Surface characteristics on commercial dental implants differentially activate macrophages in vitro and in vivo.

OBJECTIVES: Biomaterial implantation provokes an inflammatory response that controls integrative fate. M2 macrophages regulate the response to implants by resolving the inflammatory phase and recruiting progenitor cells to aid healing. We have previously shown that modified titanium (Ti) disks directly induce M2 macrophage polarization. The aim of this study was to examine macrophage response to commercially available Ti or Ti alloy implants with comparable roughness and varying hydrophilicity. MATERIAL AND METHODS: Eleven commercially available Ti (A-F) or Ti alloy (G-K) dental implants were examined in this study. Surface topography, chemistry, and hydrophilicity were characterized for each implant. To compare the immune response in vitro, human monocyte-derived macrophages were seeded on implants and secreted pro- and anti-inflammatory proteins measured. To evaluate the inflammatory response in vivo, mice were subcutaneously instrumented with clinical implants, and implant adherent macrophage populations were characterized by flow cytometry. RESULTS: Macrophages on hydrophobic Implant C produced the highest level of pro-inflammatory proteins in vitro. In contrast, hydrophilic Implant E produced the second-highest pro-inflammatory response. Implants F and K, both hydrophilics, produced the highest anti-inflammatory protein secretions. Likewise, pro-inflammatory CD80hi macrophages predominated in vivo on implants C and E, and M2 CD206 + macrophages predominated on implants F and K. CONCLUSIONS: These findings show that hydrophilicity alone is insufficient to predict the anti-inflammatory effect on macrophage polarization and that other properties-surface composition or topography-determine immune modulation. This in vivo model may be a useful screening method to compare the immunomodulatory response to clinical implants of disparate geometry or size.

Comparable responses of osteoblast lineage cells to microstructured hydrophilic titanium-zirconium and microstructured hydrophilic titanium.

OBJECTIVES: Although titanium (Ti) is commonly used for dental implants, Ti alloy materials are being developed to improve their physical material properties. Studies indicate that osteoblast differentiation and maturation of human mesenchymal stem cells (MSCs) and normal human osteoblasts (NHOsts) respond to microstructured Ti and titanium-aluminum-vanadium (Ti6Al4V) surfaces in a similar manner. The goal of this study was to determine whether this is the case for osteoblast lineage cells grown on microstructured TiZr surfaces and whether their response is affected by surface nanotexture and hydrophilicity. MATERIALS AND METHODS: Grade 4 Ti and TiZr (13-17% Zr) disks were modified by large grit sand-blasting and acid-etching with storage in saline solution, resulting in a complex microstructured and hydrophilic surface corresponding to the commercially available implants SLActive® and Roxolid® SLActive® (Institut Straumann AG, Basel, Switzerland). The subsequent Ti modSLA and TiZr modSLA surfaces were characterized and osteogenic markers were measured. RESULTS: Evaluation of physical parameters revealed that the fabrication method was capable of inducing a microstructured and hydrophilic surface on both the Ti and TiZr disks. Overall, the surfaces were similar, but differences in nanostructure morphology/density and surface chemistry were detected. On Ti modSLA and TiZr modSLA, osteoblastic differentiation and maturation markers were enhanced in both MSCs and NHOsts, while inflammatory markers decreased compared with TCPS. CONCLUSIONS: These results indicate a similar positive cell response of MSCs and NHOsts when cultured on Ti modSLA and TiZr modSLA. Both surfaces were hydrophilic, indicating the importance of this property to osteoblast lineage cells.

Dental implant surface chemistry and energy alter macrophage activation in vitro.

