Early evolution of enamel matrix proteins is reflected by pleiotropy of physiological functions.

Highly specialized enamel matrix proteins (EMPs) are predominantly expressed in odontogenic tissues and diverged from common ancestral gene. They are crucial for the maturation of enamel and its extreme complexity in multiple independent lineages. However, divergence of EMPs occured already before the true enamel evolved and their conservancy in toothless species suggests that non-canonical functions are still under natural selection. To elucidate this hypothesis, we carried out an unbiased, comprehensive phenotyping and employed data from the International Mouse Phenotyping Consortium to show functional pleiotropy of amelogenin, ameloblastin, amelotin, and enamelin, genes, i.e. in sensory function, skeletal morphology, cardiovascular function, metabolism, immune system screen, behavior, reproduction, and respiratory function. Mice in all KO mutant lines, i.e. amelogenin KO, ameloblastin KO, amelotin KO, and enamelin KO, as well as mice from the lineage with monomeric form of ameloblastin were affected in multiple physiological systems. Evolutionary conserved motifs and functional pleiotropy support the hypothesis of role of EMPs as general physiological regulators. These findings illustrate how their non-canonical function can still effect the fitness of modern species by an example of influence of amelogenin and ameloblastin on the bone physiology.

STIM1 R304W in mice causes subgingival hair growth and an increased fraction of trabecular bone.

Calcium signaling plays a central role in bone development and homeostasis. Store operated calcium entry (SOCE) is an important calcium influx pathway mediated by calcium release activated calcium (CRAC) channels in the plasma membrane. Stromal interaction molecule 1 (STIM1) is an endoplasmic reticulum calcium sensing protein important for SOCE. We generated a mouse model expressing the STIM1 R304W mutation, causing Stormorken syndrome in humans. Stim1R304W/R304W mice showed perinatal lethality, and the only three animals that survived into adulthood presented with reduced growth, low body weight, and thoracic kyphosis. Radiographs revealed a reduced number of ribs in the Stim1R304W/R304W mice. Microcomputed tomography data revealed decreased cortical bone thickness and increased trabecular bone volume fraction in Stim1R304W/R304W mice, which had thinner and more compact bone compared to wild type mice. The Stim1R304W/+ mice showed an intermediate phenotype. Histological analyses showed that the Stim1R304W/R304W mice had abnormal bone architecture, with markedly increased number of trabeculae and reduced bone marrow cavity. Homozygous mice showed STIM1 positive osteocytes and osteoblasts. These findings highlight the critical role of the gain-of-function (GoF) STIM1 R304W protein in skeletal development and homeostasis in mice. Furthermore, the novel feature of bilateral subgingival hair growth on the lower incisors in the Stim1R304W/R304W mice and 25 % of the heterozygous mice indicate that the GoF STIM1 R304W protein also induces an abnormal epithelial cell fate.

Quercitrin Nanocoated Implant Surfaces Reduce Osteoclast Activity In Vitro and In Vivo.

In this study, the effect on osteoclast activity in vitro and in vivo of titanium implants that were coated with quercitrin was evaluated. Titanium surfaces were covalently coated with the flavonoid quercitrin. The effect of the surfaces on osteoclastogenesis was first tested in vitro on RAW264.7 cells that were supplemented with receptor activator of nuclear factor kappa-B ligand (RANKL) to generate osteoclast-like cells by tartrate-resistant acid phosphatase (TRAP) inmunostaining after five days of culture, and by analysis of the mRNA expression levels of markers related to bone resorption after seven days of culture. A rabbit tibial model was used to evaluate the in vivo biological response to the implant surfaces after eight weeks of healing, analyzing the lactate dehydrogenase (LDH) and the alkaline phosphatase (ALP) activities in the wound fluid that were present at the implant interface and the peri-implant bone mRNA expression levels of several markers related to inflammation, bone resorption and osteoblast-osteoclast interaction. No differences between groups and control surfaces were found in the wound fluid analyses. Moreover, quercitrin implant surfaces significantly decreased the expression of osteoclast related genes in vitro (Trap, CalcR, Ctsk, H⁺ATPase, Mmp9) and in vivo (Ctsk, H⁺ATPase, Mmp9) as well as the expression of RankL in vivo. Moreover, quercitrin surfaces were not cytotoxic for the cells. Thus, quercitrin implant surfaces were biocompatible and decreased osteoclastogenesis in vitro and in vivo. This could be used to improve the performance of dental implants.

