Gelatin Methacrylamide Hydrogel Research Articles

Osteosarocma progression in biomimetic matrix with different stiffness: Insights from a three-dimensional printed gelatin methacrylamide hydrogel

Recent studies on osteosarcoma and matrix stiffness are still mostly performed in a 2D setting, which is distinct from in vivo conditions. Therefore, the results from the 2D models may not reflect the real effect of matrix stiffness on cell phenotype. Here, we employed a 3D bioprinted osteosarcoma model, to study the effect of matrix stiffness on osteosarcoma cells. Through density adjustment of GelMA, we constructed three osteosarcoma models with distinct matrix stiffnesses of 50, 80, and 130 kPa. In this study, we found that osteosarcoma cells proliferated faster, migrated more actively, had a more stretched morphology, and a lower drug sensitivity in a softer 3D matrix. When placed in a stiffer matrix, osteosarcoma cells secrete more MMP and VEGF, potentially to fight for survival and attract vascular invasion. Transcriptomic analysis showed that matrix stiffness could impact the signaling pathway of integrin α5-MAPK. The transplantation of 3D printed models in nude mice showed that cells encapsulated in the softer hydrogel were more likely to form subcutaneous tumors. These results suggest that matrix stiffness plays an important role in the development of osteosarcoma in a 3D environment and that inhibition of integrin α5 could block the signal transduction of matrix stiffness.

High-throughput fabrication of cell spheroids with 3D acoustic assembly devices.

Acoustic cell assembly devices are applied in cell spheroid fabrication attributed to their rapid, label-free and low-cell damage production of size-uniform spheroids. However, the spheroids yield and production efficiency are still insufficient to meet the requirements of several biomedical applications, especially those that require large quantities of cell spheroids, such as high-throughput screening, macro-scale tissue fabrication, and tissue repair. Here, we developed a novel 3D acoustic cell assembly device combined with a gelatin methacrylamide (GelMA) hydrogels for the high-throughput fabrication of cell spheroids. The acoustic device employs three orthogonal piezoelectric transducers that can generate three orthogonal standing bulk acoustic waves to create a 3D dot-array (25 × 25 × 22) of levitated acoustic nodes, enabling large-scale fabrication of cell aggregates (>13,000 per operation). The GelMA hydrogel serves as a supporting scaffold to preserve the structure of cell aggregates after the withdrawal of acoustic fields. As a result, mostly cell aggregates (>90%) mature into spheroids maintaining good cell viability. We further applied these acoustically assembled spheroids to drug testing to explore their potency in drug response. In conclusion, this 3D acoustic cell assembly device may pave the way for the scale-up fabrication of cell spheroids or even organoids, to enable flexible application in various biomedical applications, such as high-throughput screening, disease modeling, tissue engineering, and regenerative medicine.

Implantation of hydrogel-liposome nanoplatform inhibits glioblastoma relapse by inducing ferroptosis

Glioblastoma is acknowledged as the most aggressive cerebral tumor in adults. However, the efficacy of current standard therapy is seriously undermined by drug resistance and suppressive immune microenvironment. Ferroptosis is a recently discovered form of iron-dependent cell death that may have excellent prospect as chemosensitizer. The utilization of ferropotosis inducer Erastin could significantly mediate chemotherapy sensitization of Temozolomide and exert anti-tumor effects in glioblastoma. In this study, a combination of hydrogel-liposome nanoplatform encapsulated with Temozolomide and ferroptosis inducer Erastin was constructed. The αvβ3 integrin-binding peptide cyclic RGD was utilized to modify codelivery system to achieve glioblastoma targeting strategy. As biocompatible drug reservoirs, cross-linked GelMA (gelatin methacrylamide) hydrogel and cRGD-coated liposome realized the sustained release of internal contents. In the modified intracranial tumor resection model, GelMA-liposome system achieved slow release of Temozolomide and Erastin in situ for more than 14 d. The results indicated that nanoplatform (T+E@LPs-cRGD+GelMA) improved glioblastoma sensitivity to chemotherapeutic temozolomide and exerted satisfactory anti-tumor effects. It was demonstrated that the induction of ferroptosis could be utilized as a therapeutic strategy to overcome drug resistance. Furthermore, transcriptome sequencing was conducted to reveal the underlying mechanism that the nanoplatform (T+E@LPs-cRGD+GelMA) implicated in. It is suggested that GelMA-liposome system participated in the immune response and immunomodulation of glioblastoma via interferon/PD-L1 pathway. Collectively, this study proposed a potential combinatory therapeutic strategy for glioblastoma treatment.

