Advances in Animal Models and Cutting-Edge Research in Alternatives: Proceedings of the Third International Conference on 3Rs Research and Progress, Vishakhapatnam, 2022.

Animal experimentation has been integral to drug discovery and development and safety assessment for many years, since it provides insights into the mechanisms of drug efficacy and toxicity (e.g. pharmacology, pharmacokinetics and pharmacodynamics). However, due to species differences in physiology, metabolism and sensitivity to drugs, the animal models can often fail to replicate the effects of drugs and chemicals in human patients, workers and consumers. Researchers across the globe are increasingly applying the Three Rs principles by employing innovative methods in research and testing. The Three Rs concept focuses on: the replacement of animal models (e.g. with in vitro and in silico models or human studies), on the reduction of the number of animals required to achieve research objectives, and on the refinement of existing experimental practices (e.g. eliminating distress and enhancing animal wellbeing). For the last two years, Oncoseek Bio-Acasta Health, a 3-D cell culture-based cutting-edge translational biotechnology company, has organised an annual International Conference on 3Rs Research and Progress. This series of global conferences aims to bring together researchers with diverse expertise and interests, and provides a platform where they can share and discuss their research to promote practices according to the Three Rs principles. In November 2022, the 3rd international conference, Advances in Animal Models and Cutting-Edge Research in Alternatives, took place at the GITAM University in Vishakhapatnam (AP, India) in a hybrid format (i.e. online and in-person). These conference proceedings provide details of the presentations, which were categorised under five different topic sessions. It also describes a special interactive session on in silico strategies for preclinical research in oncology, which was held at the end of the first day.

Quantifying viscosity and elasticity using holographic imaging by Rayleigh wave dispersion.

Viscoelasticity is an important diagnostic parameter to investigate physiological dysfunctions in biological tissues. This Letter reports the quantification of viscoelastic parameters by Rayleigh wave tracing on the surface of tissue-mimicking phantoms using holographic imaging. The Rayleigh wave is induced by an electromechanical actuator on the surface of oil-in-gelatin phantoms and a biological tissue sample followed by holographic imaging and reconstruction of the wave. The frequency-dependent velocity dispersion is fitted to a Voigt model for the quantification of viscous and elastic moduli. The viscoelastic parameters calculated by the proposed method are validated by comparing the results from a conventional mechanical rheometer.

Fabrication and analysis of chitosan oligosaccharide based mucoadhesive patch for oromucosal drug delivery.

OBJECTIVE: Fabrication and analyses of mucoadhesive patches made from chitosan oligosaccharide for the purpose of oromucosal drug delivery. SIGNIFICANCE: The mucosal epithelium in the oral cavity, consisting of buccal and sublingual epithelium, has gained significant attention in the last decade as an alternative anatomical site for systemic drug delivery that could potentially minimize the challenges of solid oral dosage and parenteral delivery. In this study, we have fabricated and tested drug-loaded chitosan oligosaccharide-based patches for the oromucosal drug delivery. METHODS: The chitosan oligosaccharide (with and without alginate) based patches were fabricated using the conventional solvent casting method and were analyzed for their swelling capacity, hydrophilicity, anti-cancer activity, in vitro drug release, and in vivo drug release activity. The in-house developed artificial saliva was used for the swelling study. RESULTS: Alginate-containing patches showed lesser swelling ability compared to the bare chitosan oligosaccharide-based patches. The former was also found to be more hydrophobic compared to the latter one. Both the unloaded patches restricted the growth of epithelial cancer cells indicating their anti-cancer behavior. In vitro drug release indicated a super case II release pattern while in vivo study demonstrated the release of drug from the patch into the plasma indicating the purpose of the fabricated patch. CONCLUSIONS: The chitosan oligosaccharide-based mucoadhesive hydrogel patch fabricated in this study can be highly suitable for possible translational purposes.

3D Bioprinting of Tissue/Organ Models.

