Zuyong Wang

Enhancing mesenchymal stem cell response using uniaxially stretched poly(ε-caprolactone) film micropatterns for vascular tissue engineering application

Regeneration of tunica media with anisotropic architecture still remains a challenging issue for vascular tissue engineering (TE). Herein, we present the development of flexible poly(ε-caprolactone) (PCL) film micropatterns to regulate mesenchymal stem cells (MSCs) function for tunica media construction. Results showed that uniaxial thermal stretching of PCL films resulted in topographical micropatterns comprising of ridges/grooves, and improved mechanical properties, including yield stress, Youngs modulus, and fracture stress without sacrificing film elasticity. Culturing on such PCL film micropatterns, MSCs self-aligned along the ridges with a more elongated morphology as compared to that of the un-stretched film group. Moreover, MSCs obtained a contractile SMCs-like phenotype, with ordered organization of cellular stress filaments and upregulated expression of the contractile makers, including SM-α-actin, calponin, and SM-MHC. The PCL film micropatterns could be rolled into a small-diameter 3D tubular scaffold with circumferential anisotropy of ridges/grooves, and in the incorporation of MSCs, which facilitated a hybrid sandwich-like vascular wall construction with ordered cell architecture similar to that of the tunica media. These results provide insights of how geometric cues are able to regulate stem cells with desired functions and have significant implications for the designing of a functionalized vascular TE scaffold with appropriate topographical geometries for guiding tunica media regeneration with microscale control of cell alignment and genetic expression.

Direct E-jet printing of three-dimensional fibrous scaffold for tendon tissue engineering

Tissue engineering (TE) offers a promising strategy to restore diseased tendon tissue. However, a suitable scaffold for tendon TE has not been achieved with current fabrication techniques. Herein, we report the development of a novel electrohydrodynamic jet printing (E-jetting) for engineering 3D tendon scaffold with high porosity and orientated micrometer-size fibers. The E-jetted scaffold comprised tubular multilayered micrometer-size fibrous bundles, with interconnected spacing and geometric anisotropy along the longitudinal direction of the scaffold. Fiber diameter, stacking pattern, and interfiber distance have been observed to affect the structural stability of the scaffold, of which the enhanced mechanical strength can be obtained for scaffolds with thick fibers as the supporting layer. Human tenocytes showed a significant increase in cellular metabolism on the E-jetted scaffolds as compared to that on conventional electrospun scaffolds (2.7-, 2.8-, and 3.1-fold increase for 150, 300, and 600 µm interfiber distance, respectively; p < 0.05). Furthermore, the scaffolds provided structural support for human tenocytes to align with controlled orientation along the longitudinal direction of the scaffold, and promoted the expression of collagen type I. For the first time, E-jetting has been explored as a novel scaffolding approach for tendon TE, and offers a 3D fibrous scaffold to promote organized tissue reconstruction for potential tendon healing.

Dual-Microstructured Porous, Anisotropic Film for Biomimicking of Endothelial Basement Membrane

Human endothelial basement membrane (BM) plays a pivotal role in vascular development and homeostasis. Here, a bioresponsive film with dual-microstructured geometries was engineered to mimic the structural roles of the endothelial BM in developing vessels, for vascular tissue engineering (TE) application. Flexible poly(ε-caprolactone) (PCL) thin film was fabricated with microscale anisotropic ridges/grooves and through-holes using a combination of uniaxial thermal stretching and direct laser perforation, respectively. Through optimizing the interhole distance, human mesenchymal stem cells (MSCs) cultured on the PCL films ridges/grooves obtained an intact cell alignment efficiency. With prolonged culturing for 8 days, these cells formed aligned cell multilayers as found in native tunica media. By coculturing human umbilical vein endothelial cells (HUVECs) on the opposite side of the film, HUVECs were observed to build up transmural interdigitation cell-cell contact with MSCs via the through-holes, leading to a rapid endothelialization on the PCL film surface. Furthermore, vascular tissue construction based on the PCL film showed enhanced bioactivity with an elevated total nitric oxide level as compared to single MSCs or HUVECs culturing and indirect MSCs/HUVECs coculturing systems. These results suggested that the dual-microstructured porous and anisotropic film could simulate the structural roles of endothelial BM for vascular reconstruction, with aligned stromal cell multilayers, rapid endothelialization, and direct cell-cell interaction between the engineered stromal and endothelial components. This study has implications of recapitulating endothelial BM architecture for the de novo design of vascular TE scaffolds.

