Gelatin-hydroxyphenylpropionic Acid Hydrogel Research Articles

Injectable gelatin hydroxyphenyl propionic acid hydrogel protects human retinal progenitor cells (hRPCs) from shear stress applied during small-bore needle injection

Many progenitor and stem cell-based therapies in pre-clinical development and in clinical trial have failed to achieve desired outcomes, in part, due to: low cell viability; dispersion of cells from the injection site; and/or failure of the cells to differentiate and engraft into host tissue. These problems may be exacerbated by the elevated shear stress applied to the cells as they pass through a small-bore needle. Our supposition is that these issues can be mitigated by replacing the phosphate buffered saline (PBS), which is likely employed as the cell carrier, with an injectable gelatin-based hydrogel incorporating an appropriate regulatory molecule (human epidermal growth factor). The first objectives of the study were to evaluate in vitro: the effects of injection through a 31-gauge needle on viability, proliferation, and phenotypic expression of human retinal progenitor cells (hRPCs), when employing PBS as the carrier and compare it to a gelatin-based gel as a carrier. The second objective of the work, conducted in vivo, in a rat model, was to: determine the degree of engraftment of injected cells; and evaluate markers of an immune response to the xenogeneic cells.Hydroxyphenyl propionic acid was conjugated to gelatin (Gtn-HPA) to enable in situ, enzyme-mediated covalent cross-linking, with independent control over the gelation time and degree of cross-linking. After 1 and 6 culture days, hRPC exposed to shear stress showed 50% or more reduction in viability of cells in PBS compared to cells in medium and in gel. Shear stress decreased proliferation and increased apoptosis in the PBS group by 50% or more, while cells in the gel group were protected from shear-induced apoptosis and their proliferation remained 2-fold higher than the PBS group. Also, of note was the preservation of or increase in stemness markers in the gel with hEGF group compared to cells in PBS and the gel alone, with or without shear stress. In vivo studies, performed 3 days after subretinal injection into non-immunosuppressed rats revealed: a) a greater number of cells at the injection site in the gel group, with signs of engraftment of cells to the retina; and b) a significantly lower immune response in the gel group, compared to cells injected in PBS.This work demonstrates that the shear stress experienced by hRPCs in PBS as a result of injection through a small-bore needle adversely affects cell viability, proliferation, apoptosis, phenotype, in vivo retention of cells at the injection site, migration, engraftment and immune reaction. These findings suggest the use of a biomaterial hydrogel as a replacement for PBS for retinal cell therapy.

Fast three-dimensional micropatterning of PC12 cells in rapidly crosslinked hydrogel scaffolds using ultrasonic standing waves

The ability to spatially organise the microenvironment of tissue scaffolds unlocks the potential of many scaffold-based tissue engineering applications. An example application is to aid the regeneration process of peripheral nerve injuries. Herein, we present a promising approach for three-dimensional (3D) micropatterning of nerve cells in tissue scaffolds for peripheral nerve repair. In particular, we demonstrate the 3D micropatterning of PC12 cells in a gelatin-hydroxyphenylpropionic acid (Gtn-HPA) hydrogel using ultrasound standing waves (USWs). PC12 cells were first aligned in 3D along nodal planes by the USWs in Gtn-HPA hydrogel precursor solution. The precursor was then crosslinked using horseradish peroxidase (HRP) and diluted hydrogen peroxide (H2O2), thus immobilising the aligned cells within 90–120 s. This micropatterning process is cost effective and can be replicated easily without the need for complex and expensive specialised equipment. USW-aligned PC12 cells showed no adverse effect in terms of viability or ability to proliferate. To our best knowledge, this is the first report on the effect of USW alignment on neural cell differentiation. Differentiated and USW-aligned PC12 cells showed directional uniformity after 20 d, making this technique a promising alternative approach to guide the nerve regeneration process.

