Meifeng Zhu

Fabrication of highly interconnected porous silk fibroin scaffolds for potential use as vascular grafts

Silk fibroin (SF) scaffolds have been designed and fabricated for multiple organ engineering owing to SFs remarkable mechanical property, excellent biocompatibility and biodegradability, as well as its low immunogenicity. In this study, an easy-to-adopt and mild approach based on a modified freeze-drying method was developed to fabricate a highly interconnected porous SF scaffold. The physical properties of the SF scaffold, including pore morphology, pore size, porosity and compressive modulus, could be adjusted by the amount of ethanol added, the freezing temperature and the concentration of SF. Fourier transform infrared spectroscopy illustrated that treatment of the lyophilized scaffolds with 90% methanol led to a structure transition of SF from silk I (random coil) to silk II (beta-sheet), which stabilized the SF scaffolds in water. We also incorporated heparin during fabrication to obtain a heparin-loaded scaffold which possessed excellent anticoagulant property. The heparin that was incorporated into the SF scaffolds could be released in a sustain manner for approximately 7days, inhibiting the proliferation of human smooth muscle cells within the scaffold in vitro while promoting neovascularization in vivo. We therefore propose that the SF porous scaffold fabricated here may be an attractive candidate for use as a potential vascular graft for implantation based on its high porosity, excellent blood compatibility and mild fabrication process.

Integrated trilayered silk fibroin scaffold for osteochondral differentiation of adipose-derived stem cells.

Repairing osteochondral defects (OCD) remains a formidable challenge due to the high complexity of native osteochondral tissue and the limited self-repair capability of cartilage. Osteochondral tissue engineering is a promising strategy for the treatment of OCD. In this study, we fabricated a novel integrated trilayered scaffold using silk fibroin and hydroxyapatite by combining paraffin-sphere leaching with a modified temperature gradient-guided thermal-induced phase separation (TIPS) technique. This biomimetic scaffold is characterized by three layers: a chondral layer with a longitudinally oriented microtubular structure, a bony layer with a 3D porous structure and an intermediate layer with a dense structure. Live/dead and CCK-8 tests indicated that this scaffold possesses good biocompatibility for supporting the growth, proliferation, and infiltration of adipose-derived stem cells (ADSCs). Histological and immunohistochemical stainings and real-time polymerase chain reaction (RT-PCR) confirmed that the ADSCs could be induced to differentiate toward chondrocytes or osteoblasts in vitro at chondral and bony layers in the presence of chondrogenic- or osteogenic-induced culture medium, respectively. Moreover, the intermediate layer could play an isolating role for preventing the cells within the chondral and bony layers from mixing with each other. In conclusion, the trilayered and integrated osteochondral scaffolds can effectively support cartilage and bone tissue generation in vitro and are potentially applicable for OC tissue engineering in vivo.

Influence of substrate temperature on the orientation and optical properties of sputtered ZnO films

Abstract The influence of substrate temperature on the orientation and optical properties of sputtered ZnO films is studied in the paper. X-ray diffraction (XRD) demonstrates that at the substrate temperature of 350 °C, (002) and (004) diffraction peaks of ZnO films have occurred. It reveals that highly c -axis preferential orientation of ZnO film with wurtzite phase was fabricated at 350 °C. Besides, the red shift of the absorption band edge, as the substrate temperature increases, is attributed to the high O 2 vapour pressure of ZnO, which causes the vacancies in ZnO films. Meanwhile, photoluminescence (PL) spectrum of the transparent ZnO films deposited at 350 °C shows the intrinsic emission peak and a broad green emission band.

Co-Transplantation of Skin-Derived Precursors and Collagen Sponge Facilitates Diabetic Wound Healing by Promoting Local Vascular Regeneration

Background/Aims: Impaired diabetes wound healing can often lead to serious complications and remains a major health concern due to the lack of effective therapeutic approaches. Compromised angiogenesis, disrupted growth factor and cytokine activity are all attributable to diabetic wound healing impairment. The skin-derived precursors (SKPs) have been shown to differentiate into vascular and nerve cells, both of which are crucial components for wound repair. Given their easy accessibility and multipotency, the SKPs were proposed as an ideal therapeutic candidate for diabetic wound healing. Since the efficacy of cell therapy is limited by poor cell survival, collagen sponge was employed for better SKPs delivery. Methods: SKPs were isolated and transplanted directly to the wound areas of diabetic mice in the absence and presence of collagen sponge. The effects of SKPs and/or collagen sponge on diabetic wound healing were examined histologically as well as immunostaining of isolectin and α-SMA. Mechanisms via which the SKPs facilitate wound healing were then investigated by transplanting SKPs that have been pre-labelled with a fluorescence dye, Dil. Expression patterns of Dil and an SKP marker, nestin, was also examined. Results and Conclusion: Accelerated wound healing and enhanced local capillary regeneration could be observed 14 days after skin ablation from both SKPs and collagen sponge co-transplanted and collagen sponge only groups. Subsequent analyses further revealed superior pro-angiogenic effects from the SKP and collagen sponge co-delivered group, which are mainly attributable to in vivo transdifferentation and paracrine signalling of the SKPs.

