Periodontal tissue regeneration with cementogenesis after application of brain-derived neurotrophic factor in 3-wall inflamed intra-bony defect.

OBJECTIVE: The purpose of this study is to investigate regenerative process by immunohistochemical analysis and evaluate periodontal tissue regeneration following a topical application of BDNF to inflamed 3-wall intra-bony defects. BACKGROUND: Brain-derived neurotrophic factor (BDNF) plays a role in the survival and differentiation of central and peripheral neurons. BDNF can regulate the functions of non-neural cells, osteoblasts, periodontal ligament cells, endothelial cells, as well as neural cells. Our previous study showed that a topical application of BDNF enhances periodontal tissue regeneration in experimental periodontal defects of dog and that BDNF stimulates the expression of bone (cementum)-related proteins and proliferation of human periodontal ligament cells. METHODS: Six weeks after extraction of mandibular first and third premolars, 3-wall intra-bony defects were created in mandibular second and fourth premolars of beagle dogs. Impression material was placed in all of the artificial defects to induce inflammation. Two weeks after the first operation, BDNF (25 and 50 µg/mL) immersed into atelocollagen sponge was applied to the defects. As a control, only atelocollagen sponge immersed in saline was applied. Two and four weeks after the BDNF application, morphometric analysis was performed. Localizations of osteopontin (OPN) and proliferating cell nuclear antigen (PCNA)-positive cells were evaluated by immunohistochemical analysis. RESULTS: Two weeks after application of BDNF, periodontal tissue was partially regenerated. Immunohistochemical analyses revealed that cells on the denuded root surface were positive with OPN and PCNA. PCNA-positive cells were also detected in the soft connective tissue of regenerating periodontal tissue. Four weeks after application of BDNF, the periodontal defects were regenerated with cementum, periodontal ligament, and alveolar bone. Along the root surface, abundant OPN-positive cells were observed. Morphometric analyses revealed that percentage of new cementum length and percentage of new bone area of experimental groups were higher than control group and dose-dependently increased. CONCLUSION: These findings suggest that BDNF could induce cementum regeneration in early regenerative phase by stimulating proliferation of periodontal ligament cells and differentiation into periodontal tissue cells, resulting in enhancement of periodontal tissue regeneration in inflamed 3-wall intra-bony defects.

Effect of neurotrophin-4/5 on bone/cementum-related protein expressions and DNA synthesis in cultures of human periodontal ligament cells.

BACKGROUND: We studied neurotrophins (NTs) as signaling molecules for periodontal tissue regeneration and showed that nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) modulate the proliferation and differentiation of human periodontal ligament (HPL) cells in vitro. The purpose of this study was to investigate whether NT-4/5 also has the ability to regulate the function of HPL cells. METHODS: mRNA expressions of NT-4/5 and its high-affinity tyrosine kinase receptor (trk)B were analyzed in HPL cells by reverse transcription-polymerase chain reaction. We examined how NT-4/5 regulates the mRNA expression of bone/cementum-related proteins (alkaline phosphatase [ALPase], osteopontin [OPN], osteocalcin [OC], and bone morphogenetic protein [BMP]-2) in cultures of HPL cells. Moreover, the effects of NT-4/5 on calcification, the production of OPN and OC, and DNA synthesis in HPL cells were examined. RESULTS: NT-4/5 and trkB mRNA were expressed in HPL cells. NT-4/5 elevated the mRNA levels of ALPase, OPN, OC, and BMP-2 and the syntheses of OPN, OC, and DNA in HPL cells. PD98059, an extracellular signal-regulated kinase (ERK) inhibitor, obviated the increase in the mRNA levels of ALPase, OPN, OC, and BMP-2. NT-4/5 increased the levels of phosphorylated ERK1/2. Furthermore, NT-4/5 enhanced the amount of mineral deposits in cultures of HPL cells. CONCLUSION: NT-4/5, as well as BDNF and NGF, is suggested to play a role in the regulation of function of periodontal ligament cells.

Effect of neurotrophins on differentiation, calcification and proliferation in cultures of human pulp cells.

Neurotrophins (NTs) are expressed during tooth development. However, little is known about a role of NTs in differentiation of pulp cells into mineralizing cells. In this study, mRNA expressions of hard tissue-related proteins, calcification and proliferation are examined in cultures of human pulp (HP) cells. Nerve growth factor (NGF), brain derived neurotrophic factor (BDNF), neurotrophin (NT)-3 and NT-4/5 increased the mRNA levels of dentin sialophsphoprotein, alkaline phosphatase, osteopontin, type I collagen and bone morphogenetic protein-2 and mineral deposition in cultures of HP cells. The increased levels and manners varied, depending on the concentrations of NTs and hard-tissue related protein tested. On the other hand, only NGF significantly stimulated DNA synthesis in cultures of HP cells. These findings suggest that NTs characteristically regulate hard-tissue related protein expression, calcification and proliferation in pulp cells. NTs may accelerate pulp cell differentiation.

