Immunohistochemical Evaluation of Periodontal Regeneration Using a Porous Collagen Scaffold.

(1) Aim: To immunohistochemically evaluate the effect of a volume-stable collagen scaffold (VCMX) on periodontal regeneration. (2) Methods: In eight beagle dogs, acute two-wall intrabony defects were treated with open flap debridement either with VCMX (test) or without (control). After 12 weeks, eight defects out of four animals were processed for paraffin histology and immunohistochemistry. (3) Results: All defects (four test + four control) revealed periodontal regeneration with cementum and bone formation. VCMX remnants were integrated in bone, periodontal ligament (PDL), and cementum. No differences in immunohistochemical labeling patterns were observed between test and control sites. New bone and cementum were labeled for bone sialoprotein, while the regenerated PDL was labeled for periostin and collagen type 1. Cytokeratin-positive epithelial cell rests of Malassez were detected in 50% of the defects. The regenerated PDL demonstrated a larger blood vessel area at the test (14.48% ± 3.52%) than at control sites (8.04% ± 1.85%, p = 0.0007). The number of blood vessels was higher in the regenerated PDL (test + control) compared to the pristine one (p = 0.012). The cell proliferative index was not statistically significantly different in pristine and regenerated PDL. (4) Conclusions: The data suggest a positive effect of VCMX on angiogenesis and an equally high cell turnover in the regenerated and pristine PDL. This VCMX supported periodontal regeneration in intrabony defects.

Monitoring Biochemical and Structural Changes in Human Periodontal Ligaments during Orthodontic Treatment by Means of Micro-Raman Spectroscopy.

The aim of the study was to examine the biochemical and structural changes occurring in the periodontal ligament (PDL) during orthodontic-force application using micro-Raman spectroscopy ( µ -RS). Adolescent and young patients who needed orthodontic treatment with first premolar extractions were recruited. Before extractions, orthodontic forces were applied using a closed-coil spring that was positioned between the molar and premolar. Patients were randomly divided into three groups, whose extractions were performed after 2, 7, and 14 days of force application. From the extracted premolars, PDL samples were obtained, and a fixation procedure with paraformaldehyde was adopted. Raman spectra were acquired for each PDL sample in the range of 1000-3200 cm - 1 and the more relevant vibrational modes of proteins (Amide I and Amide III bands) and CH 2 and CH 3 modes were shown. Analysis indicated that the protein structure in the PDL samples after different time points of orthodontic-force application was modified. In addition, changes were observed in the CH 2 and CH 3 high wavenumber region due to local hypoxia and mechanical force transduction. The reported results indicated that µ -RS provides a valuable tool for investigating molecular interchain interactions and conformational modifications in periodontal fibers after orthodontic tooth movement, providing quantitative insight of time occurring for PDL molecular readjustment.

An unusual disordered alveolar bone material in the upper furcation region of minipig mandibles: A 3D hierarchical structural study.

Teeth are subjected to compressive loads during mastication. Under small loads the soft tissue periodontal ligament (PDL) deforms most. However when the loads increase and the PDL is highly compressed, the tooth and the alveolar bone supporting the tooth, begin to deform. Here we report on the structure of this alveolar bone in the upper furcation region of the first molars of mature minipigs. Using light microscopy and scanning electron microscopy (SEM) of bone cross-sections, we show that this bone is hypermineralized, containing abundant small pores around 1-5 µm in diameter, lacunae around 10-20 µm as well as larger spaces. This bone does not possess the typical lamellar motif or other repeating structures normally found in cortical or trabecular mammalian bone. We also use high resolution focused ion beam scanning electron microscopy (FIB-SEM) in the serial surface mode to image the 3D organization of the demineralized bone matrix. We show that the upper furcation bone matrix has a disordered isotropic structure composed mainly of individual collagen fibrils with no preferred orientation, as well as highly staining material that is probably proteoglycans. Much larger aligned arrays of collagen fibers - presumably Sharpey's fibers - are embedded in this material. This unusual furcation bone material is similar to the disordered material found in human lamellar bone. In the upper furcation region this disordered bone comprises almost all the volume excluding Sharpey's fibers. We surmise that this most unusual bone type functions to resist the repeating compressive loads incurred by molars during mastication.

Cellular and collagen reference values of gingival and periodontal ligament tissues in rats: a pilot study.

