Gelatin-hydroxyphenylpropionic Acid Research Articles

Enhanced Cellular Activity in Gelatin-Poly(Ethylene Glycol) Hydrogels without Compromising Gel Stiffness.

Adhesion and proliferation of cells are often suppressed in rigid hydrogels as gel stiffness induces mechanical stress to embedded cells. Herein, the composite hydrogel systems to facilitate high cellular activities are described, while maintaining relatively high gel stiffness. This unusual property is obtained by harmonizing gelatin-poly(ethylene glycol)-tyramine (GPT, semisynthetic polymer) and gelatin-hydroxyphenyl propionic acid conjugates (GH, natural polymer) into hydrogels. A minimum GH concentration of 50% is necessary for cells to be proliferative. GPT is utilized to improve biological stability (>1 week) and gelation time (<20 s) of the hydrogels. These results suggest that deficiency in cellular activity driven by gel stiffness could be overcome by finely tuning the material properties in the microenvironments.

In Situ Generation of Tunable Porosity Gradients in Hydrogel‐Based Scaffolds for Microfluidic Cell Culture

Compared with preformed anisotropic matrices, an anisotropic matrix that allows users to alter its properties and structure in situ after synthesis offers the important advantage of being able to mimic dynamic in vivo microenvironments, such as in tissues undergoing morphogenesis or in wounds undergoing tissue repair. In this study, porous gradients are generated in situ in a hydrogel comprising enzymatically crosslinked gelatin hydroxyphenylpropionic acid (GTN-HPA) conjugate and carboxylmethyl cellulose tyramine (CMC-TYR) conjugate. The GTN-HPA component acts as the backbone of the hydrogel, while CMC-TYR acts as a biocompatible sacrificial polymer. The hydrogel is then used to immobilize HT1080 human fibrosarcoma cells in a microfluidic chamber. After diffusion of a biocompatible cellulase enzyme through the hydrogel in a spatially controlled manner, selective digestion of the CMC component of the hydrogel by the cellulase gives rise to a porosity gradient in situ instead of requiring its formation during hydrogel synthesis as with other methods. The influence of this in situ tunable porosity gradient on the chemotactic response of cancer cells is subsequently studied both in the absence and presence of chemoattractant. This platform illustrates the potential of hydrogel-based microfluidics to mimic the 3D in vivo microenvironment for tissue engineering and diagnostic applications.

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.

In situ forming gelatin-based tissue adhesives and their phenolic content-driven properties.

The present study describes enzymatically cross-linked gelatin-based hydrogels as in situ forming tissue adhesives. A series of gelatin derivatives with different phenolic contents were synthesized by conjugating hydroxyphenyl propionic acid and tyramine to gelatin backbones. Two gelatin derivatives, gelatin-hydroxyphenyl propionic acid (GH) and gelatin-hydroxyphenyl propionic acid-tyramine (GHT) with maximum obtainable phenolic contents (146.6 μmol g-1 GH and 395.7 μmol g-1 GHT), were used to prepare gelatin-based hydrogels via horseradish peroxidase (HRP)-mediated reactions in the presence of hydrogen peroxide (H2O2). By changing the HRP and H2O2 concentrations, the gelation time, mechanical strength, and degradation rate of the hydrogels were fairly well controlled, indicating a tunable rate and degree of cross-linking. In addition, we found that an increase in phenolic content led to increased mechanical strength of the hydrogels. Lap-shear test results clearly showed that the GH and GHT hydrogels exhibited 2-3 times greater tissue adhesiveness compared to fibrin glues. On the basis of these results, we conclude that in situ forming gelatin-based hydrogels, which are both injectable and sprayable, can be used as an alternative to conventional tissue adhesives.

