• Journal of Periodontal and Implant Science
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• v.37 no.3
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• pp.497-509
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• 2007

Bone morphogenetic proteins(BMPs) are regarded as members of the transforming growth $factor-{\beta}$ superfamily with characteristic features in their amino acid sequences. A number of studies have demonstrated the biologic activities of BMPs, which include the induction of cartilage and bone formation. Recently there was a attempt to overcome a limitation of mass production, and economical efficieny of rh-BMPs. The method producing PTD by using bacteria have advantages of acquiry a mass of proteins. Hences, a new treatment which deliver protein employed by protein transduction domain(PTD) has been tried. The purpose of this study was to evaluate the bone regenerative effect of TATBMP-2 and TAT-HA2-BMP-2 employed by PTD from HlV-1 TAT protein for protein translocation in the rat calvarial model. An 8mm calvarial, critical size osteotomy defect was created in each of 32 male Spraque-Dawley rats(weight $250{\sim}300g$). The animals were divided into 4 groups of 32 animals each (4 animals/group/healing interval). The defect was treated with TATBMP-2/ACS(Absorbable collagen sponge) (TATBMP-2 0.1mg/ml), TAT-HA2-BMP-2/ACS(TAT-HA2-BMP-2 0.1mg/ml), ACS alone or left untreated for surgical control(negative control). The rats were sacrificed at 2 or 8 weeks postsurgery, and the results were evaluated histologically. The results were as follows: New bone formation were not significantly greater in the TATBMP-2/ACS group relative to negative, and positive control groups. New bone was evident at the defect sites in TAT-HA2-BMP-2/ACS group relative to negative, positive control and TATBMP-2 groups. There were a little bone regeneration in TATBMP-2 groups. While, enhanced local bone formation were observed in TAT-HA2-BMP-2 group. But, The results was not the same in all rat defects. Therefore, further investigations are required to develop a method. which disperse homogenously, and adhere to target cells.

• KSBB Journal
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• v.31 no.2
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• pp.120-125
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• 2016

Two-dimensional cultivation is typically used for cell growth, but the method reduces the characteristics of chondrocytes and stem cells, and limits culture area. Therefore, development of three-dimensional culture method is needed to mimic in vivo environment, improve quality of cells and scale-up efficiently. Improving proliferation and chondrogenesis is available by co-culture of chondrocytes and mesenchymal stem cells (MSCs) that leads to interaction between two kinds of cells. However, the co-culture has problems that permeability of sphere diminishes as aggregate size increased and ratio of two kinds of cells composing each spheres is different. In this work, co-cultivation method using controlled sphere composed of chondrocytes and MSCs was established and enhanced chondrogenesis. Periosteum-derived progenitor cells (PDPCs) that are appropriate for cell therapy source of articular cartilage were used as MSCs. Controlled spheres were formed in the hanging-drop plates and shifted for being induced chondrogenesis in 35-mm non-adhesive culture dishes at a rotation rate of 60 rpm. After inducing chondrogenesis, gene expressions related with chondrogenesis were found to be improved and it was apparent that the utilization of controlled spheres promoted chondrogenesis. As a result, available numbers of cells per unit area were increased and chondrogenic differentiation ability was improved compared to typical two-dimensional culture. This approach shows the potential in cartilage regeneration as it can provide sufficient numbers of chondrocytes.

• Journal of Embryo Transfer
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• v.27 no.3
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• pp.175-181
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• 2012

The synovial tissues are a valuable MSCs source for cartilage tissue engineering because these cells are easily obtainable by the intra-articular biopsy during diagnosis. In this study, we isolated and characterized the canine MSCs derived from synovial fluid of female and male donors. Synovial fluid was flushed with saline solution from pre and post-puberty male (cM1-sMSC and cM2-sMSC) and female (cF1-sMSC and cF2-sMSC) dogs, and cells were isolated and cultured in advanced-DMEM (A-DMEM) supplemented with 10% FBS in a humidified 5% $CO_2$ atmosphere at $38.5^{\circ}C$. The cells were evaluated for the expression of the early transcriptional factors, such as Oct3/4, Nanog and Sox2 by RT-PCR. The cells were induced under conditions conductive for adipogenic, osteogenic, and chondrogenic lineages, then evaluated by specific staining (Oil red O, von Kossa, and Alcian Blue staining, respectively) and analyzed for lineage specific markers by RT-PCR. All cell types were positive for alkaline phosphatase (AP) activity and early transcriptional factors (Oct3/4 and Sox2) were also positively detected. However, Nanog were not positively detected in all cells. Further, these MSCs were observed to differentiate into mesenchymal lineages, such as adipocytes (Oil red O staining), osteocytes (von Kossa staining), and chondrocytes (Alcian Blue staining) by cell specific staining. Lineage-specific genes (osteocyte; osteonectin and Runx2, adipocytes; PRAR-${\gamma}2$, FABP and LEP, and chondrocytes; collagen type-2 and Sox9) were also detected in all cells. In this study, we successfully established synovial fluid derived mesenchymal stem cells from female and male dogs, and determined their basic biological properties and differentiation ability. These results suggested that synovial fluid is a valuable stem cell source for cartilage regeneration therapy, and it is easily accessible from osteoarthritic knee.