In vitro tissue/organ models are useful platforms that can facilitate systematic, repetitive, and quantitative investigations of drugs/chemicals. The primary objective when developing tissue/organ models is to reproduce physiologically relevant functions that typically require complex culture systems. Bioprinting offers exciting prospects for constructing 3D tissue/organ models, as it enables the reproducible, automated production of complex living tissues. Bioprinted tissues/organs may prove useful for screening novel compounds or predicting toxicity, as the spatial and chemical complexity inherent to native tissues/organs can be recreated. In this Review, we highlight the importance of developing 3D in vitro tissue/organ models by 3D bioprinting techniques, characterization of these models for evaluating their resemblance to native tissue, and their application in the prioritization of lead candidates, toxicity testing, and as disease/tumor models.

Preparation of Corneal Tissue Matrix Bioink for 3D Bioprinting.

There are many protocols available to decellularize tissues for the preparation of bioink for 3D bioprinting purposes. Almost all the methods comprise multiple chemicals and enzymes in different combinations. Here we describe the usage of sodium chloride that enables the decellularization of corneal tissues from human and animal sources, which is a simple, rapid, and detergent-free method, unlike conventional decellularization protocols. The method described here is for cornea tissue decellularization and its digestion and bioink preparation for 3D bioprinting applications. We demonstrate the efficient decellularization of tissues by retaining the extracellular matrix.

High Throughput Bioprinting Using Decellularized Adipose Tissue-Based Hydrogels for 3D Breast Cancer Modeling.

3D bioprinting allows rapid automated fabrication and can be applied for high throughput generation of biomimetic constructs for in vitro drug screening. Decellularized extracellular matrix (dECM) hydrogel is a popular biomaterial choice for tissue engineering and studying carcinogenesis as a tumor microenvironmental mimetic. This study proposes a method for high throughput bioprinting with decellularized adipose tissue (DAT) based hydrogels for 3D breast cancer modeling. A comparative analysis of decellularization protocol using detergent-based and detergent-free decellularization methods for caprine-origin adipose tissue is performed, and the efficacy of dECM hydrogel for 3D cancer modeling is assessed. Histological, biochemical, morphological, and biological characterization and analysis showcase the cytocompatibility of DAT hydrogel. The rheological property of DAT hydrogel and printing process optimization is assessed to select a bioprinting window to attain 3D breast cancer models. The bioprinted tissues are characterized for cellular viability and tumor cell-matrix interactions. Additionally, an approach for breast cancer modeling is shown by performing rapid high throughput bioprinting in a 96-well plate format, and in vitro drug screening using 5-fluorouracil is performed on 3D bioprinted microtumors. The results of this study suggest that high throughput bioprinting of cancer models can potentially have downstream clinical applications like multi-drug screening platforms and personalized disease models.

Assessment and process optimization of high throughput biofabrication of immunocompetent breast cancer model for drug screening applications.

Recent advancements in 3D cancer modeling have significantly enhanced our ability to delve into the intricacies of carcinogenesis. Despite the pharmaceutical industry's substantial investment of both capital and time in the drug screening and development pipeline, a concerning trend persists: drug candidates screened on conventional cancer models exhibit a dismal success rate in clinical trials. One pivotal factor contributing to this discrepancy is the absence of drug testing on pathophysiologically biomimetic 3D cancer models during pre-clinical stages. Unfortunately, current manual methods of 3D cancer modeling, such as spheroids and organoids, suffer from limitations in reproducibility and scalability. In our study, we have meticulously developed 3D bioprinted breast cancer model utilizing decellularized adipose tissue-based hydrogel obtained via a detergent-free decellularization method. Our innovative printing techniques allows for rapid, high-throughput fabrication of 3D cancer models in a 96-well plate format, demonstrating unmatched scalability and reproducibility. Moreover, we have conducted extensive validation, showcasing the efficacy of our platform through drug screening assays involving two potent anti-cancer drugs, 5-Fluorouracil and PRIMA-1Met. Notably, our platform facilitates effortless imaging and gene expression analysis, streamlining the evaluation process. In a bid to enhance the relevance of our cancer model, we have introduced a heterogeneous cell population into the DAT-based bioink. Through meticulous optimization and characterization, we have successfully developed a biomimetic immunocompetent breast cancer model, complete with microenvironmental cues and diverse cell populations. This breakthrough paves the way for rapid multiplex drug screening and the development of personalized cancer models, marking a paradigm shift in cancer research and pharmaceutical development.