EMD in periodontal regenerative surgery modulates cytokine profiles: A randomised controlled clinical trial.

The enamel matrix derivative (EMD) contains hundreds of peptides in different levels of proteolytic processing that may provide a range of biological effects of importance in wound healing. The aim of the present study was to compare the effect of EMD and its fractions on the cytokine profiles from human gingival fibroblasts in vitro and in gingival crevicular fluid (GCF) in a randomized controlled split-mouth clinical study (n = 12). Levels of cytokines in cell culture medium and in GCF were measured by Luminex over a 2-week period. In the clinical study, levels of pro-inflammatory cytokines and chemokines were increased, whereas the levels of transforming growth factor-α (TGF-α) and platelet-derived growth factor-BB (PDGF-BB) were reduced. The in vitro study showed that EMD and its high and low molecular weight fractions reduced the secretion of pro-inflammatory cytokines and chemokines compared to untreated cells. EMD had an effect on levels of cytokines related to fibroplasia, angiogenesis, inflammation and chemotaxis both in vitro and in vivo, however, the anti-inflammatory effect induced by EMD observed in the in vitro study could not be confirmed clinically.

Suture materials affect peri-implant bone healing and implant osseointegration.

The aim of this study was to evaluate the effects of the remnants of two suture materials on osseointegration of titanium implants in a rabbit tibial model. Calibrated defects were prepared in the tibia of five Chinchilla rabbits. Filaments of nonresorbable (NR) nylon or resorbable (R) chitosan were placed at the bone to implant interface, whereas control sites had no suture material. After a healing period of 4 weeks, a pull-out test procedure was performed followed by enzymatic analyses of the wound fluid and relative quantification of mRNA levels for bone-related and cytokine markers from the peri-implant bone. A trend toward a reduced pull-out force was observed in the NR group (NR: 23.0 ± 12.8 N; R: 33.9 ± 11.3 N; control: 33.6 ± 24.0 N). Similarly, the bone resorption marker vacuolar type H+-ATPase was increased in the NR group compared with that in the control group (P = 0.041). The R group showed trends for lower alkaline phosphatase activity and osteocalcin expression and higher total protein content and RNA compared with the control group. In this submerged healing model, peri-implant bone healing was marginally affected by the two suture materials tested. However, there was a tendency toward better osseointegration and lower expression of bone resorption markers in the R group compared with the control group.

Proline-Rich Peptide Mimics Effects of Enamel Matrix Derivative on Rat Oral Mucosa Incisional Wound Healing.

BACKGROUND: Proline-rich peptides have been shown to promote periodontal regeneration. However, their effect on soft tissue wound healing has not yet been investigated. The aim of this study is to evaluate the effect of enamel matrix derivative (EMD), tyrosine-rich amelogenin peptide (TRAP), and a synthetic proline-rich peptide (P2) on acute wound healing after a full-thickness flap procedure in an incisional rat model. METHODS: This experimental study has a split-mouth, randomized, placebo-controlled design. Test and control wounds were created on the palatal mucosa of 54 Sprague-Dawley rats. Wounds were histologically processed, and reepithelialization, leukocyte infiltration, and angiogenesis were assessed at days 1, 3, and 7 post-surgery. RESULTS: EMD and P2 significantly promoted early wound closure at day 1 (P <0.001 and P = 0.004, respectively). EMD maintained a significant acceleration of reepithelialization at day 3 (P = 0.004). Wounds treated by EMD and P2 showed increased angiogenesis during the first 3 days of healing (P = 0.03 and 0.001, respectively). Leukocyte infiltration was decreased in EMD-treated wounds at day 1 (P = 0.03), and P2 and TRAP induced a similar effect at days 3 (P = 0.002 and P <0.0001, respectively) and 7 (P = 0.005 and P <0.001). CONCLUSION: EMD and P2 promoted reepithelialization and neovascularization in full-thickness surgical wounds on rat oral mucosa.

