Wook Chun

Coaxial structured collagen–alginate scaffolds: fabrication, physical properties, and biomedical application for skin tissue regeneration

Collagen is the most promising natural biomaterial and has been used in various tissue engineering applications for skin, bone, and cartilage because it provides good biocompatibility and low antigenicity. Although collagen is an excellent candidate material for various biomedical applications, its difficult processability and mechanical properties have remained important limitations. To overcome the problems, several methods including indirect printing combined with a sacrificing mold and low-temperature printing were suggested. However, it is difficult to fabricate precisely controlled 3D pore structure using the methods. In a previous study, we introduced a three-dimensional (3D) pore-structure-controlled collagen scaffold fabricated by a 3D dispensing system supplemented with a cryogenic and freeze-drying system. The fabricated scaffold had remarkably good cellular behaviour (cell migration and differentiation) but poor mechanical stability due to the highly porous structure consisting of micro-sized strands and poor mechanical nature of collagen. To overcome this deficiency, we designed a hybrid (core/shell) scaffold composed of an outer collagen and an inner alginate. The collagen/alginate scaffolds exhibited good structural stability (core–shell structure), increased Youngs modulus about seven times compared to pure collagen scaffold under a similar pore-structure, and resulted in good cell viability, similar to a pure collagen scaffold. In an in vivo test, the hybrid scaffold was used as a dermal substitute and provided good granulation tissue formation and rapid vascularisation.

Designed Three-Dimensional Collagen Scaffolds for Skin Tissue Regeneration

One of the challenges in tissue engineering is the development of a reproducible three-dimensional (3D) scaffold to support cell migration and infiltration. As a dermal substitute, 3D collagen scaffolds with precisely controlled pore structures were fabricated using an innovative cryogenic dispenser system. The scaffolds were composed of perpendicular, highly porous collagen strands in successive layers. The fabricated scaffolds were evaluated in an in vitro keratinocyte/fibroblast coculture test. Fibroblasts were well dispersed within the scaffold, and keratinocytes had completely migrated through the well-designed pore structure and differentiated on top of the scaffold surface. The differentiated keratinocytes generated a stratum corneum in the 3D dispensed scaffolds, similar to that in normal skin tissue.

A cryogenic direct-plotting system for fabrication of 3D collagen scaffolds for tissue engineering

The goal of tissue engineering is to repair or regenerate damaged tissue using a combination of cellular biology and materials engineering techniques. One of the challenging problems in tissue engineering is the development of a reproducible three-dimensional (3D) scaffold to support cell migration and infiltration. Although natural polymers, such as dissolved collagen or alginate, are considered ideal for this purpose, their hydrophilic properties have hindered the fabrication of designed 3D scaffold structures. To overcome this problem, we developed a novel system for the cryogenic plotting of 3D scaffolds. Using this technique, we created various 3D collagen scaffolds with designed pore structures that exhibited desired properties. The diameter of the individual collagen strands, which varied from 250 µm to 500 µm, was reproducibly dependent on processing parameters, and the final collagen scaffold showed little shrinkage (less than 12%) relative to the initial design. To evaluate the fabricated scaffold, we adapted the scaffold to regenerate skin tissue. Immunohistochemical analysis demonstrated that co-cultured keratinocytes and fibroblasts completely migrated throughout the 3D collagen scaffold and keratinocytes were well differentiated on the surface of scaffold like a human skin.

The use of AlloDerm on major burn patients: AlloDerm prevents post-burn joint contracture

In efforts to prevent and reduce joint contracture and scar formation after burn, we used the acellular human dermis (AlloDerm) as a dermal replacement in the acute stage. A total of 64 patients received AlloDerm graft selectively on joint areas during the study period from March, 2005 to July, 2007. From January to March, 2008, a total of 31 patients returned to our burn center to examine the functional results by measuring range of motion of joints. Additionally, the quality of grafted skin condition criteria of skin elasticity, scar thickness, trans-epidermal water loss, melanin and erythema level was measured in a total of 11 patients among them. By analyzing the limitation level of 55 joints excluding hand areas, we found that 24 joints (43.6%) showed no limitations, 12 joints (21.8%) showed limitations below 10%, 16 joints (29.1%) showed limitations between 10 and 19% and 3 joints (5.5%) showed limitations over 20%. The scar thickness of non-AlloDerm applied areas was 2.5+/-0.9 mm and AlloDerm applied areas was 1.8+/-0.7 mm (p = 0.396). Trans-epidermal water loss for non-AlloDerm applied areas was 20.9+/-7.7 g/h/m(2) and AlloDerm applied areas was 10.8+/-3.4 g/h/m(2) (p<0.001). Erythema value for non-AlloDerm applied areas was 436.1+/-65.8, whereas AlloDerm applied area was 394.4+/-61.2 (p<0.001). Acellular dermal matrix is a good option for treating major burns to prevent scar formation after burn and loss of joint function.

