Human bone chips release of sclerostin and FGF-23 into the culture medium: an in vitro pilot study

Abstract OBJECTIVE: Signaling molecules derived from osteocytes have been proposed as a mechanism by which autografts contribute to bone regeneration. However, there have been no studies that determined the role of osteocytes in bone grafts. MATERIAL AND METHOD: Herein, it was examined whether bone chips and demineralized bone matrix release sclerostin and FGF-23, both of which are highly expressed by osteocytes. RESULTS: Bone grafts from seven donors were placed in culture medium. Immunoassay showed that bone chips released sclerostin (median 1.0 ng/ml) and FGF-23 (median 9.8 relative units/ml) within the first day, with declining levels overtime. Demineralized bone matrix also released detectable amounts of sclerostin into culture medium, while FGF-23 remained close to the detection limit. In vitro expanded isolated bone cells failed to release detectable amounts of sclerostin and FGF-23. CONCLUSION: These results suggest that autografts but also demineralized bone matrix can release signaling molecules that are characteristically produced by osteocytes. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd. KEYWORDS: FGF-23; autologous bone; bone grafts; demineralized bone matrix; osteocytes; sclerostin

Cell-to-cell communication--periodontal regeneration.

BACKGROUND Although regenerative treatment options are available, periodontal regeneration is still regarded as insufficient and unpredictable. AIM This review article provides scientific background information on the animated 3D film Cell-to-Cell Communication - Periodontal Regeneration. RESULTS Periodontal regeneration is understood as a recapitulation of embryonic mechanisms. Therefore, a thorough understanding of cellular and molecular mechanisms regulating normal tooth root development is imperative to improve existing and develop new periodontal regenerative therapies. However, compared to tooth crown and earlier stages of tooth development, much less is known about the development of the tooth root. The formation of root cementum is considered the critical element in periodontal regeneration. Therefore, much research in recent years has focused on the origin and differentiation of cementoblasts. Evidence is accumulating that the Hertwig's epithelial root sheath (HERS) has a pivotal role in root formation and cementogenesis. Traditionally, ectomesenchymal cells in the dental follicle were thought to differentiate into cementoblasts. According to an alternative theory, however, cementoblasts originate from the HERS. What happens when the periodontal attachment system is traumatically compromised? Minor mechanical insults to the periodontium may spontaneously heal, and the tissues can structurally and functionally be restored. But what happens to the periodontium in case of periodontitis, an infectious disease, after periodontal treatment? A non-regenerative treatment of periodontitis normally results in periodontal repair (i.e., the formation of a long junctional epithelium) rather than regeneration. Thus, a regenerative treatment is indicated to restore the original architecture and function of the periodontium. Guided tissue regeneration or enamel matrix proteins are such regenerative therapies, but further improvement is required. As remnants of HERS persist as epithelial cell rests of Malassez in the periodontal ligament, these epithelial cells are regarded as a stem cell niche that can give rise to new cementoblasts. Enamel matrix proteins and members of the transforming growth factor beta (TGF-ß) superfamily have been implicated in cementoblast differentiation. CONCLUSION A better knowledge of cell-to-cell communication leading to cementoblast differentiation may be used to develop improved regenerative therapies to reconstitute periodontal tissues that were lost due to periodontitis.

Bone loss after ridge expansion with or without reflection of the periosteum.

