Comparative Study of Silk Fibroin-Based Hydrogels and Their Potential as Material for 3-Dimensional (3D) Printing.

Three-dimensional (3D) printing is regarded as a critical technology in material engineering for biomedical applications. From a previous report, silk fibroin (SF) has been used as a biomaterial for tissue engineering due to its biocompatibility, biodegradability, non-toxicity and robust mechanical properties which provide a potential as material for 3D-printing. In this study, SF-based hydrogels with different formulations and SF concentrations (1-3%wt) were prepared by natural gelation (SF/self-gelled), sodium tetradecyl sulfate-induced (SF/STS) and dimyristoyl glycerophosphorylglycerol-induced (SF/DMPG). From the results, 2%wt SF-based (2SF) hydrogels showed suitable properties for extrusion, such as storage modulus, shear-thinning behavior and degree of structure recovery. The 4-layer box structure of all 2SF-based hydrogel formulations could be printed without structural collapse. In addition, the mechanical stability of printed structures after three-step post-treatment was investigated. The printed structure of 2SF/STS and 2SF/DMPG hydrogels exhibited high stability with high degree of structure recovery as 70.4% and 53.7%, respectively, compared to 2SF/self-gelled construct as 38.9%. The 2SF/STS and 2SF/DMPG hydrogels showed a great potential to use as material for 3D-printing due to its rheological properties, printability and structure stability.

The development of injectable gelatin/silk fibroin microspheres for the dual delivery of curcumin and piperine.

The objective of this study was to develop the microspheres from gelatin (G) and silk fibroin (SF) aimed to be applied for the controlled release of curcumin and piperine. The glutaraldehyde-crosslinked G/SF microspheres at various weight blending ratios (100/0, 70/30, 50/50, and 30/70) were successfully fabricated by water in oil emulsion technique. The microspheres prepared from all compositions were in a round shape with homogeneous size distribution both in the dried (194-217 µm) and swollen states (297-367 µm). When subjected in collagenase solution at physiological condition, the G microspheres gradually degraded within 14 days while the blended G/SF microspheres, particularly at 50/50 and 30/70, were not degraded. For the release application, the microspheres were loaded with curcumin and/or piperine. It was found that the microspheres composed of SF tended to entrap curcumin and piperine with the high entrapment and loading efficiencies, possibly due to their hydrophobic interactions. The G/SF microspheres, particularly at the ratios of 50/50 and 30/70, released curcumin and piperine in a sustained manner both for the single and dual release systems. The controlled dual release of curcumin and piperine from the G/SF microspheres would prolong their half-life, provide the optimal concentrations for therapeutic effects at a target site, and improve the bioavailability of curcumin. These novel injectable microspheres dually releasing curcumin and piperine would be introduced for the treatment of diseases without the need of operation.

Silk Fibroin-Based Coatings for Pancreatin-Dependent Drug Delivery.

Triggerable coatings, such as pH-responsive polymethacrylate copolymers, can be used to protect the active pharmaceutical ingredients contained within oral solid dosage forms from the acidic gastric environment and to facilitate drug delivery directly to the intestine. However, gastrointestinal pH can be highly variable, which can reduce delivery efficiency when using pH-responsive drug delivery technologies. We hypothesized that biomaterials susceptible to proteolysis could be used in combination with other triggerable polymers to develop novel enteric coatings. Bioinformatic analysis suggested that silk fibroin is selectively degradable by enzymes in the small intestine, including chymotrypsin, but resilient to gastric pepsin. Based on the analysis, we developed a silk fibroin-polymethacrylate copolymer coating for oral dosage forms. In vitro and in vivo studies demonstrated that capsules coated with this novel silk fibroin formulation enable pancreatin-dependent drug release. We believe that this novel formulation and extensions thereof have the potential to produce more effective and personalized oral drug delivery systems for vulnerable populations including patients that have impaired and highly variable intestinal physiology.

Modification of human cancellous bone using Thai silk fibroin and gelatin for enhanced osteoconductive potential.