Silk fibroin porous scaffolds for nucleus pulposus tissue engineering.

Intervertebral discs (IVDs) are structurally complex tissue that hold the vertebrae together and provide mobility to spine. The nucleus pulposus (NP) degeneration often results in degenerative IVD disease that is one of the most common causes of back and neck pain. Tissue engineered nucleus pulposus offers an alternative approach to regain the function of the degenerative IVD. The aim of this study is to determine the feasibility of porous silk fibroin (SF) scaffolds fabricated by paraffin-sphere-leaching methods with freeze-drying in the application of nucleus pulposus regeneration. The prepared scaffold possessed high porosity of 92.38±5.12% and pore size of 165.00±8.25μm as well as high pore interconnectivity and appropriate mechanical properties. Rabbit NP cells were seeded and cultured on the SF scaffolds. Scanning electron microscopy, histology, biochemical assays and mechanical tests revealed that the porous scaffolds could provide an appropriate microstructure and environment to support adhesion, proliferation and infiltration of NP cells in vitro as well as the generation of extracellular matrix. The NP cell-scaffold construction could be preliminarily formed after subcutaneously implanted in a nude mice model. In conclusion, The SF porous scaffold offers a potential candidate for tissue engineered NP tissue.

Functional Modification of Electrospun Poly(ε-caprolactone) Vascular Grafts with the Fusion Protein VEGF–HGFI Enhanced Vascular Regeneration

Synthetic artificial vascular grafts have exhibited low patency rate and severe neointimal hyperplasia in replacing small-caliber arteries (<6 mm) because of their failure to generate a functional endothelium. In this study, small-caliber (2.0 mm) electrospun poly(ε-caprolactone) (PCL) vascular grafts were modified with a fusion protein VEGF-HGFI which consists of the class I hydrophobin (HGFI) and vascular endothelial growth factor (VEGF), via hydrophobic interactions. Immunofluorescence staining with the anti-VEGF antibody showed that VEGF-HGFI formed a protein layer on the surface of fibers in the grafts. Scanning electron microscopy (SEM) and mechanical measurements showed that VEGF-HGFI modification had no effect on the structure and mechanical properties of PCL grafts. Blood compatibility tests demonstrated a lower level of fibrinogen (FGN) absorption, platelet activation, and aggregation on the VEGF-HGFI-modified PCL mats than that on the bare PCL mats. The hemolysis rate was comparable in both the modified and bare PCL mats. In vitro culture of human umbilical vein endothelial cells (HUVECs) demonstrated that VEGF-HGFI modification could remarkably enhance nitric oxide (NO) production, prostacyclin2 (PGI2) release, and the uptake of acetylated low-density lipoprotein (Ac-LDL) by HUVECs. The healing characteristics of the modified grafts were examined in the replacement of rat abdominal aorta for up to 1 month. Immunofluorescence staining revealed that endothelialization, vascularization, and smooth muscle cell (SMC) regeneration were markedly improved in the VEGF-HGFI-modified PCL grafts. These results suggest that modification with fusion protein VEGF-HGFI is an effective method to improve the regeneration capacity of synthetic vascular grafts.

Creation of macropores in electrospun silk fibroin scaffolds using sacrificial PEO-microparticles to enhance cellular infiltration.

Electrospun scaffolds are widely used in tissue engineering; however, a common problem is the poor cell infiltration because of the small pore size and tightly packed structure of these fibrous scaffolds. To address this issue, a novel technique was developed to fabricate electrospun silk fibroin (SF) scaffolds with rather macropores and high porosity using electrospraying-generated PEO microparticles as porogen. The morphology and pore size of MPES scaffolds were evaluated by scanning electron microscopy. It was revealed that MPES scaffold had a relatively loose structure with an increase of mean pore size (i.e., approx. 30 μm of MPES vs. approx. 5 μm of traditional electrospun scaffolds (TES) and porosity (i.e., 95% vs. 84% of TES). Culture of mouse 3T3 fibroblast in TES and MPES scaffold revealed that both scaffolds could support cell attachment, spread and proliferation. Yet, cell inflitration in vitro under the static culture condition only occurred in the MPES scaffold. Subcutaneous implantation of scaffolds in rats further confirmed that the tissue ingrowth was more efficient in the MPES scaffold compared to TES scaffold. Thus, the use of PEO microparticles as porogen was a feasible and effective method for creating macroporous electrospun SF scaffold, which provided an alternative to address the limitation of cell infiltration associated with electrospun fibrous scaffold.

Three-Layered PCL Grafts Promoted Vascular Regeneration in a Rabbit Carotid Artery Model.