• Biomaterials and Biomechanics in Bioengineering
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• v.2 no.1
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• pp.23-32
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• 2015

Facile immobilization of growth factors in hyaluronic acid (HA) hydrogels using dual enzymes is reported in the paper. The hydrogels were formed by using horseradish peroxidase (HRP) and hydrogen peroxide ($H_2O_2$) and transforming growth factor-${\beta}3$ (TGF-${\beta}3$) was covalently conjugated on the hydrogels in situ using tyrosinase (Ty) without any modifications. For the preparation of hydrogels, HA was grafted with poly(ethylene glycol) (PEG), which was modified with a tyrosine. The gelation times of the HA hydrogels were ranging from 415 to 17 s and the storage moduli was dependent on the concentration of $H_2O_2$ and Ty (470-1600 Pa). A native TGF-${\beta}3$ (200 ng/mL) was readily encapsulated in the HA hydrogels and 17% of the TGF-${\beta}3$ was released over 1 month at the Ty concentration of 0.5 KU/mL, while the release was faster when 0.3 KU/mL of Ty was used for the encapsulation (27%). It can be suggested that the growth factors resident in the hydrogels for a long period of time may lead cells proliferating and differentiating, whereas the growth factors that are initially released from the hydrogels can induce the ingrowth of cells into the matrices. Therefore, the dual enzymatic methods as facile gel forming and loading of various native growth factors or therapeutic proteins could be highly promising for tissue regenerative medicines.

• Journal of Veterinary Clinics
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• v.36 no.4
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• pp.200-206
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• 2019

This study was performed to assess the anti-inflammatory and cartilage regenerative effects of platelet-rich plasma (PRP) on canine chondrocytes. Proliferation and migration assays under both normal and lipopolysaccharide (LPS)-induced inflammatory conditions were performed with various concentrations of PRP (1% to 10%). The expression levels of genes related to osteoarthritis were evaluated in the following groups: PRP group, LPS group and LPS + PRP group. mRNA expression levels were detected using real-time polymerase chain reaction (RT-PCR). Proliferation assays showed significantly enhanced proliferation in all PRP-treated groups compared with the no serum group. Compared with 10% fetal bovine serum (FBS), PRP concentrations above 3% in the normal condition and 1% to 7% PRP in the LPS-induced inflammatory condition were found to significantly promote chondrocyte proliferation. In the normal condition, all PRP-treated groups showed significantly increased cell migration compared with the no serum group. Chondrocyte migration was decreased with LPS-induced inflammation, but PRP treatment resulted in significantly enhanced migration compared with the other groups in this condition. According to RT-PCR, the LPS + PRP group showed significantly higher levels of COL1A1, IL-6, aggrecan and lower levels of $TNF-{\alpha}$, MMP-1, MMP-3 mRNA expression compared to the LPS group. The results of this study suggest that PRP application can enhance the proliferation and migration of canine chondrocytes and improve canine articular cartilage regeneration.

• Journal of Periodontal and Implant Science
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• v.29 no.4
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• pp.873-893
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• 1999