The modification of human cancellous bone (hBONE) with silk fibroin/gelatin (SF/G) using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC)/N-hydroxysuccini-mide (NHS) crosslinking was established. The SF/G solutions at a weight ratio of 50/50 and the solution concentrations of 1, 2, and 4 wt % were studied. SF/G sub-matrix was formed on the surface and inside pore structure of hBONE. All hBONE scaffolds modified with SF/G showed smaller pore sizes, less porosity, and slightly lower compressive modulus than unmodified hBONE. SF/G sub-matrix was gradually biodegraded in collagenase solution along 4 days. The hBONE scaffolds modified with SF/G, particularly at 2 and 4 wt % solution concentrations, promoted attachment, proliferation, and osteogenic differentiation of bone marrow-derived mesenchymal stem cells (MSC), comparing to the original hBONE. The highest cell number, ALP activity and calcium production were observed for MSC cultured on the hBONE scaffolds modified with 4 wt % SF/G. The mineralization was also remarkably induced in the cases of modified hBONE scaffolds as observed from the deposited calcium phosphate by EDS. The modification of hBONE with SF/G was, therefore, the promising method to enhance the osteoconductive potential of human bone graft for bone tissue engineering.

Hydrogelation of Regenerated Silk Fibroin via Gamma Irradiation.

Gamma irradiation, which is one of the more conventional sterilization methods, was used to induce the hydrogelation of silk fibroin in this study. The physical and chemical characteristics of the irradiation-induced silk fibroin hydrogels were investigated. Silk fibroin solution with a concentration greater than 1 wt% formed hydrogel when irradiated by gamma rays at a dose of 25 or 50 kGy. The hydrogel induced by 50 kGy of radiation was more thermally stable at 80 °C than those induced by 25 kGy of radiation. When compared to the spontaneously formed hydrogels, the irradiated hydrogels contained a greater fraction of random coils and a lower fraction of ß-sheets. This finding implies that gelation via gamma irradiation occurs via other processes, in addition to crystalline ß-sheet formation, which is a well-established mechanism. Our observation suggests that crosslinking and chain scission via gamma irradiation could occur in parallel with the ß-sheet formation. The irradiation-induced hydrogels were obtained when the solution concentration was adequate to support the radiation crosslinking of the silk fibroin chains. This work has, therefore, demonstrated that gamma irradiation can be employed as an alternative method to produce chemical-free, random coil-rich, and sterilized silk fibroin hydrogels for biomedical applications.

Degradation products of crosslinked silk fibroin scaffolds modulate the immune response but not cell toxicity.

Silk fibroin (SF) scaffolds have widely been used as functional materials for tissue engineering and implantation. For long-term applications, many cross-linking strategies have been developed to enhance the stability and enzymatic degradation of scaffolds. Although the biocompatibility of SF scaffolds has been investigated, less is known about the extent to which the degradation products of these scaffolds affect the host response in the long term after implantation. In this work, we first studied the effect of two different crosslinkers, namely, 1-ethyl-3-(3-dimethylaminopropyl-carbodiimide hydrochloride) (EDC) and glutaraldehyde (GA), on the topology, mechanical stability and enzymatic degradation of SF scaffolds. We found that the SF scaffolds treated with GA (GA-SF) appeared to show an increase in the sheet thickness and a higher elastic modulus when compared to that treated with EDC (EDC-SF) at a similar level of crosslinking degree. The uncrosslinked and both crosslinked SF scaffolds were completely digested by proteinase K but were not susceptible to degradation by collagenase type IV and trypsin. We next investigated the effect of the degradation of SF on the cytotoxicity, genotoxicity, and immunogenicity. The results demonstrated that the degradation products of the uncrosslinked and crosslinked SFs did not trigger cell proliferation, cell death, or genotoxicity in primary human cells, while they appeared to modulate the phenotypes of macrophages. The degradation products of GA-SF promoted pro-inflammatory phenotypes, while those from EDC-SF enhanced polarization towards anti-inflammatory macrophages. Our results demonstrated that the degradation products of SF scaffolds can mediate the immune modulation of macrophages, which can be implemented as a therapeutic strategy to control the long-term immune response during implantation.

Osteogenic differentiation of encapsulated cells in dexamethasone-loaded phospholipid-induced silk fibroin hydrogels.

