Jirut Meesane

Extraction and characterisation of pepsin‐solubilised collagens from the skin of bigeye snapper (Priacanthus tayenus and Priacanthus macracanthus)

BACKGROUND Fish collagen has been paid increasing attention as an alternative to the mammalian counterpart owing to the abundance of fish skin as a processing by-product. Generally, the low yield of collagen extracted using the typical acid solubilisation process has led to the use of mammalian pepsin as an aid for increasing the yield. Alternatively, fish pepsin, especially from tuna stomach, can be used for the extraction of pepsin-solubilised collagen (PSC). Therefore the objective of this study was to extract and characterise PSC from the skin of bigeye snapper, a fish widely used for surimi production in Thailand. RESULTS PSCs from the skin of two species of bigeye snapper, Priacanthus tayenus and Priacanthus macracanthus, were extracted with the aid of tongol tuna (Thunnus tonggol) pepsin and porcine pepsin. PSCs from the skin of both species extracted using porcine pepsin had a higher content of beta-chain but a lower content of alpha-chains compared with those extracted using tuna pepsin. All PSCs contained glycine as the major amino acid and had an imino acid (proline and hydroxyproline) content of 189-193 residues per 1000 residues. Transition temperatures of PSCs were in the range 30.6-31.3 degrees C. Fourier transform infrared spectra revealed some differences in molecular order between PSCs extracted using porcine pepsin and tuna pepsin. Nevertheless, the triple-helical structure of PSCs was not affected by pepsin digestion. Zeta potential analysis indicated that PSCs from P. tayens and P. macracanthus possessed zero net charge at pH 7.15-7.46 and 5.97-6.44 respectively. CONCLUSION Tongol tuna pepsin could be used as a replacement for mammalian pepsin in PSC extraction. However, a slight difference in PSC properties was found.

Modified silk fibroin scaffolds with collagen/decellularized pulp for bone tissue engineering in cleft palate: Morphological structures and biofunctionalities

Cleft palate is a congenital malformation that generates a maxillofacial bone defect around the mouth area. The creation of performance scaffolds for bone tissue engineering in cleft palate is an issue that was proposed in this research. Because of its good biocompatibility, high stability, and non-toxicity, silk fibroin was selected as the scaffold of choice in this research. Silk fibroin scaffolds were prepared by freeze-drying before immerging in a solution of collagen, decellularized pulp, and collagen/decellularized pulp. Then, the immersed scaffolds were freeze-dried. Structural organization in solution was observed by Atomic Force Microscope (AFM). The molecular organization of the solutions and crystal structure of the scaffolds were characterized by Fourier transform infrared (FT-IR) and X-ray diffraction (XRD), respectively. The weight increase of the modified scaffolds and the pore size were determined. The morphology was observed by a scanning electron microscope (SEM). Mechanical properties were tested. Biofunctionalities were considered by seeding osteoblasts in silk fibroin scaffolds before analysis of the cell proliferation, viability, total protein assay, and histological analysis. The results demonstrated that dendrite structure of the fibrils occurred in those solutions. Molecular organization of the components in solution arranged themselves into an irregular structure. The fibrils were deposited in the pores of the modified silk fibroin scaffolds. The modified scaffolds showed a beta-sheet structure. The morphological structure affected the mechanical properties of the silk fibroin scaffolds with and without modification. Following assessment of the biofunctionalities, the modified silk fibroin scaffolds could induce cell proliferation, viability, and total protein particularly in modified silk fibroin with collagen/decellularized pulp. Furthermore, the histological analysis indicated that the cells could adhere in modified silk fibroin scaffolds. Finally, it can be deduced that modified silk fibroin scaffolds with collagen/decellularized pulp had the performance for bone tissue engineering and a promise for cleft palate treatment.

Mimicked cartilage scaffolds of silk fibroin/hyaluronic acid with stem cells for osteoarthritis surgery: Morphological, mechanical, and physical clues