OBJECTIVE To evaluate the role of the periosteum in preserving the buccal bone after ridge splitting and expansion with simultaneous implant placement. MATERIAL AND METHODS In 12 miniature pigs, the mandibular premolars and first molars were removed together with the interdental bone septa and the buccal bone. Three months later, ridge splitting and expansion of the buccal plate was performed with simultaneous placement of two titanium implants per quadrant. Access by a mucosal flap (MF) was prepared on test sides, while a mucoperiosteal flap (MPF) with complete denudation of the buccal bone was increased on control sides. After healing periods of six and 12 weeks, the animals were sacrificed for histologic and histometric evaluation. RESULTS In the MF group, all 16 implants were osseointegrated, while in the MPF group, four of 16 implants were lost. Noticeable differences of bone levels on the implant surface and of the bone crest (BC) were found between the MF and the MPF group. Buccally after 6 weeks, the median distance between the implant shoulder (IS) and the coronal-most bone on the implant (cBIC) was for the MF group -1.42 ± 0.42 mm and for the MPF group -4.80 ± 2.72 mm (P = 0.15). The median distance between the IS and the buccal BC was -1.24 ± 0.51 mm and -2.78 ± 1.98 mm (P = 0.12) for the MF and MPF group, respectively. After 12 weeks, median IS-cBIC was -2.12 ± 0.84 mm for MF and -7.19 mm for MPF, while IS-BC was -2.08 ± 0.79 mm for MF and -5.96 mm for MPF. After 6 weeks, the median buccal bone thickness for MF and MPF was 0.01 and 0 mm (P < 0.001) at IS, 1.48 ± 0.97 mm and 0 ± 0.77 mm (P = 0.07) at 2 mm apical to IS, and 2.12 ± 1.19 mm and 1.72 ± 01.50 mm (P = 0.86) at 4 mm apical to IS, respectively. After 12 weeks, buccal bone thickness in the MF group was 0 mm at IS, 0.21 mm at 2 mm apical to IS, and 2.56 mm at 4 mm apical to IS, whereas complete loss of buccal bone was measured from IS to 4 mm apical to IS for the MPF group. CONCLUSIONS In this ridge expansion model in miniature pigs, buccal bone volume was significantly better preserved when the periosteum remained attached to the bone.

Osteoclast-like cells on deproteinized bovine bone mineral and biphasic calcium phosphate: light and transmission electron microscopical observations.

OBJECTIVES The occurrence of multinucleated giant cells (MNGCs) on bone substitute materials has been recognized for a long time. However, there have been no studies linking material characteristics with morphology of the MNGCs. The aim was to analyze the qualitative differences of MNGCs on two commercially available calcium phosphate bone substitute materials retrieved from bone defects. MATERIAL AND METHODS Six defects were prepared bilaterally in the mandibular body of three mini pigs. The defects were randomly grafted with either deproteinized bovine bone mineral (DBBM) or biphasic calcium phosphate (BCP). After a healing period of four weeks, bone blocks were embedded in LR White resin. Three consecutive sections per defect were analyzed as follows: two with light microscopy using toluidine blue and tartrate-resistant acid phosphatase (TRAP) staining and one with transmission electron microscopy. RESULTS Multinucleated giant cells appeared on both biomaterials. On BCP, MNGCs had a flat morphology and were not observed in resorption lacunae. On DBBM, the MNGCs appeared more round and were often found in shallow concavities. MNGCs on both biomaterials demonstrated a varying degree of TRAP staining, with a tendency toward higher staining intensity of MNGCs on BCP. At the ultrastructural level, signs of superficial dissolution of BCP together with phagocytosis of minor fragments were observed. MNGCs on the surface of DBBM demonstrated sealing zones and ruffled borders, both features of mature osteoclasts. CONCLUSION MNGCs demonstrated distinctly different histological features depending on the bone substitute material used. Further research is warranted to understand the clinical implications of these morphological observations.

Dimethyloxalylglycine lyophilized onto bone substitutes increase vessel area in rat calvarial defects.