OBJECTIVES: To determine the effects of dental implant surface chemistry and energy on macrophage activation in vitro. MATERIALS AND METHODS: Disks made from two clinically used implant materials (titanium [Ti], titanium zirconium alloy [TiZr]) were produced with two different surface treatments (sandblast/acid-etch [SLA], hydrophilic-SLA [modSLA]). Surface roughness, energy, and chemistry were characterized. Primary murine macrophages were isolated from 6- to 8-week-old male C57Bl/6 mice and cultured on test surfaces (Ti SLA, TiZr SLA, Ti modSLA, TiZr modSLA) or control tissue culture polystyrene. mRNA was quantified by quantitative polymerase chain reaction after 24 h of culture. Pro- (IL-1ß, IL-6, and TNF-α) and anti-inflammatory (IL-4, IL-10) protein levels were measured by ELISA after 1 or 3 days of culture. RESULTS: Quantitatively, microroughness was similar on all surfaces. Qualitatively, nanostructures were present on modSLA surfaces that were denser on Ti than on TiZr. modSLA surfaces were determined hydrophilic (high-energy surface) while SLA surfaces were hydrophobic (low-energy surface). Cells on high-energy surfaces had higher levels of mRNA from anti-inflammatory markers characteristic of M2 activation compared to cells on low-energy surfaces. This effect was enhanced on the TiZr surfaces when compared to cells on Ti SLA and Ti modSLA. Macrophages cultured on TiZr SLA and modSLA surfaces released more anti-inflammatory cytokines. CONCLUSIONS: The combination of high-energy and altered surface chemistry present on TiZr modSLA was able to influence macrophages to produce the greatest anti-inflammatory microenvironment and reduce extended pro-inflammatory factor release.

Signaling components of the 1α,25(OH)2D3-dependent Pdia3 receptor complex are required for Wnt5a calcium-dependent signaling.

Wnt5a and 1α,25(OH)2D3 are important regulators of endochondral ossification. In osteoblasts and growth plate chondrocytes, 1α,25(OH)2D3 initiates rapid effects via its membrane-associated receptor protein disulfide isomerase A3 (Pdia3) in caveolae, activating phospholipase A2 (PLA2)-activating protein (PLAA), calcium/calmodulin-dependent protein kinase II (CaMKII), and PLA2, resulting in protein kinase C (PKC) activation. Wnt5a initiates its calcium-dependent effects via intracellular calcium release, activating PKC and CaMKII. We investigated the requirement for components of the Pdia3 receptor complex in Wnt5a calcium-dependent signaling. We determined that Wnt5a signals through a CaMKII/PLA2/PGE2/PKC cascade. Silencing or blocking Pdia3, PLAA, or vitamin D receptor (VDR), and inhibition of calmodulin (CaM), CaMKII, or PLA2 inhibited Wnt5a-induced PKC activity. Wnt5a activated PKC in caveolin-1-silenced cells, but methyl-beta-cyclodextrin reduced its stimulatory effect. 1α,25(OH)2D3 reduced stimulatory effects of Wnt5a on PKC in a dose-dependent manner. In contrast, Wnt5a had a biphasic effect on 1α,25(OH)2D3-stimulated PKC activation; 50ng/ml Wnt5a caused a 2-fold increase in 1α,25(OH)2D3-stimulated PKC but higher Wnt5a concentrations reduced 1α,25(OH)2D3-stimulated PKC activation. Western blots showed that Wnt receptors Frizzled2 (FZD2) and Frizzled5 (FZD5), and receptor tyrosine kinase-like orphan receptor 2 (ROR2) were localized to caveolae. Blocking ROR2, but not FZD2 or FZD5, abolished the stimulatory effects of 1α,25(OH)2D3 on PKC and CaMKII. 1α,25(OH)2D3 membrane receptor complex components (Pdia3, PLAA, caveolin-1, CaM) interacted with Wnt5a receptors/co-receptors (ROR2, FZD2, FZD5) in immunoprecipitation studies, interactions that changed with either 1α,25(OH)2D3 or Wnt5a treatment. This study demonstrates that 1α,25(OH)2D3 and Wnt5a mediate their effects via similar receptor components and suggests that these pathways may interact.

Superposition of nanostructures on microrough titanium-aluminum-vanadium alloy surfaces results in an altered integrin expression profile in osteoblasts.

