Effects of endodontic root canal irrigants on tooth dentin revealed by infrared spectroscopy: a systematic literature review.

BACKGROUND: Root canal irrigation endodontic solutions have effects on the chemistry of dentin. Infrared spectroscopy is a non-destructive chemical characterization method where the strength of absorption often correlates with mineral or organic composition. OBJECTIVES: To survey effects of commonly used irrigation solutions on the composition of root dentin as detected by widely-available Fourier transform infrared spectroscopy (FTIR) methods. METHODS: Electronic databases were searched for articles published between 1983 to 2023. After risk of bias assessments (OHAT), studies were grouped according to effects per irrigation solution. Inclusion criteria comprised in vitro studies that used extracted human or bovine teeth, treated by irrigation solutions characterized using FTIR spectroscopy and presenting spectral data. Publications that did not share FTIR spectra were excluded. RESULTS: A wide range of concentrations, durations, and methodologies have been tested but only 30 out of 3452 studies met our inclusion criteria. Different FTIR methods were used with the Attenuated Total Reflection (ATR) variant being the most common (21 studies). Investigated solutions included sodium hypochlorite (NaOCl), ethylenediaminetetraacetic-acid (EDTA), 1-hydroxyethylidene-1-1-diphosphonic-acid (HEDP), peracetic-acid (PAA), glycolic-acid (GA), and citric-acid (CA) though most focused on NaOCl and EDTA. All solutions had detectable effects on the FTIR signature of dentin. NaOCl mainly affects the organic signatures, revealing reduced amide/phosphate ratios with increasing concentrations. EDTA mainly effects the inorganic component, increasing with time and concentration, yet glycolic acid has stronger effects than EDTA on dentin. Beyond the type of irrigant and dentin exposure durations, concentration and protocol of application had strong effects. There is a lack of studies comparing similar irrigants under conditions that mimic clinical scenarios analyzing bulk sample because FTIR of powder dentin differs from bulk measurements. SIGNIFICANCE: The ideal root-canal irrigant should combine local disinfection properties with minimal compositional effects on healthy dentin. FTIR methods appear reliable to identify important changes in root dentin chemical composition. Such information can help understand when endodontic irrigation might lead to root degradation or possibly contribute to long term failures such as vertical fractures. Awareness of chemical damage from irrigation procedures may help clinicians select procedures that reduce deleterious effects on the root canal structures.

Absorption Correction for 3D Elemental Distributions of Dental Composite Materials Using Laboratory Confocal Micro-X-ray Fluorescence Spectroscopy.

Confocal micro-X-ray fluorescence (micro-XRF) spectroscopy facilitates three-dimensional (3D) elemental imaging of heterogeneous samples in the micrometer range. Laboratory setups using X-ray tube excitation render the method accessible for diverse research fields but interpretation of results and quantification remain challenging. The attenuation of X-rays in composites depends on the photon energy as well as on the composition and density of the material. For confocal micro-XRF, attenuation severely impacts elemental distribution information, as the signal from deeper layers is distorted by superficial layers. Absorption correction and quantification of fluorescence measurements in heterogeneous composite samples have so far not been reported. Here, an absorption correction approach for confocal micro-XRF combining density information from microcomputed tomography (micro-CT) data with laboratory X-ray absorption spectroscopy (XAS) and synchrotron transmission measurements is presented. The energy dependency of the probing volume is considered during the correction. The methodology is demonstrated on a model composite sample consisting of a bovine tooth with a clinically used restoration material.

Swim training induces distinct osseous gene expression patterns in anosteocytic and osteocytic teleost fish.

The traditional understanding of bone mechanosensation implicates osteocytes, canaliculi, and the lacunocanalicular network in biomechanical adaptation. However, recent findings challenge this notion, as shown in advanced teleost fish where anosteocytic bone lacking osteocytes are nevertheless responsive to mechanical load. To investigate specific molecular mechanisms involved in bone mechanoadaptation in osteocytic and anosteocytic fish bone, we conducted a 5-min single swim-training experiment with zebrafish and ricefish, respectively. Through RNASeq analysis of fish spines, analyzed at various time points following swim training, we uncovered distinct gene expression patterns in osteocytic and anosteocytic fish bones. Notably, osteocytic fish bone exhibited an early response to mechanical load, contrasting to a delayed response observed in anosteocytic fish bones, both within 8 h following stimulation. We identified an increase in osteoblast differentiation in anosteocytic bone following training, while chordoblast activity was delayed. This temporal response suggests a time-dependent adaptation in anosteocytic bone, indicating the presence of intricate feedback networks within bone that lacks osteocytes.