The tissue engineering triad comprises the combination of cells, scaffolds and biological factors. Therefore, we prepared cell- and drug-loaded hydrogels using in situ silk fibroin (SF) hydrogels induced by dimyristoyl glycerophosphoglycerol (DMPG). DMPG is reported to induce rapid hydrogel formation by SF, facilitating cell encapsulation in the hydrogel matrix while maintaining high cell viability and proliferative capacity. In addition, DMPG can be used for liposome formulations in entrapping drug molecules. Dexamethasone (Dex) was loaded into the DMPG-induced SF hydrogels together with human osteoblast-like SaOS-2 cells, then the osteogenic differentiation of the entrapped cells was evaluated in vitro and compared to cells cultured under standard conditions. Calcium production by cells cultured in DMPG/Dex-SF hydrogels with Dex-depleted osteogenic medium was equivalent to that of cells cultured in conventional osteogenic medium containing Dex. The extended-release of the entrapped Dex by the hydrogels was able to provide a sufficient drug amount for osteogenic induction. The controlled release of Dex was also advantageous for cell viability even though its dose in the hydrogels was far higher than that in osteogenic medium. The results confirmed the possibility of using DMPG-induced SF hydrogels to enable dual cell and drug encapsulation to fulfil the practical applications of tissue-engineered constructs.

Hemocompatibility Evaluation of Thai Bombyx mori Silk Fibroin and Its Improvement with Low Molecular Weight Heparin Immobilization.

Bombyx mori silk fibroin (SF), from Nangnoi Srisaket 1 Thai strain, has shown potential for various biomedical applications such as wound dressing, a vascular patch, bone substitutes, and controlled release systems. The hemocompatibility of this SF is one of the important characteristics that have impacts on such applications. In this study, the hemocompatibility of Thai SF was investigated and its improvement by low molecular weight heparin (LMWH) immobilization was demonstrated. Endothelial cell proliferation on the SF and LMWH immobilized SF (Hep/SF) samples with or without fibroblast growth factor-2 (FGF-2) was also evaluated. According to hemocompatibility evaluation, Thai SF did not accelerate clotting time, excess stimulate complement and leukocyte activation, and was considered a non-hemolysis material compared to the negative control PTFE sheet. Platelet adhesion of SF film was comparable to that of the PTFE sheet. For hemocompatibility enhancement, LMWH was immobilized successfully and could improve the surface hydrophilicity of SF films. The Hep/SF films demonstrated prolonged clotting time and slightly lower complement and leukocyte activation. However, the Hep/SF films could not suppress platelet adhesion. The Hep/SF films demonstrated endothelial cell proliferation enhancement, particularly with FGF-2 addition. This study provides fundamental information for the further development of Thai SF as a hemocompatible biomaterial.

Impacts of Blended Bombyx mori Silk Fibroin and Recombinant Spider Silk Fibroin Hydrogels on Cell Growth.

Binary-blended hydrogels fabricated from Bombyx mori silk fibroin (SF) and recombinant spider silk protein eADF4(C16) were developed and investigated concerning gelation and cellular interactions in vitro. With an increasing concentration of eADF4(C16), the gelation time of SF was shortened from typically one week to less than 48 h depending on the blending ratio. The biological tests with primary cells and two cell lines revealed that the cells cannot adhere and preferably formed cell aggregates on eADF4(C16) hydrogels, due to the polyanionic properties of eADF4(C16). Mixing SF in the blends ameliorated the cellular activities, as the proliferation of L929 fibroblasts and SaOS-2 osteoblast-like cells increased with an increase of SF content. The blended SF:eADF4(C16) hydrogels attained the advantages as well as overcame the limitations of each individual material, underlining the utilization of the hydrogels in several biomedical applications.

Properties and antityrosinase activity of sericin from various extraction methods.

The present study investigated the chemical properties and antityrosinase activities of SS (silk sericin) extracted from different Thai silk strains via various extraction methods. Different silk strains contain distinct SS with various amino acid compositions, which are significantly influenced by the extraction method used. Urea extraction of SS was the only method that provided clearly distinguishable bands and had the most significant impact on SS conformation as illustrated by FTIR (Fourier-transform infrared) spectra. The use of urea or either acidic or alkaline chemicals in the extraction process also influenced SS thermal behaviour. With regard to biological activity, SS extracted using urea exhibited the highest antityrosinase activity, whereas alkali-degraded SS showed no inhibition of mushroom tyrosinase. Pigments, primarily flavonoids and carotenoids from silk cocoons, were also found to enhance tyrosinase inhibition of SS.

Tissue response and biodegradation of composite scaffolds prepared from Thai silk fibroin, gelatin and hydroxyapatite.

