Stability, survival, patient satisfaction, and cost-minimization of CAD/CAM versus conventional multistranded fixed retainers in orthodontic patients: a 2-year follow-up of a two-centre randomized controlled trial.

BACKGROUND: CAD/CAM (computer-aided design/computer-aided manufacturing) fixed retainers (FRs) as an alternative to multistranded FRs to maintain orthodontic treatment outcome. OBJECTIVES: The primary aim was to compare CAD/CAM versus conventional multistranded FRs in terms of stability until 2 years. Secondary outcomes were failure rates, patient satisfaction, and cost-minimization. TRIAL DESIGN: 2-arm parallel, two-centre randomized controlled trial. METHODS: Patients were randomized to CAD/CAM or conventional FRs in both arches, in a 1:1 ratio and blocks of four. Allocation concealment was secured by using sequentially numbered envelopes. Patients were blinded. FRs were bonded at the end of treatment, and patients were recalled after 12 and 24 months. First-time retainer failures were recorded and digital impressions were taken. Arch widths and lengths, as well as Little's Irregularity Index (LII), were measured. Additionally, patients answered satisfaction questionnaires. Linear mixed models were applied for measurements and patient satisfaction. Survival analyses were estimated with Kaplan-Meier curves, along with Cox-regression modelling. Cost-minimization analysis was undertaken. RESULTS: One hundred and eighty-one patients were randomized (98 in Centre 1, and 83 in Centre 2): 90 in CAD/CAM and 91 in conventional group. One hundred and fifty three patients attended T24 follow-up. There were no significant differences in LII and arch dimensions between groups for failure-free patients. Within 24 months, 34% maxillary CAD/CAM FRs and 38% maxillary conventional FRs failed, along with 42% mandibular CAD/CAM FRs and 40% mandibular conventional FRs, with no significant difference in survival between groups (hazard ratios conventional to CAD/CAM: maxillary arch: 1.20 [P = 0.46], mandibular arch: 0.98 [P = 0.94]). There were no significant differences in patient satisfaction between groups. No harms were observed. Cost-minimization analysis showed that CAD/CAM FRs were slightly cheaper than conventional FRs. CONCLUSIONS: There were no clinically significant differences in LII, arch widths, and lengths between CAD/CAM and conventional FRs after 24 months. There were no differences in failures and patient satisfaction between groups. CAD/CAM FRs were slightly cheaper than conventional FRs. TRIAL REGISTRATION: ClinicalTrials.gov NCT04389879.

Elemental composition, corrosion resistance and mechanical properties of computer-aided design and computer-aided manufacturing fixed retainers versus conventional fixed retainers.

PURPOSE: To investigate the elemental composition, corrosion resistance, and mechanical properties of computer-aided design and computer-aided manufacturing (CAD-CAM) retainers versus conventional fixed retainers (FRs). METHODS: Eight different retainer wires were investigated. Energy dispersive X-ray spectroscopy was used to determine the elemental composition. Leakage was analysed according to ISO 10271:2020 guidelines. Hardness was tested using the Vickers method with a load of 0.3 kg. The tensile force and tensile strength were evaluated. Multiple comparisons among wires of hardness, tensile force, and strength were conducted using the Welch t-test, with Bonferroni correction. RESULTS: Nickel was present in all wires. The CAD-CAM-FR wire, which contained more nickel than the other wires, had no measurable leakage. The gold-plated wires had the highest total leakage, but did not exceed the ISO standard limit. The hardness of the stainless-steel twisted wires was the highest and that of the CAD-CAM-FR wire was the lowest. The tensile strength of the CAD-CAM-FR wire was significantly lower than that of the other wires and similar to the other twisted-wire retainers. CONCLUSION: The CAD-CAM-FR wire is likely to have high corrosion resistance and flexibility due to its low hardness.

Irisin reduces orthodontic tooth movement in rats by promoting the osteogenic potential in the periodontal ligament.

