Hydrogel-based Scaffolds Research Articles

The Individual and Combined Effects of Shear, Tension, and Flexure on Aortic Heart Valve Endothelial Cells in Culture

The necessity of living engineered heart valves to treat patients with severe heart disease poses a challenge to tissue engineers. To reach such goal it is crucial to fully understand the role and the activities of valvular endothelial cells (VECs) when they face different types of mechanical stimuli. This study focuses on decomposing the roles of different mechanical stimuli on heart valve endothelial surfaces and the response of VECs in terms of morphology and phenotype change. This study utilizes soft hydrogel-based scaffolds to use as a substrate for cell culture to mimic heart valve tissue leaflet. VECs were cultured as a monolayer on the gel surface and different types of mechanical stimuli were applied. Finally, the response of cells was investigated in terms of morphology and protein expression changes. Single stimuli introduces actin fibers reorganization in VECs, change in cell morphology, and higher mesenchymal protein expression. On the other hand, combined stimuli application has lower impact on actin fibers reorganization and cell morphology change, with lower mesenchymal protein expression. When VECs face a single mechanical stimuli, they undergo transdifferentiation and transform into mesenchymal cells. However, when these cells face a combination of mechanical stimuli, the rate of transformation decreases compared to single stimuli applications. This indicates that a single stimulus induces endothelial to mesenchymal transition in VECs while the process is slower under the combination of multiple mechanical stimuli.

Nanocomposite Scaffolds for Monitoring of Drug Diffusion in Three-Dimensional Cell Environments by Surface-Enhanced Raman Spectroscopy.

Monitoring dynamic processes in complex cellular environments requires the integration of uniformly distributed detectors within such three-dimensional (3D) networks, to an extent that the sensor could provide real-time information on nearby perturbations in a non-invasive manner. In this context, the development of 3D-printed structures that can function as both sensors and cell culture platforms emerges as a promising strategy, not only for mimicking a specific cell niche but also toward identifying its characteristic physicochemical conditions, such as concentration gradients. We present herein a 3D cancer model that incorporates a hydrogel-based scaffold containing gold nanorods. In addition to sustaining cell growth, the printed nanocomposite inks display the ability to uncover drug diffusion profiles by surface-enhanced Raman scattering, with high spatiotemporal resolution. We additionally demonstrate that the acquired information could pave the way to designing novel strategies for drug discovery in cancer therapy, through correlation of drug diffusion with cell death.

Advances of Hydrogel-Based Bioprinting for Cartilage Tissue Engineering.

Bioprinting has gained immense attention and achieved the revolutionized progress for application in the multifunctional tissue regeneration. On account of the precise structural fabrication and mimicking complexity, hydrogel-based bio-inks are widely adopted for cartilage tissue engineering. Although more and more researchers have reported a number of literatures in this field, many challenges that should be addressed for the development of three-dimensional (3D) bioprinting constructs still exist. Herein, this review is mainly focused on the introduction of various natural polymers and synthetic polymers in hydrogel-based bioprinted scaffolds, which are systematically discussed via emphasizing on the fabrication condition, mechanical property, biocompatibility, biodegradability, and biological performance for cartilage tissue repair. Further, this review describes the opportunities and challenges of this 3D bioprinting technique to construct complex bio-inks with adjustable mechanical and biological integrity, and meanwhile, the current possible solutions are also conducted for providing some suggestive ideas on developing more advanced bioprinting products from the bench to the clinic.

Laminin-1 Peptides Conjugated to Fibrin Hydrogels Promote Salivary Gland Regeneration in Irradiated Mouse Submandibular Glands.

Previous studies demonstrated that salivary gland morphogenesis and differentiation are enhanced by modification of fibrin hydrogels chemically conjugated to Laminin-1 peptides. Specifically, Laminin-1 peptides (A99: CGGALRGDN-amide and YIGSR: CGGADPGYIGSRGAA-amide) chemically conjugated to fibrin promoted formation of newly organized salivary epithelium both in vitro (e.g., using organoids) and in vivo (e.g., in a wounded mouse model). While these studies were successful, the model’s usefulness for inducing regenerative patterns after radiation therapy remains unknown. Therefore, the goal of the current study was to determine whether transdermal injection with the Laminin-1 peptides A99 and YIGSR chemically conjugated to fibrin hydrogels promotes tissue regeneration in irradiated salivary glands. Results indicate that A99 and YIGSR chemically conjugated to fibrin hydrogels promote formation of functional salivary tissue when transdermally injected to irradiated salivary glands. In contrast, when left untreated, irradiated salivary glands display a loss in structure and functionality. Together, these studies indicate that fibrin hydrogel-based implantable scaffolds containing Laminin-1 peptides promote secretory function of irradiated salivary glands.