Synergistic Coassembly of Folic Acid-Based Supramolecular Polymer with a Covalent Polymer Toward Fabricating Functional Antibacterial Biomaterials.

Supramolecular biomaterials can recapitulate the structural and functional facets of the native extracellular matrix and react to biochemical cues, leveraging the unique attributes of noncovalent interactions, including reversibility and tunability. However, the low mechanical properties of supramolecular biomaterials can restrict their utilization in specific applications. Combining the advantages of supramolecular polymers with covalent polymers can lead to the fabrication of tailor-made biomaterials with enhanced mechanical properties/degradability. Herein, we demonstrate a synergistic coassembled self-healing gel as a multifunctional supramolecular material. As the supramolecular polymer component, we chose folic acid (vitamin B9), an important biomolecule that forms a gel comprising one-dimensional (1D) supramolecular polymers. Integrating polyvinyl alcohol (PVA) into this supramolecular gel alters its ultrastructure and augments its mechanical properties. A drastic improvement of complex modulus (G*) (∼3674 times) was observed in the folic acid-PVA gel with 15% w/v PVA (33215 Pa) compared with the folic acid gel (9.04 Pa). The coassembled hydrogels possessed self-healing and injectable/thixotropic attributes and could be printed into specific three-dimensional (3D) shapes. Synergistically, the supramolecular polymers of folic acid also improve the toughness, durability, and ductility of the PVA films. A nanocomposite of the gels with silver nanoparticles exhibited excellent catalytic efficiency and antibacterial activity. The folic acid-PVA coassembled gels and films also possessed high cytocompatibility, substantiated by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and live-dead assays. Taken together, the antibacterial and cell-adhesive attributes suggest potential applications of these coassembled biomaterials for tissue engineering and wound healing.

Enhancing Bone Implants: Magnesium-Doped Hydroxyapatite for Stronger, Bioactive, and Biocompatible Applications.

Hydroxyapatite (HAp) with the chemical formula Ca10(PO4)6(OH)2 is an inorganic material that exhibits morphology and composition similar to those of human bone tissues, making it highly desirable for bone regeneration applications. As one of the most biocompatible materials currently in use, HAp has undergone numerous attempts to enhance its mechanical strength. This research focuses on investigating the influence of magnesium (Mg) incorporation on the structural and mechanical properties of synthesized magnesium-doped hydroxyapatite (MgHAp) samples. Apart from its biocompatibility, Mg possesses a density and elasticity comparable to those of human bone. Therefore, incorporating Mg into HAp can be pivotal for improving bone formation. Previous studies have not extensively explored the structural changes induced by Mg substitution in HAp, which motivated us to revisit this issue. Hydrothermal synthesis technique was used to synthesize MgHAp samples with varying molar concentrations (x = 0, 0.5, 1.0, and 1.5). Theoretical simulation of HAp and MgHAp for obtaining 3D structures has been done, and theoretical X-ray diffraction (XRD) data have been compared with the experimental XRD data. Rietveld analysis revealed the alteration and deviation of lattice parameters with an increase in the Mg content, which ultimately affect the structure as well the mechanical properties of prepared samples. The findings revealed an increase in compressive stress and fracture toughness as the Mg concentration in the composition increased. Furthermore, using a finite-element analysis technique and modeling of the mechanical testing data, the von Mises stress distribution and Young's modulus values were calculated, demonstrating the similarity of the prepared samples to human cortical bone. Biocompatibility assessments using NIH-3T3 fibroblast cells confirmed the biocompatible and bioactive nature of the synthesized samples. MgHAp exhibits great potential for biomedical applications in the dental, orthopedic, and tissue engineering research fields.

