Integrated GelMA and interleukin 8-loaded liposome composite scaffold for endogenous BMSCs recruitment in bone repair.

Bone repair strategies, based on endogenous stem cell recruitment, can effectively avoid immune rejection and the low utilization of exogenous stem cells. Endogenous stem cells can be recruited to the implantation site by loading chemokines onto bone tissue-engineered scaffolds. However, challenges such as unstable chemokine activity and easy inactivation after implantation remain significant. In the present study, composite fiber scaffolds ((IL8@LIP)-GelMA) consisting of Interleukin 8 (IL8) -loaded liposomes and GelMA were constructed by electrospinning and photocrosslinking, and its ability to recruit bone marrow-derived mesenchymal stem cells (BMSCs) and immunomodulatory effect was investigated. Compared to GelMA loaded directly with IL8, scaffolds of (IL8@LIP)-GelMA demonstrated superior protection of IL8 activity, ensuring a slow and continuous release. Both in vivo and in vitro experiments demonstrated that the (IL8@LIP)-GelMA scaffolds effectively recruited BMSCs to the desired sites. Additionally, the (IL8@LIP)-GelMA scaffolds exhibited the capacity to recruit more macrophages to the implantation site. Importantly, they promoted the polarization of macrophages toward the M2 anti-inflammatory phenotype, facilitating the transition from the inflammatory stage to the tissue repair stage. Therefore, (IL8@LIP)-GelMA scaffolds show great potential for cell-free tissue engineering applications and provide insights into the loading mode of growth factors in scaffolds.

Electrofluidic control for textile-based cell culture: Identification of appropriate conditions required to integrate cell culture with electrofluidics.

Electric field-driven microfluidics, known as electrofluidics, is a novel attractive analytical tool when it is integrated with low-cost textile substrate. Textile-based electrofluidics, primarily explored on yarn substrates, is in its early stages, with few studies on 3D structures. Further, textile structures have rarely been used in cellular analysis as a low-cost alternative. Herein, we investigated novel 3D textile structures and develop optimal electrophoretic designs and conditions that are favourable for direct 3D cell culture integration, developing an integrated cell culture textile-based electrofluidic platform that was optimised to balance electrokinetic performance and cell viability requirements. Significantly, there were contrasting electrolyte compositional conditions that were required to satisfy cell viability and electrophoretic mobility requiring the development of and electrolyte that satisfied the minimum requirements of both these components within the one platform. Human dermal fibroblast cell cultures were successfully integrated with gelatine methacryloyl (GelMA) hydrogel-coated electrofluidic platform and studied under different electric fields using 5 mM TRIS/HEPES/300 mM glucose. Higher analyte mobility was observed on 2.5% GelMA-coated textile which also facilitated excellent cell attachment, viability and proliferation. Cell viability also increased by decreasing the magnitude and time duration of applied electric field with good cell viability at field of up to 20 V cm-1.

Bioprinted dermis with human adipose tissue-derived microvascular fragments promotes wound healing.

Tissue-engineered skin is an effective material for treating large skin defects in a clinical setting. However, its use is limited owing to vascular complications. Human adipose tissue-derived microvascular fragments (HaMVFs) are vascularized units that form vascular networks by rapid reassembly. In this study, we designed a vascularized bionic skin tissue using a three-dimensional (3D) bioprinter of HaMVFs and human fibroblasts encapsulated in a hybrid hydrogel composed of GelMA, HAMA, and fibrinogen. Tissues incorporating HaMVFs showed good in vitro vascularization and mechanical properties after UV crosslinking and thrombin exposure. Thus, the tissue could be sutured appropriately to the wound. In vivo, the vascularized 3D bioprinted skin promoted epidermal regeneration, collagen maturation in the dermal tissue, and vascularization of the skin tissue to accelerate wound healing. Overall, vascularized 3D bioprinted skin with HaMVFs is an effective material for treating skin defects and may be clinically applicable to reduce the necrosis rate of skin grafts.

