Calcitriol and enamel matrix derivative differentially regulated cemento-induction and mineralization in human periodontal ligament-derived cells.

BACKGROUND: Alveolar bone and cementum share many biological and developmental similarities. The mineralizing effect of calcitriol has been previously reported. Yet, its cemento-inductivity has not been confirmed. This study evaluated the potential cemento-inductivity effect of calcitriol and enamel matrix derivative (EMD) on human periodontal ligament-derived cells (hPDLCs). METHODS: The hPDLCs obtained from extracted third molars or premolars were cultured with calcitriol, or EMD. Cementogenic gene expression was examined using real-time quantitative reverse transcription polymerase chain reaction. Expression analysis also included cementoblast-specific markers, cementum protein 1 (CEMP1), cementum attachment protein (CAP), and recently reported cementoblast-enriched genes, secreted frizzled related protein 1 (SFRP1), and Dickkopf-related protein 1 (DKK1). Mineralization capacities were evaluated by alkaline phosphatase (ALP) activity, Alizarin Red, and Von Kossa staining followed by scanning electron microscope imaging and element mapping. RESULTS: Among tested conditions, 10 nM calcitriol enhanced most cementogenic gene expression, transforming growth factor-ß1, bone morphogenetic proteins (BMP-2 and BMP-4), core-binding factor subunit alpha-1/Runt-related transcription factor 2, Type I collagen, ALP, bone sialoprotein, osteopontin), osteocalcin, CEMP1, and CAP, and Wnt signaling negative modulators, SFRP1 and DKK1, along with highest ALP activity and mineralization formation in hPDLCs. However, only moderate CEMP1 protein was observed. In contrast, EMD stimulated stronger CEMP1 and CAP protein, but presented weaker mineralization capacity, hinting at the possibility that strong stimulation of mineralization might dominate cemetogenic specific factors and vice versa. CONCLUSIONS: Calcitriol demonstrated not only great osteoinductivity, but also the potential to induce cementogenic gene expression by initiating hPDLC differentiation and promoting mineralization. Compared with calcitriol, EMD promoted cemento-inductivity in hPDLCs at a later time point via highly expressed CEMP1 and CAP protein, but with less mineralization. Thus, calcitriol and EMD could provide differential enhancement of cemento-induction and mineralization, likely acting at various differentiation stages.

Calcitriol exerts a mineralization-inductive effect comparable to that of vitamin C in cultured human periodontium cells.

This study inspected whether calcitriol could exert a mineralization-inductive effect comparable to that of vitamin C in cultured human periodontium cells (hPDCs). The mRNA expression of the mineralization-related biomarkers core-binding factor subunit alpha-1 (Cbfa1), collagen 1 α1 (Col-I), alkaline phosphatase (ALP), osteopontin (OPN), bone sialoprotein (BSP), osteocalcin (OCN), vitamin D receptor (VDR), cementum protein 1 (CEMP-1), cementum attachment protein (CAP), interleukin 6 (IL-6), transforming growth factor-ß1 (TGF-ß1) and osteoprotegerin (OPG) was surveyed after incubation of hPDCs with vitamin C and calcitriol for 2 weeks. Translational expression information from ALP activity and CEMP-1 and CAP immunofluorescence assays was acquired from hPDCs at the second and third weeks. Extracellular calcifications were confirmed by von Kossa staining, Alizarin Red staining and synchrotron transmission X-ray microscopy (TXM) at the fourth and fifth weeks. It was found that both vitamin C and calcitriol not only increased mineralization-related mRNA fold-changes but also enhanced ALP activity, CEMP-1 immunofluorescence, von Kossa and Alizarin Red staining and TXM-associated calcifications. Generally, 10-8 M calcitriol displayed greater mineralization significance than 10-7 M calcitriol in the assays tested. However, vitamin C stimulated lower Cbfa1, Col-1, ALP, OPN, BSP, OCN, VDR, CEMP-1 and IL-6 mRNA fold-changes than 10-8 M calcitriol. Finally, TXM analysis indicated that a 10-8 M calcitriol treatment stimulated greater calcifications than vitamin C treatment. Therefore, the analytical results confirmed the osteo-inductive potential of vitamin C in cultured hPDCs. In contrast, 10-8 M calcitriol could potentially function as a substitute because it stimulates a greater mineralization effect than vitamin C or 10-7 M calcitriol.

