Time-dependent and somatically acquired mitochondrial DNA mutagenesis and respiratory chain dysfunction in a scleroderma model of lung fibrosis.

Reactive oxygen species (ROS) have been implemented in the etiology of pulmonary fibrosis (PF) in systemic sclerosis. In the bleomycin model, we evaluated the role of acquired mutations in mitochondrial DNA (mtDNA) and respiratory chain defects as a trigger of ROS formation and fibrogenesis. Adult male Wistar rats received a single intratracheal instillation of bleomycin and their lungs were examined at different time points. Ashcroft scores, collagen and TGFβ1 levels documented a delayed onset of PF by day 14. In contrast, increased malon dialdehyde as a marker of ROS formation was detectable as early as 24 hours after bleomycin instillation and continued to increase. At day 7, lung tissue acquired significant amounts of mtDNA deletions, translating into a significant dysfunction of mtDNA-encoded, but not nucleus-encoded respiratory chain subunits. mtDNA deletions and markers of mtDNA-encoded respiratory chain dysfunction significantly correlated with pulmonary TGFβ1 concentrations and predicted PF in a multivariate model.

Cyclooxygenase-2 and 5-lipoxygenase in dogs with chronic enteropathies.

BACKGROUND Cyclooxygenase-2 (COX-2) is a key enzyme in the synthesis of pro-inflammatory prostaglandins and 5-lipoxygenase (5-LO) is the major source of leukotrienes. Their role in IBD has been demonstrated in humans and animal models, but not in dogs with chronic enteropathies (CCE). HYPOTHESIS COX-2 and 5-LO are upregulated in dogs with CCE. ANIMALS Fifteen healthy control dogs (HCD), 10 dogs with inflammatory bowel disease (IBD), and 15 dogs with food-responsive diarrhea (FRD). METHODS Prospective study. mRNA expression of COX-2, 5-LO, IL-1b, IL-4, IL-6, TNF, IL-10 and TFG-β was evaluated by quantitative real-time RT-PCR in duodenal and colonic biopsies before and after treatment. RESULTS COX-2 expression in the colon was significantly higher in IBD and FRD before and after treatment (all P < .01). IL-1b was higher in FRD in the duodenum after treatment (P = .021). TGF-β expression was significantly higher in the duodenum of HCD compared to FRD/IBD before treatment (both P < .001) and IBD after treatment (P = .012). There were no significant differences among groups and within groups before and after treatment for IL-4, IL-6, TNF, and IL-10. There was a significant correlation between COX-2 and IL-1b in duodenum and colon before treatment in FRD and IBD, whereas 5-LO correlated better with IL-6 and TNF. IL-10 and TGF-β usually were correlated. CONCLUSIONS AND CLINICAL IMPORTANCE COX-2 is upregulated in IBD and FRD, whereas IL-1b and TGF-β seem to be important pro- and anti-inflammatory cytokines, respectively. The use of dual COX/5-LO inhibitors could be an interesting alternative in the treatment of CCE.

Nuclear Factor I-C acts as a regulator of hepatocyte proliferation at the onset of liver regeneration

BACKGROUND & AIMS: Knockout studies of the murine Nuclear Factor I-C (NFI-C) transcription factor revealed abnormal skin wound healing and growth of its appendages, suggesting a role in controlling cell proliferation in adult regenerative processes. Liver regeneration following partial hepatectomy (PH) is a well-established regenerative model whereby changes elicited in hepatocytes lead to their rapid and phased proliferation. Although NFI-C is highly expressed in the liver, no hepatic function was yet established for this transcription factor. This study aimed to determine whether NFI-C may play a role in hepatocyte proliferation and liver regeneration. METHODS: Liver regeneration and cell proliferation pathways following two-thirds PH were investigated in NFI-C knockout (ko) and wild-type (wt) mice. RESULTS: We show that the absence of NFI-C impaired hepatocyte proliferation because of plasminogen activator I (PAI-1) overexpression and the subsequent suppression of urokinase plasminogen activator (uPA) activity and hepatocyte growth factor (HGF) signalling, a potent hepatocyte mitogen. This indicated that NFI-C first acts to promote hepatocyte proliferation at the onset of liver regeneration in wt mice. The subsequent transient down regulation of NFI-C, as can be explained by a self-regulatory feedback loop with transforming growth factor beta 1 (TGF-ß1), may limit the number of hepatocytes entering the first wave of cell division and/or prevent late initiations of mitosis. CONCLUSION: NFI-C acts as a regulator of the phased hepatocyte proliferation during liver regeneration.