Subfractions of enamel matrix derivative differentially influence cytokine secretion from human oral fibroblasts.

Enamel matrix derivative is used to promote periodontal regeneration during the corrective phase of the treatment of periodontal defects. Our main goal was to analyze the bioactivity of different molecular weight fractions of enamel matrix derivative. Enamel matrix derivative, a complex mixture of proteins, was separated into 13 fractions using size-exclusion chromatography and characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and liquid chromatography-electrospray ionization-tandem mass spectrometry. Human periodontal ligament fibroblasts were treated with either enamel matrix derivative or the different fractions. Proliferation and cytokine secretion to the cell culture medium were measured and compared to untreated cells. The liquid chromatography-electrospray ionization-tandem mass spectrometry analyses revealed that the most abundant peptides were amelogenin and leucine-rich amelogenin peptide related. The fractions containing proteins above 20 kDa induced an increase in vascular endothelial growth factor and interleukin-6 secretion, whereas lower molecular weight fractions enhanced proliferation and secretion of interleukin-8 and monocyte chemoattractant protein-1 and reduced interleukin-4 release. The various molecular components in the enamel matrix derivative formulation might contribute to reported effects on tissue regeneration through their influence on vascularization, the immune response, and chemotaxis.

Flavonoid-modified surfaces: multifunctional bioactive biomaterials with osteopromotive, anti-inflammatory, and anti-fibrotic potential.

Flavonoids are small polyphenolic molecules of natural origin with antioxidant, anti-inflammatory, and antibacterial properties. Here, a bioactive surface based on the covalent immobilization of flavonoids taxifolin and quercitrin on titanium substrates is presented, using (3-aminopropyl)triethoxysilane (APTES) as coupling agent. FTIR and XPS measurements confirm the grafting of the flavonoids to the surfaces. Using 2-aminoethyl diphenylborinate (DPBA, a flavonoid-specific dye), the modified surfaces are imaged by fluorescence microscopy. The bioactivity of the flavonoid-modified surfaces is evaluated in vitro with human umbilical cord derived mesenchymal stem cells (hUC-MSCs) and human gingival fibroblasts (HGFs) and compared to that of simple flavonoid coatings prepared by drop casting. Flavonoid-modified surfaces show anti-inflammatory and anti-fibrotic potential on HGF. In addition, Ti surfaces covalently functionalized with flavonoids promote the differentiation of hUC-MSCs to osteoblasts--enhancing the expression of osteogenic markers, increasing alkaline phosphatase activity and calcium deposition; while drop-casted surfaces do not. These findings could have a high impact in the development of advanced implantable medical devices like bone implants. Given the broad range of bioactivities of flavonoid compounds, these surfaces are ready to be explored for other biomedical applications, e.g., as stent surface or tumor-targeted functionalized nanoparticles for cardiovascular or cancer therapies.

Correlation between molecular signals and bone bonding to titanium implants.

OBJECTIVES: A better understanding of the biological processes controlling osseointegration at the bone-to-implant interface is needed. The aim of this study was to examine which are the molecular and biochemical variables that are significantly related to osseointegration, using multiple regression analysis. MATERIALS AND METHODS: Titanium coins were placed into the tibial cortical bone of New Zealand White rabbits and evaluated using pull-out test after 4 and 8 weeks of healing. Correlations between pull-out and several markers from tissue fluid (Lactate dehydrogenase [LDH] and Alkaline phosphatase [ALP] activities and total protein content) and peri-implant bone tissue (total protein, RNA and DNA content, implant area covered with bone and gene expression of osteoblast, osteoclast and inflammation markers) were used to assess the importance of these parameters in bone healing and in relation to implant performance. RESULTS: Our results showed a negative correlation between the content of DNA, RNA and total protein at the peri-implant bone tissue and the pull-out force, indicating that as bone matures and implant becomes more osseointegrated, the organic content of bone decreases. The negative correlation found between pull-out force and ALP activity pointed to a delayed healing in implants with lower pull-out values and primary mineralization still ongoing. LDH activity and total protein content in the tissue fluid were as well negatively correlated with the pull-out force. Finally, a positive correlation was observed between the pull-out force and the expression of the osteoblast and the bone resorption markers, being osteocalcin and collagen-I the best predictive markers for osseointegration after 4 and 8 weeks of healing respectively. CONCLUSIONS: These results suggest that the evaluation of these markers could be relevant for the assessment of new implant surfaces for rapid bone healing and improved implant performance.