A multicrosslinked network composite hydrogel scaffold based on DLP photocuring printing for nasal cartilage repair.

Natural hydrogels are widely employed in tissue engineering and have excellent biodegradability and biocompatibility. Unfortunately, the utilization of such hydrogels in the field of three-dimensional (3D) printing nasal cartilage is constrained by their subpar mechanical characteristics. In this study, we provide a multicrosslinked network hybrid ink made of photocurable gelatin, hyaluronic acid, and acrylamide (AM). The ink may be processed into intricate 3D hydrogel structures with good biocompatibility and high stiffness properties using 3D printing technology based on digital light processing (DLP), including intricate shapes resembling noses. By varying the AM content, the mechanical behavior and biocompatibility of the hydrogels can be adjusted. In comparison to the gelatin methacryloyl (GelMA)/hyaluronic acid methacryloyl (HAMA) hydrogel, adding AM considerably enhances the hydrogel's mechanical properties while also enhancing printing quality. Meanwhile, the biocompatibility of the multicrosslinked network hydrogels and the development of cartilage were assessed using neonatal Sprague-Dawley (SD) rat chondrocytes (CChons). Cells sown on the hydrogels considerably multiplied after 7 days of culture and kept up the expression of particular proteins. Together, our findings point to GelMA/HAMA/polyacrylamide (PAM) hydrogel as a potential material for nasal cartilage restoration. The photocuring multicrosslinked network ink composed of appropriate proportions of GelMA/HAMA/PAM is very suitable for DLP 3D printing and will play an important role in the construction of nasal cartilage, ear cartilage, articular cartilage, and other tissues and organs in the future. Notably, previous studies have not explored the application of 3D-printed GelMA/HAMA/PAM hydrogels for nasal cartilage regeneration.

Injective hydrogel loaded with liposomes-encapsulated MY-1 promotes wound healing and increases tensile strength by accelerating fibroblast migration via the PI3K/AKT-Rac1 signaling pathway.

Failed skin wound healing, through delayed wound healing or wound dehiscence, is a global public health issue that imposes significant burdens on individuals and society. Although the application of growth factor is an effective method to improve the pace and quality of wound healing, the clinically approved factors are limited. Parathyroid hormone (PTH) demonstrates promising results in wound healing by promoting collagen deposition and cell migration, but its application is limited by potentially inhibitory effects when administered continuously and locally. Through partially replacing and repeating the amino acid domains of PTH(1-34), we previously designed a novel PTH analog, PTH(3-34)(29-34) or MY-1, and found that it avoided the inhibitory effects of PTH while retaining its positive functions. To evaluate its role in wound healing, MY-1 was encapsulated in liposomes and incorporated into the methacryloyl gelatin (GelMA) hydrogel, through which an injectable nanocomposite hydrogel (GelMA-MY@Lipo, or GML) was developed. In vitro studies revealed that the GML had similar properties in terms of the appearance, microstructure, functional groups, swelling, and degradation capacities as the GelMA hydrogel. In vitro drug release testing showed a relatively more sustainable release of MY-1, which was still detectable in vivo 9 days post-application. When the GML was topically applied to the wound areas of rat models, wound closure as well as tensile strength were improved. Further studies showed that the effects of GML on wound repair and tensile strength were closely related to the promotion of fibroblast migration to the wound area through the controlled release of MY-1. Mechanically, MY-1 enhanced fibroblast migration by activating PI3K/AKT signaling and its downstream molecule, Rac1, by which it increased fibroblast aggregation in the early stage and resulting in denser collagen deposition at a later time. Overall, these findings demonstrated that the nanocomposite hydrogel system promoted skin wound healing and increased tensile strength, thus offering new potential in the treatment of wound healing.

Hypoxia preconditioning of adipose stem cell-derived exosomes loaded in gelatin methacryloyl (GelMA) promote type H angiogenesis and osteoporotic fracture repair.