Behavior of transplanted bone marrow-derived mesenchymal stem cells in periodontal defects.

BACKGROUND: Recently, there have been an increased number of basic and clinical reports indicating the superior potential of bone marrow-derived mesenchymal stem cells (MSCs) for tissue regeneration. In periodontal treatment, previous animal studies indicated that autotransplantation of bone marrow MSCs into experimental periodontal defects enhanced periodontal tissue regeneration. However, mechanisms for periodontal tissue regeneration with MSCs are still unclear. The purpose of this study was to elucidate the behavior of transplanted MSCs in periodontal defects. METHODS: Bone marrow MSCs were isolated from beagle dogs, labeled with green fluorescent protein (GFP), and expanded in vitro. The expanded MSCs were mixed with atelocollagen (2% type I collagen) at final concentrations of 2 x 10(7) cells/ml and transplanted into experimental Class III periodontal defects. Localizations of GFP and proliferating cell nuclear antigen (PCNA)-positive cells were evaluated by immunohistochemical analysis. RESULTS: Four weeks after transplantation, the periodontal defects were almost regenerated with periodontal tissue. Cementoblasts, osteoblasts, osteocytes, and fibroblasts of the regenerated periodontal tissue were positive with GFP. PCNA-positive cells were present in regenerating connective tissue. CONCLUSION: These findings suggest that transplanted mesenchymal stem cells could survive and differentiate into periodontal tissue cells, resulting in enhancement of periodontal tissue regeneration.

Promotion of functioning of human periodontal ligament cells and human endothelial cells by nerve growth factor.

BACKGROUND: We have previously shown that cultured human periodontal ligament (HPL) cells produce nerve growth factor (NGF) and express mRNA of tyrosine kinase receptor (trkA), a high-affinity receptor of NGF. These findings suggest that NGF modulates the differentiation and proliferation of the periodontal ligament cells by paracrine and autocrine functions in vivo. Endothelial cells also express NGF and trkA. Therefore, NGF may regulate functions of periodontal ligament cells and endothelial cells during periodontal tissue regeneration. METHODS: Effects of NGF on expressions of bone/cementum-related proteins (osteocalcin [OC], bone sialoprotein [BSP], bone morphogenetic protein [BMP-7], core binding factor alpha [Cbfa-1], and type I collagen), calcification in HPL cells, and proliferation and mRNA expression of vascular endothelial growth factor (VEGF), an endothelial cell mitogen, in human microvascular endothelial cells (HMVECs) were examined. RESULTS: NGF elevated mRNA levels of OC, BSP, BMP-7, Cbfa-1, and type I collagen and enhanced mineral deposition in cultures of HPL cells. Furthermore, NGF stimulated mRNA expressions of VEGF-A and VEGF-B and cell proliferation in HMVEC. CONCLUSION: These findings suggest that the functional regulation of periodontal ligament cells and endothelial cells by NGF might result in the acceleration of periodontal tissue regeneration in vivo.

Brain-derived neurotrophic factor enhances periodontal tissue regeneration.

To address whether brain-derived neurotrophic factor (BDNF) could be involved in periodontal tissue regeneration, we examined the effects of BDNF on proliferation and the expression of bone (cementum)- related proteins (osteopontin, bone morphogenetic protein [BMP]-2, type I collagen, alkaline phosphatase [ALPase], and osteocalcin) in cultures of human periodontal ligament (HPL) cells, which are thought to be prerequisite for periodontal tissue regeneration, and on proliferation and angiogenesis in human endothelial cells. Furthermore, we examined the effect of BDNF on the regeneration of periodontal tissues in experimentally induced periodontal defects in dogs. BDNF elevated the expression of ALPase and osteocalcin mRNAs and increased the synthesis of osteopontin, BMP-2, and type I collagen DNA in HPL cells. BDNF stimulated mRNA expression of vascular endothelial growth factor-B and tenascin-X, and proliferation and angiogenesis in human endothelial cells. In vivo studies showed that BDNF stimulated the formation of new alveolar bone cementum and connective new fibers, which were inserted into the newly formed cementum and bone. BDNF also stimulated blood capillary formation. These findings suggest that the regulation of functioning of periodontal ligament cells and endothelial cells by BDNF results in the promotion of periodontal tissue regeneration.

[Cell transplantation for periodontal diseases. A novel periodontal tissue regenerative therapy using bone marrow mesenchymal stem cells].

