The teeth in the study were collected after routine extractions performed for reasons unrelated to the research, and each had no more than two millimetres of physiological root resorption. The sample comprised 41 primary maxillary first molars and 19 second molars, drawn from children aged roughly four to seven years. Because the teeth were fully anonymised and extracted as part of standard clinical care, the ethics committee waived individual informed consent. Every specimen received a random alphanumeric code so that the single board-certified oral and maxillofacial radiologist who made all measurements remained blinded to specimen identity. Before data collection, the examiner completed a calibration session on independent pilot teeth, and re-analysis of 20 percent of the specimens after a two-week interval showed excellent agreement, with an intraclass correlation coefficient of 0.95.

The scanning protocol itself was unusually detailed. Each tooth was mounted in a custom holder and imaged in a desktop SkyScan 1172 system operating at 85 kilovolts and 118 microamperes, with a rotation step of 0.60 degrees and an exposure time of 2600 milliseconds. Raw projections were reconstructed with beam-hardening correction and ring-artefact reduction, and all quantitative measurements were performed in dedicated analysis software. The researchers standardised their reference levels carefully: cross-sections were taken one millimetre below the canal orifice, at mid-root, and one millimetre coronal to the apical constriction. This non-destructive approach allowed the team to map canal lengths, diameters, inter-orifice distances, dentin wall thickness, isthmuses and accessory canals without cutting into or otherwise altering the precious specimens.

A conservative definition governed what counted as MB2. The researchers required a separate orifice divided from the main mesiobuccal orifice by a complete band of dentin, with the canal space traceable as a continuous lumen for at least two millimetres apical to the orifice. Incompletely separated or shorter canal spaces were classified instead as lateral ramifications. This stricter criterion probably explains why the observed prevalence of 43.3 percent, with a 95 percent confidence interval of 31.6 to 55.9 percent, sits in the middle of the previously reported range of roughly 20 to 60 percent for primary maxillary molars. Of the 26 MB2-positive teeth, the orifice arose in the coronal third in 20 cases and in the middle third in six; none appeared in the apical third.

Three-rooted anatomy dominated the sample, with 50 of the 60 teeth, or 83.3 percent, showing the classic trifurcation into mesiobuccal, distobuccal and palatal roots. The remaining ten teeth, 16.7 percent, displayed the typical two-rooted variant produced by fusion of the distobuccal and palatal roots. Even within those fused roots, both canals remained individually traceable on the micro-CT scans. The prevalence of MB2 in these baby molars is lower than the roughly 60 to 70 percent reported in cone-beam CT studies of permanent maxillary first molars, but the clinical stakes are different: primary molars must survive only a few years, yet untreated canal spaces can harbour microbial biofilms that drive chronic apical periodontitis and treatment failure.

Perhaps the most striking morphometric result concerned canal shape. Across 315 classifiable canal lumens, long-oval cross-sections accounted for 48.3 percent and oval profiles for 45.4 percent, with flattened canals making up just 6.3 percent. Not a single canal met the strict round criterion of an aspect ratio below 1.05. Shape did not differ significantly across the coronal, middle and apical root levels, although oval profiles became proportionally more frequent toward the apex. This matters clinically because rotary nickel-titanium instruments are round in cross-section and therefore cannot fully shape an oval canal, leaving buccal and lingual extensions uninstrumented where biofilm may persist. Conversely, larger or oval-path files risk stripping away dentin in roots whose walls are already dangerously thin.

That danger was quantified directly. The minimum dentin thickness at the apical level was lowest in the palatal roots, averaging just 0.10 millimetres, followed by the mesiobuccal roots at 0.12 millimetres and the distobuccal roots at 0.21 millimetres, a difference that was statistically significant. In other words, the thinnest apical walls in these teeth approach a tenth of a millimetre, leaving very little margin for error during apical preparation. The buccolingual diameter significantly exceeded the mesiodistal diameter in the mesiobuccal, MB2 and distobuccal roots at all three levels, while the palatal root showed no significant directional difference, indicating a more circular cross-section that round instruments can shape more completely.