Conditioned Medium of Demineralized Freeze-Dried Bone Activates Gene Expression in Periodontal Fibroblasts in Vitro.

BACKGROUND: Demineralized bone matrix (DBM) is used for the treatment of osseous defects. Conditioned medium from native bone chips can activate transforming growth factor (TGF)-β signaling in mesenchymal cells. The aim of the study was to determine whether processing of native bone into DBM affects the activity of the conditioned medium. METHODS: Porcine cortical bone blocks were subjected to defatting, different concentrations of hydrochloric acid and various temperatures. DBM was lyophilized, ground, and placed into culture medium. Human gingiva and periodontal fibroblasts were exposed to the respective conditioned medium (DBCM). Changes in the expression of TGF-β target genes were determined. RESULTS: DBCM altered the expression of TGF-β target genes, e.g., adrenomedullin, pentraxin 3, KN Motif And Ankyrin Repeat Domains 4, interleukin 11, NADPH oxidase 4, and BTB (POZ) Domain Containing 11, by at least five-fold. The response was observed in fibroblasts from both sources. Defatting lowered the activity of DBCM. The TGF-β receptor type I kinase inhibitor SB431542, but not the inhibitor of bone morphogenetic protein receptor dorsomorphin, blocked the effects of DBCM on gene expression. Moreover, conditioned medium obtained from commercial human DBM modulated the expression of TGF-β target genes. CONCLUSION: The findings suggest that the conditioned medium from demineralized bone matrix can activate TGF-β signaling in oral fibroblasts. KEYWORDS: TGF-beta superfamily proteins; bone; bone substitutes; bone transplantation; conditioned media; freeze drying

Antiseptic solutions modulate the paracrine-like activity of bone chips: differential impact of chlorhexidine and sodium hypochlorite

AIM: Chemical decontamination increases the availability of bone grafts; however, it is unclear whether antiseptic processing changes the biological activity of bone. MATERIALS AND METHODS: Bone chips were incubated with 4 different antiseptic solutions including (1) povidone-iodine (0.5%), (2) chlorhexidine diguluconate (0.2%), (3) hydrogen peroxide (1%) and (4) sodium hypochlorite (0.25%). After 10 minutes of incubation, changes in the capacity of the bone-conditioned medium to modulate gene expression of gingival fibroblasts was investigated. RESULTS: Conditioned medium obtained from freshly prepared bone chips increased the expression of TGF-β target genes interleukin 11 (IL11), proteoglycan4 (PRG4), NADPH oxidase 4 (NOX4), and decreased the expression of adrenomedullin (ADM), and pentraxin 3 (PTX3) in gingival fibroblasts. Incubation of bone chips with 0.2% chlorhexidine, followed by vigorously washing resulted in a bone-conditioned medium with even higher expression of IL11, PRG4, and NOX4. These findings were also found with a decrease in cell viability and an activation of apoptosis signaling. Chlorhexidine alone, at low concentrations, increased IL11, PRG4 and NOX4 expression, independent of the TGF-β receptor I kinase activity. In contrast, 0.25% sodium hypochlorite almost entirely abolished the activity of bone-conditioned medium, while the other two antiseptic solutions, 1% hydrogen peroxide and 0.5% povidone-iodine, had relatively no impact, respectively. CONCLUSION: These in vitro findings demonstrate that incubation of bone chips with chlorhexidine differentially affects the activity of the respective bone-conditioned medium compared to the other antiseptic solutions. The data further suggest that the main effects are caused by chlorhexidine remaining in the bone-conditioned medium after repeated washing of the bone chips. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved. KEYWORDS: Autografts; TGF-β; antiseptic solution; bone; bone conditioned medium; bone supernatant; chlorhexidine; hydrogen peroxide; povidone-iodine; sodium hypochlorite

Bone Conditioned Medium: Preparation and Bioassay.