This work aimed to investigate tissue responses and biodegradation, both in vitro and in vivo, of four types of Bombyx mori Thai silk fibroin based-scaffolds. Thai silk fibroin (SF), conjugated gelatin/Thai silk fibroin (CGSF), hydroxyapatite/Thai silk fibroin (SF4), and hydroxyapatite/conjugated gelatin/Thai silk fibroin (CGSF4) scaffolds were fabricated using salt-porogen leaching, dehydrothermal/chemical crosslinking and an alternate soaking technique for mineralization. In vitro biodegradation in collagenase showed that CGSF scaffolds had the slowest biodegradability, due to the double crosslinking by dehydrothermal and chemical treatments. The hydroxyapatite deposited from alternate soaking separated from the surface of the protein scaffolds when immersed in collagenase. From in vivo biodegradation studies, all scaffolds could still be observed after 12 weeks of implantation in subcutaneous tissue of Wistar rats and also following ISO10993-6: Biological evaluation of medical devices. At 2 and 4 weeks of implantation the four types of Thai silk fibroin based-scaffolds were classified as "non-irritant" to "slight-irritant", compared to Gelfoam(®) (control samples). These natural Thai silk fibroin-based scaffolds may provide suitable biomaterials for clinical applications.

The effect of sericin from various extraction methods on cell viability and collagen production.

Silk sericin (SS) can accelerate cell proliferation and attachment; however, SS can be extracted by various methods, which result in SS exhibiting different physical and biological properties. We found that SS produced from various extraction methods has different molecular weights, zeta potential, particle size and amino acid content. The MTT assay indicated that SS from all extraction methods had no toxicity to mouse fibroblast cells at concentrations up to 40 mug/mL after 24 h incubation, but SS obtained from some extraction methods can be toxic at higher concentrations. Heat-degraded SS was the least toxic to cells and activated the highest collagen production, while urea-extracted SS showed the lowest cell viability and collagen production. SS from urea extraction was severely harmful to cells at concentrations higher than 100 mug/mL. SS from all extraction methods could still promote collagen production in a concentration-dependent manner, even at high concentrations that are toxic to cells.

Dual-functional liposomes for curcumin delivery and accelerating silk fibroin hydrogel formation.

The administration of a drug-loaded implantable hydrogel at the tumor site after surgical resection is a viable approach to prevent the local recurrence or metastasis. Dimyristoyl glycerophosphorylglycerol (DMPG)-based liposomes were developed for inducing the rapid gelation of silk fibroin (SF) and delivering an anticancer drug, curcumin. Curcumin was loaded in the liposomes and the stability of curcumin was enhanced. The gelation time of liposome-induced SF hydrogels ranged from 3 min to more than 6 h. The biological activity of liposome-SF hydrogels was evaluated in vitro using L929 fibroblasts and MDA-MB-231 breast cancer cells. The release of curcumin can inhibit the growth of cancer cells. Both cells cultured on the surface of the hydrogels loaded with curcumin displayed low cell survival due to the combination of low cell attachment and cytotoxicity of curcumin. Liposome-SF hydrogels show potential as a sealant administered at the tumor site to eliminate residual cancer cells after tumor removal.

Phospholipid-induced silk fibroin hydrogels and their potential as cell carriers for tissue regeneration.

Silk fibroin (SF) hydrogels can be obtained via self-assembly, but this process takes several days or weeks, being unfeasible to produce cell carrier hydrogels. In this work, a phospholipid, namely, 1,2-dimyristoyl-sn-glycero-3-phospho-(1'-rac-glycerol) sodium salt (DMPG), was used to induce and accelerate the gelation process of SF solutions. Due to the amphipathic nature and negative charge of DMPG, electrostatic and hydrophobic interactions between the phospholipids and SF chains will occur, inducing the structural transition of SF chains to the beta sheet and consequently a rapid gel formation is observed (less than 50 min). Moreover, the gelation time can be controlled by varying the lipid concentration. To assess the potential of the hydrogels as cell carriers, several mammalian cell lines, including L929, NIH/3T3, SaOS-2, and CaSki, were encapsulated into the hydrogel. The silk-based hydrogels supported the normal growth of fibroblasts, corroborating their cytocompatibility. Interestingly, an inhibition in the growth of cancer-derived cell lines was observed. Therefore, DMPG-induced SF hydrogels can be successfully used as a 3D platform for in situ cell encapsulation, opening promising opportunities in biomedical applications, such as in cell therapies and tissue regeneration.

Effectiveness of inflammatory cytokines induced by sericin compared to sericin in combination with silver sulfadiazine cream on wound healing.

