Decellularised plant scaffolds facilitate porcine skeletal muscle tissue engineering for cultivated meat biomanufacturing.

Cultivated meat (CM) offers a sustainable and ethical alternative to conventional animal agriculture, involving cell maturation in a controlled environment. To emulate the structural complexity of traditional meat, the development of animal-free and edible scaffolds is crucial, providing vital physical and biological support during tissue development. The aligned vascular bundles of the decellularised asparagus scaffold were selected to facilitate the attachment and alignment of murine myoblasts (C2C12) and porcine adipose-derived mesenchymal stem cells (pADMSCs). Muscle differentiation was assessed through immunofluorescence staining with muscle markers, including Myosin heavy chain (MHC), Myogenin (MYOG), and Desmin. The metabolic activity of Creatine Kinase in C2C12 differentiated cells significantly increased compared to proliferated cells. Quantitative PCR analysis revealed a significant increase in Myosin Heavy Polypeptide 1 (MYH1) and MYOG expression compared to Day 0. These results highlight the application of decellularised plant scaffold (DPS) as a promising, edible material conducive to cell attachment, proliferation, and differentiation into muscle tissue. To create a CM prototype with biological mimicry, pADMSC-derived muscle and fat cells were also co-cultured on the same scaffold. The co-culture was confirmed through immunofluorescence staining of muscle markers and LipidTOX staining, revealing distinct muscle fibres and adipocytes containing lipid droplets respectively. Texture profile analysis conducted on uncooked CM prototypes and pork loin showed no significant differences in textural values. However, the pan-fried CM prototype differed significantly in hardness and chewiness compared to pork loin. Understanding the scaffolds' textural profile enhances our insight into the potential sensory attributes of CM products. DPS shows potential for advancing CM biomanufacturing.

Synthesis and fabrication of gelatin-based elastomeric hydrogels through cosolvent-induced polymer restructuring.

Hydrogels have a wide range of applications in tissue engineering, drug delivery, device fabrication for biological studies and stretchable electronics. For biomedical applications, natural polymeric hydrogels have general advantages such as biodegradability and non-toxic by products as well as biocompatibility. However, applications of nature derived hydrogels have been severely limited by their poor mechanical properties. For example, most of the protein derived hydrogels do not exhibit high stretchability like methacrylated gelatin hydrogel has ∼11% failure strain when stretched. Moreover, protein derived elastomeric hydrogels that are fabricated from low molecular weight synthetic peptides require a laborious process of synthesis and purification. Biopolymers like gelatin, produced in bulk for pharma and the food industry can provide an alternative for the development of elastomeric hydrogels. Here, we report the synthesis of ureidopyrimidinone (Upy) functionalized gelatin and its fabrication into soft elastomeric hydrogels through supramolecular interactions that could exhibit high failure strain (318.73 ± 44.35%). The hydrogels were fabricated through a novel method involving co-solvent optimization and structural transformation with 70% water content. It is anticipated that the hydrogel fabrication method involves the formation of hydrophobic cores of ureidopyrimidinone groups inside the hydrogel which introduced elastomeric properties to the resulting hydrogel.

Progress in drug-delivery systems in cardiovascular applications: stents, balloons and nanoencapsulation.

Drug-delivery systems in cardiovascular applications regularly include the use of drug-eluting stents and drug-coated balloons to ensure sufficient drug transfer and efficacy in the treatment of cardiovascular diseases. In addition to the delivery of antiproliferative drugs, the use of growth factors, genetic materials, hormones and signaling molecules has led to the development of different nanoencapsulation techniques for targeted drug delivery. The review will cover drug delivery and coating mechanisms in current drug-eluting stents and drug-coated balloons, novel innovations in drug-eluting stent technologies and drug encapsulation in nanocarriers for delivery in vascular diseases. Newer technologies and advances in nanoencapsulation techniques, such as the use of liposomes, nanogels and layer-by-layer coating to deliver therapeutics in the cardiovascular space, will be highlighted.

Scaffolds for the manufacture of cultured meat.

