Fabrication of Gelatin-Based Electrospun Composite Fibers for Anti-Bacterial Properties and Protein Adsorption

A major goal of biomimetics is the development of chemical compositions and structures that simulate the extracellular matrix. In this study, gelatin-based electrospun composite fibrous membranes were prepared by electrospinning to generate bone scaffold materials. The gelatin-based multicomponent composite fibers were fabricated using co-electrospinning, and the composite fibers of chitosan (CS), gelatin (Gel), hydroxyapatite (HA), and graphene oxide (GO) were successfully fabricated for multi-function characteristics of biomimetic scaffolds. The effect of component concentration on composite fiber morphology, antibacterial properties, and protein adsorption were investigated. Composite fibers exhibited effective antibacterial activity against Staphylococcus aureus and Escherichia coli. The study observed that the composite fibers have higher adsorption capacities of bovine serum albumin (BSA) at pH 5.32–6.00 than at pH 3.90–4.50 or 7.35. The protein adsorption on the surface of the composite fiber increased as the initial BSA concentration increased. The surface of the composite reached adsorption equilibrium at 20 min. These results have specific applications for the development of bone scaffold materials, and broad implications in the field of tissue engineering.     

Mesenchymal Stem Cells as an Alternative for Schwann Cells in Rat Spinal Cord Injury

Background: Spinal cord has a limited capacity to repair; therefore, medical interventions are necessary for treatment of injuries. Transplantation of Schwann cells has shown a great promising result for spinal cord injury (SCI). However, harvesting Schwann cell has been limited due to donor morbidity and limited expansion capacity. Furthermore, accessible sources such as bone marrow stem cells have drawn attentions to themselves. Therefore, this study was designed to evaluate the effect of bone marrow-derived Schwann cell on functional recovery in adult rats after injury. Methods: Mesenchymal stem cells were cultured from adult rats’ bone marrow and induced into Schwann cells in vitro. Differentiation was confirmed by immunocytochemistry and RT-PCR. Next, Schwann cells were seeded into collagen scaffolds and engrafted in 3 mm lateral hemisection defects. For 8 weeks, motor and sensory improvements were assessed by open field locomotor scale, narrow beam, and tail flick tests. Afterwards, lesioned spinal cord was evaluated by conventional histology and immunohistochemistry. Results: In vitro observations showed that differentiated cells had Schwann cell morphology and markers. In this study, we had four groups (n = 10 each): laminectomy, control, scaffold and scaffold + Schwann cells. Locomotor and sensory scores of cell grafted group were significantly better than control and scaffold groups. In histology, axonal regeneration and remyelination were better than control and scaffold groups. Conclusion: This study demonstrates that bone marrow-derived Schwann cells can be considered as a cell source for Schwann cells in SCI treatment. Key Words: Rats, Spinal cord injuries (SCI), Bone marrow, Schwann cells, Cell transdifferentiation     

Increased Osteogenic Potential of Pre-Osteoblasts on Three-Dimensional Printed Scaffolds Compared to Porous Scaffolds for Bone Regeneration

Background:One of the main challenges with conventional scaffold fabrication methods is the inability to control scaffold architecture. Recently, scaffolds with controlled shape and architecture have been fabricated using 3D-printing. Herein, we aimed to determine whether the much tighter control of microstructure of 3DP PLGA/β-TCP scaffolds is more effective in promoting osteogenesis than porous scaffolds produced by solvent casting/porogen leaching. Methods:Physical and mechanical properties of porous and 3DP scaffolds were studied. The response of pre-osteoblasts to the scaffolds was analyzed after 14 days. Results:The 3DP scaffolds had a smoother surface (R_a: 22 ± 3 µm) relative to the highly rough surface of porous scaffolds (R_a: 110 ± 15 µm). Water contact angle was 112 ± 4° on porous and 76 ± 6° on 3DP scaffolds. Porous and 3DP scaffolds had the pore size of 408 ± 90 and 315 ± 17 µm and porosity of 85 ± 5% and 39 ± 7%, respectively. Compressive strength of 3DP scaffolds (4.0 ± 0.3 MPa) was higher than porous scaffolds (1.7 ± 0.2 MPa). Collagenous matrix deposition was similar on both scaffolds. Cells proliferated from day 1 to day 14 by fourfold in porous and by 3.8-fold in 3DP scaffolds. ALP activity was 21-fold higher in 3DP scaffolds than porous scaffolds. Conclusion:The 3DP scaffolds show enhanced mechanical properties and ALP activity compared to porous scaffolds in vitro, suggesting that 3DP PLGA/β-TCP scaffolds are possibly more favorable for bone formation. Key Words: Alkaline phosphatase, β-tricalcium phosphate, Poly(lactic-co-glycolic) acid copolymer     

