Yunki Lee

Synthesis of Si, Mg substituted hydroxyapatites and their sintering behaviors

Si, Mg-substituted hydroxyapatites, alone and co-substituted, have been prepared to obtain biomaterials having an improved biocompatibility. From FT-IR, XRD and ICP analyses, it was confirmed that single phases of hydroxyapatite substituted by Si alone or co-substituted by Si, Mg. The XRD data indicated the absence of extra phases related to silicon and magnesium oxide or other calcium phosphate species. Si-substituted hydroxyapatite of up to 2 wt% for Si and Si, Mg co-substituted hydroxyapatite of 1 wt% for the each ion keep their original structures intact for the sintering temperatures of up to 1200 degrees C. However, it is observed that ion substitutions by an amount higher than the above ratios for each hydroxyapatite lead to destabilization of original structures of the hydroxyapatite and to the production of tricalcium phosphate and calcium phosphate silicate phases when the samples were sintered at 1100 degrees C or higher.

Synthesis and Characterizations of In Situ Cross-Linkable Gelatin and 4-Arm-PPO-PEO Hybrid Hydrogels via Enzymatic Reaction for Tissue Regenerative Medicine

In situ cross-linkable hybrid hydrogels composed of gelatin and 4-arm-polypropylene oxide-polyethylene oxide (Tetronic) was developed as an injectable scaffold for tissue regeneration. The gelatin was modified by hydroxyphenyl propionic acid (HPA) and the Tetronic was conjugated with tyramines (Tet-TA). The hydrogels were rapidly formed by mixing the polymer solutions containing horseradish peroxidase (HRP) and hydrogen peroxide (H(2)O(2)). The gelation time and mechanical properties of the hydrogels could be controlled by varying the HRP and H(2)O(2) concentrations. In vitro degradation study of the hybrid hydrogels was carried out using collagenase and the prolonged proteolytic degradation was obtained due to the presence of the Tetronic. Human dermal fibroblast (hDFB) was cultured in the hydrogel matrices to evaluate the cyto-compatibility. The encapsulated cells were shown to be highly viable and spread over the gel matrices, suggesting that the hybrid hydrogels have an excellent cyto-compatibility. The hydrogels were also subcutaneously injected in the back of mice and the results demonstrated that the hydrogels were rapidly formed at the injected site. From these results, we demonstrate that the in situ cross-linkable hydrogels formed by hybridization of gelatin and Tetronic via enzyme-mediated reactions hold great promise for use as injectable matrices for tissue regenerative medicine due to their tunable physico-chemical properties and excellent bioactivity.

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.

Horseradish peroxidase‐catalysed in situ‐forming hydrogels for tissue‐engineering applications

In situ‐forming hydrogels are an attractive class of implantable biomaterials that are used for biomedical applications. These injectable hydrogels are versatile and provide a convenient platform for delivering cells and drugs via minimally invasive surgery. Although several crosslinking methods for preparing in situ forming hydrogels have been developed over the past two decades, most hydrogels are not sufficiently versatile for use in a wide variety of tissue‐engineering applications. In recent years, enzyme‐catalysed crosslinking approaches have been emerged as a new approach for developing in situ‐forming hydrogels. In particular, the horseradish peroxidase (HRP)‐catalysed crosslinking approach has received increasing interest, due to its highly improved and tunable capacity to obtain hydrogels with desirable properties. The HRP‐catalysed crosslinking reaction immediately occurs upon mixing phenol‐rich polymers with HRP and hydrogen peroxide (H2O2) in aqueous media. Based on this unique gel‐forming feature, recent studies have shown that various properties of formed hydrogels, such as gelation time, stiffness and degradation rate, can be easily manipulated by varying the concentrations of HRP and H2O2. In this review, we outline the versatile properties of HRP‐catalysed in situ‐forming hydrogels, with a brief introduction to the crosslinking mechanisms involved. In addition, the recent biomedical applications of HRP‐catalysed in situ‐forming hydrogels for tissue regeneration are described. Copyright

Enzyme-catalyzed in situ forming gelatin hydrogels as bioactive wound dressings: effects of fibroblast delivery on wound healing efficacy

In this study, in situ forming gelatin hydrogels via horseradish peroxidase (HRP)-catalyzed cross-linking were developed to serve as bioactive wound dressings with suitable tissue adhesive properties to deliver dermal fibroblasts (DFBs). The DFB-encapsulated gelatin hydrogels with different stiffnesses, GH-soft (1.1 kPa) and GH-hard (6.2 kPa), were prepared by controlling the hydrogen peroxide (H2O2) concentrations. The GH-soft hydrogel was capable of facilitating the proliferation of DFBs and the synthesis of extracellular components, as compared to GH-hard hydrogels. In addition, the subcutaneously injected GH-soft hydrogel with bioluminescent reporter cells provided enhanced cell survival and local retention over 14 days. In vivo transplantation of DFB-encapsulated GH-soft hydrogels accelerated wound contraction, and promoted collagen deposition and neovascularization within the incisions performed on mice skin. Therefore, we expect that HRP-catalyzed in situ forming gelatin hydrogels can be useful for local delivery of cells with high viability in wounds, which holds great promise for advancing wound healing technologies and other tissue engineering applications.

