Cell Surface Accumulation of Intracellular Leucine Proline-Enriched Proteoglycan 1 Enhances Odontogenic Potential of Human Dental Pulp Stem Cells.

Primary dental pulp cells can be differentiated into odontoblast-like cells, which are responsible for dentin formation and mineralization. Successful differentiation of primary dental pulp cells can be verified using a few markers. However, odontoblast-specific cell surface markers have not been fully studied yet. LEucine PRoline-Enriched Proteoglycan 1 (LEPRE1) is a basement membrane-associated proteoglycan. LEPRE1 protein levels are increased during odontoblastic differentiation of human dental pulp cells (hDPCs). Intracellular and cell surface accumulation of this protein completely disappeared during dentin maturation and mineralization. Cell surface binding of an anti-LEPRE1 monoclonal antibody that could recognize an extracellular region was gradually increased in the odontoblastic stage. Overexpression and knockdown experiments showed that accumulation of intracellular LEPRE1 could lead to inefficient odontoblastic differentiation and that the movement of LEPRE1 from intracellular region to the cell surface was required for odontoblastic differentiation. Indeed, when LEPRE1 already located on the cell surface was blocked by the anti-LEPRE1 monoclonal antibody, odontoblastic differentiation of hDPCs was inhibited. In this study, we looked at other aspects of LEPRE1 function as a cell surface molecule rather than its known intracellular hydroxylase activity. Our results indicate that this protein has potential as a specific cell surface marker in odontoblastic differentiation.

3D Spheroid Formation Using BMP-Loaded Microparticles Enhances Odontoblastic Differentiation of Human Dental Pulp Stem Cells.

Human dental pulp stem cells (hDPSCs) are the primary cells responsible for dentin regeneration. Typically, in order to allow for odontoblastic differentiation, hDPSCs are cultured over weeks with differentiation-inducing factors in a typical monolayered culture. However, monolayered cultures have significant drawbacks including inconsistent differentiation efficiency, require a higher BMP concentration than should be necessary, and require periodic treatment with BMPs for weeks to see results. To solve these problems, we developed a 3D-cell spheroid culture system for odontoblastic differentiation using microparticles with leaf-stacked structure (LSS), which allow for the sustained release of BMPs and adequate supply of oxygen in cell spheroids. BMPs were continuously released and maintained an effective concentration over 37 days. hDPSCs in the spheroid maintained their viability for 5 weeks, and the odontoblastic differentiation efficiency was increased significantly compared to monolayered cells. Finally, dentin-related features were detected in the spheroids containing BMPs-loaded microparticles after 5 weeks, suggesting that these hDPSC-LSS spheroids might be useful for dentin tissue regeneration.

Identification of dentinogenic cell-specific surface antigens in odontoblast-like cells derived from adult dental pulp.

BACKGROUND: Odontoblast is a unique progenitor that plays a role in dentin formation. So far, the dentinogenic differentiation of dental pulp stem cells and the role of surface molecules of odontoblasts in dentinogenesis are not well known yet. In this study, we obtained odontoblast-like cells from human dental pulp cells and screened odontoblast-specific cell surface antigens by decoy immunization. METHODS: Through decoy immunization with intact odontoblast-like cells derived from human dental pulp cells, we constructed 12 monoclonal antibodies (mAbs) of IgG type, and their binding affinities for cell surface of odontoblast-like cells were analyzed by flow cytometry. Immunoprecipitation, mass spectrometry, and immunohistochemistry were performed to demonstrate odontoblast-specific antigens. Odontoblasts were sorted by these mAbs using magnetic-activated cell sorting system, and their mineralization efficiency was increased after sorting. RESULTS: We constructed 12 mAbs of IgG type, which had a strong binding affinity for cell surface antigens of odontoblast-like cells. In human adult tooth, these mAbs accumulated in the odontoblastic layer between dentin and pulp and in the perivascular region adjacent to the blood vessels in the pulp core. Cell surface expression of the antigenic molecules was increased during odontogenic cytodifferentiation and decreased gradually as dentinogenic maturation progressed. Proteomic analysis showed that two representative antigenic molecules, OD40 and OD46, had the potential to be components for cell adhesion and extracellular matrix structures. CONCLUSION: These results suggest that mAbs will be useful for detecting and separating odontoblasts from the primary pulp cells and other lineage cells and will provide information on the structures of extracellular matrix and microenvironment that appears during the dentinogenic differentiation.

Amelogenic transcriptome profiling in ameloblast-like cells derived from adult gingival epithelial cells.