Reference data are lacking on the periodontal ligament and the gingival tissue of the rat model, which would be useful for studies of new medical or biomaterial periodontal treatments. The objective of the current study was to propose cellular and collagen reference values of gingival and periodontal ligament tissues in rat, using a simple and reliable quantitative method after decalcification. Mandibular samples of ten adult Sprague-Dawley rats were used. Mild decalcification was carried out using ethylenediaminetetraacetic acid (EDTA) to preserve the morphology of tissues. Half of the samples were decalcified and the other half were not. The gingiva and the periodontal ligament were analyzed. Descriptive histology and computer-assisted image analysis were performed. The data showed that qualitatively, cellular and extracellular matrix morphologies were well preserved compared to non-decalcified periodontal soft tissue biopsies. Histomorphometrically, constitutive cellularity and the total amount of native collagen, collagen directionality and collagen anisotropy in both experimental conditions did not significantly differ. Taken together, these results suggested that EDTA decalcification did not negatively affect the studied endpoints. Moreover, this mild decalcification method allowed in situ maintenance of the periodontal soft and hard tissue integrity. The structural and compositional computerized assessment performed in the healthy periodontal soft tissue could provide reference values that will be required for future assessment on the effects of pathological, reparative and regenerative processes in rat periodontal soft tissues.

Sequential application of bFGF and BMP-2 facilitates osteogenic differentiation of human periodontal ligament stem cells.

BACKGROUND AND OBJECTIVES: Basic fibroblast growth factor (bFGF) promotes cells proliferation and chemotaxis and maintains stemness while inhibits mineralized nodule formation. Bone morphogenetic protein 2 (BMP-2) shows great potential in promoting bone formation. However, sequential application of these two growth factors on periodontal ligament stem cells (PDLSCs) has not been explored. In this study, we aimed to identify the optimal concentration and time of bFGF on PDLSCs proliferation, migration and then investigate the sequential delivery of bFGF and BMP-2 on osteogenic differentiation of PDLSCs in vitro. MATERIALS AND METHODS: Periodontal ligament stem cells were isolated by limiting dilution method. Dose-dependent additive effects of bFGF and BMP-2 on PDLSCs were detected. Cell counting assay, cell migration assay, alkaline phosphatase (ALP) activity assay, Alizarin red staining, quantitative real-time polymerase chain reaction (qRT-PCR), and western blot analysis were used to determine different application modalities of bFGF and BMP-2 on proliferation, migration, and osteogenic differentiation of PDLSCs. RESULTS: 50 ng/mL bFGF significantly promoted PDLSCs proliferation and chemotaxis while time-dependently inhibited BMP-2 induced ALP activity. Sequential application of 25 ng/mL bFGF for first 3 days and followed with 50 ng/mL BMP-2 for another 9, 18, and 25 days significantly promoted PDLSCs osteogenic differentiation. Compared with bFGF and BMP-2 simultaneous group, sequential application of bFGF and BMP-2 group significantly enhanced ALP activity, osteogenesis-related genes and proteins expression and mineral deposition. CONCLUSION: Sequential application of bFGF and BMP-2 synergistically promoted osteogenic differentiation of PDLSCs, and this sequential application modality of growth factors would provide a new strategy for periodontal regeneration.

Expression of Wnt3a, Wnt10b, ß-catenin and DKK1 in periodontium during orthodontic tooth movement in rats.

OBJECTIVE: To investigate the expression of Wnt3a, Wnt10b, ß-catenin and DKK1 in the periodontal ligament (PDL) during orthodontic tooth movement (OTM) in rats. MATERIALS AND METHODS: Nickel-titanium closed-coil springs were used to deliver an initial 50 g mesial force to the left maxillary first molars in 30 rats. The force was kept constant for 1, 3, 5, 7, 10 and 14 days until the animals were sacrificed. The right maxillary molars without force application served as control. Paraffin-embedded sections of the upper jaws were prepared for histological and immunohistochemical analyses to detect Wnt3a, Wnt10b, ß-catenin and DKK1 expression in PDL. RESULTS: Wnt3a, Wnt10b, ß-catenin and DKK1 were expressed on both the ipsilateral and contralateral sides of PDL in each group. After the application of orthodontic force, the expression of ß-catenin and DKK1 was initially increased and then decreased on both sides, with maximal levels of expression at day 7 and day 10, respectively. On the compression side, Wnt3a and Wnt10b levels started to increase at day 5, while on the tension side, these two molecules began to increase at day 1. Furthermore, the expression levels of Wnt3a, Wnt10b, and ß-catenin were much stronger on the tension side than on the compression side at any of the observation points, while DKK1 level was much higher on the compression side. CONCLUSION: Wnt3a, Wnt10b, ß-catenin and DKK1 expression may be related to the periodontal tissue remodeling following the application of an orthodontic force in rats. These observations suggest that the Wnt/ß-catenin signaling pathway may play a crucial role in periodontal tissue remodeling during OTM.