Fabrication and Characterization of Nanocomposite Hydrogel Based on Alginate/Nano-Hydroxyapatite Loaded with Linum usitatissimum Extract as a Bone Tissue Engineering Scaffold

In the current paper, we fabricated, characterized, and applied nanocomposite hydrogel based on alginate (Alg) and nano-hydroxyapatite (nHA) loaded with phenolic purified extracts from the aerial part of Linum usitatissimum (LOH) as the bone tissue engineering scaffold. nHA was synthesized based on the wet chemical technique/precipitation reaction and incorporated into Alg hydrogel as the filler via physical cross-linking. The characterizations (SEM, DLS, and Zeta potential) revealed that the synthesized nHA possess a plate-like shape with nanometric dimensions. The fabricated nanocomposite has a porous architecture with interconnected pores. The average pore size was in the range of 100–200 µm and the porosity range of 80–90%. The LOH release measurement showed that about 90% of the loaded drug was released within 12 h followed by a sustained release over 48 h. The in vitro assessments showed that the nanocomposite possesses significant antioxidant activity promoting bone regeneration. The hemolysis induction measurement showed that the nanocomposites were hemocompatible with negligible hemolysis induction. The cell viability/proliferation confirmed the biocompatibility of the nanocomposites, which induced proliferative effects in a dose-dependent manner. This study revealed the fabricated nanocomposites are bioactive and osteoactive applicable for bone tissue engineering applications.     

Effect of Dioxane and N-Methyl-2-pyrrolidone as a Solvent on Biocompatibility and Degradation Performance of PLGA/nHA Scaffolds

Background:Solvent casting/particulate leaching is one of the most conventional methods for fabricating polymer/ceramic composite scaffolds. In this method, the solvent generally affects resulting scaffold properties, including porosity and degradation rate. Methods:Herein, composite scaffolds of PLGA/nHA with different percentages of nHA (25, 35, and 45 wt. %) were prepared by the solvent casting/particle leaching combined with freeze drying. The effects of two different solvents, DIO and NMP, on morphology, porosity, bioactivity, degradation rate, and biocompatibility of the resulting scaffolds were investigated. Results:The results revealed that increasing the nHA percentages had no significant effect on the porosity and interconectivity of scaffolds (p > 0.05), whereas altering the solvent from DIO into NMP decreased the porosity from about 87% into 71%, respectively. Moreover, scaffolds of DIO illustrated the high results of cell proliferation compared to those of NMP; the cell viability of GD25 decreased from 85% to 65% for GN25. The findings also indicated that scaffolds prepared by NMP had a higher rate of losing weight in comparison to DIO. Adding nHA to PLGA had a significant effect on the bioactivity of scaffolds (p < 0.05), composite scaffolds with 45 wt % nHA had at least 30% more weight gain compared to the neat polymer scaffolds. Conclusion:The DIO scaffolds have higher rates of porosity, interconnectivity, bioactivity, and biocompatibility than NMP scaffolds due to its high evaporation rate. Key Words: Freeze drying, Porosity, Solvents     

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.     

Ximaoornatins A–C,Polyoxygenated Diterpenoids from the Hainan Soft Coral Sinularia ornata

Three complex polyoxygenated diterpenoids possessing uncommon tetradecahydro-2,13:6,9-diepoxybenzo[10]annulene scaffold, namely ximaoornatins A–C (1–3), one new eunicellin-type diterpene, litophynin K (4), and a related known compound, litophynol B (5) were isolated from the South China Sea soft coral Sinularia ornata. The structures and absolute configurations of 1–4 were established by extensive spectroscopic analysis, X-ray diffraction analysis, and/or modified Mosher’s method. A plausible biosynthetic relationship of 1 and its potential precursor 4 was proposed. In a bioassay, none of the isolated compounds showed obvious anti-inflammatory activity on LPS-induced TNF-α release in RAW264.7 macrophages and PTP1B inhibitory effects.     

