Purification and Characterization of a Novel Fibrinolytic Enzyme from Marine Bacterium Bacillus sp. S-3685 Isolated from the South China Sea.

A novel fibrinolytic enzyme, BSFE1, was isolated from the marine bacterium Bacillus sp. S-3685 (GenBank No.: KJ023685) found in the South China Sea. This enzyme, with a molecular weight of approximately 42 kDa and a specific activity of 736.4 U/mg, exhibited its highest activity at 37 °C in a phosphate buffer at pH 8.0. The fibrinolytic enzyme remained stable over a pH range of 7.5 to 10.0 and retained about 76% of its activity after being incubated at 37 °C for 2 h. The Km and Vmax values of the enzyme at 37 °C were determined to be 2.1 µM and 49.0 µmol min-1 mg-1, respectively. The fibrinolytic activity of BSFE1 was enhanced by Na+, Ba2+, K+, Co2+, Mn2+, Al3+, and Cu2+, while it was inhibited by Fe3+, Ca2+, Mg2+, Zn2+, and Fe2+. These findings indicate that the fibrinolytic enzyme isolated in this study exhibits a strong affinity for fibrin. Moreover, the enzyme we have purified demonstrates thrombolytic enzymatic activity. These characteristics make BSFE1 a promising candidate for thrombolytic therapy. In conclusion, the results obtained from this study suggest that our work holds potential in the development of agents for thrombolytic treatment.

Stem-Cell-Regenerative and Protective Effects of Squid (Symplectoteuthis oualaniensis) Skin Collagen Peptides against H2O2-Induced Fibroblast Injury.

Recently, there has been a growing interest in collagen peptides derived from marine sources for their notable ability to protect skin cells against apoptosis induced by oxidants. Therefore, the current study aimed to investigate the fundamental properties of collagen peptides, including their physicochemical, thermal, structural, stem-cell-regenerative, and skin-cell-protective effects, in comparison to commercial collagen peptides. The acid-soluble (ASC) and pepsin-soluble (PSC) collagens exhibited three distinct bands on SDS-PAGE, namely α (α1 and α2), ß, and γ chains, confirming a type I pattern. The thermal profiles obtained from TG and DSC analyses confirmed the denaturation of PSC and ASC at temperatures ranging from 51.94 to 56.4 °C and from 52.07 to 56.53 °C, respectively. The purified collagen peptides were analyzed using SDS-PAGE and MALDI-TOF mass spectrometry, revealing a mass range of 900-15,000 Da. Furthermore, the de novo peptide sequence analysis confirmed the presence of the Gly-X-Y repeating sequence in collagen peptides. Collagen peptide treatments significantly enhanced HFF-1 cell proliferation and migration compared to the control group. ELISA results confirmed the potential interactions between collagen peptides and HFF-1 cells through α2ß1, α10ß1, and α11ß1 integrin receptors. Notably, collagen peptide treatment effectively restored the proliferation of HFF-1 cells damaged by H2O2. Consequently, the advantageous characteristics of squid skin collagen peptides highlight their promising role in regenerative medicine.

Isolation and Biochemical Properties of Type II Collagen from Blue Shark (Prionace glauca) Cartilage.

Numerous studies have shown that type II collagen (CII) has a potential role in the treatment of rheumatoid arthritis. However, most of the current studies have used terrestrial animal cartilage as a source of CII extraction, with fewer studies involving marine organisms. Based on this background, collagen (BSCII) was isolated from blue shark (Prionace glauca) cartilage by pepsin hydrolysis and its biochemical properties including protein pattern, total sugar content, microstructure, amino acid composition, spectral characteristics and thermal stability were further investigated in the present study. The SDS-PAGE results confirmed the typical characteristic of CII, comprising three identical α1 chains and its dimeric ß chain. BSCII had the fibrous microstructure typical of collagen and an amino acid composition represented by high glycine content. BSCII had the typical UV and FTIR spectral characteristics of collagen. Further analysis revealed that BSCII had a high purity, while its secondary structure comprised 26.98% of ß-sheet, 35.60% of ß-turn, 37.41% of the random coil and no α-helix. CD spectra showed the triple helical structure of BSCII. The total sugar content, denaturation temperature and melting temperature of BSCII were (4.20 ± 0.03)%, 42 °C and 49 °C, respectively. SEM and AFM images confirmed a fibrillar and porous structure of collagen and denser fibrous bundles formed at higher concentrations. Overall, CII was successfully extracted from blue shark cartilage in the present study, and its molecular structure was intact. Therefore, blue shark cartilage could serve as a potential source for CII extraction with applications in biomedicine.

