MEW/PVA‐MA Hydrogel Vascular Grafts: Enhancing Hemocompatibility and Endothelialization

The field of melt electrowriting (MEW) has seen significant progress, bringing innovative advancements to the fabrication of biomaterial scaffolds, and creating new possibilities for applications in tissue engineering and beyond. Multidisciplinary collaboration across materials science, computational modeling, AI, bioprinting, microfluidics, and dynamic culture systems offers promising new opportunities to gain deeper insights into complex biological systems. As the focus shifts towards personalized medicine and reduced reliance on animal models, the multidisciplinary approach becomes indispensable. This review provides a concise overview of current strategies and innovations in controlling and optimizing cellular responses to MEW scaffolds, highlighting the potential of scaffold material, MEW architecture, and computational modeling tools to accelerate the development of efficient biomimetic systems. Innovations in material science and the incorporation of biologics into MEW scaffolds have shown great potential in adding biomimetic complexity to engineered biological systems. These techniques pave the way for exciting possibilities for tissue modeling and regeneration, personalized drug screening, and cell therapies.

Many tissues in the human body exhibit low regenerative potential. This has inspired the design of advanced biomaterials and biofabrication strategies to study the tissues in vitro or enhance their function in vivo. Because some of these tissues, such as neural and cardiac, depend on electrochemical signaling, conductive biomaterials have shown promise for improving their function in vitro and in vivo. Biofabrication techniques can be combined with these materials to generate structures mimicking the complex architecture of tissues with increasing sophistication and fidelity. However, many conductive hydrogels are precluded from biofabrication strategies since many conductive materials are toxic, their crosslinking does not enable fabrication, or their structures are unstable during the fabrication process. In addition, the fragility of hydrogels limits their utility as implantable materials for tissue replacement or regeneration. Approaches to combine the design of novel conductive or extensible hydrogels with biofabrication techniques has great potential to tackle these setbacks and improve our ability to generate complex tissues in vitro. In this work, several hydrogel formulations were designed to address the low conductivity and poor mechanical performance of hydrogels for cell encapsulation and biofabrication. Firstly, to improve the conductivity of photocrosslinkable and cell-laden gelatin methacrylate (GelMA)-based hydrogels, poly(3,4-ethylenedioxythiophene):poly(styrene sulfonate) (PEDOT:PSS) was dispersed in the GelMA hydrogels to enhance their electroactive properties, and were characterized for their physicochemical and bioactive properties. This formulation was further modified by changing the gelatin source from fish to mammalian, and by crosslinking the PEDOT:PSS with bivalent calcium ions to enable advanced biofabrication of the hydrogel with wet spinning and 3D bioprinting. Following these studies, a third hydrogel formulation was designed to improve the extensibility and elasticity of GelMA hydrogels by blending them with cysteine-containing elastin-like polypeptides (ELPs). Results showed enhanced conductivity for composite fish GelMA/PEDOT:PSS hydrogels compared to controls, but reduced cell attachment and limited viability of C2C12 myoblasts in formulations containing the highest concentration of PEDOT:PSS (0.3%), purportedly due to the interaction of anionic sulfate groups in PSS with guanidinium groups on gelatin, as well as incomplete crosslinking of the GelMA due to reduced light penetration in hydrogels containing PEDOT:PSS. It was hypothesized that neutralization of the PSS chains with a cation, as well as using visible light for crosslinking, could mitigate the interactions of PSS with gelatin and improve light penetration into the hydrogels to improve cytocompatibility. Full recovery of viability and spreading in the 3D hydrogel structures were observed as a result of these changes. In addition, the ionic crosslinking step enabled the use of the material for wet spinning and 3D bioprinting of cell-laden constructs. Despite improvements in the cytocompatibility of the GelMA/PEDOT:PSS hydrogels, their mechanical extensibility was low, limiting their use as biomaterials for many practical applications. As the third and final aim of this work, mechanically resilient hydrogels were designed and investigated for use as bioinks for 3D printing of cardiac cells. The two components of the hydrogel were GelMA and a custom elastin-like-polypeptide containing cysteine groups at either end of the peptide chain, which enabled thiol-ene crosslinking of the peptides. GelMA promoted cell attachment and viability and was enzymatically degradable, while the ELP imparted the hydrogels with significantly improved mechanical extensibility. These results represent novel improvements in the properties of a widely used hydrogel for 3D cell culture and improved biofabrication strategies that can be used for generating tissues with complex architecture.

