Hydrogel-based Scaffolds Research Articles

The Potential Applications of Stem Cell Conditioned Medium Secretome in Knee Cartilage Regeneration: A Systematic Review

Background: Articular cartilage injuries often result from trauma, genetic predisposition, and degeneration. These injuries lack inherent regeneration mechanisms due to the absence of blood vessels and limited progenitor cell entry. Osteoarthritis is characterized by gradual cartilage deterioration. Mesenchymal stem cells (MSCs), particularly their secretome including exosomes, hold promise as a regenerative therapy. This review explores the application of MSCs and their secretome to address cartilage defects.Methods: This review was conducted based on PRISMA guidelines. Animal model studies focusing on the use of stem cell secretomes for cartilage regeneration were explored. The search, encompassing PubMed, MEDLINE, the Cochrane Library, Scopus, Web of Science, and Science Direct from January 10, 2023, to July 27, 2023, was conducted utilizing Google Chrome as the search engine. Studies with outcomes based on OARSI or ICRS scores, as well as any additional outcomes related to MSC secretome utilization, cartilage regeneration, and proliferation, were included.Results: Our systematic review identified six studies using MSCs in vivo and in vitro. Synovial membrane-derived MSCs significantly enhanced cartilage regeneration by elevating chondrogenic capabilities. Hydrogel-based systems techniques and 3D-printed scaffolds have emerged for innovative delivery. Specific microRNAs, such as miR-92a-3p, have been recognized for enhancing cartilage regeneration. Strategies for the effective targeting of MSC exosomes to the precise cartilage damage site have been explored.Conclusions: The studies demonstrate the potential of MSC-derived secretomes and exosomes for knee cartilage regeneration in animal models. Further research and clinical trials are needed to refine these approaches for practical application.

Retraction Note: Strontium doped bioglass incorporated hydrogel-based scaffold for amplified bone tissue regeneration

Retraction Note: Strontium doped bioglass incorporated hydrogel-based scaffold for amplified bone tissue regeneration

A Facile Strategy for Preparing Flexible and Porous Hydrogel-Based Scaffolds from Silk Sericin/Wool Keratin by In Situ Bubble-Forming for Muscle Tissue Engineering Applications.

In the present study, it is aimed to fabricate a novel silk sericin (SS)/wool keratin (WK) hydrogel-based scaffolds using an in situ bubble-forming strategy containing an N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) coupling reaction. During the rapid gelation process, CO2 bubbles are released by activating the carboxyl groups in sericin with EDC and NHS, entrapped within the gel, creating a porous cross-linked structure. With this approach, five different hydrogels (S2K1, S4K2, S2K4, S6K3, and S3K6) are constructed to investigate the impact of varying sericin and keratin ratios. Analyses reveal that more sericin in the proteinaceous mixture reinforced the hydrogel network. Additionally, the hydrogels' pore size distribution, swelling ratio, wettability, and in vitro biodegradation rate, which are crucial for the applications of biomaterials, are evaluated. Moreover, biocompatibility and proangiogenic properties are analyzed using an in-ovo chorioallantoic membrane assay. The findings suggest that the S4K2 hydrogel exhibited the most promising characteristics, featuring an adequately flexible and highly porous structure. The results obtained by in vitro assessments demonstrate the potential of S4K2 hydrogel in muscle tissue engineering. However, further work is necessary to improve hydrogels with an aligned structure to meet the features that can fully replace muscle tissue for volumetric muscle loss regeneration.

Electrospun "Hard-Soft" Interpenetrating Nanofibrous Tissue Scaffolds Facilitating Enhanced Mechanical Strength and Cell Proliferation.