Nanocrystal residual strains and density layers enhance failure resistance in the cleithrum bone of evolutionary advanced pike fish.

Failure-resistant designs are particularly crucial for bones subjected to rapid loading, as is the case for the ambush-hunting northern pike (Esox lucius). These fish have slim and low-density osteocyte-lacking bones. As part of the swallowing mechanism, the cleithrum bone opens and closes the jaw. The cleithrum needs sufficient strength and damage tolerance, to withstand years of repetitive rapid gape-and-suck cycles of feeding. The thin wing-shaped bone comprises anisotropic layers of mineralized collagen fibers that exhibit periodic variations in mineral density on the mm and micrometer length scales. Wavy collagen fibrils interconnect these layers yielding a highly anisotropic structure. Hydrated cleithra exhibit Young's moduli spanning 3-9 GPa where the yield stress of ∼40 MPa increases markedly to exceed ∼180 MPa upon drying. This 5x observation of increased strength corresponds to a change to brittle fracture patterns. It matches the emergence of compressive residual strains of ∼0.15% within the mineral crystals due to forces from shrinking collagen layers. Compressive stresses on the nanoscale, combined with the layered anisotropic microstructure on the mm length scale, jointly confer structural stability in the slender and lightweight bones. By employing a range of X-ray, electron and optical imaging and mechanical characterization techniques, we reveal the structure and properties that make the cleithra impressively damage resistant composites. STATEMENT OF SIGNIFICANCE: By combining structural and mechanical characterization techniques spanning the mm to the sub-nanometer length scales, this work provides insights into the structural organization and properties of a resilient bone found in pike fish. Our observations show how the anosteocytic bone within the pectoral gridle of these fish, lacking any biological (remodeling) repair mechanisms, is adapted to sustain natural repeated loading cycles of abrupt jaw-gaping and swallowing. We find residual strains within the mineral apatite nanocrystals that contribute to forming a remarkably resilient composite material. Such information gleaned from bony structures that are different from the usual bones of mammals showcases how nature incorporates smart features that induce damage tolerance in bone material, an adaptation acquired through natural evolutionary processes.

Focused ion beam-SEM 3D study of osteodentin in the teeth of the Atlantic wolfish Anarhichas lupus.

The palette of mineralized tissues in fish is wide, and this is particularly apparent in fish dentin. While the teeth of all vertebrates except fish contain a single dentinal tissue type, called orthodentin, dentin in the teeth of fish can be one of several different tissue types. The most common dentin type in fish is orthodentin. Orthodentin is characterized by several key structural features that are fundamentally different from those of bone and from those of osteodentin. Osteodentin, the second-most common dentin type in fish (based on the tiny fraction of fish species out of ∼30,000 extant fish species in which tooth structure was so far studied), is found in most Selachians (sharks and rays) as well as in several teleost species, and is structurally different from orthodentin. Here we examine the hypothesis that osteodentin is similar to anosteocytic bone tissue in terms of its micro- and nano-structure. We use Focused Ion Beam-Scanning Electron Microscopy (FIB/SEM), as well as several other high-resolution imaging techniques, to characterize the 3D architecture of the three main components of osteodentin (denteons, inter-denteonal matrix, and the transition zone between them). We show that the matrix of osteodentin, although acellular, is extremely similar to mammalian osteonal bone matrix, both in general morphology and in the three-dimensional nano-arrangement of its mineralized collagen fibrils. We also document the presence of a complex network of nano-channels, similar to such networks recently described in bone. Finally, we document the presence of strings of hyper-mineralized small 'pearls' which surround the denteonal canals, and characterize their structure.

The rostral micro-tooth morphology of blue marlin, Makaira nigricans.

