Application of cryopreservation to tooth germ transplantation for root development and tooth eruption.

We cryopreserved mouse tooth germs with widely open cervical margins of the enamel organ to overcome difficulties in cryoprotectant permeation and tested their efficacy by transplanting them into recipient mice. The upper right first molar germs of 8-day-old donor mice were extracted and categorized into the following four groups according to cryopreservation time: no cryopreservation, 1 week, 1 month, and 3 months. The donor tooth germs were transplanted into the upper right first molar germ sockets of the 8-day-old recipient mice. The upper left first molars of the recipient mice were used as controls. The outcome of the transplantation was assessed at 1, 2, and 3 weeks after transplantation. Stereomicroscopic evaluation revealed that most of the transplanted teeth erupted by 3 weeks after transplantation. Micro-computed tomography analysis revealed root elongation in the transplanted groups as well as in the controls. There was no significant difference between the cryopreserved and non-cryopreserved transplanted teeth, but the roots of the cryopreserved teeth were significantly shorter than those of the control teeth. Histological examination revealed root and periodontal ligament formations in all the transplanted groups. These results suggest that the transplantation of cryopreserved tooth germs facilitates subsequent root elongation and tooth eruption.

Zbp1-positive cells are osteogenic progenitors in periodontal ligament.

Periodontal ligament (PDL) possesses a stem/progenitor population to maintain the homeostasis of periodontal tissue. However, transcription factors that regulate this population have not yet been identified. Thus, we aimed to identify a molecule related to the osteogenic differentiation of PDL progenitors using a single cell-based strategy in this study. We first devised a new protocol to isolate PDL cells from the surface of adult murine molars and established 35 new single cell-derived clones from the PDL explant. Among these clones, six clones with high (high clones, n = 3) and low (low clones, n = 3) osteogenic potential were selected. Despite a clear difference in the osteogenic potential of these clones, no significant differences in their cell morphology, progenitor cell marker expression, alkaline phosphatase activity, proliferation rate, and differentiation-related gene and protein expression were observed. RNA-seq analysis of these clones revealed that Z-DNA binding protein-1 (Zbp1) was significantly expressed in the high osteogenic clones, indicating that Zbp1 could be a possible marker and regulator of the osteogenic differentiation of PDL progenitor cells. Zbp1-positive cells were distributed sparsely throughout the PDL. In vitro Zbp1 expression in the PDL clones remained at a high level during osteogenic differentiation. The CRISPR/Cas9 mediated Zbp1 knockout in the high clones resulted in a delay in cell differentiation. On the other hand, Zbp1 overexpression in the low clones promoted cell differentiation. These findings suggested that Zbp1 marked the PDL progenitors with high osteogenic potential and promoted their osteogenic differentiation. Clarifying the mechanism of differentiation of PDL cells by Zbp1 and other factors in future studies will facilitate a better understanding of periodontal tissue homeostasis and repair, possibly leading to the development of novel therapeutic measures.

Tooth-Supporting Hard Tissue Regeneration Using Biopolymeric Material Fabrication Strategies.

The mineralized tissues (alveolar bone and cementum) are the major components of periodontal tissues and play a critical role to anchor periodontal ligament (PDL) to tooth-root surfaces. The integrated multiple tissues could generate biological or physiological responses to transmitted biomechanical forces by mastication or occlusion. However, due to periodontitis or traumatic injuries, affect destruction or progressive damage of periodontal hard tissues including PDL could be affected and consequently lead to tooth loss. Conventional tissue engineering approaches have been developed to regenerate or repair periodontium but, engineered periodontal tissue formation is still challenging because there are still limitations to control spatial compartmentalization for individual tissues and provide optimal 3D constructs for tooth-supporting tissue regeneration and maturation. Here, we present the recently developed strategies to induce osteogenesis and cementogenesis by the fabrication of 3D architectures or the chemical modifications of biopolymeric materials. These techniques in tooth-supporting hard tissue engineering are highly promising to promote the periodontal regeneration and advance the interfacial tissue formation for tissue integrations of PDL fibrous connective tissue bundles (alveolar bone-to-PDL or PDL-to-cementum) for functioning restorations of the periodontal complex.

Biomimetic collagen-sodium alginate-titanium oxide (TiO2) 3D matrix supports differentiated periodontal ligament fibroblasts growth for periodontal tissue regeneration.