Hydrogel-Based Scaffolds in Oral Tissue Engineering

Regenerative therapy in dentistry has gained interest given the complexity to restore dental and periodontal tissues with inert materials. The best approach for regeneration requires three elements for restoring functions of affected or diseased organ tissues: cells, bioactive molecules, and scaffolds. This triad is capable of modulating the processes to replace lost or damaged tissues and restore function, as it has an impact on diverse cellular processes, influencing cell behavior positively to induce the complete restoration of function and morphology of such complex tissues. Hydrogels (HG) have shown advantages as scaffolds as they are soft and elastic three-dimensional (3D) networks formed from hydrophilic homopolymers, copolymers, or macromers. Besides simple or hybrid, HG show chemical, mechanical and biological activities such as the incorporation of cells in their structures, the retention of high-water content which enhances the transportation of cell nutrients and waste, and elastic and flexible characteristics that emulate the native extracellular matrix (ECM). HG can induce changes in cellular processes such as chemotaxis, proliferation, angiogenesis, biomineralization, and expression of specific tissue biomarkers, enhancing the regeneration process. Besides some of them have anti-inflammatory and anti-bacterial effects. This review aims to show an extensive overview of the most used hydrogels in tissue engineering, emphasizing those that are studied for the regeneration of oral tissues, their biological effects, and their clinical implications. Even though most of the HG are still under investigation, some of them have been studied in vitro and in vivo with outstanding results that may lead to preclinical studies. Besides there are HG that have shown their efficacy in patients such as hyaluronan HG that enhances the healing of gingival tissue.

Preparation and in vitro Evaluation of Injectable Alginate/Thiolated Chitosan Hydrogel Scaffold for Neural Tissue Engineering

Introduction: Spinal cord injuries are one of the main causes of disability with devastating neurological consequences and secondary conflicts in other organs. Tissue engineering and regenerative medicine have been recognized as novel, promising methods in the treatment of tissue injuries, especially in neurological damage in recent decades. Hydrogels have the advantage of compatibility with damaged tissue, and injectable hydrogels can be applied in minimally invasive surgeries. This study aimed to evaluate an injectable hydrogel-based scaffold consisting of thiolated chitosan and alginate for neural tissue regeneration. Materials and Methods: In the present study, an injectable hydrogel-based containing thiolated chitosan and alginate was prepared. Microbiology and pH tests were performed. Microstructural properties and porosity of scaffold were evaluated by scanning electron microscope (SEM). The swelling /shrinkage ratio and rates of biodegradation were also conducted. Finally, the viability of L929 cells on the scaffold was assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Results: Thiolated chitosan/ alginate hydrogel had low pH with no contamination. SEM showed hydrogel had a porous microstructure with a mean pore diameter of 21.89 ± 0.32 μm which is suitable for cell culture. Furthermore, according to MTT test results, this hydrogel was biocompatible. Conclusion: Thiolated chitosan/ alginate hydrogel is convenient for application in neural tissue engineering based on its structural properties and its ability to support cell proliferation. According to the in vitro analysis, it can also be used as a scaffold to create a suitable environment for increasing cell viability.

Increased connectivity of hiPSC-derived neural networks in multiphase granular hydrogel scaffolds