The cultured meat market has been growing at an accelerated space since the first creation of cultured meat burger back in 2013. Substantial efforts have been made to reduce costs by eliminating serum in growth media and improving process efficiency by employing bioreactors. In parallel, efforts are also being made on scaffolding innovations to offer better cells proliferation, differentiation and tissue development. So far, scaffolds used in cultured meat research are predominantly collagen and gelatin, which are animal-derived. To align with cell-based meat vision i.e. environment conservation and animal welfare, plant-derived biomaterials for scaffolding are being intensively explored. This paper reviews and discusses the advantages and disadvantages of scaffold materials and potential scaffolding related to scale-up solution for the production of cultured meat.

Nanoparticles-reinforced poly-l-lactic acid composite materials as bioresorbable scaffold candidates for coronary stents: Insights from mechanical and finite element analysis.

Current generation of bioresorbable coronary scaffolds (BRS) posed thrombogenicity and deployment issues owing to its thick struts and overall profile. To this end, we hypothesize that the use of nanocomposite materials is able to provide improved material properties and sufficient radial strength for the intended application even at reduced strut thickness. The nanocomposite formulations of tantalum dioxide (Ta2O5), L-lactide functionalized (LA)-Ta2O5, hydroxyapatite (HA) and LA-HA with poly-l-lactic acid (PLLA) were evaluated in this study. Results showed that tensile modulus and strength were enhanced with non-functionalized nanofillers up until 15 wt% loading, whereas ductility was compromised. On the other hand, functionalized nanofillers/PLLA exhibited improved nanofiller dispersion which resulted higher tensile modulus, strength, and ductility. Selected nanocomposite formulations were evaluated using finite element analysis (FEA) of a stent with varying strut thickness (80, 100 and 150 µm). FEA data has shown that nanocomposite BRS with thinner struts (80-100 µm) made with 15 wt% LA-Ta2O5/PLLA and 10 wt% LA-HA/PLLA have increased radial strength, stiffness and reduced recoil compared to PLLA BRS at 150 µm. The reduced strut thickness can potentially mitigate issues such as scaffold thrombosis and promote re-endothelialisation of the vessel.

3D Hepatic Organoid-Based Advancements in LIVER Tissue Engineering.

Liver-associated diseases and tissue engineering approaches based on in vitro culture of functional Primary human hepatocytes (PHH) had been restricted by the rapid de-differentiation in 2D culture conditions which restricted their usability. It was proven that cells growing in 3D format can better mimic the in vivo microenvironment, and thus help in maintaining metabolic activity, phenotypic properties, and longevity of the in vitro cultures. Again, the culture method and type of cell population are also recognized as important parameters for functional maintenance of primary hepatocytes. Hepatic organoids formed by self-assembly of hepatic cells are microtissues, and were able to show long-term in vitro maintenance of hepato-specific characteristics. Thus, hepatic organoids were recognized as an effective tool for screening potential cures and modeling liver diseases effectively. The current review summarizes the importance of 3D hepatic organoid culture over other conventional 2D and 3D culture models and its applicability in Liver tissue engineering.

Bioengineered 3D electrospun nanofibrous scaffold with human liver cells to study alcoholic liver disease in vitro.