Fabrication of biomaterial that mimics a suitable biological microenvironment is still a major challenge in the field of periodontitis treatment. Hence, in this report, we presented for the first time the fabrication of a novel biomaterial 3D matrix using collagen combined with sodium alginate and titanium oxide (TiO2) to recreate the in-vivo microenvironment and to act as a platform for the culture of human periodontal ligament fibroblasts (HPLF) towards osteogenic differentiation. Further, we explored the changes of differentiated and undifferentiated HPLF cells in morphological and cellular level comparing 2D (standard culture plates) and 3D cell culture systems. The physicochemical parameters such as stiffness, water binding capacity, swelling, shrinkage factor, porosity and in-vitro biodegradation show the suitability of this 3D matrix to act as a scaffold for in-vitro periodontal regeneration. The differentiated HPLF cells in the 3D matrix secrete high levels of collagen, osteocalcin, alkaline phosphatase compared to the conventional 2D cell culture. Morphological analysis revealed the structural changes of HPLF cells before and after differentiation in 2D and 3D cell culture. In this study, we find that the level of osteocalcin secretion towards osteogenic differentiation was enhanced in HPLF cells by 3D matrix as compared with 2D cell culture, which demonstrates the osteogenic stimulatory potential of 3D matrix. Overall, the fabricated 3D matrix supports the differentiation of the HPLF cells into osteoblastogenic lineage cells in-vitro and is a promising approach for further investigations in in-vivo treatment of periodontal tissue impairment.

Epigenetic modifier trichostatin A enhanced osteogenic differentiation of mesenchymal stem cells by inhibiting NF-κB (p65) DNA binding and promoted periodontal repair in rats.

We wished to evaluate whether epigenetic modifiers have a beneficial effect on treating experimental periodontitis and mechanisms for regulating the cell fate of mesenchymal stem cells (MSCs) in inflammatory microenvironments. We isolated MSCs from healthy and inflamed gingival tissues to investigate whether trichostatin A (TSA) could improve osteogenic differentiation and resolve inflammation in vitro. The tissue regenerative potentials were evaluated when treated with a temperature-dependent, chitosan-scaffold-encapsulated TSA, in a rat model of periodontitis. After induction with the conditioned medium, TSA treatment increased the osteogenic differentiation potential of inflamed MSCs and healthy MSCs. In addition, interleukin-6 and interleukin-8 levels in supernatants were significantly decreased after TSA treatment. Moreover, TSA promoted osteogenic differentiation by inhibiting nuclear factor-κB (p65) DNA binding in MSCs. In rats with experimental periodontitis, 7 weeks after local injections of chitosan-scaffold-encapsulated TSA, histology and microcomputed tomography showed a significant increase in alveolar bone volume and less inflammatory infiltration compared with vehicle-treated rats. The concentrations of interferon-γ and interleukin-6 were significantly decreased in the gingival crevicular fluid after TSA treatment. This study demonstrated that TSA had anti-inflammatory properties and could promote periodontal tissue repair, which indicated that epigenetic modifiers hold promise as a potential therapeutic option for periodontal tissue repair.

Sustained Release of Two Bioactive Factors from Supramolecular Hydrogel Promotes Periodontal Bone Regeneration.

Intact and stable bone reconstruction is ideal for the treatment of periodontal bone destruction but remains challenging. In research, biomaterials are used to encapsulate stem cells or bioactive factors for periodontal bone regeneration, but, to the best of our knowledge, using a supramolecular hydrogel to encapsulate bioactive factors for their sustained release in bone defect areas to promote periodontal bone regeneration has not been reported. Herein, we used a well-studied hydrogelator, NapFFY, to coassemble with SDF-1 and BMP-2 to prepare a supramolecular hydrogel, SDF-1/BMP-2/NapFFY. In vitro and in vivo results indicated that these two bioactive factors were ideally, synchronously, and continuously released from the hydrogel to effectively promote the regeneration and reconstruction of periodontal bone tissues. Specifically, after the bone defect areas were treated with our SDF-1/BMP-2/NapFFY hydrogel for 8 weeks using maxillary critical-sized periodontal bone defect model rats, a superior bone regeneration rate of 56.7% bone volume fraction was achieved in these rats. We anticipate that our SDF-1/BMP-2/NapFFY hydrogel could replace bone transplantation in the clinic for the repair of periodontal bone defects and periodontally accelerated osteogenic orthodontics in the near future.