Osteoarthritis is a critical disease that comes from degeneration of cartilage tissue. In severe cases surgery is generally required. Tissue engineering using scaffolds with stem cell transplantation is an attractive approach and a challenge for orthopedic surgery. For sample preparation, silk fibroin (SF)/hyaluronic acid (HA) scaffolds in different ratios of SF/HA (w/w) (i.e., 100:0, 90:10, 80:20, and 70:30) were formed by freeze-drying. The morphological, mechanical, and physical clues were considered in this research. The morphological structure of the scaffolds was observed by scanning electron microscope. The mechanical and physical properties of the scaffolds were analyzed by compressive and swelling ratio testing, respectively. For the cell experiments, scaffolds were seeded and cultured with human umbilical cord-derived mesenchymal stem cells (HUMSCs). The cultured scaffolds were tested for cell viability, histochemistry, immunohistochemistry, and gene expression. The SF with HA scaffolds showed regular porous structures. Those scaffolds had a soft and elastic characteristic with a high swelling ratio and water uptake. The SF/HA scaffolds showed a spheroid structure of the cells in the porous structure particularly in the SF80 and SF70 scaffolds. Cells could express Col2a, Agg, and Sox9 which are markers for chondrogenesis. It could be deduced that SF/HA scaffolds showed significant clues for suitability in cartilage tissue engineering and in surgery for osteoarthritis.

Modified porous scaffolds of silk fibroin with mimicked microenvironment based on decellularized pulp/fibronectin for designed performance biomaterials in maxillofacial bone defect.

Maxillofacial bone defect is a critical problem for many patients. In severe cases, the patients need an operation using a biomaterial replacement. Therefore, to design performance biomaterials is a challenge for materials scientists and maxillofacial surgeons. In this research, porous silk fibroin scaffolds with mimicked microenvironment based on decellularized pulp and fibronectin were created as for bone regeneration. Silk fibroin scaffolds were fabricated by freeze-drying before modification with three different components: decellularized pulp, fibronectin, and decellularized pulp/fibronectin. The morphologies of the modified scaffolds were observed by scanning electron microscopy. Existence of the modifying components in the scaffolds was proved by the increase in weights and from the pore size measurements of the scaffolds. The modified scaffolds were seeded with MG-63 osteoblasts and cultured. Testing of the biofunctionalities included cell viability, cell proliferation, calcium content, alkaline phosphatase activity (ALP), mineralization and histological analysis. The results demonstrated that the modifying components organized themselves into aggregations of a globular structure. They were arranged themselves into clusters of aggregations with a fibril structure in the porous walls of the scaffolds. The results showed that modified scaffolds with a mimicked microenvironment of decellularized pulp/fibronectin were suitable for cell viability since the cells could attach and spread into most of the pores of the scaffold. Furthermore, the scaffolds could induce calcium synthesis, mineralization, and ALP activity. The results indicated that modified silk fibroin scaffolds with a mimicked microenvironment of decellularized pulp/fibronectin hold promise for use in tissue engineering in maxillofacial bone defects.

Physicochemical properties and responses in microcirculation of native tapioca starch-based plasma expander

Plasma expanders (PEs) such as hydroxyethyl strach are widely used for volume replacement. The plantation and production of tapioca in Thailand is abundant which may provide a new source for PEs starch with novel properties. This work investigated the properties and circulatory effects of native tapioca starch-based PE (TPE). Various formulations of mixture between native tapioca starch and 0.9% sodium chloride solution were prepared and characterized in order to obtain the proper physicochemical and rheological properties. About 1% concentration by weight per volume of TPE was compared with 6% hydroxyethyl starch 130/0.4 in 0.9% sodium chloride (HES130/0.4) using an acute hemodilution by 40% of blood volume in an animal protocol. TPE had higher turbidity and viscosity but lower colloid osmotic pressure compared with HES 130/0.4. The in vivo study demonstrated that Golden Syrian hamsters hemodiluted with TPE maintained a mean arterial blood pressure and no significant difference compared to HES 130/0.4. The arterial vasodilation and functional capillary density in the animals hemodiluted with TPE had higher values than in the animals hemodiluted with HES 130/0.4. Although the in vivo study reported positive results using this native tapioca starch-based PE, the product needs work to improve some of its physiochemical properties.

In vivo evaluation of modified silk fibroin scaffolds with a mimicked microenvironment of fibronectin/decellularized pulp tissue for maxillofacial surgery

This study aimed to carry out in vivo testing of the formation of new bone by modified silk fibroin scaffolds with a mimicked microenvironment of fibronectin/decellularized pulp in bone defects. Silk fibroin scaffolds were fabricated into three-dimensional scaffolds before being coated with fibronectin/decellularized pulp. The coated scaffolds were implanted into rabbits. Twenty-four bicortical calvarial defects in 12 rabbits were divided randomly into two groups: non-coated and coated silk fibroin scaffolds. The rabbits were sacrificed 2, 4 and 8 weeks after operation for evaluation of new bone formation. The morphology of the scaffolds, new bone formation and histology were evaluated by scanning electron microscopy, micro-CT and hematoxylin and eosin staining, respectively. The results showed that the coated silk fibroin scaffolds had a fibrillar network and crystal particles in the porous structure. The coated silk fibroin scaffolds demonstrated the ability to induce the formation of new bone with low inflammation and high vascularization. The results indicated that the modified silk fibroin scaffolds showed suitable biological performance and promise for bone regeneration in maxillofacial surgery.