The challenges posed by delayed atrophic healing and nonunion stand as formidable obstacles in osteoporotic fracture treatment. The processes of type H angiogenesis and osteogenesis emerge as pivotal mechanisms during bone regeneration. Notably, the preconditioning of adipose-derived stem cell (ADSC) exosomes under hypoxic conditions has garnered attention for its potential to augment the secretion and functionality of these exosomes. In the present investigation, we embarked upon a comprehensive elucidation of the underlying mechanisms of hypo-ADSC-Exos within the milieu of osteoporotic bone regeneration. Our findings revealed that hypo-ADSC-Exos harboured a preeminent miRNA, namely, miR-21-5p, which emerged as the principal orchestrator of angiogenic effects. Through in vitro experiments, we demonstrated the capacity of hypo-ADSC-Exos to stimulate the proliferation, migration, and angiogenic potential of human umbilical vein endothelial cells (HUVECs) via the mediation of miR-21-5p. The inhibition of miR-21-5p effectively attenuated the proangiogenic effects mediated by hypo-ADSC-Exos. Mechanistically, our investigation revealed that exosomal miR-21-5p emanating from hypo-ADSCs exerts its regulatory influence by targeting sprouly1 (SPRY1) within HUVECs, thereby facilitating the activation of the PI3K/AKT signalling pathway. Notably, knockdown of SPRY1 in HUVECs was found to potentiate PI3K/AKT activation and, concomitantly, HUVEC proliferation, migration, and angiogenesis. The culminating stage of our study involved a compelling in vivo demonstration wherein GelMA loaded with hypo-ADSC-Exos was validated to substantially enhance local type H angiogenesis and concomitant bone regeneration. This enhancement was unequivocally attributed to the exosomal modulation of SPRY1. In summary, our investigation offers a pioneering perspective on the potential utility of hypo-ADSC-Exos as readily available for osteoporotic fracture treatment.

l-Proline and GelMA hydrogel complex：An efficient antifreeze system for cell cryopreservation.

Cryopreservation of biological samples is an important technology for expanding their applications in the biomedical field. However, the quality and functionality of samples after rewarming are limited by the toxicity of commonly used cryoprotectant agents (CPAs). Here, we developed a novel preservation system by combining the natural amino acid l-proline (L-Pro) with gelatin methacryloyl (GelMA) hydrogels. Compared with dimethyl sulfoxide (DMSO), L-Pro and GelMA demonstrated excellent biocompatibility when co-culturing with cells. Cryopreservation procedures were optimized using 3T3 as model cells. The results showed that rapid cooling was the most suitable cooling procedure for L-Pro and GelMA among the three cooling procedures. Co-culturing with cells for 3 h before cryopreservation, 6 % L-Pro +7 % GelMA had the highest survival rate, reaching up to 80 %. Differential Scanning Calorimetry (DSC) analysis showed that 6 % L-Pro + 7 % GelMA lowered the freezing point of the solution to -4.2 °C and increased the unfrozen water content to 20 %. To the best of our knowledge, this is the first report of cell cryopreservation using a combination of L-Pro and GelMA hydrogels, which provides a new strategy for improving cell cryopreservation.

3D Bioprinting-Based Dopamine-Coupled Flexible Material for Nasal Cartilage Repair.