Correction: Incompatible amount of 3-D and 2-D periodontal attachments on micro-CT scanned premolars.

[This corrects the article DOI: 10.1371/journal.pone.0193894.].

Isolation of Mesenchymal Stem Cells from Human Alveolar Periosteum and Effects of Vitamin D on Osteogenic Activity of Periosteum-derived Cells.

Mesenchymal stem cells (MSCs) are present in a variety of tissues and can be differentiated into numerous cell types, including osteoblasts. Among the dental sources of MSCs, the periosteum is an easily accessible tissue, which has been identified to contain MSCs in the cambium layer. However, this source has not yet been widely studied. Vitamin D3 and 1,25-(OH)2D3 have been demonstrated to stimulate in vitro differentiation of MSCs into osteoblasts. In addition, vitamin C facilitates collagen formation and bone cell growth. However, no study has yet investigated the effects of Vitamin D3 and Vitamin C on MSCs. Here, we present a method of isolating MSCs from human alveolar periosteum and examine the hypothesis that 1,25-(OH)2D3 may exert an osteoinductive effect on these cells. We also investigate the presence of MSCs in the human alveolar periosteum and assess stem cell adhesion and proliferation. To assess the ability of vitamin C (as a control) and various concentrations of 1,25-(OH)2D3 (10-10, 10-9, 10-8, and 10-7 M) to alter key mRNA biomarkers in isolated MSCs mRNA expression of alkaline phosphatase (ALP), bone sialoprotein (BSP), core binding factor alpha-1 (CBFA1), collagen-1, and osteocalcin (OCN) are measured using real-time polymerase chain reaction (RT-PCR).

Incompatible amount of 3-D and 2-D periodontal attachments on micro-CT scanned premolars.

Micro-computed tomography (micro-CT) was employed to relate the root surface area (RSA) to the periodontal attachment levels (PALs) of extracted premolars to diagnose periodontitis. Single-rooted human maxillary and mandibular premolars 31 and 36, respectively, were surveyed by micro-CT and its associated software. RSA levels from the 1st to 10th mm, corono-apically, were analyzed using statistical t tests. The average root length (RL) and RSA of the maxillary and mandibular premolars were significantly different (p < 0.05). Both premolars demonstrated a non-significant RSA percentage comparison at the evaluated PALs. For the 30% coronal 2-D radiographic RL, the 3-D RSAs 3.77 mm and 3.99 mm apical to the cementoenamel junction (CEJ) were 39.48% and 40.65% for maxillary and mandibular premolars, respectively. At the 15% coronal 2-D RL, the 3-D RSA 2 mm apical to the CEJ of the premolars was approximately 21%. At the 50% coronal 2-D RL level, approximately 62% coronal 3-D RSA and 6.5 mm RL decreased. The amount of decrease of the RSA attachment is significant in every 2-mm measurement for both premolars. Sampling periodontal microbial pathogens based on the condition of 2-D radiographic bone and clinical attachment losses without considering 3-D RSA is potentially inadequate and may underestimate the severity of the periodontitis.

Inconsistency in the Crown-to-Root Ratios of Single-Rooted Premolars Measured by 2D and 3D Examinations.

Micro-computed tomography (micro-CT) was applied to elucidate the relationship between the three-dimensional (3D) root surface area (RSA) and two-dimensional (2D) crown-to-root ratio (CRR) of extracted teeth to classify the periodontitis and assign a periodontal/prosthetic prognosis. A total of 31 maxillary and 35 mandibular single-rooted human premolars were examined. The amount of periodontal support on the basis of 3D RSA and 2D root length (RL) at CRRs of 1:1, 5:4, 3:2, and 2:1 were analyzed. Both maxillary and mandibular premolars demonstrated a nonsignificant RSA percentage at the evaluated CRRs. The coronal 21%-22% 2D RL and the 26%-28% 3D RSA bone loss apical to the cemento-enamel junction corresponded to a CRR of 1:1, relating to mild-moderate periodontitis. The coronal 30%-31% 2D RL and the 41%-42% 3D RSA bone loss corresponded to a CRR of 5:4, correlating to severe periodontitis. More severe clinical attachment loss (CAL) was observed in the 3D RSA measurement than in the 2D RL measurement at the evaluated CRRs. The amount of CAL at the CRR of 1:1 was inadequate to assess the severity of periodontitis on the basis of the 2D RL and 3D RSA measurements.

