Demonstrating the Potential of Using Bio-Based Sustainable Polyester Blends for Bone Tissue Engineering Applications.

Healthcare applications are known to have a considerable environmental impact and the use of bio-based polymers has emerged as a powerful approach to reduce the carbon footprint in the sector. This research aims to explore the suitability of using a new sustainable polyester blend (Floreon™) as a scaffold directed to aid in musculoskeletal applications. Musculoskeletal problems arise from a wide range of diseases and injuries related to bones and joints. Specifically, bone injuries may result from trauma, cancer, or long-term infections and they are currently considered a major global problem in both developed and developing countries. In this work we have manufactured a series of 3D-printed constructs from a novel biopolymer blend using fused deposition modelling (FDM), and we have modified these materials using a bioceramic (wollastonite, 15% w/w). We have evaluated their performance in vitro using human dermal fibroblasts and rat mesenchymal stromal cells. The new sustainable blend is biocompatible, showing no differences in cell metabolic activity when compared to PLA controls for periods 1-18 days. FloreonTM blend has proven to be a promising material to be used in bone tissue regeneration as it shows an impact strength in the same range of that shown by native bone (just under 10 kJ/m2) and supports an improvement in osteogenic activity when modified with wollastonite.

Thiolene- and Polycaprolactone Methacrylate-Based Polymerized High Internal Phase Emulsion (PolyHIPE) Scaffolds for Tissue Engineering.

Highly porous emulsion templated polymers (PolyHIPEs) provide a number of potential advantages in the fabrication of scaffolds for tissue engineering and regenerative medicine. Porosity enables cell ingrowth and nutrient diffusion within, as well as waste removal from, the scaffold. The properties offered by emulsion templating alone include the provision of high interconnected porosity, and, in combination with additive manufacturing, the opportunity to introduce controlled multiscale porosity to complex or custom structures. However, the majority of monomer systems reported for PolyHIPE preparation are unsuitable for clinical applications as they are nondegradable. Thiol-ene chemistry is a promising route to produce biodegradable photocurable PolyHIPEs for the fabrication of scaffolds using conventional or additive manufacturing methods; however, relatively little research has been reported on this approach. This study reports the groundwork to fabricate thiol- and polycaprolactone (PCL)-based PolyHIPE materials via a photoinitiated thiolene click reaction. Two different formulations, either three-arm PCL methacrylate (3PCLMA) or four-arm PCL methacrylate (4PCLMA) moieties, were used in the PolyHIPE formulation. Biocompatibility of the PolyHIPEs was investigated using human dermal fibroblasts (HDFs) and human osteosarcoma cell line (MG-63) by DNA quantification assay, and developed PolyHIPEs were shown to be capable of supporting cell attachment and viability.

Delivery of Bioactive Compounds to Improve Skin Cell Responses on Microfabricated Electrospun Microenvironments.

The introduction of microtopographies within biomaterial devices is a promising approach that allows one to replicate to a degree the complex native environment in which human cells reside. Previously, our group showed that by combining electrospun fibers and additive manufacturing it is possible to replicate to an extent the stem cell microenvironment (rete ridges) located between the epidermal and dermal layers. Our group has also explored the use of novel proangiogenic compounds to improve the vascularization of skin constructs. Here, we combine our previous approaches to fabricate innovative polycaprolactone fibrous microtopographical scaffolds loaded with bioactive compounds (2-deoxy-D-ribose, 17ß-estradiol, and aloe vera). Metabolic activity assay showed that microstructured scaffolds can be used to deliver bioactive agents and that the chemical relation between the working compound and the electrospinning solution is critical to replicate as much as possible the targeted morphologies. We also reported that human skin cell lines have a dose-dependent response to the bioactive compounds and that their inclusion has the potential to improve cell activity, induce blood vessel formation and alter the expression of relevant epithelial markers (collagen IV and integrin ß1). In summary, we have developed fibrous matrixes containing synthetic rete-ridge-like structures that can deliver key bioactive compounds that can enhance skin regeneration and ultimately aid in the development of a complex wound healing device.