"Soft" hydrogel-based macroporous scaffolds have been widely used in tissue engineering and drug delivery applications due to their hydrated interfaces and macroporous structures, but have drawbacks related to their weak mechanics and often weak adhesion to cells. In contrast, "hard" poly(caprolactone) (PCL) electrospun fibrous networks have desirable mechanical strength and ductility but offer minimal interfacial hydration and thus limited capacity for cell proliferation. Herein, we demonstrate the fabrication of interpenetrating nanofibrous networks based on coelectrospun PCL and poly(oligoethylene glycol methacrylate) (POEGMA) nanofibers that exhibit the mechanical benefits of PCL but the interfacial hydration benefits of hydrogels. The electrospinning process results in partially aligned but interpenetrating fiber network with minimal internal phase separation, leading to anisotropic but strong mechanical properties even in the hydrated state; apparent ultimate tensile strengths of the swollen scaffolds ranged from 429 ± 39 kPa in the direction of fiber alignment (longitudinal) to 86 ± 25 kPa perpendicular to fiber alignment (cross-longitudinal), typical of PCL-based scaffolds and enabling efficient suture retention in different directions. However, contact angle measurements indicate hydrogel-like interfacial properties due to the presence of the interpenetrating POEGMA network. C2C12 myoblast proliferation in the PCL-POEGMA scaffolds was 50% higher than that observed on PCL-only scaffolds, a result attributed to the presence of the more hydrophilic POEGMA interpenetrating nanofiber network. Overall, this method is demonstrated to represent a facile single-step strategy to fabricate strong macroporous but still interfacially hydrophilic scaffolds for tissue engineering applications.

Matrix Stiffness of GelMA Hydrogels Regulates Lymphatic Endothelial Cells toward Enhanced Lymphangiogenesis.

Lymphatic vessel regeneration is crucial for various tissue engineering strategies, particularly in resolving inflammation and restoring tissue homeostasis. In our study, we focused on investigating how hydrogel matrix stiffness influences lymphatic endothelial cells (LECs) in promoting lymphatic vessel regeneration. Gelatin methacrylate (GelMA) was chosen as our biomaterial due to its versatility in tissue engineering and biofabrication. We fabricated GelMA hydrogels at concentrations of 5, 7.5, and 15% (w/v) with corresponding Young's modulus values of 1.55 kPa (soft matrix), 12.02 kPa (medium matrix), and 48.50 kPa (stiff matrix). Among these, the 7.5% GelMA hydrogel exhibited optimal stiffness for promoting lymphangiogenesis. LECs seeded either on the hydrogel surface or within spontaneously formed a more stable lymphatic capillary network compared with other GelMA formulations. Furthermore, we investigated the enhancement of lymphangiogenesis by incorporating VEGF-C into the GelMA hydrogel, leveraging the synergistic effects of mechanical and chemical cues. Our results underscored the critical role of FAK-phosphorylation in this process; treatment with an FAK-specific inhibitor prevented the formation of tube-like structures by LECs and attenuated the expression of lymphatic markers. Overall, our findings highlight how the mechanical and chemical cues provided by GelMA hydrogels can effectively regulate LEC behavior toward enhanced lymphangiogenesis via the integrin/FAK mechanotransduction pathway. This study proposes a promising strategy for developing hydrogel-based scaffolds or bioinks tailored to promote lymphatic vessel regeneration in therapeutic applications.

Preparation of silk fibroin-derived hydrogels and applications in skin regeneration.

To compare different methods of preparing silk fibroin hydrogels, then summarize the applications of silk fibroin hydrogel-based scaffolds in skin regeneration and finally discuss about future prospects to inspire people interested in this field. A narrative review of the relevant papers was conducted. Notably, for applications in skin regeneration, this review provides a categorized summary and discussion of studies from the past decade. Silk fibroin is a naturally occurring, biocompatible biomaterial that is easily producible. Thanks to its exceptional processability, silk fibroin has found diverse applications in skin regeneration. These applications encompass sponges, fiber fabrics, thin films, and hydrogels. Hydrogels, in particular, are noteworthy due to their water-containing network structure, closely resembling natural tissues. They provide a biomimetic three-dimensional growth environment for cells and have the capacity to incorporate growth factors. Consequently, there are abundant studies of silk fibroin hydrogel-based scaffolds in skin regeneration. Besides, some commercialized medical devices are also made of silk fibroin. Silk fibroin hydrogel could be prepared with multiple methods and it is widely used in constructing scaffolds for efficient skin regeneration. In the future, silk fibroin hydrogel-based skin scaffolds could be more biomimetic and smart.

