Natural Extracellular Matrices Research Articles

Abstract 6769: Applications of a novel fibrous extracellular matrix for advanced in vitro cancer models

Abstract Compared to traditional in vitro two-dimensional (2D) planar cell culture system, three-dimensional (3D) mammalian cell culture can better recapitulate physiological environments and the complexity of natural tissues, hence they are crucial in modern biomedical research. Extracellular matrices (ECM) play a key role in in vitro mammalian cell culture systems. The role of the ECM on in vitro 3D cell culture are: 1) to simulate an in vivo-like physiological environment, 2) support cell-cell and cell-ECM interactions, 3) promote cell adhesion, migration, and communication, 4) aid in tissue development, morphogenesis, differentiation, and functioning. Although basal membrane extracts (BME) are the most popular type of ECM supporting 3D modeling in vitro, they often have significant limitations including limited long-term stability, significant batch to batch heterogeneity, restricted cell movement, and relatively high cost for culturing many cancer cell lines. In this investigation, we employ a novel micro-fibrous scaffold that closely mimics natural ECM, providing porous and interconnected networks for 3D culturing of hepatic cells, lung cancer, breast cancer, pancreatic cancer. As revealed by optical and scanning electron microscopy, the scaffold promotes significant cell-to-cell and cell-to-matrix interactions. Moreover, we observed improved cell growth in 3D culture over longer periods than 2D culture systems for all cell types examined. We also evaluated the anticancer efficiency of different drugs including Lapatinib, Cisplatin, Docetaxel, Doxorubicin and observed that 2D cultured cells were more sensitive to drugs treatment in comparison to 3D. Furthermore, ECM was observed to support 3D growth and structure formation with cell pools of primary cells achieved from disassociated human tumors. Tumor organoids were able to form within seven days and ready for anticancer screening within 14 days. We achieved an 80% success rate in developing breast cancer organoids from patient tissues. Additionally, tissue granules were also able to be cultured under the same condition, which allowed us to investigate the invasion of cancer cells into tissue. The primary cells from human tumors formed cell clusters resembling the original tumor tissue's structure. In conclusion, the novel micro-fibrous scaffold, closely mimicking natural extracellular matrixes, promotes significant cell-to-cell and cell-to-matrix interactions, while also facilitating cell growth over extended periods providing a more physiologically relevant platform for evaluating drug responses for various cancer cell types, further showing the importance of 3D culture systems in cancer research. Citation Format: Navneet Kaur, Dazhi Yang, Jamel Ali. Applications of a novel fibrous extracellular matrix for advanced in vitro cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6769.

Advances in Hydrogels for Meniscus Tissue Engineering: A Focus on Biomaterials, Crosslinking, Therapeutic Additives.

Meniscus tissue engineering (MTE) has emerged as a promising strategy for meniscus repair and regeneration. As versatile platforms, hydrogels have gained significant attention in this field, as they possess tunable properties that allow them to mimic native extracellular matrices and provide a suitable microenvironment. Additionally, hydrogels can be minimally invasively injected and can be adjusted to match the shape of the implant site. They can conveniently and effectively deliver bioactive additives and demonstrate good compatibility with other functional materials. These inherent qualities have made hydrogel a promising candidate for therapeutic approaches in meniscus repair and regeneration. This article provides a comprehensive review of the advancements made in the research on hydrogel application for meniscus tissue engineering. Firstly, the biomaterials and crosslinking strategies used in the formation of hydrogels are summarized and analyzed. Subsequently, the role of therapeutic additives, including cells, growth factors, and other active products, in facilitating meniscus repair and regeneration is thoroughly discussed. Furthermore, we summarize the key issues for designing hydrogels used in MTE. Finally, we conclude with the current challenges encountered by hydrogel applications and suggest potential solutions for addressing these challenges in the field of MTE. We hope this review provides a resource for researchers and practitioners interested in this field, thereby facilitating the exploration of new design possibilities.

