The ideal tissue engineering (TE) strategy for ligament regeneration should recapitulate the bonecalcified cartilage -fibrocartilage -soft tissue interface. Aligned electrospun-fibers have been shown to guide the deposition of a highly organized extracellular matrix (ECM) necessary for ligament TE.However, recapitulating the different tissues observed in the bone-ligament interface using such constructs remains a challenge. This study aimed to explore how fiber alignment and growth factor stimulation interact to regulate the chondrogenic and ligamentous differentiation of mesenchymal stem cells (MSCs). Without growth factor stimulation, MSCs on aligned-microfibers showed higher levels of tenomodulin (TNMD) and aggrecan gene expression compared to MSCs on randomlyoriented fibers. MSCs on aligned-microfibers stimulated with transforming growth factor β3 (TGFβ3) formed cellular aggregates and underwent robust chondrogenesis, evidenced by increased type II collagen expression and sulphated glycosaminoglycans (sGAG) synthesis compared to MSCs on randomly-oriented scaffolds. Bone morphogenetic protein 2 (BMP2) and type I collagen gene expression were higher on randomly-oriented scaffolds stimulated with TGFβ3, suggesting this substrate was more supportive of an endochondral phenotype. In the presence of connective tissue growth factor (CTGF), MSCs underwent ligamentous differentiation, with increased TNMD expression on aligned compared to randomly aligned scaffolds. Upon sequential growth factor stimulation, MSCs expressed types I and II collagen and deposited higher overall levels of collagen compared to scaffolds stimulated with either growth factor in isolation. These findings demonstrate that modulating the alignment of microfibrillar scaffolds can be used to promote either an endochondral, chondrogenic, fibro-chondrogenic or ligamentous MSC phenotype upon presentation of appropriate biochemical cues.

Cardiac patches represent a promising strategy for the treatment of myocardial infarction (MI). Here, an electroconductive cardiac patch that conforms to the mechanics of human myocardium is fabricated. By melt electrospinning writing (MEW), it is possible to fabricate an auxetic patch that can overcome the limited range of elasticity seen in conventional square patch designs. The auxetic patches can accommodate the strains and stresses exhibited by the human myocardium during diastole and systole. It is shown that the geometry of the auxetic patches can be fine-tuned to reflect anisotropic mechanical properties. The anisotropic ratio of effective stiffness (E 1 /E 2) of the auxetic patches agrees with the directionally-dependent mechanics of the heart. Furthermore, in situ polymerization of dopedpolypyrrole (PPy) on the auxetic patches confers electroconductive properties close to those reported of human myocardium. This approach demonstrates the potential use of a rational design of PPy-coated patches for their use as cardiac patches.

Controlling the phenotype of transplanted stem cells is integral to ensuring their therapeutic efficacy. Hypoxia is a known regulator of stem cell fate, the effects of which can be mimicked using hypoxia-inducible factor (HIF) prolyl hydroxylase inhibitors such as dimethyloxalylglycine (DMOG). By releasing DMOG from mesenchymal stem cell (MSC) laden alginate hydrogels, it is possible to stabilize HIF-1α and enhance its nuclear localization. This correlated with enhanced chondrogenesis and a reduction in the expression of markers associated with chondrocyte hypertrophy, as well as increased SMAD 2/3 nuclear localization in the encapsulated MSCs. In vivo, DMOG delivery significantly reduced mineralisation of the proteoglycan-rich cartilaginous tissue generated by MSCs within alginate hydrogels loaded with TGF-β3 and BMP-2. Together these findings point to the potential of hypoxia mimicking hydrogels to control the fate of stem cells following their implantation into the body.

Abstract. Electrospinning is considered a relative simple and versatile technique to form high porosity porous scaffolds with micron to nanoscale fibers for biomedical applications. Here, electrospinning of unsaturated aliphatic polyglobalide (PGl) into well-defined fibers with an average diameter of 9 µm is demonstrated. Addition of a dithiol crosslinker and a photoinitiator to the polymer solution enabled the UV-triggered intra-crosslinking of the fibers during the spinning process. The in-situ crosslinking of the fibers resulted in amorphous material able to swell up to 14% in tetrahydrofyrane (THF) without losing the fiber morphology. Seeding Mesenchymal Stem Cells (MSCs) onto both crosslinked and non-crosslinked PGl fibers proved their compatibility with MSCs and suitability as scaffolds for cell growth and proliferation of MSCs. Moreover, the ability to directly load crosslinked PGl with hydrophobic molecules by soaking the fiber mesh in solution is shown with Rhodamine B and Indomethacin, a hydrophobic anti-inflammatory drug. This marks an advantage over conventional aliphatic polyesters and opens opportunities for the design of drug loaded polyester scaffolds for biomedical applications or tissue engineering.