Dental enamel is the highly mineralized tissue covering the tooth surface and is formed by ameloblasts. Ameloblasts have been known to be impossible to detect in adult tooth because they are shed by apoptosis during enamel maturation and tooth eruption. Owing to these, little was known about appropriate cell surface markers to isolate ameloblast-like cells in tissues. To overcome these problems, epithelial cells were selectively cultivated from the gingival tissues and used as a stem cell source for ameloblastic differentiation. When gingival epithelial cells were treated with a specified concentration of BMP2, BMP4, and TGFß-1, the expression of ameloblast-specific markers was increased, and both the MAPK and Smad signaling pathways were activated. Gingival epithelial cells differentiated into ameloblast-like cells through epithelial-mesenchymal transition. By RNA-Seq analysis, we reported 20 ameloblast-specific genes associated with cell surface, cell adhesion, and extracellular matrix function. These cell surface markers might be useful for the detection and isolation of ameloblast-like cells from dental tissues.

Effect of FGF-2, TGF-ß-1, and BMPs on Teno/Ligamentogenesis and Osteo/Cementogenesis of Human Periodontal Ligament Stem Cells.

The periodontal ligament (PDL) is the connective tissue between tooth root and alveolar bone containing mesenchymal stem cells (MSC). It has been suggested that human periodontal ligament stem cells (hPDLSCs) differentiate into osteo/cementoblast and ligament progenitor cells. The periodontitis is a representative oral disease where the PDL tissue is collapsed, and regeneration of this tissue is important in periodontitis therapy. Fibroblast growth factor-2 (FGF-2) stimulates proliferation and differentiation of fibroblastic MSCs into various cell lineages. We evaluated the dose efficacy of FGF-2 for cytodifferentiation of hPDLSCs into ligament progenitor. The fibrous morphology was highly stimulated even at low FGF-2 concentrations, and the expression of teno/ligamentogenic markers, scleraxis and tenomodulin in hPDLSCs increased in a dose dependent manner of FGF-2. In contrast, expression of the osteo/cementogenic markers decreased, suggesting that FGF-2 might induce and maintain the ligamentogenic potential of hPDLSCs. Although the stimulation of tenocytic maturation by TGF-ß1 was diminished by FGF-2, the inhibition of the expression of early ligamentogenic marker by TGF-ß1 was redeemed by FGF-2 treatment. The stimulating effect of BMPs on osteo/cementogenesis was apparently suppressed by FGF-2. These results indicate that FGF-2 predominantly differentiates the hPDLSCs into teno/ligamentogenesis, and has an antagonistic effect on the hard tissue differentiation induced by BMP-2 and BMP-4.

In Vitro and In Vivo Dentinogenic Efficacy of Human Dental Pulp-Derived Cells Induced by Demineralized Dentin Matrix and HA-TCP.

Human dental pulp cells have been known to have the stem cell features such as self-renewal and multipotency. These cells are differentiated into hard tissue by addition of proper cytokines and biomaterials. Hydroxyapatite-tricalcium phosphates (HA-TCPs) are essential components of hard tissue and generally used as a biocompatible material in tissue engineering of bone. Demineralized dentin matrix (DDM) has been reported to increase efficiency of bone induction. We compared the efficiencies of osteogenic differentiation and in vivo bone formation of HA-TCP and DDM on human dental pulp stem cells (hDPSCs). DDM contains inorganic components as with HA-TCP, and organic components such as collagen type-1. Due to these components, osteoinduction potential of DDM on hDPSCs was remarkably higher than that of HA-TCP. However, the efficiencies of in vivo bone formation are similar in HA-TCP and DDM. Although osteogenic gene expression and bone formation in immunocompromised nude mice were similar levels in both cases, dentinogenic gene expression level was slightly higher in DDM transplantation than in HA-TCP. All these results suggested that in vivo osteogenic potentials in hDPSCs are induced with both HA-TCP and DDM by osteoconduction and osteoinduction, respectively. In addition, transplantation of hDPSCs/DDM might be more effective for differentiation into dentin.

Functional efficacy of human recombinant FGF-2s tagged with (His)6 and (His-Asn)6 at the N- and C-termini in human gingival fibroblast and periodontal ligament-derived cells.