Integrating classical and fractional calculus rheological models in developing hydroxyapatite-enhanced hydrogels

This study presents a novel method for comprehending the rheological behavior of biomaterials utilized in bone regeneration. The focus is on gelatin, alginate, and hydroxyapatite nanoparticle composites to enhance their mechanical properties and osteoconductive potential. Traditional rheological models are insufficient for accurately characterizing the behavior of these composites due to their complexity and heterogeneity. To address this issue, we utilized fractional calculus rheological models, such as the Scott-Blair, Fractional Kelvin-Voigt, Fractional Maxwell, and Fractional Kelvin-Zener models, to accurately represent the viscoelastic properties of the hydrogels. Our findings demonstrate that the fractional calculus approach is superior to classical models in describing the intricate, time-dependent behaviors of the hydrogel-hydroxyapatite composites. Furthermore, the addition of hydroxyapatite not only improves the mechanical strength of hydrogels but also enhances their bioactivity. These findings demonstrate the potential of these composites in bone tissue engineering applications. The study highlights the usefulness of fractional calculus in biomaterials science, providing new insights into the design and optimization of hydrogel-based scaffolds for regenerative medicine.

Differentiation of mesenchymal stem cells into vascular endothelial cells in 3D culture: a mini review.

Mesenchymal Stem Cells, mesodermal origin and multipotent stem cells, have ability to differentiate into vascular endothelial cells. The cells are squamous in morphology, inlining, and protecting blood vessel tissue, as well as maintaining homeostatic conditions. ECs are essential in vascularization and blood vessels formation. The differentiation process, generally carried out in 2D culture systems, were relied on growth factors induction. Therefore, an artificial extracellular matrix with relevant mechanical properties is essential to build 3D culture models. Various 3D fabrication techniques, such as hydrogel-based and fibrous scaffolds, scaffold-free, and co-culture to endothelial cells were reviewed and summarized to gain insights. The obtained MSCs-derived ECs are shown by the expression of endothelial gene markers and tubule-like structure. In order to mimicking relevant vascular tissue, 3D-bioprinting facilitates to form more complex microstructures. In addition, a microfluidic chip with adequate flow rate allows medium perfusion, providing mechanical cues like shear stress to the artificial vascular vessels.

Breaking the Tumor Chronic Inflammation Balance with a Programmable Release and Multi-Stimulation Engineering Scaffold for Potent Immunotherapy.

Tumor-associated chronic inflammation severely restricts the efficacy of immunotherapy in cold tumors. Here, a programmable release hydrogel-based engineering scaffold with multi-stimulation and reactive oxygen species (ROS)-response (PHOENIX) is demonstrated to break the chronic inflammatory balance in cold tumors to induce potent immunity. PHOENIX can undergo programmable release of resiquimod and anti-OX40 under ROS. Resiquimod is first released, leading to antigen-presenting cell maturation and the transformation of myeloid-derived suppressor cells and M2 macrophages into an antitumor immune phenotype. Subsequently, anti-OX40 is transported into the tumor microenvironment, leading to effector T-cell activation and inhibition of Treg function. PHOENIX consequently breaks the chronic inflammation in the tumor microenvironment and leads to a potent immune response. In mice bearing subcutaneous triple-negative breast cancer and metastasis models, PHOENIX effectively inhibited 80% and 60% of tumor growth, respectively. Moreover, PHOENIX protected 100% of the mice against TNBC tumor rechallenge by electing a robust long-term antigen-specific immune response. An excellent inhibition and prolonged survival in PHOENIX-treated mice with colorectal cancer and melanoma is also observed. This work presents a potent therapeutic scaffold to improve immunotherapy efficiency, representing a generalizable and facile regimen for cold tumors.