In this study, a three layered poly (ε-caprolactone) (PCL) graft (tPCL) was fabricated by electrospinning PCL and electrospraying poly (ethylene oxide) (PEO), which has a thin dense inner layer, a loose middle layer, and a dense outer layer. Regular PCL grafts (rPCL) with only a dense layer were used as control. In vivo evaluation was performed in rabbit carotid artery. Enhanced cell infiltration, rapid regeneration of endothelium and smooth muscle layers, and increased elastin deposition were observed within the tPCL graft wall. After 3 months, tPCL grafts showed faster PCL degradation than the rPCL grafts. Infiltrated macrophages in the tPCL grafts secreted higher level of monocyte chemoattractant protein-1 (MCP-1) and vascular endothelial growth factor (VEGF) which enhanced vascular regeneration. In conclusion, the tPCL graft may be a useful vascular prosthesis and worth for further investigation.

Small-diameter hybrid vascular grafts composed of polycaprolactone and polydioxanone fibers

Electrospun polycaprolactone (PCL) vascular grafts showed good mechanical properties and patency. However, the slow degradation of PCL limited vascular regeneration in the graft. Polydioxanone (PDS) is a biodegradable polymer with high mechanical strength and moderate degradation rate in vivo. In this study, a small-diameter hybrid vascular graft was prepared by co-electrospinning PCL and PDS fibers. The incorporation of PDS improves mechanical properties, hydrophilicity of the hybrid grafts compared to PCL grafts. The in vitro/vivo degradation assay showed that PDS fibers completely degraded within 12 weeks, which resulted in the increased pore size of PCL/PDS grafts. The healing characteristics of the hybrid grafts were evaluated by implantation in rat abdominal aorta replacement model for 1 and 3 months. Color Doppler ultrasound demonstrated PCL/PDS grafts had good patency, and did not show aneurysmal dilatation. Immunofluorescence staining showed the coverage of endothelial cells (ECs) was significantly enhanced in PCL/PDS grafts due to the improved surface hydrophilicity. The degradation of PDS fibers provided extra space, which facilitated vascular smooth muscle regeneration within PCL/PDS grafts. These results suggest that the hybrid PCL/PDS graft may be a promising candidate for the small-diameter vascular grafts.

A macroporous heparin-releasing silk fibroin scaffold improves islet transplantation outcome by promoting islet revascularisation and survival

Islet transplantation is considered the most promising therapeutic option with the potential to cure diabetes. However, efficacy of current clinical islet transplantation is limited by long-term graft dysfunction and attrition. We have investigated the therapeutic potential of a silk fibroin macroporous (SF) scaffold for syngeneic islet transplantation in diabetic mice. The SF scaffold was prepared via lyophilisation, which enables incorporation of active compounds including cytokines, peptide and growth factors without compromising their biological activity. For the present study, a heparin-releasing SF scaffold (H-SF) in order to evaluate the versatility of the SF scaffold for biological functionalisation. Islets were then co-transplanted with H-SF or SF scaffolds in the epididymal fat pad of diabetic mice. Mice from both H-SF and SF groups achieved 100% euglycaemia, which was maintained for 1year. More importantly, the H-SF-islets co-transplantation led to more rapid reversal of hyperglycaemia, complete normalisation of glucose responsiveness and lower long-term blood glucose levels. This superior transplantation outcome is attributable to H-SF-facilitated islet revascularisation and cell proliferation since significant increase of islet endocrine and endothelial cells proliferation was shown in grafts retrieved from H-SF-islets co-transplanted mice. Better intra-islet vascular reformation was also evident, accompanied by VEGF upregulation. In addition, when H-SF was co-transplanted with islets extracted from vegfr2-luc transgenic mice in vivo, sustained elevation of bioluminescent signal that corresponds to vegfr2 expression was collected, implicating a role of heparin-dependent activation of endogenous VEGF/VEGFR2 pathway in promoting islet revascularisation and proliferation. In summary, the SF scaffolds provide an open platform as scaffold development for islet transplantation. Furthermore, given the pro-angiogenic, pro-survival and minimal post-transplantation inflammatory reactions of H-SF, our data also support the feasibility of clinical implementation of H-SF to improve islet transplantation outcome. STATEMENT OF SIGNIFICANCE 1) The silk fibroin scaffold presented in the present study provides an open platform for scaffold development in islet transplantation, with heparinisation as an example. 2) Both heparin and silk fibroin have been used clinically. The excellent in vivo therapeutic outcome reported here may therefore be clinically relevant and provide valuable insights for bench to bed translation. 3) Compared to conventional clinical islet transplantation, during which islets are injected via the hepatic portal vein, the physical/mechanical properties of silk fibroin scaffolds create a more accessible transplantation site (i.e., within fat pad), which significantly reduces discomfort. 4) Islet implantation into the fat pad also avoids an instant blood mediated inflammatory response, which occurs upon contact of islet with recipients blood during intraportal injection, and prolongs survival and function of implanted islets.