Im-Hee Jung

Isolation and characterization of human periodontal ligament (PDL) stem cells (PDLSCs) from the inflamed PDL tissue: in vitro and in vivo evaluations.

OBJECTIVES Mesenchymal stem cells (MSC) could be isolated from healthy periodontal ligaments (PDL). The aims of this study were to isolate and characterize human PDL stem cells (hPDLSCs) from inflamed PDL tissue, and to evaluate their regenerative potential. MATERIALS AND METHODS Inflamed hPDLSCs (ihPDLSCs) were isolated from the inflamed PDL tissue obtained from intra-bony defects during flap surgery, and characterized by immunohistochemical staining, colony-forming unit assay, fluorescence-activated cell sorting, and mRNA expression in comparison with healthy hPDLSCs obtained from extracted teeth for orthodontic purpose. The proliferative potential and migratory potential was evaluated, and compared with healthy hPDLSCs. Regenerative potential was assessed by an in vivo ectopic transplantation model. RESULTS ihPDLSCs were successfully isolated and characterized as MSCs. Both ihPDLSCs and hPDLSCs were successfully differentiated under osteogenic/cementogenic and adipogenic microenvironment. The proliferative potential did not differ between healthy hPDLSCs and ihPDLSCs, while the migratory capacity was significantly increased in ihPDLSCs (p<0.05). Both groups exhibited new cementum-like tissue and related PDL fibre regeneration in an in vivo transplantation model. CONCLUSION ihPDLSCs could be successfully isolated from inflamed PDL tissue, and they retained the regenerative potential for cementum and related PDL tissues.

Enhanced adipogenic differentiation and reduced collagen synthesis induced by human periodontal ligament stem cells might underlie the negative effect of recombinant human bone morphogenetic protein-2 on periodontal regeneration.

BACKGROUND AND OBJECTIVE Recombinant human bone morphogenetic protein-2 (rhBMP-2) is a potent inducer for the regeneration of mineralized tissue, but has a limited effect on the regeneration of cementum and periodontal ligament (PDL). The aim of the present study was to determine the effects of rhBMP-2 on the in vitro and in vivo biologic activity of well-characterized human PDL stem cells (hPDLSCs) and to elucidate the underlying mechanism of minimal periodontal regeneration by rhBMP-2. MATERIAL AND METHODS hPDLSCs were isolated and cultured, and then transplanted into an ectopic subcutaneous mouse model using a carrier treated either with or without rhBMP-2. Comprehensive histologic, histometric and immunohistochemical analyses were performed after an 8-wk healing period. The effects of rhBMP-2 on the adipogenic and osteogenic/cementogenic differentiation of hPDLSCs were also evaluated. The effect of rhBMP-2 on both soluble and insoluble collagen synthesis was analyzed, and the expression of mRNA and protein for collagen types I, II, III and V was assessed. RESULTS In the present study, rhBMP-2 promoted both adipogenic and osteogenic/cementogenic differentiation of hPDLSCs in vitro, and the in vivo potential of hPDLSCs to form mineralized cementum and organized PDL tissue was down-regulated following treatment with rhBMP-2. Collagen synthesis, which plays a crucial role in the regeneration of cementum and the periodontal attachment, was significantly reduced, with associated modification of the relevant mRNA and protein expression profiles. CONCLUSION In summary, the findings of the present study suggest that enhanced adipogenic differentiation and inhibition of collagen synthesis by hPDLSCs appear to be partly responsible for the minimal effect of rhBMP-2 on cementum and PDL tissue regeneration by hPDLSCs.

