Characterization and in-vitro assessment of silicon-based apatite microspheres for bone tissue engineering applications.

In the field of bone tissue engineering, silicon (Si) has been found as an essential element for bone growth. However, the use of silicon in bioceramics microspheres remains limited. In this work, different weight percentages (0.8, 1.6, and 2.4 wt %) of silicon was incorporated into hydroxyapatite and fabricated into microspheres. 2.4 wt % of Si incorporated into HAp microspheres (2.4 SiHAp) were found to enhance functional properties of the microspheres which resulted in improved cell viability of human mesenchymal stem cells (hMSCs), demonstrating rapid cell proliferation rates resulting in high cell density accumulated on the surface of the microspheres which in turn permitted better hMSCs differentiation into osteoblasts when validated by bone marker assays (Type I collagen, alkaline phosphatase, osteocalcin, and osteopontin) compared to apatite microspheres of lower wt % of Si incorporated and non-substituted HAp (2.4 SiHAp >1.6 SiHAp >0.8 SiHAp > HAp). SEM images displayed the densest cell population on 2.4 SiHAp surfaces with the greatest degree of cell stretching and bridging between neighboring microspheres. Incorporation of silicon into apatite microspheres was found to accelerate the rate and number of apatite nucleation sites formed when subjected to physiological conditions improving the interface between the microsphere scaffolds and bone forming cells, facilitating better adhesion and proliferation.

Optimizing fabrication parameters via Taguchi method for production of high yield hydroxyapatite microsphere scaffolds using Drop-on-Demand inkjet method.

Drop on demand (DOD) inkjet method is a cost-efficient way of producing hydroxyapatite (HAp) microsphere scaffolds with narrow size distribution. However, DOD fabrication parameters may influence the yield and characteristics of the microsphere scaffolds. Testing different permutations and combinations of fabrication parameters is costly and time consuming. Taguchi method could be used as a predictive tool for optimizing the key fabrication parameters to produce HAp microspheres with desired yield and properties, minimizing the number of experimental combinations to be tested. The aim of this study is to investigate the influence of the fabrication parameters on the characteristics of the microspheres formed and determine optimum parameter conditions for producing high yield HAp microsphere scaffolds with the desired properties intended to serve as potential bone substitutes. We aimed to achieve microspheres with high production yield, microsphere size of <230 µm, micropore sizes <1 µm, rough surface morphology and high sphericity. Experiments were conducted using Taguchi method with a L9 orthogonal array at three levels per parameter to determine optimum parameter values for (1) operating pressure, (2) shutter speed duration, (3) nozzle height and (4) CaCl2 concentration. Based on signal-to-noise (S/N) ratio analysis, the identified optimum parameter conditions for operating pressure, shutter speed duration, nozzle height and CaCl2 concentration to be 0.9-1.3 bar, 100 ms, 8 cm and 0.4 M respectively. The microspheres obtained had an average size of 213 µm, 0.45 µm micropore size, high sphericity index of 0.95 and high production yield of 98%. Confirmation tests and ANOVA results affirms the validity of Taguchi method in optimizing HAp microspheres with high yield, desired size, micropore size and shape. HAp microsphere scaffolds produced by optimum conditions were subjected to a 7-day in-vitro study. Cells remained viable and continued to proliferate (increased 1.2-fold) over 7 days with microspheres maintaining high cell density with cells bridging between microspheres. Alkaline phosphatase (ALP) assay increased 1.5-fold from day 1, suggesting good osteogenic potency of HAp microspheres as potential bone substitutes.

Facile Construction of a Two-in-One Injectable Micelleplex-Loaded Thermogel System for the Prolonged Delivery of Plasmid DNA.