Autologous bone grafts are widely used in oral and maxillofacial surgery, orthopedics, and traumatology. Autologous bone grafts not only replace missing bone, they also support the complex process of bone regeneration. This favorable behavior of autografts is attributed to the three characteristics: osteoconductivity, osteogenicity, and osteoinductivity. However, there is another aspect: Bone grafts release a myriad of molecules, including growth factors, which can target mesenchymal cells involved in bone regeneration. The paracrine properties of bone grafts can be studied in vitro by the use of bone-conditioned medium (BCM). Here we present a protocol on how to prepare bone-conditioned medium from native pig cortical bone, and bone that underwent thermal processing or demineralization. Cells can be directly exposed to BCM or seeded onto biomaterials, such as collagen membranes, previously soaked with BCM. We give examples for in vitro bioassays with mesenchymal cells on the expression of TGF-β regulated genes. The presented protocols should encourage to further reveal the paracrine effects of bone grafts during bone regeneration and open a path for translational research in the broad field of reconstructive surgery.

Bone-Conditioned Medium Changes Gene Expression in Bone-Derived Fibroblasts.

PURPOSE Autologous bone is used for augmentation in the course of oral implant placement. Bone grafts release paracrine signals that can modulate mesenchymal cell differentiation in vitro. The detailed genetic response of the bone-derived fibroblasts to these paracrine signals has remained elusive. Paracrine signals accumulate in bone-conditioned medium (BCM) prepared from porcine cortical bone chips. MATERIALS AND METHODS In this study, bone-derived fibroblasts were exposed to BCM followed by a whole genome expression profiling and downstream quantitative reverse transciptase polymerase chain reaction of the most strongly regulated genes. RESULTS The data show that ADM, IL11, IL33, NOX4, PRG4, and PTX3 were differentially expressed in response to BCM in bone-derived fibroblasts. The transforming growth factor beta (TGF-β) receptor 1 antagonist SB431542 blocked the effect of BCM on the expression of the gene panel, except for IL33. CONCLUSION These in vitro results extend existing evidence that cortical bone chips release paracrine signals that provoke a robust genetic response in mesenchymal cells that is not exclusively mediated via the TGF-β receptor. The present data provide further insights into the process of graft consolidation.

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.

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.

Platelet-rich concentrates differentially release growth factors and induce cell migration in vitro

