Matrix Regeneration Ability In Situ Induced by a Silk Fibroin Small-Caliber Artificial Blood Vessel In Vivo.

The success of a small-caliber artificial vascular graft in the host in order to obtain functional tissue regeneration and remodeling remains a great challenge in clinical application. In our previous work, a silk-based, small-caliber tubular scaffold (SFTS) showed excellent mechanical properties, long-term patency and rapid endothelialization capabilities. On this basis, the aim of the present study was to evaluate the vascular reconstruction process after implantation to replace the common carotid artery in rabbits. The new tissue on both sides of the SFTSs at 1 month was clearly observed. Inside the SFTSs, the extracellular matrix (ECM) was deposited on the pore wall at 1 month and continued to increase during the follow-up period. The self-assembled collagen fibers and elastic fibers were clearly visible in a circumferential arrangement at 6 months and were similar to autologous blood vessels. The positive expression rate of Lysyl oxidase-1 (LOXL-1) was positively correlated with the formation and maturity of collagen fibers and elastic fibers. In summary, the findings of the tissue regeneration processes indicated that the bionic SFTSs induced in situ angiogenesis in defects.

Motility and function of smooth muscle cells in a silk small-caliber tubular scaffold after replacement of rabbit common carotid artery.

Cell infiltration and proliferation are prerequisites for tissue regeneration and repair. The aim of the present study was to evaluate the motility and function of vascular smooth muscle cells (SMCs) in a silk-based small-caliber artificial blood vessel (SFTS) following implantation to replace the common carotid artery in rabbits. Hematoxylin and eosin (HE) staining showed a number of SMCs clearly distributed in the scaffold at 1 month, which gradually increased up to 80-90% of autologous blood vessels at 3 months and was 100% at 12 months. Smooth muscle myosin heavy chain (SM-MHC) and α-smooth muscle actin (α-SMA) are specific markers of SMCs. Real-time PCR results showed that the gene expression level of α-SMA in SFTSs was significantly down-regulated within 6 months, except in the early stage of implantation. The relative expression level of α-SMA at 12 months was five times higher than that at 3 months, indicating that SMCs phenotype transformed from synthetic to contractile. The SM-MHC+ and α-SMA+ SMCs were disorderly distributed in the scaffolds at 1 month, but became ordered along the circumference 6 months after grafting as shown by immunohistochemistry. Results indicated that the bionic SFTSs were able to induce in situ angiogenesis in defects.

Evaluation of the biomedical properties of a Ca+-conjugated silk fibroin porous material.

Hemostatic materials could reduce avertible death from bleeding during surgery and emergency treatment. To this end, silk fibroin (SF) loaded with Ca2+ (1.8, 3.6 5.4, or 7.2%, w:w) was tested as a new hemostatic material (designated as SF1.8, SF3.6, SF5.4, or SF7.2), and the Ca2+ release rate, platelet adhesion, blood coagulation, cytocompatibility, and antimicrobial properties were investigated. Platelet adhesion on SF1.8 was improved significantly compared with pure SF porous material, and increased with increasing Ca2+ concentration. For SF3.6, platelet adhesion was greater than observed for gelatin and calcium alginate porous materials, clotting occurred earlier, and the complete coagulation time was shorter. Additionally, rabbit ear wound studies revealed that the hemostatic time for SF3.6 was significantly shorter than for gelatin, and similar to that for calcium alginate. The shed blood weight was lowest when SF was loaded with 7.2% Ca2+. The SF3.6 porous material displayed no obvious cytotoxicity, and exhibited satisfactory antibacterial activity against Escherichia coli and Staphylococcus aureus.

Steady-State Behavior and Endothelialization of a Silk-Based Small-Caliber Scaffold In Vivo Transplantation.

A silk-based small-caliber tubular scaffold (SFTS), which is fabricated using a regenerated silk fibroin porous scaffold embedding a silk fabric core layer, has been proved to possess good cell compatibility and mechanical properties in vitro. In this study, the endothelialization ability and the steady-state blood flow of SFTSs were evaluated in vivo by implanting and replacing a common carotid artery in a rabbit. The results of the color doppler ultrasound and angiographies showed that the blood flow was circulated in the grafts without aneurysmal dilations or significant stenoses at any time point, and ran stronger and close to the autologous blood vessel from one month after implantation. The SFTSs presented an initial tridimensionality without being distorted or squashed. SEM and immunohistochemistry results showed that a clear and discontinuous endodermis appeared after one month of implantation; when implanted for three months, an endothelial layer fully covered the inner surface of SFTSs. RT-PCR results indicated that the gene expression level of CD31 in SFTSs was 45.8% and 75.3% by that of autologous blood vessels at 3 months and 12 months, respectively. The VEGF gene showed a high expression level that continued to increase after implantation.

Vascular Cell Co-Culture on Silk Fibroin Matrix.

Silk fibroin (SF), a natural polymer material possessing excellent biocompatibility and biodegradability, and has been widely used in biomedical applications. In order to explore the behavior of vascular cells by co-culturing on regenerated SF matrix for use as artificial blood vessels, human aorta vascular smooth muscle cells (HAVSMCs) were co-cultured with human arterial fibroblasts (HAFs) or human umbilical vein endothelial cells (HUVECs) on SF films and SF tubular scaffolds (SFTSs). Analysis of cell morphology and deoxyribonucleic acid (DNA) content showed that HUVECs, HAVSMCs and HAFs adhered and spread well, and exhibited high proliferative activity whether cultured alone or in co-culture. Immunofluorescence and scanning electron microscopy (SEM) analysis showed that HUVECs and HAFs co-existed well with HAVSMCs on SF films or SFTSs. Cytokine expression determined by reverse transcription-polymerase chain reaction (RT-PCR) indicated that the expression levels of α-smooth muscle actin (α-SMA) and smooth muscle myosin heavy chain (SM-MHC) in HAVSMCs were inhibited on SF films or SFTSs, but expression could be obviously promoted by co-culture with HUVECs or HAFs, especially that of SM-MHC. On SF films, the expression of vascular endothelial growth factor (VEGF) and platelet endothelial cell adhesion molecule-1 (CD31) in HUVECs was promoted, and the expression levels of both increased obviously when co-cultured with HAVSMCs, with the expression levels of VEGF increasing with increasing incubation time. The expression levels of VEGF and CD31 in cells co-cultured on SFTSs improved significantly from day 3 compared with the mono-culture group. These results were beneficial to the mechanism analysis on vascular cell colonization and vascular tissue repair after in vivo transplantation of SFTSs.

The effect of hirudin modification of silk fibroin on cell growth and antithrombogenicity.

Thrombus formation remains a particular challenge for small-diameter vascular grafts. In this study, the direct thrombin inhibitor hirudin (Hir) was used to modify silk fibroin films in an attempt to enhance its antithrombogenic properties. Hir was successfully attached to silk fibroin and uniformly distributed in the regenerative material. Hir-modified films showed good cytocompatibility, and supported adhesion and proliferation of fibroblasts (L929), human umbilical vascular endothelial cells (HUVECs) and human aortic smooth muscle cells (HASMCs). Proliferation of HAVSMCs was inhibited by increasing Hir concentration. Activated partial thrombin time (APTT), prothrombin time (PT) and thrombin time (TT) of Hir-modified silk fibroin tubular scaffolds (SFTSs) were all increased markedly compared with fresh rabbit blood, ethanol-treated SFTS and unmodified SFTS, demonstrating the improved antithrombogenicity of SFTSs following modification with Hir.