• Journal of Periodontal and Implant Science
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• v.25 no.2
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• pp.341-356
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• 1995

The purpose of this study was to evaluate the effect of demineralized freeze dried bone and demineralized bone gel with guided tissue regeneration treatment around titanium implants with dehisced bony defects and also evaluate space maintaining capacity of demineralized bone gel type and DFDB powder type under e-PTFE membrane. In 3 Beagle dogs, mandibular premolar was extracted and four peri-implant osteotomies were formed for dehiscence. After insertion of implants, the four peri-implant defects were treated as follows. 1) In control group. no graft material and barrier membrane were applied. 2) In experimental group.1, the site was covered only with the e-PTFE membrane. 3) In experimental group 2,received DFDB powder and covered by the e-PTFE membrane. 4) In experimental group 3, demineralized bone gel and e-PTFE membrane were used. By random selection, animals were sacrificed at 4, 8, 12 weeks. The block sectioned specimens were prepared for decalcified histologic evaluation(hematoxylin and eosin staining) and undecalcified histologic evahiation(Von Kossa's and toluidine blue staining) with light microscopy. The results of this study were as follows. 1) In control group, there was a little new bone formation and connective tissue was completely filled in the defect area. 2) Experimental group 1 showed lesser quantity of bone formation as compared to the bone grafted group. Thin vertical growth of new bone formation around implant fixture was shown. 3) Experimental group 2 showed thick bucco-lingual growth of new bone formation and grafted bone particles were almost resorbed in 12 week group. 4) In experimental group 3, most grafted bone particles were not resorbed in 12 week group and thick bucco-lingual bone formation was shown in dehisced defect base area. 5) There was no remarkable differences in space making capacity and new bone formation procedure between demineralized freeze-dried bone powder type and demineralized bone gel type.

• Biomedical Science Letters
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• v.21 no.1
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• pp.40-49
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• 2015

Mesenchymal stem cells (MSCs) have the ability to self-renew and differentiate into multi-lineage cells, thus highlighting the feasibility of using umbilical cord blood-derived MSCs (UCB-MSCs) for cell-therapy and tissueengineering. However, the low numbers of UCB-MSC derived from clinical samples requires that an ex vivo expansion step be implemented. As most stem cells reside in low oxygen tension environments (i.e., hypoxia), we cultured the UCBMSCs under 3% $O_2$ or 21% $O_2$ and the following parameters were examined: proliferation, senescence, differentiation and stem cell specific gene expression. UCB-MSCs cultured under hypoxic conditions expanded to significantly higher levels and showed less senescence compared to UCB-MSCs cultured under normoxic conditions. In regards to differentiation potential, UCB-MSCs cultured under hypoxic and normoxic conditions both underwent similar levels of osteogenesis as determined by ALP and von Kossa assay. Furthermore, UCB-MSCs cultured under hypoxic conditions exhibited higher expression of OCT4, NANOG and SOX2 genes. Moreover, cells expanded under hypoxia maintained a stem cell immnunophenotype as determined by flow cytometry. These results demonstrate that the expansion of human UCB-MSCs under a low oxygen tension microenvironment significantly improved cell proliferation and differentiation. These results demonstrate that hypoxic culture can be rapidly and easily implemented into the clinical-scale expansion process in order to maximize UCB-MSCs yield for application in clinical settings and at the same time reduce culture time while maintaining cell product quality.

• Journal of Yeungnam Medical Science
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• v.20 no.2
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• pp.142-151
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• 2003

To induce bone formation at ectopic site by tissue engineering and gene therapy, we transplanted collagen sponges containing rhBMP-7 transduced HEK 293 cells in the hypodermis of nude mice. Bone formation was investigated by histological and electron microscopic method at 3, 6, and 9 weeks after transplantation. At 9 weeks after transplantation, eosinophilic bony tissue was observed in the implanted collagen sponge and was confirmed as bone tissue by Von Kossa stain. In the transmission electron microscopic observation, the cells in newly formed bone tissue had eccentrically located nucleus and well developed rough endoplasmic reticulum (rER). Therefore, the cells were evaluated as osteoblasts. Those results suggest that it is possible to form a bone tissue in the ectopic site by transplantation of rhBMP-7 transduced HEK 293 cells. This will be contributed to push more advanced gene therapy for bone formation. However, the HEK 293 cell is unable to apply to the clinical gene therapy. Therefore it is worth to find more compatible cells for clinical application. In addition, collagen sponge is considered as an excellent scaffold and/or carrier for gene therapy and a good biomaterial for tissue engineering.

