Junseok Yeom

In situ supramolecular assembly and modular modification of hyaluronic acid hydrogels for 3D cellular engineering.

A facile in situ supramolecular assembly and modular modification of biocompatible hydrogels were demonstrated using cucurbit[6]uril-conjugated hyaluronic acid (CB[6]-HA), diaminohexane-conjugated HA (DAH-HA), and tags-CB[6] for cellular engineering applications. The strong and selective host-guest interaction between CB[6] and DAH made possible the supramolecular assembly of CB[6]/DAH-HA hydrogels in the presence of cells. Then, the 3D environment of CB[6]/DAH-HA hydrogels was modularly modified by the simple treatment with various multifunctional tags-CB[6]. Furthermore, we could confirm in situ formation of CB[6]/DAH-HA hydrogels under the skin of nude mice by sequential subcutaneous injections of CB[6]-HA and DAH-HA solutions. The fluorescence of modularly modified fluorescein isothiocyanate (FITC)-CB[6] in the hydrogels was maintained for up to 11 days, reflecting the feasibility to deliver the proper cues for cellular proliferation and differentiation in the body. Taken together, CB[6]/DAH-HA hydrogels might be successfully exploited as a 3D artificial extracellular matrix for various tissue engineering applications.

Effect of cross-linking reagents for hyaluronic acid hydrogel dermal fillers on tissue augmentation and regeneration.

A novel, biocompatible, and nontoxic dermal filler using hyaluronic acid (HA) hydrogels was successfully developed for tissue augmentation applications. Instead of using highly reactive cross-linkers such as divinyl sulfone (DVS) for Hylaform, 1,4-butanediol diglycidyl ether (BDDE) for Restylane, and 1,2,7,8-diepoxyoctane (DEO) for Puragen, HA hydrogels were prepared by direct amide bond formation between the carboxyl groups of HA and hexamethylenediamine (HMDA) with an optimized carboxyl group modification for effective tissue augmentation. The HA-HMDA hydrogels could be prepared within 5 min by the addition of HMDA to HA solution activated with 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDC) and 1-hydroxybenzotriazole monohydrate (HOBt). Five kinds of samples, a normal control, a negative control, a positive control of Restylane, adipic acid dihydrazide grafted HA (HA-ADH) hydrogels, and HA-HMDA hydrogels, were subcutaneously injected to wrinkled model mice. According to the image analysis on dorsal skin augmentation, the HA-HMDA hydrogels exhibited the best tissue augmentation effect being stable longer than 3 months. Furthermore, histological analyses after hematoxylin-eosin (H&E) and Massons trichrome staining revealed the excellent biocompatibility and safety of HA-HMDA hydrogels. The dermal thickness and the dermal collagen density in wrinkled mice after treatment with HA-HMDA hydrogels for 12 weeks were comparable to those of normal mice. Compared with HA-DVS hydrogels and Restylane, the excellent tissue augmentation by HA-HMDA hydrogels might be ascribed to the biocompatible residues of amine groups in the cross-linker of HMDA. The HA-HMDA hydrogels will be investigated further as a novel dermal filler for clinical applications.

Three-dimensional bioprinting of multilayered constructs containing human mesenchymal stromal cells for osteochondral tissue regeneration in the rabbit knee joint

The use of cell-rich hydrogels for three-dimensional (3D) cell culture has shown great potential for a variety of biomedical applications. However, the fabrication of appropriate constructs has been challenging. In this study, we describe a 3D printing process for the preparation of a multilayered 3D construct containing human mesenchymal stromal cells with a hydrogel comprised of atelocollagen and supramolecular hyaluronic acid (HA). This construct showed outstanding regenerative ability for the reconstruction of an osteochondral tissue in the knee joints of rabbits. We found that the use of a mechanically stable, host-guest chemistry-based hydrogel was essential and allowed two different types of extracellular matrix (ECM) hydrogels to be easily printed and stacked into one multilayered construct without requiring the use of potentially harmful chemical reagents or physical stimuli for post-crosslinking. To the best of our knowledge, this is the first study to validate the potential of a 3D printed multilayered construct consisting of two different ECM materials (atelocollagen and HA) for heterogeneous tissue regeneration using an in vivo animal model. We believe that this 3D printing-based platform technology can be effectively exploited for regeneration of various heterogeneous tissues as well as osteochondral tissue.