Development of an integrated device utilizing exosome-hyaluronic acid-based hydrogel and investigation of its osteogenic and angiogenic characteristics

Tissue engineering (TE) has been widely proposed as a potential solution to the global shortage of donor organs for transplantation. Organic and biocompatible materials have been explored as potential candidates to mimic natural extracellular matrices for regenerative applications. Biomimetic environments have been created using these materials, and hydrogels have shown promise in tissue regeneration due to their rapid gel-forming ability and excellent biocompatibility. Therefore, achieving comparisons between studies utilizing identical materials can prove arduous, as variations in material concentrations, sample preparation techniques, and laboratory conditions significantly influence the physicochemical characteristics of hydrogel systems. To ensure their promotion for clinical purposes, stable variables are crucial. In this regard, we have created an amalgamated and simplified device to assist in hydrogel-based systems using hyaluronic acid and alginate-loaded exosomes for regenerative medicine. The objective of this study is to examine the porosity, injectability, release of loaded exosomes, mechanical properties, and the rate of dissolution and degradation of well-known biomaterials, including alginate and hyaluronic acid, at different concentrations in a stable and controlled setting provided by a simplified integrated device. Furthermore, it aims to comprehensively assess their ex vivo and in vivo osteogenesis and vascularity-related metrics.

Stimuli-Responsive Protein Hydrogels: Their Design, Properties, and Biomedical Applications.

Protein-based hydrogels are considered ideal biomaterials due to their high biocompatibility, diverse structure, and their improved bioactivity and biodegradability. However, it remains challenging to mimic the native extracellular matrices that can dynamically respond to environmental stimuli. The combination of stimuli-responsive functionalities with engineered protein hydrogels has facilitated the development of new smart hydrogels with tunable biomechanics and biological properties that are triggered by cyto-compatible stimuli. This review summarizes the recent advancements of responsive hydrogels prepared from engineered proteins and integrated with physical, chemical or biological responsive moieties. We underscore the design principles and fabrication approaches of responsive protein hydrogels, and their biomedical applications in disease treatment, drug delivery, and tissue engineering are briefly discussed. Finally, the current challenges and future perspectives in this field are highlighted.

Silica Nanofibers with Enhanced Wettability and Mechanical Strength for Bone Tissue Engineering: Electrospinning without Polymer Carrier and Subsequent Heat Treatment

AbstractThe unique properties of silica, such as biocompatibility and the ability to promote cell growth, demonstrate favorable results in different applications such as drug delivery, biomedical applications, and tissue engineering (TE). Electrospinning has emerged as a method for creating a substrate with a high surface area and structural resemblance to natural extracellular matrices. A common method of fabricating silica nanofibers (SNFs) for TE involves hybrid silica/ polymer nanofibers, which require calcination to remove the polymer and obtain pure silica. This study aimed to use the sol‐gel precursor tetraethyl orthosilicate (TEOS) to fabricate pure SNFs through electrospinning and investigate calcination's effect on their morphology, thermal behavior, mechanical properties, wettability, and porosity. The findings indicated that the calcination process reduced Young's modulus, wettability, and porosity, which may influence the applications of SNFs in TE. Nanofibrous mats with high Young's modulus can play a crucial role in providing mechanical support for tissue repair. Additionally, high porosity and wettability can facilitate cell attachment and growth by improving nutrient and oxygen transport to the cells. This study provides valuable insights into the potential use of pure SNFs and highlighted the effects of heat treatment on their properties.

Reactive Cell Electrospinning of Anisotropically Aligned and Bilayer Hydrogel Nanofiber Networks.

Structured hydrogels that incorporate aligned nanofibrous morphologies have been demonstrated to better replicate the structures of native extracellular matrices and thus their function in guiding cell responses. However, current techniques for nanofiber fabrication are limited in their ability to create hydrogel scaffolds with tunable directional alignments and cell types/densities, as required to reproduce more complex native tissue structures. Herein, we leverage a reactive cell electrospinning technique based on the dynamic covalent cross-linking of poly(ethylene glycol methacrylate (POEGMA) precursor polymers to fabricate aligned hydrogel nanofibers that can be directly loaded with cells during the electrospinning process. The scaffolds were found to support high C2C12 myoblast viabilities greater than 85% over 14 days, with changes in the electrospinning collector allowing for the single-step fabrication of nonaligned, aligned, or cross-aligned nanofibrous networks. Cell aspect ratios on aligned scaffolds were found on average to be 27% higher compared to those on nonaligned scaffolds; furthermore, cell-loaded bilayer scaffolds with perpendicular fiber alignments showed evidence of enabling localized directional cell responses to individual layer fiber directions while avoiding delamination between the layers. This fabrication approach thus offers potential for better mimicking the structure and thus function of aligned and multilayered tissues (e.g., smooth muscle, neural, or tendon tissues).