Immunohistochemical localization of connective tissue growth factor, transforming growth factor-beta1 and phosphorylated-smad2/3 in the developing periodontium of rats.

BACKGROUND AND OBJECTIVE: Connective tissue growth factor (CTGF) is a downstream mediator of transforming growth factor-beta1 (TGF-ß1), and TGF-ß1-induced CTGF expression is regulated through the SMAD pathway. CTGF is implicated in the development of cartilage, bone and tooth. However, its expression in the developing periodontium is unclear. Therefore, we aimed to investigate the immunolocalization of CTGF, TGF-ß1 and phosphorylated SMAD2/3 (pSMAD2/3) in the developing periodontium of rats. MATERIAL AND METHODS: The maxillaries of Wistar rats, 2, 3, 7 and 12 wk of age, were used and the localization of CTGF, TGF-ß1 and pSMAD2/3 was detected using immunoperoxidase techniques. RESULTS: Hertwig' s epithelial root sheath (HERS) cells were strongly positive for CTGF and TGF-ß1, but not for pSMAD2/3. Positive staining for CTGF, TGF-ß1 and pSMAD2/3 was found in bone and periodontal ligament. In cementum, most cementoblasts associated with cellular cementum and some cementocytes stained strongly for CTGF, whereas cementoblasts associated with acellular cementum did not express CTGF. No signal for TGF-ß1 was observed in cellular and acellular cementum. In addition, most cementocytes were strongly positive for pSMAD2/3. CONCLUSION: CTGF, TGF-ß1 and pSMAD2/3 are localized in bone and periodontal ligament, but are differentially expressed in HERS and cementum. The results of our study indicate that the regulation of CTGF expression by TGF-ß1 might be cell-type specific in periodontium.

The molecular and cellular effects of ageing on the periodontal ligament.

AIM: Many in vitro studies have investigated age-related biological changes in cells comprising the periodontium but the basic question of whether the periodontium can maintain its integrity with age remains unanswered. Thus, the aim of this study was to understand how, in the absence of disease, advancing age impacts the structure of the periodontium. MATERIALS AND METHODS: Of 4, 10, 25, and 50-week-old mice were examined using histology and immunohistochemical analyses for cell proliferation, cell turnover, collagen quantity and quality, osteogenic markers, bone turnover, and cytokine expression. RESULTS: The periodontal ligament (PDL) space shows a gradual decrease in width over the lifespan of the mice. Cell proliferation as well as the quantity and quality of collagen fibres decreased with age although cell density did not appear to be altered. Osteoprogenitor markers in the PDL maintained their expression with increasing age. Alkaline phosphatase (ALP) activity decreased, but osteoclast activity increased with age. CONCLUSIONS: Ageing is associated with a decline in the quality and quantity of collagen and an increase in bone resorption, all of which can diminish the function of the periodontium even in the absence of disease.

Evaluation of BSP expression and apoptosis in the periodontal ligament during orthodontic relapse: a preliminary study.

OBJECTIVE: To examine the expression of bone sialoprotein (BSP) and apoptosis in an in vivo orthodontic relapse model. MATERIALS AND METHODS: Male mice (10-12 weeks old), either transgenic [green fluorescent protein (GFP) driven by the BSP promoter] or wild type, were used in this study. To achieve orthodontic tooth movement (OTM), maxillary right first molars were moved mesially using closed-coil springs. Animals were divided into an OTM group (14 days continuous orthodontic force - 11 animals) or Relapse group (10 days of force application followed by 4 days of relapse - 8 animals). The control group was comprised of the contralateral maxillary molars. The periodontal ligament (PDL) was analyzed in areas of compression and tension for transgenic expression, osteoclast localization, and the presence of apoptotic cells. RESULTS: There was a significant decrease in GFP-labeled cells on the compression and tension sides of the PDL in the OTM group compared with control. In the relapse group, GFP-labeled cells were significantly decreased only on the old compression side. Osteoclasts were localized on the compression side of the OTM group, whereas in the Relapse group, they were present on both sides. PDL apoptosis significantly increased on the compression side in OTM and Relapse groups. CONCLUSION: Both OTM and Relapse groups exhibited a decreased number of GFP-labeled cells in areas of compression and tension. There was significant PDL apoptosis in regions under compressive forces following OTM and to a lesser extent following relapse.