Billfish rostra potentially have several functions; however, their role in feeding is unequivocal in some species. Recent work linked morphological variation in rostral micro-teeth to differences in feeding behavior in two billfish species, the striped marlin (Kajikia audax) and the sailfish (Istiophorus platypterus). Here, we present the rostral micro-tooth morphology for a third billfish species, the blue marlin (Makaira nigricans), for which the use of the rostrum in feeding behavior is still undocumented from systematic observations in the wild. We measured the micro-teeth on rostrum tips of blue marlin, striped marlin, and sailfish using a micro-computed tomography approach and compared the tooth morphology among the three species. This was done after an analysis of video-recorded hunting behavior of striped marlin and sailfish revealed that both species strike prey predominantly with the first third of the rostrum, which provided the justification to focus our analysis on the rostrum tips. In blue marlin, intact micro-teeth were longer compared to striped marlin but not to sailfish. Blue marlin had a higher fraction of broken teeth than both striped marlin and sailfish, and broken teeth were distributed more evenly on the rostrum. Micro-tooth regrowth was equally low in both marlin species but higher in sailfish. Based on the differences and similarities in the micro-tooth morphology between the billfish species, we discuss potential feeding-related rostrum use in blue marlin. We put forward the hypothesis that blue marlin might use their rostra in high-speed dashes as observed in striped marlin, rather than in the high-precision rostral strikes described for sailfish, possibly focusing on larger prey organisms.

Deep learning to overcome Zernike phase-contrast nanoCT artifacts for automated micro-nano porosity segmentation in bone.

Bone material contains a hierarchical network of micro- and nano-cavities and channels, known as the lacuna-canalicular network (LCN), that is thought to play an important role in mechanobiology and turnover. The LCN comprises micrometer-sized lacunae, voids that house osteocytes, and submicrometer-sized canaliculi that connect bone cells. Characterization of this network in three dimensions is crucial for many bone studies. To quantify X-ray Zernike phase-contrast nanotomography data, deep learning is used to isolate and assess porosity in artifact-laden tomographies of zebrafish bones. A technical solution is proposed to overcome the halo and shade-off domains in order to reliably obtain the distribution and morphology of the LCN in the tomographic data. Convolutional neural network (CNN) models are utilized with increasing numbers of images, repeatedly validated by `error loss' and `accuracy' metrics. U-Net and Sensor3D CNN models were trained on data obtained from two different synchrotron Zernike phase-contrast transmission X-ray microscopes, the ANATOMIX beamline at SOLEIL (Paris, France) and the P05 beamline at PETRA III (Hamburg, Germany). The Sensor3D CNN model with a smaller batch size of 32 and a training data size of 70 images showed the best performance (accuracy 0.983 and error loss 0.032). The analysis procedures, validated by comparison with human-identified ground-truth images, correctly identified the voids within the bone matrix. This proposed approach may have further application to classify structures in volumetric images that contain non-linear artifacts that degrade image quality and hinder feature identification.

Acidic Osteoid Templates the Plywood Structure of Bone Tissue.

Bone is created by osteoblasts that secrete osteoid after which an ordered texture emerges, followed by mineralization. Plywood geometries are a hallmark of many trabecular and cortical bones, yet the origin of this texturing in vivo has never been shown. Nevertheless, extensive in vitro work revealed how plywood textures of fibrils can emerge from acidic molecular cholesteric collagen mesophases. This study demonstrates in sheep, which is the preferred model for skeletal orthopaedic research, that the deeper non-fibrillar osteoid is organized in a liquid-crystal cholesteric geometry. This basophilic domain, rich in acidic glycosaminoglycans, exhibits low pH which presumably fosters mesoscale collagen molecule ordering in vivo. The results suggest that the collagen fibril motif of twisted plywood matures slowly through self-assembly thermodynamically driven processes as proposed by the Bouligand theory of biological analogues of liquid crystals. Understanding the steps of collagen patterning in osteoid-maturation processes may shed new light on bone pathologies that emerge from collagen physico-chemical maturation imbalances.

A strong quick-release biointerface in mussels mediated by serotonergic cilia-based adhesion.

The mussel byssus stem provides a strong and compact mechanically mismatched biointerface between living tissue and a nonliving biopolymer. Yet, in a poorly understood process, mussels can simply jettison their entire byssus, rebuilding a new one in just hours. We characterized the structure and composition of the byssus biointerface using histology, confocal Raman mapping, phase contrast-enhanced microcomputed tomography, and advanced electron microscopy, revealing a sophisticated junction consisting of abiotic biopolymer sheets interdigitated between living extracellular matrix. The sheet surfaces are in intimate adhesive contact with billions of motile epithelial cilia that control biointerface strength and stem release through their collective movement, which is regulated neurochemically. We posit that this may involve a complex sensory pathway by which sessile mussels respond to environmental stresses to release and relocate.