Transforming growth factor-β1-loaded RADA-16 hydrogel scaffold for effective cartilage regeneration

Cartilage repair remains a major challenge in clinical trials. These current cartilage repair materials can not effectively promote chondrocyte generation, limiting their practical application in cartilage repair. In this work, we develop an implantable scaffold of RADA-16 peptide hydrogel incorporated with TGF-β1 to provide a microenvironment for stem cell-directed differentiation and chondrocyte adhesion growth. The longest release of growth factor TGF-β1 release can reach up to 600 h under physiological conditions. TGF-β1/RADA-16 hydrogel was demonstrated to be a lamellar porous structure. Based on the cell culture with hBMSCs, TGF-β1/RADA-16 hydrogel showed excellent ability to promote cell proliferation, directed differentiation into chondrocytes, and functional protein secretion. Within 14 days, 80% of hBMSCs were observed to be directed to differentiate into vigorous chondrocytes in the co-culture of TGF-β1/RADA-16 hydrogels with hBMSCs. Specifically, these newly generated chondrocytes can secrete and accumulate large amounts of collagen II within 28 days, which can effectively promote the formation of cartilage tissue. Finally, the exploration of RADA-16 hydrogel-based scaffolds incorporated with TGF-β1 bioactive species would further greatly promote the practical clinical trials of cartilage remediation, which might have excellent potential to promote cartilage regeneration in areas of cartilage damage.

A mechanical-assisted post-bioprinting strategy for challenging bone defects repair

Bioprinting that can synchronously deposit cells and biomaterials has lent fresh impetus to the field of tissue regeneration. However, the unavoidable occurrence of cell damage during fabrication process and intrinsically poor mechanical stability of bioprinted cell-laden scaffolds severely restrict their utilization. As such, on basis of heart-inspired hollow hydrogel-based scaffolds (HHSs), a mechanical-assisted post-bioprinting strategy is proposed to load cells into HHSs in a rapid, uniform, precise and friendly manner. HHSs show mechanical responsiveness to load cells within 4 s, a 13-fold increase in cell number, and partitioned loading of two types of cells compared with those under static conditions. As a proof of concept, HHSs with the loading cells show an enhanced regenerative capability in repair of the critical-sized segmental and osteoporotic bone defects in vivo. We expect that this post-bioprinting strategy can provide a universal, efficient, and promising way to promote cell-based regenerative therapy.

A Scaffold-Assisted 3D Cancer Cell Model for Surface-Enhanced Raman Scattering-Based Real-Time Sensing and Imaging.

Despite recent advances in the development of scaffold-based three-dimensional (3D) cell models, challenges persist in imaging and monitoring cell behavior within these complex structures due to their heterogeneous cell distribution and geometries. Incorporating sensors into 3D scaffolds provides a potential solution for real-time, in situ sensing and imaging of biological processes such as cell growth and disease development. We introduce a 3D printed hydrogel-based scaffold capable of supporting both surface-enhanced Raman scattering (SERS) biosensing and imaging of 3D breast cancer cell models. The scaffold incorporates plasmonic nanoparticles and SERS tags, for sensing and imaging, respectively. We demonstrate the scaffold's adaptability and modularity in supporting breast cancer spheroids, thereby enabling spatial and temporal monitoring of tumor evolution.

Mesenchymal stem cell-secretome laden photopolymerizable hydrogels for wound healing.