ACVR1 is essential for periodontium development and promotes alveolar bone formation.

OBJECTIVE: To explore the role of a BMP type I receptor (ACVR1) in regulating periodontium development, Acvr1 was conditionally disrupted in Osterix-expressing cells. METHODS: Mandibles from both control (Acvr1 fx/+; Osterix-Cre (+)/(-)) and cKO (Acvr1 fx/-; Osterix-Cre (+)/(-)) mice at postnatal day 21 (PN21) were scanned by micro-CT, followed by decalcification and histological observations. Distributions and levels of differentiation markers of fibroblasts, osteoblasts and cementocytes in the periodontium were detected by immunohistochemical (IHC) staining. RESULTS: Micro-CT results showed that bone mass and bone mineral density of the alveolar bones in the cKO mice were lower than those in the controls. Histomorphometry within the alveolar bones revealed that the lower bone mass observed in the cKO mice was caused by increased numbers and resorption activities of osteoclasts. The markers for osteoblast differentiation, Col I and DMP1, were reduced and the signals of the RANKL/OPG ratio were increased in the alveolar bones of the cKO mice compared to those of the control mice. The periodontal ligament in the cKO mice exhibited disorganized collagen fibers with weaker signals of Col I and periostin. However, there was no difference in terms of the cellular cementum between the two groups. CONCLUSION: ACVR1 is essential for normal periodontium development. ACVR1 in the osteoblasts negatively regulates osteoclast differentiation in association with the RANKL/OPG axis and thus promotes alveolar bone formation.

Are Hertwig's epithelial root sheath cells necessary for periodontal formation by dental follicle cells?

OBJECTIVE: The role of Hertwig's epithelial root sheath (HERS) cells in periodontal formation has been controversial. This study aimed to further clarify whether HERS cells participate in formation of the periodontium, and the necessity of HERS cells in differentiation of dental follicle cells (DFCs) for periodontal regeneration. DESIGN: HERS cells and DFCs were isolated and identified from post-natal 7-day Sprauge-Dawley rats. In vitro, direct co-culture of HERS cells and DFCs as well as the individual culture of HERS and DFCs were performed and followed by alizarin red staining and the quantitative real-time polymerase chain reaction analysis. For in vivo evaluation, the inactivated dentin matrix (iTDM) was fabricated. HERS cells and DFCs were seeded in combination or alone on iTDM and then transplanted into the rat omentum. Scanning electron microscope and further histological analysis were carried out. RESULTS: In vitro, mineral-like nodules were found in the culture of HERS cells alone or HERS + DFCs either by alizarin red staining or scanning electronic microscope. The mineralization and fiber-forming relevant mRNA expressions, such as bone sialoprotein, osteopontin, collagen I and collagen III in HERS + DFCs were significantly higher than that of the HERS or DFCs alone group. After transplantation in vivo, cementum and periodontal ligament-like tissues were formed in groups of HERS + DFCs and HERS alone, while no evident hard tissues and attached fibers were found in DFCs alone. CONCLUSIONS: Hertwig's epithelial root sheath cells directly participate in the formation of the periodontium, and they are essential for the differentiation of dental follicle cells to form periodontal structures. The combination use of Hertwig's epithelial root sheath cells and dental follicle cells is a promising approach for periodontal regeneration.

Nuevas tendencias en regeneración tisular: fibrina rica en plaquetas y leucocitos / New tendencies in tissue regeneration: leucocyte-rich platelet-rich fibrin

La regeneración periodontal es la reproducción o reconstitución de una parte perdida o dañada del periodonto con el fin de restaurar su arquitectura y función. En los últimos años se ha puesto de manifiesto el papel clave que juegan las plaquetas en la regeneración tisular, acelerando la cicatrización tanto de tejidos blandos como duros, mediada por la liberación de citocinas y factores de crecimiento durante un tiempo prolongado. La fibrina rica en plaquetas y leucocitos utilizada por primera vez por Choukroun en el 2001 es un concentrado de plaquetas de segunda generación que se obtiene a partir de la propia sangre del paciente, sin el empleo de aditivos, con el fin de conseguir una malla de fibrina que sirva de andamiaje para las sustancias implicadas en la regeneración. El objetivo de este trabajo es el de realizar una revisión y puesta al día en el uso de esta técnica (AU)

