Reciprocal role of PLAP-1 in HIF-1α-mediated responses to hypoxia.

OBJECTIVE: To investigate the mutual regulation of hypoxia-inducible factor (HIF)-1α activity and periodontal ligament-associated protein-1 (PLAP-1) expression in human periodontal ligament cells (HPDLs). BACKGROUND: Cellular responses to hypoxia regulate various biological events (e.g., inflammation and tissue regeneration) through activation of HIF-1α. PLAP-1, an extracellular matrix protein preferentially expressed in the periodontal ligament, plays important roles in the functions of HPDLs. Although PLAP-1 expression has been demonstrated in hypoxic regions, the involvement of PLAP-1 in responses to hypoxia has not been revealed. METHODS: HPDLs were cultured under normoxic (20% O2 ) or hypoxic (1% O2 ) conditions with or without deferoxamine mesylate (chemical hypoxia inducer) or chetomin (HIF signaling inhibitor). Expression levels of PLAP-1 and HIF-1α were examined by real-time reverse transcription-polymerase chain reaction and western blot analysis. Luciferase reporter assays of HIF-1α activity were performed using 293T cells stably transfected with a hypoxia response element (HRE)-containing luciferase vector in the presence or absence of recombinant PLAP-1 or PLAP-1 gene transfection. RESULTS: Cultivation under hypoxic conditions elevated the gene and protein expression levels of PLAP-1 in HPDLs. Deferoxamine mesylate treatment also enhanced PLAP-1 expression in HPDLs. Hypoxia-induced PLAP-1 expression was significantly suppressed in the presence of chetomin. PLAP-1-suppressed HPDLs showed increased HIF-1α accumulation in the nucleus during culture under hypoxic conditions, but not in the presence of recombinant PLAP-1. In the presence of recombinant PLAP-1, hypoxia-induced HRE activity of 293T cells was significantly suppressed in a dose-dependent manner. Transfection of the PLAP-1 gene resulted in a significant reduction of HRE activity during culture under hypoxic conditions. CONCLUSION: PLAP-1 expression is upregulated under hypoxic conditions through HIF-1α activation. Moreover, hypoxia-induced PLAP-1 expression regulates HIF-1α signaling.

Activation of periodontal ligament cell cytodifferentiation by juxtacrine signaling from cementoblasts.

BACKGROUND: New cementum forms from existing cementum during periodontal tissue regeneration, indicating that cementoblasts may interact with progenitor cells in the periodontal ligament to enhance cementogenesis. However, the molecular mechanisms of this process are currently unknown. This study aims to clarify the role of cell-cell interactions between cementoblasts and periodontal ligament cells in differentiation into cementoblasts. METHODS: To analyze the role of human cementoblast-like cells (HCEMs) on human periodontal ligament cells (HPDLs), we mixed cell suspensions of enhanced green fluorescent protein-tagged HPDLs and HCEMs, and then seeded and cultured them in single wells (direct co-cultures). We sorted co-cultured HPDLs and analyzed their characteristics, including the expression of cementum-related genes. In addition, we cultured HPDLs and HCEMs in a non-contact environment using a culture system composed of an upper insert and a lower well separated by a semi-permeable membrane (indirect co-cultures), and similar analysis was performed. Gene expression of integrin-binding sialoprotein (IBSP) in cementoblasts was confirmed in mouse periodontal tissues. We also investigated the effect of Wingless-type (Wnt) signaling on the differentiation of HPDLs into cementoblasts. RESULTS: Direct co-culture of HPDLs with HCEMs significantly upregulated the expression of cementoblast-related genes in HPDLs, whereas indirect co-culture exerted no effect. Wnt3A stimulation significantly upregulated IBSP expression in HPDLs, whereas inhibition of canonical Wnt signaling suppressed the effects of co-culture. CONCLUSION: Our results suggest that direct cell interactions with cementoblasts promote periodontal ligament cell differentiation into cementoblasts. Juxtacrine signaling via the canonical Wnt pathway plays a role in this interaction.

Periodontal Tissue Regeneration by Transplantation of Autologous Adipose Tissue-Derived Multi-Lineage Progenitor Cells With Carbonate Apatite.