Nanoparticle-hydrogel systems have recently emerged as a class of interesting hybrid materials with immense potential for several biomedical applications. Remarkably, the incorporation of nanoparticles into a hydrogel may yield synergistic benefits lacking in a singular system. However, most synthetic strategies require laborious steps to achieve the system, severely restricting the process of translational research. Herein, a facile strategy to access a two-in-one system comprising two distinct polyurethane (PU)-based micellar systems is demonstrated and applied as a novel sustained gene delivery platform, where the two PUs are synthesized similarly but with slightly different compositions. One PU forms cationic micelles that complex with plasmid DNA (pDNA), which are loaded into a thermogel formed by another PU micellar system for the prolonged release of pDNA micelleplexes. Specifically, a thermogelling multiblock PU copolymer (denoted as EPH) was synthesized via the step-growth polymerization of poly(ethylene glycol), poly(propylene glycol), and poly(3-hydroxybutyrate). By further introducing a cationic extender, 3-(dimethylamino)-1,2-propanediol, into the reaction feed, a series of cationic PUs (denoted as EPHD) with varying compositions were obtained. The EPHDs formed positively charged micelles in aqueous solutions, efficiently condensed pDNA into nano-sized micelleplexes (<200 nm) at optimized w/w ratios, and mediated transient green fluorescence protein expression in HEK293T cells at 48 h post-transfection. On the other hand, aqueous EPH solution (4 wt %) was injectable at 4 °C and rapidly gelled upon heating to 37 °C to form a stable hydrogel depot. EPHD/pDNA micelleplexes were easily loaded into EPH by mixing the solutions at 4 °C, before heating to 37 °C, leading to the resultant hydrogel system. The in vitro release study revealed that while free pDNA loaded in the thermogel was completely released in 2 weeks, the release of EPHD/pDNA micelleplexes was prolonged to at least 28 days, suggesting substantial micelleplex-hydrogel interactions. Intact, bioactive, and noncytotoxic EPHD/pDNA micelleplexes in the release media were proved by gel retardation, in vitro gene transfection, and CCK-8 cytotoxicity assay results, respectively. Collectively, this work presents a simple approach to achieving and optimizing a novel two-in-one nanoparticle-hydrogel system for the prolonged delivery of pDNA and may be promising for long-term gene delivery applications.

Silver-substituted hydroxyapatite inhibits Pseudomonas aeruginosa outer membrane protein F: A potential antibacterial mechanism.

Bone fractures are one of the most common injuries, and they have a big effect on population health worldwide. Traumatic bone injuries can be partially treated with implanting bone-graft substitutes, for example, hydroxyapatite (HA), a bioceramic that is similar materially to natural bones with good bioactivity and osteoconductivity. It could, however, be vulnerable to infections because of the way an HA-based bone graft is put in, which could be a weakness in the host's defense. This study incorporated silver (Ag) into hydroxyapatite (Ag-HA) and silicon-containing hydroxyapatite (AgSi-HA) discs to combat this implant-triggered infection. Further, we investigated the antibacterial activities and potential underlying mechanism against a gram-negative bacterium, Pseudomonas aeruginosa. We noticed that the rich calcium (Ca2+) content in HA discs could trigger the change in P. aeruginosa physiology that leads to the enhanced bacterial growth on non­silver incorporated HA discs. But the released Ag+ from Ag-HA and AgSi-HA discs caused significant damage to bacterial cells at a low concentration of 0.3 ppm. We also observed dramatic morphological changes of Ag-HA and AgSi-HA surface-attached bacteria cells. Finally, we identified a potential action mechanism - the surface-bound Ag+ from Ag-HA and AgSi-HA potently inhibited the outer membrane protein F (OprF) expression of P. aeruginosa. Collectively, our results indicate that incorporating silver ions into HA could contribute viably to excellent antibacterial activities against P. aeruginosa to prevent HA-based bone graft infection.

Biodegradation of polymers in managing plastic waste - A review.