To reflect human development, it is critical to create a substrate that can support long-term cell survival, differentiation, and maturation. Hydrogels are promising materials for 3D cultures. However, a bulk structure consisting of dense polymer networks often leads to suboptimal microenvironments that impedes nutrient exchange and cell-to-cell interaction. Herein, granular hydrogel-based scaffolds were used to support 3D human induced pluripotent stem cell (hiPSC)-derived neural networks. A custom designed 3D printed toolset was developed to extrude hyaluronic acid hydrogel through a porous nylon fabric to generate hydrogel granules. Cells and hydrogel granules were combined using a weaker secondary gelation step, forming self-supporting cell laden scaffolds. At three and seven days, granular scaffolds supported higher cell viability compared to bulk hydrogels, whereas granular scaffolds supported more neurite bearing cells and longer neurite extensions (65.52 ± 11.59 μm) after seven days compared to bulk hydrogels (22.90 ± 4.70 μm). Long-term (three-month) cultures of clinically relevant hiPSC-derived neural cells in granular hydrogels supported well established neuronal and astrocytic colonies and a high level of neurite extension both inside and beyond the scaffold. This approach is significant as it provides a simple, rapid and efficient way to achieve a tissue-relevant granular structure within hydrogel cultures.

Differentiation and proliferation of spermatogonial stem cells using a three-dimensional decellularized testicular scaffold: a new method to study the testicular microenvironment in vitro.

Successful in vitro transplantation of spermatogonial stem cells (SSCs) demands effective culture systems for SSCs proliferation and differentiation. Natural extracellular matrix (ECM) creates a microenvironment suitable for culture of stem cells. In the present study, we intended to assess the capability of the porous scaffold consisting of hyaluronic acid (HA), chitosan, and decellularized testicular matrix (DTM) as a proper niche for SSCs seeding. The testes of four NMRI mice were extracted for further detergent-based decellularization process. We isolated, cultured, and clarified neonate mouse SSC, and a three-dimensional scaffold was prepared for SSCs culture. The loaded SSCs and hydrogel-based scaffold were investigated by several studies including scanning electron microscopy (SEM), 4',6-diamidino-2-phenylindole (DAPI), 3-[4, 5-dimethyl (thiazol-2yl)-3,5diphenyl] tetrazolium bromide (MTT), Acridine orange, and Immunohistochemistry (IHC) staining. The efficiency of decellularization process was confirmed by DAPI, hematoxylin and eosin (H&E), and Masson's Trichrome staining. Acridine orange also depicted SSCs proliferation and viability. SEM approved the preservation of ECM components and also showed complex, coiled, and tubular seminiferous tubules, with intact and condensed collagenous form of the tunica albuginea. MTT test also revealed the scaffold's non-toxicity. Expression of PLZF, TP1, and TEKT1 markers also verified the capacity of SSCs proliferation on a cogel scaffold. In conclusion, cogel scaffold consisting of DTM, HA, and chitosan may provide the supporting layer for in vitro SSC differentiation and proliferation.

3D Spheroids Versus 3D Tumor-Like Microcapsules: Confinement and Mechanical Stress May Lead to the Expression of Malignant Responses in Cancer Cells.

As 2D surfaces fail to resemble the tumoral milieu, current discussions are focused on which 3D cell culture strategy may better lead the cells to express in vitro most of the malignant hints described in vivo. In this study, this question is assessed by analyzing the full genetic profile of MCF7 cells cultured either as 3D spheroids-considered as "gold standard" for in vitro cancer research- or immobilized in 3D tumor-like microcapsules, by RNA-Seq and transcriptomic methods, allowing to discriminate at big-data scale, which in vitro strategy can better resemble most of the malignant features described in neoplastic diseases. The results clearly show that mechanical stress, rather than 3D morphology only, stimulates most of the biological processes involved in cancer pathogenicity, such as cytoskeletal organization, migration, and stemness. Furthermore, cells entrapped in hydrogel-based scaffolds are likely expressing other physiological hints described in malignancy, such as the upregulated expression of metalloproteinases or the resistance to anticancer drugs, among others. According to the knowledge, this study represents the first attempt to answer which 3D experimental system can better mimic the neoplastic architecture in vitro, emphasizing the relevance of confinement in cancer pathogenicity, which can be easily achieved by using hydrogel-based matrices.

