Pill-Hoon Choung

Efficacy of Periodontal Stem Cell Transplantation in the Treatment of Advanced Periodontitis

Periodontitis is the most common cause for tooth loss in adults and advanced types affect 10–15% of adults worldwide. The attempts to save tooth and regenerate the periodontal apparatus including cementum, periodontal ligament, and alveolar bone reach to the dental tissue-derived stem cell therapy. Although there have been several periodontitis models suggested, the apical involvement of tooth root is especially challenging to be regenerated and dental stem cell therapy for the state has never been investigated. Three kinds of dental tissue-derived adult stem cells (aDSCs) were obtained from the extracted immature molars of beagle dogs (n = 8), and ex vivo expanded periodontal ligament stem cells (PDLSCs), dental pulp stem cells (DPSCs), and periapical follicular stem cells (PAFSCs) were transplanted into the apical involvement defect. As for the lack of cementum-specific markers, anti-human cementum protein 1 (rhCEMP1) antibody was fabricated and the aDSCs and the regenerated tissues were immunostained with anti-CEMP1 antibody. Autologous PDLSCs showed the best regenerating capacity of periodontal ligament, alveolar bone, and cementum as well as peripheral nerve and blood vessel, which were evaluated by conventional and immune histology, 3D micro-CT, and clinical index. The rhCEMP1 was expressed strongest in PDLSCs and in the regenerated periodontal ligament space. We suggest here the PDLSCs as the most favorable candidate for the clinical application among the three dental stem cells and can be used for treatment of advanced periodontitis where tooth removal was indicated in the clinical cases.

Graphene-incorporated chitosan substrata for adhesion and differentiation of human mesenchymal stem cells†

A simple method that uses graphene to fabricate nanotopographic substrata was reported for stem cell engineering. Graphene-incorporated chitosan substrata promoted adhesion and differentiation of human mesenchymal stem cells (hMSCs). In addition, we proposed that nanotopographic cues of the substrata could enhance cell-cell and cell-material interactions for promoting functions of hMSCs.

Nuclear Factor I-C Is Essential for Odontogenic Cell Proliferation and Odontoblast Differentiation during Tooth Root Development

Our previous studies have demonstrated that nuclear factor I-C (NFI-C) null mice developed short molar roots that contain aberrant odontoblasts and abnormal dentin formation. Based on these findings, we performed studies to elucidate the function of NFI-C in odontoblasts. Initial studies demonstrated that aberrant odontoblasts become dissociated and trapped in an osteodentin-like mineralized tissue. Abnormal odontoblasts exhibit strong bone sialoprotein expression but a decreased level of dentin sialophosphoprotein expression when compared with wild type odontoblasts. Loss of Nfic results in an increase in p-Smad2/3 expression in aberrant odontoblasts and pulp cells in the subodontoblastic layer in vivo and primary pulp cells from Nfic-deficient mice in vitro. Cell proliferation analysis of both cervical loop and ectomesenchymal cells of the Nfic-deficient mice revealed significantly decreased proliferative activity compared with wild type mice. In addition, Nfic-deficient primary pulp cells showed increased expression of p21 and p16 but decreased expression of cyclin D1 and cyclin B1, strongly suggesting cell growth arrest caused by a lack of Nfic activity. Analysis of the pulp and abnormal dentin in Nfic-deficient mice revealed an increase in apoptotic activity. Further, Nfic-deficient primary pulp cells exhibited an increase in caspase-8 and -3 activation, whereas the cleaved form of Bid was hardly detected. These results indicate that the loss of Nfic leads to the suppression of odontogenic cell proliferation and differentiation and induces apoptosis of aberrant odontoblasts during root formation, thereby contributing to the formation of short roots.

Synergistic effects of nanotopography and co-culture with endothelial cells on osteogenesis of mesenchymal stem cells.

Inspired by the aligned nanostructures and co-existence of vascular cells and stem cells in human cancellous bone, we quantitatively investigated the relative contributions of nanotopography and co-culture with human umbilical endothelial cells (HUVECs) to the osteogenesis of human mesenchymal stem cells (hMSCs). Although both nanotopography and co-culture independently enhanced the osteogenesis of hMSCs, osteogenesis was further enhanced by the two factors in combination, indicating the importance of synergistic cues in stem cell engineering. Interestingly, nanotopography provided a larger relative contribution to the osteogenesis of hMSCs than did co-culture with HUVECs. Furthermore, the osteogenesis of hMSCs was also affected by the density of parallel nanogrooves, exhibiting a maximum at a 1:3 spacing ratio, as defined as the ratio of ridge width to groove width. Analysis of (i) biochemical soluble factors, (ii) hMSC-substrate interaction and (iii) hMSC-HUVEC interaction suggests that (ii) and (iii) play a crucial role in mediating osteogenic phenotypes.