Decreased Amount of Supporting Alveolar Bone at Single-Rooted Premolars Is Under Estimated by 2D Examinations.

The purpose of this study was to relate the proportions of bone-supported root length of a 2D view into the amount of a 3D bone-attached root surface area (BA-RSA) by using a dental laser scanner examination. White-light 3D scanning technology was used to probe 36 maxillary and 35 mandibular single-rooted premolars. The bone-supported height (BSH) and BA-RSA at designated levels (95-25%) were compared using statistical t tests. The 100% BSH and BA-RSA of the maxillary/mandibular premolars were 12.6 ± 1.60 mm/13.45 ± 1.47 mm (p < 0.05) and 220.78 ± 35.31 mm2/199.51 ± 26.33 mm2 (p < 0.01), respectively. Approximately 79-80%, 59-60%, and 35-36% premolars 2D BSH remained in comparison to 75%, 50%, and 25% 3D BA-RSA preservation, respectively. However, corresponding to a 75%, 50%, and 25% 2D BSH reserve, premolars retained 67-68%, 39-41%, and 15-17% 3D BA-RSA, respectively. When taking 1.0 mm connective tissue attachment into account, 60% 3D BA-RSA and 50% 2D BSH loss were noted at the 5.1-5.4 mm clinical attachment level. Assigning a periodontal prognosis and determining the severity of periodontitis for premolars with alveolar bone loss based on 3D's or 2D's measurement is inconsistent.

Developing Stability of Posterior Mandibular Implants Placed With Osteotome Expansion Technique Compared With Conventional Drilling Techniques.

Clinical decisions regarding the stability and osseointegration of mandibular implants positioned using the bone expansion techniques are conflicting and limited. The objective was to evaluate the stability of implants placed using 2 surgical techniques, selected according to the initial width of the mandibular posterior edentulous ridge, with D3 bone density, during a 12-week period. Fifty-eight implants in 33 patients were evaluated. Thirty-two implants in 24 patients were positioned using the osteotome expansion technique, and 26 fixtures in 17 patients were installed using the conventional drilling technique. The implant stability quotient values were recorded at weeks 0, 1, 2, 3, 4, 6, 8, 10, and 12 postsurgery and evaluated using analysis of variance, independent, and paired t tests. Calibrated according to the stability reading of a 3.3-mm diameter implant, the osteotome expansion group was associated with a lower bone density than the conventional group (64.96 ± 6.25 vs 68.98 ± 5.06, P = .011). The osteotome expansion group achieved a comparable primary stability (ISQb-0, P = .124) and greater increases in secondary stability (ISQb-12, P = .07) than did the conventional technique. A D3 quality ridge with mild horizontal deficiency is expandable by using the osteotome expansion technique. Although the 2 groups presented similar implant stability quotient readings during the study period, the osteotome expansion technique showed significant improvement in secondary stability. The healing patterns for these techniques are therefore inconsistent.

Implant Stability Quotients of Osteotome Bone Expansion and Conventional Drilling Technique for 4.1 mm Diameter Implant at Posterior Mandible.