Periodontal regeneration is the reproduction or re-enactment of an injured, or lost, part of the periodontium, with the aim of repairing its architecture and main function. The key role of platelets in tissue regeneration has been demonstrated in the last few years. They accelerate healing in both the soft and hard tissues due to the liberation of cytokines and growth factors over a long period. Leucocyte-rich platelet-rich fibrin, used for the first time by Choukroun in 2001, is a second generation platelets extract that is obtained from the patient's own blood, without the need of additives. Its purpose is to attain an autologous fibrin mesh to be used for as a framework for the substances involved in bone regeneration. The purpose of this work is to present a review and an update on the use of this technique (AU)

[Comparative study of proliferative and periodontal differentiation propensity of induced pluripotent stem cells at different passages].

OBJECTIVE: To compare the proliferative and periodontal specific differentiation abilities of induced pluripotent stem cells (iPSCs) at different passages, and to investigate whether long term culturing would have a negative influence on their proliferation and specific differentiation capacity, thus providing a theoretical basis for further in-depth research on periodontal regeneration and the possible clinical applications of iPSCs. METHODS: IPSCs derived from human gingival fibroblasts at passages 5, 10, 15 and 20 were recovered and cultured in vitro. Their morphology and proliferation rates were observed respectively. We further induced the iPSCs at different passages toward periodontal tissue under the treatment of growth/differentiation factor-5 (GDF-5) for 14 days through the EB routine, then compared the periodontal differentiation propensities between the different passages of iPSCs by detecting their calcified nodules formation by Alizarin red staining and assaying their relative periodontal tissue related marker expressions by qRT-PCR and immunofluorescence staining, including bone related markers: osteocalcin (OCN), bone sialoprotein (BSP); periodontal ligament related markers: periostin, vimentin; and cementum related markers: cementum attachment protein (CAP), cementum protein 1 (CEMP1). The untreated spontaneous differentiation groups were set as negative controls respectively. RESULTS: iPSCs at different passages all showed a high proliferative capacity when cultured in vitro and turned into a spindle-like shape similar to fibroblasts upon periodontal specific differentiation. All iPSCs formed typical calcified nodules upon GDF-5 induction by Alizarin red staining in comparison to their untreated controls. The relative calcium deposition at all passages had been significantly upgraded under the treatment of GDF-5 (P5: t=2.125, P=0.003; P10: t=2.246, P=0.021; P15: t=3.754, P=0.004; P20: t=3.933, P=0.002), but no significant difference in their calcium deposition were detected within passages 5, 10, 15 and 20 (periodontal differentiation: F=2.365, P=0.109; spontaneously differentiation: F=2.901, P=0.067). Periodontal tissue related marker expressions of iPSCs at all passages had also been significantly upgraded under the treatment of GDF-5 (P<0.05), but still, no significant difference in their expression levels of periodontal tissue related proteins were detected within passages (BSP: F=0.926 7, P=0.450; vimentin: F=0.917 1, P=0.455; CEMP1: F=2.129, P=0.1367). CONCLUSION: Our results preliminarily confirmed that long term culturing won't influence the proliferation capacity and periodontal specific differentiation propensity of iPSCs, as they can still proliferate and differentiate toward periodontal cells with high efficiency upon growth factor induction after continuous passaging. Therefore, iPSCs could be recognized as a promising cell source for future possible application in periodontal tissue regeneration.

In vitro proliferation of periodontal ligament-like tissue on extracted teeth.

OBJECTIVE: Transplantation of autologous teeth is a routine component of orthodontic treatment. The aim of this study was to develop a method for the regeneration of damaged periodontal ligament (PDL) on extracted teeth using a three-dimensional culture system. DESIGN: We used the maxillary first premolars or third molars extracted from patients for orthodontic treatment. The extracted teeth were stained with toluidine blue to measure the residual PDL area. After confirming damage of the periodontal tissue on the root surface of the extracted teeth, we tried to regenerate the periodontal tissue. Other extracted teeth were inserted into a cell strainer filled with cellulose-based carrier materials to regenerate the periodontal tissue. The strainer was then placed in a 90-mm culture dish filled with culture medium and incubated at 37°C and 5% CO2 for about 1 month. The cultured teeth were observed under a stereomicroscope and examined by scanning electron microscopy (SEM), and were stained to detect alkaline phosphatase (ALP) activity. RESULT: Toluidine blue staining revealed that the residual periodontal membrane covered an average of 50.4% of the root surface area of each tooth. After culturing extracted teeth with our culture system, globular structures were found on the entire tooth root surface by stereomicroscopy, and PDL-like filamentous tissue was also detected by SEM. The entire tooth root surfaces of the cultured teeth were positive for ALP activity. CONCLUSIONS: We have developed a useful culture method to stimulate the proliferation of cells in PDL-like tissue on the roots of extracted teeth.