In Situ Cross-linking Hydrogel as a Vehicle for Retinal Progenitor Cell Transplantation

One of the current limitations of retinal transplantation of stem cells as well as other cell types is the dispersion of cells from the injection site (including loss of cells into the vitreous chamber) and low survival after transplantation. Gelatin-hydroxyphenyl propionic acid (Gtn-HPA) conjugate is a biodegradable polymer that can undergo covalent cross-linking in situ, allowing for injection of incorporated cells through a small caliber needle followed by gel formation in vivo. We tested the hypothesis that Gtn-HPA hydrogel supports survival and integration of retinal progenitor cells (RPCs) post-transplantation. In vitro compatibility and in vivo graft survival were assessed by mixing an equal volume of Gtn-HPA conjugate and RPC suspension and triggering enzyme-mediated gelation, using minute amounts of horseradish peroxidase and peroxide. Immunocytochemistry showed >80% survival of cells and minimal apoptosis for cells incorporated into Gtn-HPA, equivalent to controls grown on fibronectin-coated flasks. RPCs undergoing mitosis were seen within the three-dimensional Gtn-HPA hydrogel, but the percentage of Ki-67-positive cells was lower compared with the monolayer controls. For in vivo studies, gel–cell mixture or cell suspension in saline was trans-sclerally injected into the left eye of female Long Evans rats immunosuppressed with cyclosporine A. Grafts survived at the 1 week time point of the study, with Gtn-HPA-delivered grafts showing less inflammatory response demonstrated by anti-leukocyte staining. More eyes in the gel–cell mixture group showed surviving cells in the subretinal space compared with saline-delivered controls, while the number of cells surviving per graft was not significantly different between the two groups. This work demonstrates an injectable in situ cross-linking hydrogel as a potential vehicle for stem cell delivery in the retina.

Peroxidase-immobilized porous silica particles for in situ formation of peroxidase-free hydrogels with attenuated immune responses

Enzymatic crosslinking chemistry using horseradish peroxidase (HRP) has been widely utilized as an effective approach to fabricating injectable hydrogels with high efficiency under mild reaction conditions. However, their clinical applications are limited by the immunogenicity of the plant-derived enzyme. Herein we report the design, synthesis and characterization of HRP-immobilized porous silica particles (HRP-particles) and their use for in situ formation of HRP-free hydrogels. HRP was immobilized on aminopropyl-modified porous silica particles of 70–140 µm in diameter via poly(ethylene glycol) spacers of different molecular weights by reductive amination reaction. Two different HRP-free hydrogels based on dextran-tyramine and gelatin-hydroxyphenylpropionic acid (GHPA) conjugates were produced by passing a solution containing the conjugates and H2O2 through a syringe packed with HRP-particles. The storage modulus and gelation rate of both hydrogels were tunable by varying the contact time between the polymer solution and HRP-particles. Our in vitro study revealed that HRP-free GHPA hydrogel was less stimulatory to activated mouse macrophages than HRP-containing GHPA hydrogel with the same stiffness. Furthermore, HRP-free GHPA hydrogel exhibited remarkably lower levels of local and systemic inflammation than HRP-containing one upon subcutaneous injection in immunocompetent C57BL/6J mice. The attenuated immunogenicity of HRP-free GHPA hydrogels makes them an attractive platform for tissue engineering applications. Statement of significanceThe immunogenicity of HRP is a significant issue for clinical application of HRP-catalyzed in situ forming hydrogels. HRP-particles are developed to overcome the safety concerns by fabricating HRP-free hydrogels. The porosity of silica particles and molecular weight of poly(ethylene glycol) spacers are discovered as important factors determining the catalytic ability of HRP-particles to induce the in situ crosslinking of polymer-phenol conjugates. Although several articles speculate the potential of HRP to trigger immune responses when administered as a part of hydrogel formulation, no literature has attempted to investigate the immunogenicity of HRP-containing hydrogels in comparison with HRP-free hydrogels. Our findings suggest that the immunogenicity issue should be carefully considered before clinical translation of HRP-containing hydrogels.

Multiphoton imaging of myogenic differentiation in gelatin-based hydrogels as tissue engineering scaffolds.