The Role of Integrin Receptor's α and ß Subunits of Mouse Mesenchymal Stem Cells on the Interaction of Marine-Derived Blacktip Reef Shark (Carcharhinus melanopterus) Skin Collagen.

Marine collagen (MC) has recently attracted more attention in tissue engineering as a biomaterial substitute due to its significant role in cellular signaling mechanisms, especially in mesenchymal stem cells (MSCs). However, the actual signaling mechanism of MC in MSC growth, which is highly influenced by their molecular pattern, is poorly understood. Hence, we investigated the integrin receptors (α1ß1, α2ß1, α10ß1, and α11ß1) binding mechanism and proliferation of MCs (blacktip reef shark collagen (BSC) and blue shark collagen (SC)) compared to bovine collagen (BC) on MSCs behavior through functionalized collagen molecule probing for the first time. The results showed that BSC and SC had higher proliferation rates and accelerated scratch wound healing by increasing migratory rates of MSCs. Cell adhesion and spreading results demonstrated that MC had a better capacity to anchor MSCs and maintain cell morphology than controls. Living cell observations showed that BSC was gradually assembled by cells into the ECM network within 24 h. Interestingly, qRT-PCR and ELISA revealed that the proliferative effect of MC was triggered by interacting with specific integrin receptors such as α2ß1, α10ß1, and α11ß1 of MSCs. Accordingly, BSC accelerated MSCs' growth, adhesion, shape, and spreading by interacting with specific integrin subunits (α2 and ß1) and thereby triggering further signaling cascade mechanisms.

Comparison of Physicochemical and Structural Properties of Acid-Soluble and Pepsin-Soluble Collagens from Blacktip Reef Shark Skin.

Fish collagen has been widely used in tissue engineering (TE) applications as an implant, which is generally transplanted into target tissue with stem cells for better regeneration ability. In this case, the success rate of this research depends on the fundamental components of fish collagen such as amino acid composition, structural and rheological properties. Therefore, researchers have been trying to find an innovative raw material from marine origins for tissue engineering applications. Based on this concept, collagens such as acid-soluble (ASC) and pepsin-soluble (PSC) were extracted from a new type of cartilaginous fish, the blacktip reef shark, for the first time, and were further investigated for physicochemical, protein pattern, microstructural and peptide mapping. The study results confirmed that the extracted collagens resemble the protein pattern of type-I collagen comprising the α1, α2, ß and γ chains. The hydrophobic amino acids were dominant in both collagens with glycine and hydroxyproline as major amino acids. From the FTIR spectra, α helix (27.72 and 26.32%), ß-sheet (22.24 and 23.35%), ß-turn (21.34 and 22.08%), triple helix (14.11 and 14.13%) and random coil (14.59 and 14.12%) structures of ASC and PSC were confirmed, respectively. Collagens retained their triple helical and secondary structure well. Both collagens had maximum solubility at 3% NaCl and pH 4, and had absorbance maxima at 234 nm, respectively. The peptide mapping was almost similar for ASC and PSC at pH 2, generating peptides ranging from 15 to 200 kDa, with 23 kDa as a major peptide fragment. The microstructural analysis confirmed the homogenous fibrillar nature of collagens with more interconnected networks. Overall, the preset study concluded that collagen can be extracted more efficiently without disturbing the secondary structure by pepsin treatment. Therefore, the blacktip reef shark skin could serve as a potential source for collagen extraction for the pharmaceutical and biomedical applications.

FGFC1 Exhibits Anti-Cancer Activity via Inhibiting NF-κB Signaling Pathway in EGFR-Mutant NSCLC Cells.