Micro-Raman spectroscopy for monitoring changes in periodontal ligaments and gingival crevicular fluid.

Micro-Raman Spectroscopy is an efficient method for analyzing biological specimens due to its sensitivity to subtle chemical and structural changes. The aim of this study was to use micro-Raman spectroscopy to analyze chemical and structural changes in periodontal ligament after orthodontic force application and in gingival crevicular fluid in presence of periodontal disease. The biopsy of periodontal ligament samples of premolars extracted for orthodontic reasons and the gingival crevicular fluid samples collected by using absorbent paper cones; were analyzed by micro-Raman spectroscopy. Changes of the secondary protein structure related to different times of orthodontic force application were reported; whereas an increase of carotene was revealed in patients affected by periodontal inflammation.

A qualitative investigation of RANKL, RANK and OPG in a rat model of transient ankylosis.

INTRODUCTION: Previous studies have found ankylosis occurs as a part of the inflammatory process of aseptic root resorption initiated in a rat model. The physiologic mechanisms behind the development of dentoalveolar ankylosis and healing response are still unclear. While receptor activator of nuclear factor-κß ligand (RANKL), receptor activator of nuclear factor-κß (RANK) and osteoprotegerin (OPG) have gained momentum in the understanding of resorption, no study to date has investigated their role in dentoalveolar ankylosis. AIMS: The aims of this study were to investigate if, and when, ankylosis occurred in the rat PDL, whether the resolution of ankylosis occurred with time and, finally, to observe the expression of RANKL, RANK and OPG during the ankylotic process. MATERIALS AND METHODS: Dry ice was applied for 20 minutes to the upper right first molar crown of 15 eight-week-old, male Sprague-Dawley rats. An additional three rats served as untreated external controls. Groups of three rats were sacrificed after the thermal insult on day 0, 4, 7, 14 and 28 respectively. Each maxilla was dissected out and processed for histological examination and RANKL, OPG and RANK immunohistochemistry. RESULTS: By the use of light microscopy and H&E staining, no ankylosis was detected in the external control group and the experimental groups at days 0 and 4. On day 7, disruption within the periodontal ligament was observed in the interradicular region and the initial signs of ankylosis were seen in the form of finger-like projections extending from the alveolar bone towards the cementum. Fourteen days after the thermal insult, all animals exhibited extensive ankylosis that spanned the entire interradicular periodontal space. At 28 days, the development of ankylosis appeared to have ceased and repair was observed, together with an intact periodontal ligament in all but one rat. Positive staining results were obtained with RANKL, RANK and OPG antibodies. The expressions of RANKL, RANK and OPG were similar in the external control group, 0-, 4-, and 28-day experimental groups. In the 7- and 14-day experimental groups, RANKL, RANK and OPG were expressed in the blood vessels within the ankylotic regions. CONCLUSIONS: During the development of ankylosis and its resolution, it was concluded from their simultaneous presence that there is a complex interaction between RANKL, RANK and OPG that requires further investigation.

Acellular Extrinsic Fiber Cementum Is Invariably Present in the Superficial Layer of Apical Cementum in Mouse Molar.

The cementum is a highly mineralized tissue that covers the tooth root. The regional differences among the types of cementum, especially in the extrinsic fibers that contribute to tooth support, remain controversial. Therefore, this study used second harmonic generation imaging in conjunction with automated collagen extraction and image analysis algorithms to facilitate the quantitative examination of the fiber characteristics and the changes occurring in these fibers over time. Acellular extrinsic fiber cementum (AEFC) was invariably observed in the superficial layer of the apical cementum in mouse molars, indicating that this region of the cementum plays a crucial role in supporting the tooth. The apical AEFC exhibited continuity and fiber characteristics comparable with the cervical AEFC, suggesting a common cellular origin for their formation. The cellular intrinsic fiber cementum present in the inner layer of the apical cementum showed consistent growth in the apical direction without layering. This study highlights the dynamic nature of the cementum in mouse molars and underscores the requirement for re-examining its structure and roles. The findings of the present study elucidate the morphophysiological features of cementum and have broader implications for the maintenance of periodontal tissue health.