GelMA and Biomimetic Culture Allow the Engineering of Mineralized, Adipose, and Tumor Tissue Human Microenvironments for the Study of Advanced Prostate Cancer In Vitro and In Vivo.

Increasing evidence shows bone marrow (BM)-adipocytes as a potentially important contributor in prostate cancer (PCa) bone metastases. However, a lack of relevant models has prevented the full understandingof the effects of human BM-adipocytes in this microenvironment. It is hypothesized that the combination of tunable gelatin methacrylamide (GelMA)-based hydrogels with the biomimetic culture of human cells would offer a versatile 3D platform to engineer human bone tumor microenvironments containing BM-adipocytes. Human osteoprogenitors, adipocytes, and PCa cells are individually cultured in vitro in GelMA hydrogels, leading to mineralized, adipose, and PCa tumor 3D microtissues, respectively. Osteoblast mineralization and tumor spheroid formation are tailored by hydrogel stiffness with lower stiffnesses correlating with increased mineralization and tumor spheroid size. Upon coculture with tumor cells, BM-adipocytes undergo morphological changes and delipidation, suggesting reciprocal interactions between the cell types. When brought in vivo, the mineralized and adipose microtissues successfully form a humanized fatty bone microenvironment, presenting, for the first time, with human adipocytes. Using this model, an increase in tumor burden is observed when human adipocytes are present, suggesting that adipocytes support early bone tumor growth. The advanced platform presented here combines natural aspects of the microenvironment with tunable properties useful for bone tumor research.

A conductive gelatin methacrylamide hydrogel for synergistic therapy of osteosarcoma and potential bone regeneration

In this study, a methacrylic gelatin/oxidized dextran/montmorillonite‑strontium/polypyrrole (GOMP) hydrogel was prepared. The GOMP hydrogel had dual network structure which was formed through photoinitiator-initiated double bond polymerization and Schiff base reaction. The network structure led to a sustained release of the antitumor drug, doxorubicin (DOX). Polypyrrole introduced the conductivity and high photothermal conversion capacity to the GOMP hydrogel, which showed a photothermal conversion efficiency of 31.61 % under 808 nm laser radiation. The GOMP hydrogel had good swelling properties in solvents. Further study showed that the GOMP hydrogel had good biocompatibility and excellent biodegradability in vitro and in vivo. The experiments of in vitro tumor therapy and in vivo anti-tumor recurrence indicated that the DOX-loaded GOMP hydrogel had synergistic effects on tumor cell apoptosis based on chemotherapy and photothermal therapy. In addition, montmorillonite‑strontium (MMT-Sr) doped in the hydrogel not only improved the mechanical properties of the hydrogel but also promoted potential bone regeneration. The multifunctional DOX-loaded GOMP hydrogel with bone regeneration, photothermal therapy, and chemotherapy functions has great potential application for treating osteosarcoma.

DLP fabricating of precision GelMA/HAp porous composite scaffold for bone tissue engineering application

The hydrogel composited hydroxyapatite (HAp) scaffold shown biomimetic extracellular matrix and osteogenic activity, which is a promising bone regeneration biomaterial. 3D printing has been widely used in fabrication of customized and complex bone regeneration scaffold. However, it is still a challenge to prepare high-precision HAp reinforced composites by digital light processing (DLP) printing that can effectively promote bone regeneration. In this study, shear-thinning gelatin methacrylamide (GelMA)/hydroxyapatite (HAp) composite inks were prepared to fabricate high-precision scaffolds by DLP printing for bone repair. Firstly, GelMA hydrogels with adjustable mechanical, rheological and printing properties were synthesized to screen hydrogel concentration and degree of substitution. Then carefully selected biocompatible dispersants to reduce the agglomeration of hydroxyapatite. The actual printing resolution of the GelMA/HAp porous composite scaffolds with excellent mechanical strength through DLP 3D printing was up to about 100 μm. In vitro, the 3D printed composite scaffold significantly promoted the adhesion and proliferation of osteoblasts, as well as promoted osteogenic differentiation. In vivo, the GelMA/HAp porous composite scaffolds showed excellent ability in accelerating the formation of new bone in rabbit skull defects. The GelMA/HAp porous composite scaffolds fabricated by the composite inks with good printability, mechanical properties and osteogenic activity have fascinating potential in bone repair application.