Vascularized cranial bone grafts for mandibular and maxillary reconstruction. The parietal osteofascial flap

The authors have performed 13 cases of vascularized cranial bone grafts for reconstruction of maxillofacial defects since 1986. Two types of flaps were used: the parietal osteofascial flap pedicled to the parieto-temporal fascia based on the superficial temporal artery and the temporalis osteomuscular flap pedicled to the temporalis muscle based on the deep temporal artery. Zygomatico-orbital complex, maxilla and mandible were reconstructed and hemifacial microsomia was also treated. The results of vascularized cranial bone grafts pedicled to fascia were as good as those of grafts pedicled to muscle. There were no major complications. Two types of vascularized cranial bone grafts seem to be useful in reconstruction of maxillofacial defects with avascular recipient beds because of their good blood supply. The parietal osteofascial flap has additional advantages including easy rotation of the flap to the defect, particularly a mandibular defect, and versatile use of fascia without bulkiness for reconstruction of soft tissue defects. This flap can be designed as a full- or partial-thickness cranial bone graft with good vascularity. In this paper, our technique for mandibular and maxillary reconstruction using the parietal osteofascial flap is introduced, and the results compared with our temporalis osteomuscular flap technique are reported.

Effect of platelet-rich plasma on dental stem cells derived from human impacted third molars

AIM Platelet-rich plasma (PRP) is fabricated from autologous blood and extensively used to promote soft and hard tissue healing. In the dental field, autologous PRP is widely used combined with dental implant installation and bone graft. This study will evaluate the biologic effect of PRP on the proliferation and the differentiation of human dental stem cells, and find the key cytokines inducing these effects to estimate the clinical feasibility of PRP for dental tissue engineering. MATERIALS & METHODS Venous blood was obtained from four individuals and each PRP was fabricated. The human dental stem cells were obtained from the periodontal ligament (PDL) and dental pulp of the surgically extracted human third molars and expanded in vitro. Immunocytochemical staining and flow cytometry with STRO-1 and CD146 confirmed existence of mesenchymal stem cells in the PDL and dental pulp. The effect of PRP on the proliferation of PDL stem cells (PDLSCs) and dental pulp stem cells (DPSCs) was assessed by colony-forming ability measurement, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and bromodeoxyuridine incorporation assay. Alkaline phosphatase activity and calcium deposit were measured to evaluate the mineralization effect of PRP PDLSCs and DPSCs. Alizarin red S staining was used to detect mineral nodules. Odontogenic and osteogenic gene expressions were evaluated in the PRP-treated PDLSCs and DPSCs by real-time quantitative PCR. A protein array was performed to detect the key cytokines that have an important role in the tissue regenerative effect of PRP. RESULTS Flow cytometry cell sorting showed that the cells from human PDL and dental pulp contained mesenchymal stem cell populations. Colony-forming ability and cellular proliferation of the dental stem cells were increased at 0.5 and 1% PRP concentration but decreased at 5% concentration. Long-term treatment with 1% PRP enhanced proliferation of the human dental stem cells PDLSCs and DPSCs by 120 h and showed the most significant enhancement at 96 h. PRP also promoted mineralization differentiation of the two kinds of dental stem cells as shown by measurement of alkaline phosphatase activity and calcium deposit under mineralization conditioned media. Increased formation of mineral nodules stained with alizarin red was observed in both PDLSCs and DPSCs after treatment with 1% PRP. Real-time quantitative PCR showed higher odontogenic and osteogenic gene expressions in PRP-treated PDLSCs and DPSCs. RANTES/CCL5 and ICAM-1 were the two key cytokines that were detected in human cytokine array with PRP. CONCLUSION The appropriate concentration of the PRP treatment enhanced proliferation and mineralization differentiation of human dental stem cells. RANTES/CCL5 and ICAM-1 might play an important role in PRP-induced tissue regeneration but further study is needed to investigate the whole mechanism.

An intraoral approach to treatment of condylar hyperplasia or high condylar process fractures using the intraoral vertico-sagittal ramus osteotomy

PURPOSE The intraoral vertico-sagittal ramus osteotomy (J Craniomaxillofac Surg 20:153, 1992) can be used to reduce high condylar process fractures and recontour hyperplastic condyles while simultaneously correcting the malocclusion. This article presents the technique and reports the clinical results. PATIENTS AND METHODS A technique for removal and replantation of the condyloid process using the vertico-sagittal ramus osteotomy and rigid fixation was used to treat 23 patients with markedly displaced, high condylar process fractures and condylar hyperplasia associated with malocclusion. RESULTS The replanted condyles did not show ischemic necrosis or any functional disturbance when followed for more than 3 years. All patients showed nearly normal mouth opening, with slight mandibular deviation, usually in sixth postoperative month. CONCLUSIONS In selected patients, this technique allows intraoral accessibility to the condyle and its repositioning. The method is particularly useful to treat vertical discrepancies associated with the hyperplastic or hypoplastic condyle.