Immunolocalization of RANK and RANKL along the root surface and in the periodontal membrane of human primary and permanent teeth.

OBJECTIVE: Root resorption, impaired tooth eruption and early tooth loss have been described in relation to diseases that involve defects in the RANK-RANKL-OPG-expression. The aim of the present immunhistochemical study was to localize and compare the reactions for RANK and membrane-bound RANKL along root surfaces and in the periodontal membrane in close proximity to the root surface of human primary and permanent teeth. MATERIALS AND METHODS: The material comprised extracted human teeth (11 primary teeth and six permanent teeth) from 10 different patients. Paraffin sections were prepared of each tooth and sections of each tooth were immunohistochemically stained with antibodies specific for membrane-bound RANKL and RANK. RESULTS: The root surface and the periodontal membrane in close proximity to the root surface did not show immunoreactivity for RANKL. RANKL was only located in odontoblasts and in cells along denticles in one primary tooth. RANK was located in mononuclear cells in the pulp and in multinucleated odontoclasts along resorbed root surfaces and along resorbed dentin surfaces in the pulp in primary teeth and one permanent tooth. CONCLUSIONS: This study demonstrated RANK positivity in resorption areas in primary and permanent teeth. RANKL was positive in the pulp of one primary tooth. RANK expression in odontoclasts and RANKL expression in the pulp may indicate that RANK/RANKL play a role during resorption.

Effect of milk renewal on human periodontal ligament fibroblast viability in vitro.

Milk has been studied extensively and has gained wide acceptance as a suitable storage medium capable of maintenance of avulsed teeth that cannot be replanted immediately. The objective of this study was to evaluate whether the renewal of milk as a storage medium every 24 h for up to 120 h is able to increase its ability to maintain human periodontal ligament fibroblasts (PDLF) viability in vitro. Plates with confluent PDLF were soaked in minimum essential medium (MEM) at 37°C (positive control) and in skimmed milk (22 wells) and water (negative control) for 24, 48, 72, 96, and 120 h at 5 and 20°C. The skimmed milk was renewed every 24 h in 11 of the wells of each plate. After these periods, cell viability was determined by the tetrazolium salt-based colorimetric (MTT) assay. Data were statistically analyzed by Kruskal-Wallis and Scheffé tests (α = 5%). At 24 h, milk and MEM performed similarly. However, from 48 h onwards, MEM was significantly better than renewed and not renewed milk at both temperatures. Regardless of temperature (5 or 20°C), renewal of milk with fresh milk did not affect its ability to maintain PDLF viability.

Reduced functional loads alter the physical characteristics of the bone-periodontal ligament-cementum complex.