Periodontal tissue engineering by nano beta-tricalcium phosphate scaffold and fibroblast growth factor-2 in one-wall infrabony defects of dogs.

BACKGROUND AND OBJECTIVE: Nanoparticle bioceramics are being investigated for biomedical applications. We fabricated a regenerative scaffold comprising type I collagen and beta-tricalcium phosphate (ß-TCP) nanoparticles. Fibroblast growth factor-2 (FGF-2) is a bioeffective signaling molecule that stimulates cell proliferation and wound healing. This study examined the effects, on bioactivity, of a nano-ß-TCP/collagen scaffold loaded with FGF-2, particularly on periodontal tissue wound healing. MATERIAL AND METHODS: Beta-tricalcium phosphate was pulverized into nanosize particles (84 nm) and was then dispersed. A nano-ß-TCP scaffold was prepared by coating the surface of a collagen scaffold with a nanosize ß-TCP dispersion. Scaffolds were characterized using scanning electron microscopy, compressive testing, cell seeding and rat subcutaneous implant testing. Then, nano-ß-TCP scaffold, nano-ß-TCP scaffold loaded with FGF-2 and noncoated collagen scaffold were implanted into a dog one-wall infrabony defect model. Histological observations were made at 10 d and 4 wk postsurgery. RESULTS: Scanning electron microscopy images show that TCP nanoparticles were attached to collagen fibers. The nano-ß-TCP scaffold showed higher compressive strength and cytocompatibility compared with the noncoated collagen scaffold. Rat subcutaneous implant tests showed that the DNA contents of infiltrating cells in the nano-ß-TCP scaffold and the FGF-2-loaded scaffold were approximately 2.8-fold and 3.7-fold greater, respectively, than in the collagen scaffold. Histological samples from the periodontal defect model showed about five-fold greater periodontal tissue repair following implantation of the nano-ß-TCP scaffold loaded with FGF-2 compared with the collagen scaffold. CONCLUSION: The ß-TCP nanoparticle coating strongly improved the collagen scaffold bioactivity. Nano-ß-TCP scaffolds containing FGF-2 are anticipated for use in periodontal tissue engineering.

The parathyroid hormone-related protein is secreted during the osteogenic differentiation of human dental follicle cells and inhibits the alkaline phosphatase activity and the expression of DLX3.

The dental follicle is involved in tooth eruption and it expresses a great amount of the parathyroid hormone-related protein (PTHrP). PTHrP as an extracellular protein is required for a multitude of different regulations of enchondral bone development and differentiation of bone precursor cells and of the development of craniofacial tissues. The dental follicle contains also precursor cells (DFCs) of the periodontium. Isolated DFCs differentiate into periodontal ligament cells, alveolar osteoblast and cementoblasts. However, the role of PTHrP during the human periodontal development remains elusive. Our study evaluated the influence of PTHrP on the osteogenic differentiation of DFCs under in vitro conditions for the first time. The PTHrP protein was highly secreted after 4days of the induction of the osteogenic differentiation of DFCs with dexamethasone (2160.5pg/ml±345.7SD. in osteogenic differentiation medium vs. 315.7pg/ml±156.2SD. in standard cell culture medium; Student's t Test: p<0.05 (n=3)). We showed that the supplementation of the osteogenic differentiation medium with PTHrP inhibited the alkaline phosphatase activity and the expression of the transcription factor DLX3, but the depletion of PTHrP did not support the differentiation of DFCs. Previous studies have shown that Indian Hedgehog (IHH) induces PTHrP and that PTHrP, in turn, inhibits IHH via a negative feedback loop. We showed that SUFU (Suppressor Of Fused Homolog) was not regulated during the osteogenic differentiation in DFCs. So, neither the hedgehog signaling pathway induced PTHrP nor PTHrP suppressed the hedgehog signaling pathway during the osteogenic differentiation in DFCs. In conclusion, our results suggest that PTHrP regulates independently of the hedgehog signaling pathway the osteogenic differentiated in DFCs.

