Gonhyung Kim

Isolation and multilineage differentiation of bovine bone marrow mesenchymal stem cells

The bone marrow harbors a population of mesenchymal stem cells (MSCs) that possess the potential to differentiate into bone, cartilage, and fat, and along other tissue pathways. To date, MSCs from various species have been studied. Despite the bovine experimental model being widely used in experiments in vivo and in vitro, only a limited amount of information regarding bovine MSCs is available. The aim of this study was to isolate and induce the multilineage mesenchymal differentiation of bovine MSCs, thereby initiating further research on these cells. Bovine MSCs were isolated from eight calves, and osteogenic, chondrogenic, and adipogenic differentiation was induced by using a combination of previously reported protocols for other species. The level of differentiation was evaluated by histological examination and by analyzing the expression of tissue-specific genes by a quantitative “real time” reverse transcription/polymerase chain reaction technique. Following osteoinduction, the isolated fibroblast-like cells transformed into cuboidal cells and formed alkaline-phosphatase-positive colonies; during differentiation, these colonies transformed into mineralized nodules. In addition, osteogenesis was followed by osteocalcin and collagen type I mRNA expression. Chondrogenesis was confirmed by the demonstration of collagen type II, aggrecan, and sox9 mRNA expression in the cells stimulated by transforming growth factor β1 in monolayer culture. After being cultured in an adipogenesis-inducing medium, the MSCs responded by the accumulation of lipid vacuoles and the expression of adipocyte-specific genes. We have therefore demonstrated that cells harvested from bovine bone marrow are capable of in vitro extensive multiplication and multilineage differentiation, making them a relevant and invaluable model in the field of stem cell research.

Comparison of the cytotoxic effects of bupivacaine, lidocaine, and mepivacaine in equine articular chondrocytes

OBJECTIVE To compare the chondrotoxicity of bupivacaine, lidocaine, and mepivacaine in equine articular chondrocytes in vitro. STUDY DESIGN Prospective, experimental study. STUDY MATERIAL Equine articular chondrocytes. METHODS Primary cultured equine chondrocytes were exposed to 0.5% bupivacaine, 2% lidocaine, or 2% mepivacaine for 30 or 60 minutes. After treatment, cell viability was evaluated by trypan blue exclusion and the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay in a dose dependent manner. Apoptosis and necrosis of chondrocytes were analyzed with the double staining of Hoechst 33258 and propidium iodide using fluorescence microscopy, and the results were confirmed using flow cytometry. RESULTS After 30-minute exposure, trypan blue exclusion assay revealed that cell viability of 0.5% bupivacaine group was 28.73±8.44%, and those of 2% lidocaine and 2% mepivacaine were 66.85±6.03% and 86.27±2.00%, respectively. The viability of chondrocytes after saline treatment was 95.95±2.75%. The results of MTT assay and fluorescence microscopy had similar tendency with trypan blue assay. Each result showed that bupivacaine was the most toxic of the three local anaesthetics. Mepivacaine was less toxic than lidocaine. The results of the viability test suggest that bupivacaine and lidocaine exhibit a marked chondrotoxicity, and that this is mainly due to necrosis rather than apoptosis. CONCLUSIONS AND CLINICAL RELEVANCE Bupivacaine may induce detrimental chondrotoxicity when administered intra-articularly, especially in patients with joint disease, and we suggest that it should be used cautiously in equine practice. Mepivacaine may be an alternative to both bupivacaine and lidocaine.