Mechanism of alveolar bone loss in a collagen-induced arthritis model in mice

OBJECTIVE the aim of this study was to understand the cellular/molecular mechanisms of periodontal breakdown in a collagen-induced arthritis (CIA) model in mice to enhance the understanding of rheumatoid arthritis (RA)-associated alveolar bone loss in humans. MATERIALS AND METHODS all analyses were performed on paired samples from CIA and control group mice. Mandibles were retrieved for micro-computed tomography (micro-CT), histomorphometric analysis, and isolation of alveolar bone cells (ABCs). In vitro osteoclastogenic/osteogenic/adipogenic potentials of ABCs were evaluated and the mRNA expression of downstream effector genes was assessed. Bone formation of ABCs was assessed using an ectopic transplantation model. RESULTS histomorphometric and micro-CT data showed that alveolar bone loss was significantly increased in the CIA group (p<0.05). Osteoclastogenesis was significantly increased in the CIA group in vivo (p<0.05), with upregulated mRNA expressions of osteoclastogenesis-associated genes. Osteoblasts appeared to undergo increased apoptosis, and the bone-forming activity of ABCs concomitantly decreased with in vitro osteogenic differentiation and in vivo ectopic transplantation (p<0.05). Also, adipogenesis-associated mRNA expression was highly expressed in the CIA group, resulting in significantly enhanced adipocyte differentiation in vitro (p<0.05). CONCLUSIONS these data demonstrate that increased osteoclastic activity, decreased bone-forming activity and enhanced adipogenesis promote alveolar bone loss in a CIA model in mice, and they suggest that these mechanisms could account for the same outcome in human RA.

Induction of bone formation by Escherichia coli‐expressed recombinant human bone morphogenetic protein‐2 using block‐type macroporous biphasic calcium phosphate in orthotopic and ectopic rat models

BACKGROUND AND OBJECTIVE The potential of the Escherichia coli-expressed recombinant human bone morphogenetic protein-2 (ErhBMP-2) to support new bone formation/maturation using a block-type of macroporous biphasic calcium phosphate (bMBCP) carrier was evaluated in an orthotopic and ectopic rat model. MATERIAL AND METHODS Critical-size (Φ 8 mm) calvarial defects and subcutaneous pockets in 32 Sprague-Dawley rats received implants of rhBMP-2 (2.5 μg) in a bMBCP carrier or bMBCP alone (control). Implant sites were evaluated using histological and histometric analysis following 2- and 8-wk healing intervals (eight animals/group/interval). RESULTS   ErhBMP-2/bMBCP supported significantly greater bone formation at 2 and 8 wk (10.8% and 25.4%, respectively) than the control at 2 and 8 wk (5.3% and 14.0%, respectively) in calvarial defects (p < 0.01). Bone formation was only observed for the ErhBMP-2/bMBCP ectopic sites and was significantly greater at 8 wk (7.5%) than at 2 wk (4.5%) (p < 0.01). Appositional and endochondral bone formation was usually associated with a significant increase in fatty marrow at 8 wk. The bMBCP carrier showed no evidence of bioresorption. CONCLUSION ErhBMP-2/bMBCP induced significant bone formation in both calvarial and ectopic sites. Further study appears to be required to evaluate the relevance of the bMBCP carrier.

Optimal Medium Formulation for the Long-Term Expansion and Maintenance of Human Periodontal Ligament Stem Cells

BACKGROUND Human periodontal ligament stem cells (hPDLSCs) are promising mesenchymal stem cells that are readily accessible. However, there is as yet no consensus as to the optimal culture medium for hPDLSCs. Thus, the purpose of the present study is to determine the optimal culture medium for long-term expansion of hPDLSCs. METHODS hPDLSCs were isolated from healthy third molars, and the most widely used medium formulations in previous studies were used: 1) an α minimum essential medium-based medium formulation (MBM); and 2) a Dulbeccos minimum essential medium-based medium formulation. Passage 5 (P5) and P8 were evaluated with the two media for cell proliferation, differentiation, and immunophenotype. RESULTS hPDLSCs that were primarily cultured in MBM were far more proliferated than those grown in DBM. In general, application of the MBM for longer periods produced greater cell growth and osteogenic differentiation. Furthermore, MBM-precultured hPDLSCs exhibited a greater degree of cell proliferation and a greater production of mineralized tissue and alkaline phosphatase (ALP) activity in vitro, although the levels of both were dependent on the culture medium used. With respect to long-term expansion, the P5 hPDLSCs grew and produced the largest amount of mineralized nodules faster than the P8 hPDLSCs, but both passages exhibited a similar phenotype for stemness and ALP activity. CONCLUSION The present study indicates that the inherent capacity of hPDLSCs could be maintained until a later passage, P8 in MBM, and MBM appears to be an optimal choice for manipulating the finest and most stable hPDLSCs.