BackgroundHydrogels can serve as three-dimensional (3D) scaffolds for cell culture and be readily injected into the body. Recent advances in the image technology for 3D scaffolds like hydrogels have attracted considerable attention to overcome the drawbacks of ordinary imaging technologies such as optical and fluorescence microscopy. Multiphoton microscopy (MPM) is an effective method based on the excitation of two-photons. In the present study, C2C12 myoblasts differentiated in 3D gelatin hydroxyphenylpropionic acid (GHPA) hydrogels were imaged by using a custom-built multiphoton excitation fluorescence microscopy to compare the difference in the imaging capacity between conventional microscopy and MPM.ResultsThe physicochemical properties of GHPA hydrogels were characterized by using scanning electron microscopy and Fourier-transform infrared spectroscopy. In addition, the cell viability and proliferation of C2C12 myoblasts cultured in the GHPA hydrogels were analyzed by using Live/Dead Cell and CCK-8 assays, respectively. It was found that C2C12 cells were well grown and normally proliferated in the hydrogels. Furthermore, the hydrogels were shown to be suitable to facilitate the myogenic differentiation of C2C12 cells incubated in differentiation media, which had been corroborated by MPM. It was very hard to get clear images from a fluorescence microscope.ConclusionsOur findings suggest that the gelatin-based hydrogels can be beneficially utilized as 3D scaffolds for skeletal muscle engineering and that MPM can be effectively applied to imaging technology for tissue regeneration.

Astrocyte migration in injectable gelatin-hydroxyphenyl propionic acid matrices for neuronal guidance in spinal cord injury

Event Abstract Back to Event Astrocyte migration in injectable gelatin-hydroxyphenyl propionic acid matrices for neuronal guidance in spinal cord injury Josna Joseph1*, Kazuhide Hayakawa2*, Motoichi Kurisawa3*, Li Shan Wang3*, Wanting Niu1 and Myron Spector1 1 VA Boston Healthcare System, Brigham&Women's Hospital, Harvard Medical School, Tissue Engineering, United States 2 Massachusetts General Hospital, Neuroprotection Research laboratory, Department of Neurology& Radiology, United States 3 Institute for Bioengineering and Nanotechnology, Singapore Introduction: The glial scar that is formed around residual cavities after certain spinal cord injuries is a physical and physiological impediment to neuronal regeneration. Gelatin–hydroxyphenyl propionic acid (Gtn-HPA), a novel injectable matrix with tuneable cross-linking properties[1], could provide the missing stroma in such defects, enabling migration of astrocytes from the glial scar into the matrix-filled defect and their guidance of neurons into the lesion[2]. The principal objective of this study was to evaluate astrocyte migration and activation in an in vitro ‘tissue simulant’ model using Gtn-HPA incorporating select growth factors. Materials and Methods: Cortical astrocytes were isolated from postnatal rats, and grown in DMEM high glucose medium. Astrocyte proliferation in Gtn-HPA gels at days 2, 4 and 7 was analysed by counting live cells stained by Calcein-AM under fluorescence microscopy. A three-dimensional migration assay model was set up in 3% agarose-coated 24-well culture plates by surrounding Gtn-HPA cores (1cm diameter) containing EGF (50 or 200 ng/ul) or TGF-β1 (50 or 200 ng/ul) with an annular tissue simulant of 2 wt. % Gtn-HPA gel (cross-linked with 0.1 U/ml horseradish peroxidase and 1.2 mM H202) containing 3*105 cells/ml. Cellular migration into the growth factor-laden inner core was analysed quantitatively by counting cells and measuring the migration distance (n=5 fields) from the gel interface to the farthest cell body (n=4 gels) under phase contrast microscopy. Immunostaining of 30-mm thick cryo sections of gel construct was carried out to detect the presence of GFAP-positive astrocytes. GFAP expression by migrated cells was quantified by Western blot by lysing the gel with collagenase 1 (500U/ml). The results were tested for statistical significance by ANOVA and post-hoc tests. Results and Discussion: Astrocytes migrated in response to chemotactic gradients in Gtn-HPA gels in contrast to the blank gels which displayed virtually no migration. The migration distance was significantly higher (p<0.001) in TGF-50 ng (TGFL) group when compared to EGF and blank Gtn-HPA control group (Fig.1). The number of cells infiltrating the inner core and GFAP expression was significantly higher in the EGF-50 ng (EGFL) group (Fig.2). This indicates that astrocytes migrate well in response to TGF-β1 and are activated by EGF at low concentration, whereas higher conc. of EGF has no specific effect on GFAP expression. Conclusion: This study shows that growth factor-laden Gtn-HPA hydrogels provide a supportive environment for astrocyte migration, proliferation and activation. Gtn-HPA hydrogels laden with optimum combination of EGF and TFG-β1 could effectively guide astrocyte infiltration and subsequent neurite outgrowth in a spinal cord injury defect with diminished glial scarring. J. Joseph acknowledges Fulbright Fellowship from United States India Educational Foundation. Gtn-HPA was provided by the Institute of Bioengineering and Nanotechnology, Singapore.