• Journal of Industrial and Engineering Chemistry
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• v.67
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• pp.244-254
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• 2018

Surface modified poly ${\text\tiny{L}}$-lactic acid (PLLA) samples with hydroxyapatite (HA), heparin and bone morphogenetic protein-2 (BMP-2) mediated by polydopamine (pDA) coating (PLLA/pDA/HA/Hep/BMP-2) were prepared, and their effects on the enhancements of bone formation and osseointegration were evaluated in vitro and in vivo as compared to PLLA, PLLA/pDA/HA, and PLLA/pDA/Hep/BMP-2. The changes in surface chemical compositions, morphologies and wettabilities were observed by X-ray photoelectron spectroscopy (XPS), field-emission scanning electron microscopy (FE-SEM), atomic force microscopy (AFM) and water contact angle measurements. Pre-coating of HA particles with pDA provided uniform and homogeneous anchoring of particles to PLLA surface. In addition, the strong ionic interaction between heparin and pDA led PLLA surface readily heparinized for loading of BMP-2. In vitro experiments revealed that the levels of alkaline phosphatase (ALP) activity, calcium deposition, and osteocalcin (OCN) gene expression were higher in MG-63 human osteosarcoma cell lines grown on PLLA/pDA/HA/Hep/BMP-2 than on control PLLA, PLLA/pDA/HA, and PLLA/pDA/Hep/BMP-2. In vivo studies using micro-computed tomography (micro-CT) also showed that PLLA/pDA/HA/Hep/BMP-2 screw exhibited greatest value of bone volume (BV) and bone volume/tissue volume (BV/TV) among samples. Histological evaluations with H&E and Von Kossa staining demonstrated that a combination of HA and BMP-2 contributed to the strong osseointegration.

• Maxillofacial Plastic and Reconstructive Surgery
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• v.32 no.3
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• pp.199-206
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• 2010

This study investigated the effects of strontium on osteoblastic phenotypes of cultured human periostealderived cells. Periosteal tissues were harvested from mandible during surgical extraction of lower impacted third molar. Periosteal-derived cells were introduced into cell culture. After passage 3, the periostealderived cells were further cultured for 28 days in an osteogenic induction DMEM medium supplemented with fetal bovine serum, ascorbic acid 2-phosphate, dexamethasone and at a density of $3{\times}10^4$ cells/well in a 6-well plate. In this culture medium, strontium at different concentrations (1, 5, 10, and 100 ${\mu}g$/mL) was added. The medium was changed every 3 days during the incubation period. We examined the cellular proliferation, histochemical detection and biochemical measurements of alkaline phosphatase (ALP), the RT-PCR analysis for ALP and osteocalcin, and von Kossa staining and calcium contents in the periostealderived cells. Cell proliferation was not associated with the addition of strontium in periosteal-derived cells. The ALP activity in the periosteal-derived cells was higher in 5, 10, and 100 ${\mu}g$/ml strontium-treated cells than in untreated cells at day 14 of culture. Among the strontium-treated cells, the ALP activity was appreciably higher in 100 ${\mu}g$/ml strontium-treated cells than in 5 and 10 ${\mu}g$/ml strontium-treated cells. The levels of ALP and osteocalcin mRNA in the periosteal-derived cells was also higher in strontium-treated cells than in untreated cells at day 14 of culture. Their levels were increased in a dose-dependent manner. Von Kossa-positive mineralization nodules were strongly observed in the 1 ${\mu}g$/ml strontium-treated cells at day 21 and 28 of culture. The calcium content in the periosteal-derived cells was also higher in 1 ${\mu}g$/ml strontium-treated cells at day 28 of culture. These results suggest that low concentration of strontium stimulates the osteoblastic phenotypes of more differentiated periosteal-derived cells, whereas high concentration of strontium stimulates the osteoblastic phenotypes of less differentiated periosteal-derived cells. The effects of strontium on osteoblastic phenotypes of periosteal-derived cells appear to be associated with differentiation-extent.

