Development of Thai Silk Fibroin/Hyaluronic Acid Microspheres and the Application on Controlled Release of Curcumin

The aim of this study was to develop microspheres from Thai silk fibroin (SF) blended with hyaluronic acid (HA) for controlled release application. HA was introduced to blend with SF to enhance properties of the microspheres. The SF-based microspheres could be prepared by water in oil emulsion and glutaraldehyde crosslinking technique. The obtained microspheres contained 71 - 91% SF and 9 - 29% HA. The microspheres having higher SF content showed more stability and slower degradation rate. Then, to prove the potential of SFHA microspheres as carriers for drug delivery, curcumin was absorbed in the microspheres. All microspheres released curcumin in a controllable manner, possibly due to the hydrophobic interaction between curcumin and crystalline domain of SF. Furthermore, it was shown that HA was self-released from the microspheres. The GA-crosslinked SFHA microspheres that simultaneously and controlled released curcumin and HA could be further developed and applied as injectable drug delivery system for treatment of diseases e.g. arthritis

Effect of Silver Sulfadiazine and Metallic Ions on Properties of Thai Silk Fibroin/Gelatin Films for Anti-Bacterial Applications

This study aimed to develop Thai silk fibroin/gelatin (SF/GA) films incorporating various concentrations of silver sulfadiazine (SSD) and to investigate the effects of SSD and metallic ions (Ag(I) and Cu(II)) on chemical conformation of the SF/GA films. We found that the incorporation of SSD in the films changed the conformation of silk fibroin protein by increasing β-sheet content. The same phenomena was also observed with the other 2 metallic ions, Ag(I) and Cu(II), incorporated in the SF/GA films. This effect of metallic ions on the SF conformation transition in the SF/GA films may be similar to the phenomenon occurred during natural spinning process of the Bombyx mori silkworm. The SF/GA films incorporating SSD had therefore more stability, less water-insoluble fraction and extended degradation rate than the SF/GA films without SSD due to the higher content of stable β-sheet conformation. When cultured with L929 mouse fibroblast cells according to ISO 10993 part 5 standard, the SF/GA films incorporating SSD at all concentrations showed no cytotoxicity. The SSD released from the films also showed obvious anti-bacterial activity against Staphylococcus aureus (Gram positive) and Escherichia coli (Gram negative) bacteria. We suggested that the SF/GA films incorporating SSD may be useful for some medical applications in which anti-bacterial effect was required

Cell engineering by the internalization of bioinstructive micelles for enhanced bone regeneration

To direct precursor cells toward the osteoblastic lineage, by using an intracellular nanocarrier releasing dexamethasone. Materials & methods: Biodegradable gelatinbased micelles entrapped dexamethasone (dex-micelles). Internalization efficiency and biocompatibility of dex-micelles and their potency for in vitro osteogenic differentiation and in vivo bone regeneration were assessed. Results: Dex-micelles were internalized by rat bone marrow mesenchymal stem cells and demonstrated a pH-responsive release profile and an enhancement of 2D and 3D in vitro osteogenic differentiation. In vivo implantation of gelatin scaffolds seeded with rat bone marrow mesenchymal stem cells precultured for 24 h with dex-micelles promoted a significant enhancement of de novo bone formation in a rat ulna defect, in a dose-dependent manner. Conclusion: The proposed intracellular delivery system is a powerful tool to promote bone regeneration.The authors thank Fundacao para a Ciencia e Tecnologia and Japan Society for the Promotion of Science (JSPS) for VE Santo's PhD grant (SFRH/BD/39486/2007) and J Ratanavaraporn's post-doc fellowship, respectively. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed

Fabrication and characterisation of electrospun silk fibroin/gelatin scaffolds crosslinked with glutaraldehyde vapour

Bombyx mori silk fibroin (SF) /gelatin nanofibre mats with different blend ratios of 100/0, 90/10 and 70/30 were prepared by electrospinning and crosslinked with glutaraldehyde (GTA) vapour at room temperature. GTA was shown to induce the conformational transition of SFs from random coils to β-sheets along with increasing nanofibre diameters with the addition of gelatin into SFs. It was found that by increasing the gelatin content, crosslinking degree was enhanced from 34% for pure SF nanofibre mats to 43% for SF/gelatin counterparts at the blend ratio of 70/30, which directly affected mechanical properties, porosity, and water uptake capacity (WUC) of prepared nanofibre mats. The addition of 10 and 30 wt% gelatin into SFs improved tensile strengths of SF/gelatin nanofibre mats by 10 and 27% along with significant increases in Young’s modulus by 1.1 and 1.3 times, respectively, as opposed to plain SF counterparts. However, both porosity and WUC were found to decrease from 62 and 405% for pristine SF nanofibre mats to 47% and 232% for SF/gelatin counterparts at the blend ratio of 70/30 accordingly. To further evaluate the combined effect of GTA crosslinking and gelatin content on biological response of SF/gelatin scaffolds, the proliferation assay using 3T3 mouse fibroblast was conducted. In comparison with pure SFs, cell proliferation rate was lower for SF/gelatin constructs, which declined when the gelatin content increased. These results indicated that the adverse effect of GTA crosslinking on cell response may be ascribed to imposed changes in morphology and physiochemical properties of SF/gelatin nanofibre mats. Although crosslinking could be used to improve mechanical properties of nanofibre mats, it reduced their capacity to support the cell activity. GTA optimisation is required to further modulate the physico-chemical properties of SF/gelatin nanofibre mats in order to obtain stable materials with favourable bioactive properties and promote cellular responses for tissue engineering applications