Bioinspired Multifunctional Black Phosphorus Hydrogel with Antibacterial and Antioxidant Properties: A Stepwise Countermeasure for Diabetic Skin Wound Healing.

Cutaneous wound healing, especially diabetic wound healing, is a common clinical challenge. Reactive oxygen species (ROS) and bacterial infection are two major detrimental states that induce oxidative stress and inflammatory responses and impede angiogenesis and wound healing. A derivative of the metabolite itaconate, 4-octyl itaconate (4OI) has attracted increasing research interest in recent years due to its antioxidant and anti-inflammatory properties. In this study, 4OI-modified black phosphorus (BP) nanosheets are incorporated into a photosensitive, multifunctional gelatin methacrylamide hydrogel to produce a new photothermal therapy (PTT) and photodynamic therapy (PDT) system with antibacterial and antioxidant properties for diabetic wound regeneration. Under laser irradiation, the 4OI-BP-entrapped hydrogel enables rapid gelation, forming a membrane on wounds, and offers high PTT and PDT efficacy to eliminate bacterial infection. Without laser irradiation, BP acts as a carrier and controls the release of 4OI, with which it synergistically enhances antioxidant activity, thus alleviating excessive ROS damage to endothelial cells, promoting neovascularization, and facilitating faster diabetic wound closure. These findings indicate that 4OI-BP-entrapped multifunctional hydrogel provides a stepwise countermeasure with antibacterial and antioxidant properties for enhanced diabetic wound healing and may lead to novel therapeutic interventions for diabetic ulcers.

Surface charge-dependent osteogenic behaviors of edge-functionalized graphene quantum dots

Understanding the relationship between stem cell’ osteogenic capacity and physicochemical properties of nanomaterials is essential for designing new tissue engineering materials capable of regulating stem cell fate. It has reported that the surface potential of nanomaterials affects cell behavior, however, the underlying mechanism is still controversial and surface charge was not the only variable in the studies performed. In this work, graphene quantum dots (GQDs) were used as a simplified model for single-factor study for the effect of surface charge on osteogenic differentiation, by changing the surface charge of GQD only while keeping all other variables constant. Notably, it was found that the positively charged GQD+ exerted no impact on osteogenic differentiation, while the negatively charged GQD‾ could significantly enhance osteogenic differentiation of human mesenchymal stem cells (hMSCs) by activating the BMP/Smad signaling pathway. The negatively charged GQD‾ was then encapsulated in gelatin methacrylamide (GelMA) hydrogel via a UV light-activated photopolymerization in the presence of photoinitiator, obtaining an 3D injectable GQD‾/GelMA hydrogel scaffold. A significantly accelerated bone regeneration induced by GQD‾/GelMA hydrogel scaffolds was observed in the mouse calvarial defect model. The systematic study of the effects of surface charge on the stem cell behavior could provide much inspiration for the tissue engineering application of nanomaterials.

Bioinspired stratified electrowritten fiber-reinforced hydrogel constructs with layer-specific induction capacity for functional osteochondral regeneration

Despite significant advances in osteochondral tissue engineering, it remains challenging to successfully reconstruct native-like complex tissues organized in three-dimension with spatially varying compositional, structural and functional properties. In this contribution, inspired by the gradients in extracellular matrix (ECM) composition and collagen fiber architecture in native osteochondral tissue, we designed and fabricated a tri-layered (superficial cartilage (S), deep cartilage (D) and subchondral bone (B) layer) stratified scaffold in which a mesenchymal stem cell (MSC)-laden gelatin methacrylamide (GelMA) hydrogel with zone-specific growth factor delivery was combined with melt electrowritten triblock polymer of poly(ε-caprolactone) and poly(ethylene glycol) (PCEC) networks with depth-dependent fiber organization. Introducing PCEC fibers into the weak GelMA hydrogel contributed to a significant increase in mechanical strength. In vitro biological experiments indicated that the stratified fiber-reinforced and growth factor-loaded hydrogel construct induced the MSCs to differentiate down both the chondrogenic and osteogenic lineages and that the engineered complex exhibited cellular phenotype and matrix accumulation profiles resembling those of the native tissue. Simultaneous cartilage and subchondral bone regeneration were achieved in vivo by using the tri-layered integrated scaffold. More importantly, the inclusion of the S layer could impart the regenerated cartilage with a more lubricating and wear-resistant surface. These findings suggest that the bioinspired construct mimicking the spatial variations of native osteochondral tissue might serve as a promising candidate to enhance osteochondral regeneration.