Morphological and genetic mechanisms underlying the plasticity of the coral Porites astreoides across depths in Bermuda.

The widespread decline of shallow-water coral reefs has fueled interest in assessing whether mesophotic reefs can act as refugia replenishing deteriorated shallower reefs through larval exchange. Here we explore the morphological and molecular basis facilitating survival of planulae and adults of the coral Porites astreoides (Lamarck, 1816; Hexacorallia: Poritidae) along the vertical depth gradient in Bermuda. We found differences in micro-skeletal features such as bigger calyxes and coarser surface of the skeletal spines in shallow corals. Yet, tomographic reconstructions reveal an analogous mineral distribution between shallow and mesophotic adults, pointing to similar skeleton growth dynamics. Our study reveals patterns of host genetic connectivity and minimal symbiont depth-zonation across a broader depth range than previously known for this species in Bermuda. Transcriptional variations across life stages showed different regulation of metabolism and stress response functions, unraveling molecular responses to environmental conditions at different depths. Overall, these findings increase our understanding of coral acclimatory capability across broad vertical gradients, ultimately allowing better evaluation of the refugia potential of mesophotic reefs.

Physiological and morphological plasticity in Stylophora pistillata larvae from Eilat, Israel, to shallow and mesophotic light conditions.

Mesophotic reefs have been proposed as climate change refugia but are not synonymous ecosystems with shallow reefs and remain exposed to anthropogenic impacts. Planulae from the reef-building coral Stylophora pistillata, Gulf of Aqaba, from 5- and 45-m depth were tested ex situ for capacity to settle, grow, and acclimate to reciprocal light conditions. Skeletons were scanned by phase contrast-enhanced micro-CT to study morphology. Deep planulae had reduced volume, smaller diameter on settlement, and greater algal symbiont density. Light conditions did not have significant impact on settlement or mortality rates. Photosynthetic acclimation of algal symbionts was evident within 21-35 days after settlement but growth rate and polyp development were slower for individuals translocated away from their parental origin compared to controls. Though our data reveal rapid symbiont acclimation, reduced growth rates and limited capacity for skeletal modification likely limit the potential for mesophotic larvae to settle on shallow reefs.

The role and risks of selective adaptation in extreme coral habitats.

The alarming rate of climate change demands new management strategies to protect coral reefs. Environments such as mangrove lagoons, characterized by extreme variations in multiple abiotic factors, are viewed as potential sources of stress-tolerant corals for strategies such as assisted evolution and coral propagation. However, biological trade-offs for adaptation to such extremes are poorly known. Here, we investigate the reef-building coral Porites lutea thriving in both mangrove and reef sites and show that stress-tolerance comes with compromises in genetic and energetic mechanisms and skeletal characteristics. We observe reduced genetic diversity and gene expression variability in mangrove corals, a disadvantage under future harsher selective pressure. We find reduced density, thickness and higher porosity in coral skeletons from mangroves, symptoms of metabolic energy redirection to stress response functions. These findings demonstrate the need for caution when utilizing stress-tolerant corals in human interventions, as current survival in extremes may compromise future competitive fitness.

Scanning electron microscopy evaluation of enamel surfaces using different air-polishing powders in the orthodontic setting: an in vitro study.

PURPOSE: The aim of this in vitro study was to quantify and compare changes of the enamel surface caused by periodical use of different air-polishing powders during multibracket therapy. METHODS: Bovine high-gloss polished enamel specimens were air-polished using an AIR-FLOW® Master Piezon with maximum powder and water settings. Each specimen was blasted with sodium bicarbonate (AIR-FLOW® Powder Classic, Electro Medical Systems, Munich, Germany) and erythritol (AIR-FLOW® Powder Plus, Electro Medical Systems). Blasting duration was adapted to the powders' cleaning efficacy and corresponded to 25 air-polishing treatments in a patient with braces. A spindle apparatus ensured uniform guidance at a distance of 4 mm and a 90° angle. Qualitative and quantitative assessments were performed with the use of low vacuum scanning electron microscopy. Following external filtering and image processing, arithmetical square height (Sa) and root mean square height (Sq) were determined. RESULTS: Both prophy powders caused a significant increase in enamel roughness. Surfaces blasted with sodium bicarbonate (Sa = 64.35 ± 36.65 nm; Sq = 80.14 ± 44.80 nm) showed significantly (p < 0.001) higher roughness than samples treated with erythritol (Sa = 24.40 ± 7.42 nm; Sq = 30.86 ± 9.30 nm). The observed defects in enamel structure caused by sodium bicarbonate extended across prism boundaries. Prism structure remained intact after air-polishing with erythritol. CONCLUSION: Both applied air-polishing powders led to surface alterations. Despite shorter treatment times, sodium bicarbonate was significantly more abrasive than erythritol. Clinicians must compromise between saving time and abrasively removing healthy enamel.

