Introduction: Common in vitro cell culture systems for testing implant material immune compatibility either rely on immortal human leukocyte cell lines or isolated primary cells. Compared to in vivo conditions, this generates an environment of substantially reduced complexity, often lacking important immune cell types, such as neutrophil granulocytes and others. The aim of this study was to establish a reliable test system for in vitro testing of implant materials under in vivo-like conditions. Methods: Test materials were incubated in closed, CO2-independent, tube-based culture vessels containing a proprietary cell culture medium and human whole blood in either a static or occasionally rotating system. Multiplex cytokine analysis was used to analyze immune cell reactions. Results: To demonstrate the applicability of the test system to implant materials, three commercially available barrier membranes (polytetrafluoroethylene (PTFE), polycaprolactone (PCL) and collagen) used for dental, trauma and maxillofacial surgery, were investigated for their potential interactions with immune cells. The results showed characteristic differences between the static and rotated incubation methods and in the overall activity profiles with very low immune cell responses to PTFE, intermediate ones to collagen and strong reactions to PCL. Conclusion: This in vitro human whole blood model, using a complex organotypic matrix, is an excellent, easily standardized tool for categorizing immune cell responses to implant materials. Compared to in vitro cell culture systems used for materials research, this new assay system provides a far more detailed picture of response patterns the immune system can develop when interacting with different types of materials and surfaces.

Guided bone regeneration (GBR) is a widely used procedure that prevents the fast in-growth of soft tissues into bone defect. Among the different types of membranes, the use of collagen membranes is the gold standard. However, these membranes are implanted in tissue location where a severe acute inflammation will occur and can be negatively affected. The aim of this study was to develop a collagen-based membrane for GBR that incorporated alginate-hydroxyapatite microparticles. Membranes were manufactured using collagen type I and gelatin and alginate-hydroxyapatite microparticles. Membranes were assessed in terms of topography by scanning electron microscopy and confocal microscopy; stability by swelling after an overnight incubation in saline and enzymatic degradation against collagenase and mechanical properties by tensile tests. Furthermore, the biological response was assessed with SaOs-2 cells and THP-1 macrophages to determine alkaline phosphatase activity and inflammatory cytokine release. Our results showed that the incorporation of different percentages of these microparticles could induce changes in the surface topography. When the biological response was analyzed, either membranes were not cytotoxic to THP-1 macrophages or to SaOs-2 cells and they did not induce the release of pro-inflammatory cytokines. However, the different surface topographies did not induce changes in the macrophage morphology and the release of pro- and anti-inflammatory cytokines, suggesting that the effect of surface roughness on macrophage behavior could be dependent on other factors such as substrate stiffness and composition. Collagen-gelatin membranes with embedded alginate-hydroxyapatite microparticles increased ALP activity, suggesting a positive effect of them on bone regeneration, remaining unaffected the release of pro- and anti-inflammatory cytokines.

Meshes or reinforcement for membranes for guided bone regeneration (GBR) are currently made of titanium with high specific mechanical strength, but it is not resorbable and second surgeries are needed. In this project, it was hypothesized that titanium can be replaced by polycaprolactone (PCL), a long-term biodegradable polymer, while applying a collagen coating to provide a cell barrier effect required in GBR. Different meshes with different patterns were designed and manufactured using 3D printing. Mechanical tests of the meshes were also performed to assess pattern influence on the mechanical properties and, later, a finite element model (FEM) was set to further understand the mechanical behavior of these meshes. In order to obtain the cell barrier effect, meshes were coated with collagen previously treated with NaOH solution using different molarities (1 and 5 M), assuring a uniform and adhesive collagen coating. The results showed that the patterns influenced the mechanical properties, and the model was validated with the experimental data. Contact angle and SEM analysis confirmed the extension of the alkaline treatment to enhance PCL meshes wettability, needed for the collagen coating. Meshes treated with 5 M NaOH for 24 hours at 37 degrees C or room temperature achieved an adequate collagen adhesion that did not exhibit any toxic effect and was able to provide a fibroblast barrier up to 7 days. Finally, it was confirmed that thanks to the application of heat, the meshes can be shaped as required.