Pyranodipyran Derivatives with Tyrosyl DNA Phosphodiesterase 1 Inhibitory Activities and Fluorescent Properties from Aspergillus sp. EGF 15-0-3

Four new benzodipyran racemates, namely (±)-aspergiletals A–D (3–6), representing a rare pyrano[4,3-h]chromene scaffold were isolated together with eurotiumide G (1) and eurotiumide F (2) from the soft-coral-derived fungus Aspergillus sp. EGF 15-0-3. All the corresponding optically pure enantiomers were successfully separated by a chiral HPLC column. The structures and configurations of all the compounds were elucidated based on the combination of NMR and HRESIMS data, chiral separation, single-crystal X-ray diffraction, quantum chemical ¹³C NMR, and electronic circular dichroism calculations. Meanwhile, the structure of eurotiumide G was also revised. The TDP1 inhibitor activities and photophysical properties of the obtained compounds were evaluated. In the TDP1 inhibition assay, as a result of synergy between (+)-6 and (−)-6, (±)-6 displayed strong inhibitory activity to TDP1 with IC₅₀ values of 6.50 ± 0.73 μM. All compounds had a large Stokes shift and could be utilized for elucidating the mode of bioactivities by fluorescence imaging.     

Identification and Heterologous Expression of the Kendomycin B Biosynthetic Gene Cluster from Verrucosispora sp. SCSIO 07399

Verrucosispora sp. SCSIO 07399, a rare marine-derived actinomycete, produces a set of ansamycin-like polyketides kendomycin B–D (1–3) which possess potent antibacterial activities and moderate tumor cytotoxicity. Structurally, kendomycin B–D contain a unique aliphatic macrocyclic ansa scaffold in which the highly substituted pyran ring is connected to the quinone moiety. In this work, a type I/type III polyketide synthase (PKS) hybrid biosynthetic gene cluster coding for assembly of kendomycin B (kmy), and covering 33 open reading frames, was identified from Verrucosispora sp. SCSIO 07399. The kmy cluster was found to be essential for kendomycin B biosynthesis as verified by gene disruption and heterologous expression. Correspondingly, a biosynthetic pathway was proposed based on bioinformatics, cluster alignments, and previous research. Additionally, the role of type III PKS for generating the precursor unit 3,5-dihydroxybenzoic acid (3,5-DHBA) was demonstrated by chemical complementation, and type I PKS executed the polyketide chain elongation. The kmy cluster was found to contain a positive regulatory gene kmy4 whose regulatory effect was identified using real-time quantitative PCR (RT-qPCR). These advances shed important new insights into kendomycin B biosynthesis and help to set the foundation for further research aimed at understanding and exploiting the carbacylic ansa scaffold.     

Separacenes A–D,Novel Polyene Polyols from the Marine Actinomycete,Streptomyces sp

Separacenes A–D (1–4), novel polyene polyols, were isolated from Streptomyces sp. collected from the southern area of Jeju Island, Korea. The chemical structures of 1–4 were established by NMR, mass, UV, and IR spectroscopy as well as the modified Mosher’s method. Separacenes A–B (1–2), which share an identical planar structure but possess different relative configurations, bear tetraene units flanked by two diol moieties, whereas the stereoisomeric separacenes C–D (3–4) possess a triene moiety between two diol substructures. Separacenes A–D each contain a terminal olefinic methylene. Separacene A displayed inhibitory activity against Candida albicans isocitrate lyase and weak cytotoxicity against both the colon carcinoma cell line HCT-116 and the lung cancer cell line A549.     

Parathyrsoidins A–D,Four New Sesquiterpenoids from the Soft Coral Paralemnalia thyrsoides

Four new nardosinane-type sesquiterpenoids, parathyrsoidins A–D (1–4) were isolated from the soft coral Paralemnalia thyrsoides. The structures of parathyrsoidins A–D (1–4) were determined by extensive spectral analysis and their cytotoxicity against selected cancer cell lines as well as antiviral activity against human cytomegalovirus (HCMV) were evaluated in vitro.     