Alcohol injury induces hepatic fibrosis which gradually progresses to cirrhosis, sometimes may lead to liver cancer. Animal models are less efficient in mimicking responses of human liver cells, whereas in vitro models discussed so far are majorly based on rodent cells. In this work, a coculture of primary human hepatocytes (PHHs) with LX-2 cells was established on the unmodified (C:F_0:0), collagen-I modified (C:F_1:0), fibronectin modified (C:F_0:1) and 3:1 collagen-I to fibronectin modified (C:F_3:1) 3D electrospun fibrous scaffolds. The effect of alcohol injury was evaluated on this cell-scaffold model at 0-40 µl/ml alcohol concentrations over 14 days of culture period by using the gold standard sandwich culture as the control. Among all the culture groups, C:F_3:1 scaffold was able to maintain translational and transcriptional properties of human liver cells at all concentrations of alcohol treatment. The study reveals that, PHHs on C:F_3:1 were able to maintain ~4-fold and ~1.6-fold higher secretion of albumin than the gold standard sandwich culture on Day 3 and Day 7, respectively. When treated with alcohol, at concentrations of 20 and 40 µl/ml, albumin secretion was also observed to be higher (~2-fold) when compared to the gold standard sandwich culture. Again as expected, in C:F_3:1 culture group on 40 µl/ml alcohol treatment, albumin gene expression decreased by ~2-fold due to alcohol toxicity, whereas CYP2C9, CYP3A4, CYP2E1 and CYP1A2 gene expressions upregulated by ~3.5, ~~4, ~5 and ~15-fold, respectively in response to the alcohol injury. LX-2 cells also acquire more quiescent phenotype on C:F_3:1 scaffolds when compared to the gold standard sandwich culture upon alcohol treatment. Thus, C:F_3:1 scaffold with human liver cells was established as the potential platform to scan alcohol toxicity at varied alcohol concentrations. Thus, it can pave a promising path not only to support functional healthy human liver cells for liver tissue engineering but also to examine potential drugs to study the progression or inhibition of alcoholic liver fibrosis in vitro.

Synthesis and characterization of site selective photo-crosslinkable glycidyl methacrylate functionalized gelatin-based 3D hydrogel scaffold for liver tissue engineering.

The presented work outlined the development of a new biocompatible hydrogel material that has potential applications in soft tissue engineering. As a proof of concept, human hepatocytes were used to demonstrate the suitability of this material in providing conducive environment for cellular growth and functional development. Herein, a detailed synthesis of novel gelatin derivatives - photo-crosslinkable glycidyl methacrylate (GMA) functionalized gelatins (Gelatin-GMA), and preparation of three-dimensional (3D) hydrogel scaffolds for the encapsulated Huh-7.5 cells is reported. The Gelatin-GMA biopolymers were synthesized at two different pH values of 3.5 (acidic) and 10.5 (basic) where two different photo-crosslinkable polymers were formed utilizing -COOH & -OH groups in acidic pH, and -NH2 & -OH groups in basic pH. The hydrogels were prepared using an initiator (Irgacure I2959) in the presence of UV light. The Gelatin-GMA biopolymers were characterized using spectroscopic studies which confirmed the successful preparation of the polymer derivatives. Rheological measurement was carried out to characterize the mechanical properties and derive the mesh sizes of the 3D hydrogels. Subsequently, detailed in vitro hepatocyte compatibility and functionality studies were performed in the 3D cell seeded hydrogel platform. The 3D hydrogel design with larger mesh sizes utilizes the advantage of the excellent diffusion properties of porous platform, and enhanced cell-growth was observed, which in turn elicited favorable Huh-7.5 response. The hydrogels led to improved cellular functions such as differentiation, viability and proliferation. Overall, it showed that the Gelatin-GMA based hydrogels presented better results compared to control sample (GelMA) because of the higher mesh sizes in Gelatin-GMA based hydrogels. Additionally, the functional group studies of the two Gelatin-GMA samples revealed that the cell functionalities are almost unaffected even after the tripeptide - Arg-Gly-Asp (RGD) in Gelatin-GMA synthesized at pH 3.5 is no longer completely available.

Direct and Label-Free Cell Status Monitoring of Spheroids and Microcarriers Using Microfluidic Impedance Cytometry.