Role of PHOSPHO1 in Periodontal Development and Function.

The tooth root and periodontal apparatus, including the acellular and cellular cementum, periodontal ligament (PDL), and alveolar bone, are critical for tooth function. Cementum and bone mineralization is regulated by factors including enzymes and extracellular matrix proteins that promote or inhibit hydroxyapatite crystal growth. Orphan Phosphatase 1 (Phospho1, PHOSPHO1) is a phosphatase expressed by chondrocytes, osteoblasts, and odontoblasts that functions in skeletal and dentin mineralization by initiating deposition of hydroxyapatite inside membrane-limited matrix vesicles. The role of PHOSPHO1 in periodontal formation remains unknown and we aimed to determine its functional importance in these tissues. We hypothesized that the enzyme would regulate proper mineralization of the periodontal apparatus. Spatiotemporal expression of PHOSPHO1 was mapped during periodontal development, and Phospho1(-/-) mice were analyzed using histology, immunohistochemistry, in situ hybridization, radiography, and micro-computed tomography. The Phospho1 gene and PHOSPHO1 protein were expressed by active alveolar bone osteoblasts and cementoblasts during cellular cementum formation. In Phospho1(-/-) mice, acellular cementum formation and mineralization were unaffected, whereas cellular cementum deposition increased although it displayed delayed mineralization and cementoid. Phospho1(-/-) mice featured disturbances in alveolar bone mineralization, shown by accumulation of unmineralized osteoid matrix and interglobular patterns of protein deposition. Parallel to other skeletal sites, deposition of mineral-regulating protein osteopontin (OPN) was increased in alveolar bone in Phospho1(-/-) mice. In contrast to the skeleton, genetic ablation of Spp1, the gene encoding OPN, did not ameliorate dentoalveolar defects in Phospho1(-/-) mice. Despite alveolar bone mineralization defects, periodontal attachment and function appeared undisturbed in Phospho1(-/-) mice, with normal PDL architecture and no evidence of bone loss over time. This study highlights the role of PHOSPHO1 in mineralization of alveolar bone and cellular cementum, further revealing that acellular cementum formation is not substantially regulated by PHOSPHO1 and likely does not rely on matrix vesicle-mediated initiation of mineralization.

Periodontal regeneration with stem cells-seeded collagen-hydroxyapatite scaffold.

Re-establishing compromised periodontium to its original structure, properties and function is demanding, but also challenging, for successful orthodontic treatment. In this study, the periodontal regeneration capability of collagen-hydroxyapatite scaffolds, seeded with bone marrow stem cells, was investigated in a canine labial alveolar bone defect model. Bone marrow stem cells were isolated, expanded and characterized. Porous collagen-hydroxyapatite scaffold and cross-linked collagen-hydroxyapatite scaffold were prepared. Attachment, migration, proliferation and morphology of bone marrow stem cells, co-cultured with porous collagen-hydroxyapatite or cross-linked collagen-hydroxyapatite, were evaluated in vitro. The periodontal regeneration capability of collagen-hydroxyapatite scaffold with or without bone marrow stem cells was tested in six beagle dogs, with each dog carrying one sham-operated site as healthy control, and three labial alveolar bone defects untreated to allow natural healing, treated with bone marrow stem cells - collagen-hydroxyapatite scaffold implant or collagen-hydroxyapatite scaffold implant, respectively. Animals were euthanized at 3 and 6 months (3 animals per group) after implantation and the resected maxillary and mandibular segments were examined using micro-computed tomography scan, H&E staining, Masson's staining and histometric evaluation. Bone marrow stem cells were successfully isolated and demonstrated self-renewal and multi-potency in vitro. The porous collagen-hydroxyapatite and cross-linked collagen-hydroxyapatite had average pore sizes of 415 ± 20 µm and 203 ± 18 µm and porosity of 69 ± 0.5% and 50 ± 0.2%, respectively. The attachment, proliferation and migration of bone marrow stem cells were satisfactory on both porous collagen-hydroxyapatite and cross-linked collagen-hydroxyapatite scaffolds. Implantation of bone marrow stem cells - collagen-hydroxyapatite or collagen-hydroxyapatite scaffold in beagle dogs with experimental periodontal defects resulted in significantly enhanced periodontal regeneration characterized by formation of new bone, periodontal ligament and cementum, compared with the untreated defects, as evidenced by histological and micro-computed tomography examinations. The prepared collagen-hydroxyapatite scaffolds possess favorable bio-compatibility. The bone marrow stem cells - collagen-hydroxyapatite and collagen-hydroxyapatite scaffold - induced periodontal regeneration, with no aberrant events complicating the regenerative process. Further research is necessary to improve the bone marrow stem cells behavior in collagen-hydroxyapatite scaffolds after implantation.

