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.

GelMA/PEGDA microneedles patch loaded with HUVECs-derived exosomes and Tazarotene promote diabetic wound healing.

Clinical work and research on diabetic wound repair remain challenging globally. Although various conventional wound dressings have been continuously developed, the efficacy is unsatisfactory. The effect of drug delivery is limited by the depth of penetration. The sustained release of biomolecules from biological wound dressings is a promising treatment approach to wound healing. An assortment of cell-derived exosomes (exos) have been proved to be instrumental in tissue regeneration, and researchers are dedicated to developing biomolecules carriers with unique properties. Herein, we reported a methacrylate gelatin (GelMA) microneedles (MNs) patch to achieve transdermal and controlled release of exos and tazarotene. Our MNs patch comprising GelMA/PEGDA hydrogel has distinctive biological features that maintain the biological activity of exos and drugs in vitro. Additionally, its unique physical structure prevents it from being tightly attached to the skin of the wound, it promotes cell migration, angiogenesis by slowly releasing exos and tazarotene in the deep layer of the skin. The full-thickness cutaneous wound on a diabetic mouse model was carried out to demonstrate the therapeutic effects of GelMA/PEGDA@T + exos MNs patch. As a result, our GelMA/PEGDA@T + exos MNs patch presents a potentially valuable method for repairing diabetic wound in clinical applications.

A Patch of Detachable Hybrid Microneedle Depot for Localized Delivery of Mesenchymal Stem Cells in Regeneration Therapy.

Mesenchymal stem cells (MSCs) have been widely used for regenerative therapy. In most current clinical applications, MSCs are delivered by injection but face significant issues with cell viability and penetration into the target tissue due to a limited migration capacity. Some therapies have attempted to improve MSC stability by their encapsulation within biomaterials; however, these treatments still require an enormous number of cells to achieve therapeutic efficacy due to low efficiency. Additionally, while local injection allows for targeted delivery, injections with conventional syringes are highly invasive. Due to the challenges associated with stem cell delivery, a local and minimally invasive approach with high efficiency and improved cell viability is highly desired. In this study, we present a detachable hybrid microneedle depot (d-HMND) for cell delivery. Our system consists of an array of microneedles with an outer poly(lactic-co-glycolic) acid (PLGA) shell and an internal gelatin methacryloyl (GelMA)-MSC mixture (GMM). The GMM was characterized and optimized for cell viability and mechanical strength of the d-HMND required to penetrate mouse skin tissue was also determined. MSC viability and function within the d-HMND was characterized in vitro and the regenerative efficacy of the d-HMND was demonstrated in vivo using a mouse skin wound model.

Gelatin methacryloyl-based tactile sensors for medical wearables.

Gelatin methacryloyl (GelMA) is a widely used hydrogel with skin-derived gelatin acting as the main constituent. However, GelMA has not been used in the development of wearable biosensors, which are emerging devices that enable personalized healthcare monitoring. This work highlights the potential of GelMA for wearable biosensing applications by demonstrating a fully solution-processable and transparent capacitive tactile sensor with microstructured GelMA as the core dielectric layer. A robust chemical bonding and a reliable encapsulation approach are introduced to overcome detachment and water-evaporation issues in hydrogel biosensors. The resultant GelMA tactile sensor shows a high-pressure sensitivity of 0.19 kPa-1 and one order of magnitude lower limit of detection (0.1 Pa) compared to previous hydrogel pressure sensors owing to its excellent mechanical and electrical properties (dielectric constant). Furthermore, it shows durability up to 3000 test cycles because of tough chemical bonding, and long-term stability of 3 days due to the inclusion of an encapsulation layer, which prevents water evaporation (80% water content). Successful monitoring of various human physiological and motion signals demonstrates the potential of these GelMA tactile sensors for wearable biosensing applications.

Multicellular Co-Culture in Three-Dimensional Gelatin Methacryloyl Hydrogels for Liver Tissue Engineering.