 Silk sericin (SS) has been shown to promote collagen synthesis during wound healing, but it lacks antimicrobial activity. We investigated the effectiveness and the induction of the inflammatory mediators IL-1bß and TNF-aα by SS, silver sulfadiazine (SSD) cream, and SS in combination with SSD cream on wound healing in rats. The results show that SS at 8% w/w partially inhibits SSD antibacterial activity. Treating wounds with a combination of SS and SSD did not induce significant wound size reduction when compared to other treatments. However, SS can promote collagen production in wounds even in the presence of SSD. Wounds treated with the combination of SS and SSD cream showed higher levels of IL-1ß and TNF-α when compared to wounds treated by SS alone, but the differences were not significant. Although SS may decrease the antimicrobial effect of SSD, SS in combination with SSD cream has the benefit of promoting collagen production without generating significant levels of inflammatory cytokines..

Stability enhancement of mulberry-extracted anthocyanin using alginate/chitosan microencapsulation for food supplement application.

The stability of mulberry-extracted anthocyanin is a main concern in food supplement application. In this article, the alginate/chitosan beads were fabricated by spray drying and external gelation techniques using different processes: (1) dropping a sodium alginate solution into a CaCl2 solution containing chitosan and (2) incubating calcium alginate beads in a solution of chitosan. These beads were introduced as microcarrier to enhance the stability and bioavailability of anthocyanin. We showed that the beads fabricated by different processes could encapsulate the anthocyanin at different amounts. All alginate/chitosan beads had high swelling percentage under pH 6 and 7.4 but not completely swell at pH 1 and 4. The alginate/chitosan beads degraded in a simulated gastric fluid condition (SGF) in the faster rate than that in a simulated intestinal fluid condition (SIF). Under SGF condition, the release of anthocyanin seemed to be governed by electrostatic interaction while the release of anthocyanin under SIF condition may be manipulated by the beads' degradation. Herein, we showed that the beads produced by incubating calcium alginate beads in 0.05% chitosan solution were the most appropriate microcarriers for encapsulation of mulberry-extracted anthocyanin which showed high encapsulation efficiency and had resistance to gastric condition.

Tissue engineering of cartilage with porous polycarprolactone--alginate scaffold: the first report of tissue engineering in Thailand.

OBJECTIVE: To engineer human cartilage with porous polycaprolactone (PCL)-Alginate Scaffold. BACKGROUND: Polycaprolactone (PCL) is a prolonged degradable polymer that has good mechanical strength. The authors fabricated PCL as an ear shaped scaffold. Alginate hydrogel was used to seed chondrocyte into the PCL porous scaffold by a gel-cell seeding technique. MATERIAL AND METHOD: PCL Scaffolds were fabricated like human pinna by particle leaching technique. Chondrocyte was isolated from human rib cartilage and then cultured. The cultured chondrocyte were mixed with 1.2% alginate and b-FGF (basic-fibroblast growth factor) 5 ng/ml at a concentration of 25 x 10(6) cell/ml, then were seeded in porous PCL scaffold to make the constructs. The constructs were cultured in vitro for 1 week. Then they were implanted in subcutaneous plane of the back of six-female nude mice (5 weeks old). Two nude mice were sacrificed at 2, 3, and 6 months. Histological study was done (H&E, Alcian blue, collagen type II). RESULT: Neocartilage was formed in the porous cavity of PCL scaffold. At 2 and 3 months, neocartilage were similar to very young cartilage. At 6 months, they were mature. The delayed maturation until 6 months and the highly vascularization of neocartilage in the early phase was the effect of human b-FGF The growths of neocartilage islands in porous cavity were also observed along with degradation ofPCL inter-porous septum. CONCLUSION: This paper reports the first success of cartilage tissue engineering in Thailand.

An axial distribution of seeding, proliferation, and osteogenic differentiation of MC3T3-E1 cells and rat bone marrow-derived mesenchymal stem cells across a 3D Thai silk fibroin/gelatin/hydroxyapatite scaffold in a perfusion bioreactor.