The modern economy that is fast-moving and convenience centric has led to excessive consumption of plastic. This has unwittingly led to egregious accumulation of plastic waste polluting the environment. Unfortunately, present means of plastic waste management have all been proven as less than adequate; namely recycling, landfill and incineration. Recent focus on plastic waste management has seen the confluence of the developments in biodegradable polymers and microbial engineering strategy for more expedient decomposition of plastic waste at composting facilities. This review paper is an assimilation of current developments in the areas of biodegradable polymer as well as microbial strategy towards management of polymer waste. Advents in biodegradable polymers have been promising, especially with aliphatic polyesters and starch in blends or co-polymers of these. Microbial strategies have been pursued for the identification of microbial strains and understanding of their enzymatic degradation process on polymers. New insights in these two areas have been focused in improving the rate of degradation of plastic waste at composting facilities. Recent alignment of testing and certification standards is outlined to give intimate insights into the mechanisms and factors influencing biodegradation. Despite recent milestones, economic viability of composting plastic waste in mainstream waste facilities is still a distance away. As it remains that a polymer that is biodegradable is functionally inferior to conventional polymers. Rather, it requires a shift in consumer behaviour to accept less durable biodegradable plastic products, this will then lower the threshold for biodegradable polymers to become a commercial reality.

Surface Design for Antibacterial Materials: From Fundamentals to Advanced Strategies.

Healthcare-acquired infections as well as increasing antimicrobial resistance have become an urgent global challenge, thus smart alternative solutions are needed to tackle bacterial infections. Antibacterial materials in biomedical applications and hospital hygiene have attracted great interest, in particular, the emergence of surface design strategies offer an effective alternative to antibiotics, thereby preventing the possible development of bacterial resistance. In this review, recent progress on advanced surface modifications to prevent bacterial infections are addressed comprehensively, starting with the key factors against bacterial adhesion, followed by varying strategies that can inhibit biofilm formation effectively. Furthermore, "super antibacterial systems" through pre-treatment defense and targeted bactericidal system, are proposed with increasing evidence of clinical potential. Finally, the advantages and future challenges of surface strategies to resist healthcare-associated infections are discussed, with promising prospects of developing novel antimicrobial materials.

Silver, silicon co-substituted hydroxyapatite modulates bacteria-cell competition for enhanced osteogenic function.

Combating bacteria while promoting tissue regeneration is an aim of highest priority for employing biomaterials in orthopedics that often embroiled with pre-operative contamination. Through simulating a surgical site infection environment and an infected implant site, we showcase the ability of a functionally modified hydroxyapatite, Ag,Si-HA that permits preferential adhesion of human bone marrow derived mesenchymal stem cells (BMSCs) over co-cultured bacterial pathogen,Pseudomonas aeruginosa, by displaying immediate suppression and killing of the bacteria present with minimum cytotoxicity for 28 d. And, at the same time, Ag,Si-HA stimulates BMSCs towards osteogenic differentiation despite being within the contaminated milieu. These findings provide well-defined requirements for incorporating antibacterial properties to biomaterials in managing pre-operative contamination. In addition, it highlights the dual positive attributes of Ag,Si-HA as an effective antibacterial biomaterial and at the same time, promotes bone tissue regeneration.

Silicon-incorporated nanohydroxyapatite-reinforced poly(ε-caprolactone) film to enhance osteogenesis for bone tissue engineering applications.

Biomaterials composed of polymers and bioceramics have great prospects to repair large and complicated bone defects. Here, we developed a composite film consisting of poly(ε-caprolactone) (PCL) and silicon-substituted hydroxyapatite (Si-HA) nanoparticles to enhance the osteogenic effects of the scaffold for bone tissue engineering applications. The results showed that the Si-HA nanoparticles obtained an even distribution in the PCL matrix, resulting in a homogeneous composite film. Compared to HA-incorporated PCL film, the addition of silicon did not cause hydrophilic alterations to the film surface. With the seeding of mouse calvarial preosteoblasts (MC3T3-E1), the cells exhibited the good behaviors of adhesion and growth on the PCL/Si-HA film. Compared to the PCL/HA films, incorporation of Si-HA nanoparticles in PCL/Si-HA films showed the increased production of alkaline phosphatase (ALP) and calcium content by MC3T3-E1 cells. These results suggested the suitability of the PCL/Si-HA composite film to elicit cellular growth and functional differentiation with the potential for bone tissue engineering applications.

Effect of copper substitution on the local chemical structure and dissolution property of copper-doped ß-tricalcium phosphate.