HE@PCL/PCE Gel-Nanofiber Dressing with Robust Self-Adhesion toward High Wound-Healing Rate via Microfluidic Electrospinning Technology.

Hydrogels have attracted increasing attention in the biomedical field due to their similarity in structure and composition to natural extracellular matrices. However, they have been greatly limited by their low mechanical strength and self-adhesion for further application. Here, a gel-nanofiber material is designed for wound healing, which synergistically combines the benefits of hydrogels and nanofibers and can overcome the bottleneck of poor mechanical strength and self-adhesion in hydrogels and inadequate healing environment created by nanofibers. First, a nanofiber scaffold composed of polycaprolactone/poly(citric acid)-ε-lysine (PCL/PCE) nanofibers is fabricated via a new strategy of microfluidic electrospinning, which could provide a base for hyaluronic acid-polylysine (HE) gel growth on nanofibers. The prepared HE@PCL/PCE gel-nanofiber possesses high tensile strength (24.15 ± 1.67 MPa), excellent air permeability (656 m3/m2 h kPa), outstanding self-adhesion property, and positive hydrophilicity. More importantly, the prepared gel-nanofiber dressing shows good cytocompatibility and antibacterial properties, achieving a high wound-healing rate (92.48%) and 4.685 mm granulation growth thickness within 12 days. This material may open a promising avenue for accelerating wound healing and tissue regeneration, providing potential applications in clinical medicine.

Advances in Biomimetic Nerve Guidance Conduits for Peripheral Nerve Regeneration.

Injuries to the peripheral nervous system are a common clinical issue, causing dysfunctions of the motor and sensory systems. Surgical interventions such as nerve autografting are necessary to repair damaged nerves. Even with autografting, i.e., the gold standard, malfunctioning and mismatches between the injured and donor nerves often lead to unwanted failure. Thus, there is an urgent need for a new intervention in clinical practice to achieve full functional recovery. Nerve guidance conduits (NGCs), providing physicochemical cues to guide neural regeneration, have great potential for the clinical regeneration of peripheral nerves. Typically, NGCs are tubular structures with various configurations to create a microenvironment that induces the oriented and accelerated growth of axons and promotes neuron cell migration and tissue maturation within the injured tissue. Once the native neural environment is better understood, ideal NGCs should maximally recapitulate those key physiological attributes for better neural regeneration. Indeed, NGC design has evolved from solely physical guidance to biochemical stimulation. NGC fabrication requires fundamental considerations of distinct nerve structures, the associated extracellular compositions (extracellular matrices, growth factors, and cytokines), cellular components, and advanced fabrication technologies that can mimic the structure and morphology of native extracellular matrices. Thus, this review mainly summarizes the recent advances in the state-of-the-art NGCs in terms of biomaterial innovations, structural design, and advanced fabrication technologies and provides an in-depth discussion of cellular responses (adhesion, spreading, and alignment) to such biomimetic cues for neural regeneration and repair.

Strategies for acellular graft scaffolds as alternatives to tissue regeneration

Autologous grafts represent the gold standard for regenerated tissues after diseases, trauma or defects. However, the main problems of this strategy are availability, donor site morbidity and postoperative complications. For this reason, a new approach in tissue engineering looks at scaffolds obtained through the decellularization of native extracellular matrices as an alternative technology to circumvent these limitations. Thus, this review aims to focus on the potential for use of acellular grafts, particularly on the methods of decellularization and recellularization, and continues with their application as part of the formulation of bioinks, which would represent a breakthrough in the field of biofabrication due to their naturally bioactive composition, preserving tissue architecture and structural integrity as a potential alternative for tissue regeneration.