An Innovative Collagen-Based Cell-Printing Method for Obtaining Human Adipose Stem Cell-Laden Structures Consisting of Core–Sheath Structures for Tissue Engineering

Three-dimensional (3D) cell printing processes have been used widely in various tissue engineering applications due to the efficient embedding of living cells in appropriately designed micro- or macro-structures. However, there are several issues to overcome, such as the limited choice of bioinks and tailor-made fabricating strategies. Here, we suggest a new, innovative cell-printing process, supplemented with a core-sheath nozzle and an aerosol cross-linking method, to obtain multilayered cell-laden mesh structure and a newly considered collagen-based cell-laden bioink. To obtain a mechanically and biologically enhanced cell-laden structure, we used collagen-bioink in the core region, and also used pure alginate in the sheath region to protect the cells in the collagen during the printing and cross-linking process and support the 3D cell-laden mesh structure. To achieve the most appropriate conditions for fabricating cell-embedded cylindrical core-sheath struts, various processing conditions, including weight fractions of the cross-linking agent and pneumatic pressure in the core region, were tested. The fabricated 3D MG63-laden mesh structure showed significantly higher cell viability (92 ± 3%) compared with that (83 ± 4%) of the control, obtained using a general alginate-based cell-printing process. To expand the feasibility to stem cell-embedded structures, we fabricated a cell-laden mesh structure consisting of core (cell-laden collagen)/sheath (pure alginate) using human adipose stem cells (hASCs). Using the selected processing conditions, we could achieve a stable 3D hASC-laden mesh structure. The fabricated cell-laden 3D core-sheath structure exhibited outstanding cell viability (91%) compared to that (83%) of an alginate-based hASC-laden mesh structure (control), and more efficient hepatogenic differentiations (albumin: ∼ 1.7-fold, TDO-2: ∼ 7.6-fold) were observed versus the control. The selection of collagen-bioink and the new printing strategy could lead to an efficient way to achieve 3D cell-laden mesh structures that mimic the anatomical architecture of a patients defective region.

The effect of burn rehabilitation massage therapy on hypertrophic scar after burn: A randomized controlled trial

OBJECTIVE To evaluate the effect of burn rehabilitation massage therapy on hypertrophic scar after burn. METHOD One hundred and forty-six burn patients with hypertrophic scar(s) were randomly divided into an experimental group and a control group. All patients received standard rehabilitation therapy for hypertrophic scars and 76 patients (massage group) additionally received burn scar rehabilitation massage therapy. Both before and after the treatment, we determined the scores of visual analog scale (VAS) and itching scale and assessed the scar characteristics of thickness, melanin, erythema, transepidermal water loss (TEWL), sebum, and elasticity by using ultrasonography, Mexameter(®), Tewameter(®), Sebumeter(®), and Cutometer(®), respectively. RESULTS The scores of both VAS and itching scale decreased significantly in both groups, indicating a significant intragroup difference. With regard to the scar characteristics, the massage group showed a significant decrease after treatment in scar thickness, melanin, erythema, TEWL and a significant intergroup difference. In terms of scar elasticity, a significant intergroup difference was noted in immediate distension and gross skin elasticity, while the massage group significant improvement in skin distensibility, immediate distension, immediate retraction, and delayed distension. CONCLUSION Our results suggest that burn rehabilitation massage therapy is effective in improving pain, pruritus, and scar characteristics in hypertrophic scars after burn.

A novel cell-printing method and its application to hepatogenic differentiation of human adipose stem cell-embedded mesh structures

We report a cell-dispensing technique, using a core–shell nozzle and an absorbent dispensing stage to form cell-embedded struts. In the shell of the nozzle, a cross-linking agent flowed continuously onto the surface of the dispensed bioink in the core nozzle, so that the bioink struts were rapidly gelled, and any remnant cross-linking solution during the process was rapidly absorbed into the working stage, resulting in high cell-viability in the bioink strut and stable formation of a three-dimensional mesh structure. The cell-printing conditions were optimized by manipulating the process conditions to obtain high mechanical stability and high cell viability. The cell density was 1 × 107 mL−1, which was achieved using a 3-wt% solution of alginate in phosphate-buffered saline, a mass fraction of 1.2 wt% of CaCl2 flowing in the shell nozzle with a fixed flow rate of 0.08 mL min−1, and a translation velocity of the printing nozzle of 10 mm s−1. To demonstrate the applicability of the technique, preosteoblasts and human adipose stem cells (hASCs) were used to obtain cell-laden structures with multi-layer porous mesh structures. The fabricated cell-laden mesh structures exhibited reasonable initial cell viabilities for preosteoblasts (93%) and hASCs (92%), and hepatogenic differentiation of hASC was successfully achieved.