Advanced ADA-GEL bioink for bioprinted artificial cancer models

One of the main problems encountered in cancer pathologies is the lack of proper in vitro systems resembling the architecture of tumoral tissues. Since 2D surfaces do not represent the morpho-mechanical characteristics of malignant milieus, 3D systems are rapidly gaining interest in the field of Biomaterials for Cancer Research.In this work, we used a new kind of hydrogel-based scaffold constituted by crosslinked alginate di-aldehyde (ADA) and gelatin (GEL) to mimic the tumoral milieu in vitro by biofabrication. For this purpose, flat 2D films and 3D bioprinted scaffolds were crosslinked with solutions of calcium, barium, and combinations of the two. These structures were analyzed in terms of their chemical and mechanical properties, as well as their biological compatibility in the presence of the tumoral cell line HCT116 (colorectal cancer).Our results indicate that ADA-GEL, crosslinked with these divalent ions, can perform effectively as an artificial cancer model, matching the mechanical properties of colorectal tumors, as well as enabling cell growth in confinement. Furthermore, the use of different combinations of divalent ions allowed the tailoring of the mechanical properties of the material.The cell-loaded ADA-GEL constructs are not significantly affected by the type of crosslinking ion. The best outcome was achieved when 3D printed structures were produced with an ADA concentration of 2.5 (w/v)% and crosslinked with barium, showing remarkable results in terms of printability and mechanical properties. With this composition and barium as the crosslinking ion, a stiffness of 44.97 kPa was achieved, compared with calcium crosslinked samples, where it was only possible to achieve an elastic modulus of 21.55 kPa.This work demonstrates that ADA-GEL-based matrices are a very interesting option to perform as tumor-like scaffolds, which could be used to improve current methods of drug testing as well as for personalized medicine.

Protocol for the Use of a Novel Bioreactor System for Hydrated Mechanical Testing, Strained Sterile Culture, and Force of Contraction Measurement of Tissue Engineered Muscle Constructs.

Bioreactor systems are built as controlled environments for biological processes and utilized in the field of tissue engineering to apply mechanical, spatial, and chemical cues to developing tissue grafts. Often the systems are applied to instruct differentiation and maturation of the cells grown inside. Perhaps the most obvious targets for strain and compression-based bioreactors are mechanically active tissues, as it is hypothesized that biomimetic mechanical environments instruct immature cells to form differentiated tissues. One such tissue, skeletal muscle, has been identified as a key candidate for strain application due to the close structure-function relationship of myofibers. Here we detail the multiple uses of a custom-built bioreactor system in combination with electrospun fibrin microfibers for muscle tissue engineering. Outlined below are the methods used in the system to test the mechanical properties of hydrogel-based scaffolds in an aqueous environment, including Young’s modulus and poroelasticity. Additionally, we demonstrate the application of tensile strain to sterile cell cultures grown on electrospun scaffolds and perform end-point testing of tissue contractility with the addition of an electrode.

Meet me halfway: Are in vitro 3D cancer models on the way to replace in vivo models for nanomedicine development?

The complexity and diversity of the biochemical processes that occur during tumorigenesis and metastasis are frequently over-simplified in the traditional in vitro cell cultures. Two-dimensional cultures limit researchers' experimental observations and frequently give rise to misleading and contradictory results. Therefore, in order to overcome the limitations of in vitro studies and bridge the translational gap to in vivo applications, 3D models of cancer were developed in the last decades. The three dimensions of the tumor, including its cellular and extracellular microenvironment, are recreated by combining co-cultures of cancer and stromal cells in 3D hydrogel-based growth factors-inclusive scaffolds. More complex 3D cultures, containing functional blood vasculature, can integrate in the system external stimuli (e.g. oxygen and nutrient deprivation, cytokines, growth factors) along with drugs, or other therapeutic compounds. In this scenario, cell signaling pathways, metastatic cascade steps, cell differentiation and self-renewal, tumor-microenvironment interactions, and precision and personalized medicine, are among the wide range of biological applications that can be studied. Here, we discuss a broad variety of strategies exploited by scientists to create in vitro 3D cancer models that resemble as much as possible the biology and patho-physiology of in vivo tumors and predict faithfully the treatment outcome.

Recent Advances in Fiber-Hydrogel Composites for Wound Healing and Drug Delivery Systems.

In the last decades, much research has been done to fasten wound healing and target-direct drug delivery. Hydrogel-based scaffolds have been a recurrent solution in both cases, with some reaching already the market, even though their mechanical stability remains a challenge. To overcome this limitation, reinforcement of hydrogels with fibers has been explored. The structural resemblance of fiber–hydrogel composites to natural tissues has been a driving force for the optimization and exploration of these systems in biomedicine. Indeed, the combination of hydrogel-forming techniques and fiber spinning approaches has been crucial in the development of scaffolding systems with improved mechanical strength and medicinal properties. In this review, a comprehensive overview of the recently developed fiber–hydrogel composite strategies for wound healing and drug delivery is provided. The methodologies employed in fiber and hydrogel formation are also highlighted, together with the most compatible polymer combinations, as well as drug incorporation approaches creating stimuli-sensitive and triggered drug release towards an enhanced host response.

