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Over the past decades, hydrogels have been widely studied as biomaterials for various biomedical applications like implants, drugs and cell delivery carriers because of their high biocompatibility, high water contents and excellent permeability for nutrients and metabolites. Especially, in situ forming hydrogel systems have received much attention because of their easy application based on minimal invasive techniques. Chemical cross-linking systems fabricated using enzymatic reactions have various advantages, such as high biocompatibility and easy control of reaction rates under mild condition. In this study, we report enzyme-triggered injectable and biodegradable hydrogels composed of Tetronic-tyramine conjugates. The Tetronic-tyramine conjugates were synthesized by first reacting Tetronic with succinic anhydride and subsequent conjugation with tyramine using DCC/NHS as coupling reagents. The chemical structure of Tetronic-succinic anhydride-tyramine (Tet-SA-TA) copolymer was characterized by (1)H NMR and FTIR. The hydrogels were prepared from a Tet-SA-TA solution above 3 wt % in the presence of horseradish peroxidase (HRP) and H(2)O(2) under physiological conditions. Their mechanical property, gelation time, swelling ratio and degradation time were evaluated at different polymer, HRP, and H(2)O(2) concentrations. In addition, a cyto-compatibility study was performed using the MC3T3-E1 cell line. In the cytotoxicity test, it was clear that the Tet-SA-TA hydrogel had no apparent cytotoxicity except for the hydrogel formed with 0.25 wt % H(2)O(2) due to the cytotoxicity of residual H(2)O(2). In conclusion, the obtained results demonstrated that the Tet-SA-TA hydrogel has great potential for use as an injectable scaffold for tissue engineering and as a drug carrier for controlled drug delivery systems.

Controlled adhesion and continuous growth of human mesenchymal stem cells (hMSCs) are essential for scaffold-based delivery of hMSCs in tissue engineering applications. The main goal of this study is to develop biofunctionalized synthetic substrates to actively control adhesion, spreading, and proliferation of hMSCs. gamma-Ray irradiation was employed to graft acrylic acid (AAc) to biodegeradable poly(L-lactide-co--caprolactone) (PLCL) films. Gelatin, a natural polymer, was then immobilized on the AAc grafted PLCL film (AAc-PLCL) to induce biomimetic interactions with the cells. The graft yield of AAc increased as the irradiation dose and AAc concentration increased, and the presence of gelatin (gelatin-AAc-PLCL) following immobilization was confirmed using ESCA. To investigate cell responses, hMSCs isolated from a human mandible were cultured on the various substrates and their adhesion, spreading, and proliferation were examined. After three days of culture, the DNA concentration from the cells cultured on gelatin-AAc-PLCL film was 2.9-fold greater than that on the PLCL film. Immunofluorescent staining of hMSCs cultured on the gelatin-AAc-PLCL films demonstrated homogeneous localization of F-Actin and vinculin in their cytoplasm, while mature adhesive structure was not observed from the cells cultured on other substrates. Furthermore, the ratio of projected area of adherent single cells on gelatin-AAc-PLCL films was significantly larger (116.80 +/- 12.78%) than that on the PLCL films (30.11 +/- 5.07%). Our results suggest that gelatin-immobilized PLCL substrates may be potentially used in tissue engineering, particularly as a stem cell delivery carrier for the regeneration of target tissue.

Current advances in biomaterial fabrication techniques have broadened their application in different realms of biomedical engineering, spanning from drug delivery to tissue engineering. The success of biomaterials depends highly on the ability to modulate cell and tissue responses, including cell adhesion, as well as induction of repair and immune processes. Thus, most recent approaches in the field have concentrated on functionalizing biomaterials with different biomolecules intended to evoke cell- and tissue-specific reactions. Marine mussels produce mussel adhesive proteins (MAPs), which help them strongly attach to different surfaces, even under wet conditions in the ocean. Inspired by mussel adhesiveness, scientists discovered that dopamine undergoes self-polymerization at alkaline conditions. This reaction provides a universal coating for metals, polymers, and ceramics, regardless of their chemical and physical properties. Furthermore, this polymerized layer is enriched with catechol groups that enable immobilization of primary amine or thiol-based biomolecules via a simple dipping process. Herein, this review explores the versatile surface modification techniques that have recently been exploited in tissue engineering and summarizes polydopamine polymerization mechanisms, coating process parameters, and effects on substrate properties. A brief discussion of polydopamine-based reactions in the context of engineering various tissue types, including bone, blood vessels, cartilage, nerves, and muscle, is also provided.