FGFC1, an active compound isolated from the culture of marine fungi Stachybotrys longispora FG216, elicits fibrinolytic, anti-oxidative, and anti-inflammatory activity. We have previously reported that FGFC1 inhibited the proliferation, migration, and invasion of the non-small cell lung cancer (NSCLC) cells in vitro. However, the precise mechanisms of FGFC1 on NSCLC and its anti-cancer activity in vivo remains unclear. Hence, this study was focused to investigate the effects and regulatory mechanisms of FGFC1 on two NSCLC cell lines, EGFR-mutant PC9 (ex19del) and EGFR wild-type H1299. Results suggested that FGFC1 significantly inhibited proliferation, colony formation, as well as triggered G0/G1 arrest and apoptosis of PC9 cells in a dose- and time-dependent manner, but no obvious inhibitory effects were observed in H1299 cells. Subsequently, transcriptome analysis revealed that FGFC1 significantly down-regulated 28 genes related to the NF-κB pathway, including IL-6, TNF-α, and ICAM-1 in the PC9 cells. We further confirmed that FGFC1 decreased the expression of protein p-IKKα/ß, p-p65, p-IκB, IL-6, and TNF-α. Moreover, NF-κB inhibitor PDTC could strengthen the effects of FGFC1 on the expression of CDK4, Cyclin D1, cleaved-PARP-1, and cleaved-caspase-3 proteins, suggesting that the NF-κB pathway plays a major role in FGFC1-induced cell cycle arrest and apoptosis. Correspondingly, the nuclear translocation of p-p65 was also suppressed by FGFC1 in PC9 cells. Finally, the intraperitoneal injection of FGFC1 remarkably inhibited PC9 xenograft growth and decreased the expression of Ki-67, p-p65, IL-6, and TNF-α in tumors. Our results indicated that FGFC1 exerted anti-cancer activity in PC9 cells via inhibiting the NF-κB signaling pathway, providing a possibility for FGFC1 to be used as a lead compound for the treatment of NSCLC in the future.

Enhancing osteoblast differentiation through small molecule-incorporated engineered nanofibrous scaffold.

OBJECTIVE: This study aimed to investigate the effect of small molecules incorporated into the engineered nanofibrous scaffold to enhance the osteoblast differentiation MATERIALS AND METHODS: Poly-ε-caprolactone (PCL) nanofiber matrices with lithium chloride (LiCl) were fabricated using the electrospinning technique. Scaffolds were characterized using scanning electron microscopy (SEM) and energy-dispersive X-ray (EDX). Scaffolds were seeded with MC3T3-E1 cells and assessed using Western blots (ß-catenin), alamarBlue assay (proliferation), qPCR (osteoblast differentiation), and mineralization (Alizarin Red staining). RESULTS: We observed LiCl nanofiber scaffolds induced concentration-dependent cell proliferation that correlated with an increased ß-catenin expression indicating sustained Wnt signaling. Next, we examined osteoblast differentiation markers such as osteocalcin (OCN) and Runt-related transcription factor 2 (Runx2) and noted increased expression in LiCl nanofiber scaffolds. We also noted increased bone morphogenetic protein (BMP-2, 4, and 7) expressions suggesting activated Wnt can promote cures to further osteogenic differentiation. Finally, Alizarin Red staining demonstrated increased mineral deposition in LiCl-incorporated nanofiber scaffolds. CONCLUSIONS: Together, these results indicated that LiCl-incorporated nanofiber scaffolds enhance osteoblast differentiation. CLINICAL RELEVANCE: Small molecule-incorporated nanofibrous scaffolds are an innovative clinical tool for bone tissue engineering.

Retinol-Loaded Poly(vinyl alcohol)-Based Hydrogels as Suitable Biomaterials with Antimicrobial Properties for the Proliferation of Mesenchymal Stem Cells.

Polyvinyl alcohol (PVA) hydrogels are well-known biomimetic 3D systems for mammalian cell cultures to mimic native tissues. Recently, several biomolecules were intended for use in PVA hydrogels to improve their biological properties. However, retinol, an important biomolecule, has not been combined with a PVA hydrogel for culturing bone marrow mesenchymal stem (BMMS) cells. Thus, for the first time, the effect of retinol on the physicochemical, antimicrobial, and cell proliferative properties of a PVA hydrogel was investigated. The ability of protein (3.15 nm) and mineral adsorption (4.8 mg/mL) of a PVA hydrogel was improved by 0.5 wt.% retinol. The antimicrobial effect of hydrogel was more significant in S. aureus (39.3 mm) than in E. coli (14.6 mm), and the effect was improved by increasing the retinol concentration. The BMMS cell proliferation was more upregulated in retinol-loaded PVA hydrogel than in the control at 7 days. We demonstrate that the respective in vitro degradation rate of retinol-loaded PVA hydrogels (RPH) (75-78% degradation) may promote both antibacterial and cellular proliferation. Interestingly, the incorporation of retinol did not affect the cell-loading capacity of PVA hydrogel. Accordingly, the fabricated PVA retinol hydrogel proved its compatibility in a stem cell culture and could be a potential biomaterial for tissue regeneration.