Three-dimensional (3D) tissue models replicating liver architectures and functions are increasingly being needed for regenerative medicine. However, traditional studies are focused on establishing 2D environments for hepatocytes culture since it is challenging to recreate biodegradable 3D tissue-like architecture at a micro scale by using hydrogels. In this paper, we utilized a gelatin methacryloyl (GelMA) hydrogel as a matrix to construct 3D lobule-like microtissues for co-culture of hepatocytes and fibroblasts. GelMA hydrogel with high cytocompatibility and high structural fidelity was determined to fabricate hepatocytes encapsulated micromodules with central radial-type hole by photo-crosslinking through a digital micromirror device (DMD)-based microfluidic channel. The cellular micromodules were assembled through non-contact pick-up strategy relying on local fluid-based micromanipulation. Then the assembled micromodules were coated with fibroblast-laden GelMA, subsequently irradiated by ultraviolet for integration of the 3D lobule-like microtissues encapsulating multiple cell types. With long-term co-culture, the 3D lobule-like microtissues encapsulating hepatocytes and fibroblasts maintained over 90% cell viability. The liver function of albumin secretion was enhanced for the co-cultured 3D microtissues compared to the 3D microtissues encapsulating only hepatocytes. Experimental results demonstrated that 3D lobule-like microtissues fabricated by GelMA hydrogels capable of multicellular co-culture with high cell viability and liver function, which have huge potential for liver tissue engineering and regenerative medicine applications.

A Bioactive Gelatin-Methacrylate Incorporating Magnesium Phosphate Cement for Bone Regeneration.

Maintaining proper mechanical strength and tissue volume is important for bone growth at the site of a bone defect. In this study, potassium magnesium phosphate hexahydrate (KMgPO4·6H2O, MPC) was applied to gelma-methacrylate hydrogel (GelMA) to prepare GelMA/MPC composites (GMPCs). Among these, 5 GMPC showed the best performance in vivo and in vitro. These combinations significantly enhanced the mechanical strength of GelMA and regulated the degradation and absorption rate of MPC. Considerably better mechanical properties were noted in 5 GMPC compared with other concentrations. Better bioactivity and osteogenic ability were also found in 5 GMPC. Magnesium ions (Mg2+) are bioactive and proven to promote bone tissue regeneration, in which the enhancement efficiency is closely related to Mg2+ concentrations. These findings indicated that GMPCs that can release Mg2+ are effective in the treatment of bone defects and hold promise for future in vivo applications.

Assessment of Stiffness-Dependent Autophagosome Formation and Apoptosis in Embryonal Rhabdomyosarcoma Tumor Cells.

Remodeling of the extracellular matrix (ECM) eventually causes the stiffening of tumors and changes to the microenvironment. The stiffening alters the biological processes in cancer cells due to altered signaling through cell surface receptors. Autophagy, a key catabolic process in normal and cancer cells, is thought to be involved in mechano-transduction and the level of autophagy is probably stiffness-dependent. Here, we provide a methodology to study the effect of matrix stiffness on autophagy in embryonal rhabdomyosarcoma cells. To mimic stiffness, we seeded cells on GelMA hydrogel matrices with defined stiffness and evaluated autophagy-related endpoints. We also evaluated autophagy-dependent pathways, apoptosis, and cell viability. Specifically, we utilized immunocytochemistry and confocal microscopy to track autophagosome formation through LC3 lipidation. This approach suggests that the use of GelMA hydrogels with defined stiffness represents a novel method to evaluate the role of autophagy in embryonal rhabdomyosarcoma and other cancer cells.

Nanomaterials-combined methacrylated gelatin hydrogels (GelMA) for cardiac tissue constructs.

Among non-communicable diseases, cardiovascular diseases are the most prevalent, accounting for approximately 17 million deaths per year. Despite conventional treatment, cardiac tissue engineering emerges as a potential alternative for the advancement and treatment of these patients, using biomaterials to replace or repair cardiac tissues. Among these materials, gelatin in its methacrylated form (GelMA) is a biodegradable and biocompatible polymer with adjustable biophysical properties. Furthermore, gelatin has the ability to replace and perform collagen-like functions for cell development in vitro. The interest in using GelMA hydrogels combined with nanomaterials is increasingly growing to promote the responsiveness to external stimuli and improve certain properties of these hydrogels by exploring the incorporation of nanomaterials into these hydrogels to serve as electrical signaling conductive elements. This review highlights the applications of electrically conductive nanomaterials associated with GelMA hydrogels for the development of structures for cardiac tissue engineering, by focusing on studies that report the combination of GelMA with nanomaterials, such as gold and carbon derivatives (carbon nanotubes and graphene), in addition to the possibility of applying these materials in 3D tissue engineering, developing new possibilities for cardiac studies.

Netrin-1 co-cross-linked hydrogel accelerates diabetic wound healing in situ by modulating macrophage heterogeneity and promoting angiogenesis.