BACKGROUND: Clinical conclusions in studying the stability and osseointegration of mandibular implants positioned using the bone expansion techniques are conflicting and limited. PURPOSE: The objective was to examine the implant stability quotient (ISQ) values of mandibular posterior dental implants with 4.1 mm diameter that inserted with osteotome bone expansion technique versus conventional drilling technique during a 12-week observation period. MATERIALS AND METHODS: Twenty-four implants with 4.1mm diameter in 18 patients were included. Twelve implants in 10 patients were positioned using osteotome bone expansion technique, and 12 fixtures in 9 patients were installed using the conventional drilling technique. The ISQ values of a 3.3 mm diameter implant was measured at recipient sites (ISQb ) before final drilling or expansion technique to standardize the increased ISQ value of 4.1 mm diameter implants. The ISQ values at Weeks 0, 1, 2, 3, 4, 6, 8, 10, and 12 post-surgery were recorded. Data were analyzed by Wilcoxon rank sum test, repeated measure ANOVA, and Fisher Lest Significant Difference test. RESULTS: Calibrated according to a 3.3-mm-diameter implant, bone expansion technique was adopted for the sites with ISQ≦65 bone density, and the areas with ISQ >65 bone condition were treated with conventional drilling technique (p =.038). Both groups presented a similar healing pattern and a comparable ISQ reading from Week 0 to Week 12 (p > .05) for 4.1 mm diameter implants. However, bone expansion technique could enhance more stability when the ISQ values were calibrated by 3.3 mm diameter implant (p < .05). CONCLUSIONS: Bone expansion technique substantially increased more ISQ values from primary stability and achieved comparable primary and secondary stabilities with the conventional technique. Both groups reached a stability plateaus at Week 10.

Potential Osteoinductive Effects of Calcitriol on the m-RNA of Mesenchymal Stem Cells Derived from Human Alveolar Periosteum.

This study characterized alveolar periosteum-derived mesenchymal stem cells (P-MSCs) and examined the hypothesis that 1,25-(OH)2D3 (calcitriol) exerts osteoinductive effects on P-MSCs. The mRNA expressions of alkaline phosphatase (ALP), bone sialoprotein (BSP), core-binding factor alpha-1 (CBFA1), collagen-1 (Col-1), osteocalcin (OCN), and vitamin D3 receptor (VDR) were assessed after incubation with calcitriol for 2 weeks. Vitamin C as positive control (Vit. C-p) increased ALP and CBFA1 mRNA expression at both 1 and 2 weeks and increased BSP and Col-1 mRNA expression only at the first week. A concentration of 10-8 M calcitriol enhanced ALP, CBFA1, Col-1, and OCN mRNA expression at both weeks and BSP mRNA expression at the first week. Furthermore, 10-7 M calcitriol increased the mRNA expressions of all compounds at both weeks, except that of CBFA1 at the first week. 10-8 M calcitriol and Vit. C-p enhanced ALP activity at the second and third weeks. The results revealed that 10-9, 10-8, and 10-7 M calcitriol induced osteoinduction in alveolar P-MSCs by increasing ALP, CBFA1, Col-1, and OCN mRNA expression. A 10-7 M calcitriol yielded a higher mRNA expression than Vit. Cp on VDR and OCN mRNA expression at both weeks and on Col-1 mRNA at the second week.

Association of vitamin D3 with alveolar bone regeneration in dogs.

Designed sockets prepared on the mandibles of nine Beagle dogs were divided into three groups: Calcitriol +Alloplast, Alloplast and Empty. Five of the nine dogs received Vit.D3 and calcium supplement (Vit.D/Ca group), while the other four dogs without supplements were assigned to Non-Vit.D/Ca group. After 4 weeks, the extent of vertical ridge resorption (VRR), bone density (density), new bone formation (NBF) and implant stability quotient (ISQ) were measured. Following systemic Vit.D/Ca administration, the Empty subgroup showed significant differences from the Calcitriol + Alloplast subgroup on variants NBF/Density/VRR and the Alloplast subgroup on items NBF/Density/ISQ/VRR. Alternatively, the Calcitriol + Alloplast subgroup revealed higher values of NBF/Density/ISQ (P < 0.001) and a lower VRR value (P = 0.001) than the Alloplast subgroup. Although there were no significant differences in NBF (P = 0.349), density (P = 0.796), ISQ (P = 0.577) and VRR (0.979) comparisons on alloplast treatment between the Vit.D/Ca and Non-Vit.D/Ca groups, local application with Calcitriol + Alloplast demonstrated better NBF/Density/ISQ (P = 0.02 to <0.001) effects than which of Alloplast subgroups. Consequently, the results showed that both systemic and local vitamin D3 treatment might accelerate bone regeneration in dogs. Within the using dose, systemic vitamin D3 treatment displayed a superior stimulating effect than local vitamin D3 application did.