Beob Soo Kim

Human extracellular matrix (ECM) powders for injectable cell delivery and adipose tissue engineering.

Here, we present extracellular matrix (ECM) powders derived from human adipose tissue as injectable cell delivery carriers for adipose tissue engineering. We postulate that human adipose tissue may provide an ideal biomaterial because it contains large amounts of ECM components including collagen. Fresh human adipose tissue was obtained by a simple surgical operation (liposuction). After removing blood and oil components, the tissue was homogenized, centrifuged, freeze-dried, and ground to powders by milling. In an in vitro study, the human ECM powders were highly effective for promotion of cell attachment and proliferation for three-dimensional (3D) cell culture. In in vivo studies, suspensions of human ECM powders containing human adipose-derived stem cells (hASCs) were subcutaneously injected into nude mice. At eight weeks post-injection, numerous blood vessels were observed and the newly formed tissue exhibited adipogenesis with accumulated intracellular small lipid droplets. Overall, the grafts showed well-organized adipose tissue constructs without any signs of tissue necrosis, cystic spaces, or fibrosis. We believe that human ECM powders could act as efficient injectable biomaterials for tissue engineering and have great potential for meeting clinical challenges in regenerative medicine, particularly in relation to adipose tissue engineering.

Decellularized extracellular matrix derived from human adipose tissue as a potential scaffold for allograft tissue engineering

Decellularized tissues composed of extracellular matrix (ECM) have been clinically used to support the regeneration of various human tissues and organs. Most decellularized tissues so far have been derived from animals or cadavers. Therefore, despite the many advantages of decellularized tissue, there are concerns about the potential for immunogenicity and the possible presence of infectious agents. Herein, we present a biomaterial composed of ECM derived from human adipose tissue, the most prevalent, expendable, and safely harvested tissue in the human body. The ECM was extracted by successive physical, chemical, and enzymatic treatments of human adipose tissue isolated by liposuction. Cellular components including nucleic acids were effectively removed without significant disruption of the morphology or structure of the ECM. Major ECM components were quantified, including acid/pepsin-soluble collagen, sulfated glycosaminoglycan (GAG), and soluble elastin. In an in vivo experiment using mice, the decellularized ECM graft exhibited good compatibility to surrounding tissues. Overall results suggest that the decellularized ECM containing biological and chemical cues of native human ECM could be an ideal scaffold material not only for autologous but also for allograft tissue engineering.

Recellularization of decellularized human adipose-tissue-derived extracellular matrix sheets with other human cell types

Decellularized human extracellular matrices (ECMs) are an extremely appealing biomaterial for tissue engineering and regenerative medicine. In this study, we decellularized human adipose tissue, fabricated a thin ECM sheet and explored the potential of this human adipose-derived ECM sheet as a substrate to support the formation of tissues other than adipose tissue. Acellular ECM sheets were fabricated from human adipose tissue through successive physical and chemical treatments: homogenization, centrifugation, casting, freeze-drying and sodium dodecyl sulfate treatment. The ECM sheets exhibited good mechanical properties, despite their porous structure. They degraded quickly in the presence of collagenase and the degradation rate increased with the collagenase concentration in phosphate-buffered saline. Five different human cell types, covering a broad range of cells and applications (normal human dermal fibroblasts, human aortic smooth muscle cells, human chondrocytes, human umbilical vein endothelial cells and human adipose-derived stem cells), were seeded onto the ECM sheets. All the human cell types spread well, proliferated and were successfully integrated into the decellularized ECM sheet. Overall, the results suggest that recellularized ECM sheets are a promising substitute for defective or damaged human tissues.

Human collagen-based multilayer scaffolds for tendon-to-bone interface tissue engineering.

