Jong Won Rhie

Printing three-dimensional tissue analogues with decellularized extracellular matrix bioink

The ability to print and pattern all the components that make up a tissue (cells and matrix materials) in three dimensions to generate structures similar to tissues is an exciting prospect of bioprinting. However, the majority of the matrix materials used so far for bioprinting cannot represent the complexity of natural extracellular matrix (ECM) and thus are unable to reconstitute the intrinsic cellular morphologies and functions. Here, we develop a method for the bioprinting of cell-laden constructs with novel decellularized extracellular matrix (dECM) bioink capable of providing an optimized microenvironment conducive to the growth of three-dimensional structured tissue. We show the versatility and flexibility of the developed bioprinting process using tissue-specific dECM bioinks, including adipose, cartilage and heart tissues, capable of providing crucial cues for cells engraftment, survival and long-term function. We achieve high cell viability and functionality of the printed dECM structures using our bioprinting method.

The correlation between human adipose-derived stem cells differentiation and cell adhesion mechanism.

In recent years, research in the areas of stem cells has dramatically increased, including studies of cellular adhesion to a substrate. We sought to determine the adhesive properties of human adipose-derived stem cells (hASCs) for extracellular matrix proteins. The adhesion of hASCs to collagens and laminin was completely inhibited by a monoclonal antibody, Mab 2253, which binds to the beta1 integrin subunit. These data indicate that hASC adhesion to collagens and laminin was exclusively mediated by an integrin. Cell adhesion on fibronectin (Fn) was inhibited by the heparin-binding peptide (HBP) in the presence of Mab 2253, but not by either Mab 2253 or HBP alone. These results indicate that both the beta1 subunit and the heparan sulfate proteoglycan participated in the cell adhesion to Fn. Microscopic views showed extensive spreading of hASCs cultured on Fn, whereas the cells maintained a round shape when cultured on a heparin-binding domain (HBD) substrate. hASCs differentiated into adipocytes, which stained positive for lipid vacuoles by Oil Red-O analysis, more readily on HBD substrate than on FN substrate. These results suggest that hASCs have an adhesion mechanism for the HBD of Fn and hASC morphology is controlled by the adhesion mechanism and strongly correlated with adipogenic differentiation.

Enhancement of bone regeneration through facile surface functionalization of solid freeform fabrication-based three-dimensional scaffolds using mussel adhesive proteins

Solid freeform fabrication (SFF) is recognized as a promising tool for creating tissue engineering scaffolds due to advantages such as superior interconnectivity and highly porous structure. Despite structural support for SFF-based three-dimensional (3-D) scaffolds that can lead to tissue regeneration, lack of cell recognition motifs and/or biochemical factors has been considered a limitation. Previously, recombinant mussel adhesive proteins (MAPs) were successfully demonstrated to be functional cell adhesion materials on various surfaces due to their peculiar adhesive properties. Herein, MAPs were applied as surface functionalization materials to SFF-based 3-D polycaprolactone/poly(lactic-co-glycolic acid) scaffolds. We successfully coated MAPs onto scaffold surfaces by simply dipping the scaffolds into the MAP solution, which was confirmed through X-ray photoelectron spectroscopy and scanning electron microscopy analyses. Through in vitro study using human adipose tissue-derived stem cells (hADSCs), significant enhancement of cellular activities such as attachment, proliferation, and osteogenic differentiation was observed on MAP-coated 3-D scaffolds, especially on which fused arginine-glycine-aspartic acid peptides were efficiently exposed. In addition, we found that in vivo hADSC implantation with MAP-coated scaffolds enhanced bone regeneration in a rat calvarial defect model. These results collectively demonstrate that facile surface functionalization of 3-D scaffolds using MAP would be a promising strategy for successful tissue engineering applications.

Regulation of osteogenic differentiation of human adipose-derived stem cells by controlling electromagnetic field conditions

Many studies have reported that an electromagnetic field can promote osteogenic differentiation of mesenchymal stem cells. However, experimental results have differed depending on the experimental and environmental conditions. Optimization of electromagnetic field conditions in a single, identified system can compensate for these differences. Here we demonstrated that specific electromagnetic field conditions (that is, frequency and magnetic flux density) significantly regulate osteogenic differentiation of adipose-derived stem cells (ASCs) in vitro. Before inducing osteogenic differentiation, we determined ASC stemness and confirmed that the electromagnetic field was uniform at the solenoid coil center. Then, we selected positive (30/45 Hz, 1 mT) and negative (7.5 Hz, 1 mT) osteogenic differentiation conditions by quantifying alkaline phosphate (ALP) mRNA expression. Osteogenic marker (for example, runt-related transcription factor 2) expression was higher in the 30/45 Hz condition and lower in the 7.5 Hz condition as compared with the nonstimulated group. Both positive and negative regulation of ALP activity and mineralized nodule formation supported these responses. Our data indicate that the effects of the electromagnetic fields on osteogenic differentiation differ depending on the electromagnetic field conditions. This study provides a framework for future work on controlling stem cell differentiation.