Recent advances in additive manufacturing of patient-specific devices for dental and maxillofacial rehabilitation.

OBJECTIVES: Customization and the production of patient-specific devices, tailoring the unique anatomy of each patient's jaw and facial structures, are the new frontiers in dentistry and maxillofacial surgery. As a technological advancement, additive manufacturing has been applied to produce customized objects based on 3D computerized models. Therefore, this paper presents advances in additive manufacturing strategies for patient-specific devices in diverse dental specialties. METHODS: This paper overviews current 3D printing techniques to fabricate dental and maxillofacial devices. Then, the most recent literature (2018-2023) available in scientific databases reporting advances in 3D-printed patient-specific devices for dental and maxillofacial applications is critically discussed, focusing on the major outcomes, material-related details, and potential clinical advantages. RESULTS: The recent application of 3D-printed customized devices in oral prosthodontics, implantology and maxillofacial surgery, periodontics, orthodontics, and endodontics are presented. Moreover, the potential application of 4D printing as an advanced manufacturing technology and the challenges and future perspectives for additive manufacturing in the dental and maxillofacial area are reported. SIGNIFICANCE: Additive manufacturing techniques have been designed to benefit several areas of dentistry, and the technologies, materials, and devices continue to be optimized. Image-based and accurately printed patient-specific devices to replace, repair, and regenerate dental and maxillofacial structures hold significant potential to maximize the standard of care in dentistry.

Enhanced redifferentiation of chondrocytes on microperiodic silk/gelatin scaffolds: toward tailor-made tissue engineering.

Direct-write assembly allows rapid fabrication of complex three-dimensional (3D) architectures, such as scaffolds simulating anatomical shapes, avoiding the need for expensive lithographic masks. However, proper selection of polymeric ink composition and tailor-made viscoelastic properties are critically important for smooth deposition of ink and shape retention. Deposition of only silk solution leads to frequent clogging due to shear-induced ß-sheet crystallization, whereas optimized viscoelastic property of silk-gelatin blends facilitate the flow of these blends through microcapillary nozzles of varying diameter. This study demonstrates that induction of controlled changes in scaffold surface chemistry, by optimizing silk-gelatin ratio, can govern cell proliferation and maintenance of chondrocyte morphology. Microperiodic silk-gelatin scaffolds can influence postexpansion redifferentiation of goat chondrocytes by enhancing Sox-9 gene expression, aggregation, and driving cartilage matrix production, as evidenced by upregulation of collagen type II and aggrecan expression. The strategy for optimizing redifferentiation of chondrocytes can offer valuable consideration in scaffold-based cartilage repair strategies.

Decellularized extracellular matrix and silk fibroin-based hybrid biomaterials: A comprehensive review on fabrication techniques and tissue-specific applications.

Biomaterials play a fundamental role in tissue engineering by providing biochemical and physical cues that influence cellular fate and matrix development. Decellularized extracellular matrix (dECM) as a biomaterial is distinguished by its abundant composition of matrix proteins, such as collagen, elastin, fibronectin, and laminin, as well as glycosaminoglycans and proteoglycans. However, the mechanical properties of only dECM-based constructs may not always meet tissue-specific requirements. Recent advancements address this challenge by utilizing hybrid biomaterials that harness the strengths of silk fibroin (SF), which contributes the necessary mechanical properties, while dECM provides essential cellular cues for in vitro studies and tissue regeneration. This review discusses emerging trends in developing such biopolymer blends, aiming to synergistically combine the advantages of SF and dECM through optimal concentrations and desired cross-linking density. We focus on different fabrication techniques and cross-linking methods that have been utilized to fabricate various tissue-engineered hybrid constructs. Furthermore, we survey recent applications of such biomaterials for the regeneration of various tissues, including bone, cartilage, trachea, bladder, vascular graft, heart, skin, liver, and other soft tissues. Finally, the trajectory and prospects of the constructs derived from this blend in the tissue engineering field have been summarized, highlighting their potential for clinical translation.