The ultimate goal of periodontal therapy is the regeneration of periodontal tissue and repair of function. For more than a decade there have been many efforts to develop materials and bioactive molecule(such as growth factor and differentiation factors) to promote periodontal wound healing. Among the bioactive molecules, bone morphogenetic protein(BMP) was studied for periodontal wound healing. Since Urist demonstrated that demineralized bone matrix could induce the formation of cartilage and bone in ectopic site, many studies on BMP have been reported. Among those BMPs, it was reported that rhBMP-2 enhanced the healing of bone defects in animal studies and clinical studies. However, its efficacy in periodontal regeneration, especially 1-wall intrabony defects is still unknown. The purpose of this study was to examine the effect of rhBMP-2/ACS on the epithelial migration, gingival connective tissue adhesion, cementum formation, alveolar bone regeneration in intrabony defects of dogs. Four millimeter deep and four millimeter wide 1-wall defects were surgically created in the mesial aspects of the 3rd incisors. The test group received rhBMP-2/ACS with a flap procedure and the control underwent buffer/ACS with a flap procedure. Histologic analysis after 8 weeks of healing revealed the following results: 1. The length of epithelial growth(the distance from alveolar crest to the apical end of JE) was $0.9{\pm}1.5mm$ in the control group and $1.2{\pm}1.4mm$ in the test group. There was no statistically significant difference between the two groups. 2. The length of connective tissue adhesion was $2.4{\pm}1.3mm$ in the control group and $1.2{\pm}1.1mm$ in the test group. The control group showed significantly enhanced adhesion(P<0.05). 3. The length of new cementum was $0.9{\pm}1.0mm$ in the control group and $1.7{\pm}0.8mm$ in the test group. The test group showed significantly enhanced cementum regeneration(P<0.05). 4. The length of new bone height was $1.9{\pm}0.6mm$ in the control group and $2.4{\pm}0.9mm$ in the test group. There was no statistically significant difference between the two groups. 5. The new bone area was $4.7{\pm}1.7mm^2$ in the control group and $8.0{\pm}2.0mm^2$ in the test group. The test group showed significantly enhanced bone formed area(P<0.05). 6. The new bone density was $73.0{\pm}8.6%$ in the control group and $66.6{\pm}15.3%$ in the test group. There was no statistically significant difference between the two groups. These results suggest that the use of rhBMP-2 in 1-wall intrabony defects has significant effect on new cementum and new bone formation area, but doesn't have any significant effect on the prevention of junctional epithelium migration and new bone formation height.

• Journal of Veterinary Clinics
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• v.17 no.1
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• pp.121-128
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• 2000

The effects of cryosurgery on tear production and histological changes of the third eyelid were studied in dogs. Clinically normal 12 mixed breeds weighing 2∼6 kg were divided into three groups and treated as follows; 45 seconds double freeze-thaw treated group, 60 seconds double freeze-thaw treated group and 90 seconds double freeze-thaw treated group. The significant decrease of the tear production after cryosurgery was shown in all groups throughout the observed periods(p<0.05). However, there was no difference among groups. The main complications after cryosurgery were chemosis and conjunctival injection. Other complications such as eyelid edema, eyelid depigmentation and keratitis were more preominent in group III compared to those of groups I and II. On histopathological examination, chronic inflammatory changes and regeneration of the third eyelid glands were noted in group I and predominated loss of the third eyelid glands and necrosis of hyaline cartilage were observed in group III However, such changes were less appeared in group II. The results of this study suggested that double freeze-thaw cryosurgery for 60 seconds on the third eyelid glands would be the most effective method for treating tear staining syndrome.

• Journal of the Korean Society of Manufacturing Process Engineers
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• v.13 no.3
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• pp.35-41
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• 2014

Poly(g-glutamic acid) (g-PGA) is a very promising biodegradable polymer that is produced by microorganism of Bacillus subtilis. Because g-PGA is water-soluble, anionic, biodegradable, and even edible, its potential applications have been studied from an industrial standpoint. In this study, we fabricated porous g-PGA foams by means of a freeze-solvent extraction method for tissue-engineering applications. Porous g-PGA foams were chemically cross-linked using a hexamethylene diisocyanate solution. An aqueous basic solution was used to neutralize g-PGA foam for cell culturing. During an in vitro cell culture study, it was observed that primary rabbit ear chondrocytes were well at tached and spread over the surface oft hree-dimensional cross-linkedg-PGA foam. From these results, it is concluded that cross-linkedg-PGA foam is aprom is in gmaterial for tissue-engineering applications, especially those pertaining to the regeneration of human cartilage.

• The Journal of Pediatrics of Korean Medicine
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• v.24 no.1
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• pp.117-125
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• 2010

Objectives The purpose of this study is to examine the effect of chondrocyte re-differentiation in using ukmijihwang-Tang gamibang aqua-acupuncture. Methods In this study, chondrocytes were extracted from New Zealand white rabbit's knee joint, and cultured in monolayer after collagenase treatment. In the third passage, after the mRNA transcript of the type II collagen significantly reduced, diluted Yukmijihwang-Tang gamibang were added to cultured of chondrocyte, and its effect on the mRNA expression of type II collagen was quantitatively evaluated. Results As a result of treatment with Yukmijihwang-Tang gamibang in vitro for 48 hours, the mRNA expression of type II collagen was up-regulated. In addition, the result of H&E-staining in vivo indicated that chondrocyte-like tissues were formed in repairing injured cartilages after 12 weeks of treatment with Yukmijihwang-Tang gamibang. Conclusions These results indicated that Yukmijihwang-Tang gamibang was effective on the recovery of chondrocyte phenotype, and could be used for cartilage regeneration in arthritic diseases. However, more clinical study of Oriental medical treatment for this case might be also needed.

• Proceedings of the Korean Society of Veterinary Clinics Conference
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• 2007.10a
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• pp.577-577
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• 2007