OBJECTIVES: Positive effects of irisin on osteogenic differentiation of periodontal ligament (PDL) cells have been identified previously, this study aims to examine its effect on orthodontic tooth movement (OTM) in vivo. MATERIALS AND METHODS: The maxillary right first molars of male Wistar rats (n = 21) were moved mesially for 14 days, with submucosal injection of two dosages of irisin (0.1 or 1 µg) or phosphate-buffered saline (control) every third day. OTM was recorded by feeler gauge and micro-computed tomography (µCT). Alveolar bone and root volume were analysed using µCT, and plasma irisin levels by ELISA. Histological characteristics of PDL tissues were examined, and the expression of collagen type I, periostin, osteocalcin (OCN), von Willebrand factor (vWF) and fibronectin type III domain-containing protein 5 (FNDC5) in PDL was evaluated by immunofluorescence staining. RESULTS: Repeated 1 µg irisin injections suppressed OTM on days 6, 9, and 12. No significant differences were observed in OTM in the 0.1 µg irisin group, or in bone morphometric parameters, root volume or plasma irisin, compared to control. Resorption lacunae and hyalinization were found at the PDL-bone interface on the compression side in the control, whereas they were scarce after irisin administration. The expression of collagen type I, periostin, OCN, vWF, and FNDC5 in PDL was enhanced by irisin administration. LIMITATIONS: The feeler gauge method may overestimate OTM. CONCLUSIONS: Submucosal irisin injection reduced OTM by enhancing osteogenic potential of PDL, and this effect was more significant on the compression side.

A single amino acid deletion in the ER Ca2+ sensor STIM1 reverses the in vitro and in vivo effects of the Stormorken syndrome-causing R304W mutation.

Stormorken syndrome is a multiorgan hereditary disease caused by dysfunction of the endoplasmic reticulum (ER) Ca2+ sensor protein STIM1, which forms the Ca2+ release-activated Ca2+ (CRAC) channel together with the plasma membrane channel Orai1. ER Ca2+ store depletion activates STIM1 by releasing the intramolecular "clamp" formed between the coiled coil 1 (CC1) and CC3 domains of the protein, enabling the C terminus to extend and interact with Orai1. The most frequently occurring mutation in patients with Stormorken syndrome is R304W, which destabilizes and extends the STIM1 C terminus independently of ER Ca2+ store depletion, causing constitutive binding to Orai1 and CRAC channel activation. We found that in cis deletion of one amino acid residue, Glu296 (which we called E296del) reversed the pathological effects of R304W. Homozygous Stim1 E296del+R304W mice were viable and phenotypically indistinguishable from wild-type mice. NMR spectroscopy, molecular dynamics simulations, and cellular experiments revealed that although the R304W mutation prevented CC1 from interacting with CC3, the additional deletion of Glu296 opposed this effect by enabling CC1-CC3 binding and restoring the CC domain interactions within STIM1 that are critical for proper CRAC channel function. Our results provide insight into the activation mechanism of STIM1 by clarifying the molecular basis of mutation-elicited protein dysfunction and pathophysiology.

Recombinant irisin enhances the extracellular matrix formation, remodeling potential, and differentiation of human periodontal ligament cells cultured in 3D.

BACKGROUND: Irisin is expressed in human periodontal ligament (hPDL), and its administration enhances growth, migration and matrix deposition in hPDL cells cultured in monolayers in vitro. OBJECTIVES: To identify whether irisin affects the gene expression patterns directing the morphology, mechanical properties, extracellular matrix (ECM) formation, osteogenic activity and angiogenic potential in hPDL cell spheroids cultured in 3D. MATERIALS AND METHODS: Spheroids of primary human hPDL cells were generated in a rotational 3D culture system and treated with or without irisin. The gene expression patterns were evaluated by Affymetrix microarrays. The morphology of the spheroids was characterized using histological staining. Mechanical properties were quantified by nanoindentation. The osteogenic and angiogenic potential of spheroids were assessed through immunofluorescence staining for collagen type I, periostin fibronectin and von Willebrand factor (vWF), and mRNA expression of osteogenic markers. The secretion of multiple myokines was evaluated using Luminex immunoassays. RESULTS: Approximately 1000 genes were differentially expressed between control and irisin-treated groups by Affymetrix. Several genes related to ECM organization were differentially expressed, and multiple deubiquitinating enzymes were upregulated in the irisin-exposed samples analyzed. These represent cellular and molecular mechanisms indicative of a role for irisin in tissue remodeling. Irisin induced a rim-like structure on the outer region of the hPDL spheroids, ECM-related protein expression and the stiffness of the spheroids were enhanced by irisin. The expression of osteogenic and angiogenetic markers was increased by irisin. CONCLUSIONS: Irisin altered the morphology in primary hPDL cell-derived spheroids, enhanced its ECM deposition, mechanical properties, differentiation and remodeling potential.

Stability, survival, and patient satisfaction with CAD/CAM versus conventional multistranded fixed retainers in orthodontic patients: a 6-month follow-up of a two-centre randomized controlled clinical trial.