3D bioprinting of tissue constructs employing dual crosslinking of decellularized extracellular matrix hydrogel.

Bioprinted tissues are currently being utilized for drug and cosmetic screening mostly, but the long-term goal is to achieve human scale functional tissues and organs for transplantation. Hence, recapitulating the multiscale architecture, 3D structures, and complexity of native tissues is the key to produce bioengineered tissues/organs. Decellularized extracellular matrix (dECM)-based biomaterials are widely being used as bioinks for 3D bioprinting for tissue engineering applications. Their potential to provide excellent biocompatibility for the cells drove researchers to use them extensively. However, the decellularization process involves many detergents and enzymes which may contribute to their loss of mechanical properties. Moreover, thermal gelation of dECM-based hydrogels is typically slow which affects the shape fidelity, printability, and physical properties while printing complex structures with 3D printing. But, thermally gelled dECM hydrogels provide excellent cell viability and functionality. To overcome this, a novel dual crosslinking of unmodified dECM has been proposed in this study to render shape fidelity and enhance cell viability and functionality. The dECM-based bioink can be initially polymerized superficially on exposure to light to achieve immediate stability and can attain further stability upon thermal gelation. This dual crosslinking mechanism can maintain the microenvironment of the structure, hence allowing the printing of stable flexible structures. Optimized concentrations of novel photo crosslinkers have been determined and printing of a few complex-shaped anatomical structures has been demonstrated. This approach of fabricating complex scaffolds employing dual crosslinking can be used for the bioprinting of different complex tissue structures with tissue-specific dECM based bioinks.

Strategic Replication of the Hepatic Zonation In Vitro Employing a Biomimetic Approach.

The varied functions of the liver are dependent on the metabolic heterogeneity exhibited by the hepatocytes within the liver lobule spanning the porto-central axis. This complex phenomenon plays an important role in maintaining the physiological homeostasis of the liver. Standard in vitro culture models fail to mimic this spatial heterogeneity of hepatocytes, assuming a homogeneous population of cells, which leads to inaccurate translation of results. Here, we demonstrate the development of an in vitro model of hepatic zonation by mimicking the microarchitecture of the liver using a 3D printed mini bioreactor and decellularized liver matrix to provide the native microenvironmental cues. There was a differential expression of hypoxic and metabolic markers across the developed mini bioreactor, showing the establishment of gradients of oxygen, Wnt/ß-catenin pathway, and other metabolic pathways. The model also showed the establishment of zone-dependent toxicity on treatment with acetaminophen. The developed model would thus be a promising avenue in the field of tissue engineering for understanding the liver physiology and pathophysiology and for drug screening to evaluate the potential of new pharmaceutical interventions.

Newer approaches to dry eye therapy: Nanotechnology, regenerative medicine, and tissue engineering.

Definitive treatment of dry eye disease (DED), one of the commonest ocular surface disorders, has remained elusive despite several recent advances in better diagnostics and the introduction of newer therapeutic molecules. The current treatment paradigms rely heavily on lubricating eye drops and anti-inflammatory agents that may need to be used long-term and are mainly palliative. Research is ongoing not only for a curative treatment option but also to improve the potency and efficacy of existing drug molecules through better formulations and delivery platforms. In the past two decades, significant advancement has been made in terms of preservative-free formulations, biomaterials such as nanosystems and hydrogels, stem cell therapy, and creation of a bioengineered lacrimal gland. This review comprehensively summarizes the newer approaches to DED treatment, which are biomaterials such as nanosystems, hydrogels, and contact lenses for drug delivery, cell and tissue-based regenerative therapy for damaged lacrimal gland and ocular surface, and tissue engineering for developing artificial lacrimal gland. Also, their potential efficacies in animal models or in vitro studies and possible limitations are discussed. The ongoing research looks promising and needs to be supported with clinical efficacy and safety studies for human use.