The inter-orifice measurements added another layer of clinical relevance. The shortest mean separation between any pair of canal orifices was between MB1 and MB2, at just 1.12 millimetres, while the greatest was between MB1 and the palatal canal, at 5.54 millimetres. Such tight proximity substantially increases the risk of overlooking MB2 during access cavity preparation, particularly given its 43.3 percent prevalence. The team also mapped isthmuses, the narrow pulp-filled connections between adjacent canals: these were present in the coronal third in 28.3 percent of specimens, in the middle third in 23.3 percent, and in the apical third in only 6.7 percent, with the distobuccal-palatal region the most frequent site. Accessory canals appeared in all four canal types, most often in the distobuccal root at 43.3 percent and least often in MB2 at 10.0 percent, and were most commonly located in the apical third.

The authors are careful to frame the practical implications as anatomically informed considerations rather than validated clinical recommendations, since no treatment outcomes were tested in this laboratory study. They suggest that magnification with dental loupes or an operating microscope may aid MB2 detection, that circumferential instrumentation may suit the oval mesiobuccal and distobuccal canals, and that apical preparation should be conservative in the palatal and mesiobuccal roots where the walls are thinnest. Cone-beam CT is not a realistic clinical option for primary molars because of radiation considerations and the teeth’s limited remaining lifespan, so the value of this ex vivo characterisation lies in supplying the prior anatomical knowledge clinicians need during conventional treatment.

The study has clear limitations. Its in vitro design cannot be extrapolated directly to clinical outcomes, rare configurations such as four-rooted molars may be under-represented in a sample of 60 teeth, and micro-CT findings do not transfer directly to the two-dimensional radiographs used in everyday practice. The specimens came from two universities in Egypt and do not represent the entire Egyptian population, and because the sample contained markedly unequal numbers of first and second molars, a robust tooth-type comparison was not feasible. The authors call for future work correlating these three-dimensional anatomical findings with clinical pulpectomy outcomes and validating the MB2 diagnostic criteria across multiple operators and imaging systems. Even so, the message for paediatric dentistry is vivid: inside every small baby molar lies a landscape of hidden canals, ribbon-like isthmuses and paper-thin walls that only high-resolution imaging could reveal.

Subject of Research: Three-dimensional root and canal anatomy of maxillary primary molars assessed by micro-computed tomography

Article Title: Micro-computed tomography analysis of root and canal morphology in maxillary primary molars

Article References: Eldehna, A. M., Elzahar, S., Ahmad, A. H., El Kharadly, D. Y., Salem, N. A., Abdel-Ghany, D. M., Abd-Allah, N. F., Abd-Allah, M. F., Ahmed, H. M. A., & Abdelkafy, H. (2026). Micro-computed tomography analysis of root and canal morphology in maxillary primary molars. Scientific Reports, 16(1), Article 33592. https://doi.org/10.1038/s41598-026-71739-0

Image Credits: AI Generated

DOI: 10.1038/s41598-026-71739-0

Keywords: micro-CT, primary molars, MB2 canal, root canal anatomy, pulpectomy, paediatric dentistry, dentin thickness, isthmus, accessory canals, canal morphology, dental imaging, Micro-computed

The investigation used a micro-Multiphysics Agent-based model, or micro-MPA, developed to connect the mechanics of bone tissue with the cellular and molecular events that shape regeneration. The framework combines three domains: micro-finite element analysis for local mechanical strain energy density, micro-finite difference calculations for cytokine reaction, diffusion and decay, and discrete agents representing cells that migrate, divide, differentiate and die. Osteocytes act as the principal mechanical sensors, changing production of sclerostin, receptor activator of nuclear factor kappa-B ligand and osteoprotegerin according to their mechanical environment. Low mechanical strain promotes signals associated with osteoclast recruitment, whereas sufficiently strong stimulation can suppress sclerostin and permit osteoblast-mediated bone formation.

The model was based on longitudinal micro-computed tomography data from female PolgA mice with a mitochondrial mutation that produces premature ageing. A 0.5-millimetre osteotomy was stabilised with an external fixator. After the protocol-defined bridging point at approximately three weeks after surgery, animals received either habitual loading through the fixator or an individualised cyclic loading regimen at 10 hertz for 3,000 cycles three times per week. Weekly scans at a 10.5-micrometre isotropic resolution tracked mineralised tissue across the defect. The young cohort underwent surgery at 12 weeks and was evaluated at 19 weeks, while the aged cohort underwent surgery at 35 weeks and was evaluated at 42 weeks.