• The korean journal of orthodontics
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• v.32 no.2 s.91
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• pp.129-142
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• 2002

The purpose of this study was to evaluate the effects of caffeine and calcium on the activities of the osteoblastic cell from mouse calvaria. The author cultured osteoblastic cells obtained from the mouse calvaria and were divided into three groups : the caffeine-treated, the calcium-treated and the combine-treated group. In caffeine-treated group, the cell toxicity was measured by MTT assay at 1, 2 and 4 days after treatment of caffeine. In all groups, the densities of the mineralized bone nodules were measured by imaging analyzer after Von Kossa staining. The alkaline phosphotase (ALP) activities were measured at 2, 7, 14, 21 and 28 days and the interleukin-1 ${\beta}$ activities at 48 hours after treatment of caffeine and calcium. The measurements were statistically executed with ANOVA test and the results were as follows. 1. The cellular toxicity of the caffeine increased with the concentration of caffeine during the incubation period. 2. The maximum densities of mineralization were observed at 0.2 mM caffeine-treated group, 1.2 mM calcium-treated group, 0.1 mM caffeine and 1.8 mM calcium-treated group. 3. The activities of ALP were peaked at 14 days at calcium-treated group as no-treated. But, the activities of ALP increased with concentrations of caffeine at caffeine-treated group. At combine-treated group, the act of ALP were peaked at 24 days at 1.2 mM, 1.8 mM calcium-treated group, But decreased at 2.5 mM calcium-treated group. 4. The activites of the IL-1 ${\beta}$ were increased significantly at 0.2 mM caffeine-treated group, 1.8 mM calcium-treated group and 0.1 mM caffeine and 1.8 mM calcium-treated group. But, they were decreased at all groups of high concentration.

• Maxillofacial Plastic and Reconstructive Surgery
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• v.31 no.3
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• pp.198-206
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• 2009

Purpose: The aims of this study were to assess the in vitro co-culturing pattern of isolated skin-derived precursor cells (SKPs) with a mixed demineralized bone (DMB) and fibrin glue scaffold and to evaluate in vivo osteogenesis after transplantation of autogenous SKPs with a these mixed scaffold in the animal's mandibular defects. Materials and Methods: We isolated SKPs from the ears of adult 4 miniature pigs. The isolated SKPs were co-cultured with a mixed DMB and fibrin glue scaffold in a non-osteogenic medium for 1, 2, and 4 weeks. Histological characteristics of in vitro co-cultured cells and scaffold were evaluated. $1{\times}10^7\;cells/100\;{\mu}l$ of autogenous porcine SKPs were grafted into the mandibular defects with a DMB and fibrin glue scaffold. In the control sites, only a scaffold was grafted, without SKPs. After two animals each were euthanized at 2 and 4 weeks after grafting, the in vivo osteogenesis was evaluated with histolomorphometric and osteocalcin immunohistochemical studies. Results: Homogeneously shaped skin-derived cells were isolated from porcine ear skin after 3 or 4 weeks of primary culture. In vitro osteogenic differentiation of SKPs was observed after co-culturing with a DMB and fibrin glue scaffold in a non-osteogenic medium. Von Kossa-positive bone minerals were also noted in the co-cultured medium at 4 weeks. As the culture time progressed, the number of observable cells increased. Trabecular new bone formation and osteocalcin expression were more pronounced in the SKP-grafted group compared to the control group. Conclusion: These findings suggest that autogenous SKP grafting with a DMB and fibrin glue scaffold can serve as a useful alternative to bone grafting technique.

• Journal of Periodontal and Implant Science
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• v.25 no.1
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• pp.45-55
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• 1995

치주질환의 치유에 있어서 치주인대 세포의 증식과 분화는 매우 중요하다. 몇몇 학자들에 의해 치주인대 세포의 증식과 분화에 영향을 주는 platelet derived growth나 fibronectin과 같은 growth factor에 대한 연구가 있었다. 이 연구는 홍삼 총사포닌이 치주인대 세포에 미치는 세포독성과 세포의 성장 및 분화에 미치는 영향을 규명하고자 사람의 치주인대 세포를 분리, 배양하여 실험하였다. 총사포닌이 치주인대 세포에 미치는 세포독성을 특정하기 위해 여러가지 농도의 총사포닌을 세포배양액에 첨가하여 1주일 배양후의 결과와 단일 농도($1{\mu}g/ml$})하에서의 세포 성장을 혈구계산반을 사용하여 관찰하였다. 치주인대 세포가 조골세포양세포로의 분화과정에 사포닌이 영향을 미치는 것을 관찰하기 위해 개개의 총사포닌 농도)0.1,1,$10{\mu}g/ml}$)를 세포배양액에 첨가하여 배양하였다. 치주인대 세포가 조골세포의 표현형으로 분화되는데 미치는 총사포닌의 영향을 알아보기 위하여 총사포닌의 단일 농도($1{\mu}g/ml$)하에서 $50{\mu}g/ml$ ascorbic acid와 10mM ${\beta}-glycerophosphate$를 배양액에 첨가하여 배양후 von Kossa's staining을 시행하여 생성된 골결절을 관찰하였다. 이상의 실험에서 얻어진 결과는 아래와 같다. 1. 각각의 농도를 투여한 결과, $1{\mu}g/ml$의 총사포닌에 의해서 세포독성이 유의성있게 증가하였다. 2. 0.01,0.01,1,$10{\mu}g/ml$의 총사포닌을 세포배양액에 첨가한 다음 7일 후의 고나찰시 cell viability가 실험농도 모두에서 유의성있게 증가하였다. 3. 0.1,1,10,$100{\mu}g/ml$의 농도에서 유외성있는 세포 증식이 있었다. 4. $1{\mu}g/ml$의 총사포닌을 세포배양액에 첨가한 다음 1,3,5,7,9일의 관찰시 시간경과에 따라 유의성있는 세포 증식이 있었다. 5. $10{\mu}g/ml$의 총사포닌을 세포배양액에 첨가시 ALP activity가 대조군에 비해 유외성있게 증가하였다. 6. $1{\mu}g/ml$의 총사포닌으로 배양된 치주인대 세포내에서 ALP positive cell이 관찰되었다. 7. $1{\mu}g/ml$의 총사포닌으로 배양된 치주인대 세포내에서 골결절 형성이 관찰되었다.