Background Common in vitro cell culture systems for testing implant material immune-compatibility either employ immortal human leukocyte cell lines or use isolated primary cells. Compared to in vivo conditions, this generates an environment of substantially reduced complexity, often lacking important immune cell types, such as neutrophil granulocytes and others. This paper describes an innovative human whole blood culture model for in vitro testing of implant materials under in vivo -like conditions. The major goal of this culture model was to maintain as much of the naturally inherent complexity of immune cell interactions as possible and to avoid errors often caused by stressful conditions during cell preparation. Methods A closed, CO 2 -independent, tube-based culture vessel was used, containing one milliliter of freshly drawn human blood for each sample. The cultures were occasionally rotated to increase immune cell contacts with the test materials. Immune cell responses were examined by multiplexed cytokine analysis. Results Three different types of commercially available implant materials i.e. barrier membranes, used for dental, trauma and maxillofacial surgery, were examined for their potential interactions with immune cells. The barrier membranes were either of synthetic (i.e. the polymers polytetrafluoroethylene, PTFE, and polycaprolactone, PCL), or of natural origin (porcine collagen membrane). The results identified characteristic differences in the overall activity profiles with very low immune cell responses for PTFE, intermediate ones for collagen, and strong reactions towards PCL. Conclusions This innovative human whole blood in vitro model, using a complex, organotypic matrix and all immune cells available in peripheral blood, is an excellent, easy to standardize tool to categorize immune cell responses to implant materials. Compared to in vitro cell culture systems used for material research, this new assay system provides a far more detailed picture of response patterns the immune system is able to develop when interacting with different types of materials and surfaces.

Capacitive–resistive energy transfer therapy (CRet) is used to improve the rehabilitation of different injuries. This study aimed to evaluate and compare the changes in temperature and current flow during different CRet applications on upper and lower molars and incisors, with and without implants, on ten cryopreserved corpses. Temperatures were taken on molars and incisors with invasive devices and skin temperature was taken with a digital thermometer at the beginning and after treatments. Four interventions: 15 VA capacitive hypothermic (CAPH), 8 watts resistive (RES8), 20 watts resistive (RES20) and 75 VA capacitive (CAP75) were performed for 5 min each. All treatments in this study generated current flow (more than 0.00005 A/m 2 ) and did not generate a significant temperature increase (p > 0.05). However, RES20 application slightly increased surface temperature on incisors without implants (p = 0.010), and molar with (p = 0.001) and without implant (p = 0.008). Also, CAP75 application increased surface temperature on molars with implant (p = 0.002) and upper incisor with implant (p = 0.001). In conclusion, RES8 and CAPH applications seem to be the best options to achieve current flow without an increase in temperature on molars and incisors with and without implants.

Collagen-based scaffolds hold great potential for tissue engineering, since they closely mimic the extracellular matrix. We investigated tissue integration of an engineered porous collagen-elastin scaffold developed for soft tissue augmentation. After implantation in maxillary submucosal pouches in 6 canines, cell invasion (vimentin), extracellular matrix deposition (collagen type I) and scaffold degradation (cathepsin k, tartrate-resistant acid phosphatase (TRAP), CD86) were (immuno)-histochemically evaluated. Invasion of vimentin(+) cells (scattered and blood vessels) and collagen type I deposition within the pores started at 7 days. At 15 and 30 days, vimentin(+) cells were still numerous and collagen type I increasingly filled the pores. Scaffold degradation was characterized by collagen loss mainly occurring around 15 days, a time point when medium-sized multinucleated cells peaked at the scaffold margin with simultaneous labeling for cathepsin k, TRAP, and CD86. Elastin was more resistant to degradation and persisted up to 90 days in form of packages well-integrated in the newly formed soft connective tissue. In conclusion, this collagen-based scaffold maintained long-enough volume stability to allow an influx of blood vessels and vimentin(+) fibroblasts producing collagen type I, that filled the scaffold pores before major biomaterial degradation and collapse occurred. Cathepsin k, TRAP and CD86 appear to be involved in scaffold degradation.