Thrombospondin-2 secreted by human umbilical cord blood-derived mesenchymal stem cells promotes chondrogenic differentiation

Increasing evidence indicates that the secretome of mesenchymal stem cells (MSCs) has therapeutic potential for the treatment of various diseases, including cartilage disorders. However, the paracrine mechanisms underlying cartilage repair by MSCs are poorly understood. Here, we show that human umbilical cord blood‐derived MSCs (hUCB‐MSCs) promoted differentiation of chondroprogenitor cells by paracrine action. This paracrine effect of hUCB‐MSCs on chondroprogenitor cells was increased by treatment with synovial fluid (SF) obtained from osteoarthritis (OA) patients but was decreased by SF of fracture patients, compared to that of an untreated group. To identify paracrine factors underlying the chondrogenic effect of hUCB‐MSCs, the secretomes of hUCB‐MSCs stimulated by OA SF or fracture SF were analyzed using a biotin label‐based antibody array. Among the proteins increased in response to these two kinds of SF, thrombospondin‐2 (TSP‐2) was specifically increased in only OA SF‐treated hUCB‐MSCs. In order to determine the role of TSP‐2, exogenous TSP‐2 was added to a micromass culture of chondroprogenitor cells. We found that TSP‐2 had chondrogenic effects on chondroprogenitor cells via PKCα, ERK, p38/MAPK, and Notch signaling pathways. Knockdown of TSP‐2 expression on hUCB‐MSCs using small interfering RNA abolished the chondrogenic effects of hUCB‐MSCs on chondroprogenitor cells. In parallel with in vitro analysis, the cartilage regenerating effect of hUCB‐MSCs and TSP‐2 was also demonstrated using a rabbit full‐thickness osteochondral‐defect model. Our findings suggested that hUCB‐MSCs can stimulate the differentiation of locally presented endogenous chondroprogenitor cells by TSP‐2, which finally leads to cartilage regeneration. Stem Cells 2013;31:2136–2148

A Viscoelastic Chitosan-Modified Three-Dimensional Porous Poly(L-Lactide-co-ε-Caprolactone) Scaffold for Cartilage Tissue Engineering

Biomaterials have been playing important roles in cartilage regeneration. Although many scaffolds have been reported to enhance cartilage regeneration, none of the scaffolds available are optimal regarding mechanical properties, integration with host cartilage and providing proper micro-environment for chondrocyte attachment, proliferation and differentiation. In the current study, chitosan-modified poly(L-lactide-co-ε-caprolactone) (PLCL) scaffolds were fabricated to simulate the main biochemical components of cartilage, as well as their interaction with the aim to endow them with viscoelasticity similar to native cartilage. Porous PLCL scaffolds were fabricated with porogen-leaching, freeze-extraction and freeze-gelation before chitosan was cross-linked. The acquired porous scaffolds had pore sizes ranging from 200 to 500 μm and about 85% porosity with good interconnection between individual pores. Chitosan was successfully cross-linked to PLCL scaffolds, as validated by ninhydrin staining and X-ray photoelectron spectroscopy (XPS). The viscoelasticity of the scaffolds was similar to that of bovine cartilage and they had a relatively good recovery ratio from compression deformation, while the Youngs modulus was one order of magnitude less than cartilage. Not only could the chitosan-modified PLCL scaffolds promote cell adhesion and proliferation, but also they could significantly enhance excretion of aggrecan and type-II collagen, as testified by both histology and quantitative PCR, compared with PLCL scaffolds. With the fabrication of biomimetic scaffolds, it is possible to make scaffolds for cartilage tissue engineering, which are not only biocompatible, but also have mechanical properties similar to native cartilage.

Dexamethasone induces apoptosis in proliferative canine tendon cells and chondrocytes.

Dexamethasone (Dexa) has been commonly used in humans and domestic animals, particularly in the treatment of tendon injuries and cartilage degeneration. However, it is often associated with tendon rupture and impaired tendon and cartilage healing. In the present study, we investigated Dexas in vitro effects on the growth of cell proliferation and the induction of apoptosis in canine Achilles tendon cells and chondrocytes. Cell proliferation after treatment with Dexa for two to six days was quantified by a 2,3-bis{2-methoxy-4-nitro-5-sulfophenyl}-2H-tetrazolium-5-carboxyanilide inner salt assay (XTT). The results showed that Dexa could inhibit the proliferation of tendon cells and chondrocytes at increasing concentrations (0.1-50 microg/ml) compared with untreated cells. Cell apoptosis was induced by Dexa, as evidenced by the typical nuclear apoptosis using Hoechst 33258 staining. Dexa increased the apoptosis of canine tendon cells and chondrocytes in a time-dependent manner. In canine tendon cells and chondrocytes that were treated with 25 and 50 microg/ml concentration of Dexa, the number of condensed apoptotic nuclei was significantly increased. In addition, culturing with Dexa and the glucocorticoid receptor blocker, mifepristone, significantly arrested apoptosis of tendon cells and chondrocytes. Based on our in vitro data, we hypothesized that in vivo treatment with glucocorticoids may diminish the proliferation of tendon and cartilage cells by increasing apoptosis and suppressing the proliferation. Our findings suggest that Dexa could be used with caution in dogs with articular or tendon problems.