Substitution of inorganic ions into ß-tricalcium phosphate (ß-TCP) is a well-known approach for facilitating biological functions of bioceramics. However, the dissolution mechanism of those ß-TCPs is still under intensive debates. In the present study, the effect of copper substitution into ß-TCP crystal structure on the local chemical structure and dissolution property of the copper-doped ß-TCP (CuTCP) was investigated to clarify the dissolution mechanism of ß-TCP. A copper-dependent decrease in the dissolution rate of CuTCP with time was observed. The 1H → 31P nuclear magnetic resonance (NMR) spectra of 10 mol% copper-doped ß-TCP after the dissolution test demonstrated an amorphous hydrated layer on the surface of ß-TCP core particles, which contained hydroxyapatite and dicalcium phosphate dihydrate and anhydrate. As such, all the dissolution curves could be curve-fitted by a heterogeneous dissolution model composing of fast and slow dissolution components. Overall, dissolution mechanism could be proposed as follows: the CuTCP particles initially dissolved by hydrolysis based on the fast dissolution component. Subsequently, the amorphous hydrated layers were formed on their surface, and caused the diffusion-controlled dissolution. As the result, the slow dissolution component would be dominant, and led to the decreased dissolution rate. STATEMENT OF SIGNIFICANCE: Understanding the dissolution mechanism of copper doped ß-tricalcium phosphate (CuTCP) is crucial for designing an angiogenetic controlled copper release CuTCP for therapeutic biomaterials. However, dissolution mechanism of ß-TCP or CuTCP is still under intensive debates. This study demonstrated for the first time, that amorphous hydrated layers were formed on the CuTCP particle surface during its dissolution process, which caused a diffusion-controlled dissolution, and decreased the dissolution rate of CuTCP. This work not only provided a novel dissolution mechanism of ß-TCP or CuTCP, but also a new finding for designing an angiogenetic controlled copper release CuTCP for therapeutic biomaterials.

Beta-cyclodextrin modified mesoporous bioactive glass nanoparticles/silk fibroin hybrid nanofibers as an implantable estradiol delivery system for the potential treatment of osteoporosis.

Osteoporosis, a systemic skeletal disease prevalent in elderly women, is associated with post-menopausal estrogen deficiency. Although systemic administration of exogenous estradiol (E2) reduced fragility fractures, the treatment has adverse effects. Localized delivery technologies of E2 could be utilized to circumvent the systemic adverse effects of systemic administration. In this study, a localized E2 delivery system is developed. Mesoporous bioactive glass nanoparticles (MBGNPs) with inherent osteogenic properties are modified with ß-cyclodextrin (CD-MBGNPs) to enhance their affinity for E2. To ensure mechanical stability and integrity, E2 loaded CD-MBGNPs are further electrospun with silk fibroin (SF) to produce a nanofibrous mesh (E2@CD-MBGNPs/SF). The incorporation of MBGNPs in SF enhances in vitro apatite formation and sustains the constant release of E2. Moreover, osteoblast proliferation and differentiation markers such as alkaline phosphatase activity, collagen 1 and osteocalcin expression of MC3T3-E1 are augmented in CD-MBGNPs/SF and E2@CD-MBGNPs/SF as compared to SF nanofibers. On the other hand, osteoclast DNA, tartrate resistant acid phosphatase activity and multinucleated cell formation are reduced in E2@CD-MBGNPs/SF as compared to CD-MBGNPs/SF and SF. Hence the presence of CD-MBGNPs in SF stimulates osteoblast function whereas E2 incorporation in CD-MBGNPs/SF reduces osteoclast activity. This is the first report to develop CD-MBGNPs/SF as a localized delivery system for hydrophobic molecules such as estradiol to treat osteoporosis.

Fabrication of chelate-setting α-tricalcium phosphate cement using sodium citrate and sodium alginate as mixing solution and its in vivo osteoconductivity.