A short inflammatory phase and fast ingrowth of blood vessels and mesenchymal cells are essential for tissue integration of a biomaterial. Macrophages play a key role in this process. We investigated invasion of macrophages, blood vessels, and proliferating cells into a highly porous and volume-stable collagen matrix (VCMX) used for soft tissue augmentation around teeth and dental implants. The biomaterial was implanted in submucosal pouches in the canine maxilla, and the tissue response was analyzed at six different time points. Immunohistochemistry was done for proliferating cells (PCNA), macrophages (MAC387), multinucleated giant cells (CD86), and blood vessels (TGM2). Blood rapidly filled the VCMX pores. During the first week, MAC387+ cells populated the VCMX pores, blood vessels and PCNA+ cells invaded the VCMX, and CD86+ scattered cells were observed. At 15 days, MAC387+ cells were scanty, blood vessels had completely invaded the VCMX, the number of proliferating cells peaked, and fibroblasts appeared. At 30 days, MAC387+ were absent, the numbers of proliferating and CD86+ cells had declined, while blood vessel and fibroblast numbers were high. At 90 days, residual VCMX was well-integrated in soft connective tissue. In conclusion, the VCMX elicited a short inflammatory phase followed by rapid tissue integration.

Background Guided Bone Regeneration (GBR) techniques are nowadays essential procedures in implant dentistry where barrier membranes are crucial to avoid soft tissue ingrowth. Not all membranes behave in the same way, as they differ in origin and structure. It is therefore important to understand membranes behavior and properties in order to select the needed biomaterial for each possible situation. Aim/Hypothesis To date no study has evaluated and compared physico-chemical properties of various families of barrier membranes. The aim of this study was to evaluate the physico-chemical properties of various barrier membranes. Material and Methods Five different origin membranes were tested for this study. Porcine Peritoneum (BioGide, (Geistlich Pharma AG, Wolhusen, Switzerland)), Bovine Cross-linked Collagen (Cytoplast RTX, (Osteogenics Biomedical, Lubbock, TX, USA), Porcine Cross-linked Collagen (Ossix Plus, (Datum Dental, Israel)), Bovine Collagen (Implosorb, (Bioimplon GmbH, Gieflen)), Bovine Collagen + Hyaluronic Acid (Imploflex, (Bioimplon GmbH, Gieflen)) and Polycaprolactone (Osteoguide, (Genoss, Korea)). All membranes were tested in terms of tension, stiffness, absorption ability, pH and wettability. All data were translated into radial graphics showing the differences and strongest and lowest potential for each membrane. A physico-chemical index was then calculated. Results All membranes showed similar low tension and little stiffness. Porcine origin membranes had greater wettability and hydration, whereas bovine origin barrier membranes had a lower hydration and wettability, especially if mixed with hyaluronic acid. Synthetic membranes had a very stable pH and were very little stiff, which can be of importance in some GBR techniques. Conclusion and Clinical Implications The wide variety of barrier membranes opens a debate in which the practitioner should select the ideal barrier membrane for each clinical situation. Different materials show singular potentials depending on their tissue origin. More studies regarding adsorption, integration and degradation of barrier membranes are needed in order to understand their behavior.

Purpose: The aim of this retrospective study was to determine the prevalence of early implant failure using a single implant system and to identify the factors contributing to early implant failure. Methods: Patients who received implant treatment with a single implant system (Luna, Shinhung, Seoul, Korea) at Dankook University Dental Hospital from 2015 to 2017 were enrolled. The following data were collected for analysis: sex and age of the patient, seniority of the surgeon, diameter and length of the implant, position in the dental arch, access approach for sinus-floor elevation, and type of guided bone regeneration (GBR) procedure. The effect of each predictor was evaluated using the crude hazard ratio and the adjusted hazard ratio (aHR) in univariate and multivariate Cox regression analyses, respectively. Results: This study analyzed 1,031 implants in 409 patients, who comprised 169 females and 240 males with a median age of 54 years (interquartile range [IQR], 47-61 years) and were followed up fora median of 7.2 months (IQR, 5.6-9.9 months) after implant placement. Thirty-five implants were removed prior to final prosthesis delivery, and the cumulative survival rate in the early phase at the implant level was 95.6%. Multivariate regression analysis revealed that seniority of the surgeon (residents: aHR=2.86; 95% confidence interval [CI], 1.37-5.94) and the jaw in which the implant was placed (mandible: aHR=2.31; 95% CI, 1.12-4.76) exerted statistically significant effects on early implant failure after adjusting for sex, age, dimensions of the implant, and type of GBR procedure (preoperative and/or simultaneous) (p< 0.05). Conclusions: Prospective studies are warranted to further elucidate the factors contributing to early implant failure. In the meantime, surgeons should receive appropriate training and carefully select the bone bed in order to minimize the risk of early implant failure.