3D high-precision melt electro written polycaprolactone modified with yeast derived peptides for wound healing

The lack of transplantable tissues and organs as well as the limitations of synthetic implants highlight the need for tissue-engineered constructs to obtain safe, long-lasting, and limitless tissue replacements. Scaffolds for cardiovascular applications, such as for a tissue-engineered vascular graft (TEVG), are thus highly required. For TEVGs, tubular scaffolds should support the formation of confluent endothelial layers in particular under dynamic conditions to prevent thrombosis and maintain hemostasis. For that purpose, a porous and highly diffusible scaffold structure is necessary to allow optimal cell adhesion as well as oxygen and nutrient exchange with the surrounding tissue. Here, we present a three-dimensional-printed scaffold made by a combination of fused deposition modeling (FDM) and melt electrowriting (MEW) out of polycaprolactone that enables monolayer formation and alignment of endothelial cells in the direction of medium flow under a shear stress of up to 10 dyn cm-2. Pore size and coating with human fibrin were optimized to enable confluent endothelial layers on the printed scaffold structures. Cell orientation and shape analysis showed a characteristic alignment and elongation of the tested endothelial cells with the direction of flow after dynamic cultivation. In contrast, melt electrospun scaffolds based on the same CAD design under comparable printing and cultivation conditions were not sufficient to form gapless cell layers. Thus, the new scaffold fabricated by MEW/FDM approach appears most suitable for TEVGs as a template for the innermost vascular wall layer, the tunica intima.

Bioengineered grafts have the potential to overcome the limitations of autologous and non-resorbable synthetic vessels as vascular substitutes. However, one of the challenges in creating these living grafts is to induce and maintain multiple cell phenotypes with a biomimetic organization. Our biomimetic grafts with heterotypic design hold promises for functional neovessel regeneration by guiding the layered cellular and tissue organization into a native-like structure. In this study, a perfusable two-compartment bioreactor chamber was designed for the further maturation of these vascular grafts, with a compartmentalized exposure of the graft’s luminal and outer layer to cell-specific media. We used the system for a co-culture of endothelial colony forming cells and multipotent mesenchymal stromal cells (MSCs) in the vascular grafts, produced by combining electrospinning and melt electrowriting. It was demonstrated that the targeted cell phenotypes (i.e. endothelial cells (ECs) and vascular smooth muscle cells (vSMCs), respectively) could be induced and maintained during flow perfusion. The confluent luminal layer of ECs showed flow responsiveness, as indicated by the upregulation of COX-2, KLF2, and eNOS, as well as through stress fiber remodeling and cell elongation. In the outer layer, the circumferentially oriented, multi-layered structure of MSCs could be successfully differentiated into vSM-like cells using TGFβ, as indicated by the upregulation of αSMA, calponin, collagen IV, and (tropo)elastin, without affecting the endothelial monolayer. The cellular layers inhibited diffusion between the outer and the inner medium reservoirs. This implies tightly sealed cellular layers in the constructs, resulting in truly separated bioreactor compartments, ensuring the exposure of the inner endothelium and the outer smooth muscle-like layer to cell-specific media. In conclusion, using this system, we successfully induced layer-specific cell differentiation with a native-like cell organization. This co-culture system enables the creation of biomimetic neovessels, and as such can be exploited to investigate and improve bioengineered vascular grafts.