OBJECTIVES: The primary aim of this two-arm parallel two-centre randomized controlled trial was to compare computer-aided design and computer-aided manufacturing (CAD/CAM) versus conventional multistranded fixed retainers (FRs) in terms of stability over 6 months. Secondary outcomes were failure rates and patient satisfaction. METHODS: Patients were randomized to CAD/CAM or conventional FRs in both arches, in 1:1 ratio and blocks of four. Allocation concealment was secured by using sequentially numbered envelopes. Patients were blinded. Retainers were bonded at the end of orthodontic treatment (T0), and patients were recalled after 1 (T1), 3 (T3), and 6 (T6) months. First-time retainer failures were recorded and digital impressions were taken. Arch widths and lengths, as well as Little's Irregularity Index (LII), were measured. Additionally, patients answered satisfaction questionnaires. Linear mixed models were applied for measurements and patient satisfaction. Survival analyses were estimated with Kaplan-Meier curves, along with Cox-regression modelling. RESULTS: One hundred and eighty-one patients were randomized (98 in Centre 1, and 83 in Centre 2): Ninety in the CAD/CAM group and 91 in the conventional group. Three subjects dropped out at baseline, as they did not attend any of the follow-up appointments.168 patients attended the T6 visit. There were no significant differences in arch dimensions between T0 and T6, whilst the LII was different only in the CAD/CAM group (mean difference: 0.2 mm; 95% confidence interval: 0.1 to 0.4; P < 0.001). Within 6 months, 39 upper retainers (19 out of 88 CAD/CAM and 20 out of 90 conventional retainers) and 52 lower retainers failed (26 out of 88 CAD/CAM and 26 out of 90 conventional retainers), with no significant difference between the survival of both types of retainers (hazard ratios conventional to CAD/CAM: upper arch: 0.99 [P =0.99], lower arch: 0.93 [P = 0.80]). There were no significant changes in patient satisfaction between the groups. No harms were observed. CONCLUSIONS: There were no clinically significant differences in LII, arch widths and lengths between CAD/CAM and conventional retainers after 6 months. There was no difference in failures and in patient satisfaction between both types of FRs. REGISTRATION: ClinicalTrials.gov NCT04389879.

FNDC5/irisin is expressed and regulated differently in human periodontal ligament cells, dental pulp stem cells and osteoblasts.

OBJECTIVE: To examine the expression and regulation of fibronectin type III domain-containing protein 5/irisin (FNDC5/irisin) in primary human periodontal ligament (hPDL) cells, dental pulp stem cells (hDPCs) and osteoblasts (hOBs). METHODS: FNDC5/irisin was identified in sections of paraffin embedded rat maxillae, cryo-sections of 3D cultured spheroids hPDL cells, hDPCs and hOBs, 2D cultured hPDL cells, hDPCs and hOBs by immunohistochemistry. The expression of FNDC5/irisin was identified by qPCR, followed by sequencing of the qPCR product. Regulation of FNDC5/irisin expression in hPDL cells, hDPCs and hOBs were evaluated after administration of different concentrations of irisin and all-trans retinoic acid (ATRA). qPCR and ELISA were used to identify expression and secretion of FNDC5/irisin in odontoblast-like differentiation of hDPCs. RESULTS: FNDC5/irisin was confirmed to be present in rat periodontium and dental pulp regions, as well as in 2D and 3D cultured hPDL cells, hDPCs and hOBs. BLAST analyses verified the generated nucleotide alignments matched human FNDC5/irisin. FNDC5/irisin gene expression was enhanced during odontoblast-like differentiation of hDPCs whereas the secretion of the protein was decreased compared to control. The protein signals in rat periodontal and pulpal tissues were higher than that of alveolar bone, and the expression of FNDC5/irisin was differently regulated by recombinant irisin and ATRA in hPDL cells and hDPCs compared to hOBs. CONCLUSIONS: FNDC5/irisin expression was verified in rodent periodontium and dental pulp, and in hPDL cells, hDPCs and hOBs. The FNDC5/irisin expression was regulated by recombinant irisin and ATRA. Finally, expression and secretion of FNDC5/irisin were affected during odontoblast-like differentiation of hDPCs.

Irisin promotes growth, migration and matrix formation in human periodontal ligament cells.