OBJECTIVE:Polyetheretherketone (PEEK) is a popular synthetic thermoplastic polymer for medical applications, but its clinical use suffers from several limitations. Therefore, the aim was to compare the soft tissue response to dental implant closure caps made of PEEK or titanium as evaluated by the occurrence of multinucleated giant cells (MNGCs).MATERIAL AND METHODS:Forty-two implants were placed in the maxilla of seven miniature pigs. While commercially pure titanium (Ti) implants had a Ti closure cap, ceramic implants made of either zirconia (Zr) or alumina-toughened zirconia (Zr + Al) received a PEEK closure cap. Histomorphometry was performed to evaluate the number of small and large MNGCs being in contact with the PEEK or the Ti in different compartments of the implant systems.RESULTS:No histological signs of inflammation were noticed, and MNGCs were observed on both PEEK and Ti closure caps and on all three implant types. Significantly higher numbers of MNGCs were found on closure caps made of PEEK than on closure caps made of Ti on the external closure cap surface facing both soft (p = 0.0008 for PEEK on Zr and p = 0.0016 for PEEK on Zr + Al) and hard tissues (p = 0.016 for PEEK on Zr and p = 0.003 for PEEK on Zr + Al) as well as in the internal closure cap surface (p = 0.014 for PEEK on Zr and p = 0.0088 for PEEK on Zr + Al). No statistically significant differences in the number of MNGCs were observed on the three implant types.CONCLUSIONS:Significantly more MNGCs were in contact with PEEK than with Ti closure caps.

OBJECTIVES:Guided bone / tissue regeneration (GBR/GTR) procedures are necessary to improve conditions for implant placement. These techniques in turn can be enhanced by using growth factors (GFs) such as bone morphogenetic protein (BMP-2) and platelet-derived growth factor (PDGF) to accelerate regeneration. The aim of the present systematic review was to evaluate the GF loading and release kinetics of barrier membranes.STUDY DESIGN:A total of 138 articles were screened in PubMed databases, and 31 meeting the inclusion criteria were included in the present systematic review.RESULTS:All the articles evaluated bio-resorbable membranes, especially collagen or polymer-based membranes. In most studies, the retention and release kinetics of osteogenic GFs such as BMP-2 and PDGF were widely investigated. Growth factors were incorporated to the membranes by soaking and incubating the membranes in GF solution, followed by lyophilization, or mixing in the polymers before evaporation. Adsorption onto the membranes depended upon the membrane materials and additional reagents such as heparin, cross-linkers and GF concentration. Interestingly, most studies showed two phases of GF release from the membranes: a first phase comprising a burst release (about 1 day), followed by a second phase characterized by slower release. Furthermore, all the studies demonstrated the controlled release of sufficient concentrations of GFs from the membranes for bioactivities.CONCLUSIONS:The adsorption and release kinetics varied among the different materials, forms and GFs. The combination of membrane materials, GFs and manufacturing methods should be considered for optimizing GBR/GTR procedures.

Barrier membranes are essential biomaterials for guided bone regeneration. Due to different origin and structure of barrier membranes, singular mechanical properties and clinical behaviors can be expected. It is important to understand the physic and chemical properties of barrier membranes to select the needed biomaterial for each clinical situation. To date, no study has evaluated and compared the physicochemical properties of various families of barrier membranes. The aim of this study is to evaluate the physicochemical properties of various barrier membranes. Fifteen membranes of different origin were tested in this study. Membranes were divided into biological or synthetic origin and grouped in natural allogenic collagen, natural xenogenic collagen, cross-linked collagen and synthetic membranes. Physicochemical properties were evaluated in terms of tension, stiffness, absorption ability, pH and wettability. For the tension tests, all membranes showed similar low tension and low stiffness, especially after a 4-min hydration, except for bone laminas that showed a greater stiffness particularly in a dry status. Regarding wettability and hydration of the barrier membranes, porcine origin membranes had greater hydration; wettability was also superior in porcine derived barrier membranes and showed a faster absorption of the drop on the rough surfaces. All membranes had a stable pH, having the synthetic membranes the most stable pH when compared to physiologic. The wide variety of barrier membranes opens a debate in which the practitioner should select the adequate barrier membrane for each clinical situation. Different materials show singular potentials depending on their tissue origin making them suitable for specific clinical indications. More studies regarding adsorption, integration and degradation of barrier membranes are needed to understand their behavior.