Mesenchymal stem cell-derived secretome represents an emerging acellular therapeutic which possess significant opportunity for clinical applications due to its anti-inflammatory, immunomodulatory, and wound healing properties. However, maintaining therapeutic efficacy and ensuring stability of cell-based products is challenging, requiring a robust delivery method. Therefore, we designed a hydrogel-based scaffold loaded with CK Cell Technologies' proprietary Mesenchymal stem cell-secretome for controlled release treatment of acute and chronic wounds. We incorporated both conditioned media (CM) and extracellular vesicles (EVs) into gelatin methacryloyl (GelMA) hydrogels and demonstrated how we can tune the diffusive release of the EVs from them. To demonstrate viability of the approach, we developed a wound healing scratch assay where we see in situ release of CM and EVs promote enhanced migration of human dermal fibroblasts (hDFs). We see the colocalization of these EVs in the fibroblasts using fluorescent microscopy. Finally, as a surrogate for in vivo neovascularization, we conducted an in vitro tube formation assay for the MSC-secretome using matrigel-embedded human microvascular endothelial cells. By adding CM and EVs, we observe an increase in tubulogenesis. Collectively, our data demonstrates by tuning the GelMA properties, we can influence the controlled release of the MSC-secretome for a wound dressing and bandage application for chronic and acute wounds.

Assessing the efficacy of curcumin-loaded alginate hydrogel on skin wound healing: A gene expression analysis.

Skin tissue engineering has gained significant attention as a promising alternative to traditional treatments for skin injuries. In this study, we developed 3D hydrogel-based scaffolds, Alginate, incorporating different concentrations of Curcumin and evaluated their properties, including morphology, swelling behavior, weight loss, as well as hemo- and cytocompatibility. Furthermore, we investigated the therapeutic potential of Alginate hydrogel containing different amounts of Curcumin using an in vitro wound healing model. The prepared hydrogels exhibited remarkable characteristics, SEM showed that the pore size of hydrogels was 134.64μm with interconnected pores, making it conducive for cellular infiltration and nutrient exchange. Moreover, hydrogels demonstrated excellent biodegradability, losing 63.5% of its weight over 14 days. In addition, the prepared hydrogels had a stable release of curcumin for 3days. The results also show the hemocompatibility of prepared hydrogels and a low amount of blood clotting. To assess the efficacy of the developed hydrogels, 3T3 fibroblast growth was examined during various incubation times. The results indicated that the inclusion of Curcumin at a concentration of 0.1mg/mL positively influenced cellular behavior. The animal study showed that Alginate hydrogel containing 0.1mg/mL curcumin had high wound closure(more than 80%) after 14 days. In addition, it showed up-regulation of essential wound healing genes, including TGFβ1 and VEGF, promoting tissue repair and angiogenesis. Furthermore, the treated group exhibited down-regulation of MMP9 gene expression, indicating a reduction in matrix degradation and inflammation. The observed cellular responses and gene expression changes substantiate the therapeutic efficacy of prepared hydrogels. Consequently, our study showed the healing effect of alginate-based hydrogel containing Curcumin on skin injuries.

Bioprinting: Mechanical Stabilization and Reinforcement Strategies in Regenerative Medicine.

Bioprinting describes the printing of biomaterials and cell-laden or cell-free hydrogels with various combinations of embedded bioactive molecules. It encompasses the precise patterning of biomaterials and cells to create scaffolds for different biomedical needs. There are many requirements that bioprinting scaffolds face, and it is ultimately the interplay between the scaffold's structure, properties, processing, and performance that will lead to its successful translation. Among the essential properties that the scaffolds must possess-adequate and appropriate application-specific chemical, mechanical, and biological performance-the mechanical behavior of hydrogel-based bioprinted scaffolds is the key to their stable performance in vivo at the site of implantation. Hydrogels that typically constitute the main scaffold material and the medium for the cells and biomolecules are very soft, and often lack sufficient mechanical stability, which reduces their printability and, therefore, the bioprinting potential. The aim of this review article is to highlight the reinforcement strategies that are used in different bioprinting approaches to achieve enhanced mechanical stability of the bioinks and the printed scaffolds. Enabling stable and robust materials for the bioprinting processes will lead to the creation of truly complex and remarkable printed structures that could accelerate the application of smart, functional scaffolds in biomedical settings. Impact statement Bioprinting is a powerful tool for the fabrication of 3D structures and scaffolds for biomedical applications. It has gained tremendous attention in recent years, and the bioink library is expanding to include more and more material combinations. From the practical application perspective, different properties need to be considered, such as the printed structure's chemical, mechanical, and biological performances. Among these, the mechanical behavior of the printed constructs is critical for their successful translation into the clinic. The aim of this review article is to explore the different reinforcement strategies used for the mechanical stabilization of bioinks and bioprinted structures.