3D cellular spheroids/microcarriers (100 µm-1 mm) are widely used in biomanufacturing, and non-invasive biosensors are useful to monitor cell quality in bioprocesses. In this work, a novel microfluidic approach for label-free and continuous-flow monitoring of single spheroid/microcarrier (hydrogel and Cytodex) based on electrical impedance spectroscopy using co-planar Field's metal electrodes is reported. Through numerical simulation and experimental validation, two unique impedance signatures (|ZLF | (60 kHz), |ZHF | (1 MHz)) which are optimal for spheroid growth and viability monitoring are identified. Using a closed-loop recirculation system, it is demonstrated that |ZLF | increases with breast cancer (MCF-7) spheroid biomass, while higher opacity (impedance ratio |ZHF |/|ZLF |) indicates cell death due to compromised cell membrane. Anti-cancer drug (paclitaxel)-treated spheroids also exhibit lower |ZLF | with increased cell dissociation. Interestingly, impedance characterization of adipose-derived mesenchymal stem cell differentiation on Cytodex microcarriers reveals that adipogenic cells (higher intracellular lipid content) exhibit higher impedance than osteogenic cells (more conductive due to calcium ions) for both microcarriers and single cell level. Taken together, the developed platform offers great versatility for multi-parametric analysis of spheroids/microcarriers at high throughput (≈1 particle/s), and can be readily integrated into bioreactors for long-term and remote monitoring of biomass and cell quality.

Bioactive micropatterned platform to engineer myotube-like cells from stem cells.

Skeletal muscle has the capacity to repair and heal itself after injury. However, this self-healing ability is diminished in the event of severe injuries and myopathies. In such conditions, stem cell-based regenerative treatments can play an important part in post-injury restoration. We herein report the development of a bioactive (integrin-ß1antibody immobilized) gold micropatterned platform to promote human mesenchymal stem cell (hMSC) differentiation into myotube-like cells. hMSCs grown on bioactive micropattern differentiated into myotube-like cells within two weeks. Furthermore, the up-regulation of myogenic markers, multi-nucleated state with continuous actin cytoskeleton and the absence of proliferation marker confirmed the formation of myotube-like cells on bioactive micropattern. The prominent expression of elongated integrin-ß1(ITG-ß1) focal adhesions and the development of anisotropic stress fibers in those differentiated cells elucidated their importance in stem cell myogenesis. Together, these findings delineate the synergistic role of engineered cell anisotropy and ITG-ß1-mediated signaling in the development of myotube-like cells from hMSCs.

Commercialization of Plant-Based Meat Alternatives.

Plant-based meat alternatives are a sustainable source of proteins that can match the taste and texture, color, and nutritional profile of specific types of meat. Here we highlight the product focus, the geographical spread of companies, and the funding landscape along with the critical challenges facing plant-based meat alternatives.

Bioresorbable Polymeric Scaffold in Cardiovascular Applications.

Advances in material science and innovative medical technologies have allowed the development of less invasive interventional procedures for deploying implant devices, including scaffolds for cardiac tissue engineering. Biodegradable materials (e.g., resorbable polymers) are employed in devices that are only needed for a transient period. In the case of coronary stents, the device is only required for 6-8 months before positive remodelling takes place. Hence, biodegradable polymeric stents have been considered to promote this positive remodelling and eliminate the issue of permanent caging of the vessel. In tissue engineering, the role of the scaffold is to support favourable cell-scaffold interaction to stimulate formation of functional tissue. The ideal outcome is for the cells to produce their own extracellular matrix over time and eventually replace the implanted scaffold or tissue engineered construct. Synthetic biodegradable polymers are the favoured candidates as scaffolds, because their degradation rates can be manipulated over a broad time scale, and they may be functionalised easily. This review presents an overview of coronary heart disease, the limitations of current interventions and how biomaterials can be used to potentially circumvent these shortcomings in bioresorbable stents, vascular grafts and cardiac patches. The material specifications, type of polymers used, current progress and future challenges for each application will be discussed in this manuscript.

Collagen-I and fibronectin modified three-dimensional electrospun PLGA scaffolds for long-term in vitro maintenance of functional hepatocytes.