Modulation of chondrocyte functions and stiffness-dependent cartilage repair using an injectable enzymatically crosslinked hydrogel with tunable mechanical properties

We developed an injectable hydrogel system to evaluate the effect of hydrogel stiffness on chondrocyte cellular functions in a three-dimensional (3D) environment and its subsequent influence on ectopic cartilage formation and early-stage osteochondral defect repair in a rabbit model. The hydrogels, composed of gelatin-hydroxyphenylpropionic acid (Gtn-HPA) conjugate, were formed using oxidative coupling of HPA moieties catalyzed by hydrogen peroxide (H2O2) and horseradish peroxidase (HRP). The storage modulus (G′) of the hydrogels, which was tunable by changing the H2O2 and Gtn-HPA concentrations, ranged from 570 Pa to 2750 Pa. It was found that the cellular functions of chondrocytes encapsulated in hydrogels, including cell proliferation, biosynthesis of collagen and sulfated glycosaminoglycans (sGAG), as well as gene expression of type I (Col-I) and type II collagen (Col-II), were strongly affected by the stiffness of the hydrogels. Of note, chondrocytes cultured within the Gtn-HPA hydrogel of medium stiffness (G′ = 1000 Pa) produced highest level of sGAG production, as well as highest ratio of Col-II to Col-I gene expression among the Gtn-HPA hydrogels of different stiffness. Consistent with the results from in vitro and in vivo ectopic cartilage formation, osteochondral defect repair in a rabbit model showed stiffness-dependent tissue repair, with defects implanted with chondrocytes in hydrogels of medium stiffness having markedly more hyaline cartilage formation, smoother surface and better integration with adjacent cartilage, compared to defects treated with hydrogels of low or high stiffness. These results suggest that the tunable stiffness of Gtn-HPA hydrogels modulates chondrocyte cellular functions, and has a dramatic impact on cartilage tissue histogenesis and repair.

The effect of injectable gelatin-hydroxyphenylpropionic acid hydrogel matrices on the proliferation, migration, differentiation and oxidative stress resistance of adult neural stem cells

Transplanted or endogenous neural stem cells often lack appropriate matrix in cavitary lesions in the central nervous system. In this study, gelatin-hydroxyphenylpropionic acid (Gtn-HPA), which could be enzymatically crosslinked with independent tuning of crosslinking degree and gelation rate, was explored as an injectable hydrogel for adult neural stem cells (aNSCs). The storage modulus of Gtn-HPA could be tuned (449–1717 Pa) to approximate adult brain tissue. Gtn-HPA was cytocompatible with aNSCs (yielding high viability >93%) and promoted aNSC adhesion. Gtn-HPA demonstrated a crosslinking-based approach for preconditioning aNSCs and increased the resistance of aNSCs to oxidative stress, improving their viability from 8-15% to 84% when challenged with 500 μM H2O2. In addition, Gtn-HPA was able to modulate proliferation and migration of aNSCs in relation to the crosslinking degree. Finally, Gtn-HPA exhibited bias for neuronal cells. In mixed differentiation conditions, Gtn-HPA increased the proportion of aNSCs expressing neuronal marker β-tubulin III to a greater extent than that for astrocytic marker glial fibrillary acidic protein, indicating an enhancement in differentiation towards neuronal lineage. Between neuronal and astrocytic differentiation conditions, Gtn-HPA also selected for higher survival in the former. Overall, Gtn-HPA hydrogels are promising injectable matrices for supporting and influencing aNSCs in ways that may be beneficial for brain tissue regeneration after injuries.