Diabetic wounds, characterized by prolonged inflammation and impaired vascularization, are a serious complication of diabetes. This study aimed to design a gelatin methacrylate (GelMA) hydrogel for the sustained release of netrin-1 and evaluate its potential as a scaffold to promote diabetic wound healing. The results showed that netrin-1 was highly expressed during the inflammation and proliferation phases of normal wounds, whereas it synchronously exhibited aberrantly low expression in diabetic wounds. Neutralization of netrin-1 inhibited normal wound healing, and the topical application of netrin-1 accelerated diabetic wound healing. Mechanistic studies demonstrated that netrin-1 regulated macrophage heterogeneity via the A2bR/STAT/PPARγ signaling pathway and promoted the function of endothelial cells, thus accelerating diabetic wound healing. These data suggest that netrin-1 is a potential therapeutic target for diabetic wounds.

Dandelion-derived vesicles-laden hydrogel dressings capable of neutralizing Staphylococcus aureus exotoxins for the care of invasive wounds.

Delayed wound healing caused by bacterial infection remains a major challenge in clinical treatment. Exotoxins incorporated in bacterial extracellular vesicles play a key role as the disease-causing virulence factors. Safe and specific antivirulence agents are expected to be developed as an effective anti-bacterial infection strategy, instead of single antibiotic therapy. Plant-derived extracellular vesicle-like nanoparticles have emerged as promising therapeutic agents for skin diseases, but the elucidations of specific mechanisms of action and clinical transformation still need to be advanced. Here, dandelion-derived extracellular vesicle-like nanoparticles (TH-EVNs) are isolated and exert antivirulence activity through specifically binding to Staphylococcus aureus (S. aureus) exotoxins, thereby protecting the host cell from attack. The neutralization of TH-EVNs against exotoxins has considerable binding force and stability, showing complete detoxification effect in vivo. Then gelatin methacryloyl hydrogel is developed as TH-EVNs-loaded dressing for S. aureus exotoxin-invasive wounds. Hydrogel dressings demonstrate good physical and mechanical properties, thus achieving wound retention and controlled release of TH-EVNs, in addition to promoting cell proliferation and migration. In vivo results show accelerated re-epithelialization, promotion of collagen maturity and reduction of inflammation after treatment. Collectively, the developed TH-EVNs-laden hydrogel dressings provide a potential therapeutic approach for S. aureus exotoxin- associated trauma.

Live cell pool and rare cell isolation using Enrich TROVO system.

Although several technologies have been developed to isolate cells of interest from a heterogenous sample, clogging and impaired cell viability limit such isolation. We have developed the Enrich TROVO system as a novel, nonfluidic technology to sort live cells. The TROVO system combines imaging-based cell selection and photo-crosslinking of (gelatin methacrylate) gelMA-hydrogel to capture cells. After capture, cells are released by enzymatic digestion of the hydrogel and then retrieved for downstream analysis or further cell culturing. The system can capture cells with a recovery rate of 48% while maintaining 90% viability. Moreover, TROVO can enrich rare cells 506-fold with 93% efficiency using single step isolation from a 1:104 cell mixture, and can also capture one target cell from 1 million cells, reaching an enrichment ratio of 9128. In addition, 100% purity and 49% recovery rate can be achieved by a following negative isolation process. Compared to existing technologies, the TROVO system is clog-resistant, highly biocompatible, and can process a wide range of sample sizes.

Photo-crosslinkable hydrogel incorporated with bone matrix particles for advancements in dentin tissue engineering.

The objective of this study was to create injectable photo-crosslinkable biomaterials, using gelatin methacryloyl (GelMA) hydrogel, combined with a decellularized bone matrix (BMdc) and a deproteinized (BMdp) bovine bone matrix. These were intended to serve as bioactive scaffolds for dentin regeneration. The parameters for GelMA hydrogel fabrication were initially selected, followed by the incorporation of BMdc and BMdp at a 1% (w/v) ratio. Nano-hydroxyapatite (nHA) was also included as a control. A physicochemical characterization was conducted, with FTIR analysis indicating that the mineral phase was complexed with GelMA, and BMdc was chemically bonded to the amide groups of gelatin. The porous structure was preserved post-BMdc incorporation, with bone particles incorporated alongside the pores. Conversely, the mineral phase was situated inside the pore opening, affecting the degree of porosity. The mineral phase did not modify the degradability of GelMA, even under conditions of type I collagenase-mediated enzymatic challenge, allowing hydrogel injection and increased mechanical strength. Subsequently, human dental pulp cells (HDPCs) were seeded onto the hydrogels. The cells remained viable and proliferative, irrespective of the GelMA composition. All mineral phases resulted in a significant increase in alkaline phosphatase activity and mineralized matrix deposition. However, GelMA-BMdc exhibited higher cell expression values, significantly surpassing those of all other formulations. In conclusion, our results showed that GelMA-BMdc produced a porous and stable hydrogel, capable of enhancing odontoblastic differentiation and mineral deposition when in contact with HDPCs, thereby showing potential for dentin regeneration.