In Vitro Effects of Low-Intensity Pulsed Ultrasound Stimulation on the Osteogenic Differentiation of Human Alveolar Bone-Derived Mesenchymal Stem Cells for Tooth Tissue Engineering

Ultrasound stimulation produces significant multifunctional effects that are directly relevant to alveolar bone formation, which is necessary for periodontal healing and regeneration. We focused to find out effects of specific duty cycles and the percentage of time that ultrasound is being generated over one on/off pulse period, under ultrasound stimulation. Low-intensity pulsed ultrasound ((LIPUS) 1 MHz) with duty cycles of 20% and 50% was used in this study, and human alveolar bone-derived mesenchymal stem cells (hABMSCs) were treated with an intensity of 50 mW/cm2 and exposure time of 10 min/day. hABMSCs exposed at duty cycles of 20% and 50% had similar cell viability (O.D.), which was higher (*P < 0.05) than that of control cells. The alkaline phosphatase (ALP) was significantly enhanced at 1 week with LIPUS treatment in osteogenic cultures as compared to control. Gene expressions showed significantly higher expression levels of CD29, CD44, COL1, and OCN in the hABMSCs under LIPUS treatment when compared to control after two weeks of treatment. The effects were partially controlled by LIPUS treatment, indicating that modulation of osteogenesis in hABMSCs was related to the specific stimulation. Furthermore, mineralized nodule formation was markedly increased after LIPUS treatment than that seen in untreated cells. Through simple staining methods such as Alizarin red and von Kossa staining, calcium deposits generated their highest levels at about 3 weeks. These results suggest that LIPUS could enhance the cell viability and osteogenic differentiation of hABMSCs, and could be part of effective treatment methods for clinical applications.

Development of water‐insoluble chitosan patch scaffold to repair traumatic tympanic membrane perforations

Perforated tympanic membranes (TM) and otitis media can be managed with a paper patch or tympanoplasty. However, a paper patch is not biocompatible and tympanoplasty requires complex aseptic surgical procedures. A novel biocompatible patch with a water-insoluble chitosan as the main component was prepared. Optimal mechanical characteristics of a water-insoluble chitosan patch scaffold (CPS) was approximately 40 microm in thickness, 7 MPa in tensile strength, and 107% in percent elongation, even though the characteristics varied significantly depending on the concentrations of chitosan and glycerol. SEM of the CPSs showed a very smooth surface as compared with that of the paper patches. These CPSs showed no cytotoxicity and had a stimulating effect on the proliferation of TM cells in in vitro study. In in vivo study, 4 (21.1%) and 17 (89.5%) TMs out of 19 adult rats with CPSs showed no perforations at 1 and 2 weeks, respectively. However, left control TMs showed healing of 0 (0%) at 1 week and 18 (94.7%) at 2 weeks. TEM findings of regenerated eardrums using CPSs showed thinner, smoother, and more compact tissues than spontaneously healed eardrums. A CPS was more effective than spontaneous healing to repair traumatic TM perforations.

Synergistic activity of fibronectin and fibroblast growth factor receptors on neuronal adhesion and neurite extension through extracellular signal-regulated kinase pathway.

Fibroblast growth factor (FGF) is an important modulator of cell growth and differentiation of various cells including neuron. Cells need to adhere specifically to cellular and extracellular components of their environment to carry out diverse physiological functions. Here, we examined whether fibronectin (FN) and FGF can cooperate for neuronal adhesion and neurite outgrowth. Using recombinant FN peptide (FNIII9-10), we found that FNIII9-10-mediated adhesion promotes the effect of FGF1 on neurite outgrowth of PC12 cells, while FGF1 enhances the FNIII9-10-mediated neuronal adhesion of PC12 cells. This collaboration of FNIII9-10 and FGF1 was the result of the sustained activation of extracellular signal-regulated kinase (ERK)-type MAP kinase. Finally, the synergistic activity of FGF1 and FN was inhibited by PD98059, an MEK inhibitor. Taken together, these findings indicate that FN-mediated signaling can collaborate with FGFRs signaling for neurite outgrowth through selective activation of ERK-type MAP kinase in PC12 cells, and suggest that FN and FGF act in concert to regulate cell differentiation in the nervous system.