Attributes of Nanomaterials and Nanotopographies for Improved Bone Tissue Engineering and Regeneration.

Bone tissue engineering (BTE) is a multidisciplinary area that can solve the limitation of conventional grafting methods by developing viable and biocompatible bone replacements. The three essential components of BTE, i.e., Scaffold material and Cells and Growth factors altogether, facilitate support and guide for bone formation, differentiation of the bone tissues, and enhancement in the cellular activities and bone regeneration. However, there is a scarcity of the appropriate materials that can match the mechanical property as well as functional similarity to native tissue, considering the bone as hard tissue. In such scenarios, nanotechnology can be leveraged upon to achieve the desired aspects of BTE, and that is the key point of this review article. This review article examines the significant areas of nanotechnology research that have an impact on regeneration of bone: (a) scaffold with nanomaterials helps to enhance physicochemical interactions, biocompatibility, mechanical stability, and attachment; (b) nanoparticle-based approaches for delivering bioactive chemicals, growth factors, and genetic material. The article begins with the introduction of components and healing mechanisms of bone and the factors associated with them. The focus of this article is on the various nanotopographies that are now being used in scaffold formation, by describing how they are made, and how these nanotopographies affect the immune system and potential underlying mechanisms. The advantages of 4D bioprinting in BTE by using nanoink have also been mentioned. Additionally, we have investigated the importance of an in silico approach for finding the interaction between drugs and their related receptors, which can help to formulate suitable systems for delivery. This review emphasizes the role of nanoscale approach and how it helps to increase the efficacy of parameters of scaffold as well as drug delivery system for tissue engineering and bone regeneration.

Human cornea-derived extracellular matrix hydrogel for prevention of post-traumatic corneal scarring: A translational approach.

Corneal scarring and opacification are a significant cause of blindness affecting millions worldwide. The current standard of care for corneal blindness is corneal transplantation, which suffers from several drawbacks. One alternative approach that has shown promise is the use of xenogeneic corneal extracellular matrix (ECM), but its clinical applicability is challenging due to safety concerns. This study reports the innovative use of human cornea-derived ECM to prevent post-traumatic corneal scarring. About 30 - 40% of corneas donated to the eye banks do not meet the standards defined for clinical use and are generally discarded, although they are completely screened for their safety. In this study, human cornea-derived decellularized ECM hydrogel was prepared from the non-transplantation grade human cadaveric corneas obtained from an accredited eye-bank. The prepared hydrogel was screened for its efficacy against corneal opacification following an injury in an animal model. Our in vivo study revealed that, the control collagen-treated group developed corneal opacification, while the prophylactic application of human cornea-derived hydrogel effectively prevented corneal scarring and opacification. The human hydrogel-treated corneas were indistinguishable from healthy corneas and comparable to those treated with the xenogeneic bovine corneal hydrogel. We also demonstrated that the application of the hydrogel retained the biological milieu including cell behavior, protein components, optical properties, curvature, and nerve regeneration by remodeling the corneal wound after injury. The hydrogel application is also sutureless, resulting in faster corneal healing. We envision that this human cornea-derived ECM-based hydrogel has potential clinical application in preventing scarring from corneal wounding. STATEMENT OF SIGNIFICANCE: There are significant challenges surrounding corneal regeneration after injury due to extensive scarring. Although there is substantial research on corneal regeneration, much of it uses synthetic materials with chemical cross-linking methods or xenogeneic tissue-based material devices which have to undergo exhaustive safety analysis before clinical trials. Herein, we demonstrate the potential application of a human corneal extracellular matrix hydrogel without any additional materials for scarless corneal tissue regeneration, and a method to reduce the wasting of donated allogenic corneal tissue from eye banks. We found no difference in efficacy between the usage of human tissues compared to xenogeneic sources. This may help ease clinical translation and can be used topically without sutures as an outpatient procedure.