The topographic effect of zinc oxide nanoflowers on osteoblast growth and osseointegration.

Cell-material interactions are one of the most important factors to be considered for the applications of biomaterials to tissue engineering fi elds. Cellular responses are dependent on the bio-interphases with various topologies such as nanopits, nanorods, nanogrates, and nanotubes, etc. [ 1–3 ] Cells interact with specifi c binding motifs in adhesion proteins via cell surface receptors, such as integrins with non-covalently associated α and β chains. There have been extensive research efforts to investigate the topographic effect of biomaterials with various microand nano-structures on cell-material interactions. The fabrication of nanostructures on biomaterials improves their biocompatibility more effectively with well-defi ned morphologies than the conventional chemical or physicochemical surface treatments. [ 4 , 5 ] It was reported that aligned groove patterns led to an increase in endothelial cell and osteoblast adhesions. [ 2 ] In the same way, nanostructures were fabricated and assessed with human foreskin fi broblasts. The results showed that these architectures were advantageous to cause the discreet contact guidance for fi lopodia extension. [ 4 ] Despite wide investigations on the interactions between cells and nanoscale topographic surfaces of biomaterials, the effect of nanofl ower structure on cell adhesion, proliferation, and growth has not been reported yet. ZnO nanostructures have been widely studied for various electronic applications due to their unique piezoelectric, semiconducting, and catalytic properties with a high electron mobility and a wide band-gap. [ 6 ] Recently, they have been also exploited for biomedical applications such as implantable biomedical nanosensors, and restorative and prosthodontic applications in dentistry. [ 7 , 8 ] The biodegradability, biocompatibility, and biosafety of ZnO nano-structures were reported in cellular levels elsewhere. [ 9 , 10 ] Furthermore, the topographic effect and the cytotoxicity of ZnO nanorods were extensively studied to control the cell adhesion and macrophage responses for tissue engineering applications. [ 11 , 12 ] In this work, we investigated the topographic effect of ZnO nanofl owers on MC3T3-E1 osteoblast

3D Tissue Engineered Supramolecular Hydrogels for Controlled Chondrogenesis of Human Mesenchymal Stem Cells

Despite a wide investigation of hydrogels as an artificial extracellular matrix, there are few scaffold systems for the facile spatiotemporal control of mesenchymal stem cells (MSCs). Here, we report 3D tissue engineered supramolecular hydrogels prepared with highly water-soluble monofunctionalized cucurbit[6]uril-hyaluronic acid (CB[6]-HA), diaminohexane conjugated HA (DAH-HA), and drug conjugated CB[6] (drug-CB[6]) for the controlled chondrogenesis of human mesenchymal stem cells (hMSCs). The mechanical property of supramolecular HA hydrogels was modulated by changing the cross-linking density for the spatial control of hMSCs. In addition, the differentiation of hMSCs was temporally controlled by changing the release profiles of transforming growth factor-β3 (TGF-β3) and/or dexamethasone (Dexa) from the hydrolyzable Dexa-CB[6]. The effective chondrogenic differentiation of hMSCs encapsulated in the monoCB[6]/DAH-HA hydrogel with TGF-β3 and Dexa-CB[6] was confirmed by biochemical glycosaminoglycan content analysis, real-time quantitative PCR, histological, and immunohistochemical analyses. Taken together, we could confirm the feasibility of cytocompatible monoCB[6]/DAH-HA hydrogels as a platform scaffold with controlled drug delivery for cartilage regeneration and other various tissue engineering applications.

Supramolecular hydrogels for long-term bioengineered stem cell therapy.