INTRODUCTION: Since 3D printing can be used to design implants according to the specific conditions of patients, it has become an emerging technology in tissue engineering and regenerative medicine. How to improve the mechanical, elastic and adhesion properties of 3D-printed photocrosslinked hydrogels is the focus of cartilage tissue repair and reconstruction research. MATERIALS AND METHODS: We established a strategy for toughening hydrogels by mixing GelMA-DOPA (GD), which is prepared by coupling dopamine (DA) with GelMA, with HAMA, bacterial cellulose (BC) to produce composite hydrogels (HB-GD). HB-GD hydrogel scaffolds were characterized in vitro by scanning electron microscopy (SEM), Young's modulus, swelling property and rheological property tests. And biocompatibility and chondrogenic ability were tested by live/dead staining, DNA quantitative analysis and immunofluorescence staining. Combined with 3D bioprinting technology, mouse chondrocytes (ADTC5) were added to form a biological chain to construct an in vitro model, and the feasibility of the model for nasal cartilage regeneration was verified by cytology evaluation. RESULTS: With the increase of GD concentration, the toughness of the composite hydrogel increased (47.0 ± 2.7 kPa (HB-5GD)-158 ± 3.2 kPa (HB-20GD)), and it had excellent swelling properties, rheological properties and printing properties. The HB-GD composite hydrogel promoted the proliferation and differentiation of ATDC5. Cells in 3D printed scaffolds had higher survival rates (> 95%) and better protein expression than the encapsulated cultures. CONCLUSION: The HB-10GD hydrogel can be made into a porous scaffold with precise shape, good internal pore structure, high mechanical strength and good swelling rate through extrusion 3D printing. NO LEVEL ASSIGNED: This journal requires that authors assign a level of evidence to each submission to which Evidence-Based Medicine rankings are applicable. This excludes Review Articles, Book Reviews, and manuscripts that concern Basic Science, Animal Studies, Cadaver Studies, and Experimental Studies. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266.

Recent advances on 3D-bioprinted gelatin methacrylate hydrogels for tissue engineering in wound healing: A review of current applications and future prospects.

Advancements in 3D bioprinting, particularly the use of gelatin methacrylate (GelMA) hydrogels, are ushering in a transformative era in regenerative medicine and tissue engineering. This review highlights the pivotal role of GelMA hydrogels in wound healing and skin regeneration. Its biocompatibility, tunable mechanical properties and support for cellular proliferation make it a promising candidate for bioactive dressings and scaffolds. Challenges remain in optimizing GelMA hydrogels for clinical use, including scalability of 3D bioprinting techniques, durability under physiological conditions and the development of advanced bioinks. The review covers GelMA's applications from enhancing wound dressings, promoting angiogenesis and facilitating tissue regeneration to addressing microbial infections and diabetic wound healing. Preclinical studies underscore GelMA's potential in tissue healing and the need for further research for real-world applications. The future of GelMA hydrogels lies in overcoming these challenges through multidisciplinary collaboration, advancing manufacturing techniques and embracing personalized medicine paradigms.

Preparation and characterization of propolis reinforced eggshell membrane/ GelMA composite hydrogel for biomedical applications.

Gelatin methacrylate-based hydrogels (GelMA) were widely used in tissue engineering and regenerative medicine. However, to manipulate their various chemical and physical properties and create high-efficiency hydrogels, different materials have been used in their structure. Eggshell membrane (ESM) and propolis are two nature-derived materials that could be used to improve the various characteristics of hydrogels, especially structural and biological properties. Hence, the main purpose of this study is the development of a new type of GelMA hydrogel containing ESM and propolis, for use in regenerative medicine. In this regard, in this study, after synthesizing GelMA, the fragmented ESM fibers were added to it and the GM/EMF hydrogel was made using a photoinitiator and visible light irradiation. Finally, GM/EMF/P hydrogels were prepared by incubating GM/EMF hydrogels in the propolis solution for 24 h. After various structural, chemical, and biological characterizations, it was found that the hydrogels obtained in this study offer improved morphological, hydrophilic, thermal, mechanical, and biological properties. The developed GM/EMF/P hydrogel presented more porosity with smaller and interconnected pores compared to the other hydrogels. GM/EMF hydrogels due to possessing EMF showed compressive strength up to 25.95 ± 1.69 KPa, which is more than the compressive strength provided by GM hydrogels (24.550 ± 4.3 KPa). Also, GM/EMF/P hydrogel offered the best compressive strength (44.65 ± 3.48) due to the presence of both EMF and propolis. GM scaffold with a contact angle of about 65.41 ± 2.199 θ showed more hydrophobicity compared to GM/EMF (28.67 ± 1.58 θ), and GM/EMF/P (26.24 ± 0.73 θ) hydrogels. Also, the higher swelling percentage of GM/EMF/P hydrogels (343.197 ± 42.79) indicated the high capacity of this hydrogel to retain more water than other scaffolds. Regarding the biocompatibility of the fabricated structures, MTT assay results showed that GM/EMF/P hydrogel significantly (p-value < 0.05) supported cell viability. Based on the results, it seems that GM/EMF/P hydrogel could be a promising biomaterial candidate for use in various fields of regenerative medicine.