Peripheral nerve injuries cause different degrees of nerve palsy and function loss. Due to the limitations of autografts, nerve tissue engineering (TE) scaffolds incorporated with various neurotrophic factors and cells have been investigated to promote nerve regeneration. However, the molecular mechanism is still poorly understood. In this study, we co-cultured Schwann cells (SCs) and rat adrenal pheochromocytoma (PC-12) cells on 50% degrees of methacryloyl substitution gelatin methacrylate (GelMA) scaffold. The SCs were encapsulated within the GelMA, and PC-12 cells were on the surface. A 5% GelMA was used as the co-culture scaffold since it better supports SCs proliferation, viability, and myelination and promotes higher neurotrophic factors secretion than 10% GelMA. In the co-culture, PC-12 cells demonstrated a higher cell proliferation rate and axonal extension than culturing without SCs, indicating that the secretion of neurotrophic factors from SCs can stimulate PC-12 growth and axonal outgrowth. The mRNA level for neurotrophic factors of SCs in 5% GelMA was further evaluated. We found significant upregulation when compared with a 2D culture, which suggested that this co-culture system could be a potential scaffold to investigate the mechanism of how SCs affect neuronal behaviors.

Rapid formation of a confluent endothelial monolayer is the key to the success of small‐diameter vascular grafts, which is significantly important for treating dangerous and even sometimes deadly vascular disorders. However, the difficulty to homogenously locate endothelial cells onto the lumen of small‐diameter tubular scaffolds makes 3D endothelialization greatly challenging. Here, novel shape‐morphing scaffolds enabling programmed deformation from planar shapes to small‐diameter tubular shapes are designed and developed by combining biocompatible shape memory polymer and electrospun nanofibrous membrane. Endothelial cells can be conveniently seeded and attached on the 2D surface of the scaffolds and subsequently self‐rolled into 3D organization at physiological temperature. Endothelial cell responses and functions are varied on the shape‐morphing scaffolds with different nanofibrous electrospun membranes as the inner layer, arisen from the inducement of scaffolds with different morphological, physical, and biochemical characteristics. Owing to excellent properties of the nanofibrous membrane fabricated by the coelectrospinning of poly‐ε‐caprolactone (PCL) and gelatin methacrylate (GelMA), the shape‐morphing scaffolds with a nanofibrous PCL/GelMA inner layer support desirable homogeneous endothelial cell attachment as well as the rapid formation of biomimetic cell–scaffold interaction and cell–cell interaction under the 3D cell culture condition, therefore offering a visible approach for facile 3D endothelialization.

This study investigates the fabrication of a small-diameter bilayer vascular graft, which is an inner layer fabricated from melt-electrowriting (MEW) thermoplastic polyurethane (TPU) scaffold and an outer co-electrospun layer made of heparinized polycaprolactone (PCL)/polyurethane (PU)/gelatin, aimed at mimicking the extracellular matrix (ECM). The bilayer structure exhibited good flexibility, mechanical stability, and anti-thrombogenic properties, overcoming the drawbacks of vascular grafts, such as high kink radius and tendency toward thrombosis. MTT assays proved cytocompatibility, showing an increase in cell proliferation over 7 days, the optical density of the bilayer vascular graft increased from on day to on day , respectively, due to its fibrous structure and hydrophilic properties. Live/dead and SEM assays confirmed cell viability, attachment, and endothelial layer formation on the scaffold, which provides long-term graft patency. The bilayer graft with integrated MEW structure provided the balanced mechanical and kink-radius properties (ultimate tensile strength , Young’s modulus , suture retention ) with a low kink radius (), surpassing the mechanical properties of coronary artery. A heparin release profile of 70% after 4 weeks was obtained, thus increasing anticoagulant effects. This combination of synthetic (TPU, PCL, PU) and natural (gelatin) polymers yields a biocompatible, structurally stable vascular graft, which efficiently supports endothelialization, and thus has good potential for clinical vascular applications.

