Engineering a microparticle-loaded rough membrane for guided bone regeneration modulating osteoblast response without inducing inflammation.

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.

A Human Whole Blood Culture System Reveals Detailed Cytokine Release Profiles of Implant Materials.

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.

Comparison of resistive capacitive energy transfer therapy on cadaveric molars and incisors with and without implants.

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/m2) 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.

Tissue Integration and Degradation of a Porous Collagen-Based Scaffold Used for Soft Tissue Augmentation.

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.

Blood absorption capacity of different xenograft bone substitutes. An in-vitro study.

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.

Tissue Response to a Porous Collagen Matrix Used for Soft Tissue Augmentation.

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.

Early implant failure: a retrospective analysis of contributing factors.

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 for a 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.

Physicochemical characterization of barrier membranes for bone regeneration.

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.

Soft tissue response to dental implant closure caps made of either polyetheretherketone (PEEK) or titanium.

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.

Adsorption and release kinetics of growth factors on barrier membranes for guided tissue/bone regeneration: A systematic review.

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.

On the search of the ideal barrier membrane for guided bone regeneration.

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.

Bone-conditioned medium contributes to initiation and progression of osteogenesis by exhibiting synergistic TGF-ß1/BMP-2 activity.

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-ß1 (TGF-ß1) 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-ß1 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-ß1/BMP-2 activity. Consequently, in cells that were co-stimulated with recombinant TGF-ß1 and BMP-2, we showed a significant stimulatory and dose-dependent effect of TGF-ß1 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.

Diagnostic accuracy of clinical parameters to monitor peri-implant conditions: A matched case-control study.

BACKGROUND: The 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. METHODS: A 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. RESULTS: In 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. CONCLUSIONS: The 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.

Mechanical characteristics of the maxillary sinus Schneiderian membrane ex vivo.

OBJECTIVES: 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. MATERIAL AND METHODS: Sixteen Schneiderian membranes were collected from 11 cadaver heads treated with Thiel's embalming method. The samples were processed and analyzed clinically and histologically. One-dimensional maximum elongation until perforation and two-dimensional resistance to ball penetration were performed after the biopsy. Data was analyzed by using the Wilcoxon rank test and the Spearman's rank correlation. RESULTS: The histological SMT was 1.36 ± 0.42 mm, whereas the clinical thickness was 0.27 ± 0.21 mm, yielding statistical significance (p = 0.000). The resistance under ball penetration was 0.59 ± 0.43 N and the mean maximum elongation in the one-dimension test 11.19 ± 7.14 mm. Expressed in percentage, the mean stretch was 241.36 ± 227.97% (range 31.5 up to 947%). A weak positive correlation was found between the ball penetration test and the SMT (r = 0.10, p = 0.711), while a weak negative correlation was found between stretching test and the SMT (r = -0.021, p = 0.94). CONCLUSIONS: Mechanical tests seem to indicate that SMT might not significantly predispose to Schneiderian membrane perforation. Hence, other anatomical and operator's factors should be considered of surpassing importance. CLINICAL RELEVANCE: Thinner SM might be more prone to perforation when detaching it from the maxillary sinus antrum; however, a thick membrane is not prevented to tear, as their resistance under elastic forces is not higher than thinner ones.

Morphologic Patterns of the Atrophic Posterior Maxilla and Clinical Implications for Bone Regenerative Therapy.

Regenerative therapies are commonly needed prior to implant placement in the posterior maxilla following tooth extraction. The aim of this study was to investigate the anatomical features of the atrophic posterior maxilla that might affect surgical approach selection and technique and risk of intra- and postoperative complications. Cone beam computed tomography files were screened to identify pristine atrophic maxillary ridges in need of bone augmentation (ridge height < 10 mm). Ridge height and width and lateral wall thickness (LWT) were measured at different levels, and palatonasal recess and lateral wall morphology were calculated. The influence of site was assessed using generalized estimation equations. The linear correlation between parameters was estimated with Pearson coefficient, and principal component analysis (PCA) was performed to establish patterns of multiple correlations. In the 433 edentulous sites and 6,062 measurements analyzed, the strongest correlations corresponded to LWT measurements at the different levels. Most of the parameters examined presented linearity (P < .001), allowing establishment of four main morphologic patterns according to anatomical features. The anatomical structures of the atrophic posterior maxilla follow linear patterns that might predict complications due to an anatomical feature and thus success in regenerative therapy.