Stable hydrogel adhesion to polydimethylsiloxane enables cyclic mechanical stimulation of 3D-bioprinted smooth muscle constructs.

During normal urination, smooth muscle cells (SMCs) in the lower urinary tract (LUT) are exposed to mechanical signals that have a critical impact on tissue structure and function. Nevertheless, the mechanisms underlying the maintenance of the contractile phenotype of SMCs remain poorly understood. This is due, in part, to a lack of studies that have examined the effects of mechanical loading using three-dimensional (3D) models. In this study, surface modifications of polydimethylsiloxane (PDMS) membrane were evaluated to investigate the effects of cyclic mechanical stimulation on SMC maturation in 3D constructs. Commercially available cell stretching plates were modified with amino or methacrylate groups to promote adhesion of 3D constructs fabricated by bioprinting. After 6 days of stimulation, the effects of mechanical stimulation on the expression of contractile markers at the mRNA and protein levels were analyzed. Methacrylate-modified surfaces supported stable adhesion of the 3D constructs to the membrane and facilitated cyclic mechanical stimulation, which significantly increased the expression of contractile markers at the mRNA and protein levels. These effects were found to be mediated by activation of the p38 MAPK pathway, as inhibition of this pathway abolished the effects of stimulation in a dose-dependent manner. These results provide valuable insights into the role of mechanical signaling in maintaining the contractile phenotype of bladder SMCs, which has important implications for the development of future treatments for LUT diseases.

Nanoparticle-Reinforced Tough Hydrogel as a Versatile Platform for Pharmaceutical Drug Delivery: Preparation and in Vitro Characterization.

Natural polymer-based hydrogels are excellent for encapsulating hydrophilic drugs, but they are mechanically weak and degrade easily. In this communication, we exploit the electrostatic interaction between nanosilicates (nSi) and gelatin methacrylate (GelMA) to form a mechanically tough nanocomposite hydrogel for pharmaceutical drug delivery. These hydrogels, prepared at subzero temperatures to form cryogels, displayed macroporous structures, which favors cell infiltration. The designed tough cryogel also showed a slower rate of degradation. Furthermore, we encapsulated the small molecule metformin and sustained the drug release under physiological conditions. Cryogel-loaded metformin reduced the effect of endothelial cell injury caused by nutrient deprivation in vitro. Finally, we hypothesize that this versatile nanocomposite material will find use in diverse biomedical applications.

Bioprinted PDLSCs with high-concentration GelMA hydrogels exhibit enhanced osteogenic differentiation in vitro and promote bone regeneration in vivo.

