Highly compliant biomimetic scaffolds for small diameter tissue-engineered vascular grafts (TEVGs) produced via melt electrowriting (MEW).

Biofabrication approaches toward the development of tissue-engineered vascular grafts (TEVGs) have been widely investigated. However, successful translation has been limited to large diameter applications, with small diameter grafts frequently failing due to poor mechanical performance, in particular mismatched radial compliance. Herein, melt electrowriting (MEW) of poly(ϵ-caprolactone) has enabled the manufacture of highly porous, biocompatible microfibre scaffolds with physiological anisotropic mechanical properties, as substrates for the biofabrication of small diameter TEVGs. Highly reproducible scaffolds with internal diameter of 4.0 mm were designed with 500 and 250µm pore sizes, demonstrating minimal deviation of less than 4% from the intended architecture, with consistent fibre diameter of 15 ± 2µm across groups. Scaffolds were designed with straight or sinusoidal circumferential microfibre architecture respectively, to investigate the influence of biomimetic fibre straightening on radial compliance. The results demonstrate that scaffolds with wave-like circumferential microfibre laydown patterns mimicking the architectural arrangement of collagen fibres in arteries, exhibit physiological compliance (12.9 ± 0.6% per 100 mmHg), while equivalent control geometries with straight fibres exhibit significantly reduced compliance (5.5 ± 0.1% per 100 mmHg). Further mechanical characterisation revealed the sinusoidal scaffolds designed with 250µm pores exhibited physiologically relevant burst pressures of 1078 ± 236 mmHg, compared to 631 ± 105 mmHg for corresponding 500µm controls. Similar trends were observed for strength and failure, indicating enhanced mechanical performance of scaffolds with reduced pore spacing. Preliminaryin vitroculture of human mesenchymal stem cells validated the MEW scaffolds as suitable substrates for cellular growth and proliferation, with high cell viability (>90%) and coverage (>85%), with subsequent seeding of vascular endothelial cells indicating successful attachment and preliminary endothelialisation of tissue-cultured constructs. These findings support further investigation into long-term tissue culture methodologies for enhanced production of vascular extracellular matrix components, toward the development of the next generation of small diameter TEVGs.

Photo-Cross-Linkable, Injectable, and Highly Adhesive GelMA-Glycol Chitosan Hydrogels for Cartilage Repair.

Hydrogels provide a promising platform for cartilage repair and regeneration. Although hydrogels have shown some efficacy, they still have shortcomings including poor mechanical properties and suboptimal integration with surrounding cartilage. Herein, hydrogels that are injectable, cytocompatible, mechanically robust, and highly adhesive to cartilage are developed. This approach uses GelMA-glycol chitosan (GelMA-GC) that is crosslinkable with visible light and photoinitiators (lithium acylphosphinate and tris (2,2'-bipyridyl) dichlororuthenium (II) hexahydrate ([RuII(bpy)3 ]2+ and sodium persulfate (Ru/SPS)). Ru/SPS-cross-linked hydrogels have higher compressive and tensile modulus, and most prominently higher adhesive strength with cartilage, which also depends on inclusion of GC. Tensile and push-out tests of the Ru/SPS-cross-linked GelMA-GC hydrogels demonstrate adhesive strength of ≈100 and 46 kPa, respectively. Hydrogel precursor solutions behave in a Newtonian manner and are injectable. After injection in focal bovine cartilage defects and in situ cross-linking, this hydrogel system remains intact and integrated with cartilage following joint manipulation ex vivo. Cells remain viable (>85%) in the hydrogel system and further show tissue regeneration potential after three weeks of in vitro culture. These preliminary results provide further motivation for future research on bioadhesive hydrogels for cartilage repair and regeneration.

GelMA-glycol chitosan hydrogels for cartilage regeneration: The role of uniaxial mechanical stimulation in enhancing mechanical, adhesive, and biochemical properties.