The hidden secrets of soluble RANKL in bone biology.

The discovery of cytokine tumor necrosis factor (TNF) in the 20th century revealed numerous secrets about organ development. In particular, the functions identified for the receptor activator of nuclear factor kappa-ß (NF-κß) ligand (also known as the RANKL/osteoprotegerin ligand (OPGL) or RANK ligand/TNFSF11) in the homeostasis of skeletal structure, function and regulation were not anticipated. Empirical evidence established the receptor-ligand interaction of RANKL with RANK in osteoclast formation. Reverse signaling of RANKL triggers NF-κß for the degradation of ß-catenin to inhibit bone formation. There is also evidence that RANKL modifies the behavior of other cells in the bone microenvironment, including osteoblasts, chondrocytes, endothelial cells and lymphocytes during normal (homeostatic) and diseased (osteoimmune) states. Two forms of RANKL, i.e., soluble and membrane-bound RANKL, are produced by bone cells. Even though soluble RANKL (sRANKL) and membrane-bound RANKL (mRANKL) both stimulate osteoclast formation in vitro, their biological roles are different. mRANKL triggers osteoclastogenesis by binding to RANK through cell-cell interaction; however, sRANKL released from osteogenic cells binds to RANK without cell-cell interaction. This review attempts to hypothesize how sRANKL functions biologically in bone and explore how this hypothesis might influence future research.

Natural Triterpenoids Isolated from Akebia trifoliata Stem Explants Exert a Hypoglycemic Effect via α-Glucosidase Inhibition and Glucose Uptake Stimulation in Insulin-Resistant HepG2 Cells.

The inhibition of α-glucosidase activity is a prospective approach to attenuate postprandial hyperglycemia in the treatment of type 2 diabetes mellitus (T2DM). Herein, the inhibition of α-glucosidase by three compounds T1 -T3 of Akebia trifoliata stem, namely hederagenin (T1 ), 3-epiakebonoic acid (T2 ), and arjunolic acid (T3 ) were investigated using enzyme kinetics and molecular docking analysis. The three triterpenoids exhibited excellent inhibitory activities against α-glucosidase. T1 -T3 showed the strongest inhibition with IC50 values of 42.1±5.4, 19.6±3.2, and 11.2±2.3 µM, respectively, compared to the acarbose positive control (IC50 =106.3±8.2). Enzyme inhibition kinetics showed that triterpenoids T1 -T3 demonstrated competitive, mixed, and noncompetitive-type inhibition against α-glucosidase, respectively. The inhibition constant (Ki ) values were 21.21, 7.70, and 3.18 µM, respectively. Docking analysis determined that the interaction of ligands T1 -T3 and α-glucosidase was mainly forced by hydrogen bonds and hydrophobic interactions, which could result in improved binding to the active site of the target enzyme. The insulin resistant (IR)-HepG2 cell model used in this study (HepG2 cells exposed to 10-7  M insulin for 24 h) and glucose uptake assays showed that compounds T1 -T3 had no cytotoxicity with concentrations ranging from 6.25 to 25 µM and displayed significant stimulation of glucose uptake in IR-HepG2 cells. Thus, triterpenoids T1 -T3 showed dual therapeutic effects of α-glucosidase inhibition and glucose uptake stimulation and could be used as potential medicinal resources to investigate new antidiabetic agents for the prevention or treatment of diabetes.

IL-6 exhibits both cis- and trans-signaling in osteocytes and osteoblasts, but only trans-signaling promotes bone formation and osteoclastogenesis.