This study evaluates the in vitro biocompatibility of an injectable and biodegradable polymeric network based on poly(propylene fumarate) (PPF) and the cross-linking agent PPF-diacrylate (PPF-DA). Using a methyl tetrazolium (MTT) assay, the effect of the concentrations of PPF and PPF-DA on the cytotoxicity of its unreacted macromers, cross-linked networks, and degradation products was examined. The influence of network structure properties on cell viability and attachment to the cross-linked material was also investigated. The unreacted macromers exhibited a time- and dose-dependent cytotoxic response that increased with more PPF-DA in the mixture. Conversely, the cross-linked networks formed with more PPF-DA did not demonstrate an adverse response because increases in conversion and cross-linking density prevented the extraction of toxic products. Fibroblast attachment was observed on the PPF/PPF-DA networks with the highest double bond conversions. The degradation products, obtained from the complete breakdown of the networks in basic conditions, displayed a dose-dependent cytotoxic response. These results show that there are concerns regarding the biocompatibility of injectable, biodegradable PPF/PPF-DA networks but also sheds light onto potential mechanisms to reduce the cytotoxic effects.

Currently, oligo[poly(ethylene glycol) fumarate] (OPF) hydrogels are being investigated as an injectable and biodegradable system for tissue engineering applications. In this study, cytotoxicity of each component of the OPF hydrogel formulation and the resulting cross-linked network was examined. Specifically, OPF synthesized with poly(ethylene glycol) (PEG) of different molecular weights (MW), the cross-linking agent [PEG-diacrylate (PEG-DA)], and the redox initiator pair [ammonium persulfate (APS) and ascorbic acid (AA)] were evaluated for cytotoxicity at 2 and 24 h using marrow stromal cells (MSCs) as model cells. The effect of leachable byproducts of OPF hydrogels on cytotoxicity was also investigated. Upon exposure to various concentrations of OPF for 2 h, greater than 50% of the MSCs were viable, regardless of OPF molecular weight or concentration in the media. After 24 h, the MSCs maintained more than 75% viability except for OPF concentrations higher than 25% (w/v). When examining the cross-linking agent, PEG-DA of higher MW (3400) demonstrated significantly higher viability compared to PEG-DA with MW 575 at all concentrations tested. Considering initiators, when MSCs were exposed to AA and APS, as well as the combination of AA and APS, higher viability was observed at lower concentrations. Once cross-linked, the leachable products from the OPF hydrogels had minimal adverse effects on the viability of MSCs (percentage of live cells was higher than 90% regardless of hydrogel types). The results suggest that, after optimization of cross-linking parameters, OPF-based hydrogels hold promise as novel injectable scaffolds or cell carriers in tissue engineering.

A novel macromer, oligo(poly(ethylene glycol) fumarate) (OPF), was synthesized by the reaction between poly(ethylene glycol) (PEG) of molecular weight 1000 (PEG 1.0K) and fumaryl chloride. The oligo(PEG fumarate) (OPF 1.0K) was modified with a peptide known to modulate cellular functions, Gly-Arg-Gly-Asp (GRGD), after being activated with 4-nitrophenyl chloroformate (NPC). The determined yield of the GRGD modification in 0.1 M sodium bicarbonate buffer of pH 8.3 was 83% as determined by NMR measurements. The OPF 1.0K and the OPF 1.0K modified with GRGD were cross-linked with an unsaturated biodegradable polyester, poly(propylene fumarate) (PPF), by photopolymerization. The cross-linked PPF was characterized by Fourier transform infrared (FT-IR) spectroscopy and contact angle measurements. The equilibrium contact angle of water on the cross-linked PPF surface decreased with the incorporation of OPF 1.0K and the OPF 1.0K modified with GRGD. The results suggest that the OPF macromer can be used for the preparation of functionalized networks incorporating cell adhesion specific sequences.