In vivo cartilage formation using chondrogenic-differentiated human adipose-derived mesenchymal stem cells mixed with fibrin glue.

Human adipose-derived mesenchymal stem cells (MSCs) were differentiated into chondrogenic MSCs, and fibrin glue was used together to explore the feasibility of whether cartilages can be generated in vivo by injecting the differentiated cells. Mesenchymal stem cells extracted from human adipose were differentiated into chondrogenic MSCs, and such differentiated cells mixed with fibrin glue were injected subcutaneously into the back of the nude mouse. In addition to visual evaluation of the tissues formed after 4, 8, and 12 weeks, hematoxylin-eosin staining, Masson trichrome staining, measurement of glycosaminoglycan concentration using dimethylmethylene blue, agreecan through reverse transcriptase-polymerase chain reaction, type II collagen, and expression of SOX-9 were verified. Moreover, the results were compared with 2 groups of controls: 1 control group that received only injection of chondrogenic-differentiated MSC and the supporting control group that received only fibrin glue injection. For the experimental group, cartilage-like tissues were formed after 4, 8, and 12 weeks. Formation of cartilage tissues was not observed in any of 4, 8, and 12 weeks of the control group. The supporting control group had only a small structure formation after 4 weeks, but the formed structure was completely decomposed by the 8th and 12th weeks. The range of staining dramatically increased with time at 4, 8, and 12 weeks in Masson trichrome staining. The concentration of glycosaminoglycan also increased with time. The increased level was statistically significant with more than 3 times more after 8 weeks compared with 4 weeks and more than 2 times more after 12 weeks compared with 8 weeks. Also, in reverse transcriptase-polymerase chain reaction at 4, 8, and 12 weeks, all results expressed a cartilage-specific gene called aggrecan, type II collagen, and SOX-9. The study verified that the chondrogenic-differentiated MSCs derived from human adipose tissues with fibrin glue can proliferate and form new cartilage. Our findings suggest that formation of cartilages in vivo is possible.

The Stem Cell Potential and Multipotency of Human Adipose Tissue-Derived Stem Cells Vary by Cell Donor and Are Different from Those of Other Types of Stem Cells

Human adipose tissue-derived mesenchymal stem cells (AT-MSCs) from various sites are applied in tissue engineering and cell therapy. The condition of AT-MSCs depends on the donors age, body mass index (BMI), and gender. AT-MSCs from 66 human donors were analyzed, and the cells were sorted according to donor age (10-19 years: n = 1; 20-29 years: n = 5; 30-39 years: n = 12; 40-49 years: n = 22; 50-59 years: n = 12; 60-69 years: n = 9, and 70 years or older: n = 5), BMI (under 25, 25-30, and over 30), and gender (19 males and 48 females). Additionally, AT-MSCs were compared to bone marrow MSCs and chorionic tissue-derived MSCs. We measured the MSC yield, growth rate, colony-forming units, multipotency, and surface antigens. AT-MSC proliferation was greater in cells isolated from individuals aged less than 30 years compared to the proliferation of AT-MSCs from those over 50 years old. BMI was correlated with osteogenic differentiation potency; increased BMI enhanced osteogenesis. Adipogenic differentiation was more strongly induced in cells isolated from donors aged less than 30 years compared to those isolated from other age groups. Also, a BMI above 30 was associated with enhanced adipogenic differentiation compared to cells isolated from individuals with a BMI below 25. Bone marrow MSCs were strongly induced to differentiate along both osteogenic and adipogenic lineages, whereas AT-MSCs predominantly differentiated into the chondrogenic lineage. Therefore, the type of regeneration required and variations among potential donors must be carefully considered when selecting MSCs for use in applied tissue engineering or cell therapy.

Platelet supernatant promotes proliferation of auricular chondrocytes and formation of chondrocyte mass.

&NA; Recently proposed procedures for in vitro generation of new cartilage may be difficult to perform in humans because so many chondrocytes are needed for tissue engineering. In this study the authors investigated new, efficient, low‐cost techniques for the isolation and culture of chondrocytes from the ear cartilage of the rabbit. They performed a low‐density monolayer culture with a low concentration (0.5%, 1%) of human platelet supernatant and observed cell proliferation (seeding efficiency, deoxyribonucleic acid synthesis), matrix synthesis (glycosaminoglycan synthesis), and the expression of type I and type II collagen (reverse transcriptase polymerase chain reaction). Seeding efficiency was increased in 1% of platelet supernatant‐treated cultures by two to three times compared with untreated controls. One percent platelet supernatant had increased the incorporation of [3H]‐thymidine by 1.9 to 2.5 times at 72 hours compared with controls. Glycosaminoglycan synthesis was increased in platelet supernatant‐treated chondrocytes at 96 hours compared with controls. Chondrocytes treated with 1% platelet supernatant showed a decreased expression of the type II collagen gene. Supplementation with a high concentration (10%) of the platelet supernatant provided the conditions for the in vitro chondrocyte mass formation. These results indicate that proliferation and matrix synthesis of auricular chondrocytes is stimulated by a low concentration of platelet supernatant. On the other hand, chondrocytes were immobilized by a high concentration of platelet supernatant. Platelet supernatant may be useful as an inexpensive autologous source of multiple growth factors to enhance chondrocyte proliferation, and also may play the role of scaffold for chondrocytes. Additional investigation is underway to generate culture conditions that promote the differentiation as well as the proliferation of chondrocytes. Yang SY, Ahn ST, Rhie JW, Lee KY, Choi JH, Lee BJ, Oh GT. Platelet supernatant promotes proliferation of auricular chondrocytes and formation of chondrocyte mass. Ann Plast Surg 2000;44:405‐411

