Gelatin-hydroxyphenylpropionic Acid Research Articles

Gelatin microgel-coated balloon catheter with enhanced delivery of everolimus for long-term vascular patency

In-stent restenosis (ISR) after percutaneous coronary intervention is a major reason for limited long-term patency due to complex neointimal proliferation caused by vascular injury. Drug-coated balloon (DCB) has been developed to treat various cardiovascular diseases including ISR by providing anti-proliferative drugs into blood vessel tissues. However, a significant proportion of the drug is lost during balloon tracking, resulting in ineffective drug delivery to the target region. In this study, we report an everolimus-coated balloon (ECB) using everolimus-loaded gelatin-hydroxyphenyl propionic acid microgel (GM) with enhanced everolimus delivery to vascular walls for long-term patency. GM with high drug loading (> 97%) was simply prepared by homogenizing enzyme-mediated crosslinked hydrogels. The optimal condition to prepare GM-coated ECB (GM-ECB) was established by changing homogenization time and ethanol solvent concentration (30 ∼ 80%). In vitro sustained everolimus release for 30 d, and cellular efficacy using smooth muscle cells and vascular endothelial cells were evaluated. Additionally, an in vivo drug transfer levels of GM-ECB using rabbit femoral arteries were assessed with reduced drug loss and efficient drug delivery capability. Finally, using ISR-induced porcine models, effective in vivo vascular patency 4 weeks after treatment of ECBs was also confirmed. Thus, this study strongly demonstrates that GM can be used as a potential drug delivery platform for DCB application. Statement of significanceWe report an ECB using everolimus-loaded GM prepared by homogenization of enzymatic cross-linked hydrogel. GM showed efficient drug loading (> 97 %) and controllable size. GM-ECB exhibited potential to deliver everolimus in a sustained manner to target area with drug efficacy and viability against SMC and EC. Although GM-ECB had much lower drug content compared to controls, animal study demonstrated enhanced drug transfer and reduced drug loss of GM-ECB due to the protection of encapsulated drugs by GM, and the possible interaction between GM and endothelium. Finally, vascular patency and safety were assessed using ISR-induced porcine models. We suggest an advanced DCB strategy to alleviate rapid drug clearance by bloodstream while improving drug delivery for a long-term vascular patency.

A bioinspired gelatin-hyaluronic acid-based hybrid interpenetrating network for the enhancement of retinal ganglion cells replacement therapy

Biomaterial-based cell replacement approaches to regenerative medicine are emerging as promising treatments for a wide array of profound clinical problems. Here we report an interpenetrating polymer network (IPN) composed of gelatin-hydroxyphenyl propionic acid and hyaluronic acid tyramine that is able to enhance intravitreal retinal cell therapy. By tuning our bioinspired hydrogel to mimic the vitreous chemical composition and mechanical characteristics we were able to improve in vitro and in vivo viability of human retinal ganglion cells (hRGC) incorporated into the IPN. In vivo vitreal injections of cell-bearing IPN in rats showed extensive attachment to the inner limiting membrane of the retina, improving with hydrogels stiffness. Engrafted hRGC displayed signs of regenerating processes along the optic nerve. Of note was the decrease in the immune cell response to hRGC delivered in the gel. The findings compel further translation of the gelatin-hyaluronic acid IPN for intravitreal cell therapy.

An Injectable Multifunctional Dual-Phase Bead-Reinforced Gelatin Matrix Permissive of Mesenchymal Stem Cell Infiltration for Musculoskeletal Soft Tissue Repair.

This study develops a novel strategy for regenerative therapy of musculoskeletal soft tissue defects using a dual-phase multifunctional injectable gelatin-hydroxyphenyl propionic acid (Gtn-HPA) composite. The dual-phase gel consists of stiff, degradation-resistant, ≈2-mm diameter spherical beads made from 8 wt% Gtn-HPA in a 2 wt% Gtn-HPA matrix. The results of a 3D migration assay show that both the cell number and migration distance in the dual-phase gel system are comparable with the 2 wt% mono-phase Gtn-HPA, but notably significantly higher than for 8 wt% mono-phase Gtn-HPA (into which few cells migrated). The results also show that the dual phase gel system has degradation resistance and a prolonged growth factor release profile comparable with 8 wt% mono-phase Gtn-HPA. In addition, the compressive modulus of the 2 wt% dual-phase gel system incorporating the 8 wt% bead phase is nearly four-fold higher than the 2wt% mono-phase gel (5.3 ± 0.4 kPa versus 1.5 ± 0.06 kPa). This novel injectable dual-phase Gtn-HPA composite thus combines the advantages of low-concentration Gtn-HPA (cell migration) with high-concentration Gtn-HPA (stiffness, degradation resistance, slower chemical release kinetics) to facilitate effective reparative/regenerative processes in musculoskeletal soft tissue.