Interleukin 6 (IL-6) supports development of bone-resorbing osteoclasts by acting early in the osteoblast lineage via membrane-bound (cis) or soluble (trans) receptors. Here, we investigated how IL-6 signals and modifies gene expression in differentiated osteoblasts and osteocytes and determined whether these activities can promote bone formation or support osteoclastogenesis. Moreover, we used a genetically altered mouse with circulating levels of the pharmacological IL-6 trans-signaling inhibitor sgp130-Fc to determine whether IL-6 trans-signaling is required for normal bone growth and remodeling. We found that IL-6 increases suppressor of cytokine signaling 3 (Socs3) and CCAAT enhancer-binding protein δ (Cebpd) mRNA levels and promotes signal transducer and activator of transcription 3 (STAT3) phosphorylation by both cis- and trans-signaling in cultured osteocytes. In contrast, RANKL (Tnfsf11) mRNA levels were elevated only by trans-signaling. Furthermore, we observed soluble IL-6 receptor release and ADAM metallopeptidase domain 17 (ADAM17) sheddase expression by osteocytes. Despite the observation that IL-6 cis-signaling occurs, IL-6 stimulated bone formation in vivo only via trans-signaling. Although IL-6 stimulated RANKL (Tnfsf11) mRNA in osteocytes, these cells did not support osteoclast formation in response to IL-6 alone; binucleated TRAP+ cells formed, and only in response to trans-signaling. Finally, pharmacological, sgp130-Fc-mediated inhibition of IL-6 trans-signaling did not impair bone growth or remodeling unless mice had circulating sgp130-Fc levels > 10 µg/ml. At those levels, osteopenia and impaired bone growth occurred, reducing bone strength. We conclude that high sgp130-Fc levels may have detrimental off-target effects on the skeleton.

Resveratrol and Celastrol Loaded Collagen Dental Implants Regulate Periodontal Ligament Fibroblast Growth and Osteoclastogenesis of Bone Marrow Macrophages.

Collagen is widely used for dental therapy in several ways such as films, 3D matrix, and composites, besides traditional Chinese medicine (TCM), has been used in tissue regeneration and wound healing application for centuries. Hence, the present study was targeted for the first time to fabricate collagen film with TCM such as resveratrol and celastrol in order to investigate the human periodontal ligament fibroblasts (HPLF) growth and bone marrow macrophages (BMM) derived osteoclastogenesis. Further, the physicochemical, mechanical and biological activities of collagen-TCM films crosslinked by glycerol and EDC-NHS (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide-N-hydroxysulfosuccinimide) were investigated. Collagen film characterization was significantly regulated by the nature of plasticizers like hydrophobic and degree of polarity. Interestingly, the collagen film's denaturation temperature was increased by EDC-NHS than glycerol. FT-IR data confirmed the functional group changes due to chemical interaction of collagen with TCM. Morphological changes of HPLF cells cultured in control and collagen films were observed by SEM. Importantly, the addition of resveratrol upregulated the proliferation of HPLF cells, while osteoclastogenesis of BMM cells treated with mCSF-RANKL was significantly downregulated by celastrol. Accordingly, the collagen-TCM film could be an interesting material for dental regeneration, and especially it is a therapeutic target to restrain the elevated bone resorption during osteoporosis.

Evaluation of Marine Diindolinonepyrane in Vitro and in Vivo: Permeability Characterization in Caco-2 Cells Monolayer and Pharmacokinetic Properties in Beagle Dogs.