Breast Cancer Cell Type and Biomechanical Properties of Decellularized Mouse Organs Drives Tumor Cell Colonization.

Tissue engineering has emerged as an indispensable tool for the reconstruction of organ-specific environments. Organ-derived extracellular matrices (ECM) and, especially, decellularized tissues (DCL) are recognized as the most successful biomaterials in regenerative medicine, as DCL preserves the most essential organ-specific ECM properties such as composition alongside biomechanics characterized by stiffness and porosity. Expansion of the DCL technology to cancer biology research, drug development, and nanomedicine is pending refinement of the existing DCL protocols whose reproducibility remains sub-optimal varying from organ to organ. We introduce a facile decellularization protocol universally applicable to murine organs, including liver, lungs, spleen, kidneys, and ovaries, with demonstrated robustness, reproducibility, high purification from cell debris, and architecture preservation, as confirmed by the histological and SEM analysis. The biomechanical properties of as-produced DCL organs expressed in terms of the local and total stiffness were measured using our facile methodology and were found well preserved in comparison with the intact organs. To demonstrate the utility of the developed DCL model to cancer research, we engineered three-dimensional tissue constructs by recellularization representative decellularized organs and collagenous hydrogel with human breast cancer cells of pronounced mesenchymal (MDA-MB-231) or epithelial (SKBR-3) phenotypes. The biomechanical properties of the DCL organs were found pivotal to determining the cancer cell fate and progression. Our histological and scanning electron microscopy (SEM) study revealed that the larger the ECM mean pore size and the smaller the total stiffness (as in lung and ovary), the more proliferative and invasive the mesenchymal cells became. At the same time, the low local stiffness ECMs (ranged 2.8-3.6 kPa) did support the epithelial-like SKBR-3 cells' viability (as in lung and spleen), while stiff ECMs did not. The total and local stiffness of the collagenous hydrogel was measured too low to sustain the proliferative potential of both cell lines. The observed cell proliferation patterns were easily interpretable in terms of the ECM biomechanical properties, such as binding sites, embedment facilities, and migration space. As such, our three-dimensional tissue engineering model is scalable and adaptable for pharmacological testing and cancer biology research of metastatic and primary tumors, including early metastatic colonization in native organ-specific ECM.

Decellularized extracellular matrix-based 3D nanofibrous scaffolds functionalized with polydopamine-reduced graphene oxide for neural tissue engineering

One of the exciting prospects of using decellularized extracellular matrices (ECM) lies in their biochemical profile of preserved components, many of which are regeneration-permissive. Herein, a decellularized ECM from adipose tissue (adECM) was explored to design a scaffolding strategy for the challenging repair of the neural tissue. Targeting the recreation of the nano-scaled architecture of native ECM, adECM was first processed into nanofibers by electrospinning to produce bidimensional platforms. These were further shaped into three-dimensional (3D) nanofibrous constructs by gas foaming. The conversion into a 3D microenvironment of nanofibrous walls was assisted by blending the adECM with lactide-caprolactone copolymers, wherein tuning the adECM/copolymer ratio along with the amount of caprolactone in the copolymer led to modulating the mechanical properties towards soft, yet structurally stable, 3D constructs. In view of boosting their performance to guide neural stem cell fate, adECM-based platforms were doped with a bioinspired surface modification relying on polydopamine-functionalized reduced graphene oxide (PDA-rGO). These adECM-based 3D constructs revealed a permissive microenvironment for neural stem cells (NSCs) to adhere, grow, and migrate throughout the microporosity, owing to the synergy between the unique biochemical features of the adECM and the nanofibrous architecture. NSC responded differently depending on the adECM-based architecture–nanofibrous bidimensional, or 3D design. The 3D spatial arrangement of the nanofibers – induced by the gas foaming – exhibited a remarkable effect on NSCs’ phenotype determination and neurite formation, thereby reinforcing the critical importance of engineering scaffolds with multiple length-scale architecture. Furthermore, PDA-rGO promoted the differentiation of NSC towards the neuronal lineage. Specifically in 3D, it significantly increases the levels of Tuj1 and MAP2 a/b isoforms, confirming its effectiveness in boosting neuronal differentiation and neuritogenesis.