A major goal of periodontal therapy is to reconstruct healthy periodontium destroyed by periodontal diseases. Basic studies have revealed that transplantation of mesenchymal stem cells (MSC) into periodontal defects promotes regeneration of periodontal tissue. We have developed a novel method for periodontal therapy using MSC. Human bone marrow cells are obtained from the iliac crest and expanded in vitro at Cell and Tissue Engineering Center in Hiroshima University Hospital. MSC are, then, isolated and mixed with Atelocollagen at final concentrations of 2 x 10(7) cells/mL. These MSC in Atelocollagen are transplanted into periodontal osseous defects at the periodontal surgery. The results in all seven patients who received the own MSC transplantation have shown good clinical course. Further basic studies and the continuous clinical trial are needed to prove the effectiveness of the clinical application.

Enhancement of periodontal tissue regeneration by transplantation of bone marrow mesenchymal stem cells.

BACKGROUND: The use of suitable cells transplanted into periodontal osseous defects appears to be a powerful strategy to promote periodontal tissue regeneration. Mesenchymal stem cells (MSCs) isolated from bone marrow have the potential for multilineage differentiation. The purpose of this study was to examine whether auto-transplantation of MSCs into periodontal osseous defects would be useful for periodontal tissue regeneration. METHODS: Bone marrow MSCs were isolated from beagle dogs and expanded in vitro. The expanded MSCs were mixed with atelocollagen (2% type I collagen) at final concentrations of 2 x 10(6), 5 x 10(6), 1 x 10(7), or 2 x 10(7) cells/ml, and auto-transplanted into experimental Class III defects. Atelocollagen alone was implanted into the defects as a control. Periodontal tissue healing was evaluated by histological and morphometric analyses 1 month after transplantation. RESULTS: The defects were regenerated with cementum, periodontal ligament, and alveolar bone in the MSC-atelocollagen groups. Less periodontal tissue regeneration was observed in the control group compared to the MSC-atelocollagen groups. Morphometric analysis revealed that the percentage of new cementum length in the 5 x 10(6) and 2 x 10(7) cells/ml groups and the percentage of new bone area in the 2 x 10(7) cells/ml group were significantly higher than in the control group (P < 0.01). CONCLUSION: These findings suggest that auto-transplantation of bone marrow mesenchymal stem cells is a novel option for periodontal tissue regeneration.

Expression of endothelins and their receptors in cells from human periodontal tissues.

OBJECTIVE: The present study investigated the presence of ET-1 in gingival crevicular fluid (GCF) from patients with periodontitis, and the expression of endothelins (ETs) and their receptors mRNA in cultured cells from human periodontal tissues. BACKGROUND: ET was originally discovered as a potent vasoconstrictive peptide from endothelial cells. It has been reported that ETs are produced by various cells besides endothelial cells. ETs are related to inflammatory and sclerotic lesions, such as arteriolosclerosis and hepatic cirrhosis. Therefore, ETs may be involved in periodontal disease. However, the roles of ETs in development and progression of periodontal disease are not clear. METHODS: ET-1 released from the cultured cells was measured by enzyme-linked immunosorbent assay. mRNA expressions for ETs and their receptors were examined by reverse transcription-polymerase chain reaction and Northern blotting analysis. RESULTS: ET-1 levels in GCF from patients with periodontitis were higher than those from healthy subjects. Human gingival keratinocytes (HGK) expressed mRNA for ETs and their receptors, ET-Ar and ET-Br. ET-1 mRNA expression and ET-1 peptide production from HGK were enhanced by interleukin-1beta and tumor necrosis factor-alpha. CONCLUSIONS: These results suggest that ET-1 plays a significant role in periodontal disease.

Immunohistochemical characteristics of epithelial cell rests of Malassez during cementum repair.

To clarify the roles of epithelial cell rests of Malassez (ECRM) during periodontal repair, experimental root resorption was induced in rats and then the ECRM that existed in periodontal ligament during cementum repair was investigated using morphological and immunohistochemical approaches. At day 7, after mechanical injury, root resorption was observed and ECRM were present adjacent to the site of resorption lacunae. They were observed in periodontal ligament adjacent to site of the resorption lacunae. These ECRM were immunoreactive for bone morphogenetic protein-2. During the stage of early cementum repair, the ECRM were immunoreactive for osteopontin and ameloblastin. They strongly reacted to proliferating cell nuclear antigen. In uninjured control sections, ECRM located in the periodontal ligament adjacent to cementum were not immunoreactive for any antibodies. These findings suggested that ECRM may be related to cementum repair by activating their potential to secrete matrix proteins which have been expressed in tooth development.