BACKGROUND:Commercially available xenograft blocks, claim to have adequate characteristics to interact with biological media and thus permitting biological fluid absorption. The objective of this in vitro study was to compare the blood absorption capacity of four different xenograft block materials of different composition of collagen and porosity. MATERIAL AND METHODS:Four brands of xenograft block materials were used (NuOss®, Bio-Oss®, Osteobiol® and Smartbone®). Five samples of each brand were analyzed, making a total of 20 tests. Human blood was used as the absorption liquid for the present experiment. The time period, in which the block remains in contact with the blood, was registered at 30 seconds (T1), 60 seconds (T2) and 5 minutes (T3). The xenograft blocks were evaluated according to their absorption capacity. RESULTS:The absorption capacity of the different biomaterials were statistical significant different (p<0,001) at T1, T2 and T3 time points. At 30 seconds, Smartbone® absorbed significantly less blood than NuOss® and Bio-Oss®, however, without differences comparing with Osteobiol®. The NuOss®, Bio-Oss® and Osteobiol® did not register any significant difference between them. At 60 seconds, the Smartbone® absorbed significantly less blood than the other biomaterials. CONCLUSIONS:The NuOss® was significantly superior than Osteobiol®, but without differences relatively with Bio-Oss®. Also the Bio-Oss® and Osteobiol® did not register any difference between them. At 5 minutes, the Smatbone® continued to significantly absorbed less blood than any other biomaterial, nevertheless, NuOss®, Bio-Oss® and Osteobiol® not register again any significant difference between them. Despite of small sample size, it can be concluded that NuOss® was superior, in terms of blood absorption capacity, comparing with the other block biomaterials at 30 seconds, 60 seconds and 5 minutes. However, more investigation in a clinical setting are needed to know the clinical implications of the absorption capacity of such biomaterials. Key words:Blood absorption, osteoconduction, xenograft, bone regeneration.

Since bone grafting materials were introduced for clinical use, guided tissue and bone regeneration has become a standard for bone and periodontal augmentation procedures requiring space provision. Over the past 20 years, a number of advancements have been made in terms of membrane development and utilization. The aim of this chapter is to discuss the original concept for utilizing polytetrafluoroethylene (PTFE) nonresorbable membranes and present more recent novel advancements in the field. Today, synthetic and natural,barrier membranes are being utilized more frequently because of their improved mechanical properties and degradation rates.,More recent trends have further favored the incorporation of bone-inducing agents such as osteoconductive calcium phosphates and/or bioactive growth factors into membranes to promote faster bone formation. This,, chapter explores the number of barrier membranes currently available on thee market and highlights their advantages and disadvantages. Furthermore, it provides insight into future developments in membrane fabrication and the use of autologous platelet-derived clots as bioactive membranes and touches on current studies investigating the optimization of membranes as potential next-generation barriers in regenerative dentistry.

It has been speculated that certain Schneiderian membrane thickness (SMT) might be more prone to perforation. This investigation was aimed at studying the mechanical characteristics of the Schneiderian membrane under one- and two-dimensional tests and their correlation to the histological SMT in human samples.