3D printing novel in vitro cancer cell culture model systems for lung cancer stem cell study

Two-dimensional (2D) in vitro cell cultures and laboratory animals have been used traditionally as the gold-standard preclinical cancer model systems. However, for cancer stem cell (CSC) studies, they exhibit notable limitations on simulating native environment, which depreciate their translatability for clinical development purposes. In this study, different three-dimensional (3D) printing platforms were used to establish novel 3D cell cultures enriched in CSCs from non-small cell lung cancer (NSCLC) patients and cell lines. Rigid scaffolds with an elevated compressive modulus and uniform pores and channels were produced using different filaments. Hydrogel-based scaffolds were printed with a more irregular distribution of pores and a lower compressive modulus. As a 3D model of reference, suspension spheroid cultures were established. Therein, cancer cell lines exhibited enhanced proliferation profiles on rigid scaffolds compared to the same cells grown on either hydrogel scaffolds or tumor spheres. Meanwhile, primary cancer cells grew considerably better on hydrogel scaffolds or in tumor sphere culture, compared to cells grown on rigid scaffolds. Gene expression analysis confirmed that tumor spheres and cells seeded on hydrogel scaffolds significantly overexpress most of stemness and invasion promoters tested compared to control cells grown in 2D culture. A different phenomenon was observed within cells growing on the rigid scaffolds, where fewer significant variations in gene expression were detected. Our findings provide strong evidence for the advantageous usage of 3D printed models, especially those which use GelMA-PEGDA hydrogels as the primary scaffold material, for studying lung CSCs. The results demonstrated that the 3D printed scaffolds were better to mimic tumor complexity and regulate cancer cell behavior than in vivo 2D culture models.

Porous scaffolds with the structure of an interpenetrating polymer network made by gelatin methacrylated nanoparticle-stabilized high internal phase emulsion polymerization targeted for tissue engineering.

The interlacing of biopolymers and synthetic polymers is a promising strategy to fabricate hydrogel-based tissue scaffolds to biomimic a natural extracellular matrix for cell growth. Herein, open-cellular macroporous 3D scaffolds with a semi-interpenetrating network were fabricated through high internal phase emulsion templating. The scaffolds are prepared by (I) the curing of PEG diacrylate (PEGDAC) and gelatin methacrylate (GelMA) in the continuous aquatic phase of a coconut oil-in-water emulsion stabilized by GelMA nanoparticles, and (II) the removal of the internal phase. The effect of the main contributing parameters such as pH, GelMA content, and GelMA/PEGDAC weight ratio on the emulsion features was investigated systematically. Due to the isoelectric point of GelMA at around pH 6, the GelMA particle (aggregation) size decreased at both sides of pH from 1000 to 100–140 nm because of the increased number of positive and negative charges on GelMA. These GelMA nanoparticles were able to produce stable emulsions with narrowly distributed small emulsion droplets. Moreover, the stability and emulsion droplet size were enhanced and increased, respectively, with GelMA content increasing and GelMA/PEGDAC weight ratio decreasing. These trends lie in the prevented coalescence phenomenon caused by the improved viscosity and likely partially formed network by GelMA chains in the continuous phase. Hence, the following formulation was selected for scaffold preparation: φoil = 74%, pH = 12, GeMA = 4 wt%, and GelMA/PEGDAC = 10/8. Then, PCL in different contents was infiltrated into the scaffold to balance hydrophilicity and hydrophobicity. The cell culture assay proved that the scaffold with a pore size of 60–180 μm and containing 51.2 wt% GelMA, 10.3 wt% PEG, and PCL 27.2 wt% provided a suitable microenvironment for mouse fibroblast cell (L929) adhesion, growth, and spreading. These results show that this strategy suggests promising culture for tissue engineering applications.