Recent studies of new surface modifications that superimpose well-defined nanostructures on microrough implants, thereby mimicking the hierarchical complexity of native bone, report synergistically enhanced osteoblast maturation and local factor production at the protein level compared to growth on surfaces that are smooth, nanorough, or microrough. Whether the complex micro/nanorough surfaces enhance the osteogenic response by triggering similar patterns of integrin receptors and their associated signaling pathways as with well-established microrough surfaces, is not well understood. Human osteoblasts (hOBs) were cultured until confluent for gene expression studies on tissue culture polystyrene (TCPS) or on titanium alloy (Ti6Al4V) disks with different surface topographies: smooth, nanorough, microrough, and micro/nanorough surfaces. mRNA expression of osteogenesis-related markers such as osteocalcin (BGLAP) and bone sialoprotein (BSP), bone morphogenetic protein 2 (BMP2), BMP4, noggin (NOG) and gremlin 1 (GREM1) were all higher on microrough and micro/nanorough surfaces, with few differences between them, compared to smooth and nanorough groups. Interestingly, expression of integrins α1 and α2, which interact primarily with collagens and laminin and have been commonly associated with osteoblast differentiation on microrough Ti and Ti6Al4V, were expressed at lower levels on micro/nanorough surfaces compared to microrough ones. Conversely, the αv subunit, which binds ligands such as vitronectin, osteopontin, and bone sialoprotein among others, had higher expression on micro/nanorough surfaces concomitantly with regulation of the ß3 mRNA levels on nanomodified surfaces. These results suggest that the maturation of osteoblasts on micro/nanorough surfaces may be occurring through different integrin engagement than those established for microrough-only surfaces.

CD4+ and CD8+ T cells reduce inflammation and promote bone healing in response to titanium implants.

T cells are adaptive immune cells essential in pathogenic response, cancer, and autoimmune disorders. During the integration of biomaterials with host tissue, T cells modify the local inflammatory environment by releasing cytokines that promote inflammatory resolution following implantation. T cells are vital for the modulation of innate immune cells, recruitment and proliferation of mesenchymal stem cells (MSCs), and formation of functional tissue around the biomaterial implant. We have demonstrated that deficiency of αß T cells promotes macrophage polarization towards a pro-inflammatory phenotype and attenuates MSC recruitment and proliferation in vitro and in vivo. The goal of this study was to understand how CD4+ and CD8+ T cells, subsets of the αß T cell family, impact the inflammatory response to titanium (Ti) biomaterials. Deficiency of either CD4+ or CD8+ T cells increased the proportion of pro-inflammatory macrophages, lowered anti-inflammatory macrophages, and diminished MSC recruitment in vitro and in vivo. In addition, new bone formation at the implantation site was significantly reduced in T cell-deficient mice compared to T cell-competent mice. Deficiency of CD4+ T cells exacerbated these effects compared to CD8+ T cell deficiency. Our results show the importance of CD4+ and CD8+ T cells in modulating the inflammatory response and promoting new bone formation in response to modified Ti implants. STATEMENT OF SIGNIFICANCE: CD4+ and CD8+ T cells are essential in modulating the peri-implant microenvironment during the inflammatory response to biomaterial implantation. This study shows that deficiency of either CD4+ or CD8+ T cell subsets altered macrophage polarization and reduced MSC recruitment and proliferation at the implantation site.

Membrane estrogen signaling enhances tumorigenesis and metastatic potential of breast cancer cells via estrogen receptor-α36 (ERα36).

Protein kinase C (PKC) signaling can be activated rapidly by 17ß-estradiol (E(2)) via nontraditional signaling in ERα-positive MCF7 and ERα-negative HCC38 breast cancer cells and is associated with tumorigenicity. Additionally, E(2) has been shown to elicit anti-apoptotic effects in cancer cells counteracting pro-apoptotic effects of chemotherapeutics. Supporting evidence suggests the existence of a membrane-associated ER that differs from the traditional receptors, ERα and ERß. Our aim was to identify the ER responsible for rapid PKC activation and to evaluate downstream effects, such as proliferation, apoptosis, and metastasis. RT-PCR, Western blot, and immunofluorescence were used to determine the presence of ER splice variants in multiple cell lines. E(2) effects on PKC activity were measured with and without ER-blocking antibodies. Cell proliferation was determined by [(3)H]thymidine incorporation, and cell viability was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, (MTT) whereas apoptosis was determined by DNA fragmentation and TUNEL. Quantitative RT-PCR and sandwich ELISA were used to determine the effects on metastatic factors. The role of membrane-dependent signaling in cancer cell invasiveness was examined using an in vitro assay. The results indicate the presence of an ERα splice variant, ERα36, in ERα-positive MCF7 and ERα-negative HCC38 breast cancer cells, which localized to plasma membranes and rapidly activated PKC in response to E(2), leading to deleterious effects such as enhancement of proliferation, protection against apoptosis, and enhancement of metastatic factors. These findings propose ERα36 as a novel target for the development of therapies that can prevent progression of breast cancer in the primary tumor as well as during metastasis.