Expression of Caveolin-1 in Periodontal Tissue and Its Role in Osteoblastic and Cementoblastic Differentiation In Vitro.

It has been previously reported that caveolin-1 (Cav-1) knockout mice exhibit increased bone size and stiffness. However, the expression and role of Cav-1 on periodontal tissue is poorly understood. The aim of this study was to investigate the immunohistochemical expression of Cav-1 in the mouse periodontium and explore the role of Cav-1 on osteoblastic and cementoblastic differentiation in human periodontal ligament cells (hPDLCs), cementoblasts, and osteoblasts. To reveal the molecular mechanisms of Cav-1 activity, associated signaling pathways were also examined. Immunolocalization of Cav-1 was studied in mice periodontal tissue. Differentiation was evaluated by ALP activity, alizarin red S staining, and RT-PCR for marker genes. Signal transduction was analyzed using Western blotting and confocal microscopy. Cav-1 expression was observed in hPDLCs, cementoblasts, and osteoblasts of the periodontium both in vivo and in vitro. Inhibition of Cav-1 expression by methyl-ß-cyclodextrin (MßCD) and knockdown of Cav-1 by siRNA promoted osteoblastic and cementoblastic differentiation by increasing ALP activity, calcium nodule formation, and mRNA expression of differentiation markers in hPDLCs, cementoblasts, and osteoblasts. Osteogenic medium-induced BMP-2 and BMP-7 expression, and phosphorylation of Smad1/5/8 were enhanced by MßCD and siRNA knockdown of Cav-1, which was reversed by BMP inhibitor noggin. MßCD and Cav-1 siRNA knockdown increased OM-induced AMPK, Akt, GSK3ß, and CREB phosphorylation, which were reversed by Ara-A, a specific AMPK inhibitor. Moreover, OM-induced activation of p38, ERK, JNK, and NF-κB was enhanced by Cav-1 inhibition. This study demonstrates, for the first time, that Cav-1 is expressed in developing periodontal tissue and in vitro in periodontal-related cells. Cav-1 inhibition positively regulates osteoblastic differentiation in hPDLCs, cementoblasts, and osteoblasts via BMP, AMPK, MAPK, and NF-κB pathway. Thus, Cav-1 inhibition may be a novel molecular target for therapeutic approaches in periodontitis or osteolytic disease.

Biologic Agents for Periodontal Regeneration and Implant Site Development.

The advancement of molecular mediators or biologic agents has increased tremendously during the last decade in periodontology and dental implantology. Implant site development and reconstruction of the lost periodontium represent main fields in which these molecular mediators have been employed and investigated. Different growth factors trigger different reactions in the tissues of the periodontium at various cellular levels. Proliferation, migration, and differentiation constitute the main target areas of these molecular mediators. It was the purpose of this comprehensive review to describe the origin and rationale, evidence, and the most current understanding of the following biologic agents: Recombinant Human Platelet-Derived Growth Factor-BB (rhPDGF-BB), Enamel Matrix Derivate (EMD), Platelet-Rich Plasma (PRP) and Platelet-Rich Fibrin (PRF), Recombinant Human Fibroblast Growth Factor-2 (rhFGF-2), Bone Morphogenic Proteins (BMPs, BMP-2 and BMP-7), Teriparatide PTH, and Growth Differential Factor-5 (GDF-5).

Cementum proteins: role in cementogenesis, biomineralization, periodontium formation and regeneration.