BACKGROUND Platelet-rich concentrates are used as a source of growth factors to improve the healing process. The diverse preparation protocols and the gaps in knowledge of their biological properties complicate the interpretation of clinical results. QUESTIONS/PURPOSES In this study we aimed to (1) analyze the concentration and kinetics of growth factors released from leukocyte- and platelet-rich fibrin (L-PRF), leukocyte- and platelet-rich plasma (L-PRP), and natural blood clot during in vitro culture; (2) investigate the migration of mesenchymal stem cells (MSCs) and human umbilical vein endothelial cells (HUVECs) as a functional response to the factors released; and (3) uncover correlations between individual growth factors with the initial platelet/leukocyte counts or the induced cell migration. METHODS L-PRF, L-PRP, and natural blood clot prepared from 11 donors were cultured in vitro for 28 days and media supernatants collected after 8 hours and 1, 3, 7, 14, and 28 days. Released transforming growth factor β1 (TGF-β1), vascular endothelial growth factor (VEGF), insulin growth factor (IGF-1), platelet-derived growth factor AB (PDGF-AB), and interleukin-1β (IL-1β) were measured in the supernatants with enzyme-linked immunosorbent assay. Migration of MSC and HUVEC induced by the supernatants was evaluated in Boyden chambers. RESULTS More TGF-ß1 was released (mean ± SD in pg/mL of blood) from L-PRF (37,796 ± 5492) compared with L-PRP (23,738 ± 6848; p < 0.001) and blood clot (3739 ± 4690; p < 0.001), whereas more VEGF and IL-1ß were released from blood clot (1933 ± 704 and 2053 ± 908, respectively) compared with both L-PRP (642 ± 208; p < 0.001 and 273 ± 386; p < 0.001, respectively) and L-PRF (852 ± 376; p < 0.001 and 65 ± 56, p < 0.001, respectively). No differences were observed in IGF-1 and PDGF-AB released from any of the concentrates. TGF-β1 release peaked at Day 7 in L-PRF and at 8 hours and Day 7 in L-PRP and 8 hours and Day 14 in blood clot. In all concentrates, main release of VEGF occurred between 3 and 7 days and of IL-1β between Days 1 and 7. IGF-1 and PDGF-AB were released until Day 1 in L-PRP and blood clot, in contrast to sustained release over the first 3 days in L-PRF. The strongest migration of MSC occurred in response to L-PRF, and more HUVEC migration was seen in L-PRF and blood clot compared with L-PRP. TGF-β1 correlated with initial platelet counts in L-PRF (Pearson r = 0.66, p = 0.0273) and initial leukocyte counts in L-PRP (Pearson r = 0.83, p = 0.0016). A positive correlation of IL-1β on migration of MSC and HUVEC was revealed (Pearson r = 0.16, p = 0.0208; Pearson r = 0.31, p < 0.001). CONCLUSIONS In comparison to L-PRP, L-PRF had higher amounts of released TGF-β1, a long-term release of growth factors, and stronger induction of cell migration. Future preclinical studies should confirm these data in a defined injury model. CLINICAL RELEVANCE By characterizing the biologic properties of different platelet concentrates in vitro, we may gain a better understanding of their clinical effects and develop guidelines for specific future applications.

NonViral Gene Delivery of Growth and Differentiation Factor 6 (GDF6) to whole bovine Intervertebral Disc

Question: Low back pain is an increasing global health problem, which is associated with intervertebral disc (IVD) damage and de- generation. Major changes occur in the nucleus pulposus (NP), with the degradation of the extracellular matrix (ECM) [1]. Further studies showed that growth factors from the transforming growth factor (TGF) and bone morphogenic proteins (BMP) family may induce chondrogenic differentiation of mesenchymal stem cells (MSC) [2]. Focusing on non-viral gene therapies and their possible translation into the clinics, we investigated if GDF6 (syn. BMP13 or CDMP2) can induce regeneration of degraded NP. We hypothesized that IVD transfected with plasmid over-expressing GDF6 also up-regulates other NP- and chondrogenic cell markers and enhances ECM deposition. Methods: Bovine IVD cells were isolated by pronase/collagenase II overnight digestion. After monolayer expansion up to passage 3, cells were transfected with the plasmid pGDF6 (RG211366, Origene, SF) or with green fluorescence protein (GFP) control using the NeonÒ transfection system (Invitrogen, Basel), both equipped with a Cy- tomegalovirus (CMV) promotor to induce over-expression. We tested a range of yet unpublished parameters for each of the primary disc cells to optimize efficiency. To test a non-viral gene therapy applied directly to 3D whole organ culture, bovine IVDs were harvested from fresh tails obtained from the abattoir within 5 h post-mortem [3]. Discs were then pre-incubated for 24 h in high glucose Dulbecco’s Modified Eagle Medium and 5 % fetal calf serum. Each disc was transfected by injection of 5 lg of plasmid GDF6 (Origene, RG211366) into the center by 25G needle and using Hamilton sy- ringe. Electroporation was performed using 2-needle array electrode or tweezertrodes; 8 pulses at 200mv/cm with an interval of 10 ms were applied using ECM830 Square Wave Electroporation System (Harvard Apparatus, MA) (Fig. 1). After transfection discs were cultured for 72 h to allow expression of GFP or GDF6. Discs were then fixed, cryosectioned and analysed by immunofluorescence against GDF6. Results: We successfully transfected bovine NP and AF cells in monolayer culture with the two plasmids using a 1,400 V, 20 ms and 2 pulses with a *25 % efficiency using 0.15 M cells and 3 lg DNA (Fig. 1). Organ IVD culture transfection revealed GFP6 positive staining in the centre of the disc using 2-needle array electrode. Results from tweezertrodes did not show any GFP posi- tive cells. Conclusions: We identified novel parameters to successfully transfect primary bovine IVD cells. For transfection of whole IVD explants electroporation parameters need to be further optimized. Acknowledgments: This study was supported by the Lindenhof Foundation ‘‘Forschung und Lehre’’ (Project no. 13-02-F). References 1. Roughly PJ (2004) Spine (Phila) 29:2691–2699 2. 3. Clarke LE, McConell JC, Sherratt MJ, Derby B, Richardson SM, Hoyland JA (2014) Arthritis Res Ther 16:R67 Chan SC, Gantenbein-Ritter B (2012) J Vis Exp 60(60):e3490