Cell-Laden Gelatin Methacryloyl Bioink for the Fabrication of Z-Stacked Hydrogel Scaffolds for Tissue Engineering

Hydrogel-based scaffolds have been widely used to fabricate artificial tissues capable of replacing tissues and organs. However, several challenges inherent in fabricating tissues of large size and complex morphology using such scaffolds while ensuring cell viability remain. To address this problem, we synthesized gelatin methacryloyl (GelMA) based bioink with cells for fabricating a scaffold with superior characteristics. The bioink was grafted onto a Z-stacking bioprinter that maintained the cells at physiological temperature during the printing process, without exerting any physical pressure on the cells. Various parameters, such as the bioink composition and light exposure time, were optimized. The printing accuracy of the scaffolds was evaluated using photorheological studies. The internal morphology of the scaffolds at different time points was analyzed using electron microscopy. The Z-stacked scaffolds were fabricated using high-speed printing, with the conditions optimized to achieve high model reproducibility. Stable adhesion and high proliferation of cells encapsulated within the scaffold were confirmed. We introduced various strategies to improve the accuracy and reproducibility of Z-stack GelMA bioprinting while ensuring that the scaffolds facilitated cell adhesion, encapsulation, and proliferation. Our results demonstrate the potential of the present method for various applications in tissue engineering.

Hydrogels and Dentin-Pulp Complex Regeneration: From the Benchtop to Clinical Translation.

Dentin–pulp complex is a term which refers to the dental pulp (DP) surrounded by dentin along its peripheries. Dentin and dental pulp are highly specialized tissues, which can be affected by various insults, primarily by dental caries. Regeneration of the dentin–pulp complex is of paramount importance to regain tooth vitality. The regenerative endodontic procedure (REP) is a relatively current approach, which aims to regenerate the dentin–pulp complex through stimulating the differentiation of resident or transplanted stem/progenitor cells. Hydrogel-based scaffolds are a unique category of three dimensional polymeric networks with high water content. They are hydrophilic, biocompatible, with tunable degradation patterns and mechanical properties, in addition to the ability to be loaded with various bioactive molecules. Furthermore, hydrogels have a considerable degree of flexibility and elasticity, mimicking the cell extracellular matrix (ECM), particularly that of the DP. The current review presents how for dentin–pulp complex regeneration, the application of injectable hydrogels combined with stem/progenitor cells could represent a promising approach. According to the source of the polymeric chain forming the hydrogel, they can be classified into natural, synthetic or hybrid hydrogels, combining natural and synthetic ones. Natural polymers are bioactive, highly biocompatible, and biodegradable by naturally occurring enzymes or via hydrolysis. On the other hand, synthetic polymers offer tunable mechanical properties, thermostability and durability as compared to natural hydrogels. Hybrid hydrogels combine the benefits of synthetic and natural polymers. Hydrogels can be biofunctionalized with cell-binding sequences as arginine–glycine–aspartic acid (RGD), can be used for local delivery of bioactive molecules and cellularized with stem cells for dentin–pulp regeneration. Formulating a hydrogel scaffold material fulfilling the required criteria in regenerative endodontics is still an area of active research, which shows promising potential for replacing conventional endodontic treatments in the near future.

Evolution of hydrogels for cartilage tissue engineering of the knee: A systematic review and meta-analysis of clinical studies.

In recent years, studies have boosted our knowledge about the biology and disorders of articular cartilage. In this regard, the design of hydrogel-based scaffolds has advanced to improve cartilage repair. However, the efficacy of knee cartilage repair using hydrogels remains unclear. The aim of systematic review and meta-analysis was to scrutinize the efficiency of hydrogel-based therapy in correcting cartilage defects of knee (femoral condyle, patella, tibia plateau and trochlea). The search was conducted in PubMed to gather articles published from 2004/1/1 to 2019/10/01, addressing the effects of implant of hydrogel on knee joint cartilage regeneration. The Cochrane Collaboration's tool for estimating the risk of bias was applied to check the quality of articles. The clinical data for meta-analysis was recorded using the visual analog scale (VAS), Lysholm score, WOMAC, and IKDC. The guidelines of Cochrane Handbook for Systematic Reviews of Interventions were utilized to conduct the review and meta-analysis in the RevMan 5.3 software. The search resulted in 50 clinical trials that included 2846 patients, 986 of whom received cell-based hydrogel implants while 1860 patients used hydrogel without cell. There were significant differences comparing the pain scores based on the VAS (MD: -2.97; 95% CI: -3.15 to -2.79, P<0.00001) and WOMAC (MD: -25.22; 95% CI: -31.22 to -19.22, P<0.00001) between pre- and post-treatment with hydrogels. Furthermore, there were significant improvements in the functional scores based on the IKDC total score (MD: 30.67; P<0.00001) and the Lysholm knee scale (MD: 29.26; 95% CI: 26.74 to 31.78, P<0.00001). According to the Lysholm and IKDC score and after cumulative functional analysis, there was a significant improvement in this parameter (MD: 29.25; 95% CI: 27.26 to 31.25, P<0.00001). This meta-analysis indicated clinically and statistically significant improvements in the pain score (VAS and WOMAC) and the functional score (IKDC and Lysholm) after the administration of hydrogel compared to pretreatment status. So, the current evidence shows the efficiency of hydrogel-based therapy in correcting and repairing knee cartilage defects.