Gelatin–siloxane nanoparticles to deliver nitric oxide for vascular cell regulation: Synthesis, cytocompatibility, and cellular responses

Nitric oxide (NO) is an important mediator in cardiovascular system to regulate vascular tone and maintain tissue homeostasis. Its role in vascular cell regulation makes it promising to address the post-surgery restenosis problem. However, the application of NO is constrained by its high reactivity. Here, we developed a novel NO-releasing gelatin-siloxane nanoparticle (GS-NO NP) to deliver NO effectively for vascular cell regulation. Results showed that gelatin-siloxane nanoparticles (GS NPs) could be synthesized via sol-gel chemistry with a diameter of ∼200 nm. It could be modified into GS-NO NPs via S-nitrosothiol (RSNO) modification. The synthesized GS-NO NPs could release a total of ∼0.12 µmol/mg NO sustainably for 7 days following a first-order exponential profile. They showed not only excellent cytocompatibility, but also rapid intracellularization within 2 h. GS-NO NPs showed inhibition of human aortic smooth muscle cell (AoSMC) proliferation and promotion of human umbilical vein endothelial cell (HUVEC) proliferation in a dose-dependent manner, which is an important approach to prevent restenosis. With GS-NO NP dose at 100 µg/mL, the proliferation of AoSMCs could be slowed down whereas the growth of HUVECs was significantly promoted. We concluded that GS-NO NPs could have potential to be used as a promising nano-system to deliver NO for vascular cell regulation.

Uniformly-dispersed nanohydroxapatite-reinforced poly(ε-caprolactone) composite films for tendon tissue engineering application

Regeneration of injuries at tendon-to-bone interface (TBI) remains a challenging issue due to the complex tissue composition involving both soft tendon tissues and relatively hard bone tissues. Tissue engineering using polymeric/ceramic composites has been of great interest to generate scaffolds for tissues healing at TBI. Herein, we presented a novel method to blend polymers and bioceramics for tendon tissue engineering application. A homogeneous composite comprising of nanohydroxyapatite (nHA) particles in poly(ε-caprolactone) (PCL) matrix was obtained using a combination of solvent and mechanical blending process. X-ray diffraction analysis showed that the as-fabricated PCL/nHA composite film retained phase-pure apatite and semi-crystalline properties of PCL. Infrared spectroscopy spectra confirmed that the PCL/nHA composite film exhibited the characteristics functional groups of PCL and nHA, without alteration to the chemical properties of the composite. The incorporation of nHA resulted in PCL/nHA composite film with improved mechanical properties such as Youngs Modulus and ultimate tensile stress, which were comparable to that of the native human rotator tendon. Seeding with human tenocytes, cells attached on the PCL/nHA composite film, and after 14days of culturing, these cells could acquire elongated morphology without induced cytotoxicity. PCL/nHA composite film could also result in increased cell metabolism with prolonged culturing, which was comparable to that of the PCL group and higher than that of the nHA group. All these results demonstrated that the developed technique of combining solvent and mechanical blending could be applied to fabricate composite films with potential for tendon tissue engineering applications.

Uniaxially Stretched Flexible Surface Plasmon Resonance Film for Versatile Surface Enhanced Raman Scattering Diagnostics

Surface-enhanced Raman scattering (SERS) spectroscopy affords a rapid, highly sensitive, and nondestructive approach for label-free and fingerprint diagnosis of a wide range of chemicals. It is of great significance to develop large-area, uniform, and environmentally friendly SERS substrates for in situ identification of analytes on complex topological surfaces. In this work, we demonstrate a biodegradable flexible SERS film via irreversibly and longitudinally stretching metal deposited biocompatible poly(ε-caprolactone) film. This composite film after stretching shows surprising phenomena: three-dimensional and periodic wave-shaped microribbons array embedded with a high density of nanogaps functioning as hot-spots at an average gap size of 20 nm and nanogrooves array along the stretching direction. The stretched polymer surface plasmon resonance film gives rise to more than 10 times signal enhancement in comparison with that of the unstretched composite film. Furthermore, the SERS signals with high uniformity exhibit good temperature stability. The polymer SPR film with excellent flexibility and transparency can be conformally attached onto arbitrary nonplanar surfaces for in situ detection of various chemicals. Our results pave a new way for next-generation flexible SERS detection means, as well as enabling its huge potentials toward green wearable devices for point-of-care diagnostics.

Estradiol-Loaded Poly(ε-caprolactone)/Silk Fibroin Electrospun Microfibers Decrease Osteoclast Activity and Retain Osteoblast Function

Estrogen, a steroid hormone, plays an important role in modulating osteoclast proliferation and development. Estrogen deficiency boosts osteoclast activity, leading to osteoporosis in elderly women. In this study, 17-ß estradiol (E2)-loaded poly(ε-caprolactone) (PCL)/silk fibroin (SF) electrospun microfibers were developed as a proposed localized E2 delivery system to treat osteoporotic fractures. PCL is a synthetic polymer known for its biocompatibility and excellent mechanical properties. The bioactivity of PCL was enhanced by mixing it with a natural SF polymer that has low immunogenicity and inherent bioactivity. Different ratios of PCL/SF blends were electrospun and characterized by scanning electron microscopy, Fourier transform infrared spectroscopy, and water contact angle measurement. PCL and SF at a ratio of 50:50 (PCL50/SF50) augmented cell proliferation of murine preosteoblast MC3T3-E1 cells and murine preosteoclast RAW 264.7 cells. Hence, PCL50/SF50 was selected and mixed with three concentrations of E2 to produce electrospun fiber mesh (0.1% E2@PCL/SF, 1% E2@PCL/SF, and 5% E2@PCL/SF). Sustained release of E2 was obtained for about 3 weeks at higher E2 concentration 5% E2@PCL/SF. An E2-loaded PCL50/SF50 elecrospun microfiber (1% E2@PCL/SF and 5% E2@PCL/SF) reduced tartrate-resistant acid phosphate activity, total DNA, and multinucleated cell formation of osteoclasts. On the other hand, the alkaline phosphatase activity and collagen I expression of osteoblasts were retained on all E2-loaded electrospun microfibers. The E2@PCL/SF system shows potential to be used for localized E2 delivery for the treatment of osteoporotic fractures.