Fibroblast growth factor (FGF) is a multifunctional growth factor that induces cell proliferation, survival, migration, and differentiation in various cell types and tissues. With these biological functions, FGF-2 has been evaluated for clinical use in the regeneration of damaged tissues. The expression of hFGF-2 in Escherichia coli and a purification system using the immobilized metal affinity chromatography (IMAC) is well established to generate a continuous supply of FGF-2. Although hexa-histidine tag (H6) is commonly used for IMAC purification, hexa-histidine-asparagine tag (HN6) is also efficient for purification as it is easily exposed on the surface of the protein. In this study, four different tagging constructs of hFGF-2 based on tag positions and types (H6-FGF2, FGF2-H6, HN6-FGF2, and FGF2-HN6) were designed and expressed under the inducible T7 expression system in E. coli. The experimental conditions of expression and purification of each recombinant protein were optimized. The effective dosages of the recombinant proteins were determined based on the increase of cell proliferation in human gingival fibroblast. ED50s of H6-FGF2, FGF2-H6, HN6-FGF2, and FGF2-HN6 were determined (4.42 ng/ml, 3.55 ng/ml, 3.54 ng/ml, and 4.14 ng/ml, respectively) and found to be comparable to commercial FGF-2 (3.67 ng/ml). All the recombinant hFGF-2s inhibit the osteogenic induction and mineralization in human periodontal ligament-derived cells. Our data suggested that biological activities of the recombinant hFGF-2 are irrelevant to types and positions of tags, but may have an influence on the expression efficiency and solubility.

A monoclonal antibody recognizes undifferentiation-specific carbohydrate moieties expressed on cell surface of the human dental pulp cells.

Human dental pulp cells are obtained from dental pulp tissue, and have the ability to form dentin and a pulp-like complex. Although adult stem cells have been identified from the primary culture by using specific cell surface markers, the identity of surface markers for the purification of stem cells within the dental pulp population are still unclear. Previously, we had constructed monoclonal antibodies against the undifferentiated cell-specific surface markers of human dental pulp cells (hDPCs) by performing decoy immunization. Among them, a monoclonal antibody against the cell surface antigen of the undifferentiated hDPCs (named UPSA-1) was purified and its heavy and light chain consensus regions were analyzed. The cell surface binding affinity of UPSA-1 mAb on the undifferentiated hDPCs was stronger than that on the differentiated cells. When tunicamycin was applied to hDPSCs during culture, the cell surface binding affinity of the antibody was dramatically decreased, and dentinogenic differentiation was reduced. The purified UPSA-1 antigen band resulting from immunoprecipitation disappeared or shifted down on the SDS-PAGE by deglycosylation. These data suggested that glycosylation on the cell surface might be a marker of an undifferentiated state, and that UPSA-1 mAb might be useful for identifying the carbohydrate moiety on the cell surface of undifferentiated pulp cells.

Indirect co-culture of stem cells from human exfoliated deciduous teeth and oral cells in a microfluidic platform.

Oral epithelial-mesenchymal interactions play a key role in tooth development and assist differentiation of dental pulp. Many epithelial and mesenchymal factors in the microenvironment influence dental pulp stem cells to differentiate and regenerate. To investigate the interaction between oral cells during differentiation, we designed a microfluidic device system for indirect co-culture. The system has several advantages, such as consumption of low reagent volume, high-throughput treatment of reagents, and faster mineralization analysis. In this study, stem cells from human exfoliated deciduous teeth were treated with media cultured with human gingival fibroblasts or periodontal ligament stem cells. When human exfoliated deciduous teeth was incubated in media cultured in human gingival fibroblasts and human periodontal ligament stem cells under the concentration gradient constructed by the microfluidic system, no remarkable change in human exfoliated deciduous teeth mineralization efficiency was detected. However, osteoblast gene expression levels in human exfoliated deciduous teeth incubated with human gingival fibroblasts media decreased compared to those in human exfoliated deciduous teeth treated with human periodontal ligament stem cells media, suggesting that indirect co-culture of human exfoliated deciduous with human gingival fibroblasts may inhibit osteogenic cytodifferentiation. This microfluidic culture device allows a co-culture system set-up for sequential treatment with co-culture media and differentiation additives and facilitated the mineralization assay in a micro-culture scale.

Three Different Methods in Deformity Correction of Degenerative Flat Back: A Single Surgeon's Experience with 64 Consecutive Cases.

