Wojong Yang

Effects of Natural Cartilaginous Extracellular Matrix on Chondrogenic Potential for Cartilage Cell Transplantation

Autologous chondrocyte transplantation (ACT) has been established to contribute cartilage regeneration over the past years; however, many obstacles need to be overcome. Recently, newer ACT technique involves cotransplantation of chondrocytes and biomaterial. Although various proposed intelligent biomaterials exist, many of them remain insufficient and controversial. In this study, we aimed to examine the effects of natural extracellular matrix (ECM) to the proliferation rate and differentiation on the chondrocytes. We first derived a natural ECM sheet from 10-μm-thick frozen sections of porcine knee cartilages. We then cultured the chondrocytes derived from a rabbits knee on a dish precoated with the natural ECM. Then we assessed differentiation and chondrogenic potential of the cells compared with those grown in untreated culture dishes. We characterized the gene expression of chondrogenic markers, such as collagen type II, SOX-9, and aggrecan, as well as the level of ECM protein with the use of reverse-transcription polymerase chain reaction analysis. The cells cultured with the ECM sheet showed highest chondrogenic potential and differentiation. Therefore, we can induce good chondrogenesis by with the use of a natural ECM sheet on the culture dish. The readily available and easy-to-handle thin ECM sheets create an environment that promotes efficient cartilage regeneration. Our data suggest that this natural ECM scaffold improved the chondrogenic differentiation of the cells in vitro by providing a favorable microenvironment.

Applications and Implications of Heparin and Protamine in Tissue Engineering and Regenerative Medicine

Drug repositioning is one of the most rapidly emerging fields of study. This concept is anchored on the principle that diseases have similar damaged or affected signaling pathways. Recently, drugs have been repositioned not only for their alternative therapeutic uses but also for their applications as biomaterials in various fields. However, medical drugs as biomaterials are rarely focused on in reviews. Fragmin and protamine have been recently the sources of increasing attention in the field of tissue engineering and regenerative medicine. Fragmin and protamine have been manufactured primarily as a safe antidote for the circulating heparin. Lately, these drugs have been utilized as either micro- or nanoparticle biomaterials. In this paper, we will briefly describe the concept of drug repositioning and some of the medical drugs that have been repurposed for their alternative therapeutic uses. Also, this will feature the historical background of the studies focused on fragmin/protamine micro/nanoparticles (F/P M/NPs) and their applications as biomaterials in tissue engineering, stem cell therapy, and regenerative medicine.

Artificial Islets From Hybrid Spheroids of Three Pancreatic Cell Lines

Pancreatic islets have been the focus of recent studies exploring the pathologic mechanisms of diabetes mellitus as well as more effective and radical treatments for this disease. Islet transplantation is a promising therapeutic strategy; however, isolation of pancreatic islets for this purpose has been challenging, because the technique is time consuming and technically difficult, and tissue handling can be variable. Pseudo-islets can be used as an alternative to naïve islets, but require cellular sources or artificial materials. In this study, pancreas-derived cells were used to generate pseudo-islets. Because the pancreas is composed of a variety of cell types, namely α cells, β cells, δ cells, and other pancreatic cells that perform different functions, we used 3 different cell lines-NIT-1 (a β-cell line), α TC1 clone 6 (an α-cell line), and TGP52 (a pancreatic epithelial-like cell line)-which we cocultured in nonadhesive culture plates to produce hybrid cellular spheroids. These pseudo-islets had an oval shape and were morphologically similar to naïve islets; additionally, they expressed and secreted the pancreatic hormones insulin, glucagon, and somatostatin, as confirmed by reverse-transcription polymerase chain reaction and enzyme-linked immunosorbent assay. The results demonstrate that pseudo-islets that mimic naïve islets can be successfully generated by a coculture method. These artificial islets can potentially be used for in vitro tests related to diabetes mellitus, specifically, in drug discovery or for investigating pathology. Moreover, they can be useful for examining basic questions pertaining to cell-cell interactions and tissue development.

Effect of Cryopreservation and Cell Passage Number on Cell Preparations Destined for Autologous Chondrocyte Transplantation

Autologous chondrocyte transplantation (ACT) is an effective and safe therapy for repairing articular cartilage defects and requires cell preservation and subculture before transplantation. We compared the effects of cryopreservation and passaging on cell viability, proliferation, and maintenance of the function of chondrocytes and synovium-derived mesenchymal stem cells (MSCs) used as sources for ACT. These cells were isolated from the knee joints of rabbits and were cultured, passaged serially, and divided into 2 groups that were either cryopreserved or not. The morphology, viability, gene expression, and differentiation potential of the 2 groups were compared. Maintenance of the potential to undergo chondrogenic differentiation was determined with the use of a 3-dimensional culture method. Passaging and cryopreservation significantly affected the ability of chondrocytes to maintain their morphology, express chondrogenic genes, and differentiate. In contrast, synovium-derived cells were not affected by passaging and cryopreservation. Our results may serve as the foundation for the application of passaged and cryopreserved chondrocyte or other source cells of MSCs in ACT.