BACKGROUND AND OBJECTIVE: Adaptive properties of the bone-periodontal ligament-tooth complex have been identified by changing the magnitude of functional loads using small-scale animal models, such as rodents. Reported adaptive responses as a result of lower loads due to softer diet include decreased muscle development, change in structure-function relationship of the cranium, narrowed periodontal ligament space, and changes in the mineral level of the cortical bone and alveolar jaw bone and in the glycosaminoglycans of the alveolar bone. However, the adaptive role of the dynamic bone-periodontal ligament-cementum complex to prolonged reduced loads has not been fully explained to date, especially with regard to concurrent adaptations of bone, periodontal ligament and cementum. Therefore, in the present study, using a rat model, the temporal effect of reduced functional loads on physical characteristics, such as morphology and mechanical properties and the mineral profiles of the bone-periodontal ligament-cementum complex was investigated. MATERIAL AND METHODS: Two groups of 6-wk-old male Sprague-Dawley rats were fed nutritionally identical food with a stiffness range of 127-158 N/mm for hard pellet or 0.3-0.5 N/mm for soft powder forms. Spatio-temporal adaptation of the bone-periodontal ligament-cementum complex was identified by mapping changes in the following: (i) periodontal ligament collagen orientation and birefringence using polarized light microscopy, bone and cementum adaptation using histochemistry, and bone and cementum morphology using micro-X-ray computed tomography; (ii) mineral profiles of the periodontal ligament-cementum and periodontal ligament-bone interfaces by X-ray attenuation; and (iii) microhardness of bone and cementum by microindentation of specimens at ages 6, 8, 12 and 15 wk. RESULTS: Reduced functional loads over prolonged time resulted in the following adaptations: (i) altered periodontal ligament orientation and decreased periodontal ligament collagen birefringence, indicating decreased periodontal ligament turnover rate and decreased apical cementum resorption; (ii) a gradual increase in X-ray attenuation, owing to mineral differences, at the periodontal ligament-bone and periodontal ligament-cementum interfaces, without significant differences in the gradients for either group; (iii) significantly (p < 0.05) lower microhardness of alveolar bone (0.93 ± 0.16 GPa) and secondary cementum (0.803 ± 0.13 GPa) compared with the higher load group insert bone = (1.10 ± 0.17 and cementum = 0.940 ± 0.15 GPa, respectively) at 15 wk, indicating a temporal effect of loads on the local mineralization of bone and cementum. CONCLUSION: Based on the results from this study, the effect of reduced functional loads for a prolonged time could differentially affect morphology, mechanical properties and mineral variations of the local load-bearing sites in the bone-periodontal ligament-cementum complex. These observed local changes in turn could help to explain the overall biomechanical function and adaptations of the tooth-bone joint. From a clinical translation perspective, our study provides an insight into modulation of load on the complex for improved tooth function during periodontal disease and/or orthodontic and prosthodontic treatments.

Immunohistochemical expression of heat shock proteins in the mouse periodontal tissues due to orthodontic mechanical stress.

The histopathology of periodontal ligament of the mouse subjected to mechanical stress was studied. Immunohistochemical expressions of HSP27 and p-HSP27 were examined. Experimental animals using the maxillary molars of ddY mouse by Waldo method were used in the study. A separator was inserted to induce mechanical stress. After 10 minutes, 20 minutes, 1 hour, 3 hours, 9 hours and 24 hours, the regional tissues were extracted, fixed in 4% paraformaldehyde and 0.05M phosphate-buffered fixative solution. Paraffin sections were made for immunohistochemistry using HSP27 and p-HSP27. In the control group, the periodontal ligament fibroblasts expressed low HSP27 and p-HSP27. However, in the experimental group, periodontal ligament fibroblasts expressed HSP27 10 minutes after mechanical load application in the tension side. The strongest expression was detected 9 hours after inducing mechanical load. p-HSP27 was also expressed in a time-dependent manner though weaker than HSP27. The findings suggest that HSP27 and p-HSP27 were expressed for the maintenance of homeostasis of periodontal ligament by the activation of periodontal ligament fibroblasts on the tension side. It also suggests that these proteins act as molecular chaperones for osteoblast activation and maintenance of homeostasis.

A novel serum-free medium for the isolation, expansion and maintenance of stemness and tissue-specific markers of primary human periodontal ligament cells.

PURPOSE: Periodontal ligament (PDL) cell cultures are classically maintained in serum-containing media. However, unwanted side-effects of these conditions on cellular and molecular characteristics demand a serum-free alternative. Even though these limitations are well known and efforts for the development of adequate serum-free alternatives have been made, these approaches for replacement remained unsuccessful so far. This study aimed at developing a well-defined, serum-free formulation supporting both isolation from tissue samples and efficient expansion of PDL cells. Here, of particular focus was the perpetuation of tissue-characteristic markers detectable in primary tissues and of stemness features. BASIC PROCEDURES: Primary PDL cell cultures from generally healthy human donors (n = 3) were maintained in basal media N2B27 and E6 together with different concentrations of growth and attachment factors. Cell proliferation was recorded via microscopy and WST assay. Gene expression of RUNX2, Periostin, ALP, CD73, CD90, CD105, CD45, SOX10 and SOX2 was compared to primary PDL explants via qRT-PCR. Immunocytochemistry was performed for anti-CD105, SSEA-3, CD271, HNK1. Serum-containing sDMEM medium served as control. MAIN FINDINGS: N2B27 medium substituted with 25 ng/mL EGF, 25 ng/mL IGF1, 0.5 mg/mL Fetuin plus gelatine coating (designated N2B27-PDLsf) emerged as potent serum-free formulation ensuring adequate culture isolation and expansion. Here, PDL primary tissue signature markers RUNX2 and Periostin remained stable in N2B27-PDLsf compared to controls (229.0-fold ±101.0 and 83.2-fold ±9.6 increase). Additionally, stemness markers ALP and CD105 were significantly upregulated on transcriptional, and CD105 and SOX2 on protein level. PRINCIPAL CONCLUSIONS: This investigation identified a novel serum-free medium for the isolation, and expansion of primary human PDL cells with constantly high proliferation rates. Here, purity and stemness properties are maintained. Thus, N2B27-PDLsf represents a valid replacement for serum-containing media in PDL cultures.

