Sang-Hyug Park

Potential of Fortified Fibrin/Hyaluronic Acid Composite Gel as a Cell Delivery Vehicle for Chondrocytes

Numerous treatment methods have been applied for use in cartilage repair, including abrasion, drilling, and microfracture. Although chondrocyte transplantation is the preferred treatment, it has some shortcomings, such as difficulty of application (large and posterior condylar regions), poor chondrocyte distribution, and potential cell leakage from the defect region. The cell delivery system of the tissue engineering technique can be used to overcome these shortcomings. We chose fibrin/hyaluronan (HA) composite gel as an effective cell delivery system to resolve these issues. Both components are derived from natural extracellular matrix. In the first trial, fortified fibrin/HA composite gels with rabbit chondrocytes were tested by implantation in nude mice. At 4 weeks, glycosaminoglycan contents in the fibrin/HA composite (0.186 +/- 0.006 mg/mg) were significantly higher than those in the presence of fibrin alone (0.153 +/- 0.017 mg/mg). As a next step, we applied the fibrin/HA composite gel to animal cartilage defects using full thickness cartilage defect rabbit models. The fibrin/HA composite gel with rabbit chondrocytes (allogenic) was implanted into the experimental group, and the control group was implanted with the fibrin/HA composite gel alone. Implanted chondrocytes with the fibrin/HA composite showed improved cartilage formation. In conclusion, the key to successful regeneration of cartilage is to provide the repair site with a sufficient supply of chondrogenic cells with a suitable delivery vehicle to ensure maximal differentiation and deposition of the proper extracellular matrix. This study suggests the feasibility of tissue-engineered cartilage formation using fibrin/HA composite gel.

Mechanical Stimulation by Ultrasound Enhances Chondrogenic Differentiation of Mesenchymal Stem Cells in a Fibrin‐Hyaluronic Acid Hydrogel

Chondrogenic differentiation and cartilage tissue formation derived from stem cells are highly dependent on both biological and mechanical factors. This study investigated whether or not fibrin-hyaluronic acid (HA) coupled with low-intensity ultrasound (LIUS), a mechanical stimulation, produces an additive or synergistic effect on the chondrogenesis of rabbit mesenchymal stem cells (MSCs) derived from bone marrow. For the purpose of comparison, rabbit MSCs were first cultured in fibrin-HA or alginate hydrogels, and then subjected to chondrogenic differentiation in chondrogenic-defined medium for 4 weeks in the presence of either transforming growth factor-beta3 (TGF-β3) (10 ng/mL) or LIUS treatment (1.0 MHz and 200 mW/cm(2) ). The resulting samples were evaluated at 1 and 4 weeks by histological observation, chemical assays, and mechanical analysis. The fibrin-HA hydrogel was found to be more efficient than alginate in promoting chondrogenesis of the MSCs by producing a larger amount of sulfated glycosaminoglycans (GAGs) and collagen, and engineered constructs made with the hydrogel demonstrated higher mechanical strength. At 4 weeks of tissue culture, the chondrogenesis of the MSCs in fibrin-HA were shown to be further enhanced by treatment with LIUS, as observed by analyses for the amounts of GAGs and collagen, and mechanical strength testing. In contrast, TGF-β3, a well-known chondrogenic inducer, showed a marginal additive effect in the amount of collagen only. These results revealed that LIUS further enhanced chondrogenesis of the MSCs cultured in fibrin-HA, in vitro, and suggested that the combination of fibrin-HA and LIUS is a useful tool in constructing high-quality cartilage tissues from MSCs.

Synthesis of Arg–Gly–Asp (RGD) Sequence Conjugated Thermo-Reversible Gel via the PEG Spacer Arm as an Extracellular Matrix for a Pheochromocytoma Cell (PC12) Culture

Adhesion molecules composed of Gly–Arg–Gly–Asp–Ser (GRGDS) peptides and cell recognition ligands were inculcated into thermo-reversible hydrogel composed of N-isopropylacrylamide, with a small amount of succinyl poly(ethylene glycol) (PEG) acrylate (MW 3400) used as a biomimetic extracellular matrix (ECM). The GRGDS-containing p(NiPAAm-co-PEG) copolymer gel was studied in vitro for its ability to promote cell spreading and to increase the viability of cells by introducing PEG spacers. Hydrogel lacking the adhesion molecules proved to be a poor ECM for adhesion, permitting only a 20% spread of the seeded cells after 10 days. When PEG spacer arms, immobilized by a peptide linkage, had been integrated into the hydrogel, conjugation of RGD promoted cell spread by 600% in a 10-day trial. In addition, in a serum-free medium, only GRGDS peptides conjugated with the spacer arm were able to promote cell spread. In terms of the cell viability, GRGDS peptides conjugated with the PEG-carrying copolymer gel specifically mediated cell spread. This result supports the theory that specific recognition is the result of interaction between the integrin families on the fibroblast, and the RGD sequence on the p(NiPAAm-co-PEG) copolymer gel.

Three dimensional plotted extracellular matrix scaffolds using a rapid prototyping for tissue engineering application

Naturally derived biomaterials are rarely used in advanced rapid prototyping technology despite their superior biocompatibility. The main problem of natural material plotting is the high sensitivity of materials concentration and viscosity on the plotting nozzle. The aim of the current study was to develop a three dimensional (3D) plotting system capable of dispensing extracellular matrix (ECM)-c (ECM powder blended collagen) and manufacture various shapes of ECM-c scaffolds to apply for irregular defects. We had adapted a powder-based plotting approach to print the stable 3D construct using only cartilage derived ECM materials. This study successfully developed the plotting method for high viscous ECM-c material and showed the 3D plotted scaffolds with high interconnected pores as well as complex shape. Furthermore, cell culture results proved that plotted ECM-c scaffolds were able to provide a suitable environment for cell attachment, proliferation and chondrogenesis. This study shows the 3D printing feasibility of ECM natural material has demonstrated as a first time. We believe our results will offer a meaningful step toward the 3D scaffold printing based on natural ECM materials for future organ printing.