OBJECTIVE: The objective of the study was to examine the effect of irisin on human periodontal ligament cells (hPDLCs) growth, migration and osteogenic behaviour in vitro. MATERIALS AND METHODS: Primary hPDLCs and human osteoblasts (hOBs), used as positive controls, were cultured with irisin (10 and 100 ng/ml), and effect on cell proliferation was evaluated with 5-bromo-2`-deoxyuridine incorporation at 1, 2, and 3 days, and on migration capacity was investigated by scratch assay at 2, 6, and 24 h. Osteogenic behaviour was assessed with alkaline phosphatase activity, immunoassay at 3, 7, 14, and 21 days, and confocal laser scanning microscopy at 21 days. Mineralization was examined by Alizarin red staining at 21 days. Data were compared group wise using ANOVA tests. RESULTS: Irisin induced increased proliferation of primary hPDLCs and hOBs at all time points compared to untreated controls. This was confirmed by scratch assay where irisin enhanced migration of both hPDLCs and hOBs after 6 and 24 h compared to controls. Irisin treatment promoted osteogenic behaviour of both cell types by enhancement of extracellular matrix formation. In hPDLCs irisin increased expression of type I collagen, secretion of osteoblastogenesis related proteins osteocalcin and leptin, and calcium deposition/mineralization compared to controls at 21 days. In addition, to enhance calcium deposition/mineralization in hOBs, irisin increased expression of periostin, and secretion of osteoblastogenesis related proteins osteopontin, alkaline phosphatase and osteocalcin, as compared to controls at 21 days. CONCLUSIONS: Primary hPDLCs responded to irisin treatment with enhanced cell growth, migration, and matrix formation in vitro.

STIM1 R304W in mice causes subgingival hair growth and an increased fraction of trabecular bone.

Calcium signaling plays a central role in bone development and homeostasis. Store operated calcium entry (SOCE) is an important calcium influx pathway mediated by calcium release activated calcium (CRAC) channels in the plasma membrane. Stromal interaction molecule 1 (STIM1) is an endoplasmic reticulum calcium sensing protein important for SOCE. We generated a mouse model expressing the STIM1 R304W mutation, causing Stormorken syndrome in humans. Stim1R304W/R304W mice showed perinatal lethality, and the only three animals that survived into adulthood presented with reduced growth, low body weight, and thoracic kyphosis. Radiographs revealed a reduced number of ribs in the Stim1R304W/R304W mice. Microcomputed tomography data revealed decreased cortical bone thickness and increased trabecular bone volume fraction in Stim1R304W/R304W mice, which had thinner and more compact bone compared to wild type mice. The Stim1R304W/+ mice showed an intermediate phenotype. Histological analyses showed that the Stim1R304W/R304W mice had abnormal bone architecture, with markedly increased number of trabeculae and reduced bone marrow cavity. Homozygous mice showed STIM1 positive osteocytes and osteoblasts. These findings highlight the critical role of the gain-of-function (GoF) STIM1 R304W protein in skeletal development and homeostasis in mice. Furthermore, the novel feature of bilateral subgingival hair growth on the lower incisors in the Stim1R304W/R304W mice and 25 % of the heterozygous mice indicate that the GoF STIM1 R304W protein also induces an abnormal epithelial cell fate.

Strontium enhances proliferation and osteogenic behavior of periodontal ligament cells in vitro.

BACKGROUND AND OBJECTIVE: Strontium (Sr) enhances osteogenic differentiation of certain multipotent cells. Periodontal ligament cells (PDLCs) are known to be multipotent, and Sr might be useful in periodontal bone tissue engineering. This study investigates the effect of high concentration of Sr on the proliferation and osteogenic behavior of PDLCs in vitro. MATERIAL AND METHODS: Primary human PDLCs were cultured in MEM + 10% FBS without (Ctrl) or with Sr in four diverse concentrations: Sr1, 11.3 × 10-3  mg/L, human serum physiological level; Sr2, 13 mg/L, typical human serum level after strontium ranelate treatment; Sr3, 130 mg/L, and Sr4, 360 mg/L. The spreading area (2, 4, 6, 24 hours), proliferation rate (1, 3, 7 days), osteogenic behavior (alkaline phosphatase - ALP activity, 7 and 14 days; expression of osteogenic genes, ALP, Runt-related transcription factor 2 - RUNX2, osteopontin - OPN, osteocalcin - OCN, and osteoprotegerin -OPG, 1, 3, 7, 14, 21 days), and formation of mineralized nodules (14 and 21 days) of the PDLCs were assessed. Data were compared group- and period-wise using ANOVA tests. RESULTS: Periodontal ligament cells cultured with Sr4 showed increased spreading area (after 4 hours), proliferation rate (from 3 days), and OCN and OPN (from 7 days) gene expression as compared to Ctrl, Sr1, Sr2, and Sr3. Sr4 also led to lower ALP activity (from 7 days), ALP (from 3 days), and RUNX2 (at 7 and 14 days) gene expression, together with more evident formation of mineralized nodules, compared to Ctrl, Sr1, Sr2, and Sr3. CONCLUSION: Periodontal ligament cells responded to Sr4 with increased cellular proliferation and osteogenic behavior in vitro.