A marine fibrinolytic compound was studied for use in thrombolytic therapy. Firstly, the absorption and transportation characteristics of 2,5-BHPA (2,5-BHPA:2,5-Bis-[8-(4,8-dimethyl-nona-3,7-dienyl)-5,7-dihydroxy-8-methyl-3-keto-1,2,7,8-tertahydro-6H-pyran[a]isoindol-2-yl]-pentanoic acid, a novel pyran-isoindolone derivative with bioactivity isolated from a rare marine microorganism in our laboratory) in the human Caco-2 cells monolayer model were investigated. We collected 2,5-BHPA in the cells to calculate the total recovery, and its concentration was analyzed by LC/MS/MS (Liquid Chromatography/ Mass Spectrum/ Mass Spectrum). The results showed that 2,5-BHPA has low permeability and low total recoveries in the Caco-2 cells membrane. Pharmacokinetics and tissue distribution of 2,5-BHPA were investigated in beagle dogs using HPLC (High Performance Liquid Chromatography) after intravenous administration of three different doses (7.5, 5.0, 2.5 mg·kg-1). Pharmacokinetic data indicated that 2,5-BHPA fitted well to a two-compartment model. Elimination half-lives (T1/2) were 49 ± 2, 48 ± 2, and 49 ± 2 min, respectively; the peak concentrations (Cmax) were 56.48 ± 6.23, 48.63 ± 5.53, and 13.64 ± 2.76 µg·mL-1, respectively; clearance rates (CL) were 0.0062 ± 0.0004, 0.0071 ± 0.0008, and 0.0092 ±0.0006 L·min-1·kg-1, respectively; mean retention times (MRT) were 28.17 ± 1.16, 26.23 ± 0.35, and 28.66 ± 0.84 min, respectively. The low penetrability of 2,5-BHPA indicated that the intravenous route of administration is more appropriate than the oral route. Meanwhile, 2,5-BHPA showed a good pharmacokinetic profile in beagle dogs. The tissue distribution showed that 2,5-BHPA could quickly distribute into the heart, intestines, liver, stomach, spleen, lungs, testicles, urine, intestine, kidneys, brain, and feces. The concentration of 2,5-BHPA was higher in the liver and bile. Interestingly, 2,5-BHPA was detected in the brain. Taken together, the above results suggested that our work might be beneficial in the development of agents for thrombolytic treatment.

Fish Collagen Surgical Compress Repairing Characteristics on Wound Healing Process In Vivo.

The development of biomaterials with the potential to accelerate wound healing is a great challenge in biomedicine. In this study, four types of samples including pepsin soluble collagen sponge (PCS), acid soluble collagen sponge (ACS), bovine collagen electrospun I (BCE I) and bovine collagen electrospun II (BCE II) were used as wound dressing materials. We showed that the PCS, ACS, BCE I and BCE II treated rats increased the percentage of wound contraction, reduced the inflammatory infiltration, and accelerated the epithelization and healing. PCS, ACS, BCE I, and BCE II significantly enhanced the total protein and hydroxyproline level in rats. ACS could induce more fibroblasts proliferation and differentiation than PCS, however, both PCS and ACS had a lower effect than BCE I and BCE II. PCS, ACS, BCE I, and BCE II could regulate deposition of collagen, which led to excellent alignment in the wound healing process. There were similar effects on inducing the level of cytokines including EGF, FGF, and vascular endothelial marker CD31 among these four groups. Accordingly, this study disclosed that collagens (PCS and ACS) from tilapia skin and bovine collagen electrospun (BCE I and BCE II) have significant bioactivity and could accelerate wound healing rapidly and effectively in rat model.

Evaluation of Differentiated Bone Cells Proliferation by Blue Shark Skin Collagen via Biochemical for Bone Tissue Engineering.

Collagen from a marine resource is believed to have more potential activity in bone tissue engineering and their bioactivity depends on biochemical and structural properties. Considering the above concept, pepsin soluble collagen (PSC) and acid soluble collagen (ASC) from blue shark (Prionace glauca) skin were extracted and its biochemical and osteogenic properties were investigated. The hydroxyproline content was higher in PSC than ASC and the purified collagens contained three distinct bands α1, α2, and ß dimer. The purity of collagen was confirmed by the RP-HPLC profile and the thermogravimetric data showed a two-step thermal degradation pattern. ASC had a sharp decline in viscosity at 20⁻30 °C. Scanning electron microscope (SEM) images revealed the fibrillar network structure of collagens. Proliferation rates of the differentiated mouse bone marrow-mesenchymal stem (dMBMS) and differentiated osteoblastic (dMC3T3E1) cells were increased in collagen treated groups rather than the controls and the effect was dose-dependent, which was further supported by higher osteogenic protein and mRNA expression in collagen treated bone cells. Among two collagens, PSC had significantly increased dMBMS cell proliferation and this was materialized through increasing RUNX2 and collagen-I expression in bone cells. Accordingly, the collagens from blue shark skin with excellent biochemical and osteogenic properties could be a suitable biomaterial for therapeutic application.

Proliferative and Osteogenic Supportive Effect of VEGF-Loaded Collagen-Chitosan Hydrogel System in Bone Marrow Derived Mesenchymal Stem Cells.