Platelet-Derived Growth Factor Stimulated Migration of Bone Marrow Mesenchymal Stem Cells into an Injectable Gelatin-Hydroxyphenyl Propionic Acid Matrix.

Bone marrow mesenchymal stem cells (bMSCs) are responsible in the repair of injured tissue through differentiation into multiple cell types and secretion of paracrine factors, and thus have a broad application profile in tissue engineering/regenerative medicine, especially for the musculoskeletal system. The lesion due to injury or disease may be a closed irregular-shaped cavity deep within tissue necessitating an injectable biomaterial permissive of host (endogenous) cell migration, proliferation and differentiation. Gelatin-hydroxyphenyl propionic acid (Gtn-HPA) is a natural biopolymer hydrogel which is covalently cross-linked by horseradish peroxidase (HRP) and hydrogen peroxide (H2O2) in situ and can be delivered to the lesion by needle injection. Growth factors and cytokines can be directly incorporated into the gel or into nano- and micro-particles, which can be employed for sustained release of biomolecules while maintaining their bioactivity. In this study, we selected polyelectrolyte complex nanoparticles (PCNs) prepared with dextran sulfate and chitosan as the carrier for platelet-derived growth factor (PDGF)-BB and stromal cell-derived factor (SDF)-1α, which have been tested effectively in recruiting stem cells. Our in vitro results showed a high degree of viability of bMSCs through the process of Gtn-HPA covalent cross-linking gelation. The Gtn-HPA matrix was highly permissive of bMSC migration, proliferation, and differentiation. PDGF-BB (20 ng/mL) directly incorporated into the gel and, alternatively, released from PCNs stimulated bMSC migration and proliferation. There were only small differences in the results for the direct incorporation of PDGF into the gel compared with its release from PCNs, and for increased doses of the growth factor (200 ng/mL and 2 µg/mL). In contrast, SDF-1α elicited an increase in migration and proliferation only when released from PCNs; its effect on migration was notably less than PDGF-BB. The in vitro results demonstrate that PDGF-BB substantially increases migration of bMSCs into Gtn-HPA and their proliferation in the gel, and that these benefits can be derived from incorporation of a relatively low dose of the growth factor directly into the gel. These findings commend the use of Gtn-HPA/PDGF-BB as an injectable therapeutic agent to treat defects in musculoskeletal tissues.

Controlling Growth Factor Diffusion by Modulating Water Content in Injectable Hydrogels.

Recent advancements in the delivery of therapeutics for retinal diseases include the development of injectable hydrogels, networks of one or more hydrophilic polymers that contain a high-volume fraction of water. These systems are of particular interest due to their biocompatibility, permeability to water-soluble metabolites, and function as minimally invasive injectable delivery vehicles. Recently, hydrogels for ophthalmic applications have been developed that display a controlled release of factors necessary for cellular survival and proliferation. Understanding the relationship between the volume water fraction and the physical, chemical, and diffusion properties of the hydrogel scaffold could aid in the improvement of existing drug delivery treatments for retinal regeneration. In this study, we compared the diffusion and release of human epidermal growth factor (hEGF) encapsulated in different injectable homogenous and heterogenous hydrogels, namely gelatin-hydroxyphenyl propionic acid (Gtn-HPA) and hyaluronic acid-tyramine (HA-Tyr)-based hydrogels. These experimental results were compared with the measured stiffness and water content of these hydrogels and applied to different diffusion theories of polymers to determine the model of best fit. We find that the normalized diffusion and release of hEGF increases with free water content in injectable hydrogels: ranging from 0.176 at 41% free water in HA-Tyr to 0.2 at 53% free water in Gtn-HPA, whereas it decreases with hydrogel stiffness: 600 Pa for Gtn-HPA and 1440 Pa for HA-Tyr. Further, we compared our experimental data with theoretical diffusion models. We found that homogeneous theoretical models, notably the hydrodynamic model (giving a normalized diffusion close to 0.2), provide the most suitable explanation for the measured solute diffusion coefficient. Impact statement Diffusion in a three-dimensional system is a key factor in designing new hydrogel-based materials. It allows to control and predict diffusion in implants and delivery systems. However, very little is done to explore and test the diffusion since it is a complex process. Many models can predict solute diffusion; however, practical application using these models has not yet been done. We have shown the variation of these models in a practical extent, which could have a tremendous impact on designing biomaterial for biological application as it allows one to understand the diffusion of injected drugs and growth factors.