Chemotactic recruitment of adult neural progenitor cells into multifunctional hydrogels providing sustained SDF‐1α release and compatible structural support

Without chemotactic cues and structural support, cavitary brain lesions typically fail to recruit endogenous neural progenitor cells (NPCs). Toward resolving this, we engineered multifunctional biomaterials comprising injectable gelatin-hydroxyphenylpropionic acid (Gtn-HPA) hydrogels and dextran sulfate/chitosan polyelectrolyte complex nanoparticles (PCNs) that delivered stromal cell-derived factor-1α (SDF-1α). Over 7 d of interface with in vitro tissue simulant containing adult rat hippocampal NPCs (aNPCs) and their neuronal progeny, Gtn-HPA/SDF-1α-PCN hydrogels promoted chemotactic recruitment to enhance infiltration of aNPCs by 3- to 45-fold relative to hydrogels that lacked SDF-1α or vehicles to sustain SDF-1α release. When cross-linked with 0.85-0.95 mM HO, Gtn-HPA/SDF-1α-PCN hydrogels provided optimally permissive structural support for migration of aNPCs. Specific matrix metalloproteinase (MMP) inhibitors revealed that 42, 30, and 55% of cell migration into Gtn-HPA/SDF-1α-PCN hydrogels involved MMP-2, 3, and 9, respectively, demonstrating the hydrogels to be compatible toward homing endogenous NPCs, given their expression of similar MMPs. Interestingly, PCNs utilized FGF-2 found in situ to induce chemokinesis, potentiate SDF-1α chemotactic recruitment, and increase proliferation of recruited cells, which collectively orchestrated a higher number of migrated aNPCs. Overall, Gtn-HPA/SDF-1α-PCN hydrogels prove to be promising biomaterials for injection into cavitary brain lesions to recruit endogenous NPCs and enhance neural tissue repair/regeneration.

Controlling Fibroblast Proliferation with Dimensionality-Specific Response by Stiffness of Injectable Gelatin Hydrogels

We report an injectable hydrogel system with tunable stiffness for controlling the proliferation rate of human fibroblasts (HFF-1) in both two-dimensional (2D) and three-dimensional (3D) culture environments for potential use as a wound dressing material. The hydrogel composed of gelatin–hydroxyphenylpropionic acid (Gtn–HPA) conjugate was formed by the oxidative coupling of HPA moieties catalyzed by hydrogen peroxide (H2O2) and horseradish peroxidase (HRP). The stiffness of the hydrogels was controlled well by varying the H2O2 concentration. The effects of hydrogel stiffness on the proliferation rate of HFF-1 in both 2D and 3D were investigated. We found that the proliferation rate of HFF-1 using Gtn–HPA hydrogels was strongly dependent on the hydrogel stiffness, with a dimensionality-specific response. In the 2D studies, the HFF-1 exhibited a higher proliferation rate when the stiffness of the hydrogel was increased. In contrast, the HFF-1 cultured inside the hydrogel remained non-proliferative for 12 days before a stiffness-dependent proliferation profile was shown. The proliferation rate decreased with an increase in stiffness of the hydrogel in a 3D culture environment, unlike in a 2D environment.

Enzymatically cross-linked gelatin-phenol hydrogels with a broader stiffness range for osteogenic differentiation of human mesenchymal stem cells

An injectable hydrogel system, composed of gelatin–hydroxyphenylpropionic acid (Gtn–HPA) conjugates chemically cross-linked by an enzyme-mediated oxidation reaction, has been designed as a biodegradable scaffold for tissue engineering. In light of the role of substrate stiffness on cell differentiation, we herein report a newly improved Gtn hydrogel system with a broader range of stiffness control that uses Gtn–HPA–tyramine (Gtn–HPA–Tyr) conjugates to stimulate the osteogenic differentiation of human mesenchymal stem cells (hMSCs). The Gtn–HPA–Tyr conjugate was successfully synthesized through a further conjugation of Tyr to Gtn–HPA conjugate by means of a general carbodiimide/active ester-mediated coupling reaction. Proton nuclear magnetic resonance and UV–visible measurements showed a higher total phenol content in the Gtn–HPA–Tyr conjugate than that content in the Gtn–HPA conjugate. The Gtn–HPA–Tyr hydrogels were formed by the oxidative coupling of phenol moieties catalyzed by hydrogen peroxide (H2O2) and horseradish peroxidase (HRP). Rheological studies revealed that a broader range of storage modulus (G′) of Gtn–HPA–Tyr hydrogel (600–26,800Pa) was achieved using different concentrations of H2O2, while the G′ of the predecessor Gtn–HPA hydrogels was limited to the range of 1000 to 13,500Pa. The hMSCs on Gtn–HPA–Tyr hydrogel with G′ greater than 20,000 showed significantly up-regulated expressions of osteocalcin and runt-related transcription factor 2 (RUNX2) on both the gene and protein level, with the presence of alkaline phosphatase, and the evidence of calcium accumulation. These studies with the Gtn–HPA–Tyr hydrogel with G′ greater than 20,000 collectively suggest the stimulation of the hMSCs into osteogenic differentiation, while these same observations were not found with the Gtn–HPA hydrogel with a G′ of 13,500.