Cell recruiting chemokine-loaded sprayable gelatin hydrogel dressings for diabetic wound healing.

UNLABELLED In this study, we developed horseradish peroxidase (HRP)-catalyzed sprayable gelatin hydrogels (GH) as a bioactive wound dressing that can deliver cell-attracting chemotactic cytokines to the injured tissues for diabetic wound healing. We hypothesized that topical administration of chemokines using GH hydrogels might improve wound healing by inducing recruitment of the endogenous cells. Two types of chemokines (interleukin-8; IL-8, macrophage inflammatory protein-3α; MIP-3α) were simply loaded into GH hydrogels during in situ cross-linking, and then their wound-healing effects were evaluated in streptozotocin-induced diabetic mice. The incorporation of chemokines did not affect hydrogels properties including swelling ratio and mechanical stiffness, and the bioactivities of IL-8 and MIP-3α released from hydrogel matrices were stably maintained. In vivo transplantation of chemokine-loaded GH hydrogels facilitated cell infiltration into the wound area, and promoted wound healing with enhanced re-epithelialization/neovascularization and increased collagen deposition, compared with no treatment or the GH hydrogel alone. Based on our results, we suggest that cell-recruiting chemokine-loaded GH hydrogel dressing can serve as a delivery platform of various therapeutic proteins for wound healing applications. STATEMENT OF SIGNIFICANCE Despite development of materials combined with therapeutic agents for diabetic wound treatment, impaired wound healing by insufficient chemotactic responses still remain as a significant problem. In this study, we have developed enzyme-catalyzed gelatin (GH) hydrogels as a sprayable dressing material that can deliver cell-attracting chemokines for diabetic wound healing. The chemotactic cytokines (IL-8 and MIP-3α) were simply loaded within hydrogel during in situ gelling, and wound healing efficacy of chemokine-loaded GH hydrogels was investigated in STZ-induced diabetic mouse model. These hydrogels significantly promoted wound-healing efficacy with faster wound closure, neovascularization, and thicker granulation. Therefore, we expect that HRP-catalyzed in situ forming GH hydrogels can serve as an injectable/sprayable carrier of various therapeutic agents for wound healing applications.

Enhanced Patency and Endothelialization of Small-Caliber Vascular Grafts Fabricated by Coimmobilization of Heparin and Cell-Adhesive Peptides

The clinical utility of a small-caliber vascular graft is still limited, owing to the occlusion of graft by thrombosis and restenosis. A small-caliber vascular graft (diameter, 2.5 mm) fabricated by electrospinning with a polyurethane (PU) elastomer (Pellethane) and biofunctionalized with heparin and two cell-adhesive peptides, GRGDS and YIGSR, was developed for the purpose of preventing the thrombosis and restenosis through antithrombogenic activities and endothelialization. The vascular grafts showed slightly reduced adhesion of platelets and significantly decreased adsorption of fibrinogen. In vitro studies demonstrated that peptide treatment on a vascular graft enhanced the attachment of human umbilical vein endothelial cells (HUVECs), and the presence of heparin and peptides on the graft significantly increased the proliferation of HUVECs. In vivo implantation of heparin/peptides coimmobilized graft (PU-PEG-Hep/G+Y) and PU (control) grafts was performed using an abdominal aorta rabbit model for 60 days followed by angiographic monitoring and explanting for histological analyses. The patency was significantly higher for the modified PU grafts (71.4%) compared to the PU grafts (46.2%) at 9 weeks after implantation. The nontreated PU grafts showed higher levels of α-SMA expression compared to the modified grafts, and for both samples, the proximal and distal regions expressed higher levels compared to the middle region of the grafts. Moreover, immobilization of heparin and peptides and adequate porous structure were found to play important roles in endothelialization and cellular infiltration. Our results strongly encourage that the development of small-caliber vascular grafts is feasible.

Hierarchical self-assembly of magnetic nanoclusters for theranostics: Tunable size, enhanced magnetic resonance imagability, and controlled and targeted drug delivery.