Extracellular matrix (ECM) proteins are important regulators of cellular behaviour in the native environment. It has been established that ECM proteins - collagen-I and fibronectin - are present in liver extracellular matrix and regulate specific functions of primary hepatocytes. While scaffolds grafted with the individual ECM protein have shown support for hepatocyte functional properties in vitro, the synergistic effects of both ECM proteins remain to be explored. Such studies are even more limited when three-dimensional (3D) scaffolds are involved. In the current work, the fabrication of a series of highly porous poly(lactic-co-glycolic acid) (PLGA) 3D electrospun scaffolds, simultaneously modified with both collagen-I and fibronectin, has been demonstrated. Different ratios of collagen-I to fibronectin were optimized to study the synergistic effects of the proteins in supporting the viability and functional properties of Huh-7.5 cells. The ratio of collagen-I to fibronectin at 3:1 was found to provide the most efficient chemisorption on the 3D scaffolds. At this ratio, the total protein content that can be grafted on the scaffolds was the highest and the most homogeous. This led to remarkable enhancement of cell seeding efficiency as well as proliferation. Most importantly, liver specific genes such as albumin and cytochrome P450 enzymes i.e. CYP3A4 and CYP3A7 were significantly upregulated by ~12.5, 7 and 4.5 fold respectively, as compared to unmodified PLGA scaffolds after 28 days of culture. Compared to single-protein modified scaffolds, scaffolds modified with 3:1 collagen to fibronectin result in a rise of the albumin gene expression of cultured cells by ~8 to 10 fold, whereas CYP3A4 gene expression improved by ~5 to 7 fold and CYP3A7 gene expression improved by ~4 to 4.5 fold after a long culture period of 28 days. Albumin secretion was improved by ~4 fold compared to unmodified PLGA scaffolds, ~3 fold compared to collagen-I modified culture groups and ~2 fold compared to fibronectin modified culture groups. The multi-protein modified scaffolds, at the optimum ratio, were able to significantly enhance functional properties of the liver cells. This simple yet highly functioning platform would be useful for in vitro culture of liver cells for both drug screening as well as translational purposes.

Robust Fabrication of Composite 3D Scaffolds with Tissue-Specific Bioactivity: A Proof-of-Concept Study.

The basic requirement of any engineered scaffold is to mimic the native tissue extracellular matrix (ECM). Despite substantial strides in understanding the ECM, scaffold fabrication processes of sufficient product robustness and bioactivity require further investigation, owing to the complexity of the natural ECM. A promising bioacive platform for cardiac tissue engineering is that of decellularized porcine cardiac ECM (pcECM, used here as a soft tissue representative model). However, this platform's complexity and batch-to-batch variability serve as processing limitations in attaining a robust and tunable cardiac tissue-specific bioactive scaffold. To address these issues, we fabricated 3D composite scaffolds (3DCSs) that demonstrate comparable physical and biochemical properties to the natural pcECM using wet electrospinning and functionalization with a pcECM hydrogel. The fabricated 3DCSs are non-immunogenic in vitro and support human mesenchymal stem cells' proliferation. Most importantly, the 3DCSs demonstrate tissue-specific bioactivity in inducing spontaneous cardiac lineage differentiation in human induced pluripotent stem cells (hiPSC) and further support the viability, functionality, and maturation of hiPSC-derived cardiomyocytes. Overall, this work illustrates the technology to fabricate robust yet tunable 3D scaffolds of tissue-specific bioactivity (with a proof of concept provided for cardiac tissues) as a platform for basic materials science studies and possible future R&D application in regenerative medicine.

Layer-by-layer ultraviolet assisted extrusion-based (UAE) bioprinting of hydrogel constructs with high aspect ratio for soft tissue engineering applications.