AIM Pharmacological inhibitors of prolyl hydroxylases, also termed hypoxia-mimetic agents (HMAs), when repeatedly injected can support angiogenesis and bone regeneration. However, the possible role of HMA loaded onto bone substitutes to support angiogenesis and bone regeneration under diabetic condition is unknown. The capacity of HMA loaded onto deproteinized bovine bone mineral (DBBM) to support angiogenesis and bone formation was examined in diabetic Wistar rats. METHODS Diabetes was induced by intraperitoneal injection of streptozotocin. The HMA dimethyloxalylglycine (DMOG) and desferrioxamine (DFO) were lyophilized onto DBBM. Calvarial defects were created with a trephine drill and filled with the respective bone substitutes. After 4 weeks of healing, the animals were subjected to histological and histomorphometric analysis. RESULTS In this report, we provide evidence that DMOG loaded onto DBBM can support angiogenesis in vivo. Specifically, we show that DMOG increased the vessel area in the defect site to 2.4% ± 1.3% compared with controls 1.1% ± 0.48% (P = 0.012). There was a trend toward an increased vessel number in the defect site with 38.6 ± 17.4 and 31.0 ± 10.3 in the DMOG and the control group (P = 0.231). The increase in angiogenesis, however, did not translate into enhanced bone formation in the defect area with 9.2% ± 7.1% and 8.4% ± 5.6% in DMOG and control group, respectively. No significant changes were caused by DFO. CONCLUSIONS The results suggest that DMOG loaded onto DBBM can support angiogenesis, but bone formation does not increase accordingly in a type 1 diabetic rat calvarial defect model at the indicated time point.

In vitro adhesion of fibroblastic cells to titanium alloy discs treated with sodium hydroxide.

OBJECTIVE Adhesion of osteogenic cells on titanium surfaces is a prerequisite for osseointegration. Alkali treatment can increase the hydrophilicity of titanium implant surfaces, thereby supporting the adhesion of blood components. However, it is unclear if alkali treatment also supports the adhesion of cells with a fibroblastic morphology to titanium. MATERIALS AND METHODS Here, we have used a titanium alloy (Ti-6AL-4V) processed by alkali treatment to demonstrate the impact of hydrophilicity on the adhesion of primary human gingival fibroblast and bone cells. Also included were the osteosarcoma and fibroblastoma cell lines, MG63 and L929, respectively. Cell adhesion was determined by scanning electron microscopy. We also measured viability, proliferation, and protein synthesis of the adherent cells. RESULTS Alkali treatment increased the adhesion of gingival fibroblasts, bone cells, and the two cell lines when seeded onto the titanium alloy surface for 1 h. At 3 h, no significant changes in cell adhesion were observed. Cells grown for 1 day on the titanium alloy surfaces processed by alkali treatment behave similarly to untreated controls with regard to viability, proliferation, and protein synthesis. CONCLUSION Based on these preliminary In vitro findings, we conclude that alkali treatment can support the early adhesion of cells with fibroblastic characteristics to a titanium alloy surface.

Peri-implant soft tissue colour around titanium and zirconia abutments: a prospective randomized controlled clinical study

OBJECTIVES To objectively determine the difference in colour between the peri-implant soft tissue at titanium and zirconia abutments. MATERIALS AND METHODS Eleven patients, each with two contralaterally inserted osteointegrated dental implants, were included in this study. The implants were restored either with titanium abutments and porcelain-fused-to-metal crowns, or with zirconia abutments and ceramic crowns. Prior and after crown cementation, multi-spectral images of the peri-implant soft tissues and the gingiva of the neighbouring teeth were taken with a colorimeter. The colour parameters L*, a*, b*, c* and the colour differences ΔE were calculated. Descriptive statistics, including non-parametric tests and correlation coefficients, were used for statistical analyses of the data. RESULTS Compared to the gingiva of the neighbouring teeth, the peri-implant soft tissue around titanium and zirconia (test group), showed distinguishable ΔE both before and after crown cementation. Colour differences around titanium were statistically significant different (P = 0.01) only at 1 mm prior to crown cementation compared to zirconia. Compared to the gingiva of the neighbouring teeth, statistically significant (P < 0.01) differences were found for all colour parameter, either before or after crown cementation for both abutments; more significant differences were registered for titanium abutments. Tissue thickness correlated positively with c*-values for titanium at 1 mm and 2 mm from the gingival margin. CONCLUSIONS Within their limits, the present data indicate that: (i) The peri-implant soft tissue around titanium and zirconia showed colour differences when compared to the soft tissue around natural teeth, and (ii) the peri-implant soft tissue around zirconia demonstrated a better colour match to the soft tissue at natural teeth than titanium.