Bi-layered micro-fibre reinforced hydrogels for articular cartilage regeneration

Articular cartilage has limited capacity for regeneration and when damaged cannot be repaired with currently available metallic or synthetic implants. We aim to bioengineer a microfibre-reinforced hydrogel that can capture the zonal depth-dependent mechanical properties of native cartilage, and simultaneously support neo-cartilage formation. With this goal, a sophisticated bi-layered microfibre architecture, combining a densely distributed crossed fibre mat (superficial tangential zone, STZ) and a uniform box structure (middle and deep zone, MDZ), was successfully manufactured via melt electrospinning and combined with a gelatin–methacrylamide hydrogel. The inclusion of a thin STZ layer greatly increased the composite construct’s peak modulus under both incongruent (3.2-fold) and congruent (2.1-fold) loading, as compared to hydrogels reinforced with only a uniform MDZ structure. Notably, the stress relaxation response of the bi-layered composite construct was comparable to the tested native cartilage tissue. Furthermore, similar production of sulphated glycosaminoglycans and collagen II was observed for the novel composite constructs cultured under mechanical conditioning w/o TGF-ß1 supplementation and in static conditions w/TGF-ß1 supplementation, which confirmed the capability of the novel composite construct to support neo-cartilage formation upon mechanical stimulation. To conclude, these results are an important step towards the design and manufacture of biomechanically competent implants for cartilage regeneration. Statement of SignificanceDamage to articular cartilage results in severe pain and joint disfunction that cannot be treated with currently available implants. This study presents a sophisticated bioengineered bi-layered fibre reinforced cell-laden hydrogel that can approximate the functional mechanical properties of native cartilage. For the first time, the importance of incorporating a viable superficial tangential zone (STZ) – like structure to improve the load-bearing properties of bioengineered constructs, particularly when in-congruent surfaces are compressed, is demonstrated. The present work also provides new insights for the development of implants that are able to promote and guide new cartilaginous tissue formation upon physiologically relevant mechanical stimulation.

Controlling Adult Stem Cell Behavior Using Nanodiamond-Reinforced Hydrogel: Implication in Bone Regeneration Therapy

Nanodiamonds (NDs) have attracted considerable attention as drug delivery nanocarriers due to their low cytotoxicity and facile surface functionalization. Given these features, NDs have been recently investigated for the fabrication of nanocomposite hydrogels for tissue engineering. Here we report the synthesis of a hydrogel using photocrosslinkable gelatin methacrylamide (GelMA) and NDs as a three-dimensional scaffold for drug delivery and stem cell-guided bone regeneration. We investigated the effect of different concentration of NDs on the physical and mechanical properties of the GelMA hydrogel network. The inclusion of NDs increased the network stiffness, which in turn augmented the traction forces generated by human adipose stem cells (hASCs). We also tested the ability of NDs to adsorb and modulate the release of a model drug dexamethasone (Dex) to promote the osteogenic differentiation of hASCs. The ND-Dex complexes modulated gene expression, cell area, and focal adhesion number in hASCs. Moreover, the integration of the ND-Dex complex within GelMA hydrogels allowed a higher retention of Dex over time, resulting in significantly increased alkaline phosphatase activity and calcium deposition of encapsulated hASCs. These results suggest that conventional GelMA hydrogels can be coupled with conjugated NDs to develop a novel platform for bone tissue engineering.

Gelatin methacrylamide hydrogel with graphene nanoplatelets for neural cell-laden 3D bioprinting.