We have developed an autologous transplantation method using adipose tissue-derived multi-lineage progenitor cells (ADMPCs) as a method of periodontal tissue regeneration that can be adapted to severe periodontal disease. Our previous clinical study confirmed the safety of autologous transplantation of ADMPCs and demonstrated its usefulness in the treatment of severe periodontal disease. However, in the same clinical study, we found that the fibrin gel used as the scaffold material might have caused gingival recession and impaired tissue regeneration in some patients. Carbonate apatite has a high space-making capacity and has been approved in Japan for periodontal tissue regeneration. In this study, we selected carbonate apatite as a candidate scaffold material for ADMPCs and conducted an in vitro examination of its effect on the cellular function of ADMPCs. We further performed autologous ADMPC transplantation with carbonate apatite as the scaffold material in a model of one-wall bone defects in beagles and then analyzed the effect on periodontal tissue regeneration. The findings showed that carbonate apatite did not affect the cell morphology of ADMPCs and that it promoted proliferation. Moreover, no effect on secretor factor transcription was found. The results of the in vivo analysis confirmed the space-making capacity of carbonate apatite, and the acquisition of significant new attachment was observed in the group involving ADMPC transplantation with carbonate apatite compared with the group involving carbonate apatite application alone. Our results demonstrate the usefulness of carbonate apatite as a scaffold material for ADMPC transplantation.

Periodontal tissue regeneration by transplantation of autologous adipose tissue-derived multi-lineage progenitor cells.

Periodontitis is a chronic inflammatory disease that destroys tooth-supporting periodontal tissue. Current periodontal regenerative therapies have unsatisfactory efficacy; therefore, periodontal tissue engineering might be established by developing new cell-based therapies. In this study, we evaluated the safety and efficacy of adipose tissue-derived multi-lineage progenitor cells (ADMPC) autologous transplantation for periodontal tissue regeneration in humans. We conducted an open-label, single-arm exploratory phase I clinical study in which 12 periodontitis patients were transplanted with autologous ADMPCs isolated from subcutaneous adipose tissue. Each patient underwent flap surgery during which autologous ADMPCs were transplanted into the bone defect with a fibrin carrier material. Up to 36 weeks after transplantation, we performed a variety of clinical examinations including periodontal tissue inspection and standardized dental radiographic analysis. A 36-week follow-up demonstrated no severe transplantation-related adverse events in any cases. ADMPC transplantation reduced the probing pocket depth, improved the clinical attachment level, and induced neogenesis of alveolar bone. Therapeutic efficiency was observed in 2- or 3-walled vertical bone defects as well as more severe periodontal bone defects. These results suggest that autologous ADMPC transplantation might be an applicable therapy for severe periodontitis by inducing periodontal regeneration.

Hypoxia stimulates collagen hydroxylation in gingival fibroblasts and periodontal ligament cells.

BACKGROUND: Cellular responses to hypoxia regulate various biological events, including angiogenesis and extracellular matrix metabolism. Collagen is a major component of the extracellular matrix in periodontal tissues and its coordinated production is essential for tissue homeostasis. In this study, we investigated the effects of hypoxia on collagen production in human gingival fibroblasts (HGFs) and human periodontal ligament cells (HPDLs). METHODS: HGFs and HPDLs were cultured under either normoxic (20% O2 ) or hypoxic (1% O2 ) conditions. Nuclear expression of hypoxia-inducible factor-1α (HIF-1α) was determined by western blotting. Peri-cellular expression of type I collagen was examined by immunocytochemistry analysis. Synthesis of type I collagen was evaluated by measuring the concentration of procollagen type I C-peptide (PIP) in culture supernatant using enzyme-linked immunosorbent assay. Expression of collagen hydroxylase enzymes prolyl 4-hydroxylase alpha polypeptide 1 (P4HA1) and 2-oxoglutarate 5-dioxygenase 2 (PLOD2) was determined by RT-qPCR and western blotting. The roles of these enzymes were analyzed using siRNA transfection. RESULTS: Cultivation under hypoxic conditions stimulated type I collagen production via HIF-1α in both cell types. Interestingly, hypoxic conditions did not affect collagen 1a1 or 1a2 gene expression but upregulated that of P4HA1 and PLOD2. Additionally, suppressing P4HA1 significantly decreased the levels of hypoxia-induced procollagen type I C-peptide, a product of stable triple helical collagen, in the supernatant. In contrast, PLOD2 suppression decreased cross-linked collagen expression in the pericellular region. CONCLUSION: Our results suggest that hypoxia activates collagen synthesis in HGFs and HPDLs by upregulating hydroxylases P4HA1 and PLOD2 in an HIF-1α-dependent manner.