Conditioned medium from fresh and demineralized bone enhances osteoclastogenesis in murine bone marrow cultures

OBJECTIVES Osteoclasts rapidly form on the surface of bone chips at augmentation sites. The underlying molecular mechanism, however, is unclear. Soluble factors released from bone chips in vitro have a robust impact on mesenchymal cell differentiation. Whether these soluble factors change the differentiation of hematopoietic cells into osteoclasts remains unknown. METHODS Osteoclastogenesis, the formation of tartrate-resistant acid phosphatase-positive multinucleated cells, was studied with murine bone marrow cultures exposed to RANKL and M-CSF, and conditioned medium from fresh (BCM) and demineralized bone matrix (DCM). Histochemical staining, gene and protein expression, as well as viability assays were performed. RESULTS This study shows that BCM had no impact on osteoclastogenesis. However, when BCM was heated to 85°C (BCMh), the number of tartrate-resistant acid phosphatase-positive multinucleated cells that developed in the presence of RANKL and M-CSF approximately doubled. In line with the histochemical observations, there was a trend that BCMh increased expression of osteoclast marker genes, in particular the transcription factor c-fos. The expression of c-fos was significantly reduced by the TGF-β receptor I antagonist SB431542. DCM significantly stimulated osteoclastogenesis, independent of thermal processing. CONCLUSIONS These data demonstrate that activated BCM by heat and DBM are able to stimulate osteoclastogenesis in vitro. These in vitro results support the notion that the resorption of autografts may be supported by as yet less defined paracrine mechanisms.

An Assessment of the Risk of Bias in Randomized Controlled Trial Reports Published in Prosthodontic and Implant Dentistry Journals

PURPOSE The objective of this study was to assess the risk of bias of randomized controlled trials (RCTs) published in prosthodontic and implant dentistry journals. MATERIALS AND METHODS The last 30 issues of 9 journals in the field of prosthodontic and implant dentistry (Clinical Implant Dentistry and Related Research, Clinical Oral Implants Research, Implant Dentistry, International Journal of Oral & Maxillofacial Implants, International Journal of Periodontics and Restorative Dentistry, International Journal of Prosthodontics, Journal of Dentistry, Journal of Oral Rehabilitation, and Journal of Prosthetic Dentistry) were hand-searched for RCTs. Risk of bias was assessed using the Cochrane Collaboration's risk of bias tool and analyzed descriptively. RESULTS From the 3,667 articles screened, a total of 147 RCTs were identified and included. The number of published RCTs increased with time. The overall distribution of a high risk of bias assessment varied across the domains of the Cochrane risk of bias tool: 8% for random sequence generation, 18% for allocation concealment, 41% for masking, 47% for blinding of outcome assessment, 7% for incomplete outcome data, 12% for selective reporting, and 41% for other biases. CONCLUSION The distribution of high risk of bias for RCTs published in the selected prosthodontic and implant dentistry journals varied among journals and ranged from 8% to 47%, which can be considered as substantial.

Reporting quality of randomised controlled trials published in prosthodontic and implantology journals

The purpose of this study was to examine the reporting quality of randomised controlled trials (RCTs) published in prosthodontic and implantology journals. Thirty issues of nine journals in prosthodontics and implant dentistry were searched for RCTs, covering the years 2005-2012: The Journal of Prosthetic Dentistry, Journal of Oral Rehabilitation, The International Journal of Prosthodontics, The International Journal of Periodontics & Restorative Dentistry, Clinical Oral Implants Research, Clinical Implant Dentistry & Related Research, The International Journal of Oral & Maxillofacial Implants, Implant Dentistry and Journal of Dentistry. The reporting quality was assessed using a modified Consolidated Standards of Reporting Trials (CONSORT) statement checklist. Data were analysed using descriptive statistics followed by univariable and multivariable examination of statistical associations (α = 0·05). A total of 147 RCTs were identified with a mean CONSORT score of 69·4 (s.d. = 9·7). Significant differences were found among journals with the Journal of Oral Rehabilitation achieving the highest score (80·6, s.d. = 5·5) followed by Clinical Oral Implants Research (73·7, s.d. = 8·3). Involvement of a statistician/methodologist was significantly associated with increased CONSORT scores. Overall, the reporting quality of RCTs in major prosthodontic and implantology journals requires improvement. This is of paramount importance considering that optimal reporting of RCTs is an important prerequisite for clinical decision-making.