LCST/UCST behavior of polysaccharides for hydrogel fabrication.

Hydrogel-based scaffolds play a crucial role in widespread biotechnological applications by providing physicochemical stability to loaded cells or therapeutic agents, interacting with organismal microenvironments, and controlling cargo release. Polysaccharides are regarded as attractive candidates among substrate materials because of their high water-retaining capacity, reactive functional groups, ease of gelation, low immunogenicity, biodegradability, and biocompatibility. However, employing polysaccharide-based hydrogel scaffolds for practical use in response to ongoing physiological and pathological changes within the human body, such as insufficient mechanical strength, uncertain degradation, and uncontrollable release patterns, is challenging. Several physically noncovalent or chemically covalent crosslinking strategies have been utilized to modify the physicochemical properties and biofunctionality of polysaccharide-based hydrogels. Among them, thermo-responsive gelation systems have been considered a promising approach for fabricating advanced scaffolds, referred to as 'stimuli-responsive' or 'smart' hydrogels. This is because of the sol-gel transition with a single trigger, requiring no further environmental or chemical intervention, and in situ and reversible gelation under ambient physiological temperature changes in a minimally invasive manner. This review highlights the classification, reaction mechanisms, characteristics, and advanced studies on thermo-responsive polysaccharides exploited in various biomedical fields.

Protein Isolation from 3D Hydrogel Scaffolds.

Protein isolation is an essential tool in cell biology to characterize protein abundance under various experimental conditions. Several protocols exist, tailored to cell culture or tissue sections, and have been adapted to particular downstream analyses (e.g., western blotting or mass spectrometry). An increasing trend in bioengineering and cell biology is to use three-dimensional (3D) hydrogel-based scaffolds for cell culture. In principle, the same protocols can be used to extract protein from hydrogel-based cell and tissue constructs. However, in practice the yield and quality of the recovered protein pellet is often substantially lower when using standard protocols and requires tuning of multiple steps, including the selected lysis buffer and the scaffold homogenization strategy, as well as the methods for protein purification and reconstitution. We present here specific protocols tailored to common 3D hydrogels to help researchers using hydrogel-based 3D cell culture improve the quantity and quality of their extracted protein. We focus on three materials: protease-degradable PEG-based hydrogels, collagen hydrogels, and alginate hydrogels. We discuss how the protein extraction procedure can be adapted to the scaffold of interest (degradable or non-degradable gels), proteins of interests (soluble, matrix-bound, or phosphoproteins), and downstream biochemical assays (western blotting or mass spectrometry). With the growing interest in 3D cell culture, the protocols presented should be useful to many researchers in cell biology, protein science, biomaterials, and bioengineering communities. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Isolating proteins from PEG-based hydrogels Basic Protocol 2: Isolating proteins from collagen hydrogels Basic Protocol 3: Isolating proteins from alginate hydrogels Alternate Protocol: Isolating protein from alginate gels using EDTA to dissolve the gel Support Protocol: Isolating protein and RNA simultaneously from the same samples.