GelMA hydrogel as a scaffold to enhance the survival and differentiation of human induced lateral ganglionic eminence precursor cells.

Cell reprogramming holds enormous potential to revolutionize our understanding of neurological and neurodevelopmental disorders, as well as enhance drug discovery and regenerative medicine. We have developed a direct cell reprogramming technology that allows us to generate lineage-specific neural cells. To extend our technology, we have investigated the incorporation of directly reprogrammed human lateral ganglionic eminence precursor cells (hiLGEPs) in a 3-dimensional (3D) matrix. Hydrogels are one of the most promising bio-scaffolds for 3D cell culture, providing cells with a supportive environment to adhere, proliferate, and differentiate. In particular, gelatin methacryloyl (GelMA) hydrogels have been used for a variety of 3D biomedical applications due to their biocompatibility, enzymatic cleavage, cell adhesion and tunable physical characteristics. This study therefore investigated the effect of GelMA hydrogel encapsulation on the survival and differentiation of hiLGEPs, both in vitro and following ex vivo transplantation into a quinolinic acid (QA) lesion rat organotypic slice culture model. We demonstrate, for the first time, that the encapsulation of hiLGEPs in GelMA hydrogel significantly enhances the survival and generation of DARPP32+ striatal neurons both in vitro and following ex vivo transplant. Furthermore, GelMA-encapsulated hiLGEPs were predominantly located away from the reactive astrocyte network that forms following QA lesioning, suggesting GelMA provides a protective barrier for cells in regions of inflammatory activation. Overall, these results indicate that GelMA hydrogel has the potential to act as a 3D bio-scaffold to augment the viability and differentiation of hiLGEPs for research and translation of pharmaceutical development and regenerative medicine.

Anisotropic hydrogel scaffold by flow-induced stereolithography 3D printing technique.

Essential organs, such as the heart and liver, contain a unique porous network that allows oxygen and nutrients to be exchanged, with distinct random to ordered regions displaying varying degrees of strength. A novel technique, referred to here as flow-induced lithography, was developed. This technique generates tunable anisotropic three-dimensional (3D) structures. The ink for this bioprinting technique was made of titanium dioxide nanorods (Ti) and kaolinite nanoclay (KLT) dispersed in a GelMA/PEGDA polymeric suspension. By controlling the flow rate, aligned particle microstructures were achieved in the suspensions. The application of UV light to trigger the polymerization of the photoactive prepolymer freezes the oriented particles in the polymer network. Because the viability test was successful in shearing suspensions containing cells, the flow-induced lithography technique can be used with both acellular scaffolds and cell-laden structures. Fabricated hydrogels show outstanding mechanical properties resembling human tissues, as well as significant cell viability (> 95 %) over one week. As a result of this technique and the introduction of bio-ink, a novel approach has been pioneered for developing anisotropic tissue implants utilizing low-viscosity biomaterials.

Platelet-membrane camouflaged cerium nanoparticle-embedded gelatin methacryloyl hydrogel for accelerated diabetic wound healing.

Impaired angiogenesis and excessive inflammation are major factors contributing to delayed wound healing in diabetic patients. This study presents the development of a novel multifunctional hydrogel, Pltm@CNPs/Gel, which incorporates platelet membrane camouflaged cerium nanoparticles into a gelatin methacryloyl matrix. The Pltm@CNPs/Gel nanocomposite hydrogel was characterized and tested for its effects on platelet activation, coagulation, cell viability, anti-oxidation, and anti-inflammation in vitro. Moreover, we evaluated the wound healing potential of the hydrogel in a diabetic rat model. Our findings demonstrate that the Pltm@CNPs/Gel hydrogel possesses anti-oxidative and anti-inflammatory properties. Furthermore, it accelerates diabetic wound healing by promoting neovascularization, cell proliferation, and collagen fiber organization. This study highlights the potential of the Pltm@CNPs/Gel hydrogel as a therapeutic option for diabetic wound healing and its promising applications as a diabetic wound dressing candidate.