Digital vs. conventional implant prosthetic workflows: a cost/time analysis.

OBJECTIVES The aim of this prospective cohort trial was to perform a cost/time analysis for implant-supported single-unit reconstructions in the digital workflow compared to the conventional pathway. MATERIALS AND METHODS A total of 20 patients were included for rehabilitation with 2 × 20 implant crowns in a crossover study design and treated consecutively each with customized titanium abutments plus CAD/CAM-zirconia-suprastructures (test: digital) and with standardized titanium abutments plus PFM-crowns (control conventional). Starting with prosthetic treatment, analysis was estimated for clinical and laboratory work steps including measure of costs in Swiss Francs (CHF), productivity rates and cost minimization for first-line therapy. Statistical calculations were performed with Wilcoxon signed-rank test. RESULTS Both protocols worked successfully for all test and control reconstructions. Direct treatment costs were significantly lower for the digital workflow 1815.35 CHF compared to the conventional pathway 2119.65 CHF [P = 0.0004]. For subprocess evaluation, total laboratory costs were calculated as 941.95 CHF for the test group and 1245.65 CHF for the control group, respectively [P = 0.003]. The clinical dental productivity rate amounted to 29.64 CHF/min (digital) and 24.37 CHF/min (conventional) [P = 0.002]. Overall, cost minimization analysis exhibited an 18% cost reduction within the digital process. CONCLUSION The digital workflow was more efficient than the established conventional pathway for implant-supported crowns in this investigation.

The virtual patient in dental medicine

OBJECTIVES The aim of this Short Communication was to present a workflow for the superimposition of intraoral scan (IOS), cone-beam computed tomography (CBCT), and extraoral face scan (EOS) creating a 3D virtual dental patient. MATERIAL AND METHODS As a proof-of-principle, full arch IOS, preoperative CBCT, and mimic EOS were taken and superimposed to a unique 3D data pool. The connecting link between the different files was to detect existing teeth as constant landmarks in all three data sets. RESULTS This novel application technique successfully demonstrated the feasibility of building a craniofacial virtual model by image fusion of IOS, CBCT, and EOS under 3D static conditions. CONCLUSIONS The presented application is the first approach that realized the fusion of intraoral and facial surfaces combined with skeletal anatomy imaging. This novel 3D superimposition technique allowed the simulation of treatment planning, the exploration of the patients' expectations, and the implementation as an effective communication tool. The next step will be the development of a real-time 4D virtual patient in motion.

CAD/CAM fabrication accuracy of long- vs. short-span implant-supported FDPs

OBJECTIVE To compare the precision of fit of long-span vs. short-span implant-supported screw-retained fixed dental prostheses (FDPs) made from computer-aided-design/computer-aided-manufactured (CAD/CAM) titanium and veneered with ceramic. The null hypothesis was that there is no difference in the vertical microgap between long-span and short-span FDPs. MATERIALS AND METHODS CAD/CAM titanium frameworks for an implant-supported maxillary FDP on implants with a flat platform were fabricated on one single master cast. Group A consisted of six 10-unit FDPs connected to six implants (FDI positions 15, 13, 11, 21, 23, 25) and group B of six 5-unit FDPs (three implants, FDI positions 21, 23, 25). The CAD/CAM system from Biodenta Swiss AG (Berneck, Switzerland) was used for digitizing (laser scanner) the master cast and anatomical CAD of each framework separately. The frameworks were milled (CAM) from a titanium grade V monobloc and veneered with porcelain. Median vertical distance between implant and FDP platforms from the non-tightened implants (one-screw test on implant 25) was calculated from mesial, buccal, and distal scanning electron microscope measurements. RESULTS All measurements showed values <40 μm. Total median vertical microgaps were 23 μm (range 2-38 μm) for group A and 7 μm (4-24 μm) for group B. The difference between the groups was statistically significant at implant 21 (P = 0.002; 97.5% CI -27.3 to -4.9) and insignificant at implant 23 (P = 0.093; -3.9 to 1.0). CONCLUSIONS CAD/CAM fabrication including laboratory scanning and porcelain firing was highly precise and reproducible for all long- and short-span FDPs. While all FDPs showed clinically acceptable values, the short-span FDPs were statistically more precise at the 5-unit span distance.