Primary radiation damage in bone evolves via collagen destruction by photoelectrons and secondary emission self-absorption.

X-rays are invaluable for imaging and sterilization of bones, yet the resulting ionization and primary radiation damage mechanisms are poorly understood. Here we monitor in-situ collagen backbone degradation in dry bones using second-harmonic-generation and X-ray diffraction. Collagen breaks down by cascades of photon-electron excitations, enhanced by the presence of mineral nanoparticles. We observe protein disintegration with increasing exposure, detected as residual strain relaxation in pre-stressed apatite nanocrystals. Damage rapidly grows from the onset of irradiation, suggesting that there is no minimal 'safe' dose that bone collagen can sustain. Ionization of calcium and phosphorous in the nanocrystals yields fluorescence and high energy electrons giving rise to structural damage that spreads beyond regions directly illuminated by the incident radiation. Our findings highlight photoelectrons as major agents of damage to bone collagen with implications to all situations where bones are irradiated by hard X-rays and in particular for small-beam mineralized collagen fiber investigations.

Molecular differences in collagen organization and in organic-inorganic interfacial structure of bones with and without osteocytes.

Bone is a fascinating biomaterial composed mostly of type-I collagen fibers as an organic phase, apatite as an inorganic phase, and water molecules residing at the interfaces between these phases. They are hierarchically organized with minor constituents such as non-collagenous proteins, citrate ions and glycosaminoglycans into a composite structure that is mechanically durable yet contains enough porosity to accommodate cells and blood vessels. The nanometer scale organization of the collagen fibrous structure and the mineral constituents in bone were recently extensively scrutinized. However, molecular details at the lowest hierarchical level still need to be unraveled to better understand the exact atomic-level arrangement of all these important components in the context of the integral structure of the bone. In this report, we unfold some of the molecular characteristics differentiating between two load-bearing (cleithrum) bones, one from sturgeon fish, where the matrix contains osteocytes and one from pike fish where the bone tissue is devoid of these bone cells. Using enhanced solid-state NMR measurements, we underpin disparities in the collagen fibril structure and dynamics, the mineral phases, the citrate content at the organic-inorganic interface and water penetrability in the two bones. These findings suggest that different strategies are undertaken in the erection of the mineral-organic interfaces in various bones characterized by dissimilar osteogenesis or remodeling pathways and may have implications for the mechanical properties of the particular bone. STATEMENT OF SIGNIFICANCE: Bone boasts unique interactions between collagen fibers and mineral phases through interfaces holding together this bio-composite structure. Over evolution, fish have gone from mineralizing their bones aided by certain bone cells called osteocytes, like tetrapod, to mineralization without these cells. Here, we report atomic level differences in collagen fiber cross linking and organization, porosity of the mineral phases and content of citrate molecules at the bio-mineral interface in bones from modern versus ancient fish. The dissimilar structural features may suggest disparate mechanical properties for the two bones. Fundamental level understanding of the organic and inorganic components in bone and the interfacial interactions holding them together is essential for successful bone repair and for treating better tissue pathologies.

Lacunae rostralis: A new structure on the rostrum of sailfish Istiophorus platypterus.

Recent comparative studies of billfishes (Istiophoridae and Xiphiidae) have provided evidence of differences in the form and function of the rostra (bill) among species. Here, we report the discovery of a new structure, lacuna rostralis, on the rostra of sailfish Istiophorus platypterus, which is absent on the rostra of swordfish Xiphias gladius, striped marlin Kajikia audax and blue marlin Makaira nigricans. The lacunae rostralis are small cavities that contain teeth. They were found on the ventral rostrum surface of all I. platypterus specimens examined and dorsally in half of them. Ventrally, the lacunae rostralis were most prominent in the mid-section of the rostrum. Dorsally, they occurred closer to the tip. The density of lacunae rostralis increased towards the rostrum tip but, because they are smaller in size, the percentage of rostrum coverage decreased. The teeth located within the lacunae rostralis were found to be different in size, location and orientation from the previously identified micro-teeth of billfish. We propose two potential functions of the lacunae rostralis that both relate to the use of the bill in feeding: mechanoreception of prey before tapping it with the bill and more efficient prey handling via the creation of suction, or physical grip.