Developments in tissue engineering techniques have allowed for the creation of biocompatible, non-immunogenic alternative vascular grafts through the decellularization of existing tissues. With an ever-growing number of patients requiring life-saving vascular bypass grafting surgeries, the production of functional small diameter decellularized vascular scaffolds has never been more important. However, current implementations of small diameter decellularized vascular grafts face numerous clinical challenges attributed to premature graft failure as a consequence of common failure mechanisms such as acute thrombogenesis and intimal hyperplasia resulting from insufficient endothelial coverage on the graft lumen. This review summarizes some of the surface modifying coating agents currently used to improve the re-endothelialization efficiency and endothelial cell persistence in decellularized vascular scaffolds that could be applied in producing a better patency small diameter vascular graft. A comprehensive search yielding 192 publications was conducted in the PubMed, Scopus, Web of Science, and Ovid electronic databases. Careful screening and removal of unrelated publications and duplicate entries resulted in a total of 16 publications, which were discussed in this review. Selected publications demonstrate that the utilization of surface coating agents can induce endothelial cell adhesion, migration, and proliferation therefore leads to increased re-endothelialization efficiency. Unfortunately, the large variance in methodologies complicates comparison of coating effects between studies. Thus far, coating decellularized tissue gave encouraging results. These developments in re-endothelialization could be incorporated in the fabrication of functional, off-the-shelf alternative small diameter vascular scaffolds.

In clinical practice, synthetic vascular grafts are advantageous due to their immediate availability but are burdened by high failure rates in small-diameter settings because of thrombogenicity, infections, and intimal hyperplasia (IH). A mismatch in compliance between graft and host vessel has been identified as a major contributor to the development of IH. Here, we propose a design strategy to fabricate polymeric small-diameter vascular graft scaffolds with programmable compliance based on a helical microfiber architecture via melt electrowriting (MEW). By controlling the fiber winding angle, this design strategy exploits, for the first time, the mechanical structure-function relationship of MEW scaffolds to enable tailored compliance covering the physiological range of arteries and veins. This concept is complemented by an integrated microporous MEW graft wall, potentially enabling in situ tissue engineering to combine the advantages of synthetic (off-the-shelf) and autologous (living) grafts. Leveraging this, a gradient is introduced in the fiber architecture to achieve arteriovenous grafts matching the compliance of the target vessels at their ends (arterial vs. venous compliance) with a continuous smooth transitional region in between. The potential for clinical translation is demonstrated in vitro by assessing suture-retention strength, anti-kinking properties, burst pressure, and cannulation behavior.

Construction of dentin-on-a-chip based on microfluidic technology and tissue engineering

Highly porous scaffolds with a high surface area can be designed and fabricated via melt electrowriting (MEW). Here, the study introduces morphological features onto the MEW microfibers via a hydrogel coating of phase‐separated poly(2‐hydroxyethyl methacrylate) (pHEMA). This coating is achieved by capturing phase‐separated droplets of pHEMA onto poly(ε‐caprolactone) (PCL) microfibers via dip‐coating, resulting in a hydrogel coating with webbed structures across pores of the MEW scaffold. Excess pHEMA droplets are removed and phase separation is quenched by washing in water, and then functionalized by dipping the pHEMA coated scaffold into a buffered peptide solution. It is demonstrated that a cysteine‐terminated peptide sequence (Cys‐Gly‐Arg‐Gly‐Asp‐Ser‐Gly (CG‐RGD‐SG)) promotes fibroblast adhesion on the hydrogel‐coated MEW scaffolds compared to unmodified pHEMA and compared to scrambled peptide sequence. Due to the protein‐resistant nature of pHEMA, the hydrogel‐coated scaffolds show less cell attachment than non‐coated PCL scaffolds, while RGD‐functionalized pHEMA scaffolds achieve 2.8‐fold increase in cell attachment (p = 0.02) when compared to non‐functionalized pHEMA. The study therefore presents a platform that combines PCL scaffolds of microscale fibers with a phase‐separated pHEMA hydrogel coating that maintains the high porosity of MEW scaffolds yet increases surface area and, importantly, introduces the capability for tailoring cell attachment via peptide functionalization.