Silica nanoparticles increase human adipose tissue-derived stem cell proliferation through ERK1/2 activation

Background Silicon dioxide composites have been found to enhance the mechanical properties of scaffolds and to support growth of human adipose tissue-derived stem cells (hADSCs) both in vitro and in vivo. Silica (silicon dioxide alone) exists as differently sized particles when suspended in culture medium, but it is not clear whether particle size influences the beneficial effect of silicon dioxide on hADSCs. In this study, we examined the effect of different sized particles on growth and mitogen-activated protein kinase signaling in hADSCs. Methods Silica gel was prepared by a chemical reaction using hydrochloric acid and sodium silicate, washed, sterilized, and suspended in serum-free culture medium for 48 hours, and then sequentially filtered through a 0.22 μm filter (filtrate containing nanoparticles smaller than 220 nm; silica NPs). hADSCs were incubated with silica NPs or 3 μm silica microparticles (MPs), examined by transmission electron microscopy, and assayed for cell proliferation, apoptosis, and mitogen-activated protein kinase signaling. Results Eighty-nine percent of the silica NPs were around 50–120 nm in size. When hADSCs were treated with the study particles, silica NPs were observed in endocytosed vacuoles in the cytosol of hADSCs, but silica MPs showed no cell entry. Silica NPs increased the proliferation of hADSCs, but silica MPs had no significant effect in this regard. Instead, silica MPs induced slight apoptosis. Silica NPs increased phosphorylation of extracellular signal-related kinase (ERK)1/2, while silica MPs increased phosphorylation of p38. Silica NPs had no effect on phosphorylation of Janus kinase or p38. Pretreatment with PD98059, a MEK inhibitor, prevented the ERK1/2 phosphorylation and proliferation induced by silica NPs. Conclusion Scaffolds containing silicon dioxide for tissue engineering may enhance cell growth through ERK1/2 activation only when NPs around 50–120 nm in size are included, and single component silica-derived NPs could be useful for bioscaffolds in stem cell therapy.

Endothelial Differentiation and Vasculogenesis Induced by Three-Dimensional Adipose-Derived Stem Cells

Recently, an angiogenic therapy based on adipose‐derived stem cells (ASCs) in an ischemic model has been reported. This study demonstrates the differentiation of human ASCs (hASCs) into endothelial cells clusters by culturing the cells in the form of three dimensional cell masses (3DCMs), which is based on the adherent activity of ASCs for a substrate. The 3DCM composed of hASCs induced hypoxic conditions and expressed angiogenic factors, such as vascular endothelial growth factor and interleukin‐8. From immunochemical staining analysis, the 3DCMs of hASCs were CD31+, KDR+, and CD34+, whereas monolayer‐cultured hASCs were negative for the these markers. To evaluate the ability of vasculature to form within 3DCMs, the 3DCMs were mixed in Matrigel/fibrin gel and injected into mice. Mature tubular microvessels perfused with blood were observed in the 3DCM/gel 20 days after injection, but not in the gel alone or hASC/gel mixture. Vasculature formed in the 3DCM/gel was recognized by antibodies against human α‐smooth muscle actin, KDR, CD31, and CD34, but not by antibodies against murine antigens. These results suggest that the vasculatures originated from the embedded human cells. The 3DCMs of hASCs could function as a source of vascular cells for neovascularization, and could also be co‐implanted with other cell types for regenerative medicine. Anat Rec, 2013.

Single Transconjunctival Incision and Two-point Fixation for the Treatment of Noncomminuted Zygomatic Complex Fracture

The ultimate goal in treating zygomatic complex fracture is to obtain an accurate, stable reduction while minimizing external scars and functional deformity. The present authors present our experiences with a single transconjunctival incision and two-point (inferior orbital rim and frontozygomatic suture) fixation in 53 patients with zygomatic complex fracture which were not comminuted. All patients had transconjunctival approaches with lateral canthal extensions, and six out of 53 patients also had an additional small (about less than 2 cm) gingivobuccal incision to achieve an accurate reduction. There were 3 minor complications, and the overall esthetics and functional results were satisfactory with a long term follow-up. Our method has the following advantages in the reduction of zygomatic complex fracture; It leaves only an inconspicuous lateral canthal scar. In addition, it provides excellent simultaneous visualization of the inferior orbital rim and frontozygomatic suture area. Hence, two-point fixation through a single incision can be performed with a satisfactory stability.