Nervous system is extremely complex which leads to rare regrowth of nerves once injury or disease occurs. Advanced 3D bioprinting strategy, which could simultaneously deposit biocompatible materials, cells and supporting components in a layer-by-layer manner, may be a promising solution to address neural damages. Here we presented a printable nano-bioink composed of gelatin methacrylamide (GelMA), neural stem cells, and bioactive graphene nanoplatelets to target nerve tissue regeneration in the assist of stereolithography based 3D bioprinting technique. We found the resultant GelMA hydrogel has a higher compressive modulus with an increase of GelMA concentration. The porous GelMA hydrogel can provide a biocompatible microenvironment for the survival and growth of neural stem cells. The cells encapsulated in the hydrogel presented good cell viability at the low GelMA concentration. Printed neural construct exhibited well-defined architecture and homogenous cell distribution. In addition, neural stem cells showed neuron differentiation and neurites elongation within the printed construct after two weeks of culture. These findings indicate the 3D bioprinted neural construct has great potential for neural tissue regeneration.

Gelatin-methacrylamide gel loaded with microspheres to deliver GDNF in bilayer collagen conduit promoting sciatic nerve growth.

In this study, we fabricated glial cell-line derived neurotrophic factor (GDNF)-loaded microspheres, then seeded the microspheres in gelatin-methacrylamide hydrogel, which was finally integrated with the commercial bilayer collagen membrane (Bio-Gide®). The novel composite of nerve conduit was employed to bridge a 10 mm long sciatic nerve defect in a rat. GDNF-loaded gelatin microspheres had a smooth surface with an average diameter of 3.9±1.8 μm. Scanning electron microscopy showed that microspheres were uniformly distributed in both the GelMA gel and the layered structure. Using enzyme-linked immunosorbent assay, in vitro release studies (pH 7.4) of GDNF from microspheres exhibited an initial burst release during the first 3 days (18.0%±1.3%), and then, a prolonged-release profile extended to 32 days. However, in an acidic condition (pH 2.5), the initial release percentage of GDNF was up to 91.2%±0.9% within 4 hours and the cumulative release percentage of GDNF was 99.2%±0.2% at 48 hours. Then the composite conduct was implanted in a 10 mm critical defect gap of sciatic nerve in a rat. We found that the nerve was regenerated in both conduit and autograft (AG) groups. A combination of electrophysiological assessment and histomorphometry analysis of regenerated nerves showed that axonal regeneration and functional recovery in collagen tube filled with GDNF-loaded microspheres (GM + CT) group were similar to AG group (P>0.05). Most myelinated nerves were matured and arranged densely with a uniform structure of myelin in a neat pattern along the long axis in the AG and GM + CT groups, however, regenerated nerve was absent in the BLANK group, left the 10 mm gap empty after resection, and the nerve fiber exhibited a disordered arrangement in the collagen tube group. These results indicated that the hybrid system of bilayer collagen conduit and GDNF-loaded gelatin microspheres combined with gelatin-methacrylamide hydrogels could serve as a new biodegradable artificial nerve guide for nerve tissue engineering.

Biomimetic gelatin methacrylamide hydrogel scaffolds for bone tissue engineering.

Bone tissue engineering is an exciting research area that develops functional strategies for the most challenging bone related clinical issues. Among the numerous materials used in this area, biomimetic materials have been developed amazingly over the past decades. In this study, biomimetic gelatin methacrylamide (Bio-GelMA) hydrogel scaffolds have been fabricated to mimic both the physical architecture and chemical composition of the natural bone extracellular matrix (ECM) by using the thermally induced phase separation (TIPS) technique, to provide three-dimensional templates and extracellular matrix microenvironments. Adipose derived stem cells (ADSCs) also play a pivotal role in osteogenesis when co-cultured with Bio-GelMA at days 7, 14, and 21. The effects of cell-biomaterial interactions such as adhesion, proliferation, and osteogenic differentiation were systematically investigated. The results showed that Bio-GelMA significantly enhanced cell attachment and viability, alkaline phosphatase (ALP) activity, and mineral deposition, as well as mRNA expression levels of osteogenic genes of ADSCs. In a subcutaneous model, H&E staining and dual immunofluorescent staining differed significantly. More importantly, in a critical-size rat calvarial bone defect model, the results of Micro-CT, H&E staining and Masson trichrome staining confirmed that the combination of the Bio-GelMA and ADSCs could enhance osteogenesis significantly. Altogether, the observations prove that the Bio-GelMA scaffolds can act as cell carriers for ADSCs, promote greater osteogenic differentiation of ADSCs and may have great potential in future clinical applications.