Effect of (-)-epigallocatechin-3-gallate on maintaining the periodontal ligament cell viability of avulsed teeth: a preliminary study

Purpose Avulsed tooth can be completely recovered, if sound periodontal ligament (PDL) of tooth is maintained. Although a lot of storage solutions have been explored for the better storage of avulsed tooth, there is a shortcoming that the preservation time is much short. On the other hand, there has been studies that (-)-epigallocatechin-3-gallate (EGCG), the most abundant polyphenol in green tea, which is related to the anti inflammatory, antioxygenic, and antibacterial effects, allows the successful preservations of tissues and cells. This study evaluated the effect of EGCG on avulsed-teeth preservation of Beagle dogs for a period of time. Methods The atraumatically extracted teeth of Beagle dogs were washed and preserved with 0/10/100 µM of EGCG at the time of immediate, period 1 (4 days in EGCG-contained media and additional 1 day in EGCG-free media), period 2 (8 days in EGCG-contained media and additional 2 days in EGCG-free media) and period 3 (12 days in EGCG-contained media and additional 2 days in EGCG-free media). Then, the cell viabilities of preserved teeth was calculated by dividing optical density (OD) of 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay with OD of eosin assay to eliminate the measurement errors caused by the different tissue volumes. Results From the results, the immediately analyzed group presented the highest cell viability, and the rate of living cells on teeth surface decreased dependent on the preservation period. However, the 100 µM of EGCG-treated group showed statistically significant positive cell activity than EGCG-free groups throughout preservation periods. Conclusions Our findings showed that 100 µM EGCG could maintain PDL cell viability of extracted tooth. These results suggest that although EGCG could not be a perfect additive for tooth preservation, it is able to postpone the period of tooth storage. However, further in-depth studies are required for more plausible use of EGCG.

Regeneration of rabbit calvarial defects using cells-implanted nano-hydroxyapatite coated silk scaffolds

BackgroundThe aim of this study was to characterize the efficacy of nano-hydroxyapatite-coated silk fibroin constructs as a scaffold for bone tissue engineering and to determine the osteogenic effect of human dental pulp and periodontal ligament derived cells at an early stage of healing in rabbits. 3D silk fibroin constructs were developed and coated using nano-hydroxyapatite crystals. Dental pulp and periodontal ligament cells from extracted human third molars were cultured and seeded onto the silk scaffolds prior to in vivo implantation into 8 male New Zealand White rabbits. Four circular windows 8 mm in diameter were created in the calvarium of each animal. The defects were randomly allocated to the groups; (1) silk scaffold with dental pulp cells (DPSS), (2) silk scaffold with PDL cells (PDLSS), (3) normal saline-soaked silk scaffold (SS), and (4) empty control. The animals were sacrificed 2 (n = 4) or 4 weeks (n = 4) postoperatively. The characteristics of the silk scaffolds before and after cell seeding were analyzed using SEM. Samples were collected for histologic and histomorphometic analysis. ANOVA was used for statistical analysis.ResultHistologic view of the experimental sites showed well-maintained structure of the silk scaffolds mostly unresorbed at 4 weeks. The SEM observations after cell-seeding revealed attachment of the cells onto silk fibroin with production of extracellular matrix. New bone formation was observed in the 4 week groups occurring from the periphery of the defects and the silk fibers were closely integrated with the new bone. There was no significant difference in the amount of bone formation between the SS group and the DPSS and PDLSS groups.ConclusionWithin the limitations of this study, silk scaffold is a biocompatible material with potential expediency as an osteoconductive scaffold in bone tissue engineering. However, there was no evidence to suggest that the addition of hDPCs and hPDLCs to the current rabbit calvarial defect model can produce an early effect in augmenting osteogenesis.

EGF enhances low-invasive cancer cell invasion by promoting IMP-3 expression

The initiation and progression of cancer is closely associated with the tumor microenvironment. The overexpression of oncogenes during tumor growth and progression by stromal stimuli can affect the aggressiveness of the cancer. In this study, in vitro and in vivo studies were performed to examine the role of stromal epidermal growth factor (EGF) in enhancing the invasive potential of in low-invasive cancer. EGF was tested in order to elucidate the specific molecules that participate in increasing the invasive potential of low-invasive cancer cells. EGF stimulation enhanced cancer invasion in an EGF receptor (EGFR)-dependent manner. EGF induced insulin-like growth factor-II mRNA-binding protein-3 (IMP-3) and podoplanin (PDPN) expression, which play an important role in oral squamous cell carcinoma (OSCC) cell invasion. An apparent tumor mass was not observed in the mouse xenograft; however, multiple tumor microfoci were seen in mice injected with IMP-3-overexpressing cells. These results show that EGF stimulates IMP-3 expression, thereby increasing cancer invasion and tumor progression.