Electrical polarization of titanium surfaces for the enhancement of osteoblast differentiation.

Electrical stimulation has been used clinically to promote bone regeneration in cases of fractures with delayed union or nonunion, with several in vitro and in vivo reports suggesting its beneficial effects on bone formation. However, the use of electrical stimulation of titanium (Ti) implants to enhance osseointegration is less understood, in part because of the few in vitro models that attempt to represent the in vivo environment. In this article, the design of a new in vitro system that allows direct electrical stimulation of osteoblasts through their Ti substrates without the flow of exogenous currents through the media is presented, and the effect of applied electrical polarization on osteoblast differentiation and local factor production was evaluated. A custom-made polycarbonate tissue culture plate was designed to allow electrical connections directly underneath Ti disks placed inside the wells, which were supplied with electrical polarization ranging from 100 to 500 mV to stimulate MG63 osteoblasts. Our results show that electrical polarization applied directly through Ti substrates on which the cells are growing in the absence of applied electrical currents may increase osteoblast differentiation and local factor production in a voltage-dependent manner.

Contribution of αß T cells to macrophage polarization and MSC recruitment and proliferation on titanium implants.

Physiochemical cues like topography and wettability can impact the inflammatory response and tissue integration after biomaterial implantation. T cells are essential for immunomodulation of innate immune cells and play an important role in the host response to biomaterial implantation. This study aimed to understand how CD4+ and CD8+ T cell subsets, members of the αß T cell family, polarize in response to smooth, rough, or rough-hydrophilic titanium (Ti) implants and whether their presence modulates immune cell crosstalk and mesenchymal stem cell (MSC) recruitment following biomaterial implantation. Post-implantation in mice, we found that CD4+ and CD8+ T cell subsets polarized differentially in response to modified Ti surfaces. Additionally, mice lacking αß T cells had significantly more pro-inflammatory macrophages, fewer anti-inflammatory macrophages, and reduced MSC recruitment in response to modified Ti post-implantation than αß T cell -competent mice. Our results demonstrate that T cell activation plays a significant role during the inflammatory response to implanted biomaterials, contributing to macrophage polarization and MSC recruitment and proliferation, and the absence of αß T cells compromises new bone formation at the implantation site. STATEMENT OF SIGNIFICANCE: T cells are essential for immunomodulation and play an important role in the host response to biomaterial implantation. Our results demonstrate that T cells actively participate during the inflammatory response to implanted biomaterials, controlling macrophage phenotype and recruitment of MSCs to the implantation site.

Reduction of neutrophil extracellular traps accelerates inflammatory resolution and increases bone formation on titanium implants.

Neutrophils are the most abundant immune cells in the blood and the first cells to be recruited to the biomaterial implantation site. Neutrophils are fundamental in recruiting mononuclear leukocytes to mount an immune response at the injury site. Neutrophils exert significant pro-inflammatory effects through the release of cytokines and chemokines, degranulation and release of myeloperoxidase (MPO) and neutrophil elastase (NE), and the production of large DNA-based networks called neutrophil extracellular traps (NETs). Neutrophils are initially recruited and activated by cytokines and pathogen- and damage-associated molecular patterns, but little is known about how the physicochemical composition of the biomaterial affects their activation. This study aimed to understand how ablating neutrophil mediators (MPO, NE, NETs) affected macrophage phenotype in vitro and osseointegration in vivo. We discovered that NET formation is a crucial mediator of pro-inflammatory macrophage activation, and inhibition of NET formation significantly suppresses macrophage pro-inflammatory phenotype. Furthermore, reducing NET formation accelerated the inflammatory phase of healing and produced greater bone formation around the implanted biomaterial, suggesting that NETs are essential regulators of biomaterial integration. Our findings emphasize the importance of the neutrophil response to implanted biomaterials and highlight innate immune cells' regulation and amplification signaling during the initiation and resolution of the inflammatory phase of biomaterial integration. STATEMENT OF SIGNIFICANCE: Neutrophils are the most abundant immune cells in blood and are the first to be recruited to the injury/implantation site where they exert significant pro-inflammatory effects. This study aimed to understand how ablating neutrophil mediators affected macrophage phenotype in vitro and bone apposition in vivo. We found that NET formation is a crucial mediator of pro-inflammatory macrophage activation. Reducing NET formation accelerated the inflammatory phase of healing and produced greater appositional bone formation around the implanted biomaterial, suggesting that NETs are essential regulators of biomaterial integration.