Fabrication and Characterization of π-π Stacking Peptide-Contained Double Network Hydrogels.

Since the physical properties are similar to native extracellular matrices, double network (DN) hydrogels have been studied extensively in the tissue engineering. However, the double chemical crosslinked DN hydrogel is limited by poor fatigue resistance. π-π stacking is a non-covalent bonding interaction, which is essential to maintain and self-assemble the three-dimensional structure of biological proteins and nucleic acids. In this study, a robust polyethylene glycol diacrylate (PEGDA)/FFK hybrid DN hydrogel was prepared by Michael addition and π-π stacking. The hybrid DN hydrogels with π-π stacking interactions have excellent mechanical strength and fatigue resistance. The DN FFK/PEGDA hydrogels reveal great biocompatibility and hemocompatibility. The DN hydrogels containing π-π stacking have the potential to fabricate robust hybrid DN hydrogels in drug release and tissue engineering.

Application of cell laden hydrogels with temporally tunable stiffness in biomedical research.

Extracellular matrix (ECM) is a dynamic and complex environment regulating the cell fate and behavior. It is characterized by biophysical and biochemical properties specific for each tissue. Interestingly, hydrogels can serve as exceptional artificial cellular microenvironments as they can be designed to mimic the key features of the native ECM. They are valuable tools to understand how cells respond to complex microenvironments in normal and pathologic conditions. However, unlike the highly dynamic structure of ECM, nearly all of the conventional hydrogel platforms are primarily static and lack the dynamic properties of native extracellular matrices. Thus, it is necessary to develop dynamic hydrogels to better understand the mechanisms by which dynamic changes of ECM contribute to biological processes. Stiffness is one of the significant dynamic components of ECM which must be appropriately mimicked over time in vitro. In this review, we cover recent advances in engineering strategies to make cell laden hydrogels with temporally tunable stiffness. We also highlight the applications of these hydrogel systems in biomedicine focusing on specific examples in cancer, cardiovascular system, tissue fibrosis and stem cell research. Finally, the challenges regarding the development and application of cell laden hydrogels with temporally tunable stiffness are proposed.

Hydrogels provide microenvironments to mesenchymal stem cells for craniofacial bone regeneration: Review.

The anatomical and physiological architecture of the craniofacial bone is intricate. Hence, the exact management of osteogenesis is necessary for the regeneration of the deficiencies that present in this area. Stem-based tissue engineering approaches, as opposed to conventional surgical intervention, induce bone growth with minimal postoperative risk and expense. Mesenchymal stem/stromal cells (MSC)'s pluripotent differentiation potential, anti-inflammatory and immunomodulatory properties underpin its versatility as a therapeutic agent in bone tissues. Inspired by the native stem cell niche, hydrogels are preferred choices to mediate cells and adapt to 3-D environment because of their outstanding swelling capabilities and similarity to natural extracellular matrices (ECMs). Due to their remarkable biocompatibility and capacity for stimulating bone regeneration, bone regeneration hydrogels have also received a great deal of interest. This review explores the opportunities of MSC based regenerative skeletal therapies, introduces the application of hydrogel scaffolds as artificial bone microenvironments for stem cells to explore its usage in craniofacial bone tissue engineering.