Moldable and injectable calcium-phosphate cements (CPCs) are material candidates for bone replacement applications. In the present study, we examined the effectiveness of sodium alginate and sodium citrate additives to the liquid phase of CPC, in improving its handling property as well as mechanical strength. The use of these additives enhanced the handling property significantly, in terms of consistency as compared to CPC without additives due to the liquefying effect caused by the adsorption of citrate ions on the cement particles. Sodium alginate and sodium citrate were added to CPC, which was set by the chelate-bonding capability of inositol phosphate, and was composed of mainly α-tricalcium phosphate (α-TCP) phase (>90%). The compressive strength of the CPC containing sodium alginate and sodium citrate was 3.4 ± 0.3 MPa, which was significantly higher than cement without additives. Furthermore, this cement exhibited favorable osteoconductivity and bioresorbability, and remained the α-TCP phase after 4-week implantation in a pig tibiae model. These results suggested that the cement is a potential candidate as a bioresorbable paste-like artificial bone. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 2361-2370, 2018.

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.

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.

Injectable chelate-setting hydroxyapatite cement prepared by using chitosan solution: Fabrication, material properties, biocompatibility, and osteoconductivity.

An injectable chelate-setting hydroxyapatite cement (IP6-HAp), formed by chelate-bonding capability of inositol phosphate (IP6), was developed. The effects of ball-milling duration of starting HAp powder and IP6 concentration on the material properties such as injectability and mechanical strength of the cement were examined. The cement powder was prepared by ball-milling the as-synthesized HAp powder for 5 min using ZrO2 beads with a diameter of 10 mm, followed by another 60 min with ZrO2 beads with a diameter of 2 mm, and thereafter surface-modified with 5000 ppm of IP6 solution. Injectable cement was then fabricated with this HAp powder and 2.5 mass% chitosan as a mixing solution, with a setting time of 36.3 ± 4.7 min and a compressive strength of 19.0 ± 2.1 MPa. The IP6-HAp cements prepared with chitosan showed favorable biocompatibility in vitro using an osteoblast cell model, and osteoconductivity in vivo using a pig tibia model.

Nanomaterial scaffolds to regenerate musculoskeletal tissue: signals from within for neovessel formation.

Current treatments for musculoskeletal disease and injury are restricted with the usage of autografts and allografts. Tissue engineering that applies the principles of biology and engineering to develop functional substitutes has potential promise of therapeutic regeneration for musculoskeletal tissues. However, engineering sizable tissues needs a vascular network to supply cells with nutrients, oxygen and signals after implantation. For this purpose, recent developments on therapeutic nanomaterials have been explored in delivering different vessel-inductive growth factors, small biomolecules and ions for scalable engineering into vascularizable scaffolds. Here, we provide an overview on the current efforts, and propose future perspectives for precise regulation on vascularization processes and musculoskeletal tissue functionality.

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.

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 tissue's 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 Young's 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.

Mechanically-enhanced three-dimensional scaffold with anisotropic morphology for tendon regeneration.

Tissue engineering has showed promising results in restoring diseased tendon tissue functions. Herein, a hybrid three-dimensional (3D) porous scaffold comprising an outer portion rolled from an electrohydrodynamic jet printed poly(ɛ-caprolactone) (PCL) fiber mesh, and an inner portion fabricated from uniaxial stretching of a heat-sealed PCL tube, was developed for tendon tissue engineering (TE) application. The outer portion included three layers of micrometer-scale fibrous bundles (fiber diameter: ~25 µm), with an interconnected spacing and geometric anisotropy along the scaffold length. The inner portion showed orientated micro-ridges/grooves in a parallel direction to that of the outer portion. Owning to the addition of the inner portion, the as-fabricated scaffold exhibited comparable mechanical properties to those of the human patellar tendon in terms of Young's modulus (~227 MPa) and ultimate tensile stress (~50 MPa). Compared to the rolled electrospun fibers, human tenocytes cultured in the tendon scaffolds showed increased cellular metabolism. Furthermore, the 3D tendon scaffold resulted in up-regulated cell alignment, cell elongation and formation of collagen type I. These results demonstrated the potential of mechanically-enhanced 3D fibrous scaffold for applications in tendon TE, with desired cell alignment and functional differentiation.

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 film's 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.