BACKGROUNDThe aim of this case-control study was to estimate the diagnostic accuracy of the standard clinical parameters in diagnosing healthy peri-implant tissues, peri-implant mucositis, and peri-implantitis.METHODSA case-control study was designed to compare the clinical parameters used in the diagnosis of peri-implant diseases such as: probingdepth (PD), bleeding on probing (BOP), mucosal redness (MR), suppuration (SUP), and plaque index (PI). Furthermore, the influence of patient- (sex, age) and implant-related variables (implant neck configuration, time in function after loading) were evaluated to investigate the association with the clinical findings. The inferential analysis consisted of estimation by generalized estimating equations (GEE) of multilevel logistic regression models.RESULTSIn total, 1,572 sites were evaluated around 262 implants from 141 patients. Sites with implant mucositis showed significant levels of BOP (OR = 3.56), MR (OR = 7.66) and PD (OR = 1.48) compared to healthy sites. The specificity was 90.3% while the sensitivity was only 43.6%. Likewise, sites exhibiting peri-implantitis showed significant levels of BOP (OR = 2.32), MR (OR = 7.21), PD (OR = 2.43) and SUP (OR = 6.81) compared to healthy sites. Again, the multiple logistic regressions showed high specificity (92.1%) but modest sensitivity (52.5%). PD was the only diagnostic marker displaying significance comparing peri-implant mucositis and peri-implantitis sites (OR = 1.76). Moreover, tissue-level compared to bone-level implants were less associated with SUP+ (OR = 0.20), and PI (OR = 0.36) and demonstrated statistical significance. In addition, age, sex, and function time significantly influenced the tested clinical parameters.CONCLUSIONSThe diagnosis of peri-implant diseases cannot rely solely upon individual clinical parameters but rather require a combination of criteria. The clinical parameters, particularly probing depth, might accurately discern between diagnoses among peri-implant conditions. Nevertheless, the specificity of the clinical parameters surpasses the sensitivity in the detection of peri-implant diseases, validating its potential use as a diagnostic tool.

Guided bone regeneration (GBR) often utilizes a combination of autologous bone grafts, deproteinized bovine bone mineral (DBBM), and collagen membranes. DBBM and collagen membranes pre-coated with bone-conditioned medium (BCM) extracted from locally harvested autologous bone chips have shown great regenerative potential in GBR. However, the underlying molecular mechanism remains largely unknown. Here, we investigated the composition of BCM and its activity on the osteogenic potential of mesenchymal stromal cells. We detected a fast and significant ( P < 0.001) release of transforming growth factor-β1 (TGF-β1) from autologous bone within 10 min versus a delayed bone morphogenetic protein-2 (BMP-2) release from 40 min onwards. BCMs harvested within short time periods (10, 20, or 40 min), corresponding to the time of a typical surgical procedure, significantly increased the proliferative activity and collagen matrix production of BCM-treated cells. Long-term (1, 3, or 6 days)-extracted BCMs promoted the later stages of osteoblast differentiation and maturation. Short-term-extracted BCMs, in which TGF-β1 but no BMP-2 was detected, reduced the expression of the late differentiation marker osteocalcin. However, when both growth factors were present simultaneously in the BCM, no inhibitory effects on osteoblast differentiation were observed, suggesting a synergistic TGF-β1/BMP-2 activity. Consequently, in cells that were co-stimulated with recombinant TGF-β1 and BMP-2, we showed a significant stimulatory and dose-dependent effect of TGF-β1 on BMP-2-induced osteoblast differentiation due to prolonged BMP signaling and reduced expression of the BMP-2 antagonist noggin. Altogether, our data provide new insights into the molecular mechanisms underlying the favorable outcome from GBR procedures using BCM, derived from autologous bone grafts.

BACKGROUND:GBRs are essential procedures in implant dentistry and periodontology where barrier membranes play an important role by isolating soft tissue and allowing bone to grow. Not all membranes function the same way, as they differ from their origin and structure, it is important to understand how membranes behave and differ one from others in order to achieve a predictable treatment.MATERIAL AND METHODS:A systematic search on Medline by two independent reviewers was performed for articles published until July 2017 reporting the characteristics or properties of barrier membranes. The question that preceded the search was designed according to PICO rules.RESULTS:A total of 124 articles were initially identified from electronic searching. After abstract/full-text review, 21 were included for a systematic review. According to the extracted data and article analysis, barrier membranes should fulfill the following criteria in order to success: biocompatibility, space maintaining, occlusive function, easy - handling and a bioactivation friendly property. With the development of new biomaterials and surfaces, a great advance in this area is expected.CONCLUSIONS:It has been clearly described that biocompatibility is the most important requirement to take into account when choosing a membrane, but other factors such as space maintaining capacity, cell oclusiveness, easy handling and bioactivation friendly materials are the ones that will fulfill our necessities. Key words:Barrier membrane, guided bone regeneration, dental implantology, oral surgery, collagen membrane, biomaterial.