Effects of Antiseptic Solutions Commonly Used in Dentistry on Bone Viability, Bone Morphology, and Release of Growth Factors

PURPOSE Antiseptic solutions are commonly used in dentistry for a number of sterilization procedures, including harvesting of bone chips, irrigation of extraction sockets, and sterilization of osteonecrotic bone. Despite its widespread use, little information is available regarding the effects of various antiseptic solutions on bone cell viability, morphology, and the release of growth factors. MATERIALS AND METHODS The antiseptic solutions included 1) 0.5% povidone iodine (PI), 2) 0.2% chlorhexidine diguluconate (CHX), 3) 1% hydrogen peroxide (H2O2), and 4) 0.25% sodium hypochlorite (HYP). Bone samples collected from porcine mandibular cortical bone were rinsed in the antiseptic solutions for 10 minutes and assessed for cell viability using an MTS assay and protein release of transforming growth factor (TGF-β1), bone morphogenetic protein 2 (BMP2), vascular endothelial growth factor (VEGF), interleukin (IL)-1β, and receptor activator of nuclear factor κB ligand (RANKL) using an enzyme-linked immunosorbent assay at 15 minutes and 4 hours after rinsing. RESULTS After antiseptic rinsing, changes to the surface protein content showed marked alterations, with an abundant protein layer remaining on CHX-rinsed bone samples. The amount of surface protein content gradually decreased in the following order: CHX, H2O2, PI, and HYP. A similar trend was also observed for the relative cell viability from within bone samples after rinsing, with up to 6 times more viable cells found in the CHX-rinsed bone samples than in the HYP- and PI-rinsed samples. An analysis of the growth factors found that both HYP and PI had significantly lower VEGF and TGF-β1 protein release from bone samples at 15 minutes and 4 hours after rinsing compared with CHX and H2O2. A similar trend was observed for RANKL and IL-1β protein release, although no change was observed for BMP2. CONCLUSIONS The results from the present study have demonstrated that antiseptic solutions present with very different effects on bone samples after 10 minutes of rinsing. Rinsing with CHX maintained significantly higher cell viability and protein release of growth factors potent to the bone remodeling cycle.

A novel organ-slice culturing system to simulate meniscal repair: Proof of concept using a synovium-based pool of meniscoprogenitor cells.

Meniscal injuries can occur secondary to trauma or be instigated by the changes in knee-joint function that are associated with aging, osteo- and rheumatoid arthritis, disturbances in gait and obesity. Sixty per cent of persons over 50 years of age manifest signs of meniscal pathology. The surgical and arthroscopic measures that are currently implemented to treat meniscal deficiencies bring only transient relief from pain and effect but a temporary improvement in joint function. Although tissue-engineering-based approaches to meniscal repair are now being pursued, an appropriate in-vitro model has not been conceived. The aim of this study was to develop an organ-slice culturing system to simulate the repair of human meniscal lesions in vitro. The model consists of a ring of bovine meniscus enclosing a chamber that represents the defect and reproduces its sequestered physiological microenvironment. The defect, which is closed with a porous membrane, is filled with fragments of synovial tissue, as a source of meniscoprogenitor cells, and a fibrin-embedded, calcium-phosphate-entrapped depot of the meniscogenic agents BMP-2 and TGF-ß1. After culturing for 2 to 6 weeks, the constructs were evaluated histochemically and histomorphometrically, as well as immunohistochemically for the apoptotic marker caspase 3 and collagen types I and II. Under the defined conditions, the fragments of synovium underwent differentiation into meniscal tissue, which bonded with the parent meniscal wall. Both the parent and the neoformed meniscal tissue survived the duration of the culturing period without significant cell losses. The concept on which the in-vitro system is based was thus validated. This article is protected by copyright. All rights reserved.