The use of hydrogel (HG) in regenerative medicine is an emerging field and thus several approaches have been proposed recently to find an appropriate hydrogel system. In this sense, this study developed a novel HG system using collagen, chitosan, and VEGF composites for culturing mesenchymal stem cells (MSCs), and investigated their ability for osteogenic differentiation and mineral deposition. Our results showed that the HG loaded with 100 ng/mL VEGF (HG-100) significantly supported the proliferation of undifferentiated MSCs, the fibrillary filament structure (HE stain), mineralization (alizarin red S and von Kossa stain), alkaline phosphatase, and the osteogenesis of differentiated MSCs compared to other hydrogels (loaded with 25 and 50 ng/mL VEGF) and control (without hydrogel). HG-100 showed a higher VEGF releasing rate from day 3 to day 7 than other HGs, which substantially supports the proliferative and osteogenic properties of HG-100. However, the HGs did not increase the cell growth in differentiated MSCs on days 14 and 21 due to the confluence state (reach stationary phase) and cell loading ability, regardless of the VEGF content. Similarly, the HGs alone did not stimulate the osteogenesis of MSCs; however, they increased the osteogenic ability of MSCs in presence of osteogenic supplements. Accordingly, a fabricated HG with VEGF could be used as an appropriate system to culture stem cells for bone and dental regeneration.

Protein Adsorption, Calcium-Binding Ability, and Biocompatibility of Silver Nanoparticle-Loaded Polyvinyl Alcohol (PVA) Hydrogels Using Bone Marrow-Derived Mesenchymal Stem Cells.

Several approaches have evolved to facilitate the exploration of hydrogel systems in biomedical research. In this sense, poly(vinyl alcohol) (PVA) has been widely used in hydrogel (HG) fabrication for several therapeutic applications. The biological properties of PVA hydrogels (PVA-HGs) are highly dependent on their interaction with protein receptors and extracellular matrix (mainly calcium) deposition, for which there is not enough evidence from existing research yet. Thus, for the first time, the functional properties, like protein and mineral interactions, related to the proliferation of mesenchymal stem cells (MSCs) by silver nanoparticle (AgNP)-loaded PVA hydrogels (AgNPs-PVA-HGs) were investigated in the present study. The UV absorption spectrum and TEM microscopic results showed a maximum absorbance of synthesized AgNPs at 409 nm, with an average particle size of 14.5 ± 2.5 nm, respectively. The functional properties, such as the calcium-binding and the protein adsorption of PVA-HG, were accelerated by incorporating AgNPs; however, the swelling properties of the HGs were reduced by AgNPs, which might be due to the masking of the free functional groups (hydroxyl groups of PVA) by AgNPs. SEM images showed the presence of AgNPs with a more porous structure in the HGs. The proliferative effect of MSCs increased over culture time from day 1 to day 7, and the cell proliferative effect was upregulated by HGs with more pronounced AgNPs-PVA-HG. In addition, both HGs did not produce any significant cytotoxicity in the MSCs. The histological (bright light and H&E staining) and fluorescence microscopic images showed the presence of a cytoskeleton and the fibrillar structure of the MSCs, and the cells adhered more firmly to all HGs. More fibrillar bipolar and dense fibrillar structures were seen in the day 1 and day 7 cultures, respectively. Interestingly, the MSCs cultured on AgNPs-PVA-HG produced extracellular matrix deposition on day 7. Accordingly, the present results proved the biocompatibility of AgNPs-PVA-HG as a suitable system for culturing mammalian stem cells for regenerative tissue applications.

Collagen from Iris squid grafted with polyethylene glycol and collagen peptides promote the proliferation of fibroblast through PI3K/AKT and Ras/RAF/MAPK signaling pathways.