OBJECTIVES: We aimed to explore the osteogenic potential of periodontal ligament stem cells (PDLSCs) in bioprinted methacrylate gelatine (GelMA) hydrogels in vitro and in vivo. MATERIALS AND METHODS: PDLSCs in GelMA hydrogels at various concentrations (3%, 5%, and 10%) were bioprinted. The mechanical properties (stiffness, nanostructure, swelling, and degradation properties) of bioprinted constructs and the biological properties (cell viability, proliferation, spreading, osteogenic differentiation, and cell survival in vivo) of PDLSCs in bioprinted constructs were evaluated. Then, the effect of bioprinted constructs on bone regeneration was investigated using a mouse cranial defect model. RESULTS: Ten percent GelMA printed constructs had a higher compression modulus, smaller porosity, lower swelling rate, and lower degradation rate than 3% GelMA. PDLSCs in bioprinted 10% GelMA bioprinted constructs showed lower cell viability, less cell spreading, upregulated osteogenic differentiation in vitro, and lower cell survival in vivo. Moreover, upregulated expression of ephrinB2 and EphB4 protein and their phosphorylated forms were found in PDLSCs in 10% GelMA bioprinted constructs, and inhibition of eprhinB2/EphB4 signalling reversed the enhanced osteogenic differentiation of PDLSCs in 10% GelMA. The in vivo experiment showed that 10% GelMA bioprinted constructs with PDLSCs contributed to more new bone formation than 10% GelMA constructs without PDLSCs and constructs with lower GelMA concentrations. CONCLUSIONS: Bioprinted PDLSCs with high-concentrated GelMA hydrogels exhibited enhanced osteogenic differentiation partially through upregulated ephrinB2/EphB4 signalling in vitro and promoted bone regeneration in vivo, which might be more appropriate for future bone regeneration applications. CLINICAL RELEVANCE: Bone defects are a common clinical oral problem. Our results provide a promising strategy for bone regeneration through bioprinting PDLSCs in GelMA hydrogels.

GelMA Hydrogel as a Promising Delivery System for Osthole in the Treatment of Rheumatoid Arthritis: Targeting the miR-1224-3p/AGO1 Axis.

Rheumatoid arthritis (RA) is a multifaceted, chronic, progressive autoimmune disease. This study aims to explore the potential benefits of an enhanced drug delivery system utilizing optimized Gelatin Methacryloyl (GelMA) vectors in RA management. We evaluated the levels of miR-1124-3p and AGO1 in RA tissues and cell lines using qPCR, WB, and immunofluorescence. The effects of osthole on inflammatory response and joint morphology were determined by qPCR, H&E staining, and micro-CT. The data showed that miR-1224-3p was downregulated in RA tissues and HUM-iCell-s010RA cells, while the overexpression of miR-1224-3p in HUM-iCell-s010RA cells reduced the expression of IL-6 and IL-1ß. Luciferase assay demonstrated that AGO1 was a direct target gene of miR-1224-3p. Additionally, osthole treatment increased miR-1224-3p levels and decreased AGO1 expression. The release data showed that osthole loaded on GelMA was released at a slower rate than free osthole. Further studies in a mouse model of CIA confirmed that osthole-loaded GelMA was more effective in attenuating osteopenia in RA as well as alleviating autoimmune arthritis. These findings suggest that osthole can regulate the miR-1224-3p/AGO1 axis in RASFs cells and has the potential to be developed as a clinical anti-RA drug. GelMA could provide a new approach to long-term RA treatment.

Properties and Printability of the Synthesized Hydrogel Based on GelMA.

Gelatin methacryloyl (GelMA) has recently attracted increasing attention. Unlike other hydrogels, it allows for the adjustment of the mechanical properties using such factors as degree of functionalization, concentration, and photocrosslinking parameters. In this study, GelMA with a high degree of substitution (82.75 ± 7.09%) was synthesized, and its suitability for extrusion printing, cytocompatibility, and biocompatibility was studied. Satisfactory printing quality was demonstrated with the 15% concentration hydrogel. The high degree of functionalization led to a decrease in the ability of human adipose-derived stem cells (ADSCs) to adhere to the GelMA surface. During the first 3 days after sowing, proliferation was observed. Degradation in animals after subcutaneous implantation was slowed down.

Understanding the Molecular Conformation and Viscoelasticity of Low Sol-Gel Transition Temperature Gelatin Methacryloyl Suspensions.