Influence of root canal disinfectants on growth factor release from dentin

INTRODUCTION During dentinogenesis, growth factors become entrapped in the dentin matrix that can later be released by demineralization. Their effect on pulpal stem cell migration, proliferation, and differentiation could be beneficial for regenerative endodontic therapies. However, precondition for success, as for conventional root canal treatment, will be sufficient disinfection of the root canal system. Various irrigation solutions and intracanal dressings are available for clinical use. The aim of this study was 2-fold: to identify a demineralizing solution suitable for growth factor release directly from dentin and to evaluate whether commonly used disinfectants for endodontic treatment will compromise this effect. METHODS Dentin disks were prepared from extracted human teeth and treated with EDTA or citric acid at different concentrations or pH for different exposure periods. The amount of transforming growth factor-β1 (TGF-β1), fibroblast growth factor 2, and vascular endothelial growth factor were quantified via enzyme-linked immunosorbent assay and visualized by gold labeling. Subsequently, different irrigation solutions (5.25% sodium hypochloride, 0.12% chlorhexidine digluconate) and intracanal dressings (corticoid-antibiotic paste, calcium hydroxide: water-based and oil-based, triple antibiotic paste, chlorhexidine gel) were tested, and the release of TGF-β1 was measured after a subsequent conditioning step with EDTA. RESULTS Conditioning with 10% EDTA at pH 7 rendered the highest amounts of TGF-β1 among all test solutions. Fibroblast growth factor 2 and vascular endothelial growth factor were detected after EDTA conditioning at minute concentrations. Irrigation with chlorhexidine before EDTA conditioning increased TGF-β1 release; sodium hypochloride had the opposite effect. All tested intracanal dressings interfered with TGF-β1 release except water-based calcium hydroxide. CONCLUSIONS Growth factors can be released directly from dentin via EDTA conditioning. The use of disinfecting solutions or medicaments can amplify or attenuate this effect.

Effect of Enamel Matrix Derivative Liquid on Osteoblast and Periodontal Ligament Cell Proliferation and Differentiation.

BACKGROUND Enamel matrix derivatives (EMDs) have been used clinically for more than a decade for the regeneration of periodontal tissues. The aim of the present study is to analyze the effect on cell growth of EMDs in a gel carrier in comparison to EMDs in a liquid carrier. EMDs in a liquid carrier have been shown to adsorb better to bone graft materials. METHODS Primary human osteoblasts and periodontal ligament (PDL) cells were exposed to EMDs in both gel and liquid carriers and compared for their ability to induce cell proliferation and differentiation. Alizarin red staining and real-time polymerase chain reaction for expression of genes encoding collagen 1, osteocalcin, and runt-related transcription factor 2, as well as bone morphogenetic protein 2 (BMP2), transforming growth factor (TGF)-β1, and interleukin (IL)-1β, were assessed. RESULTS EMDs in both carriers significantly increased cell proliferation of both osteoblasts and PDL cells in a similar manner. Both formulations also significantly upregulated the expression of genes encoding BMP2 and TGF-β1 as well as decreased the expression of IL-1β. EMDs in the liquid carrier further retained similar differentiation potential of both osteoblasts and PDL cells by demonstrating increased collagen and osteocalcin gene expression and significantly higher alizarin red staining. CONCLUSIONS The results from the present study indicate that the new formulation of EMDs in a liquid carrier is equally as potent as EMDs in a gel carrier in inducing osteoblast and PDL activity. Future study combining EMDs in a liquid carrier with bone grafting materials is required to further evaluate its potential for combination therapies.