Alginate hydrogel enriched with enamel matrix derivative to target osteogenic cell differentiation in TiO2 scaffolds.

The purpose of bone tissue engineering is to employ scaffolds, cells, and growth factors to facilitate healing of bone defects. The aim of this study was to assess the viability and osteogenic differentiation of primary human osteoblasts and adipose tissue-derived mesenchymal stem cells from various donors on titanium dioxide (TiO2) scaffolds coated with an alginate hydrogel enriched with enamel matrix derivative. Cells were harvested for quantitative reverse transcription polymerase chain reaction on days 14 and 21, and medium was collected on days 2, 14, and 21 for protein analyses. Neither coating with alginate hydrogel nor alginate hydrogel enriched with enamel matrix derivative induced a cytotoxic response. Enamel matrix derivative-enriched alginate hydrogel significantly increased the expression of osteoblast markers COL1A1, TNFRSF11B, and BGLAP and secretion of osteopontin in human osteoblasts, whereas osteogenic differentiation of human adipose tissue-derived mesenchymal stem cells seemed unaffected by enamel matrix derivative. The alginate hydrogel coating procedure may have potential for local delivery of enamel matrix derivative and other stimulatory factors for use in bone tissue engineering.

Simvastatin coating of TiO2 scaffold induces osteogenic differentiation of human adipose tissue-derived mesenchymal stem cells.

Bone tissue engineering requires an osteoconductive scaffold, multipotent cells with regenerative capacity and bioactive molecules. In this study we investigated the osteogenic differentiation of human adipose tissue-derived mesenchymal stem cells (hAD-MSCs) on titanium dioxide (TiO2) scaffold coated with alginate hydrogel containing various concentrations of simvastatin (SIM). The mRNA expression of osteoblast-related genes such as collagen type I alpha 1 (COL1A1), alkaline phosphatase (ALPL), osteopontin (SPP1), osteocalcin (BGLAP) and vascular endothelial growth factor A (VEGFA) was enhanced in hAD-MSCs cultured on scaffolds with SIM in comparison to scaffolds without SIM. Furthermore, the secretion of osteoprotegerin (OPG), vascular endothelial growth factor A (VEGFA), osteopontin (OPN) and osteocalcin (OC) to the cell culture medium was higher from hAD-MSCs cultured on scaffolds with SIM compared to scaffolds without SIM. The TiO2 scaffold coated with alginate hydrogel containing SIM promote osteogenic differentiation of hAD-MSCs in vitro, and demonstrate feasibility as scaffold for hAD-MSC based bone tissue engineering.

Enhanced in vitro osteoblast differentiation on TiO2 scaffold coated with alginate hydrogel containing simvastatin.

The aim of this study was to develop a three-dimensional porous bone graft material as vehicle for simvastatin delivery and to investigate its effect on primary human osteoblasts from three donors. Highly porous titanium dioxide (TiO2) scaffolds were submerged into simvastatin containing alginate solution. Microstructure of scaffolds, visualized by scanning electron microscopy and micro-computed tomography, revealed an evenly distributed alginate layer covering the surface of TiO2 scaffold struts. Progressive and sustained simvastatin release was observed for up to 19 days. No cytotoxic effects on osteoblasts were observed by scaffolds with simvastatin when compared to scaffolds without simvastatin. Expression of osteoblast markers (collagen type I alpha 1, alkaline phosphatase, bone morphogenetic protein 2, osteoprotegerin, vascular endothelial growth factor A and osteocalcin) was quantified using real-time reverse transcriptase-polymerase chain reaction. Secretion of osteoprotegerin, vascular endothelial growth factor A and osteocalcin was analysed by multiplex immunoassay (Luminex). The relative expression and secretion of osteocalcin was significantly increased by cells cultured on scaffolds with 10 µM simvastatin when compared to scaffolds without simvastatin after 21 days. In addition, secretion of vascular endothelial growth factor A was significantly enhanced from cells cultured on scaffolds with both 10 nM and 10 µM simvastatin when compared to scaffolds without simvastatin at day 21. In conclusion, the results indicate that simvastatin-coated TiO2 scaffolds can support a sustained release of simvastatin and induce osteoblast differentiation. The combination of the physical properties of TiO2 scaffolds with the osteogenic effect of simvastatin may represent a new strategy for bone regeneration in defects where immediate load is wanted or unavailable.