• Polymer(Korea)
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• v.27 no.3
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• pp.226-234
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• 2003

Ipriflavone (IP), a non-hormonal isoflavone derivative, has been shown to interfere with bone remodeling by inhibiting bone resorption and stimulating bone formation. IP consistently increased the amount of Ca incorporated into the cell layer by mesenchymal stem cells (MSCs). In this study, we developed the novel IP loaded poly(L-lactide-co-glycolide) (PLGA) scaffolds for the possibility of the application of the tissue engineered bone. IP/PLGA scaffo1ds were prepared by solvent casting/salt leaching method and were characterized by porosimeter, scanning electron microscopy, determination of residual salt amount, differential scanning calorimetry, and X-ray diffractometer, respectively. IP/PLGA scaffolds were implanted into the back of athymic nude mouse to observe the effect of IP on the osteoinduction compared with control PLGA scaffo1ds. Thin sections were cut from paraffin embedded tissues and histological sections were stained H&E, von Kossa, and immunohistochemical staining for Type I collagen and osteocalcin. It can be observed that the porosity was above 91.7% and the pore size was above 101 $\mu\textrm{m}$. Control scaffo1d and IP/PLGA scaffo1ds of 50% IP were implanted on the back of athymic nude mouse to observe the effect of IP on the induction of cells proliferation for 9 weeks. The evidence of calcification, osteoblast, and osteoid from the undifferentiated stem cells in the subcutaneous sites and other soft connective tissue sites having a preponderance of stem cells has been observed. From these results, it seems that IP plays an important role for bone induction in IP/PLCA scaffolds.

• Journal of Chest Surgery
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• v.46 no.1
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• pp.1-13
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• 2013

Background: Glutaraldehyde (GA) is a widely used cross-linking agent for improving mechanical properties and resistance to enzymatic degradation of collagenous tissue, but it has several drawbacks such as calcification and cytotoxicity. The aim of this study was to find the alternative effective cross-linking methods to GA. Materials and Methods: Bovine pericardium was processed with GA with ethanol+octanol and glycine detoxification, and polyethylene glycol (PG) space filler, dimethyl 3,3'-dithiobispropionimidate (DTBP), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) treatment, and the physical fixation of ultraviolet irradiation were done. The biologic material properties of variously treated pericardial tissues were assessed by biochemical, mechanical and histological tests. Treated pericardial tissues were also implanted subcutaneously or intramuscularly into the rabbit for 10 weeks to assess the xenoreactive antibody response of immunoglobulin G and M, their anti-calcification effect. Results: The biochemical and mechanical properties of EDC fixed pericardial tissues were comparable to the GA fixed tissue. The cytotoxicity was lowest in space filler treated GA fixed group. In rabbit subcutaneous or intramuscular implantation models, decellularization, space filler, EDC treatment group showed significantly lower calcium content than GA only and DTBP treatment group (p<0.05, analysis of variance). The titer of anti $Gal{\alpha}1-3Gal{\beta}1$-4GlcNAc-R antibodies did not change in the postimplantation serial enzyme-linked immunosorbent assay. Hematoxylin and eosin and von Kossa staining showed that decellularization, space filler, EDC, and ultraviolet treatment had less inflammatory cell infiltration and calcium deposits. Conclusion: The decellularization process, PG filler, and EDC treatments are good alternative cross-linking methods compared to GA only fixation and primary amine of DTBP treatment for cardiovascular xenograft preservation in terms of the collagen cross-linking stability and in vivo anti-calcification effects.