Photocrosslinkable Gelatin Hydrogel for Epidermal Tissue Engineering.

Natural hydrogels are promising scaffolds to engineer epidermis. Currently, natural hydrogels used to support epidermal regeneration are mainly collagen- or gelatin-based, which mimic the natural dermal extracellular matrix but often suffer from insufficient and uncontrollable mechanical and degradation properties. In this study, a photocrosslinkable gelatin (i.e., gelatin methacrylamide (GelMA)) with tunable mechanical, degradation, and biological properties is used to engineer the epidermis for skin tissue engineering applications. The results reveal that the mechanical and degradation properties of the developed hydrogels can be readily modified by varying the hydrogel concentration, with elastic and compressive moduli tuned from a few kPa to a few hundred kPa, and the degradation times varied from a few days to several months. Additionally, hydrogels of all concentrations displayed excellent cell viability (>90%) with increasing cell adhesion and proliferation corresponding to increases in hydrogel concentrations. Furthermore, the hydrogels are found to support keratinocyte growth, differentiation, and stratification into a reconstructed multilayered epidermis with adequate barrier functions. The robust and tunable properties of GelMA hydrogels suggest that the keratinocyte laden hydrogels can be used as epidermal substitutes, wound dressings, or substrates to construct various in vitro skin models.

Endochondral bone formation in gelatin methacrylamide hydrogel with embedded cartilage-derived matrix particles

The natural process of endochondral bone formation in the growing skeletal system is increasingly inspiring the field of bone tissue engineering. However, in order to create relevant-size bone grafts, a cell carrier is required that ensures a high diffusion rate and facilitates matrix formation, balanced by its degradation. Therefore, we set out to engineer endochondral bone in gelatin methacrylamide (GelMA) hydrogels with embedded multipotent stromal cells (MSCs) and cartilage-derived matrix (CDM) particles. CDM particles were found to stimulate the formation of a cartilage template by MSCs in the GelMA hydrogel in vitro. In a subcutaneous rat model, this template was subsequently remodeled into mineralized bone tissue, including bone-marrow cavities. The GelMA was almost fully degraded during this process. There was no significant difference in the degree of calcification in GelMA with or without CDM particles: 42.5 ± 2.5% vs. 39.5 ± 8.3% (mean ± standard deviation), respectively. Interestingly, in an osteochondral setting, the presence of chondrocytes in one half of the constructs fully impeded bone formation in the other half by MSCs. This work offers a new avenue for the engineering of relevant-size bone grafts, by the formation of endochondral bone within a degradable hydrogel.

Gelatin hydrogels formed by orthogonal thiol-norbornene photochemistry for cell encapsulation.

Covalently cross-linked gelatin hydrogels have received considerable attention in biomedical fields due to the inherent bioactivity of gelatin and the stability of covalent bonds linking the gelatin chains. Derivatives of gelatin, such as gelatin-methacrylamide (GelMA), can be cross-linked into covalent hydrogels through radical-mediated chain-growth photopolymerization. However, accumulating evidence suggests that chain-growth polymerized hydrogels may not be ideal for the encapsulation of cells and proteins prone to radical-mediated damage. The formation of heterogeneous kinetic chains following chain-growth polymerization of (meth)acrylates or (meth)acrylamides may also hinder molecular transport or alter cell-cell/cell-material interactions. This study presents a new synthesis route for preparing norbornene-functionalized gelatin (GelNB) that could be used to form orthogonally cross-linked gelatin-based hydrogels via a thiol-ene photo-click reaction. GelNB was synthesized through reacting gelatin with carbic anhydride in aqueous buffered solution, and the degree of norbornene substitution was controlled by adjusting the reaction time and the solution pH value. GelNB hydrogels were prepared by step-growth thiol-ene photopolymerization using multifunctional thiols as gel cross-linkers and the degree of GelNB hydrogel cross-linking was tuned by adjusting the thiol concentration, GelNB content, or cross-linker functionality. The cytocompatibility of orthogonally cross-linked GelNB hydrogels were demonstrated by in situ photo-encapsulation of human mesenchymal stem cells (hMSCs). When compared with the chain-growth GelMA hydrogels, the orthogonally cross-linked GelNB hydrogel promoted a faster and higher degree of cell spreading.