Biofunctionalization of hydrogel-based scaffolds for vascular tissue regeneration

Congenital and acquired tissular losses due to disease or trauma are a major world health problem. Regenerative therapy aims to fix damaged tissues by directing the natural capacity of a host organism to use biofunctionalized artificial tissue scaffolds. These three-dimensional (3D) scaffolds can be customized with cells and/or bioactive molecules to induce cellular homing and angiogenesis, essential to ensure successful tissue regeneration. Hydrogels (HGs) scaffolds are networks of hydrophilic homopolymers, copolymers, and/or macromers with chemical and biological activities that enhance their cell colonization. The use of HGs in regenerative medicine has shown to be advantageous since HGs can be prepared under clinical-grade conditions and tailored to the specific needs of the replaced tissue. They can be made to emulate native extracellular matrices (ECMs) including physical, mechanical, and chemical cues and resilience properties. These customized HGs can reproduce the natural hygroscopic capacity of the original tissue which improves cellular anchoring, nutrition, and waste disposal. They can enable host molecular and cellular modification conducive to a natural cellular microenvironment, modifying the properties of the scaffold, and improving chemotaxis, cell adhesion, migration, proliferation, differentiation, and angiogenesis; HGs can be created and biofunctionalized with linked growth factors and synthetic peptides tailored to positively influence scaffold colonization and functional biocompatibility. This review aims to collect the most relevant information regarding biofunctionalization of HGs used for vascular tissue regeneration, their biological effects, and their clinical implications. While most biofunctionalized HGs are still under investigation, some of them have been studied in vitro, ex vivo, and in vivo with promising results. In this regard, in vivo studies have shown that biofunctionalized scaffolds with peptides such as chitosan hydrogel with LL-37 promotes angiogenesis and healing of pressure ulcers. Also, the GHK tripeptide is widely used in trials focused on guided tissue remodeling.

An injectable, self-healing and degradable hydrogel scaffold as a functional biocompatible material for tissue engineering applications

Injectable hydrogels derived from natural extracellular matrices exhibit excellent adhesion to endothelial cells in vitro and are ideal for many biomedical applications. However, their applicability in vivo is limited by the risk of infection or immunogenicity, and the current injectables also suffer from degradation, viscosity, and drug release. In this study, a multifunctional hydrogel scaffold (COB hydrogels) was constructed by incorporating bioactive glass nanoparticles with a Schiff base crosslinking-based hydrogel composed of carboxymethyl chitosan and oxidized cellulose. The incorporation of nanoparticles not only shortened the gelation time of the COB hydrogels, but also enhanced the performance of the hydrogel in terms of function, such as drug loading capacity. The prepared hydrogels also have self-healing ability, injectability, drug loading and sustained release, antibacterial properties and biocompatibility. In addition, given their no cytotoxicity and mild inflammation in vivo, the hydrogel scaffolds will be important for tissue engineering and drug delivery applications.

Structuring supramolecular hyaluronan hydrogels via peptide self-assembly for modulating the cell microenvironment.

The use of synthetic extracellular matrices (ECMs) in fundamental in vitro cell culture studies has been instrumental for investigating the interplay between cells and matrix components. To provide cells with a more native environment in vitro, it is desirable to design matrices that are biomimetic and emulate compositional and structural features of natural ECMs. Here, the supramolecular fabrication of peptide-hyaluronan (HA) hydrogels is presented as potential ECM surrogates, combining native HA and rationally designed cationic amphipatic peptides [(KI)nK, lysine (K), isoleucine (I), n​=​2-6] whose mechanical properties and microstructure are tunable by the peptide sequence. (KI)nK peptides adopt β-sheet configuration and self-assemble into filamentous nanostructures triggered by pH or ionic strength. The self-assembly propensity of (KI)nK peptides increases with the sequence length, forming single phase hydrogels (shorter peptides) or with phase separation (longer peptides) in presence of the anionic polyelectrolyte HA through electrostatic complexations. The gel phase formed in (KI)nK-HA complexes exhibits viscoelastic behavior and triggers the formation of human mesenchymal stem cell (MSC) spheroids which disassemble over the time. It is anticipated that these (KI)nK-HA hydrogels with tunable physical and biochemical properties offer a promising platform for in vitro applications and in stem cell therapy.