Time-lapse submicrometer particle motion reveals residual strain evolution and damaging stress relaxation in clinical resin composites sealing human root canals.

Polymer based composites are widely used for treatment, for example as biofilm resistant seals of root canal fillings. Such clinical use, however, fails more frequently than other dental composite restorations, due to stress-related misfits. The reason for this is that the biomaterials used are inserted as viscous masses that may bond to the substrate, yet shrinkage stresses arising during setting of the cross-linking polymer, work against durable adhesion. Here we combine phase contrast enhanced time-lapse radiography (radioscopy), digital image correlation (DIC) and submicrometer resolution phase-contrast enhanced microtomography (PCE-CT), to reveal the spatial and temporal dynamics of composite polymerization and strain evolution. Radioscopy of cavities located in the upper part of human root canals demonstrates how the composite post-gelation "densification" is dominated by viscous flow with quantifiable motion of both particles and entrapped voids. Thereafter, these composites enter a "stress-relaxation" stage and exhibit several structural adaptations, induced by residual shrinkage stresses. Consequently critical alterations to the final biomaterial geometry emerge: (i) entrapped bubbles expand; (ii) microscopic root filling pull-out occurs; (iii) the cavity walls deform inwards, and (iv) occasionally delamination ensues, propagating out from the root canal filling along buried restoration-substrate interfaces. Our findings shed new light on the interactions between confined spaces and biomedical composites that cross-link in situ, highlighting the crucial role of geometry in channeling residual stresses. They further provide new insights into the emergence of structural flaws, calling attention to the need to find new treatment options. STATEMENT OF SIGNIFICANCE: This work quantifies recurring spatial and temporal material redistribution in composites used clinically to fill internal spaces in teeth. This knowledge is important for both promoting biomaterial resistance against potentially pathologic biofilms and for improving structural capacity to endure years of mechanical function. Our study demonstrates the significant role of geometry and the need for improved control over stress raisers to develop better treatment protocols and new space filling materials. The use of high-brilliance X-rays for time-lapse imaging at submicrometer resolution provides dynamic information about the damaging effects of stress relaxation due to polymerization shrinkage.

Microfluidic-like fabrication of metal ion-cured bioadhesives by mussels.

To anchor in seashore habitats, mussels fabricate adhesive byssus fibers that are mechanically reinforced by protein-metal coordination mediated by 3,4-dihydroxyphenylalanine (DOPA). The mechanism by which metal ions are integrated during byssus formation remains unknown. In this study, we investigated the byssus formation process in the blue mussel, Mytilus edulis, combining traditional and advanced methods to identify how and when metals are incorporated. Mussels store iron and vanadium ions in intracellular metal storage particles (MSPs) complexed with previously unknown catechol-based biomolecules. During adhesive formation, stockpiled secretory vesicles containing concentrated fluid proteins are mixed with MSPs within a microfluidic-like network of interconnected channels where they coalesce, forming protein-metal bonds within the nascent byssus. These findings advance our understanding of metal use in biological materials with implications for next-generation metallopolymers and adhesives.

Combined responses of primary coral polyps and their algal endosymbionts to decreasing seawater pH.

With coral reefs declining globally, resilience of these ecosystems hinges on successful coral recruitment. However, knowledge of the acclimatory and/or adaptive potential in response to environmental challenges such as ocean acidification (OA) in earliest life stages is limited. Our combination of physiological measurements, microscopy, computed tomography techniques and gene expression analysis allowed us to thoroughly elucidate the mechanisms underlying the response of early-life stages of corals, together with their algal partners, to the projected decline in oceanic pH. We observed extensive physiological, morphological and transcriptional changes in surviving recruits, and the transition to a less-skeleton/more-tissue phenotype. We found that decreased pH conditions stimulate photosynthesis and endosymbiont growth, and gene expression potentially linked to photosynthates translocation. Our unique holistic study discloses the previously unseen intricate net of interacting mechanisms that regulate the performance of these organisms in response to OA.