In cell culture, a perfusion bioreactor provides effective transportation of nutrients, oxygen, and waste removal to and from the core of the scaffold. In addition, it provides mechanical stimuli for enhancing osteogenic differentiation. In this study, we used an axial distribution of cell numbers, alkaline phosphatase (ALP) enzyme activity, and calcium content across 4 cross-sections of 10mm thick scaffold, made of Thai silk fibroin (SF)/gelatin (G)/hydroxyapatite (HA), as a tool to evaluate the suitable perfusion flow rate. These evaluations cover all cellular developmental phases starting from seeding, to proliferation, and later osteogenic differentiation. Mouse pre-osteoblastic MC3T3-E1 cell lines were used as a cell model during seeding and proliferation. The bioreactor seeded scaffold provided more uniform cell distribution across the scaffold compared to centrifugal and agitation seeding, while the overall number of adhered cells from bioreactor seeding was slightly lower than agitation seeding. The dynamic culture using 1 ml/min perfusion flow rate (initial shear stress of 0.1 dyn/cm(2)) enabled statistically higher MC3T3-E1 proliferation, ALP activity, and calcium deposition than those observed in the static-culturing condition. However, the perfusion flow rate of 1 ml/min seemed not to be enough for enhancing ALP expression across all sections of the scaffold. Rat bone marrow derived stromal cells (rMSC) were used in the detachment test and osteogenic differentiation. It was found that perfusion flow rate of 5 ml/min caused statistically higher cell detachment than that of 1 and 3 ml/min. The perfusion flow rate of 3 ml/min gave the highest rMSC osteogenic differentiation on a SF/G/HA scaffold than other flow rates, as observed from the significantly highest number of ALP enzyme activity and the calcium content without any significant cell growth. In addition, all of these parameters were evenly distributed across all scaffold sections.

Thai Silk Fibroin/Gelatin Sponges for the Dual Controlled Release of Curcumin and Docosahexaenoic Acid for Anticancer Treatment.

In this study, curcumin and/or docosahexaenoic acid (DHA) were encapsulated in Thai silk fibroin/gelatin (SF/G) sponges, prepared at different blending ratios, aimed to be applied as a controlled release system for localized cancer therapy. The SF/G sponges were fabricated by freeze-drying and glutaraldehyde cross-linking techniques. Physicochemical properties of the SF/G sponges were characterized. Then, curcumin and/or DHA were loaded in the sponges by physical adsorption. The encapsulation efficiency and the in vitro release of curcumin and/or DHA from the sponges were evaluated. SF/G sponges could encapsulate curcumin and/or DHA at high encapsulation efficiency. The highly cross-linked and slowly degrading SF/G (50/50) sponge released curcumin and/or DHA at the slowest rate. The in vitro cytotoxicity of the sponges against noncancer cells (L929 mouse fibroblast) and anticancer of curcumin and/or DHA released from the sponges against cervical cancer cells (CaSki) were tested. All sponges were not toxic to L929 mouse fibroblast. The mixed curcumin­DHA at the ratio of 1:4 had the highest inhibiting effect on the growth of CaSki, comparing with the release of curcumin or DHA alone. SF/G sponges could be a potential carrier for dual release of curcumin and DHA for anticancer effect.

A vascular patch prepared from Thai silk fibroin and gelatin hydrogel incorporating simvastatin-micelles to recruit endothelial progenitor cells.

Delayed re-endothelialization is one of the major disadvantages in synthetic vascular grafts, especially in small-diameter grafts (inner diameter <6 mm), leading to thrombosis and stenosis of the grafts. Simvastatin, a serum cholesterol-lowering drug, has promotional effects on endothelial progenitor cell (EPC) mobilization from bone marrow and recruitment to sites of vascular injury exhibiting acceleration of re-endothelialization. In this study, we prepared double-layer vascular patches from Thai silk fibroin/gelatin with gelatin hydrogel incorporating simvastatin-micelles (SM) for sustained release of simvastatin to recruit circulation EPCs. To enhance simvastatin solubility, simvastatin was entrapped in micelles of l-lactic acid oligomer-grafted gelatin. The drug loading efficiency was at 4.1 ± 0.5 µg/mg micelles. SM had a chemoattractive effect on EPCs comparable to nonmodified simvastatin. Gelatin hydrogel incorporating SM at 100 µM of simvastatin (GSM100) could enhance in vitro EPC activities of adhesion and proliferation. In vitro results showed the initial cell adhesion of 86%, specific growth rate of 15.33×10(-3) h(-1), and population doubling time of 46.21 h. In vivo implantation of the patches incorporating SM significantly increased the recruitment of circulating EPCs. From the results of immunofluorescence staining, they demonstrated the complete re-endothelialization on the implanted patches containing SM at 2 weeks after implantation in rat carotid arteries. The gelatin hydrogel incorporating SM could be an effective inner layer of multifunctional vascular grafts to accelerate re-endothelialization in vascular tissue engineering.