Development of chitosan-tripolyphosphate non-woven fibrous scaffolds for tissue engineering application.

The fibrous scaffolds are promising for tissue engineering applications because of their close structural resemblance with native extracellular matrix. Additionally, the chemical composition of scaffold is also an important consideration as they have significant influences on modulating cell attachment, morphology and function. In this study, chitosan-tripolyphosphate (TPP) non-woven fibrous scaffolds were prepared through wetspinning process. Interestingly, at physiological pH these scaffolds release phosphate ions, which have significant influences on cellular function. For the first time, cell viability in presence of varying concentration of sodium TPP solution was analyzed and correlated with the phosphate release from the scaffolds during 30 days incubation period. In vitro degradation of the chitosan-TPP scaffolds was higher than chitosan scaffolds, which may be due to decrease in crystallinity as a result of instantaneous ionic cross-linking during fiber formation. The scaffolds with highly interconnected porous structure present a remarkable cytocompatibility for cell growing, and show a great potential for tissue engineering applications.

Formulation of Dermal Tissue Matrix Bioink by a Facile Decellularization Method and Process Optimization for 3D Bioprinting toward Translation Research.

Decellularized extracellular matrices (ECMs) are being extensively used for tissue engineering purposes and detergents are predominantly used for this. A facile detergent-free decellularization method is developed for dermal matrix and compared it with the most used detergent-based decellularization methods. An optimized, single-step, cost-effective Hypotonic/Hypertonic (H/H) Sodium Chloride (NaCl) solutions-based method is employed to decellularize goat skin that resulted in much higher yield than other methods. The ECM composition, mechanical property, and cytocompatibility are evaluated and compared with other decellularization methods. Furthermore, this H/H-treated decellularized dermal ECM (ddECM) exhibits a residual DNA content of <50 ng mg-1  of dry tissue. Moreover, 85.64 ± 3.01% of glycosaminoglycans and 65.53 ± 2.9% collagen are retained compared to the native tissue, which is higher than the ddECMs prepared by other methods. The cellular response is superior in ddECM (H/H) than other ddECMs prepared by detergent-based methods. Additionally, a bioink is formulated with the ddECM (H/H), showing good shear thinning and shear recovery properties. Process optimization in terms of print speed, flow rate, and viscosity is done to obtain a bioprinting window for ddECM bioink. The printed constructs with optimized parameters have adequate mechanical and cell adhesive properties and excellent isotropic cellular alignment.

Smooth muscle matrix bioink promotes myogenic differentiation of encapsulated adipose-derived stem cells.

Hydrogels derived from decellularized extracellular matrices (dECM) can mimic the biochemical composition of the native tissue. They can also act as a template to culture reseeded cells in vitro. However, detergent-based decellularization methods are known to alter the biochemical compositions, thereby compromising the bioactive potential of dECM. This study proposes a facile detergent-free method to achieve dECM from smooth muscle tissue. We have used the muscle layer of caprine esophageal tissue and decellularized using hypo and hyper-molar sodium chloride solutions alternatingly. Then, a hydrogel was prepared from this decellularized smooth muscle matrix (dSMM) and characterized thoroughly. A comparative analysis of the dSMM prepared with our protocol with the existing detergent-based protocol suggests successful and comparable decellularization with minimal residual DNA content. Interestingly, an 8.78-fold increase in sulfated glycosaminoglycans content and 1.62-fold increased collagen content indicated higher retention of ECM constituents with NaCl-based decellularization strategy. Moreover, the dSMM gel induces differentiation of the encapsulated adipose-derived mesenchymal stem cells toward smooth muscle cells (SMCs) as observed by their expression of alpha-smooth muscle actin and smooth muscle myosin heavy chain, the hallmarks of SMCs. Finally, we optimized the process parameter for productive bioprinting with this dSMM bioink and fabricated 3D muscle constructs. Our results suggest that dSMM has the potential to be used as a bioink to engineer personalized esophageal tissues.