Inhibitory effect of saliva on osteoclastogenesis in vitro requires toll-like receptor 4 signaling.

OBJECTIVES: Saliva can suppress osteoclastogenesis, but the underlying mechanism has not been discovered yet. Considering that endotoxins suppress osteoclastogenesis in bone marrow cultures and that saliva contains endotoxins, it was reasonable to hypothesize that the impact of saliva on osteoclastogenesis requires toll-like receptor 4 signaling. MATERIAL AND METHODS: To test this hypothesis, we blocked toll-like receptor 4 signaling with TAK-242 in the presence of saliva in murine bone marrow cultures. Osteoclastogenesis was evaluated based on gene expression analysis and histochemical staining for tartrate-resistant acid phosphatase. Resorption was performed on dentine. RESULTS: We report that TAK-242 reversed the inhibitory effect of fresh sterile saliva on the formation of multinucleated cells that stained positive for tartrate-resistant acid phosphatase. In line with this finding, TAK-242 increased the expression of the osteoclast functional genes cathepsin K, calcitonin receptor, and tartrate-resistant acid phosphatase in the presence of saliva. TAK-242 also supported the expression of NFATc1, the master regulator of osteoclastogenesis, as well as DC-STAMP and Atp6v0d2, both being cell fusion genes. In support of the hypothesis, depletion of saliva from endotoxin partially reversed the inhibitory effect on osteoclastogenesis. Moreover, salivary pellicle on plastic and titanium did not affect osteoclastogenesis. CONCLUSION: Inhibition of toll-like receptor 4 signaling revealed that saliva can contribute to innate immunity by preventing hematopoietic progenitors to become osteoclasts. CLINICAL RELEVANCE: Saliva can activate pattern recognition receptor signaling through endotoxins and other stress factors, indicating the demand for macrophages rather than for osteoclasts.

Collagen barrier membranes adsorb growth factors liberated from autogenous bone chips.

OBJECTIVE: Collagen membranes serve as barriers to separate bone grafts from soft tissues. Bone grafts harvested with a bone scraper release growth factors activating transforming growth factor-ß (TGF-ß) signaling in mesenchymal cells. The aim of the present pilot study was to determine whether collagen membranes adsorb molecules from bone-conditioned medium (BCM) with the capacity to provoke the expression of TGF-ß target genes in vitro. MATERIALS AND METHODS: Collagen membranes were soaked in aqueous extracts from fresh and demineralized bone chips placed in cell culture medium. Recombinant human TGF-ß1 served as control. Gingival fibroblasts were seeded onto the washed collagen membranes and evaluated for the expression of adrenomedullin, pentraxin 3, interleukin 11, and proteoglycan 4. Cell viability and morphology with phalloidin staining were also determined. RESULTS: Incubation of collagen membranes with BCM for at least one minute caused fibroblasts to decrease the expression of adrenomedullin and pentraxin 3, and to increase the expression of interleukin 11 and proteoglycan 4. Four different membrane treatments - incubated with recombinant TGF-ß1, pre-wetted with saline solution, exposed to UV light, and dry out and stored one week at room temperature - also provoked significant changes in gene expression. Likewise, conditioned medium from demineralized bone chips caused gene expression changes. BCM did not alter the viability or morphology of gingival fibroblasts. CONCLUSIONS: These findings demonstrate that collagen membranes rapidly adsorb the TGF-ß activity released from bone chips, a molecular process that might contribute to guided bone regeneration.

Osteogain improves osteoblast adhesion, proliferation and differentiation on a bovine-derived natural bone mineral.

BACKGROUND: The use of enamel matrix derivative (EMD) has been shown to facilitate periodontal regeneration by histologically resulting in formation of cementum, periodontal ligament and bone. Recently, a new liquid carrier system for EMD has been introduced with better physicochemical properties specifically designed for bone graft mixing (Osteogain). The aim of this study was to investigate the combination of Osteogain with a bovine-derived natural bone mineral (NBM) on osteoblast migration, adhesion, proliferation and differentiation. MATERIALS AND METHODS: Undifferentiated mouse ST2 stromal bone marrow cells were seeded onto 1)NBM particles alone or 2)NBM + Osteogain. Samples were compared for cell migration at 8 h, cell adhesion at 4 h, cell proliferation at 1, 3 and 5 days and real-time PCR at 3 and 14 days for genes encoding runt-related transcription factor 2 (Runx2), collagen1alpha2 (COL1a2), alkaline phosphatase (ALP) and osteocalcin (OCN). Furthermore, alizarin red staining was utilized to investigate the mineralization at 14 days. RESULTS: Osteogain significantly upregulated cell adhesion over twofold onto NBM particles and promoted cell proliferation at 3 and 5 days after seeding. Furthermore, the combination of NBM with Osteogain significantly upregulated genes encoding Runx2, ALP, COL1a2 and OCN (from 1.5- to 3-fold) and increased alizarin red staining over 3 fold at 14 days when compared to NBM particles alone. CONCLUSION: Pre-coating Osteogain onto NBM bone grafting particles significantly increased cell adhesion, proliferation and differentiation of osteoblasts in vitro. Future animal studies are now necessary to further investigate the regenerative potential of Osteogain in combination with a bone grafting material prior to clinical use for bone regeneration.