UNLABELLED Nanoparticle-based imaging and therapy are of interest for theranostic nanomedicine. In particular, superparamagnetic iron oxide (SPIO) nanoparticles (NPs) have attracted much attention in cancer imaging, diagnostics, and treatment because of their superior imagability and biocompatibility (approved by the Food and Drug Administration). Here, we developed SPIO nanoparticles (NPs) that self-assembled into magnetic nanoclusters (SAMNs) in aqueous environments as a theranostic nano-system. To generate multi-functional SPIO NPs, we covalently conjugated β-cyclodextrin (β-CD) to SPIO NPs using metal-adhesive dopamine groups. Polyethylene glycol (PEG) and paclitaxel (PTX) were hosted in the β-CD cavity through high affinity complexation. The core-shell structure of the magnetic nanoclusters was elucidated based on the condensed SPIO core and a PEG shell using electron microscopy and the composition was analyzed by thermogravimetric analysis (TGA). Our results indicate that nanocluster size could be readily controlled by changing the SPIO/PEG ratio in the assemblies. Interestingly, we observed a significant enhancement in magnetic resonance contrast due to the large cluster size and dense iron oxide core. In addition, tethering a tumor-targeting peptide to the SAMNs enhanced their uptake into tumor cells. PTX was efficiently loaded into β-CDs and released in a controlled manner when exposed to competitive guest molecules. These results strongly indicate that the SAMNs developed in this study possess great potential for application in image-guided cancer chemotherapy. STATEMENT OF SIGNIFICANCE In this study, we developed multi-functional SPIO NPs that self-assembled into magnetic nanoclusters (SAMNs) in aqueous conditions as a theranostic nano-system. The beta-cyclodextrin (β-CD) was immobilized on the surfaces of SPIO NPs and RGD-conjugated polyethylene glycol (PEG) and paclitaxel (PTX) were hosted in the β-CD cavity through high affinity complexation. We found that nanocluster size could be readily controlled by varying the SPIO/PEG ratio in the assemblies, and also demonstrated significant improvement of the functional nanoparticles for theranostic systems; enhanced magnetic resonance, improved cellular uptake, and efficient PTX loading and sustained release at the desired time point. These results strongly indicate that the SAMNs developed in this study possess great potential for application in image-guided cancer chemotherapy.

Targeted doxorubicin nanotherapy strongly suppressing growth of multidrug resistant tumor in mice

The rational design of nanomedicine to treat multidrug resistant (MDR) tumors in vivo is described in the study. We prepared multifunctionalized Pluronic micelles that are already well-established to be responsive to low pH and redox in order to systemically deliver doxorubicin (DOX) to MDR tumors. Folic acids (FAs) were introduced on the micelle surface as tumor-targeting molecules. In vitro, the DOX-loaded micelles exerted high cytotoxicity in the DOX-resistant cells by bypassing MDR efflux. Cellular uptake studies clearly demonstrated that FA-conjugated DOX micelles (FA/DOX micelles) were efficiently internalized and accumulated in the MDR cells. In vivo studies indicated significant efficacy of FA/DOX micelles for MDR tumors in mice, and that the volume of tumors was 3 times smaller in this group than that of tumors in the free DOX group, and 8 times smaller than the tumors in the saline group. To the best of our knowledge, this methodology has been recognized to have significantly high efficacy, compared to previously reported DOX nanoparticle formulations. This superior anti-tumor efficacy of FA/DOX micelles in MDR tumor-bearing mice can be attributed to FA-targeted and -mediated endocytosis, inhibition of MDR effect, and subsequent DOX release triggered by dual stimuli (low pH and redox) inside the tumor. Given the promise of the multifunctional micelle mediated delivery on inhibition of MDR tumor growth, FA/DOX micelle platform is a much sought after goal for cancer chemotherapy, especially for cancers resistant to anticancer drugs.

In situ forming gelatin hydrogels by dual-enzymatic cross-linking for enhanced tissue adhesiveness

In situ forming hydrogels show promise as therapeutic implants and carriers in a wide range of biomedical applications. They can easily seal or fill damaged tissue, thereby functioning as cell/drug delivery vehicles or hemostats. In this regard, strong and stable adhesion to the surrounding tissue is considered an important parameter to improve the in vivo performance of in situ forming hydrogels. In this study, tissue-adhesive gelatin-based hydrogels were prepared by dual-enzymatic cross-linking, where horseradish peroxidase (HRP) and tyrosinase (Tyr) were used. Tyr was employed to convert phenol groups of gelatin derivatives into o-quinone, which can readily react with nucleophiles (e.g., amines or thiols) on tissue surfaces, thereby resulting in strong tissue adhesion. Incorporating Tyr (0.25 kU mL-1) did not affect the gelation rate or mechanical strength of HRP-cross-linked hydrogels. Importantly, the dual-enzymatically cross-linked hydrogels (GH/HRP/Tyr) exhibited significantly improved adhesive strength (34 kPa), which was superior to single HRP-cross-linked hydrogels (GH/HRP; 19 kPa) and commercially available fibrin glues (7 kPa). These dual-enzymatically cross-linked gelatin-based hydrogels with strong adhesiveness could act as promising bio-adhesives for tissue-regeneration applications.