Elastic porous microspheres/extracellular matrix hydrogel injectable composites releasing dual bio-factors enable tissue regeneration.

Injectable biomaterials have garnered increasing attention for their potential and beneficial applications in minimally invasive surgical procedures and tissue regeneration. Extracellular matrix (ECM) hydrogels and porous synthetic polymer microspheres can be prepared for injectable administration to achieve in situ tissue regeneration. However, the rapid degradation of ECM hydrogels and the poor injectability and biological inertness of most polymeric microspheres limit their pro-regenerative capabilities. Here, we develop a biomaterial system consisting of elastic porous poly(l-lactide-co-ε-caprolactone) (PLCL) microspheres mixed with ECM hydrogels as injectable composites with interleukin-4 (IL-4) and insulin-like growth factor-1 (IGF-1) dual-release functionality. The developed multifunctional composites have favorable injectability and biocompatibility, and regulate the behavior of macrophages and myogenic cells following injection into muscle tissue. The elicited promotive effects on tissue regeneration are evidenced by enhanced neomusle formation, vascularization, and neuralization at 2-months post-implantation in a male rat model of volumetric muscle loss. Our developed system provides a promising strategy for engineering bioactive injectable composites that demonstrates desirable properties for clinical use and holds translational potential for application as a minimally invasive and pro-regenerative implant material in multiple types of surgical procedures.

Sequence-structure characterization of recombinant polypeptides derived from silk fibroin heavy chain.

The molecular conformation of a biomedical material plays a major role in the stability, bioactivity and controlled release of drugs. In order to identify the impact of fragments derived from Bombyx mori silk fibroin on their structures and to develop a new strategy for controlling drug release, we designed several hydrophobic-hydrophilic recombinants (GS16F1, GS16F4, and GS16F8), and investigated their molecular conformations and conformational changes induced by different storage temperatures and pH values. The results showed that the α-helix characteristic peaks were prominent in the fresh freeze-dried powder with increasing F1 repeats. During storage at 4 °C, 37 °C or 60 °C, the ß-turns (especially in GS16F8) and α-helixes turned into ß-sheets. The ß-sheet content in the polypeptides increased with increasing temperature and F1 repeats. Following induction by different pH values, their molecular conformations changed significantly, but not the same as that of powder storage. The content of ß-sheets was GS16F1 > GS16F4 > GS16F8 near the isoelectric point of each polypeptide. With increasing pH value, the ß-sheet content of GS16F1 decreased more slowly compared with GS16F4 and GS16F8. These results were satisfactory for structural regulation in the field of drug controlled release research.

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.

An RGD-Containing Peptide Derived from Wild Silkworm Silk Fibroin Promotes Cell Adhesion and Spreading.

Arginine-Glycine-Aspartate (RGD) tripeptide can promote cell adhesion when present in the amino acid of proteins such as fibronectin. In order to demonstrate the bioactivity of an RGD-containing silk protein, a gene encoding the RGD motif-containing peptide GSGAGGRGDGGYGSGSS (⁻RGD⁻) derived from nonmulberry silk was designed and cloned, then multimerised and inserted into a commercial pGEX expression vector for recombinant expression of (⁻RGD⁻)n peptides. Herein, we focus on two glutathione-S-transferase (GST)-tagged fusion proteins, GST⁻(⁻RGD⁻)4 and GST⁻(⁻RGD⁻)8, which were expressed in Escherichia coli BL21, purified by GST affinity chromatography, and analyzed with sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and mass spectrometry (MS). Target peptides (⁻RGD⁻)4 and (⁻RGD⁻)8 (6.03 and 11.5 kDa) were cleaved from the GST-tag by thrombin digestion, as verified with MS and SDS-PAGE. Isoelectric point analysis confirmed that target peptides were expressed and released in accordance with the original design. Target peptides self-assembled into a mainly α-helical structure, as determined by circular dichroism spectroscopy. Furthermore, (⁻RGD⁻)4 and (⁻RGD⁻)8 modified mulberry silk fibroin films were more effective for rapid cell adhesion, spreading and proliferative activity of L929 cells than some chemically synthesized RGD peptides modified and mulberry silk lacking the RGD motif.