Neurites from PC12 cells are connected to each other by synapse-like structures

PC12 cells have been used as a model of sympathetic neurons. Nerve growth factor (NGF), basic fibroblast growth factor (bFGF), and cAMP induce neurite outgrowth from PC12 cells. cAMP induced a greater number of neurites than did NGF. In particular, we attempted to elucidate whether PC12 cell neurites, induced by several factors including NGF, bFGF, and cAMP, form synapses, and whether each neurite has presynaptic and postsynaptic properties. Using scanning electron microscopy (SEM) and transmission electron microscopy (TEM), we observed that neurites are connected to each other. The connected regions presented dense core vesicles and a clathrin‐coated membrane invagination. In addition, typical maker proteins for axon and dendrite were identified by an immuno‐staining method. Tau‐1, an axonal marker in neurons, was localized at a high concentration in the terminal tips of neurites from PC12 cells, which were connected to neurite processes containing MAP‐2, a dendritic marker in neurons. Furthermore, neurites containing SV2 and synaptotagmin, markers of synaptic vesicles, were in contact with neurites harboring drebrin, a marker of the postsynaptic membrane, suggesting that neurites from PC12 cells induced by NGF, bFGF, and cAMP may form synapse‐like structures. Tat‐C3 toxin, a Rho inhibitor, augmented neurite outgrowth induced by NGF, bFGF, and cAMP. Tat‐C3 toxin together with neurotrophins also exhibited synapse‐like structures between neurites. However, it remains to be studied whether RhoA inhibition plays a role in the formation of synapse‐like structures in PC12 cells. Synapse 64:765–772, 2010.

Inhalation injury in burn patients: Establishing the link between diagnosis and prognosis

This study was to re-evaluate inhalation injury as a prognostic factor in burn patients and to determine the factors that should be considered when refining the definition of inhalation injury. A total of 192 burn patients (152 men, 40 women; mean age, 46.1±13.8 years) who were suspected to have an inhalation injury and underwent bronchoscopy between January 2010 and June 2012 were included in this prospective observational study. All patients underwent bronchoscopy within 24h of sustaining the burn. The bronchoscopic findings were classified as normal, mild, moderate, and severe. Mechanical ventilation was administered, when required. Age, percentage of TBSA burned, ABSI score, requirement of mechanical ventilation and PF ratio, but not inhalation injury, COHb level, and bronchoscopic grades, significantly differed between the survivors and non-survivors (p<0.05). Mechanical ventilation (adjusted odds ratio [OR]: 9.787) and severe inhalation injury on bronchoscopy (adjusted OR: 45.357) were independent predictors of mortality on multivariate logistic regression analysis. Inhalation injury diagnosed through history does not predict mortality from burns. Other components such as severity of inhalation injury determined using bronchoscopy, and administration of mechanical ventilation might help predict the morbidity and mortality of burn patients with inhalation injury and all of the factors should be considered when the definition of inhalation injury is refined.

Inflammatory cytokines and their prognostic ability in cases of major burn injury.

Background Major burn injuries induce inflammatory responses and changes in the levels of various cytokines. This study was conducted to assess early changes in the serum levels of inflammatory cytokines after burn injury, identify cytokines associated with mortality, and characterize correlations among cytokines. Methods Blood samples of 67 burn patients were collected on days 1 and 3 after burn injury, and the concentrations of 27 cytokines were measured using the Bio-Plex Suspension Array System (Bio-Rad Laboratories, USA). Blood samples of 25 healthy subjects were used as controls. We analyzed statistical differences in the concentrations of each cytokine between the control and patient groups, between day 1 and day 3, and between survival and nonsurvival groups. Correlations among 27 cytokines were analyzed. Results Median concentrations of granulocyte colony-stimulating factor (G-CSF), granulocyte macrophage colony-stimulating factor (GM-CSF), interleukin 1 receptor antagonist (IL-1RA), interleukin 6 (IL-6), interleukin 8 (IL-8), interleukin 10 (IL-10), interleukin 15 (IL-15), monocyte chemoattractant protein-1 (MCP-1), macrophage inflammatory protein 1β (MIP-1β), and vascular endothelial growth factor (VEGF) were significantly higher in burn patients than in controls. IL-1RA, IL-6, and MCP-1 levels were significantly higher in the nonsurvival group than in the survival group on day 1 after burn injury. Correlation analysis of 27 cytokines showed different relationships with one another. Stronger correlations among interferon γ (IFN-γ), IL-2, IL-4, IL-7, IL-12p70, and IL-17 were found. Conclusions IL-1RA, IL-6, and MCP-1 may be used as prognostic indicators of mortality in burn patients and the increase in cytokine concentrations is induced by interactions within a complex network of cytokine-related pathways.