One of the major challenges in the field of soft tissue engineering using bioprinting is fabricating complex tissue constructs with desired structure integrity and mechanical property. To accomplish such requirements, most of the reported works incorporated reinforcement materials such as poly(ϵ-caprolactone) (PCL) polymer within the 3D bioprinted constructs. Although this approach has made some progress in constructing soft tissue-engineered scaffolds, the mechanical compliance mismatch and long degradation period are not ideal for soft tissue engineering. Herein, we present a facile bioprinting strategy that combines the rapid extrusion-based bioprinting technique with an in-built ultraviolet (UV) curing system to facilitate the layer-by-layer UV curing of bioprinted photo-curable GelMA-based hydrogels to achieve soft yet stable cell-laden constructs with high aspect ratio for soft tissue engineering. GelMA is supplemented with a viscosity enhancer (gellan gum) to improve the bio-ink printability and shape fidelity while maintaining the biocompatibility before crosslinking via a layer-by-layer UV curing process. This approach could eventually fabricate soft tissue constructs with high aspect ratio (length to diameter) of ≥ 5. The effects of UV source on printing resolution and cell viability were also studied. As a proof-of-concept, small building units (3D lattice and tubular constructs) with high aspect ratio are fabricated. Furthermore, we have also demonstrated the ability to perform multi-material printing of tissue constructs with high aspect ratio along both the longitudinal and transverse directions for potential applications in tissue engineering of soft tissues. This layer-by-layer ultraviolet assisted extrusion-based (UAE) Bioprinting may provide a novel strategy to develop soft tissue constructs with desirable structure integrity.

Epithelial-mesenchymal transition of cancer cells using bioengineered hybrid scaffold composed of hydrogel/3D-fibrous framework.

Cancer cells undergoing epithelial-mesenchymal transition (EMT) acquire stem cell-like phenotype associated with malignant behaviour, chemoresistance, and relapse. Current two-dimensional (2D) in-vitro culture models of tumorigenesis are inadequate to replicate the complexity of in-vivo microenvironment. Therefore, the generation of functional three-dimensional (3D) constructs is a fundamental prerequisite to form multi-cellular tumour spheroids for studying basic pathological mechanisms. In this study, we focused on two major points (i) designing and fabrication of 3D hybrid scaffolds comprising electrospun fibers with cancer cells embedded within hydrogels, and (ii) determining the potential roles of 3D hybrid scaffolds associated with EMT in cancer progression and metastasis. Our findings revealed that 3D hybrid scaffold enhances cell proliferation and induces cancer cells to undergo EMT, as demonstrated by significant up-regulation of EMT associated transcriptional factors including Snail1, Zeb1, and Twist2; and mesenchymal markers whereas epithelial marker, E-Cadherin was downregulated. Remarkably, this induction is independent of cancer cell-type as similar results were obtained for breast cancer cells, MDA-MB-231 and gastric cancer cells, MKN74. Moreover, the hybrid scaffolds enrich aggressive cancer cells with stem cell properties. We showed that our 3D scaffolds could trigger EMT of cancer cells which could provide a useful model for studying anticancer therapeutics against metastasis.

4D printing and stimuli-responsive materials in biomedical aspects.

Three-dimensional (3D) printing has revolutionized the world manufacturing production. In biomedical applications, however, 3D printed constructs fell short of expectations mainly due to their inability to adequately mimic the dynamic human tissues. To date, most of the 3D printed biomedical structures are largely static and inanimate as they lack the time-dependant dimension. To adequately address the dynamic healing and regeneration process of human tissues, 4D printing emerges as an important development where "time" is incorporated into the conventional concept of 3D printing as the fourth dimension. As such, additive manufacturing (AM) evolves from 3D to 4D printing and in the process putting stimulus-responsive materials in the limelight. In this review, the state-of-the-art efforts in integrating the time-dependent behaviour of stimulus-responsive materials in 4D printing will be discussed. In addition, current literatures on the interactions between various types of stimuli (categorized under physical, chemical and biological signals) with the associated stimulus-responsive materials will be the major focus in this review. Lastly, potential usage of 4D printing in biomedical applications will also be discussed, followed by technical considerations as well as outlook for future discoveries. STATEMENT OF SIGNIFICANCE: In this Review, we have demonstrated the significance of 4D printing in biomedical applications, in which "time" has been incorporated into the conventional concept of 3D printing as the 4th dimension. As such, 4D printing differentiates and evolves from 3D printing using stimulus-responsive materials which can actively respond to external stimuli and more sophisticated "hardware"-printer which can achieve multi-printing via mathematical-predicted designs that are programmed to consider the transformation of 3D constructs over time. The emphasize will be on the interactions between various types of stimuli (categorized under physical, chemical and biological signals) with the associated stimulus-responsive materials, followed by technical considerations as well as outlook for future discoveries.