STUDY DESIGN: Retrospective study. PURPOSE: To evaluate the radiological and clinical results of three different methods in the deformity correction of a degenerative flat back. OVERVIEW OF LITERATURE: There are no comparative studies about different procedures in the treatment of degenerative flat back. METHODS: Sixty-four patients who consecutively underwent corrective surgery for degenerative flat back were reviewed. The operations were performed by three different methods: posterior-only (group P, n=20), one-stage anterior-posterior (group AP, n=12), and two-stage anterior-posterior with iliac screw fixation (group AP-I, n=32). Medical and surgical complications were examined and radiological and clinical results were compared. RESULTS: The majority of medical and surgical complications were found in group AP (5/12) and group P (7/20). The sagittal vertical axes were within normal range immediately postoperatively in all groups, but only group AP-I showed normal sagittal alignment at the final follow-up. Postoperative lumbar lordosis was also significantly higher in group AP-I than in group P or group AP and the finding did not change through the last follow-up. The Oswestry disability index was significantly lower in groups AP and AP-I than in group P at the final follow-up. Meanwhile, the operating time was the longest in group AP-I, and total amount of blood loss was larger in group AP-I and group AP than in group P. CONCLUSIONS: Anterior-posterior correction showed better clinical results than posterior-only correction. Two-staged anterior-posterior correction with iliac screw fixation showed better radiological results than posterior-only or one-staged anterior-posterior correction. Two-staged anterior-posterior correction with iliac screw fixation also showed a lower complication rate than one-staged anterior-posterior correction.

Immunomic Screening of Cell Surface Molecules on Undifferentiated Human Dental Pulp Stem Cells.

Human adult dental pulp tissue is a source of adult stem cells that have a potential to differentiate into various tissues, although the primary cell suspensions cultured from pulp tissue are mixtures of both stem cell and nonstem cell populations with heterogeneous phenotypes and various differentiation efficiencies. Therefore, cell surface protein markers on dental pulp stem cells are critical for detection and purification of stem cell populations. Yet, little is known about the cell surface molecules that are specifically associated with the undifferentiated and progenitor state of human adult dental pulp stem cells (hDPSCs). Presently, cell surface proteins expressed on hDPSCs were assessed by screening surface molecules specifically expressed on dentinogenic progenitors. Using a decoy immunization strategy, a set of monoclonal antibodies (MAbs) was generated against undifferentiated pulp progenitor cells. Forty-five hybridomas produced MAbs that interacted weakly, if at all, to differentiated pulp cells. Of these, 19 MAbs (18 IgG, 1 IgM) recognized surface molecules on undifferentiated hDPSCs. By multicolor flow cytometric analysis, 40%-60% of newly identified MAb-positive cells were demonstrated to be positive for the CD44 and CD90 mesenchymal markers. When MAb-positive cells were sorted from the heterogeneous pulp cell suspension, mineralization efficiency was increased three to five times compared with MAb-negative cells. The results suggest that the decoy immunization is an efficient method for isolation of MAbs against dentinogenic progenitors. These MAbs will be helpful for identification and enrichment of hDPSCs for efficient dentin regeneration.

Heterotopic ossification following lumbar total disc replacement.

The main goal of total disc replacement (TDR) is to preserve motion. Despite reports of good clinical outcomes, various degrees of heterotopic ossification after TDR have been reported. The purpose of this study was to investigate the prevalence and its clinical relevance of heterotopic ossification. We evaluated 65 consecutive patients (82 segments) with mean follow-up duration of 45 months (range, 12-88 months). Two kinds of prosthesis, ProDisc® for 75 segments (91.5%) and CHARITE™ for seven segments (8.5%), were used. Patients with heterotopic ossification were compared with those without heterotopic ossification with regard to segmental flexion-extension ROM, VAS and ODI. We analysed the occurrence site by nine zones. Heterotopic ossification was detected in 25 out of 82 segments (30.5%) at a mean follow-up of 17 months. According to McAfee's classification, there was Class-I heterotopic ossification in eight segments (9.8%), Class-II in 12 segments (14.6%), and Class-III in five segments (6.1%). There was no Class-IV heterotopic ossification. There were no significant differences in the segmental ROM, VAS and ODI between the patients with Class-I or Class-II heterotopic ossification and those without heterotopic ossification The segmental ROM in the patients with Class-III heterotopic ossification was significantly decreased compared with the patients without heterotopic ossification (p = 0.018). But VAS and ODI were not significantly different compared with those of patients with no heterotopic ossification. Most heterotopic ossification (82.5%) was detected in the anterior and posterior aspects. In conclusion, most of the heterotopic ossification (Classes I and II) did not affect segmental ROM and clinical outcomes such as pain or function. In Class-III heterotopic ossification segmental ROM was decreased, but it did not affect clinical outcomes.