Immune cell response to orthopedic and craniofacial biomaterials depends on biomaterial composition.

Materials for craniofacial and orthopedic implants are commonly selected based on mechanical properties and corrosion resistance. The biocompatibility of these materials is typically assessed in vitro using cell lines, but little is known about the response of immune cells to these materials. This study aimed to evaluate the inflammatory and immune cell response to four common orthopedic materials [pure titanium (Ti), titanium alloy (TiAlV), 316L stainless steel (SS), polyetheretherketone (PEEK)]. Following implantation into mice, we found high recruitment of neutrophils, pro-inflammatory macrophages, and CD4+ T cells in response to PEEK and SS implants. Neutrophils produced higher levels of neutrophil elastase, myeloperoxidase, and neutrophil extracellular traps in vitro in response to PEEK and SS than neutrophils on Ti or TiAlV. Macrophages co-cultured on PEEK, SS, or TiAlV increased polarization of T cells towards Th1/Th17 subsets and decreased Th2/Treg polarization compared to Ti substrates. Although SS and PEEK are considered biocompatible materials, both induce a more robust inflammatory response than Ti or Ti alloy characterized by high infiltration of neutrophils and T cells, which may cause fibrous encapsulation of these materials. STATEMENT OF SIGNIFICANCE: Materials for craniofacial and orthopedic implants are commonly selected based on their mechanical properties and corrosion resistance. This study aimed to evaluate the immune cell response to four common orthopedic and craniofacial biomaterials: pure titanium, titanium-aluminum-vanadium alloy, 316L stainless steel, and PEEK. Our results demonstrate that while the biomaterials tested have been shown to be biocompatible and clinically successful, the inflammatory response is largely driven by chemical composition of the biomaterials.

BMP2 induces osteoblast apoptosis in a maturation state and noggin-dependent manner.

Large doses of bone morphogenetic protein 2 (BMP2) are used clinically to induce bone formation in challenging bone defects. However, complications after treatment include swelling, ectopic bone formation, and adjacent bone resorption. While BMP2 can be effective, it is important to characterize the mechanism of the deleterious effects to optimize its use. The aim of this study was to determine the effect of BMP2 on apoptosis in osteoblast lineage cells and to determine the role of the BMP inhibitor Noggin in this process. Human mesenchymal stem cells (MSCs), immature osteoblast-like MG63 cells, and mature normal human osteoblasts (NHOst) were treated with BMP2. A model system of increased endogenous BMP signaling was created by silencing Noggin (shNOG-MG63). Finally, the BMP pathway regulating apoptosis in NHOst was examined using BMP signaling inhibitors (5Z-7-oxozeaenol, dorsomorphin, H-8). Apoptosis was characterized by caspase-3, BAX/BCL2, p53, and DNA fragmentation. BMP2 induced apoptosis in a cell-type dependent manner. While the effect was minor in MSCs, MG63 cells had modest increases and NHOst cells had robust increases apoptosis after BMP2 treatment. Apoptosis was significantly higher in shNOG-MG63 than MG63 cells. 5Z-7-oxozeaenol and dorsomorphin eliminated the BMP2-induced increase in DNA fragmentation in NHOst, suggesting roles for TAB/TAK1 and Smad signaling. These results indicate that the apoptotic effect of BMP2 is dependent on cell maturation state, inducing apoptosis in committed osteoblasts through Smad and TAB/TAK1 signaling, and is regulated by Noggin. Dose and delivery must be optimized in therapeutic applications of BMP2 to minimize complications.

Interrelationship of cranial suture fusion, basicranial development, and resynostosis following suturectomy in twist1(+/-) mice, a murine model of Saethre-Chotzen syndrome.