Synthetic hydrogels have been extensively investigated as artificial extracellular matrices (ECMs) for tissue engineering in vitro and in vivo. Crucial challenges for such hydrogels are sustaining long-term cytocompatible encapsulation and providing appropriate cues at the right place and time for spatio-temporal control of the cells. Here, in situ supramolecularly assembled and modularly modified hydrogels for long-term engineered mesenchymal stem cell (eMSC) therapy are reported using cucurbit[6]uril-conjugated hyaluronic acid (CB[6]-HA), diaminohexane conjugated HA (DAH-HA), and drug-conjugated CB[6] (drug-CB[6]). The eMSCs producing enhanced green fluorescence protein (EGFP) remain alive and emit the fluorescence within CB[6]/DAH-HA hydrogels in mice for more than 60 d. Furthermore, the long-term expression of mutant interleukin-12 (IL-12M) by eMSCs within the supramolecular hydrogels results in effective inhibition of tumor growth with a significantly enhanced survival rate. Taken together, these findings confirm the feasibility of supramolecular HA hydrogels as 3D artificial ECMs for cell therapies and tissue engineering applications.

Effect of osteoconductive hyaluronate hydrogels on calvarial bone regeneration

BackgroundWithout exploitation of possibly immunogenic and carcinogenic bone morphogenetic protein, we developed simple but clinically feasible artificial bone graft using osteoconductive hyaluronate (HA) hydrogels and bioactive MegaGen synthetic bone (MGSB).MethodsHA hydrogels were synthesized by the crosslinking reaction between carboxyl groups of HA and amine groups of gelatin (GEL). Then, artificial bone grafts were prepared by mixing MGSB with HA-GEL hydrogels. The bone regeneration by the MGSB/HA-GEL hydrogel complex was assessed in the skull of New Zealand white male rabbits in 4 and 8 weeks.ResultsHA hydrogels were synthesized by the crosslinking reaction between carboxyl groups of HA and amine groups of gelatin (GEL). Then, artificial bone grafts were prepared by mixing MGSB with HA-GEL hydrogels. In vitro proliferation of preosteogenic cells was enhanced with increasing molecular weight of HA. In addition, histological analysis of dissected tissues with hematoxylin and eosin staining confirmed the effective in vivo bone regeneration by the MGSB/HA-GEL hydrogel complex. The MGSB/HA-GEL hydrogels were well resorbed and partially substituted to the lamellar bone after implantation for 8 weeks.ConclusionsThe novel artificial bone graft of MGSB/HA-GEL hydrogel complex for effective bone regeneration might be clinically feasible for further development.

Genetically engineered mesenchymal stem cell therapy using self-assembling supramolecular hydrogels

Stem cell therapy has attracted a great deal of attention for treating intractable diseases such as cancer, stroke, liver cirrhosis, and ischemia. Especially, mesenchymal stem cells (MSCs) have been widely investigated for therapeutic applications due to the advantageous characteristics of long life-span, facile isolation, rapid proliferation, prolonged transgene expression, hypo-immunogenicity, and tumor tropism. MSCs can exert their therapeutic effects by releasing stress-induced therapeutic molecules after their rapid migration to damaged tissues. Recently, to improve the therapeutic efficacy, genetically engineered MSCs have been developed for therapeutic transgene expression by viral gene transduction and non-viral gene transfection. In general, the number of therapeutic cells for injection should be more than several millions for effective cell therapy. Adequate carriers for the controlled delivery of MSCs can reduce the required cell numbers and extend the duration of therapeutic effect, which provide great benefits for chronic disease patients. In this review, we describe genetic engineering of MSCs, recent progress of self-assembling supramolecular hydrogels, and their applications to cell therapy for intractable diseases and tissue regeneration.

X-ray ablation of hyaluronan hydrogels: Fabrication of three-dimensional microchannel networks

We present a simple and highly versatile protocol for polymer ablation: hard x-ray irradiation makes it possible to rapidly depolymerize hyaluronan hydrogels and fabricate three-dimensional network of microchannels. Photodynamic and photochemical analyses show that x-ray irradiation directly cleaves the polymer backbone and the total dose controls the degradation kinetics. This nonthermal ablation protocol may offer opportunities for processing organic polymers and biological materials.