Destruction of the periodontium is normally associated with periodontal disease, although many other factors, such as trauma, aging, infections, orthodontic tooth movement and systemic and genetic diseases, can contribute to this process. Strategies (such as guided tissue regeneration) have been developed to guide and control regeneration using bioresorbable membranes and bone grafts. Although effective to a certain point, these strategies have the problem that they are not predictable and do not completely restore the architecture of the original periodontium. To achieve complete repair and regeneration it is necessary to recapitulate the developmental process with complete formation of cementum, bone and periodontal ligament fibers. Detailed knowledge of the biology of cementum is key for understanding how the periodontium functions, identifying pathological issues and for developing successful therapies for repair and regeneration of damaged periodontal tissue. It is the purpose of this review to focus on the role of cementum and its specific components in the formation, repair and regeneration of the periodontium. As cementum is a matrix rich in growth factors that could influence the activities of various periodontal cell types, this review will examine the characteristics of cementum, its composition and the role of cementum components, especially the cementum protein-1, during the process of cementogenesis, and their potential usefulness for regeneration of the periodontal structures in a predictable therapeutic manner.

Wnt5a regulates dental follicle stem/progenitor cells of the periodontium.

INTRODUCTION: Dental follicle gives rise to one or several tissues of the periodontium including the periodontal ligament, cementum and/or alveolar bone. Whether Wnt5a is expressed in the postnatal periodontium or regulates dental follicle stem/progenitor cells is unknown. METHODS: Dental follicle stem/progenitor cells were isolated from postnatal day 1 (p1) to p11 from rat mandibular first molars. Immunolocalization mapped Wnt5a expression in the alveolar bone, periodontal ligament, and the developing ameloblast and odontoblast layers. Mononucleated and adherent cells were isolated from p7 dental follicle. Wnt5a was overexpressed in dental follicle stem/progenitor cells to study their proliferation, osteogenic differentiation and migration behavior, with subpopulations of native dental follicle stem/progenitor cells as controls, using real-time PCR (Taqman), Lenti-viral transfection, Western blotting and immunofluorescence. RESULTS: Wnt5a was expressed consistently in p1 to p11 rat peridontium. Native, p7 dental follicle stem/progenitor cells had modest ability to mineralize in the tested 14 days. Even in chemically defined osteogenesis medium, dental follicle stem/progenitor cells only showed modest mineralization. Upon addition of 300 ng/mL Wnt5a protein in osteogenesis medium, dental follicle stem/progenitor cells displayed mineralization that was still unremarkable. Chemically induced or Wnt5a-induced mineralization of dental follicle cells only occurred sparsely. Combination of Wnt5a with 100 ng/mL BMP2 finally prompted dental follicle stem/progenitor cells to produce robust mineralization with elevated expression of Runx2, alkaline phosphatase, collagen 1α1 and osteocalcin. Thus, native dental follicle stem/progenitor cells or some of their fractions may be somewhat modest in mineralization. Strikingly, Wnt5a protein significantly augmented RANKL ligand, suggesting putative regulatory roles of dental follicle stem/progenitor cells for the monocyte/osteoclast lineage and potential involvement in alveolar bone remodeling and/or resorption. P-Jnk1/2 was activated in Wnt5a overexpressed dental follicle cells; conversely, exposure to SP600125, a c-Jun N-terminal kinase (JNK) inhibitor attenuated Runx2, collagen 1α1 and osteocalcin expression either in the presence or absence of Wnt5a. Wnt5a overexpression in dental follicle stem/progenitor cells significantly reduced their proliferation rates, but robustly augmented their migration capacity. CONCLUSIONS: These findings provide a glimpse of Wnt5a's putative roles in dental follicle stem/progenitor cells and the periodontium with implications in periodontal disease, tooth eruption, dental implant bone healing and orthodontic tooth movement.

Regeneration of the periodontium for preservation of the damaged tooth.

The population of the world grows every year, and life expectancy tends to increase. Thus, long-term preservation of teeth in aged individuals is an urgent issue. The main causes of tooth loss are well known to be periodontitis, caries, fractures, and orthodontic conditions. Although implant placement is a widely accepted treatment for tooth loss, most patients desire to preserve their own teeth. Many clinicians and researchers are therefore challenged to treat and preserve teeth that are irreversibly affected by deep caries, periodontitis, fractures, and trauma. Tissue engineering techniques are beneficial in addressing this issue; stem cells, signal molecules, and scaffolds are the main elements of such techniques. In this review, we describe these three elements with respect to their validation for regeneration of the periodontium and focus particularly on the potency of diverse scaffolds. In addition, we provide a short overview of the ongoing studies of 4-methacryloxyethyl trimellitate anhydride/methyl methacrylate-tri-n-butyl-borane resin including calcium chloride or hydroxyapatite for periodontium regeneration.