Tomato based gelatin methacryloyl hydrogel as an effective natural and low-cost scaffold for accelerative wound healing.

Oxidative stress and infection are the main reasons for postponement of wound healing rate. They can potentially lead to serious inflammation and eventually lead to a longer and more painful recovery phase. Although wound dressings based on synthetic materials with antioxidative property have been proved to exhibit remarkable effect in controlling ROS level and improving wound healing, issues, such as high cost in raw materials, complicated procedures, usage of various toxic additives, and potential allergies, have significantly confined further clinical applications. In this study, a novel type of tissue engineering scaffold, based on tomatoes (Solanum lycopersicon) and gelatin methacryloyl (GelMA), was prepared via facile lyophilization and photo cross-link method (SL/GelMA). By taking advantages of various antioxidative components, such as carotenoids, flavonoids, phenolic acids, vitamin E, and vitamin C in tomatoes, SL/GelMA can effectively regulate ROS level, relieve the oxidative stress in wound bed, promote cell migration and angiogenesis, contribute to collagen deposition, and thus accelerate the rate of wound enclosure. Along with its high biocompatibility and low allergic potential, we believe that the food-derived wound dressing with facile preparation method, easy accessibility, and high cost-effectiveness can be translated for clinical treatments of various chronic wounds.

Gelatin methacryloyl (GelMA) loaded with concentrated hypoxic pretreated adipose-derived mesenchymal stem cells(ADSCs) conditioned medium promotes wound healing and vascular regeneration in aged skin.

BACKGROUND: Aging skin is characterized by a disturbed structure and lack of blood supply, which makes it difficult to heal once injured. ADSCs secrete large amounts of cytokines, which promote wound healing and vascular regeneration through paracrine secretion, and the number of cytokines can be elevated by hypoxic pretreating. However, the components of ADSCs are difficult to retain in wounds. Gelatin methacrylate (GelMA) is a photopolymerizable hydrogel synthesized from gelatin and has recently emerged as a potentially attractive material for tissue engineering applications. GelMA loaded with concentrated hypoxic pretreated ADSCs conditioned medium could provide a new method of treating wounds in aged skin. METHODS: Primary ADSCs were isolated from human adipose tissue and characterized by flow cytometry and differentiation test. ADSCs in passages 4-6 were pretreated in the hypoxic and normoxic environments to collect conditioned medium, the conditioned medium was then concentrated to prepare concentrated ADSCs conditioned medium(cADSC-CM)(the one collected from ADSCs under hypoxia was called hypo-CM ,and the one from normoxia was called nor-CM). The concentration of cytokines was detected. After treated with cADSC-CM, the abilities of proliferation, migration, and tube formation of human umbilical vascular endothelial cells (HUVECs) were assayed, and Akt/mTOR and MAPK signal pathway was detected using western blotting. GelMA+hypo-CM hydrogel was prepared, and a comprehensive evaluation of morphology, protein release efficiency, degradation rate, mechanical properties, and rheology properties were performed. Full-thickness skin wounds were created on the backs of 20-month-old mice. After surgery, GelMA, GelMA+F12, GelMA+hypo-CM, and GelMA+nor-CM were applied to the wound surface respectively. H&E, Masson, and immunohistochemistry staining were performed, and a laser Doppler perfusion imager was used to evaluate the blood perfusion. The student's t-test was used for analysis between two groups and a one-way analysis of variance (ANOVA) was used for analysis among multi groups. RESULTS: Our results revealed that 1) wounds in aged skin healed more slowly than that in young skin and exhibited poorer perfusion; 2) hypoxic pretreated ADSCs secreted more cytokines including VEGF by activating HIF1α; 3) hypo-CM promoted proliferation and migration of HUVECs through VEGF/Akt/mTOR and MAPK signal pathway; 4) GelMA-hypoCM accelerated wound healing and angiogenesis in aged skin in vivo. CONCLUSION: GelMA loaded with concentrated hypoxic pretreated adipose-derived mesenchymal stem cells conditioned medium could accelerate wound healing in aged skin by promoting angiogenesis.

3D Bioprinting of an Endothelialized Liver Lobule-like Construct as a Tumor-Scale Drug Screening Platform.