Enzyme-Crosslinked Electrospun Fibrous Gelatin Hydrogel for Potential Soft Tissue Engineering.

Soft tissue engineering has been seeking ways to mimic the natural extracellular microenvironment that allows cells to migrate and proliferate to regenerate new tissue. Therefore, the reconstruction of soft tissue requires a scaffold possessing the extracellular matrix (ECM)-mimicking fibrous structure and elastic property, which affect the cell functions and tissue regeneration. Herein, an effective method for fabricating nanofibrous hydrogel for soft tissue engineering is demonstrated using gelatin–hydroxyphenylpropionic acid (Gel–HPA) by electrospinning and enzymatic crosslinking. Gel–HPA fibrous hydrogel was prepared by crosslinking the electrospun fibers in ethanol-water solution with an optimized concentration of horseradish peroxidase (HRP) and H2O2. The prepared fibrous hydrogel held the soft and elastic mechanical property of hydrogels and the three-dimensional (3D) fibrous structure of electrospun fibers. It was proven that the hydrogel scaffolds were biocompatible, improving the cellular adhesion, spreading, and proliferation. Moreover, the fibrous hydrogel showed rapid biodegradability and promoted angiogenesis in vivo. Overall, this study represents a novel biomimetic approach to generate Gel–HPA fibrous hydrogel scaffolds which have excellent potential in soft tissue regeneration applications.

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.

MSC-Encapsulating in Situ Cross-Linkable Gelatin Hydrogels To Promote Myocardial Repair.

Current stem cell-based therapy for cardiac repair and regeneration after myocardial infarction (MI) is not readily translatable into clinical scenarios due to the low retention and survival of the transplanted cells. Here, we evaluated a simple and feasible design of gelatin-hydroxyphenyl propionic acid (GH) hydrogel as an in situ cross-linkable and injectable cell delivery platform for cardiac tissue regeneration. The GH hydrogel exhibited improved cell retention and survival in vitro and in vivo when encapsulating mouse bone marrow-derived mesenchymal stem cells (MSCs) that were used as promising therapeutic candidates for stem cell therapy. Moreover, we demonstrated that MSC-encapsulating GH hydrogels led to a significant improvement in cardiac functional metrics, such as the fractional shortening (FS), ejection fraction (EF), and end-systolic volume (ESV). Similarly, MSC-encapsulating GH hydrogels induced favorable effects in the cardiac structures of the infarcted heart, producing less fibrosis and thicker infarcted walls. These results suggest that GH hydrogels can be used as an instructive cell delivery platform to provide a suitable microenvironment for transplanted cells; therefore, their in vivo applications combined with MSCs may provide great potential for repair and regeneration of injured cardiac tissues after MI.

Enzymatically Crosslinkable Hyaluronic Acid-Gelatin Hybrid Hydrogels as Potential Bioinks for Tissue Regeneration

Hydrogels that mimic the composition and properties of the extracellular matrix have attracted great attention as potential polymeric scaffolds for tissue engineering applications. In this study, an injectable hydrogel composed of hyaluronic acid and gelatin was developed via the oxidative coupling reaction using horseradish peroxidase (HRP). The hydrogel was prepared by mixing two phenol-conjugated polymer solutions, hyaluronic acid-tyramine (HA-TA) and gelatin-hydroxyphenyl propionic acid (GH), in the presence of HRP and hydrogen peroxide (H202). The gelation rate and mechanical properties of composite hydrogel were controlled by adjusting the HRP and H202 concentrations, respectively Compared to the pure HA-TA hydrogels, the stability and cellular behaviors of composite hydrogel improved significantly In addition, the injectable hydrogel system showed good performance in 3D printing with high cell viability after one day of printing. The results suggest that enzymatically crosslinked HA-TA and GH hybrid (HA-TA/GH) composite of HA-TA and GH hydrogel has the potential as a material for tissue engineering and 3D printing biofabrication.