For biomedical applications, gelatin is usually modified with methacryloyl groups to obtain gelatin methacryloyl (GelMA), which can be crosslinked by a radical reaction induced by low wavelength light to form mechanically stable hydrogels. The potential of GelMA hydrogels for tissue engineering has been well established, however, one of the main disadvantages of mammalian-origin gelatins is that their sol-gel transitions are close to room temperature, resulting in significant variations in viscosity that can be a problem for biofabrication applications. For these applications, cold-water fish-derived gelatins, such as salmon gelatin, are a good alternative due to their lower viscosity, viscoelastic and mechanical properties, as well as lower sol-gel transition temperatures, when compared with mammalian gelatins. However, information regarding GelMA (with special focus on salmon GelMA as a model for cold-water species) molecular conformation and the effect of pH prior to crosslinking, which is key for fabrication purposes since it will determine final hydrogel's structure, remains scarce. The aim of this work is to characterize salmon gelatin (SGel) and salmon methacryloyl gelatin (SGelMA) molecular configuration at two different acidic pHs (3.6 and 4.8) and to compare them to commercial porcine gelatin (PGel) and methacryloyl porcine gelatin (PGelMA), usually used for biomedical applications. Specifically, we evaluated gelatin and GelMA samples' molecular weight, isoelectric point (IEP), their molecular configuration by circular dichroism (CD), and determined their rheological and thermophysical properties. Results showed that functionalization affected gelatin molecular weight and IEP. Additionally, functionalization and pH affected gelatin molecular structure and rheological and thermal properties. Interestingly, the SGel and SGelMA molecular structure was more sensitive to pH changes, showing differences in gelation temperatures and triple helix formation than PGelMA. This work suggests that SGelMA presents high tunability as a biomaterial for biofabrication, highlighting the importance of a proper GelMA molecular configuration characterization prior to hydrogel fabrication.

Methacrylated Gelatin as a Scaffold for Mechanically Isolated Stromal Vascular Fraction for Cutaneous Wound Repair.

Mechanically processed stromal vascular fraction (mSVF) is a highly interesting cell source for regenerative purposes, including wound healing, and a practical alternative to enzymatically isolated SVF. In the clinical context, SVF benefits from scaffolds that facilitate viability and other cellular properties. In the present work, the feasibility of methacrylated gelatin (GelMA), a stiffness-tunable, light-inducible hydrogel with high biocompatibility is investigated as a scaffold for SVF in an in vitro setting. Lipoaspirates from elective surgical procedures were collected and processed to mSVF and mixed with GelMA precursor solutions. Non-encapsulated mSVF served as a control. Viability was measured over 21 days. Secreted basic fibroblast growth factor (bFGF) levels were measured on days 1, 7 and 21 by ELISA. IHC was performed to detect VEGF-A, perilipin-2, and CD73 expression on days 7 and 21. The impact of GelMA-mSVF on human dermal fibroblasts was measured in a co-culture assay by the same viability assay. The viability of cultured GelMA-mSVF was significantly higher after 21 days (p < 0.01) when compared to mSVF alone. Also, GelMA-mSVF secreted stable levels of bFGF over 21 days. While VEGF-A was primarily expressed on day 21, perilipin-2 and CD73-positive cells were observed on days 7 and 21. Finally, GelMA-mSVF significantly improved fibroblast viability as compared with GelMA alone (p < 0.01). GelMA may be a promising scaffold for mSVF as it maintains cell viability and proliferation with the release of growth factors while facilitating adipogenic differentiation, stromal cell marker expression and fibroblast proliferation.

A Microfluidic Device to Fabricate One-Step Cell Bead-Laden Hydrogel Struts for Tissue Engineering.