BackgroundCell-scaffold based therapies have the potential to offer an efficient osseous regenerative treatment and PCL has been commonly used as a scaffold, however its effectiveness is limited by poor cellular retention properties. This may be improved through a porous scaffold structure with efficient pore arrangement to increase cell entrapment. To facilitate this, melt electrowriting (MEW) has been developed as a technique able to fabricate cell-supporting scaffolds with precise micro pore sizes via predictable fibre deposition. The effect of the scaffold’s architecture on cellular gene expression however has not been fully elucidated.MethodsThe design and fabrication of three different uniform pore structures (250, 500 and 750 μm), as well as two offset scaffolds with different layout of fibres (30 and 50%) and one complex scaffold with three gradient pore sizes of 250–500 - 750 μm, was performed by using MEW. Calcium phosphate modification was applied to enhance the PCL scaffold hydrophilicity and bone inductivity prior to seeding with osteoblasts which were then maintained in culture for up to 30 days. Over this time, osteoblast cell morphology, matrix mineralisation, osteogenic gene expression and collagen production were assessed.ResultsThe in vitro findings revealed that the gradient scaffold significantly increased alkaline phosphatase activity in the attached osteoblasts while matrix mineralization was higher in the 50% offset scaffolds. The expression of osteocalcin and osteopontin genes were also upregulated compared to other osteogenic genes following 30 days culture, particularly in offset and gradient scaffold structures. Immunostaining showed significant expression of osteocalcin in offset and gradient scaffold structures.ConclusionsThis study demonstrated that the heterogenous pore sizes in gradient and fibre offset PCL scaffolds prepared using MEW significantly improved the osteogenic potential of osteoblasts and hence may provide superior outcomes in bone regeneration applications.

Biocompatible fluorescent nanodiamonds (FNDs) were introduced into polycaprolactone (PCL) – the golden standard material in melt electrowriting (MEW). MEW is an advanced additive manufacturing technique capable of depositing high-resolution micrometric fibres. Due to the high printing precision, MEW finds growing interest in tissue engineering applications. Here, we introduced fluorescent nanodiamonds (FNDs) into polycaprolactone prior to printing to fabricate scaffolds for biomedical applications with improved mechanical properties. Further FNDs offer the possibility of their real-time degradation tracking. Compared to pure PCL scaffolds, the functionalized ones containing 0.001 wt% of 70 nm-diameter nanodiamonds (PCL-FNDs) showed increased tensile moduli (1.25 fold) and improved cell proliferation during 7-day cell cultures (2.00 fold increase). Furthermore, the addition of FNDs slowed down the hydrolytic degradation process of the scaffolds, accelerated for the purpose of the study by addition of the enzyme lipase to deionized water. Pure PCL scaffolds showed obvious signs of degradation after 3 h, not observed for PCL-FNDs scaffolds during this time. Additionally, due to the nitrogen-vacancy (NV) centers present on the FNDs, we were able to track their amount and location in real-time in printed fibres using confocal microscopy. This research shows the possibility for high-resolution life-tracking of MEW PCL scaffolds’ degradation.

ABSTRACTCurrently, one of the major challenges faced by small‐diameter vascular grafts is the mismatch in compliance between grafts and natural blood vessels. Herein, hybrid manufacturing technology combining the pros of melt electrospinning (MES) and melt electrowriting (MEW) is utilized to fabricate small‐diameter vascular grafts, enabling the fabrication of grafts that exhibit both physiological compliance and appropriate mechanical properties. By leveraging their customizable structure and high‐precision printing capabilities, these MEW layers significantly enhance the overall mechanical performance of the hybrid vascular grafts, as indicated by the following values: S‐W Squ. (6.63% ± 0.19%/100 mmHg), S‐W Ser. (7.00% ± 0.52%/100 mmHg), S‐W 30° (7.95% ± 0.28%/100 mmHg), S‐W 45° (9.82% ± 0.41%/100 mmHg), and S‐W 60° (9.68% ± 0.42%/100 mmHg). Notably, these varying compliance degrees are specifically tailored to meet the requirements of different blood vessel types. This MEW‐based hybrid printing method will open new avenues and address the challenges in the fabrication of small‐diameter vascular grafts.