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.

Decellularized liver matrix as substrates for rescue of acute hepatocytes toxicity

More and more scholars regard the lesion of liver cirrhosis as a series of progressive clinical stages. Besides, liver cirrhosis is a late stage of scarring (fibrosis) of the liver, and has long been a common cause of death for the global adult population, thus, the treatment of liver cirrhosis is a key point investigated in the biomedical field. Here, we propose a novel hypothesis; if decellularized liver matrix (DLM) is possible injected into the injurant-induced fiberized liver via the hepatic portal vein to thoroughly repair the liver, it may be an effective therapeutic method for liver fibrosis or even liver cirrhosis. This study mixed rat DLM with gelatin-hydroxyphenylpropionic acid (Glt-HPA) to form a three-dimensional structure to simulate the in vivo liver environment, and cultured the primary rat hepatocytes in it. Afterward, the hepatocytes were treated using D-galactosamine (GaIN), CHCl3 , and CCl4 -containing medium to simulate the toxin-mediated liver fibrosis in vitro. Finally, they were cultured in a DLM-containing medium to observe the viability and functions of the damaged hepatocytes, and the hypothesis of this research was proved, meaning that Glt-HPA-DLM acting on damaged hepatocytes may repair them. Results have shown that Glt-HPA-DLM was effective for hepatocytes culture and repaired injured hepatocytes from GaIN, CHCl3 , and CCl4 (albumin synthesis was increased by 219, 108, and 12%, respectively, whereas relative lactate dehydrogenase activity was reduced by 38, 68, and 67%, after 5 days of culture, separately). This research shows promising effects against hepatic fibrosis and may have potential for liver cirrhosis in vivo.

Printing in situ tissue sealant with visible-light-crosslinked porous hydrogel

To reflect the rapidly growing interest in producing tissue sealants using various chemical/physical processes, we report an approach using visible light to control the crosslinking of 3D printable hydrogels as in situ tissue sealant. Gelatin-hydroxyphenylpropionic acid conjugate (Gtn-HPA) is shown to crosslink effectively within 30 s under visible light in the presence of [RuII(bpy)3]2+ and sodium persulphate, which is sufficiently rapid for surgery use. Porous structure can be also introduced by including carboxylmethyl cellulose-tyramine (CMC-Tyr) as a precursor. The detailed parameters involved in the hydrogel formation, including irradiation time and distance, are investigated in this study. The results suggested that a longer exposure time would result in a hydrogel with higher crosslinking density, while sufficient photocrosslinking can be achieved using a routine visible light source at a distance of 100 mm. Surface morphology of the photocrosslinked hydrogels are studied using scanning electron microscopy (SEM) and environmental SEM with results confirming the expected porosity. The tensile strength of the photocrosslinked hydrogels has been tested for both non-porous and porous samples. Notably, the adhesive strengths (adhesion) of the photocrosslinked hydrogels was demonstrated to be significantly higher compared to that of commercial fibrin glue. Finally, a prototype of hand-held applicator has been developed and demonstrated to print out Gtn-HPA/CMC-Tyr hydrogel of designed properties with controlled spatial resolution. The development of both material and applicator in this study provides a promising tissue sealant solution for wound closure in future surgical procedures.

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.

Tonsil-derived mesenchymal stem cell-embedded in situ crosslinkable gelatin hydrogel therapy recovers postmenopausal osteoporosis through bone regeneration.