Human monocyte responses to lipopolysaccharide and 9-cis retinoic acid after laparoscopic surgery for colon cancer.

Surgery, even modern minimal invasive laparoscopic surgery, induces an initial inflammatory and acute phase response which is followed by a period of immunosuppression rendering surgical patients more susceptible to infection. Here, we aimed to study changes in monocyte inflammatory responses and inflammatory modulation mechanisms following laparoscopic colorectal surgery for colon cancer. Blood samples were collected from 19 colon cancer patients before, directly after and daily for 3 days following surgery. Blood cells were exposed ex vivo to bacterial lipopolysaccharide (LPS) or the inflammatory modulator 9-cis retinoic acid (9cisRA). In blood samples taken prior to surgery, we found significant pro-inflammatory responses to LPS, indicating classical monocyte activation. Directly after surgery, LPS induced significantly less early pro-inflammatory cytokines and monocyte/granulocyte-attracting chemokines. The LPS-mediated release of interleukin (IL)-1ß was still significantly attenuated 3 days after surgery. In patient monocytes collected after surgery, we found increased levels of suppressors of cytokine signaling (SOCS)1 and SOCS3 mRNA, reported to be associated with polarization towards resolving macrophages. The retinoic acid isomer 9cisRA, reported to attenuate LPS-mediated inflammatory responses and alter chemokine responses in cultured monocytes, had a similar effect in patient blood. Three days after surgery, 9cisRA still attenuated pro-inflammatory responses, but the induction of monocyte chemoattractive protein (MCP)-1/CCL2 mRNA in monocytes was reduced. This study indicates changes in monocyte responses that last for at least 3 days after laparoscopic surgery.

9-cis retinoic acid inhibits inflammatory responses of adherent monocytes and increases their ability to induce classical monocyte migration.

Patients with vitamin A/retinol deficiency are shown to be prone to infections and to suffer from increased inflammation, effects which can be remedied by vitamin A supplements. We aimed to study how human monocytes from the peripheral venous blood of healthy donors acted within the initial hours after adherence and exposure to bacterial endotoxin in the presence or absence of the 9-cis-isomer of retinoic acid (9cisRA). We found that adherent human monocytes were dominated by the CD14dimCD16+ subtype. Pretreatment with 9cisRA for 1 h significantly decreased lipopolysaccharide (LPS)-induced mRNA expression and protein release of tumor necrosis factor (TNF)α, interleukin (IL)-6 and chemokine ligands (CCL)3 and CCL4. In contrast, treatment with 9cisRA rapidly enhanced the production of monocyte chemoattractive protein/CCL2. 9cisRA treatment also led to enhanced migration of classical CD14high monocytes in a transwell in vitro system. We conclude that 9cisRA treatment of human adherent monocytes attenuates the inflammatory responses to LPS and induces the attraction of classical monocytes, a feature which may help explain why supplements administered to vitamin A-deficient patients counteract inflammation and increases the ability to fight infections.

Ameloblastin upstream region contains structural elements regulating transcriptional activity in a stromal cell line derived from bone marrow.

Ameloblastin (AMBN) was originally described as a tooth-specific extracellular matrix protein, but current data have shown that AMBN is present in many different tissues of mesenchymal origin. The identification of regulatory elements in the promoter region of the Ambn gene would assist in identifying potential mesenchymal-specific transcriptional factors. In this study we subcloned a 3,788-bp region upstream (and a 54-bp region downstream) of the mouse Ambn transcriptional start site into a LacZ reporter construct and called this construct 3788-Ambn-lacZ. In silico analysis of the 3,788-bp Ambn promoter region identified 50 potential cis-regulatory elements, 29 of which are known to be functional in cell populations of mesenchymal origin. The reporter construct was activated in transfected bone marrow cells, and the promoter activity was induced in cell cultures following addition of recombinant AMBN, interferon-γ, serotonin, or dexamethasone. We discuss the relative significance of the potential cis-acting gene-regulatory elements of Ambn in relation to bone morphogenesis. Knowledge of Ambn gene-regulatory elements will be of importance when developing strategies for bone repair and replacement in a clinical surgical setting.

