Jordi Caballé-Serrano

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.

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 this study is 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 obtained from DBM (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 [4-(4-(benzo[d][1,3]dioxol-5-yl)-5-(pyridin-2-yl)-1H-imidazol-2-yl)benzamide] 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 DBCM can activate TGF-β signaling in oral fibroblasts.

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 remains unclear whether antiseptic processing changes the biological activity of bone. MATERIALS AND METHODS Bone chips were incubated with four 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 min. of incubation, changes in the capacity of the bone-conditioned medium (BCM) 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 BCM with even higher expression of IL11, PRG4 and NOX4. These findings were also detected with a decrease in cell viability and an activation of apoptosis signalling. 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 BCM, whereas 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 BCM compared to the other antiseptic solutions. The data further suggest that the main effects are caused by chlorhexidine remaining in the BCM after repeated washing of the bone chips.

Saliva Suppresses Osteoclastogenesis in Murine Bone Marrow Cultures

Saliva can reach mineralized surfaces in the oral cavity; however, the relationship between saliva and bone resorption is unclear. Herein, we examined whether saliva affects the process of osteoclastogenesis in vitro. We used murine bone marrow cultures to study osteoclast formation. The addition of fresh sterile saliva eliminated the formation of multinucleated cells that stained positive for tartrate-resistant acid phosphatase (TRAP). In line with the histochemical staining, saliva substantially reduced gene expression of cathepsin K, calcitonin receptor, and TRAP. Addition of saliva led to considerably decreased gene expression of receptor activator of nuclear factor kappa-B (RANK) and, to a lesser extent, that of c-fms. The respective master regulators of osteoclastogenesis (c-fos and NFATc1) and the downstream cell fusion genes (DC-STAMP and Atp6v0d2) showed decreased expression after the addition of saliva. Among the costimulatory molecules for osteoclastogenesis, only OSCAR showed decreased expression. In contrast, CD40, CD80, and CD86—all costimulatory molecules of phagocytic cells—were increasingly expressed with saliva. The phagocytic capacity of the cells was confirmed by latex bead ingestion. Based on these in vitro results, it can be concluded that saliva suppresses osteoclastogenesis and leads to the development of a phagocytic cell phenotype.

Thermal processing of bone: in vitro response of mesenchymal cells to bone-conditioned medium

The autoclaving, pasteurization, and freezing of bone grafts to remove bacteria and viruses, and for preservation, respectively, is considered to alter biological properties during graft consolidation. Fresh bone grafts release paracrine-like signals that are considered to support tissue regeneration. However, the impact of the autoclaving, pasteurization, and freezing of bone grafts on paracrine signals remains unknown. Therefore, conditioned medium was prepared from porcine cortical bone chips that had undergone thermal processing. The biological properties of the bone-conditioned medium were assessed by examining the changes in expression of target genes in oral fibroblasts. The data showed that conditioned medium obtained from bone chips that had undergone pasteurization and freezing changed the expression of adrenomedullin, pentraxin 3, BTB/POZ domain-containing protein 11, interleukin 11, NADPH oxidase 4, and proteoglycan 4 by at least five-fold in oral fibroblasts. Bone-conditioned medium obtained from autoclaved bone chips, however, failed to change the expression of the respective genes. Also, when bone-conditioned medium was prepared from fresh bone chips, autoclaving blocked the capacity of bone-conditioned medium to modulate gene expression. These in vitro results suggest that pasteurization and freezing of bone grafts preserve the release of biologically active paracrine signals, but autoclaving does not.

TGF-βRI kinase activity mediates Emdogain-stimulated in vitro osteoclastogenesis.

ObjectivesEmdogain, containing an extract of fetal porcine enamel matrix proteins, is a potent stimulator of in vitro osteoclastogenesis. The underlying molecular mechanisms are, however, unclear.Material and methodsHere, we have addressed the role of transforming growth factor-beta receptor type 1 (TGF-βRI) kinase activity on osteoclastogenesis in murine bone marrow cultures.ResultsInhibition of TGF-βRI kinase activity with SB431542 abolished the effect of Emdogain on osteoclastogenesis induced by receptor activator of nuclear factor kappa-B ligand or tumor necrosis factor-alpha. SB431542 also suppressed the Emdogain-mediated increase of OSCAR, a co-stimulatory protein, and dendritic cell-specific transmembrane protein and Atp6v0d2, the latter two being involved in cell fusion. Similar to transforming growth factor-beta1 (TGF-β), Emdogain could not compensate for the inhibition of IL-4 and IFNγ on osteoclast formation. When using the murine macrophage cell line RAW246.7, SB431542 and the smad-3 inhibitor SIS3 blocked Emdogain-stimulated expression of the transcription factor NFATc1.ConclusionsTaken together, the data suggest that TGF-βRI kinase activity is necessary to mediate in vitro effects of Emdogain on osteoclastogenesis.Clinical relevanceBased on these in vitro data, we can speculate that at least part of the clinical effects of Emdogain on osteoclastogenesis is mediated via TGF-β signaling.

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.

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.

Bone-conditioned medium contributes to initiation and progression of osteogenesis by exhibiting synergistic TGF-β1/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-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.Regenerative therapy: A cell culture medium that promotes bone formation‘Bone-conditioned medium’ could improve oral bone regeneration therapy by promoting the proliferation and maturation of bone-forming cells. Building on recent research demonstrating the benefits of using cell culture medium prepared with bone chips (BCM) in such treatments, researchers led by Maria Asparuhova of the University of Bern, Switzerland, set out to elucidate the medium’s mechanisms. The team found that BCM incubated with bone chips for short periods—as little as ten minutes—contained heightened levels of signaling protein TGF-β1, which enhanced mouse bone marrow cell proliferation while downregulating maturation. BCM incubated for longer periods also generated increased levels of another protein, BMP-2, which boosted the maturation of bone-forming cells. This study reveals a sequential role of these two factors in oral bone development, and the potential physiological actions of BCM when used in regenerative therapies.

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

ObjectivesSaliva 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 methodsTo 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.ResultsWe 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.ConclusionInhibition of toll-like receptor 4 signaling revealed that saliva can contribute to innate immunity by preventing hematopoietic progenitors to become osteoclasts.Clinical relevanceSaliva can activate pattern recognition receptor signaling through endotoxins and other stress factors, indicating the demand for macrophages rather than for osteoclasts.