Migration and Phenotype Control of Human Dermal Fibroblasts by Electrospun Fibrous Substrates.

Electrospun fibrous matrices, mimicking extracellular matrix (ECM) hierarchical structures, are potential scaffolds for wound healing. To design functional scaffolds, it is important to explore the interactions between scaffold topographic features and cellular responses, especially directional migration and phenotypic changes, which are critical functional aspects during wound healing. Here, accelerated and persistent migration of human dermal fibroblasts (HDFs) is observed on fibers with aligned orientation. Furthermore, aligned fibers can induce fibroblast-to-myofibroblast differentiation of HDFs. During wound healing, the presence of myofibroblasts advances wound repair by rendering contractile force and ECM deposition within the early and middle courses, but its continuous persistence in the later event may not be desired due to the contribution in pathological scarring. To tune the balance, it is noted in this work that the introduction of matricellular protein angiopoietin-like 4 (ANGPTL4) is capable of reversing the phenotypic alteration induced by aligned fibers, in a time-dependent manner. These results indicate fibrous matrices with oriented configuration are functional in mediating directional cell migration and phenotypic change. The discoveries further suggest that tissue-engineered fibrous grafts with precise alignment modulation and ANGPTL4 releasing properties may thus be promising to promote wound repair with minimizing scar formation.

Microbial transglutaminase induced controlled crosslinking of gelatin methacryloyl to tailor rheological properties for 3D printing.

Gelatin methacryloyl (GelMA) is a versatile biomaterial that has been shown to possess many advantages such as good biocompatibility, support for cell growth, tunable mechanical properties, photocurable capability, and low material cost. Due to these superior properties, much research has been carried out to develop GelMA as a bioink for bioprinting. However, there are still many challenges, and one major challenge is the control of its rheological properties to yield good printability. Herein, this study presents a strategy to control the rheology of GelMA through partial enzymatic crosslinking. Unlike other enzymatic crosslinking strategies where the rheological properties could not be controlled once reaction takes place, we could, to a large extent, keep the rheological properties stable by introducing a deactivation step after obtaining the optimized rheological properties. Ca2+-independent microbial transglutaminase (MTGase) was introduced to partially catalyze covalent bond formation between chains of GelMA. The enzyme was then deactivated to prevent further uncontrolled crosslinking that would render the hydrogel not printable. After printing, a secondary post-printing crosslinking step (photo crosslinking) was then introduced to ensure long-term stability of the printed structure for subsequent cell studies. Biocompatibility studies carried out using cells encapsulated in the printed structure showed excellent cell viability for at least 7 d. This strategy for better control of rheological properties of GelMA could more significantly enhance the usability of this material as bioink for bioprinting of cell-laden structures for soft tissue engineering.

A facile method for fabricating a three-dimensional aligned fibrous scaffold for vascular application.

Vascular graft replacement remains the optimal treatment option for many vascular diseases despite advances in endovascular surgery. In this study, we proposed the use of surface topographical cues to align and maintain the phenotype of vascular smooth muscle cells (vSMCs) which were reported as one of the vital limitations for successful graft replacement. An auxiliary electrospinning setup has been developed to collect circumferentially aligned fibres on a 3D tubular format; this micro-architecture was found to be similar to the tunica media layer of blood vessels. The presence of aligned fibres served as a signaling modality to induce cell alignment and the maintenance of the contractile phenotype. vSMCs cultured on the 3D aligned fibrous substrate were found to exhibit better cell proliferation ability and enhanced cell-shape directionality. The functional expression of the two representative intracellular contractile proteins (i.e. α-SMA and MHC) was found to exhibit definitive markers that are orderly organized as microfilament bundles. Collectively, the result suggests a possibility of adapting the 3D aligned tubular scaffold to enhance and regulate cell function along with the additional tunability of scaffold diameter and thicknesses for tailoring to the needs of individual patients or future ex vivo studies.