Cell-laden structures are widely applied for a variety of tissue engineering applications, including tissue restoration. Cell-to-cell interactions in bioprinted structures are important for successful tissue restoration, because cell-cell signaling pathways can regulate tissue development and stem cell fate. However, the low degree of cell-cell interaction in conventional cell-laden bioprinted structures is challenging for the therapeutic application of this modality. Herein, a microfluidic device with cell-laden methacrylated gelatin (GelMa) bioink and alginate as a matrix hydrogel is used to fabricate a functional hybrid structure laden with cell-aggregated microbeads. This approach effectively increases the degree of cell-to-cell interaction to a level comparable to cell spheroids. The hybrid structure is obtained using a one-step process without the exhausting procedure. It consists of cell bead fabrication and an extrusion process for the cell-bead laden structure. Different flow rates are appropriately selected to develop cell-laden struts with homogeneously distributed cell beads for each hydrogel in the process. The hybrid struts exhibit significantly higher cellular activities than those of conventional alginate/GelMa struts, which are bioprinted using similar cell densities and bioink formulations. Furthermore, hybrid struts with adipose stem cells are implanted into mice, resulting in significantly higher myogenesis in comparison to normally bioprinted struts.

3D Bioprinted GelMA-Nanoclay Hydrogels Induce Colorectal Cancer Stem Cells Through Activating Wnt/ß-Catenin Signaling.

Cancer stem cells (CSCs) are a rare cell population in tumors that are responsible for tumor recurrence and metastasis. They are a priority as therapeutic targets, however, assays targeting CSCs have been limited by expanding and maintaining CSCs in vitro. Here, the authors find that gelatin methacryloyl (GelMA)-nanoclay hybrid hydrogels can induce and enrich colorectal CSCs assisted by three-dimensional (3D) bioprinting. The presence of the nanoclay increases the printability, Young's modulus, pore size, and cytocompatibility of the hydrogels. Bioprinted GelMA-nanoclay hydrogels promote the formation of spheroids expressing elevated levels of the stemness markers LGR5, CD133, CD26, and SOX2. Cancer cells grown in GelMA-nanoclay hydrogel possess higher self-renewal and differentiation capacity in vitro and higher tumorigenic capacity in vivo. GelMA-nanoclay hydrogels induce CSCs by stimulating the activation of the Wnt/ß-catenin signaling pathway. Further studies demonstrate that spheroids from GelMA-nanoclay hydrogels possess increased stemness, higher consistency, yield, and sensitivity to the anti-CSC compounds compared to the classic CSC-enrichment model. Collectively, this study may provide a valuable biomaterial and method for inducing and enriching CSCs, to facilitate the effective CSC-targeting drug screening.

In vitro and in vivo evaluation of 3D constructs engineered with human iPSC-derived chondrocytes in gelatin methacryloyl hydrogel.

Articular cartilage defects have limited healing potential and, when left untreated, can lead to osteoarthritis. Tissue engineering focuses on regenerating the damaged joint surface, preferably in an early stage. Here, we investigate the regenerative potential of three-dimensional (3D) constructs consisting of human induced pluripotent stem cell (iPSC)-derived chondrocytes in gelatin methacryloyl (GelMA) hydrogel for stable hyaline cartilage production. iPSC-derived chondrocytes are encapsulated in GelMA hydrogel at low (1 × 107 ml-1 ) and high (2 × 107 ml-1 ) density. In a conventional medium, GelMA hydrogel supports the chondrocyte phenotype, as opposed to cells cultured in 3D in absence of hydrogel. Moreover, encapsulated iPSC-derived chondrocytes preserve their in vivo matrix formation capacity after 21 days in vitro. In differentiation medium, hyaline cartilage-like tissue forms after 21 days, demonstrated by highly sulfated glycosaminoglycans and collagen type II. Matrix deposition is delayed at low encapsulation density, corroborating with lower transcript levels of COL2A1. An ectopic assay in nude mice demonstrates further maturation of the matrix deposited in vitro. Direct ectopic implantation of iPSC-derived chondrocyte-laden GelMA, without in vitro priming, also generates hyaline cartilage-like tissue, albeit less mature. Since it is unclear what maturity upon implantation is desired for joint surface regeneration, this is an attractive technology to generate immature and more mature hyaline cartilage-like tissue.