scaffolds. The main purpose in the current study is formation of electrical excitable 3D scaffolds by appending polyaniline (PANI) to biocompatible polymers. In this paper, an attempt was made to develop conductive nanofibrous scaffolds, which can simultaneously present both electrical and topographical cues to cells. By using a proper 3D structure, two kinds of conductive scaffolds are compared with a non-conductive scaffold. The 3D nanofibrous core-sheath scaffolds, which are conductive, were prepared with nanorough sheath and aligned core. Two different sheath polymers, including poly(lactic-co-glycolic acid) PLGA and PLGA/PANI, with identical PCL/PANI cores were fabricated. Nanofibers of PCL and PLGA blends with PANI have fiber diameters of 234±60.8 nm and 770±166.6 nm, and conductivity of 3.17×10^-5 S/cm and 4.29×10^-5 S/cm, respectively. The cell proliferation evaluation of nerve cells on these two conductive scaffolds and previous non-conductive scaffolds (PLGA) indicate that the first conductive scaffold (PCL/ PANI-PLGA) could be more effective for nerve tissue regeneration. Locomotor scores of grafted animals by developed scaffolds showed significant performance of non-conductive 3D scaffolds. Moreover, the animal studies indicated the ability of two new types of conductive scaffolds as spinal cord regeneration candidates.     

Characterization of methacrylated type-I collagen as a dynamic, photoactive hydrogel

Type-I collagen is an attractive scaffold material for tissue engineering due to its ability to self-assemble into a fibrillar hydrogel, its innate support of tissue cells through bioactive adhesion sites, and its biodegradability. However, a lack of control of material properties has hampered its utility as a scaffold. We have modified collagen via the addition of methacrylate groups to create collagen methacrylamide (CMA) using a synthesis reaction that allows retention of fundamental characteristics of native collagen, including spontaneous fibrillar self-assembly and enzymatic biodegradability. This method allows for a rapid, five-fold increase in storage modulus upon irradiation with 365 nm light. Fibrillar diameter of CMA was not significantly different from native collagen. Collagenolytic degradability of uncrosslinked CMA was minimally reduced, while photocrosslinked CMA was significantly more resistant to degradation. Live/Dead staining demonstrated that a large majority (71%) of encapsulated mesenchymal stem cells remained viable 24 h after photocrosslinking, which further increased to 81% after 72 h. This material represents a novel platform for creating mechanically heterogeneous environments.     

豆渣酶解工艺及其水解液发酵抗性菌株筛选

从工业化生产实际出发,探讨了豆渣的高效水解技术、酵母菌株的选育及其培养工艺。不同工艺条件下,采用纤维素酶和普鲁兰酶水解豆渣,以葡萄糖为标准测量水解后料液中糖含量,得出水解豆渣的最佳工艺条件:温度55℃,料液比为1∶24,先用普鲁兰酶水解,调至pH5.0,再用纤维素酶水解,调至pH5.5,普鲁兰酶3 h,纤维素酶1 h,时间比为3∶1,加酶总量为3%,普鲁兰酶∶纤维素酶为3∶1。用此条件对豆渣进行水解得到水解液,料液中含糖量最高位为160.7 mg·kg~(-1)。用含量为35%的水解液去培养抗性菌株,筛选的抗性菌株C发酵效果较好,酵母含量达18.49 g·L~(-1)。     

茶叶β-葡萄糖苷酶亲和层析纯化与性质研究

以化学合成的β-葡萄糖苷酶的抑制剂β-葡萄糖脒为配体,通过N-糖苷键交联到支持物上,使糖脒N-糖苷键一侧的氨基保持带正电荷的活化状态,合成了一种β-葡萄糖苷酶的亲和层析树脂。这种合成的亲和层析树脂在弱酸条件下非常稳定,能选择性地吸附β-葡萄糖苷酶,并能多次重复使用。利用这种亲和层析树脂,在pH6和pH5条件下,从薮北种茶树叶片中纯化出两种分子量分别为63和75kDa的β-葡萄糖苷酶单体酶。以对硝基苯酚β-葡萄糖苷为底物,研究了两种酶的性质。TLC分析结果表明,这两种β-葡萄糖苷酶都能水解(Z)-3-己烯醇、苯甲醇和苯乙腈的β-D-葡萄糖苷。