Collagens from marine sources have been used widely in food, cosmetics and tissue engineering application due to their excellent functional and biological properties. In the present study, a novel protein, collagen from iris squid skin (SSC) was characterized, grafted with polyethylene-glycol (PEG) and Acid-Green 20 (AG) and was investigated the molecular signaling pathways in L-929 fibroblast cells along with their structural peptide analogs. SDS-PAGE and IR spectrum of SSC analysis showed the typical structure of type I collagen. The fibroblast proliferation was evaluated for SSC, SSC grafted PEG (SSC-PEG) and their structural analogs including Gly-Pro-Leu-Gly-Leu-Leu (PEP1), Gly-Pro-Leu-Gly-Leu-Leu-Gly-Phe-Leu (PEP2), Gly-Pro-Leu-Gly-Leu-Leu-Gly-Phe-Leu-Gly-Pro-Leu (PEP3) and Gly-Pro-Leu-Gly-Leu-Leu-Gly-Phe-Leu-Gly-Pro-Leu-Gly-Leu-Ser (PEP4). The optimal concentration of SSC and its derivative was 0.07 µ mol/L. The fibroblast growth-promoting factors were promoted by all the treatment groups by accelerating the PI3K/AKT and Ras/RAF/MAPK signaling pathways in L-929 cells, and inhibiting the secretion of apoptotic factors. Compared to the control group, mRNA and protein expression of AKT in the PI3K/AKT and Ras in Ras/RAF/MAPK signaling pathway were accelerated significantly by PEP4, respectively, while the Bax value was significantly lower (P < 0.01). The promoting effect of PEP1, PEP2, PEP3 and PEP4 on L-929 cells was closely related to the length of the peptides. Therefore, this study disclosed that PEP1, PEP2, PEP3 and PEP4 were novel analogs that greatly promote the proliferation of L-929 cells through PI3K/AKT and Ras/RAF/MAPK signaling pathways.

Fabrication, Physical-Chemical and Biological Characterization of Retinol-Loaded Poly(vinyl Alcohol) Electrospun Fiber Mats for Wound Healing Applications.

Nowadays, there exists a huge interest in producing innovative, high-performance, biofunctional, and cost-efficient electrospun biomaterials based on the association of biocompatible polymers with bioactive molecules. Such materials are well-known to be promising candidates for three-dimensional biomimetic systems for wound healing applications because they can mimic the native skin microenvironment; however, many open questions such as the interaction mechanism between the skin and the wound dressing material remain unclear. Recently, several biomolecules were intended for use in combination with poly(vinyl alcohol) (PVA) fiber mats to improve their biological response; nevertheless, retinol, an important biomolecule, has not been combined yet with PVA to produce tailored and biofunctional fiber mats. Based on the abovementioned concept, the present work reported the fabrication of retinol-loaded PVA electrospun fiber mats (RPFM) with a variable content of retinol (0 ≤ Ret ≤ 25 wt.%), and their physical-chemical and biological characterization. SEM results showed that fiber mats exhibited diameters distribution ranging from 150 to 225 nm and their mechanical properties were affected with the increasing of retinol concentrations. In addition, fiber mats were able to release up to 87% of the retinol depending on both the time and the initial content of retinol. The cell culture results using primary mesenchymal stem cell cultures proved the biocompatibility of RPFM as confirmed by their effects on cytotoxicity (low level) and proliferation (high rate) in a dose-dependent manner. Moreover, the wound healing assay suggested that the optimal RPFM with retinol content of 6.25 wt.% (RPFM-1) enhanced the cell migratory activity without altering its morphology. Accordingly, it is demonstrated that the fabricated RPFM with retinol content below the threshold 0 ≤ Ret ≤ 6.25 wt.% would be an appropriate system for skin regenerative application.

Characterization of Immunogenicity Associated with the Biocompatibility of Type I Collagen from Tilapia Fish Skin.

Collagen from fish has been proven to have a low antigenicity that has no difference in the genetic codes compared with mammalian-based collagen. This study was designed to investigate the impact of tilapia skin collagen on immunogenicity and biocompatibility in vivo and in vitro. The structural characteristics of both acid-soluble and pepsin-soluble collagen (ASC and PSC), determined using SDS-PAGE and atomic force microscopy imaging experiments, revealed that the collagen had the basic characteristics of type I collagen (COL-I). The in vitro biocompatibility of the collagens showed good cell proliferation against human foreskin fibroblast (HFF-1) cells. PSC and ASC were considered to be almost non-hemolytic biomaterials with favorable blood compatibility in hemolysis tests. The in vivo antigenicity of the collagen in an ICR mouse model evoked an acceptable specific inflammatory response compared to bovine collagen. The implant's position had developed a complete granulation tissue and the sponge disappeared after 8 weeks. The level of cytokines produced by the COL-I immune response was much lower than bovine collagen, which indicated the appropriate implantable property and biodegradability of the collagens. In conclusion, the tilapia COL-I has a lower immunogenicity with better compatibility than bovine COL-I and is a potential alternative to conventional mammalian collagens in biomedical uses.