Role of Polymer Concentration on the Release Rates of Proteins from Single- and Double-Network Hydrogels

Controlled delivery of proteins has immense potential for the treatment of various human diseases, but effective strategies for their delivery are required before this potential can be fully realized. Recent research has identified hydrogels as a promising option for the controlled delivery of therapeutic proteins, owing to their ability to respond to diverse chemical and biological stimuli, as well as their customizable properties that allow for desired delivery rates. This study utilized alginate and chitosan as model polymers to investigate the effects of hydrogel properties on protein release rates. The results demonstrated that polymer properties, concentration, and crosslinking density, as well as their responses to pH, can be tailored to regulate protein release rates. The study also revealed that hydrogels may be combined to create double-network hydrogels to provide an additional metric to control protein release rates. Furthermore, the hydrogel scaffolds were also found to preserve the long-term function and structure of encapsulated proteins before their release from the hydrogels. In conclusion, this research demonstrates the significance of integrating porosity and response to stimuli as orthogonal control parameters when designing hydrogel-based scaffolds for therapeutic protein release.

Polyglycerol-Based Hydrogel as Versatile Support Matrix for 3D Multicellular Tumor Spheroid Formation.

Hydrogel-based artificial scaffolds are essential for advancing cell culture models from 2D to 3D, enabling a more realistic representation of physiological conditions. These hydrogels can be customized through crosslinking to mimic the extracellular matrix. While the impact of extracellular matrix scaffolds on cell behavior is widely acknowledged, mechanosensing has become a crucial factor in regulating various cellular functions. cancer cells' malignant properties depend on mechanical cues from their microenvironment, including factors like stiffness, shear stress, and pressure. Developing hydrogels capable of modulating stiffness holds great promise for better understanding cell behavior under distinct mechanical stress stimuli. In this study, we aim to 3D culture various cancer cell lines, including MCF-7, HT-29, HeLa, A549, BT-474, and SK-BR-3. We utilize a non-degradable hydrogel formed from alpha acrylate-functionalized dendritic polyglycerol (dPG) and thiol-functionalized 4-arm polyethylene glycol (PEG) via the thiol-Michael click reaction. Due to its high multivalent hydroxy groups and bioinert ether backbone, dPG polymer was an excellent alternative as a crosslinking hub and is highly compatible with living microorganisms. The rheological viscoelasticity of the hydrogels is tailored to achieve a mechanical stiffness of approximately 1 kPa, suitable for cell growth. Cancer cells are in situ encapsulated within these 3D network hydrogels and cultured with cell media. The grown tumor spheroids were characterized by fluorescence and confocal microscopies. The average grown size of all tumoroid types was ca. 150 µm after 25 days of incubation. Besides, the stability of a swollen gel remains constant after 2 months at physiological conditions, highlighting the nondegradable potential. The successful formation of multicellular tumor spheroids (MCTSs) for all cancer cell types demonstrates the versatility of our hydrogel platform in 3D cell growth.

3D printing viscoelastic hydrogel-based scaffolds with a swelling-dependent gate for cartilage injury regeneration

Regeneration of articular cartilage remains a great challenge due to its poor self-healing capacity and weak stability. The microfracture surgery (MF) can provide bone marrow mesenchymal stem cells (BMSCs) and is commonly used in clinical practice, but its therapeutic effect is still limited by the lack of effective adhesion matrix and the uncontrollable tendency to develop fibrosis and even mineralization. In this study, a small molecular drug-loaded viscoelastic hydrogel-based 3D printing bilayer scaffold with a swelling-dependent gate was successfully developed to address these limitations. The scaffold consisted of a lower layer with a smaller pore size and an upper matrix with larger pore size. The former acted as a swelling-dependent gate to switch pore size, which initially was on-state allowing the infiltration of BMSCs, and eventually transformed into off-state serving as a barrier blocking excessive blood support from the bone marrow after swelling-induced closure of pores. This barrier structure cooperated with fatty acid inhibitor-suppressed hypertrophy of neo-cartilage matrix. The latter provided a biocompatible and viscoelastic matrix to guide MSCs toward stable chondrogenic commitment. By integrating structural design with the inhibitor, the bilayer scaffold provides a promising platform for future clinical applications of MF surgery in cartilage regeneration.