Stretching modulates oxytalan fibers in human periodontal ligament cells.

MATERIAL AND METHODS: We subjected periodontal ligament fibroblasts to stretching strain to examine the effects on their formation of oxytalan fibers in cell/matrix layers. RESULTS: Stretching increased the levels of fibrillin-1 and fibrillin-2 by 25% relative to the control, but did not affect the gene expression level of either type of fibrillin. Immunofluorescence and immunogold electron microscopy analysis revealed that bundles of oxytalan fibers became thicker under stretching conditions. CONCLUSION: These results suggest that tension strain functionally regulates microfibril assembly in periodontal ligament fibroblasts and thus may contribute to the homeostasis of oxytalan fibers in periodontal ligaments.

Identification of N-methyl-D-aspartate receptor subunit in human periodontal ligament fibroblasts: potential role in regulating differentiation.

BACKGROUND: Periodontal ligament fibroblasts (PDLFs), which can be differentiated into osteoblasts, are crucial cells for the regeneration of the periodontal tissue. Although N-methyl-D-aspartate (NMDA) receptors were reported to be involved in bone formation by affecting osteoblasts, the existence and function of NMDA receptors in PDLFs have not been confirmed. The purpose of this study was to examine the expression of NMDA receptors and their role in human PDLFs. METHODS: Human PDLFs were cultured and evaluated to identify the subunits of NMDA receptors (NR) by reverse transcription-polymerase chain reaction, Western blot analysis, and immunocytochemistry. Then, the cells were assigned to four different groups: a control media group, a control media with NMDA receptor antagonist group, a differentiation media group, and a differentiation media with NMDA receptor antagonist group. Cell proliferation assay, alkaline phosphatase (ALP) activity analysis, and mineralization assay were performed to determine whether NMDA receptors affected the function of PDLFs. RESULTS: NR1, NR2B, and NR2D were detected in human PDLFs. There was no statistically significant difference in proliferation among the groups. However, the NMDA receptor antagonist-treated group showed a significant reduction in ALP activity (P <0.05). Moreover, the NMDA receptor antagonist-supplemented group presented no mineralization. CONCLUSIONS: This study revealed the existence of NMDA receptors in human PDLFs and specified their subunits. Moreover, NMDA receptors had a significant influence on the differentiation and mineralization of human PDLFs but did not affect their proliferation. These results suggest that NMDA receptors may play an important role in the differentiation and mineral tissue formation of human PDLFs.

Decellularized human periodontal ligament for periodontium regeneration.

Regenerating the periodontal ligament (PDL) is a crucial factor for periodontal tissue regeneration in the presence of traumatized and periodontally damaged teeth. Various methods have been applied for periodontal regeneration, including tissue substitutes, bioactive materials, and synthetic scaffolds. However, all of these treatments have had limited success in structural and functional periodontal tissue regeneration. To achieve the goal of complete periodontal regeneration, many studies have evaluated the effectiveness of decellularized scaffolds fabricated via tissue engineering. The aim of this study was to fabricate a decellularized periodontal scaffold of human tooth slices and determine its regeneration potential. We evaluated two different protocols applied to tooth slices obtained from human healthy third molars. The extracellular matrix scaffold decellularized using sodium dodecyl sulfate and Triton X-100, which are effective in removing nuclear components, was demonstrated to preserve an intact structure and composition. Furthermore, the decellularized scaffold could support repopulation of PDL stem cells near the cementum and expressed cementum and periodontal-ligament-related genes. These results show that decellularized PDL scaffolds of human teeth are capable of inducing the proliferation and differentiation of mesenchymal stem cells, thus having regeneration potential for use in future periodontal regenerative tissue engineering.