Relationship of Disease Progression and Plasma Histamine Concentrations in 11 Dogs with Mast Cell Tumors

Plasma histamine concentrations (PHCs) were measured serially over 9 months or until death in 11 dogs with mast cell tumors (MCTs). Eight dogs had grossly visible disease and the other 3 dogs had microscopic disease. Initial PHCs in the dogs with gross disease were significantly higher than PHCs in healthy dogs (median, 0.73 ng/mL and 0.19 ng/mL respectively; P < .009), whereas initial PHCs in dogs with microscopic disease showed no difference from controls. Seven dogs subsequently had progressive increases in PHC, and developed hyperhistaminemia (median, 14.0 ng/mL; range, 5.11-30.1 ng/nL). These 7 dogs died from MCTs, and 1 had general weakness with rapid lysis of a large tumor burden after radiation therapy. PHCs of the other 4 dogs were less than 1 ng/mL during the study. These 4 dogs were still alive with adequate control of the tumor at the conclusion of the study. Four of the 11 dogs initially had gastrointestinal (G1) signs, which abated soon after administration of histamine-2 (H-2) blockers. No significant difference was found between PHCs in dogs with GI signs and those without GI signs (median, 0.86 ng/mL and 0.35 ng/mL. respectively). Thereafter, 7 dogs had serious GI complications for which H-2 blocker therapy was ineffective. PHCs in these 7 dogs were extremely high (median, 12.2 ng/mL; range, 3.42-30.1 ng/nL). Results of this study demonsrated that PHC was one factor related to disease progression, and indicated that marked hyperhistaminemia was associated with the GI signs refractory to H-2 blocker therapy in dogs with MCTs.

Osteogenic evaluation of calcium phosphate scaffold with drug-loaded poly (lactic-co-glycolic acid) microspheres in beagle dogs

The goal of this study was to develop a bioactive hydroxyapatite (HA) scaffold as a calcium phosphatebased bioceramic using drug-loaded polymeric microspheres for bone regeneration. Dexamethasone (DEX) as a model bioactive molecule and poly (lactic-co-glycolic acid) (PLGA) microspheres as a carrier were employed. Polyethyleneimine was coated on DEX-loaded PLGA microsphere surfaces, resulting in a net positively-charged surface. With such modification of the PLGA microsphere surfaces, DEX-loaded PLGA microspheres were immobilized on the negatively charged HA scaffold surfaces. The release profile of DEX over a 4-week immersion study indicated an initial burst release followed by a sustained release. In vivo evaluation of the defects filled with DEX-loaded HA scaffolds indicated that new bone formation was enhanced when compared to defects that were either unfilled or filled only with HA scaffold. This innovative platform for bioactive molecule delivery more potently induced osteogenesis in vivo, which may be exploited in implantable bone graft substitutes for stem cell therapy or improved in vivo performance.