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.

The role of stiffness of gelatin–hydroxyphenylpropionic acid hydrogels formed by enzyme-mediated crosslinking on the differentiation of human mesenchymal stem cell

We report the stimulation of neurogenesis and myogenesis of human mesenchymal stem cells (hMSCs) on the surfaces of biodegradable hydrogels with different stiffness. The hydrogels were composed of gelatin–hydroxyphenylpropionic acid (Gtn–HPA) conjugate were formed using the oxidative coupling of phenol moieties catalyzed by hydrogen peroxide (H 2O 2) and horseradish peroxidase (HRP). The storage modulus of the hydrogels was readily tuned from 600 to 12800 Pa. It was found that the stiffness of the hydrogel strongly affected the cell attachment, focal adhesion, migration and proliferation rate of hMSCs. The hMSCs on stiffer surfaces have a larger spreading area, more organized cytoskeletons, more stable focal adhesion, faster migration and a higher proliferation rate. The gene expression related to the extracellular matrix and adhesion molecules also differed when the cells were cultured on hydrogels with different stiffness. The differentiation of hMSCs on the surface of the hydrogel was closely linked to the hydrogel stiffness. The cells on a softer hydrogel (600 Pa) expressed more neurogenic protein markers, while cells on a stiffer hydrogel (12000 Pa) showed a higher up-regulation of myogenic protein markers.

Injectable biodegradable hydrogels with tunable mechanical properties for the stimulation of neurogenesic differentiation of human mesenchymal stem cells in 3D culture

We report an injectable hydrogel scaffold system with tunable stiffness for controlling the proliferation rate and differentiation of human mesenchymal stem cells (hMSCs) in a three-dimensional (3D) context in normal growth media. The hydrogels composed of gelatin-hydroxyphenylpropionic acid (Gtn–HPA) conjugate were formed using the oxidative coupling of HPA moieties catalyzed by hydrogen peroxide (H 2O 2) and horseradish peroxidase (HRP). The stiffness of the hydrogels was readily tuned by varying the H 2O 2 concentration without changing the concentration of polymer precursor. We found that the hydrogel stiffness strongly affected the cell proliferation rates. The rate of hMSC proliferation increased with the decrease in the stiffness of the hydrogel. Also, the neurogenesis of hMSCs was controlled by the hydrogel stiffness in a 3D context without the use of any additional biochemical signal. These cells which were cultured in hydrogels with lower stiffness for 3 weeks expressed much more neuronal protein markers compared to those cultured within stiffer hydrogels for the same period of time.

Cell immobilization in gelatin–hydroxyphenylpropionic acid hydrogel fibers

Gelatin–hydroxyphenylpropionic acid (Gtn–HPA) hydrogels are highly porous and biodegradable materials. Herein we report a fiber spinning method that can produce cell-seeded solid and hollow hydrogel fibers by enzymatically cross-linking Gtn–HPA in solutions flowing within a capillary tube. The cell-immobilized hydrogel fibers, with feature sizes down to 20 μm, are formed as a result of continuous cross-linking of cell-mixed hydrogel precursors in a multiphase laminar flow. This fiber formation process is mild enough to retain the cell viability. The continuous fiber formation, simultaneous cell encapsulation, as well as versatile combination of fiber structures provided by this approach make it a promising and effective technique for the preparation of cell-seeded hydrogel scaffolds and carriers for tissue engineering.