Screening of In-Vitro Anti-Inflammatory and Antioxidant Activity of Sargassum ilicifolium Crude Lipid Extracts from Different Coastal Areas in Indonesia

Sargassum brown seaweed is reported to exhibit several biological activities which promote human health, such as anticancer, antimicrobial, antidiabetic, anti-inflammatory, and antioxidant activity. This study aimed to investigate the anti-inflammatory and antioxidant activity of crude lipid extracts of Sargassum ilicifolium obtained from four different coastal areas in Indonesia, namely Awur Bay–Jepara (AB), Pari Island–Seribu Islands (PI), Sayang Heulang Beach–Garut (SHB), and Ujung Genteng Beach–Sukabumi (UGB). Results showed that treatment of RAW 264.7 macrophage cells with UGB and AB crude lipid extracts (12.5–50 µg/mL) significantly suppressed the nitric oxide production after lipopolysaccharide stimulation, both in pre-incubated and co-incubated cell culture model. The anti-inflammatory effect was most marked in the pre-incubated cell culture model. Both two crude lipid extracts showed 2,2-diphenyl-1-picrylhydrazyl radical scavenging activity and high ferric reducing antioxidant power, which were amounted to 36.93–37.87 µmol Trolox equivalent/g lipid extract and 681.58–969.81 µmol FeSO₄/g lipid extract, respectively. From this study, we can conclude that crude lipid extract of tropical S. ilicifolium can be further developed as a source of anti-inflammatory and antioxidant agent.     

Expanding the Repertoire of Spongian-16-One Derivatives in Australian Nudibranchs of the Genus Goniobranchus and Evaluation of Their Anatomical Distribution

Extracts of the mantle and viscera of the Indo-Pacific nudibranchs Goniobranchus aureopurpureus and Goniobranchus sp. 1 afforded 11 new diterpenoids (1–11), all of which possess a tetracyclic spongian-16-one scaffold with extensive oxidation at C-6, C-7, C-11, C-12, C-13, and/or C-20. The structures and relative configuration were investigated by NMR experiments, while X-ray crystallography provided the absolute configuration of 1, including a 2′S configuration for the 2-methylbutanoate substituent located at C-7. Dissection of animal tissue revealed that the mantle and viscera tissues differed in their metabolite composition with diterpenes 1–11 present in the mantle tissue of the two nudibranch species.     

Six New Diterpene Glycosides from the Soft Coral Lemnalia bournei

A chemical study on the extracts of soft coral Lemnalia bournei resulted in the isolation and identification of six new bicyclic diterpene glycosides including three new lemnaboursides E–G (1–3), and three new lemnadiolboursides A–C (4–6), along with three known lemnaboursides (7–9). Their structures were elucidated by detailed spectroscopic analysis, ECD analysis, chemical methods, and comparison with the literature data. Lemnadiolboursides A–C (4–6) contained a lemnal-1(10)-ene-7,12-diol moiety compared with the lemnaboursides. All these compounds were evaluated for antibacterial activity; cell growth inhibition of A549, Hela, HepG2, and CCRF-CEM cancer cell lines; and inhibition of LPS-induced NO production in RAW264.7 macrophages. The results indicated that compounds 1, 2, and 4–6 exhibited antibacterial activity against Staphylococcus aureus and Bacillus subtilis (MIC 4–16 μg/mL); compounds 1–9 displayed low cytotoxicity on the CCRF-CEM cell lines (IC₅₀ 10.44–27.40 µM); and compounds 1, 2, and 5 showed weak inhibition against LPS-induced NO production (IC₅₀ 21.56–28.06 μM).     

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.     

Antitumor and Antimicrobial Activity of Some Cyclic Tetrapeptides and Tripeptides Derived from Marine Bacteria

Marine derived cyclo(Gly-l-Ser-l-Pro-l-Glu) was selected as a lead to evaluate antitumor-antibiotic activity. Histidine was chosen to replace the serine residue to form cyclo(Gly-l-His-l-Pro-l-Glu). Cyclic tetrapeptides (CtetPs) were then synthesized using a solution phase method, and subjected to antitumor and antibiotic assays. The benzyl group protected CtetPs derivatives, showed better activity against antibiotic-resistant Staphylococcus aureus in the range of 60–120 μM. Benzyl group protected CtetPs 3 and 4, exhibited antitumor activity against several cell lines at a concentration of 80–108 μM. However, shortening the size of the ring to the cyclic tripeptide (CtriP) scaffold, cyclo(Gly-l-Ser-l-Pro), cyclo(Ser-l-Pro-l-Glu) and their analogues showed no antibiotic or antitumor activity. This phenomenon can be explained from their backbone structures.     