3D cell culture models replicating the complexity of cell-cell interactions and biomimetic extracellular matrix (ECM) are novel approaches for studying liver cancer, including in vitro drug screening or disease mechanism investigation. Although there have been advancements in the production of 3D liver cancer models to serve as drug screening platforms, recreating the structural architecture and tumor-scale microenvironment of native liver tumors remains a challenge. Here, using the dot extrusion printing (DEP) technology reported in our previous work, we fabricated an endothelialized liver lobule-like construct by printing hepatocyte-laden methacryloyl gelatin (GelMA) hydrogel microbeads and HUVEC-laden gelatin microbeads. DEP technology enables hydrogel microbeads to be produced with precise positioning and adjustable scale, facilitating the construction of liver lobule-like structures. The vascular network was achieved by sacrificing the gelatin microbeads at 37 °C to allow HUVEC proliferation on the surface of the hepatocyte layer. Finally, we used the endothelialized liver lobule-like constructs for anti-cancer drug (Sorafenib) screening, and stronger drug resistance results were obtained when compared to either mono-cultured constructs or hepatocyte spheroids alone. The 3D liver cancer models presented here successfully recreate liver lobule-like morphology, and may have the potential to serve as a liver tumor-scale drug screening platform.

Electrospun PCL Nerve Conduit Filled with GelMA Gel for CNTF and IGF-1 Delivery in Promoting Sciatic Nerve Regeneration in Rat.

Neural tissue engineering is an essential strategy to repair long-segment peripheral nerve defects. Modification of the nerve conduit is an effective way to improve the local microenvironment of the injury site and facilitate nerve regeneration. However, the concurrent release of multiple growth cues that regulate the activity of Schwann cells and neurons remains a challenge. The present study involved the fabrication of a composite hydrogel, specifically methacrylate-anhydride gelatin-ciliary neurotrophic factor/insulin-like growth factor-1 (GelMA-CNTF/IGF-1), with the aim of providing a sustained release of CNTF and IGF-1. The GelMA-CNTF/IGF-1 hydrogels exhibited a swelling rate of 10.2% following a 24 h incubation in vitro. In vitro, GelMA hydrogels demonstrated a high degree of efficiency in the sustained release of CNTF and IGF-1 proteins, with a release rate of 85.9% for CNTF and 90.9% for IGF-1 shown at day 28. In addition, the GelMA-CNTF/IGF-1 composite hydrogel promoted the proliferation of Schwann cells and the production of nerve growth factor (NGF), connective tissue growth factor (CTGF), fibronectin, and laminin and also considerably promoted the axonal growth of neurons. Furthermore, GelMA-CNTF/IGF-1 hydrogels were loaded into PCL electrospun nerve conduits to repair 15 mm sciatic nerve defects in rats. In vivo studies indicated that PCL-GelMA-CNTF/IGF-1 could efficiently accelerate the regeneration of the rat sciatic nerve, promote the formation of the myelin sheath of new axons, promote the electrophysiological function of regenerated nerves, and eventually improve the recovery of motor function in rats. Overall, the PCL-GelMA-CNTF/IGF-1 scaffold presents an attractive new approach for generating an optimal therapeutic alternative for peripheral nerve restoration.

Novel 3D-printing bilayer GelMA-based hydrogel containing BP,ß-TCP and exosomes for cartilage-bone integrated repair.

The integrated repair of cartilage and bone involves the migration and differentiation of cells, which has always been a difficult problem to be solved. We utilize the natural biomaterial gelatin to construct gelatin methacryloyl (GelMA), a hydrogel scaffold with high cell affinity. GelMA is mixed with different components to print a bi-layer porous hydrogel scaffold with different modulus and composition in upper and lower layers through three-dimensional (3D) printing technology. The upper scaffold adds black phosphorus (BP) and human umbilical cord mesenchymal stem cells (hUMSCs) exosomes (exos) in GelMA, which has a relatively lower elastic modulus and is conducive to the differentiation of BMSCs into cartilage. In the lower scaffold, in addition to BP and hUMSCs exos,ß-tricalcium phosphate (ß-TCP), which has osteoconductive and osteoinductive effects, is added to GelMA. The addition ofß-TCP significantly enhances the elastic modulus of the hydrogel scaffold, which is conducive to the osteogenic differentiation of bone marrow mesenchymal stem cells(BMSCs).In vitroexperiments have confirmed that the bi-layer scaffolds can promote osteogenesis and chondrogenic differentiation respectively. And in the rabbit cartilage-bone injury model, MRI and micro-CT results show that the 3D printed bi-layer GelMA composite scaffold has a repair effect close to normal tissue.