In situ forming microporous gelatin methacryloyl hydrogel scaffolds from thermostable microgels for tissue engineering.

Converting biopolymers to extracellular matrix (ECM)‐mimetic hydrogel‐based scaffolds has provided invaluable opportunities to design in vitro models of tissues/diseases and develop regenerative therapies for damaged tissues. Among biopolymers, gelatin and its crosslinkable derivatives, such as gelatin methacryloyl (GelMA), have gained significant importance for biomedical applications due to their ECM‐mimetic properties. Recently, we have developed the first class of in situ forming GelMA microporous hydrogels based on the chemical annealing of physically crosslinked GelMA microscale beads (microgels), which addressed several key shortcomings of bulk (nanoporous) GelMA scaffolds, including lack of interconnected micron‐sized pores to support on‐demand three‐dimensional‐cell seeding and cell–cell interactions. Here, we address one of the limitations of in situ forming microporous GelMA hydrogels, that is, the thermal instability (melting) of their physically crosslinked building blocks at physiological temperature, resulting in compromised microporosity. To overcome this challenge, we developed a two‐step fabrication strategy in which thermostable GelMA microbeads were produced via semi‐photocrosslinking, followed by photo‐annealing to form stable microporous scaffolds. We show that the semi‐photocrosslinking step (exposure time up to 90 s at an intensity of ~100 mW/cm2 and a wavelength of ~365 nm) increases the thermostability of GelMA microgels while decreasing their scaffold forming (annealing) capability. Hinging on the tradeoff between microgel and scaffold stabilities, we identify the optimal crosslinking condition (exposure time ~60 s) that enables the formation of stable annealed microgel scaffolds. This work is a step forward in engineering in situ forming microporous hydrogels made up from thermostable GelMA microgels for in vitro and in vivo applications at physiological temperature well above the gelatin melting point.

Faithful scanning electron microscopic (SEM) visualization of 3D printed alginate-based scaffolds

The morphological characterization of 3D printed hydrogel-based scaffolds is essential for monitoring their size, shape, surface texture and internal structure. Among other microscopic techniques, Scanning Electron Microscopy (SEM) is capable of visualizing nearly all kinds of materials at different length scales, with exceptional precision, if investigation under vacuum is possible. However, due to the high water content of hydrogel-based scaffolds and the connected volume change after drying, special preparation techniques are necessary to stabilize the 3D architecture when imaged by SEM. Here we present a straightforward cryo-SEM technique to visualize 3D printed hydrogel-based alginate scaffolds. By use of a homemade cryo-SEM holder and plunge-freezing in liquid ethane, scaffolds are visualized from the top and cross-sectional view at different magnifications. The proposed method is compared with SEM imaging in different modes (cyro-SEM, conventional SEM, ESEM) following other commonly used sample preparation techniques, such as plunging in liquid nitrogen, air-drying, freeze-drying and plunging in liquid ethane after graded dehydration. These approaches, except ESEM and cryo-SEM after plunging in liquid nitrogen, lead to shrinkage, deformation, distortion or disintegration of the scaffolds and consequently give rise to artifacts in imaging. The presented results indicate that cryo-SEM after plunging in liquid ethane allows for the most faithful and time-efficient visualization of 3D printed alginate-based scaffolds.