Osteogenic gene array of osteoblasts cultured on a novel osteoinductive biphasic calcium phosphate bone grafting material.

OBJECTIVES: Recently, novel biphasic calcium phosphate (BCP) scaffolds have emerged as a new class of bone grafts with osteoinductive potential demonstrating the ability to form ectopic bone in extra-skeletal sites. The aim of the present study was to perform an osteogenic gene array to target possible genes responsible for eliciting the changes in cell expression responsible for inducing osteoblast differentiation. MATERIALS AND METHODS: Human MG63 osteoblast-like cells were seeded for 24 h on tissue culture plastic or osteoinductive BCP particles and analyzed for upregulated genes using an osteogenesis super-array. Osteoblast-related genes including those transcribed during bone mineralization, bone metabolism, cell growth and differentiation, as well as gene products representing extracellular matrix molecules, transcription factors, and cell adhesion molecules were investigated. RESULTS: An upregulation of genes transcribing biglycan (1.7-fold), bone morphogenetic proteins 1, 2, 4, 6, and 7 (1.5-2.1-fold), various collagen isoforms including 1a1, 1a2, 2a1, and 5a1 (1.73-2.72-fold), colony stimulating factor 2 (2.59-fold), fibroblast growth factor receptor 2 (1.79-fold), fibronectin (2.56-fold), integrin alpha 1, 2, and 3 (1.82-2.24-fold), SOX9 (2.75-fold), transforming growth factor beta receptor 2 (1.72-fold), vitamin D (1.89-fold), and vascular endothelial growth factor A and B (2.00, 1.75-fold) were all significantly (p < 0.05) increased on BCP particles when compared to control tissue culture plastic. CONCLUSION: In summary, a number of activated genes were involved in bone formation following osteoblast attachment to BCP particles. The involvement of key chondrogenic genes hints that bone grafts capable of spontaneously inducing ectopic bone formation may implicate endochondral ossification. Further investigations in the triggered pathways involved in the process of ectopic bone formation are necessary to understand the key inductive properties of these novel osteoinductive BCP particles. CLINICAL RELEVANCE: Novel osteoinductive BCP particles demonstrate a wide range of significant increases over several key molecules implicated in osteogenesis that may be implicated in their ability to form ectopic bone formation.

High-dose irradiation of bone chips preserves the in vitro activity of bone-conditioned medium.

Extracorporeal irradiation sterilizes resected tumor bone used as autografts in reconstruction surgery. Therapeutic irradiation is a standard technique in head and neck cancer therapy that aims to preserve organ function. Bone irradiation has a complex, mostly inhibitory, effect on remodeling and regeneration, although the underlying mechanisms are still not fully understood. It remains unclear if extracorporeal irradiation affects the paracrine-like activity of the corresponding autografts. We recently reported that bone-conditioned medium from autogenous bone chips contains a number of factors that might affect cell activity. In the present study, we investigated the effects of extracorporeal irradiation of porcine cortical bone chips on the activity of the corresponding bone-conditioned medium. The effects of bone-conditioned medium on the expressions of transforming growth factor-beta (TGF-ß) target genes in oral fibroblasts were assessed. Bone-conditioned medium from bone chips exposed to a total radiation dose up to 120 Gy did not affect expressions of TGF-ß target genes, including adrenomedullin, BTB/POZ domain-containing protein 11, proteoglycan 4, NADPH oxidase 4, and interleukin 11, in oral fibroblasts. In conclusion, bone irradiation does not alter the capability of the corresponding bone-conditioned medium to provoke a robust fibroblastic cell response in vitro. (J Oral Sci 58, 325-331, 2016).