Nanoparticle-based bioactive agent release systems for bone and cartilage tissue engineering

The inability to deliver bioactive agents locally in a transient but sustained manner is one of the challenges on the development of bio-functionalized scaffolds for tissue engineering (TE) and regenerative medicine. The mode of release is especially relevant when the bioactive agent is a growth factor (GF), because the dose and the spatiotemporal release of such agents at the site of injury are crucial to achieve a successful outcome. Strategies that combine scaffolds and drug delivery systems have the potential to provide more effective tissue regeneration relative to current therapies. Nanoparticles (NPs) can protect the bioactive agents, control its profile, decrease the occurrence and severity of side effects and deliver the bioactive agent to the target cells maximizing its effect. Scaffolds containing NPs loaded with bioactive agents can be used for their local delivery, enabling site-specific pharmacological effects such as the induction of cell proliferation and differentiation, and, consequently, neo-tissue formation. This review aims to describe the concept of combining NPs with scaffolds, and the current efforts aiming to develop highly multi-functional bioactive agent release systems, with the emphasis on their application in TE of connective tissues.POLARIS (REGPOT-CT2012-316331-POLARIS), RL3 – TECT – NORTE-01-0124-FEDER-000020, co-financed by North Portugal Regional Operational Programme (ON.2 – O Novo Norte), under the National Strategic Reference Framework (NSRF), through the European Regional Development Fund (ERDF), the OsteoGraphy (PTDC/EME-MFE/2008) and MaxBone (PTDC/SAU-ENB/115179/2009) project

Local suppression of pro-inflammatory cytokines and the effects in BMP-2-induced bone regeneration.

The objective of this study is to investigate the effect of local inflammation suppression on the bone regeneration. Gelatin hydrogels incorporating mixed immunosuppressive triptolide-micelles and bone morphogenic protein-2 (BMP-2) were prepared. The controlled release of both the triptolide and BMP-2 from the hydrogels was observed under in vitro and in vivo conditions. When either J774.1 macrophage-like or MC3T3-E1 osteoblastic cells were cultured in the hydrogels incorporating mixed 2.5, 5 or 10 mg of triptolide-micelles and BMP-2, the expression level of pro- and anti-inflammatory cytokines including interleukin (IL)-6 and IL-10 was down-regulated, but the alkaline phosphatase (ALP) activity was promoted compared with those of hydrogels incorporating BMP-2 without triptolide-micelles. When implanted into a critical-sized bone defect of rats, the hydrogels incorporating mixed 2.5 or 5 mg of triptolide-micelles and BMP-2 showed significantly lower number of neutrophils, lymphocytes, macrophages or dendritic and mast cells infiltrated into the defect, and lower expression level of IL-6, TNF-α, and IL-10 than those incorporating BMP-2 without triptolide-micelles. The reduced local inflammation responses at the defects implanted with the hydrogels incorporating mixed 2.5 or 5 mg of triptolide-micelles and BMP-2 subsequently enhanced the bone regeneration thereat. It is concluded that the proper local modulation of inflammation responses is a promising way to achieve the enhanced bone regeneration

Synergistic effects of the dual release of stromal cell-derived factor-1 and bone morphogenetic protein-2 from hydrogels on bone regeneration.

著者版のタイトルと出版社版のタイトルに差異ありThe objective of this study is to evaluate the activity of gelatin hydrogels incorporating combined stromal cell-derived factor-1 (SDF-1) and bone morphogenetic protein-2 (BMP-2) on the in vivo bone regeneration at an ulna critical-sized defect and subcutaneous site of rats, and compared with that of those incorporating either SDF-1 or BMP-2. The similar release profile of SDF-1 and BMP-2 from the hydrogels was observed with or without the combination of BMP-2 and SDF-1, respectively. An enhanced bone regeneration by the hydrogels incorporating combined SDF-1 and BMP-2 was observed. In addition, the implantation of hydrogels incorporating combined SDF-1 and BMP-2 enhanced the expression level of CXC chemokine cell-surface receptor-4 (Cxcr4), Runt-related factor-2 (Runx2), and Osteocalcin genes. The experiments with green fluorescent protein (GFP)-positive Chimeric mice revealed that the recruitment of bone marrow-derived cells was promoted and a vascular-like structure together with strong accumulation of CD31- and CD34-positive cells was observed at the site of hydrogels incorporating combined SDF-1 and BMP-2 implanted. In addition, a large fraction of CD29- and CD44-positive non-hematopoietic cells was detected. It is concluded that the combined release of SDF-1 and BMP-2 enhanced the recruitment of osteogenic cells and angiogenesis, resulting in the synergistic effect on bone regeneration