Oxygenated Acyclic Diterpenes with Anticancer Activity from the Irish Brown Seaweed Bifurcaria bifurcata

Brown alga Bifurcaria bifurcata is a prolific source of bioactive acyclic (linear) diterpenes with high structural diversity. In the continuation of our investigations on Irish brown algae, we undertook an in-depth chemical study on the n-hexanes and chloroform subextracts of B. bifurcata that led to isolation of six new (1–6) and two known (7–8) acyclic diterpenes. Chemical structures of the compounds were elucidated by a combination of 1D and 2D NMR, HRMS, FT-IR, [α]_D and vibrational circular dichroism (VCD) spectroscopy. Compounds 1–8, as well as three additional linear diterpenes (9–11), which we isolated from the same seaweed before, were tested against the human breast cancer cell line (MDA-MB-231). Several compounds moderately inhibited the growth of the MDA-MB-231 cell line with IC₅₀ values ranging from 11.6 to 32.0 μg/mL. The present study carried out on the lipophilic extracts of the Irish B. bifurcata shows the enormous capacity of this seaweed to produce a large palette of acyclic diterpenes with diverse oxygenation and substitution patterns and promising bioactivities.     

Fabrication of Silk Scaffold Containing Simvastatin-Loaded Silk Fibroin Nanoparticles for Regenerating Bone Defects

Background:In the present study, a tissue engineered SF scaffold containing simvastatin-loaded SFNPs were used to stimulate the regeneration of the defected bone.Methods:At first, the porous SF scaffold was prepared using freeze-drying. Then simvastatin-loaded SFNPs were made by dissolvation method and embedded in the SF scaffold. Afterwards, the scaffold and the NPs were characterized in terms of physicochemical properties and the ability to release the simvastatin small molecule.Results:The results exhibited that the SF scaffold had a porous structure suitable for releasing the small molecule and inducing the proliferation and attachment of osteoblast cells. SFNPs containing simvastatin had spherical morphology and were 174 ± 4 nm in size with -24.5 zeta potential. Simvastatin was also successfully encapsulated within the SFNPs with 68% encapsulation efficiency. Moreover, the small molecule revealed a sustained release profile from the NPs during 35 days. The results obtained from the in vitro cell-based studies indicated that simvastatin-loaded SFNPs embedded in the scaffold had acceptable capacity to promote the proliferation and ALP production of osteoblast cells while inducing osteogenic matrix precipitation. Conclusion:The SF scaffold containing simvastatin-loaded SFNPs could have a good potential to be used as a bone tissue-engineered construct.     

Antimicrobial Activity of the Marine Alkaloids,Clathrodin and Oroidin,and Their Synthetic Analogues

Marine organisms produce secondary metabolites that may be valuable for the development of novel drug leads as such and can also provide structural scaffolds for the design and synthesis of novel bioactive compounds. The marine alkaloids, clathrodin and oroidin, which were originally isolated from sponges of the genus, Agelas, were prepared and evaluated for their antimicrobial activity against three bacterial strains (Enterococcus faecalis, Staphylococcus aureus and Escherichia coli) and one fungal strain (Candida albicans), and oroidin was found to possess promising Gram-positive antibacterial activity. Using oroidin as a scaffold, 34 new analogues were designed, prepared and screened for their antimicrobial properties. Of these compounds, 12 exhibited >80% inhibition of the growth of at least one microorganism at a concentration of 50 µM. The most active derivative was found to be 4-phenyl-2-aminoimidazole 6h, which exhibited MIC₉₀ (minimum inhibitory concentration) values of 12.5 µM against the Gram-positive bacteria and 50 µM against E. coli. The selectivity index between S. aureus and mammalian cells, which is important to consider in the evaluation of a compound’s potential as an antimicrobial lead, was found to be 2.9 for compound 6h.