The interrelationships among suture fusion, basicranial development, and subsequent resynostosis in syndromic craniosynostosis have yet to be examined. The objectives of this study were to determine the potential relationship between suture fusion and cranial base development in a model of syndromic craniosynostosis and to assess the effects of the syndrome on resynostosis following suturectomy. To do this, posterior frontal and coronal suture fusion, postnatal development of sphenooccipital synchondrosis, and resynostosis in Twist1(+/+) (WT) and Twist1(+/-) litter-matched mice (a model for Saethre-Chotzen syndrome) were quantified by evaluating µCT images with advanced image-processing algorithms. The coronal suture in Twist(+/-) mice developed, fused, and mineralized at a faster rate than that in normal littermates at postnatal days 6-30. Moreover, premature fusion of the coronal suture in Twist1(+/-) mice preceded alterations in cranial base development. Analysis of synchondrosis showed faster mineralization in Twist(+/-) mice at postnatal days 25-30. In a rapid resynostosis model, there was an inability to fuse both the midline posterior frontal suture and craniotomy defects in 21-day-old Twist(+/-) mice, despite having accelerated mineralization in the posterior frontal suture and defects. This study showed that dissimilarities between Twist1(+/+) and Twist1(+/-) mice are not limited to a fused coronal suture but include differences in fusion of other sutures, the regenerative capacity of the cranial vault, and the development of the cranial base.

Canonical Wnt signaling enhances pro-inflammatory response to titanium by macrophages.

Biomaterial characteristics like surface roughness and wettability can determine the phenotype of macrophages following implantation. We have demonstrated that inhibiting Wnt ligand secretion abolishes macrophage polarization in vitro and in vivo; however, the role of canonical Wnt signaling in macrophage activation in response to physical and chemical biomaterial cues is unknown. The aim of this study was to understand whether canonical Wnt signaling affects the response of macrophages to titanium (Ti) surface roughness or wettability in vitro and in vivo. Activating canonical Wnt signaling increased expression of toll-like receptors and interleukin receptors and secreted pro-inflammatory cytokines and reduced anti-inflammatory cytokines on Ti, regardless of surface properties. Inhibiting canonical Wnt signaling reduced pro-inflammatory cytokines on all Ti surfaces and increased anti-inflammatory cytokines on rough or rough-hydrophilic Ti. In vivo, activating canonical Wnt signaling increased total macrophages, pro-inflammatory macrophages, and T cells and decreased anti-inflammatory macrophages on both smooth and rough-hydrophilic implants. Functionally, canonical Wnt activation increases pro-inflammatory macrophage response to cell and cell-extracellular matrix lysates. These results demonstrate that activating canonical Wnt signaling primes macrophages to a pro-inflammatory phenotype that affects their response to Ti implants in vitro and in vivo.

Disrupted development from head to tail: Pervasive effects of postnatal restricted resources on neurobiological, behavioral, and morphometric outcomes.

When a maternal rat nurtures her pups, she relies on adequate resources to provide optimal care for her offspring. Accordingly, limited environmental resources may result in atypical maternal care, disrupting various developmental outcomes. In the current study, maternal Long-Evans rats were randomly assigned to either a standard resource (SR) group, provided with four cups of bedding and two paper towels for nesting material or a limited resource (LR) group, provided with a quarter of the bedding and nesting material provided for the SR group. Offspring were monitored at various developmental phases throughout the study. After weaning, pups were housed in same-sex dyads in environments with SRs for continued observations. Subsequent behavioral tests revealed a sex × resource interaction in play behavior on PND 28; specifically, LR reduced play attacks in males while LR increased play attacks in females. A sex × resource interaction was also observed in anxiety-related responses in the open field task with an increase in thigmotaxis in LR females and, in the social interaction task, females exhibited more external rears oriented away from the social target. Focusing on morphological variables, tail length measurements of LR males and females were shorter on PND 9, 16, and 21; however, differences in tail length were no longer present at PND 35. Following the behavioral assessments, animals were perfused at 56 days of age and subsequent immunohistochemical assays indicated increased glucocorticoid receptors in the lateral habenula of LR offspring and higher c-Fos immunoreactivity in the basolateral amygdala of SR offspring. Further, when tail vertebrae and tail tendons were assessed via micro-CT and hydroxyproline assays, results indicated increased trabecular separation, decreased bone volume fraction, and decreased connectivity density in bones, along with reduced collagen concentration in tendons in the LR animals. In sum, although the restricted resources only persisted for a brief duration, the effects appear to be far-reaching and pervasive in this early life stress animal model.

Protein-disulfide isomerase-associated 3 (Pdia3) mediates the membrane response to 1,25-dihydroxyvitamin D3 in osteoblasts.