Untreated osteochondral defects are a leading cause of osteoarthritis, a condition that places a heavy burden on both patients and orthopedic surgeons. Although tissue engineering has shown promise for creating mechanically similar cartilage-like constructs, their integration with cartilage remains elusive. Therefore, a formulation of biodegradable, biocompatible biomaterial with sufficient mechanical and adhesive properties for cartilage repair is required. To accomplish this, we prepared biocompatible, photo-curable, mechanically robust, and highly adhesive GelMA-glycol chitosan (GelMA-GC) hydrogels. GelMA-GC hydrogels had a modulus of 283 kPa and provided a biocompatible environment (>70% viability of embedded chondrocytes) in long-term culture within a bovine cartilage ring. The adhesive strength of bovine chondrocyte-laden GelMA-GC hydrogel to bovine cartilage increased from 38 to 52 kPa over four weeks of culture. Moreover, intermittent uniaxial mechanical stimulation enhanced the adhesive strength to ∼60 kPa, indicating that the cartilage-hydrogel integration could remain secure and functional under dynamic loading conditions. Furthermore, gene expression data and immunofluorescence staining revealed the capacity of chondrocytes in GelMA-GC hydrogel to synthesize chondrogenic markers (COL2A1 and ACAN), suggesting the potential for tissue regeneration. The promising in vitro results of this work motivate further exploration of the potential of photo-curable GelMA-GC bioadhesive hydrogels for cartilage repair and regeneration.

Semi-Synthetic Click-Gelatin Hydrogels as Tunable Platforms for 3D Cancer Cell Culture.

Basement membrane extracts (BME) derived from Engelbreth-Holm-Swarm (EHS) mouse sarcomas such as Matrigel® remain the gold standard extracellular matrix (ECM) for three-dimensional (3D) cell culture in cancer research. Yet, BMEs suffer from substantial batch-to-batch variation, ill-defined composition, and lack the ability for physichochemical manipulation. Here, we developed a novel 3D cell culture system based on thiolated gelatin (Gel-SH), an inexpensive and highly controlled raw material capable of forming hydrogels with a high level of biophysical control and cell-instructive bioactivity. We demonstrate the successful thiolation of gelatin raw materials to enable rapid covalent crosslinking upon mixing with a synthetic poly(ethylene glycol) (PEG)-based crosslinker. The mechanical properties of the resulting gelatin-based hydrogels were readily tuned by varying precursor material concentrations, with Young's moduli ranging from ~2.5 to 5.8 kPa. All hydrogels of varying stiffnesses supported the viability and proliferation of MDA-MB-231 and MCF-7 breast cancer cell lines for 14 and 21 days of cell culture, respectively. Additionally, the gelatin-based hydrogels supported the growth, viability, and osteogenic differentiation of patient-derived preosteoblasts over 28 days of culture. Collectively, our data demonstrate that gelatin-based biomaterials provide an inexpensive and tunable 3D cell culture platform that may overcome the limitations of traditional BMEs.

Biofabrication of small diameter tissue-engineered vascular grafts.

Current clinical treatment strategies for the bypassing of small diameter (<6 mm) blood vessels in the management of cardiovascular disease frequently fail due to a lack of suitable autologous grafts, as well as infection, thrombosis, and intimal hyperplasia associated with synthetic grafts. The rapid advancement of 3D printing and regenerative medicine technologies enabling the manufacture of biological, tissue-engineered vascular grafts (TEVGs) with the ability to integrate, remodel, and repair in vivo, promises a paradigm shift in cardiovascular disease management. This review comprehensively covers current state-of-the-art biofabrication technologies for the development of biomimetic TEVGs. Various scaffold based additive manufacturing methods used in vascular tissue engineering, including 3D printing, bioprinting, electrospinning and melt electrowriting, are discussed and assessed against the biomechanical and functional requirements of human vasculature, while the efficacy of decellularization protocols currently applied to engineered and native vessels are evaluated. Further, we provide interdisciplinary insight into the outlook of regenerative medicine for the development of vascular grafts, exploring key considerations and perspectives for the successful clinical integration of evolving technologies. It is expected that continued advancements in microscale additive manufacturing, biofabrication, tissue engineering and decellularization will culminate in the development of clinically viable, off-the-shelf TEVGs for small diameter applications in the near future. STATEMENT OF SIGNIFICANCE: Current clinical strategies for the management of cardiovascular disease using small diameter vessel bypassing procedures are inadequate, with up to 75% of synthetic grafts failing within 3 years of implantation. It is this critically important clinical problem that researchers in the field of vascular tissue engineering and regenerative medicine aim to alleviate using biofabrication methods combining additive manufacturing, biomaterials science and advanced cellular biology. While many approaches facilitate the development of bioengineered constructs which mimic the structure and function of native blood vessels, several challenges must still be overcome for clinical translation of the next generation of tissue-engineered vascular grafts.