We investigated therapeutic potential of human tonsil-derived mesenchymal stem cells (TMSC) subcutaneously delivered to ovariectomized (OVX) mice for developing more safe and effective therapy for osteoporosis. TMSC were isolated from tonsil tissues of children undergoing tonsillectomy, and TMSC-embedded in situ crosslinkable gelatin-hydroxyphenyl propionic acid hydrogel (TMSC-GHH) or TMSC alone were delivered subcutaneously to the dorsa of OVX mice. After 3 months, three-dimensionally reconstructed micro-computed tomographic images revealed better recovery of the femoral heads in OVX mice treated with TMSC-GHH. Serum osteocalcin and alkaline phosphatase were also recovered, indicating bone formation only in TMSC-GHH-treated mice, and absence in hypercalcemia or other severe macroscopic deformities showed biocompatibility of TMSC-GHH. Additionally, visceral fat reduction effects by TMSC-GHH further supported their therapeutic potential. TMSC provided therapeutic benefits toward osteoporosis only when embedded in GHH, and showed potential as a supplement or alternative to current therapies.

Sustained release of parathyroid hormone via in situ cross-linking gelatin hydrogels improves the therapeutic potential of tonsil-derived mesenchymal stem cells for hypoparathyroidism.

Biomimetic parathyroid regeneration with sustained release of parathyroid hormone (PTH) into the blood stream is a considerable challenge in hypoparathyroidism treatment. We recently reported that tonsil-derived mesenchymal stem cells (TMSCs), if these cells were both differentiated in vitro before implantation and incorporated into a scaffold Matrigel, are a good cell source for parathyroid regeneration in a parathyroidectomized (PTX) animal model. Here, we present a new strategy for improved clinical application that enhances the sustained release of PTH by controlling mechanical stiffness using in situ-forming gelatin-hydroxyphenyl propionic acid (GH) hydrogels (GHH). Differentiated TMSCs (dTMSCs) embedded in a GHH with a strength of 4.4kPa exhibited the best sustained release of PTH and were the most effective in hypoparathyroidism treatment, showing improved blood calcium homeostasis compared with Matrigel-embedded dTMSCs. Interestingly, undifferentiated control TMSCs (cTMSCs) also released PTH in a sustained manner if incorporated into GHH. Collectively, these findings may establish a new paradigm for parathyroid regeneration that could ultimately evolve into an improved clinical application. Copyright © 2017 John Wiley & Sons, Ltd.

In Situ Forming and H2O2-Releasing Hydrogels for Treatment of Drug-Resistant Bacterial Infections.

Various types of commercialized wound dressings (e.g., films, foams, gels, and nanofiber meshes) have been clinically used as a physical barrier against bacterial invasion and as wound-healing materials. Although these dressings can protect the wounded tissue from the external environment, they cannot treat the wounds that are already infected with bacteria. Herein, we report in situ H2O2-releasing hydrogels as an active wound dressing with antibacterial properties for treatment of drug-resistant bacterial infection. In this study, H2O2 was used for two major purposes: (1) in situ gel formation via a horseradish peroxidase (HRP)/H2O2-triggered cross-linking reaction, and (2) antibacterial activity of the hydrogel via its oxidative effects. We found that there were residual H2O2 in the matrix after in situ HRP-catalyzed gelling, and varying the feed amount of H2O2 (1-10 mM; used to make hydrogels) enabled control of H2O2 release kinetics within a range of 2-509 μM. In addition, although the gelatin-hydroxyphenyl propionic acid (GH) gel called "GH 10" (showing the greatest H2O2 release, 509 μM) slightly decreased cell viability (to 82-84%) of keratinocyte (HaCaT) and fibroblast (L-929) cells in in vitro assays, none of the hydrogels showed significant cytotoxicity toward tissues in in vivo skin irritation tests. When the H2O2-releasing hydrogels that promote in vivo wound healing, were applied to various bacterial strains in vitro and ex vivo, they showed strong killing efficiency toward Gram-positive bacteria including Staphylococcus aureus, S. epidermidis, and clinical isolate of methicillin-resistant S. aureus (MRSA, drug-resistant bacteria), where the antimicrobial effect was dependent on the concentration of the H2O2 released. The present study suggests that our hydrogels have great potential as an injectable/sprayable antimicrobial dressing with biocompatibility and antibacterial activity against drug-resistant bacteria including MRSA for wound and infection treatment.

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.

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.