Protein-disulfide isomerase-associated 3 (Pdia3) is a multifunctional protein hypothesized to be a membrane receptor for 1,25(OH)(2)D(3). In intestinal epithelium and chondrocytes, 1,25(OH)(2)D(3) stimulates rapid membrane responses that are different from genomic effects via the vitamin D receptor (VDR). In this study, we show that 1,25(OH)(2)D(3) stimulates phospholipase A(2) (PLA(2))-dependent rapid release of prostaglandin E(2) (PGE(2)), activation of protein kinase C (PKC), and regulation of bone-related gene transcription and mineralization in osteoblast-like MC3T3-E1 cells (WT) via a mechanism involving Pdia3. Pdia3 was present in caveolae based on co-localization with lipid rafts and caveolin-1. In Pdia3-silenced (Sh-Pdia3) cells, 1,25(OH)(2)D(3) failed to stimulate PKC and PGE(2) responses; in Pdia3-overexpressing cells (Ov-Pdia3), responses to 1,25(OH)(2)D(3) were augmented. Downstream mediators of Pdia3, PLA(2)-activating protein (PLAA) and arachidonic acid, stimulated similar PKC activation in wild-type, Sh-Pdia3, and Ov-Pdia3 cells supporting the hypothesis that Pdia3 mediates the membrane action of 1,25(OH)(2)D(3). Treatment of MC3T3-E1 cells with 1,25(OH)(2)D(3) for 9 min stimulated rapid phosphorylation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) and increased expression of alkaline phosphatase, MMP-13, and osteopontin but decreased expression of osteocalcin, osteoprotegerin (mRNA and protein), and smad2. These effects were attenuated in Sh-Pdia3 cells. Sh-Pdia3 cells produced higher numbers of von Kossa-positive nodules and alizarin red-positive nodules compared with WT cells with or without 1,25(OH)(2)D(3) treatment whereas Ov-Pdia3 did not show any mineralization. Our data suggest Pdia3 is an important initiator of 1,25(OH)(2)D(3)-stimulated membrane signaling pathways, which have both genomic and non genomic effects during osteoblast maturation.

Enhancement of surface wettability via the modification of microtextured titanium implant surfaces with polyelectrolytes.

Micrometer- and submicrometer-scale surface roughness enhances osteoblast differentiation on titanium (Ti) substrates and increases bone-to-implant contact in vivo. However, the low surface wettability induced by surface roughness can retard initial interactions with the physiological environment. We examined chemical modifications of Ti surfaces [pretreated (PT), R(a) ≤ 0.3 µm; sand blasted/acid etched (SLA), R(a) ≥ 3.0 µm] in order to modify surface hydrophilicity. We designed coating layers of polyelectrolytes that did not alter the surface microstructure but increased surface ionic character, including chitosan (CHI), poly(L-glutamic acid) (PGA), and poly(L-lysine) (PLL). Ti disks were cleaned and sterilized. Surface chemical composition, roughness, wettability, and morphology of surfaces before and after polyelectrolyte coating were examined by X-ray photoelectron spectroscopy (XPS), contact mode profilometry, contact angle measurement, and scanning electron microscopy (SEM). High-resolution XPS spectra data validated the formation of polyelectrolyte layers on top of the Ti surface. The surface coverage of the polyelectrolyte adsorbed on Ti surfaces was evaluated with the pertinent SEM images and XPS peak intensity as a function of polyelectrolyte adsorption time on the Ti surface. PLL was coated in a uniform thin layer on the PT surface. CHI and PGA were coated evenly on PT, albeit in an incomplete monolayer. CHI, PGA, and PLL were coated on the SLA surface with complete coverage. The selected polyelectrolytes enhanced surface wettability without modifying surface roughness. These chemically modified surfaces on implant devices can contribute to the enhancement of osteoblast differentiation.

Substrate stiffness induces neutrophil extracellular trap (NET) formation through focal adhesion kinase activation.