A multi-material coating containing chemically-modified apatites for combined enhanced bioactivity and reduced infection via a drop-on-demand micro-dispensing technique

Prevention of infection and enhanced osseointegration are closely related, and required for a successful orthopaedic implant, which necessitate implant designs to consider both criteria in tandem. A multi-material coating containing 1:1 ratio of silicon-substituted hydroxyapatite and silver-substituted hydroxyapatite as the top functional layer, and hydroxyapatite as the base layer, was produced via the drop-on-demand micro-dispensing technique, as a strategic approach in the fight against infection along with the promotion of bone tissue regeneration. The homogeneous distribution of silicon-substituted hydroxyapatite and silver-substituted hydroxyapatite micro-droplets at alternate position in silicon-substituted hydroxyapatite-silver-substituted hydroxyapatite/hydroxyapatite coating delayed the exponential growth of Staphylococcus aureus for up to 24 h, and gave rise to up-regulated expression of alkaline phosphatase activity, type I collagen and osteocalcin as compared to hydroxyapatite and silver-substituted hydroxyapatite coatings. Despite containing reduced amounts of silicon-substituted hydroxyapatite and silver-substituted hydroxyapatite micro-droplets over the coated area than silicon-substituted hydroxyapatite and silver-substituted hydroxyapatite coatings, silicon-substituted hydroxyapatite-silver-substituted hydroxyapatite/hydroxyapatite coating exhibited effective antibacterial property with enhanced bioactivity. By exhibiting good controllability of distributing silicon-substituted hydroxyapatite, silver-substituted hydroxyapatite and hydroxyapatite micro-droplets, it was demonstrated that drop-on-demand micro-dispensing technique was capable in harnessing the advantages of silver-substituted hydroxyapatite, silicon-substituted hydroxyapatite and hydroxyapatite to produce a multi-material coating along with enhanced bioactivity and reduced infection.

In-vivo evaluation of subcutaneously implanted cell-loaded apatite microcarriers for osteogenic potency

Cell-loaded apatite microcarriers present as potential scaffolds for direct in-vivo delivery of cells post-expansion to promote bone regeneration. The objective of this study was to evaluate the osteogenic potency of human foetal mesenchymal stem cells (hfMSC)-loaded apatite microcarriers when implanted subcutaneously in a mouse model. This was done by examining for ectopic bone formation at 2 weeks, 1 month and 2 months, which were intended to coincide with the inflammation, healing and remodelling phases, respectively. Three histological examinations including haematoxylin and eosin staining to examine general tissue morphology, Masson’s trichrome staining to identify tissue type, and Von Kossa staining to examine extent of tissue mineralisation were performed. In addition, immunohistochemistry assay of osteopontin was conducted to confirm active bone formation by the seeded hfMSCs. Results showed a high level of tissue organisation and new bone formation, with active bone remodelling being observed at the end of 2 months, and an increase in tissue density, organisation, and mineralisation could also be observed for hfMSC-loaded apatite microcarriers. Various cell morphology resembling that of osteoblasts and osteoclasts could be seen on the surfaces of the hfMSC-loaded apatite microcarriers, with presence of woven bone tissue formation being observed at the intergranular space. These observations were consistent with evidence of ectopic bone formation, which were absent in group containing apatite microcarriers only. Overall, results suggested that hfMSC-loaded apatite microcarriers retained their osteogenic potency after implantation, and provided an effective platform for bone tissue regeneration.Graphical Abstract

Tailoring of poly(vinyl alcohol) hydrogels properties by incorporation of crosslinked acrylic acid

The present paper aims to study the possibility to modify the properties of poly (vinyl alcohol) (PVA) hydrogel incorporated with crosslinked acrylic acid (AAc) by UV irradiation in order to extend the nature of PVA as a biomaterial for many applications in regenerative medicine such as a scaffold that provide structural integrity to tissue constructs, control drug and protein delivery to tissues and cultures. The chemical components in modified hydrogels have been determined by Fourier Transform Infrared Spectroscopy (FTIR) and confirmed the successful crosslinking of AAc within the hydrogels. The inter-morphology has been studied by Scanning Electronic Microscopy (SEM) and demonstrated a uniform micro-pore distribution through the entire modified hydrogels. These results, in combination with the good compatibility of PVA and P(AAc), suggest that PVA/P(AAc) hydrogel is a promising biomaterial for tissue engineering regenerative medicine application.