The anterior cruciate ligament (ACL) of the knee is vital for proper joint function and is commonly ruptured during sports injuries or car accidents. Due to a lack of intrinsic healing capacity and drawbacks with allografts and autografts, there is a need for a tissue engineered ACL replacement. Our group has previously used aligned sheets of electrospun polycaprolactone nanofibers to develop solid cylindrical bundles of longitudinally aligned nanofibers. We have shown that these nanofiber bundles support cell proliferation and elongation and the hierarchical structure and material properties are similar to the native human ACL. It is possible to combine multiple nanofiber bundles to create a scaffold that attempts to mimic the macro-scale structure of the ACL. The goal of this work was to develop a hierarchical bioactive scaffold for ligament tissue engineering using connective tissue growth factor (CTGF) conjugated nanofiber bundles and evaluate the behavior of mesenchymal stem cells (MSCs) on these scaffolds in vitro and in vivo. CTGF was immobilized onto the surface of individual nanofiber bundles or scaffolds consisting of multiple nanofiber bundles. The conjugation efficiency and the release of conjugated CTGF was assessed using X-ray photoelectron spectroscopy, assays, and immunofluorescence staining. Scaffolds were seeded with MSCs and maintained in vitro for 7 days (individual nanofiber bundles), in vitro for 21 days (scaled-up scaffolds of 20 nanofiber bundles) or in vivo for 6 weeks (small scaffolds of 4 nanofiber bundles) and ligament specific tissue formation was assessed in comparison to non-CTGF conjugated control scaffolds. Results showed that CTGF conjugation encouraged cell proliferation and ligament specific tissue formation in vitro and in vivo. The results suggest that hierarchical electrospun nanofiber bundles conjugated with CTGF are a scalable and bioactive scaffold for ACL tissue engineering.

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Electrospun fibers offer tremendous potential for tendon and ligament tissue engineering, yet developing porous scaffolds mimicking the size, stiffness and strength of human tissues remains a challenge. Previous studies have rolled, braided, or stacked electrospun sheets to produce threedimensional (3D) scaffolds with tailored sizes and mechanical properties. A common limitation with such approaches is the development of low porosity scaffolds that impede cellular infiltration into the body of the implant, thereby limiting their regenerative potential. Here, we demonstrate how varying the rotational speed of the collecting mandrel during the electrospinning of poly(ε-caprolactone) (PCL) can be used to limit inter-fiber fusion (or fiber welding). Increasing the fraction of unfused fibers reduced the flexural rigidity of the electrospun sheets, which in turn allowed us to bundle the fibers into 3D scaffolds with similar dimensions to the human anterior cruciate ligament (ACL). These unfused fibers allowed for higher levels of porosity (up to 95%) that facilitated the rapid migration of mesenchymal stem cells (MSCs) into the body of the scaffolds. Mechanical testing demonstrated that the fiber-bundles possessed a Young's modulus approaching that of the native human ACL. The scaffolds were also capable of supporting the differentiation of MSCs towards either the fibrocartilage or ligament/tendon lineage. This novel electrospinning strategy could be used to produce mechanically functional, yet porous, scaffolds for a wide range of biomedical applications.

A key goal of functional cartilage tissue engineering is to develop constructs with mechanical properties approaching those of the native tissue. Herein we describe a number of tests to characterize the mechanical properties of tissue engineered cartilage. Specifically, methods to determine the equilibrium confined compressive (or aggregate) modulus, the equilibrium unconfined compressive (or Young's) modulus, and the dynamic modulus of tissue engineered cartilaginous constructs are described. As these measurements are commonly used in both the articular cartilage mechanics literature and the cartilage tissue engineering literature to describe the mechanical functionality of cartilaginous constructs, they facilitate comparisons to be made between the properties of native and engineered tissues.