Neutrophils predominate the early inflammatory response to tissue injury and implantation of biomaterials. Recent studies have shown that neutrophil activation can be regulated by mechanical cues such as stiffness or surface wettability; however, it is not known how neutrophils sense and respond to physical cues, particularly how they form neutrophil extracellular traps (NET formation). To examine this, we used polydimethylsiloxane (PDMS) substrates of varying physiologically relevant stiffness (0.2-32 kPa) and examined the response of murine neutrophils to untreated surfaces or to surfaces coated with various extracellular matrix proteins recognized by integrin heterodimers (collagen, fibronectin, laminin, vitronectin, synthetic RGD). Neutrophils on higher stiffness PDMS substrates had increased NET formation and higher secretion of pro-inflammatory cytokines and chemokines. Extracellular matrix protein coatings showed that fibronectin induced the most NET formation and this effect was stiffness dependent. Synthetic RGD peptides induced similar levels of NET formation and pro-inflammatory cytokine release than the full-length fibronectin protein. To determine if the observed NET formation in response to substrate stiffness required focal adhesion kinase (FAK) activity, which is down stream of integrin activation, FAK inhibitor PF-573228 was used. Inhibition of FAK using PF-573228 ablated the stiffness-dependent increase in NET formation and pro-inflammatory molecule secretion. These findings demonstrate that neutrophils regulate NET formation in response to physical and mechanical biomaterial cues and this process is regulated through integrin/FAK signaling.

Control of innate immune response by biomaterial surface topography, energy, and stiffness.

As the focus of implantable biomaterials has shifted from bioinert implants to bioactive designs, recent research has highlighted the complex interactions between cell physiologic systems and material properties, particularly physical cues. From the cells known to interact with implanted biomaterials, the response of the immune system has been a critical target of study recently. Here, we review studies characterizing the response of innate immune cells to various material cues, particularly of those at the surface of implanted materials.The innate immune system consists of cell types with various roles in inflammation. Neutrophils and macrophages serve both phagocytic and signaling roles, especially early in the inflammatory phase of biomaterial implantation. These cell types ultimately dictate the outcome of implants as chronic inflammation, fibrosis, or integration. Other cell types like dendritic cells, mast cells, natural killer cells, and innate lymphoid cells may also serve an immunomodulatory role in the biomaterial context. This review highlights recent advances in our understanding of the role of innate immunity in the response to implantable biomaterials as well as key mechanobiological findings in innate immune cells underpinning these advances. STATEMENT OF SIGNIFICANCE: This review highlights recent advances in the understanding of the role of innate immunity in the response to implantable biomaterials, especially in neutrophils and macrophages, as well as key mechanobiological findings in innate immune cells underpinning these advances. Here we discuss how physicochemical properties of biomaterials control innate immune cell behavior.

E-cigarette Aerosol Mixtures Inhibit Biomaterial-Induced Osseointegrative Cell Phenotypes.

OBJECTIVES: Smoking is a known contributor to the failure of dental implants. Despite a decline in cigarette use, the popularity of e-cigarettes has exploded. However, little is known about how e-cigarettes affect the biologic response to implants. This study examines the effect of e-cigarette aerosol mixtures (ecig-AM) on macrophage activation and osteoblastogenesis of mesenchymal stem cells (MSCs) in response to titanium (Ti) implant surfaces. METHODS: Ecig-AMs were prepared by bubbling aerosol through PBS. Human-derived MSCs or murine-derived macrophages were plated on smooth, rough-hydrophobic, or rough-hydrophilic Ti surfaces in media supplemented with ecig-AM. In macrophages, expression of inflammatory markers was measured by qPCR and macrophage immunophenotype characterized by flow cytometry after 24 hours of exposure. In MSCs, expression of osteogenic markers and inflammatory cytokines was measured by qPCR and ELISA, while alkaline phosphatase activity (ALP) was determined by colorimetric assay. RESULTS: Ecig-AM polarized primary macrophages into a pro-inflammatory state with higher effect on ecig-AM with flavorants and nicotine. Metabolic activity of MSCs decreased in a concentration dependent fashion and was stronger in ecig-AM containing nicotine. MSCs reduced expression of osteogenic markers in response to ecig-AM, but increased RANKL secretion, particularly at the highest ecig-AM concentrations. The effect of ecig-AM exposure was lessened when macrophages or MSCs were cultured on rough-hydrophilic substrates. SIGNIFICANCE: Ecig-AM activated macrophages into a pro-inflammatory phenotype and impaired MSC-to-osteoblast differentiation in response to Ti implant surfaces. These effects were potentiated by flavorants and nicotine, suggesting that e-cigarette use may compromise the osseointegration of dental implants.