A systematic review on the innervation of peri-implant tissues with special emphasis on the influence of implant placement and loading protocols

OBJECTIVES To systematically review the available literature on the influence of dental implant placement and loading protocols on peri-implant innervation. MATERIAL AND METHODS The database MEDLINE, Cochrane, EMBASE, Web of Science, LILACS, OpenGrey and hand searching were used to identify the studies published up to July 2013, with a populations, exposures and outcomes (PEO) search strategy using MeSH keywords, focusing on the question: Is there, and if so, what is the effect of time between tooth extraction and implant placement or implant loading on neural fibre content in the peri-implant hard and soft tissues? RESULTS Of 683 titles retrieved based on the standardized search strategy, only 10 articles fulfilled the inclusion criteria, five evaluating the innervation of peri-implant epithelium, five elucidating the sensory function in peri-implant bone. Three included studies were considered having a methodology of medium quality and the rest were at low quality. All those papers reported a sensory innervation around osseointegrated implants, either in the bone-implant interface or peri-implant epithelium, which expressed a particular innervation pattern. Compared to unloaded implants or extraction sites without implantation, a significant higher density of nerve fibres around loaded dental implants was confirmed. CONCLUSIONS To date, the published literature describes peri-implant innervation with a distinct pattern in hard and soft tissues. Implant loading seems to increase the density of nerve fibres in peri-implant tissues, with insufficient evidence to distinguish between the innervation patterns following immediate and delayed implant placement and loading protocols. Variability in study design and loading protocols across the literature and a high risk of bias in the studies included may contribute to this inconsistency, revealing the need for more uniformity in reporting, randomized controlled trials, longer observation periods and standardization of protocols.

Immediate implant placement with simultaneous guided bone regeneration in the esthetic zone: 10-year clinical and radiographic outcomes.

AIM To associate the dimension of the facial bone wall with clinical, radiological, and patient-centered outcomes at least 10 years after immediate implant placement with simultaneous guided bone regeneration in a retrospective study. MATERIAL AND METHODS Primary endpoint was the distance from the implant shoulder (IS) to the first bone-to-implant contact (IS-BIC10y ). Secondary endpoints included the facial bone thickness (BT10y ) 2, 4, and 6 mm apical to the IS, and the implant position. At baseline, the horizontal defect width (HDWBL ) from the implant surface to the alveolar wall was recorded. At recall, distance from the IS to the mucosal margin (IS-MM10y ), degree of soft tissue coverage of the mesial and distal aspects of the implants (PISm10y , PISd10y ; Papilla Index), pocket probing depth (PPD10y ), and patient-centered outcomes were determined. Width of the keratinized mucosa (KM), Full-Mouth Plaque and Bleeding Score (FMPS, FMBS) were available for both time points. RESULTS Of the 20 patients who underwent immediate implant placement with simultaneous guided bone regeneration and transmucosal healing, nine males and eight females with a median age of 62 years (42 min, 84 max) were followed up for a median period of 10.5 y (min 10.1 max 11.5). The 10-year implant survival rate was 100%. Multivariate regression analysis revealed a correlation of the IS-BIC10y , controlled for age and gender, with four parameters: HDWBL (P = 0.03), KMBL -10 (P = 0.02), BT10 4 mm (P = 0.01), and BT10 6 mm (P = 0.01). CONCLUSION Within the conditions of the present study, the horizontal defect width was the main indicator for the vertical dimension of the facial bone. The facial bone dimension was further associated with a reduction in the width of the keratinized mucosa and the dimension of the buccal bone.