The natural tendon-to-bone region has a gradient in structure and composition, which is translated into a spatial variation of chemical, physical, and biological properties. This unique transitional tissue between bone and tendon is not normally recreated during natural bone-to-tendon healing. In this study, we have developed a human collagen-based multilayer scaffold mimicking the tendon-to-bone region. The scaffold consists of four different layers with the following composition gradient: (a) a tendon layer composed of collagen; (b) an uncalcified fibrocartilage layer composed of collagen and chondroitin sulfate; (c) a calcified fibrocartilage layer composed of collagen and less apatite; (d) a bone layer composed of collagen and apatite. The chemical, physical, and mechanical properties of the scaffold were characterized by a scanning electron microscope, porosimeter, universal tensile machine, Fourier transform infrared spectrometer, energy dispersive X-ray analysis apparatus, and thermogravimetric analysis apparatus. The multilayer scaffold provided a gradual transition of the physical, chemical, and mechanical environment and supported the adhesion and proliferation of human fibroblasts, chondrocytes, and osteoblasts toward each corresponding matrix. Overall, our results suggest the feasibility of a human collagen-based multilayer scaffold for regeneration of hard-to-soft interface tissues.

In Vitro Cartilage Tissue Engineering Using Adipose-Derived Extracellular Matrix Scaffolds Seeded with Adipose-Derived Stem Cells

Extracellular matrix (ECM) secreted from the resident cell of tissue is an ideal biomaterial evolved by nature. Cartilage is also built from well-organized ECM components in a gel-like structure with a high collagen and proteoglycan content. Here, we explored cartilage tissue engineering using ECM scaffolds seeded with stem cells. Both scaffolds and stem cells were isolated from human adipose tissue, which is abundant and easily harvested in the human body. The human ECM scaffolds contained various endogenous bioactive factors, including transforming growth factor-beta1 (TGF-β1, 8782±4989 pg/g, dry ECM), insulin growth factor-1 (13319±1388 pg/g, dry ECM), basic fibroblast growth factor (82373±9572 pg/g, dry ECM), and vascular endothelial growth factor (25647±2749 pg/g, dry ECM). A composite of ECM and stem cells was prepared and cultured in chondrogenic medium (with 10 ng/mL TGF-β1 or not) for 45 days. The volumes and weights of the composites increased during culture and the surface gradually became smooth. Cell viability remained high throughout the 45 days of in vitro culture. Composites showed the formation of cartilage-like tissue with the synthesis of cartilage-specific proteins such as collagen and glycosaminoglycan. Important chondrogenic markers were expressed including Sox-9, aggrecan, and collagen type II and XI. These results demonstrate that a cell/ECM composite containing endogenous bioactive factors could provide biochemical cues for the promotion of cartilage formation.

Electrochemical endotoxin sensors based on TLR4/MD-2 complexes immobilized on gold electrodes.

Even low concentrations of endotoxins can be life-threatening. As such, continuous effort has been directed toward the development of sensitive and specific endotoxin detection systems. In this paper, we report the design and fabrication of a new electrochemical endotoxin sensor based on a human recombinant toll-like receptor 4 (rhTLR4) and myeloid differentiation-2 (MD-2) complex. The rhTLR4/MD-2 complex, which specifically binds to endotoxin, was immobilized on gold electrodes through a self-assembled monolayer (SAM) technique involving the use of dithiobis(succinimidyl undecanoate) (DSU). The surface topography of the electrodes at each fabrication stage was characterized with a nanosurface profiler and atomic force microscope (AFM). The electrochemical signals generated from interactions between the rhTLR4/MD-2 complex and the endotoxin were characterized by cyclic voltammetry (CV) and differential pulse voltammetry (DPV). A linear relationship between the peak current and endotoxin concentration was obtained in the range of 0.0005 to 5 EU/mL with a correlation coefficient (R(2)) of 0.978. The estimated limit of detection (LOD) was fairly low, 0.0002 EU/mL. The rhTLR4/MD-2 based sensors exhibited no current responses to dipalmitoylphosphatidylcholine (DPPC) bearing two lipid chains, which is structurally similar to endotoxin, indicating the high specificity of the sensors to endotoxin.

Human collagen isolated from adipose tissue.