Effect of Preservation Conditions on Cartilage Tissue for Cell Transplantation

Autologous chondrocyte transplantation involves isolating chondrocytes from a patients cartilage tissue. Storage conditions such as storage time and temperature are important for the quality of the isolated cells. However, few studies have focused on variables for optimum tissue preservation, and there is neither an established method for storing cartilage nor reliable reports on how different conditions affect the isolated chondrocytes. Therefore, in the present study, we stored cartilage in various preservation solutions, under different temperatures, and for varying durations and determined their effects on the characteristics and viability of isolated chondrocytes. We assessed chondrocyte viability with the use of a cell proliferation assay and determined their chondrogenic potential with the use of reverse-transcription polymerase chain reaction, enzyme-linked immunosorbent assay, and glycosaminoglycan assays. Our results demonstrated that cartilage tissue stored in a preservation medium composed of dimethyl sulfoxide, fetal bovine serum, and Dulbecco Modified Eagle Medium at a ratio of 1:2:7 (v/v) or stored with a commercially available preservation solution generated greater numbers of chondrocytes when the storage temperature was -80°C than when it was 4°C. The viability of isolated cells decreased with time at 4°C, whereas these values remained constant for tissues stored at -80°C. Our data suggest that an optimal method for preserving cartilage tissue is required to ensure the quality of cells used for transplantation.

New Culture Medium Concepts for Cell Transplantation

BACKGROUND Before cell or tissue transplantation, cells or tissues have to be maintained for a certain period in vitro using culture medium and methods. Most culture media contain substances such as pH indicators and buffers. It is not known whether some of these substances are safe for subsequent application in the transplantation of cells or tissues into the human body. We investigated culture media and methods with respect to the safety of the components in future transplantation applications. METHODS A modified culture medium--medical fluid-based culture medium (FCM)--was designed by using various fluids and injectable drugs that are already currently permitted for use in clinical medicine. Medium components necessary for optimal cell growth were obtained from approved drugs. FCM was manufactured with adjusted final concentrations of the medium components similar to those in commercial Dulbeccos modified Eagles medium (DMEM). In particular, 1029.40 mg/L amino acids, approximately 88.85 mg/L vitamins, 13,525.77 mg/L inorganic salts, and 4500 mg/L D-glucose comprise the high-glucose FCM. Next, human fat synovium-derived mesenchymal stem cells and rat H9c2 (2-1) cells were cultured under 2 conditions: (1) DMEM-high glucose (HG), an original commercial medium, and (2) optimized FCM-HG. We assessed the morphologies and proliferation rates of these cells. RESULTS We observed that FCM-HG was able to induce the growth of FS-MSC and commercially available H9c2 cell. The morphologies and proliferation patterns of these cells cultured under FCM-HG showed no differences compared with cells grown in DMEM-HG. CONCLUSION Our data suggest that FCM, which we developed for the first time according to the concept of drug repositioning, was a useful culture medium, especially in cultured cells intended for human cell transplantation.

Fragmin/Protamine Microparticle Carriers as a Drug Repositioning Strategy for Cell Transplantation

BACKGROUND The importance of drug repositioning has been gaining attention in the last few years, allowing existing pharmaceutical products to be reevaluated for potential alternative therapeutic applications. The purpose of this study was to evaluate the effects of fragmin/protamine microparticles (F/P MPs) on cell aggregates under the concept of drug repositioning. METHODS Mesenchymal stem cells (MSCs) and embryonic rat heart-derived cardiac H9C2 cells were mixed with D-PBS, basal medium, fragmin, protamine, and F/P MPs to manufacture aggregates intended for cell transplantation. To evaluate their adhesive properties as cell carriers, we injected combinations of MSC aggregates into cartilage tissue, observing their leakage from the implantation site. RESULTS Our data demonstrated that MSCs and H9C2 cells mixed with F/P MPs rapidly produced large, viscous cellular aggregates. F/P MPs were bound to the surface of MSCs and H9C2 cells; thus, F/P MPs induced the formation of F/P MP-cell aggregates. Cell aggregates were prevented from leaking from the transplanted site. CONCLUSION Aggregation induced by F/P MPs may improve the efficiency of cell therapy, a novel method for transplantation.

Stem cell therapy status in veterinary medicine

The stem cell therapy in veterinary medicine continues to grow both experimentally and clinically. Application of adipose-derived mesenchymal stem cell (MSC), umbilical cord blood-derived MSC, bone marrow-derived MSC and skeletal muscle-derived MSC for treatment of various diseases are currently in use for several species. However, the differential efficacies of various approaches are still being investigated. In stem cell therapy, common disease animal models include mouse, rat, rabbit, swine, canine, caprine, equine and dolphin are used. Whereas for clinical models, canine, equine are dolphin are used. We need attention and participation from the researchers of tissue engineering and regenerative medicine, and now it is necessary to develop mutual understanding and cooperation between veterinary and human medicine fields. This review is focused on some of the recent status of stem cell therapy in the veterinary medicine.

Novel supplier of mesenchymal stem cell: subacromial bursa.

Mesenchymal stem cells (MSCs) are multipotent stromal elements that can differentiate into a variety of cell types. MSCs are good sources of therapeutic cells for degenerative diseases. For these reason, many researchers have focused on searching for other sources of MSCs. To obtain MSCs for clinical use requires surgery of the donor that therefore can induce donor morbidity, since the common sources at present are bone marrow and adipose tissues. In this study, we investigated the existence of MSCs in postoperative discarded tissues. Subacromial bursal tissues were obtained from the shoulders of 3 injured patients. The cells from the bursa tissues were isolated through treatment with collagenase. The isolated cells were then seeded and expanded by serial passaging under normal culture system. To evaluate MSC characteristics of the cells, their MSC markers were confirmed by mRNA and protein expression. Multipotent ability was assessed using differentiation media and immunohistochemistry. Cells from the bursa expressed MSCs markers-CD29, CD73, CD90, and PDGFRB (platelet-derived growth factor receptor-beta). Moreover, as to their multipotency, bursal cells differentiated into adipocytes (fat cells), osteocytes (bone cells), and chondrocytes (cartilage cells). In summary, we showed that MSCs could be generated from the subacromial bursa, which is medical waste after surgery.