Peripheral arterial disease is a common manifestation of systemic atherosclerosis, which results in more serious consequences of ischemic events in peripheral tissues such as the lower extremities. Cell therapy has been tested as a treatment for peripheral ischemia that functions by inducing angiogenesis in the ischemic region. However, the poor survival and engraftment of transplanted cells limit the efficacy of cell therapy. In order to overcome such challenges, we applied genetically engineered cell sheets using a cell-interactive and thermosensitive hydrogel and nonviral polymer nanoparticles. C2C12 myoblast sheets were formed on Tetronic-tyramine (Tet-TA)-RGD hydrogel prepared through a highly efficient and noncytotoxic enzymatic reaction. The myoblast sheets were then transfected with vascular endothelial growth factor (VEGF) plasmids using poly(β-amino ester) nanoparticles to increase the angiogenic potential of the sheets. The transfection increased the VEGF expression and secretion from the C2C12 sheets. The enhanced angiogenic effect of the VEGF-transfected C2C12 sheets was confirmed using an in vitro capillary formation assay. More importantly, the transplantation of the VEGF-transfected C2C12 sheets promoted the formation of capillaries and arterioles in ischemic muscles, attenuated the muscle necrosis and fibrosis progressed by ischemia, and eventually prevented ischemic limb loss. In conclusion, the combination of cell sheet engineering and genetic modification can provide more effective treatment for therapeutic angiogenesis.

In this study, thermosensitive hydrogels incorporated with multiple cell-interactive factors were developed as a substrate to form monolayer of human umbilical vein endothelial cells (HUVECs) that can be detached and transferrable to target sites as a cell-sheet in response to temperature change. The cell adhesive peptide (RGD) and growth factor (bFGF) covalently incorporated within the hydrogel significantly enhanced adhesion and proliferation of HUVECs, allowing for the formation of their confluent monolayer. Meanwhile, the precisely controllable change in the size of the hydrogels was observed by a repeated increase and decrease in temperature from 37 to 4 °C. By exploiting this unique behavior, the detachment and transfer of HUVEC sheet confluently cultured at 37 °C was rapidly induced within 10 min by expansion of the hydrogels when the temperature was decreased to 4 °C. The transferred cell sheet was highly viable and maintained robust cell−cell junction. Finally, the process of cell sheet transfer was directly applied onto an ischemic injury in the hind limb of mice. The transplanted HUVECs as a sheet retarded tissue necrosis over 14 days in comparison with that of direct injection of the same number of cells. Our results suggest that the developed multifunctional Tetronic-tyramine hydrogels could serve as an ideal substrate to modulate the formation of an endothelial cell layer that could potentially be utilized to treat peripheral arterial disease.

Most polymeric vascular prosthetic materials have low patency rate for replacement of small diameter vessels (

A novel hydrogel system based on oligo(poly(ethylene glycol) fumarate) (OPF) is currently being investigated as an injectable carrier for marrow stromal cells (MSCs) for orthopedic tissue engineering applications. This hydrogel is cross-linked using the redox radical initiators ammonium persulfate (APS) and ascorbic acid (AA). In this study, two different persulfate oxidizing agents (APS and sodium persulfate (NaPS)) with three reducing agents derived from ascorbic acid (AA, sodium ascorbate (Asc), and magnesium ascorbate-2-phosphate (Asc-2)) and their combinations were examined to determine the relationship between pH, exposure time, and cytotoxicity for rat MSCs. In addition, gelation times for specific combinations were determined using rheometry. pH and cell viability data after 2 h for combinations ranging from 10 to 500 mM in each reagent showed that there was a smaller pH change and a corresponding higher viability at lower concentrations, regardless of the reagents used. At 10 mM, there was less than a 1.5 unit drop in pH and greater than 90% viability for all initiator combinations examined. However, MSC viability was significantly reduced with concentrations of 100 mM and higher of the initiator combinations. At 100 mM, exposure to NaPS/Asc-2 resulted in significantly more live cells than exposure to APS/AA or NaPS/Asc, but at this concentration, NaPS/Asc-2 exhibited significantly longer OPF gelation onset times than APS/AA. At all combination concentrations, exposure time (10 min vs 2 h) did not significantly affect MSC viability. These data indicate that final pH and/or radical formation have a large impact on MSC viability and that multiple, intertwined testing procedures are required for identification of appropriate initiators for cell encapsulation applications.