Freeze-Thawed Hybridized Preparation with Biomimetic Self-Assembly for a Polyvinyl Alcohol/Collagen Hydrogel Created for Meniscus Tissue Engineering

Freeze-thawed hybridized preparation and the biomimetic self-assembly technique were used to fabricate hydrogel as tissue engineered scaffolds for meniscus tissue. Because of the advantages of both techniques, they were hybridized together as an interesting preparation for hydrogel. Three molecular weights (high, medium, and low) of PVA were prepared in a biomimetic solution before formation into hydrogel by freeze-thawing. The most suitable molecular weight PVA for hydrogel formation was chosen to be mixed with collagen. PVA, PVA/collagen, and collagen were prepared in biomimetic solutions and freeze-thawed into hydrogels. The hydrogels were analyzed and characterized by FTIR, DSC, and SEM. FTIR characterization indicated that high molecular weight PVA formed molecular interaction better than the other molecular weights, and PVA molecules formed molecular interaction with collagen molecules via –OH and C=O groups. DSC characterization showed that the hybridized preparation of freeze-thawing and biomimetic self-assembly kept the characteristics of PVA and collagen. SEM analysis demonstrated that the morphological formation of PVA/collagen was hybridized during freeze-thawing and collagen self-assembly. The morphological structure was organized into a porous network structure. The porous structure showed a rough wall that was formed by the hybridized structure of the crystal domain dispersed in amorphous and collagen self-assembly.

TiC-Coated Carbon Black Particles as a Bioactive Ceramic Compound for Application of Bone Tissue Engineering

Titanium carbide-coated carbon black particles (TCBs) were synthesized via reaction of metallic titanium powder with carbon black powder in a molten potassium chloride at 900°C for 4 hours in argon atmosphere to study their biological properties to be used as a bioactive ceramic compound for bone formation. Phase compositions and morphologies were characterized by electron diffraction spectroscopy (EDS) and scanning electron microscope (SEM). Core-shell structure was examined by transmission electron microscope (TEM). Biofunctionalities were considered by cell proliferation and protein synthesis. titanium and carbon peak were found, on an EDS curve of the as-synthesized powder, indicating the complete reaction. The shape and size of as-synthesized powder and as-received carbon black powder are similar, as it is noticed the template growth mechanism of the reaction. However, the surface morphology of as-synthesized powder is different because of its rough surface. Core-shell structure of the as-synthesized powder was revealed by TEM that an ultra-thin layer of titanium carbide was successfully coated on a carbon black particle. Biological testing showed, as compared to a control, a significant enhancement in cell proliferation and protein synthesis of the as-synthesized TCBs suggesting a good candidate as a bioactive compound for biomedical applications.

Preparation and Characterization of an In Situ Hydrogel of Self-Assembly Type I Collagen from Shark Skin/Methylcellulose for Central Nerve System Regeneration

Central nerve system degeneration is a crucial problem for many patients. To use an in situ hydrogel formation is an attractive method to treat that problem. An in situ hydrogel was developed for central nerve system regeneration. An acid soluble collagen (ASC) and pepsin soluble collagen (PSC) from the shark skin of the brownbanded bamboo shark (Chiloscyllium punctatum) were used to produce hybridized hydrogels by the biomimetic approach. Collagen was mixed with methylcellulose and used 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) as a crosslinker. The hydrogels had various ratios of collagen:methylcellulose: 100:0, 70:30, 50:50, 30:70, and 0:100. Structural, molecular, and morphological organization were characterized and observed by differential scanning calorimetry (DSC), Fourier transform infrared spectroscopy (FT-IR), and scanning electron microscopy (SEM). The DSC results showed that the peak of denatured collagen fibril shifted higher in a 30:70 ratio of collagen:methylcellulose in both ASC and PSC. The FT-IR results indicated that the structure of hydrogels from both ASC and PSC were organized into complex structures. The SEM results demonstrated that the collagen fibril networks were formed in both ASC and PSC hydrogels. The results indicated that the samples containing collagen promise to be an in situ hydrogel for central nerve regeneration.