Fabrication of Topographically Controlled Electrospun Scaffolds to Mimic the Stem Cell Microenvironment in the Dermal-Epidermal Junction.

The use of microfabrication techniques for the development of innovative constructs for tissue regeneration is a growing area of research. This area comprises both manufacturing and biological approaches for the development of smart materials aiming to control and direct cell behavior to enhance tissue healing. Many groups have focused their efforts on introducing complexity within these innovative constructs via the inclusion of nano- and microtopographical cues mimicking physical and biological aspects of the native stem cell niche. Specifically, in the area of skin tissue engineering, seminal work has reported replicating the microenvironments located in the dermal-epithelial junction, which are known as rete ridges. The rete ridges are key for both stem cell control and the physiological performance of the skin. In this work, we have introduced complexity within electrospun membranes to mimic the morphology of the rete ridges in the skin. We designed and tested three different patterns, characterized them, and explored their performance in vitro, using 3D skin models. One of the studied patterns (pattern B) was shown to aid in the development of an in vitro rite-ridgelike skin model that resulted in the expression of relevant epithelial markers such as collagen IV and integrin ß1. In summary, we have developed a new skin model including synthetic rete-ridgelike structures that replicate both morphology and function of the native dermal-epidermal junction and that offer new insights for the development of smart skin tissue engineering constructs.

Tuning Electrospun Substrate Stiffness for the Fabrication of a Biomimetic Amniotic Membrane Substitute for Corneal Healing.

Corneal blindness is the fourth most common cause of vision impairment worldwide with a high incidence in global south countries. A recently developed surgical technique for treating corneal blindness is simple limbal epithelial transplantation (SLET), which uses small pieces of healthy limbal tissue (limbal explants) delivered to the damaged eye using the human amniotic membrane (AM) as a carrier. SLET relies on the use of tissue banks for the AM that reduces the availability of the technique. Replacing the AM with a synthetic membrane is key to making SLET more accessible to those who need it. Previous research has demonstrated the suitability of electrospun poly(lactide-co-glycolide) (PLGA) scaffolds as AM substitutes, and here, we report how these membranes can be tailored to mimic fundamental AM mechanical properties. To modify the stiffness of PLGA electrospun membranes, we explored different electrospinning solvent systems (1,1,1,3,3,3,-hexafluoroisopropanol (HFIP), dichloromethane (DCM), chloroform, and N,N-dimethylformamide (DMF)) and the use of plasticizers (PEG400 and glycerol). PEG400 was found to reduce stiffness from 60 MPa to around 4 MPa, approaching the values shown by the native AM. The biocompatibility of membranes with and without PEG400 was found to be comparable, and cell outgrowth from rabbit/porcine explants was successfully observed on the materials after 3 weeks. This research underpins the manufacture of next-generation fibrous biomimetic membranes that will ultimately be used as amniotic membrane substitutes for biomedical applications including SLET.

The Emerging Potential of Extracellular Vesicles in Cell-Free Tissue Engineering and Regenerative Medicine.

Extracellular vesicles (Evs) are membrane-enclosed vesicles secreted by all cell types that mediate cell-cell communication via their protein, lipid, carbohydrate, and nucleic acid (RNA, DNA) cargo. EVs are involved in a multitude of physiological processes, including development, cell differentiation, and angiogenesis, and have been implicated in tissue repair. Thus, they have been suggested to offer opportunities for the development of novel cell-free tissue engineering (TE) approaches. In this review, we provide an overview of current understanding and emerging applications of EVs in TE and address opportunities and challenges for clinical translation. In addition, we discuss systemic and local routes of delivery of EVs and the advantages and disadvantages of different biomaterials in providing a substrate for the sustained release of EVs in vivo. Impact statement Extracellular vesicles (EVs) are nanoscale, membrane-bound vesicles released by most, if not all, cells in the body. They are implicated in a wide range of physiological processes and diseases ranging from cancer to neurodegeneration, and hold huge potential as mediators of tissue regeneration. This has led to an explosion of interest in using EVs in a variety of tissue engineering applications. In this review, we provide an overview of current progress in the field and highlight the opportunities and challenges of harnessing the potential of EVs in regenerative medicine.