Enamel matrix derivative stimulates expression and secretion of resistin in mesenchymal cells.

In this study we wanted to identify the effect of enamel matrix derivative (EMD) on adipocytokines, so-called adipokines. Primary human cells of mesenchymal origin (osteoblasts, periodontal ligament cells, mesenchymal stem cells, and pulp cells) and hematopoietic origin (monocytes) were incubated with EMD. The levels of adipokines in cell culture medium were quantified using the Lincoplex human adipocyte panel (Luminex) and by real-time PCR of mRNA isolated from cell lysates. Rats were injected with 2 mg of EMD or saline intramuscularly every third day for 14 d. Blood samples were taken before and after injections, and the level of resistin in rat plasma was measured by ELISA. We found a dramatic increase in the secretion of resistin from mesenchymal stem cells, and verified this result in all the cells of mesenchymal origin tested. However, we observed no significant changes in the amount of resistin secreted from monocytes exposed to EMD compared with the control. Injections of EMD significantly enhanced the circulating levels of resistin in rats, and EMD also significantly enhanced the activity of the resistin promoter in transfected mesenchymal stem cells, indicating a direct effect on resistin expression. Our results indicate that resistin may play a role in mediating the biological effect of EMD in mesenchymal tissues.

Ameloblastin expression during craniofacial bone formation in rats.

Based on previous results showing the expression of ameloblastin (Ambn; amelin) in the formation of mesenchymal dental hard tissues, we investigated its presence during bone development. Immunohistochemistry (IHC), in situ hybridization (ISH), and reverse transcription-polymerase chain reaction (RT-PCR) were used to investigate the expression of ameloblastin protein and mRNA during craniofacial development in rats. Tissue samples were collected on embryonic day 18 and from days 2-28 postnatally. IHC revealed the expression of ameloblastin during bone formation at embryonic and early postnatal stages with different patterns of expression in intramembranous and endochondral ossification. In intramembranous ossification, ameloblastin expression was detected in the superficial layer of the condensed vascularized primitive connective tissue and in the cellular layer covering the surface of the newly formed woven bone. In endochondral ossification, ameloblastin was expressed within the extracellular matrix of the cartilage templates and in the perichondrium. Between days 2 and 28 the expression decreased markedly, concordant with the maturation of the bone, and disappeared after completion of bone remodeling. The results obtained by IHC were confirmed by ISH and RT-PCR, showing the expression of ameloblastin mRNA during craniofacial bone formation. This study indicates the expression of the putative dental protein ameloblastin during craniofacial bone development in rats.

Adiponectin and its receptors are expressed in bone-forming cells.

Adiponectin has until now been considered to be synthesized and secreted exclusively by the adipose tissue, and is reported to influence energy homeostasis and insulin sensitivity. It is also known that body weight is positively correlated with increased bone mineral density and decreased fracture risk. The mechanisms explaining this relation, however, are not completely understood. We report a link between adiponectin and bone homeostasis by demonstrating transcription, translation, and secretion of adiponectin, as well as expression of its receptors, AdipoR1 and AdipoR2, in bone-forming cells. We show that adiponectin and the receptors are expressed in primary human osteoblasts from femur and tibia. The phenotype of bone cells was confirmed by the high expression levels of alkaline phosphatase, collagen type 1, osteocalcin, and CD44, and the formation of mineralization nodules. Immunostaining with monoclonal antibodies also demonstrated the presence of adiponectin in human osteosarcoma cells and normal osteoblasts. Both mRNA expression and secretion of adiponectin to the medium increased during differentiation of human osteoblasts in culture. The adiponectin mRNA level increases in osteoblasts cultured 3 and 7 days in the presence of dietary fatty acids and supplementation of culture medium with recombinant adiponectin enhances the proliferation of murine osteoblasts. The regulation and detailed function of adiponectin in bone still remains obscure, but our findings suggest a functional role in bone homeostasis. If so, adiponectin may provide an important signal linking fat and body weight to bone density.