Decellularization of renal fibrosis by cyclic hydrostatic pressure to restore renal function

Natural extracellular matrices (ECM) are currently being studied as an alternative source for organ transplantation or as new solutions to treat end-stage renal disease. As the gap between donors and patients in need of an organ transplant continues to widen, research in regenerative medicine seeks to provide different strategies for treatment Tissue and organ failure is currently one of the biggest health issues our society faces [1]. Arising from disease or trauma, complete treatment typically requires the repair or replace the affected organ However, the unavailability of adequate organs for transplantation to meet the existing demand has resulted in major organ shortage crisis. As a result, there has been a major increase in the number of patients [2]. The stem cell therapy fails because of the toxic environment in the organ, the implanted cells unable to regenerate. Afterwards researchers are trying to prepare the lab made artificial organ in the laboratory with the help of artificial scaffold and the tissue specific stem cells. Then tried for the transplantation of animal organs. To overcome this organ shortage challenge is the use of organ-derived scaffolds obtained via decellularization techniques by removing all viable cells from native tissues and retaining 3D structure of natural extracellular matrix (ECM) and vascular network for regeneration. This is the most adequate method where scaffold thoroughly decellularizes, free of endotoxin or bacterial contamination. An appropriate macro and microstructure to support physiological function, intact vascular tree, specific location cues.

Tunable and Compartmentalized Multimaterial Bioprinting for Complex Living Tissue Constructs.

Recapitulating inherent heterogeneity and complex microarchitectures within confined print volumes for developing implantable constructs that could maintain their structure in vivo has remained challenging. Here, we present a combinational multimaterial and embedded bioprinting approach to fabricate complex tissue constructs that can be implanted postprinting and retain their three-dimensional (3D) shape in vivo. The microfluidics-based single nozzle printhead with computer-controlled pneumatic pressure valves enables laminar flow-based voxelation of up to seven individual bioinks with rapid switching between various bioinks that can solve alignment issues generated during switching multiple nozzles. To improve the spatial organization of various bioinks, printing fidelity with the z-direction, and printing speed, self-healing and biodegradable colloidal gels as support baths are introduced to build complex geometries. Furthermore, the colloidal gels provide suitable microenvironments like native extracellular matrices (ECMs) for achieving cell growths and fast host cell invasion via interconnected microporous networks in vitro and in vivo. Multicompartment microfibers (i.e., solid, core-shell, or donut shape), composed of two different bioink fractions with various lengths or their intravolume space filled by two, four, and six bioink fractions, are successfully printed in the ECM-like support bath. We also print various acellular complex geometries such as pyramids, spirals, and perfusable branched/linear vessels. Successful fabrication of vascularized liver and skeletal muscle tissue constructs show albumin secretion and bundled muscle mimic fibers, respectively. The interconnected microporous networks of colloidal gels result in maintaining printed complex geometries while enabling rapid cell infiltration, in vivo.

Biointerfacial Analysis of HEK293 Cells in Contact with Hydrogels based on Poly‐N‐Isopropylacrylamide and Copolymers

AbstractSeveral researches have demonstrated that synthetic hydrogels can mimic the mechanical and physicochemical properties of native extracellular matrices and act as cell‐scaffold. The biointerface can influence over tissue activities such as adhesion, signaling, cell–cell communications, and proliferation. In this work, the behavior of human embryonic kidney cells (HEK293) in contact with hydrogel surfaces based on poly‐N‐isopropylacrylamide (PNIPAM) and copolymers is studied. Ionic and neutral hydrogel surfaces are synthesized by free radical polymerization and characterized by Fourier‐transform infrared spectroscopy, swelling capacity, and wettability at culture conditions. Viability, proliferation, and bioadhesive capacity are analyzed by 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide and Neutral Red assays, [3H]‐thymidine technique, Acridine Orange and Hoechst dye. Noncytotoxic and proliferative effects are observed in all cases. HEK293 cells are mainly adapted and adhered on neutral and low ionic charge surfaces showing typical cell morphology, and normal mitotic rates. While, lower adhesion, abnormal nuclear and cytoplasmic morphologies with mitotic/fragmentation processes are observed after contact with high ionic surfaces. The cellular cycle after cultive on PNIPAM, assessed by flow cytometry, is not affected regarding control surfaces (polystyrene). Therefore, hydrogel surfaces with neutral and low ionic charges can be apt to in vitro tissue development and possible applications of biomedical treatments.