Fabrication of an osteochondral graft with using a solid freeform fabrication system

Current approaches for the engineering of osteochondral grafts are associated with poor tissue formation and compromised integration at the interface between the cartilage and bone layers. Many researchers have attempted to provide osteochondral grafts of combined cartilage and bone for osteochondral repair to help overcome the limitations of standard procedures. Solid freeform fabrication is recognized as a promising tool for creating tissue engineering scaffolds due to advantages such as superior interconnectivity and a highly porous structure. This study aimed to develop a three-dimensional plotting system to enable the manufacturing of a biphasic graft consisting cartilage and subchondral bone for application to osteochondral defects. The material advantages of both synthetic (poly L lactide-co-polyglycolide) and natural (alginate) polymers were combined for a supporting frame and cell printing. Specifically, in order to promote the maturity of the osteochondral graft in our study, cartilage-derived ECM (cECM) or hydroxyapatate (HA) substances blended with alginate was plotted together with human fetal cartilage-derived progenitor cells in the cartilage or subchondral bone layer under a multi-nozzle deposition system. Notably, a plotted biphasic graft shows good integration between cartilage and subchondral bone layers without structural separation. Furthermore, the non-toxicity of the cECM and HA substances were proved from a live/dead assay of plotted cell-laden alginate. A fabricated osteochondral graft with cECM and HA substances showed dominant cartilage and bone tissue formation in a differentiation assay. Future studies should be done to modify the alginate physical properties for long-lasting structural stability.

Fabrication and Preliminary Test Results of A MEMS Cell Stimulator for Stem Cell Research

This paper presents the fabrication and preliminary test results of a MEMS cell stimulator, which has never been tried and reported. It provides a new tool of cell stimulation, culture and analysis for stem cell research. MEMS cell stimulator is designed to apply compressive pressure to the hMSCs (human mesenchymal stem cells) for inducing chondrogenesis. MEMS cell stimulator is based on the pneumatic actuator with a flexible diaphragm. It consists of air chamber and cell-media chamber. hMSCs under the cyclic compressive stimulation for one week are observed and examined. The results show the cyclic mechanical stimulation makes the different phenomenon of the cultured hMSCs in cell growth and membrane. This is an important result in terms of making a feasibility of MEMS cell stimulator to provide the reduction of the necessary quantity of stem cells, process cost and increases the throughput

Effect of joint mimicking loading system on zonal organization into tissue-engineered cartilage

Cartilage tissue engineering typically involves the combination of a biodegradable polymeric support material with chondrocytes. The culture environment in which cell–material constructs are created and stored is an important factor. The aim of the present study was to investigate the effects of combined stimuli on cartilage zonal organization which is important to maintain cartilage functions such as lubrication and cushion. For that purpose, we developed a joint mimicking loading system which was composed of compression and shear stress. To mimic the joint loading condition, we manufactured a stimuli system that has a device similar to the shape of a femoral condyle in human knee. The fibrin/hyaluronic acid mixture with chondrocytes were dropped into support made of silicon, and placed under the device. The cartilage explants were stimulated with the joint mimicking loading system for 1 hour per day over the course of 4 weeks. The amounts of GAG and collagen in the stimulated tissue were more than that of the static cultured tissue. Cells and collagen were arranged horizontally paralleled to the surface by stimuli, while it did not happen in the control group. The results of this study suggests that mechanical load exerting in the joint play a crucial role in stimulation of extracellular matrix (ECM) production as well as its functional rearrangement.

Effects of the polarity and waveform of the stimulus pulse train on the electrically-elicited pressure sensation

This study was designed to investigate effects of the polarity and waveform of the electrical stimulus pulse train on the electrically-elicited tactile sensation in terms of the sensory activation threshold. The experiments were conducted on 49 healthy subjects for 1) different polarities (anodic and cathodic), and 2) different waveforms (monophasic and biphasic). The findings included that 1) the tactile sensation became adapted to the constant stimulation, 2) the polarity of the pulse train did not make a significant difference in the activation threshold, 3) the required stimulation intensity for the biphasic waveform was almost twice higher than the monophasic waveform, 4) the activation threshold values showed a poor inter-individual consistency, but a strong intra-individual consistency, and 5) our stimulation protocol could be used for eliciting a pressure sensation.

Comparing of various collagens for application of cartilage tissue engineering

Mammalian collagens have been used as a base material for collagen matrices in tissue engineering applications. However, collagens of aquatic animals and human sources can potentially be utilized as a safe and viable substitute, because collagen products of bovine origin have been shown to be contaminated with some diseases. In the present study, we prepared and investigated collagen materials from several sources (bovine skin, porcine skin, amniotic membrane and starfish) as matrix biomaterials. Detailed investigations on their physicochemical and biological properties, such as amino acid composition, thermal transition temperature, molar mass, IR spectra, and cell response, suggested strong relations between their amino acid composition and intermolecular structure, thermal property, and cell response. Selectively, an amniotic membrane collagen scaffold was evaluated for cartilage tissue engineering in three types of three-dimensional 3D culture (sponge, gel and micro bead forms) and compared with a bovine matrix. Results showed that amniotic membrane collagen has a potential as an alternative source of collagen for use in tissue engineering.