Comparative Effects of Human Neural Stem Cells and Oligodendrocyte Progenitor Cells on the Neurobehavioral Disorders of Experimental Autoimmune Encephalomyelitis Mice

Since multiple sclerosis (MS) is featured with widespread demyelination caused by autoimmune response, we investigated the recovery effects of F3.olig2 progenitors, established by transducing human neural stem cells (F3 NSCs) with Olig2 transcription factor, in myelin oligodendrocyte glycoprotein- (MOG-) induced experimental autoimmune encephalomyelitis (EAE) model mice. Six days after EAE induction, F3 or F3.olig2 cells (1 × 106/mouse) were intravenously transplanted. MOG-injected mice displayed severe neurobehavioral deficits which were remarkably attenuated and restored by cell transplantation, in which F3.olig2 cells were superior to its parental F3 cells. Transplanted cells migrated to the injured spinal cord, matured to oligodendrocytes, and produced myelin basic proteins (MBP). The F3.olig2 cells expressed growth and neurotrophic factors including brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), ciliary neurotrophic factor (CNTF), and leukemia inhibitory factor (LIF). In addition, the transplanted cells markedly attenuated inflammatory cell infiltration, reduced cytokine levels in the spinal cord and lymph nodes, and protected host myelins. The results indicate that F3.olig2 cells restore neurobehavioral symptoms of EAE mice by regulating autoimmune inflammatory responses as well as by stimulating remyelination and that F3.olig2 progenitors could be a candidate for the cell therapy of demyelinating diseases including MS.

Comparison between open and closed methods of herniorrhaphy in calves affected with umbilical hernia

Umbilical hernias in calves commonly present to veterinary clinics, which are normally secondary to failure of the normal closure of the umbilical ring, and which result in the protrusion of abdominal contents into the overlying subcutis. The aim of this study was to compare the suitability of commonly-used herniorrhaphies for the treatment of reducible umbilical hernia in calves. Thirty-four clinical cases presenting to the Veterinary Teaching Hospital, Chittagong Veterinary and Animal Sciences University, Chittagong, Bangladesh from July 2004 to July 2007 were subjected to comprehensive study including history, classification of hernias, size of the hernial rings, presence of adhesion with the hernial sacs, postoperative care and follow-up. They were reducible, non-painful and had no evidence of infection present on palpation. The results revealed a gender influence, with the incidence of umbilical hernia being higher in female calves than in males. Out of the 34 clinical cases, 14 were treated by open method of herniorrhaphy and 20 were treated by closed method. Complications of hernia were higher (21%) in open method-treated cases than in closed method-treated cases (5%). Hernia recurred in three calves treated with open herniorrhaphy within 2 weeks of the procedure, with swelling in situ and muscular weakness at the site of operation. Shorter operation time and excellent healing rate (80%) were found in calves treated with closed herniorrhaphy. These findings suggest that the closed herniorrhaphy is better than the commonly-used open method for the correction of reducible umbilical hernia in calves.

Traumatic and Non-traumatic Osteonecrosis in the Femoral Head of a Rabbit Model.

Osteonecrosis of the femoral head is an idiopathic, debilitating and progressive disease. A number of traumatic or non-traumatic animal models have been reported for research on osteonecrosis. This study was performed to compare the efficacy of femoral head osteonecrosis in rabbits by traumatic and non-traumatic methods. Twenty-seven New Zealand White rabbits were divided into three experimental groups, nine heads each. Two groups were surgically induced into osteonecrosis; a steel cerclage wire was ligated tightly around the neck of the right femoral head (Group W), and the femoral neck was tied with a cerclage wire in the same way as in the W group, and burned by attachment of an electrode tip to the wire and then the wire was removed (Group B). The other group was induced into osteonecrosis with a single intra-muscular injection of 20 mg/kg methyl-prednisolone acetate single injection (Group M). In the control group, the left femoral head of animals in group W and B was used. After two weeks, rabbits were sacrificed and the femoral head and neck were collected. Osteonecrosis of the femoral head was evaluated by radiography, histology and immunohistology methods. Osteonecrosis lesions in the femoral head were identified in traumatic models of groups W and B. Cartilage degeneration in the superficial layer and TUNEL positive cells in the femoral head were detected more in Group B than in Group W. These findings revealed that short-term induced osteonecrosis of the femoral head was effectively achieved by cautery around the femoral neck.