Collagen, the most abundant protein in vertebrates, is a useful biomaterial in pharmaceutical and medical industries. So far, most collagen has been extracted from animals and cadavers. Herein, we suggest human adipose tissue, which is routinely abandoned after liposuction, as a plentiful source of human collagen. In this study, human collagen was obtained from adipose tissue through two successive major steps: (i) extraction of the extracellular matrix (ECM) by pulverization, centrifugation, alkaline, and alcohol treatment; (ii) isolation of collagen from ECM by pepsin treatment in dilute acetic acid. The purified human adipose‐derived collagen was characterized by Fourier transform infrared spectroscopy, polyacrylamide gel electrophoresis, amino acid analysis, and circular dichroism spectroscopy. The extracted collagen showed a typical triple helix structure, good thermal stability due to abundant imino acids, and high solubility at acidic pH. The collagen greatly facilitated the adhesion and proliferation of human adipose‐derived stem cells and normal human dermal fibroblasts on polystyrene plates. These results suggest that human adipose tissue obtained by liposuction can provide human collagen for use in cosmetics, pharmaceutics, and medicine.

Improvement of Stem Cell Viability in Hyaluronic Acid Hydrogels Using Dextran Microspheres

Although hyaluronic acid (HA) has been widely used in clinics as an injectable biomaterial, it may not be appropriate as an injectable stem cell carrier because highly hydrophilic HA hydrogels provide an unfavorable environment in which the encapsulated stem cells are likely to be constrained to a round shape, thereby losing their native morphology. Herein, we hypothesized that dextran microspheres (DMs) can improve stem cell viability in HA hydrogels because they can act as substrates for stem cell adhesion, spreading and proliferation. DMs with a mean diameter of 80 μm were mixed with HA hydrogels. Human adipose-derived stem cells (hASCs) were isolated from human adipose tissue and seeded into the DM-incorporated HA hydrogels. When compared with the hydrogels alone, the number of viable cells was significantly increased in the presence of the DMs. Initially, hASCs appeared to be round in the HA hydrogels. At 12 h after seeding, the hASCs apparently attached onto the DMs and became slightly flattened. One day after seeding, the hASCs seemed to spread onto the surface of the DMs. Fluorescence micrography of live and dead cells confirmed that the cell viability was significantly improved by use of the DMs in HA hydrogels. Overall results demonstrated that the microsphere/hydrogel composite supported stem cell survival and spreading. These characteristics show the potential for use of the composite in cell-delivery and tissue-engineering applications.

Facile Preparation of Biodegradable Glycol Chitosan Hydrogels Using Divinyladipate as a Crosslinker

Biodegradable, pH-sensitive, glycol chitosan (GC) hydrogels were prepared using divinyl adipate (DVA) as a crosslinker and acetic acid as a catalyst. DVA has highly reactive double vinyl ester groups and GC contains a high density of hydroxyl groups, with two in every glucosamine unit. The transesterification reaction between vinyl esters and hydroxyl groups produced crosslinked GC hydrogels. The initial crosslinking reaction was monitored by measuring the viscosity of the reaction mixture. When DVA was added to the GC solution and heated to 50 °C, the viscosity of the GC solution gradually increased, implying a crosslinking reaction and hydrogel formation. A new peak from the ester group was observed in the FTIR spectra of the GC hydrogels, confirming the crosslinking reaction. The synthesized GC hydrogel showed pH-dependent water absorbency, mainly due to the presence of amine groups (-NH2) at the C-2 position of the glucosamine unit of GC. The water absorbency greatly increased at acidic pH and slightly decreased at alkaline pH. The GC hydrogel gradually degraded in 37°C water due to hydrolysis of the ester bonds, which were intermolecular crosslinking sites. A red dye, 5-carboxyltetramethyl-rhodamine (CTMR), was entrapped in the GC hydrogels as a model compound. CTMR was released from GC hydrogels in two steps: an initial burst release mainly due to desorption and diffusion, and a second sustained release possibly due to gradual degradation.