Mechanical boundary conditions were first calibrated in young animals through a hierarchical grid search designed to reproduce bone-volume fraction in both habitual and cyclic loading conditions. Habitual loading matched held-out young control animals with a 3.8% root mean square error. Calibrating cyclic loading against the young loading group produced a 4.5% error and reproduced the prominent increase in lateral bone formation, while remaining close to the experimental medial response. In the calibrated mechanical environment, the fixator did more than stabilise the fracture: it shared part of the applied force with the healing tissue and introduced bending that shielded the lateral cortex during habitual loading. Additional cyclic loading partly offset this asymmetry by generating high strain in the lateral callus.

Once these conditions were fixed, the researchers tested two biological scenarios in the aged animals. In the first, native cortical osteocytes and osteocytes newly embedded in the callus both received a degenerated phenotype. In the second, the native cortex retained its aged parameters, but the callus osteocytes received the youthful baseline. The degenerated state doubled both the assumed sclerostin production rate and the strain-energy-density threshold required for mechanosensitivity. These were parameter assumptions adapted from descriptions of age-related impairment, not values directly measured in the mice. Osteocyte density and embedding rate were also held constant across conditions, so the comparison isolated selected aspects of cellular function rather than differences in cell number.

The distinction between the two scenarios was pronounced under cyclic loading. With degenerated callus osteocytes, the simulation produced widespread resorption and little formation despite elevated local strain energy. At week 42, it predicted a bone-volume fraction of 49.0% plus or minus 6.2% in the defect centre, compared with 58.4% plus or minus 8.1% in vivo. The aged loading dataset therefore showed significant deviation between simulation and observation. The model also generated a transient bone-volume peak that was absent from the experimental data. By contrast, the rejuvenated-callus scenario predicted 56.2% plus or minus 8.3% in the defect centre, close to the measured value, and its longitudinal bone-volume curves did not differ detectably from the experimental curves.

Cell-level outputs offered a mechanistic explanation for this difference. In the degenerated scenario, osteocytes exposed to substantial mechanical stimulation continued to produce elevated levels of sclerostin, suppressing osteoblast recruitment even where strain exceeded the threshold for osteogenic differentiation. The model’s qualitative outputs suggest that sclerostin dysregulation was the more proximal driver, although the study did not separately test the effects of sclerostin production and the elevated mechanosensitivity threshold. In the rejuvenated scenario, cyclic loading lowered sclerostin within the lateral callus and created a permissive environment for osteoblasts. Bone formed only where strain was sufficient, reproducing the location and scale of the experimental anabolic response while leaving the adjacent aged cortex comparatively unresponsive.

The findings support an “osteochronology” interpretation of skeletal ageing: the functional age of newly generated tissue may differ from that of the organism that produced it. The aged callus in this model behaved like a locally young unit embedded within an aged skeleton, able to detect and translate mechanical signals even though the surrounding cortex retained a quiescent state. The authors describe this as in silico evidence that the regenerative potential of a fracture callus is modulated by the functional capacity of its embedded osteocytes rather than being determined solely by the systemic environment. However, the model cannot establish that the same tissue-age separation occurs naturally in all older animals or in human fracture healing.

Several limitations constrain the conclusion. PolgA mice exhibit accelerated ageing driven by mitochondrial dysfunction and may not reproduce the full range of skeletal changes associated with natural ageing. The model treats osteocyte function as a binary choice between rejuvenated and degenerated states, although real cell populations are likely to occupy a continuum. Revascularisation was omitted because all included defects had bridged before the simulated period, leaving reduced perfusion as an untested alternative explanation for reduced native-cortex responsiveness. The mechanical conditions calibrated in young animals were assumed to apply to aged animals, although differences in body weight, activity and load partitioning could alter the true stimulus. Statistical comparisons were also underpowered, with four simulated animals per aged group, and a non-significant difference should not be interpreted as proof of equivalence.

The computational setup adds further caveats. Tissue stiffness was derived from mineral density through a linear relationship, so regions with equal density received identical elastic properties despite possible differences in collagen organisation. Osteoclast activity and the remodelling surface were simplified, producing visual discrepancies from the experimental micro-CT structures even when total bone-volume fraction was predicted accurately. The model also represented a single calibrated mechanical boundary condition for each cohort, rather than fitting the effective axial-to-bending ratio of each animal. Within those constraints, the study provides a testable explanation for a striking observation: a smaller aged callus and higher local strains were not sufficient to produce the load-driven formation observed in vivo when osteocytes were modelled as degenerated, whereas assigning the callus a youthful functional phenotype substantially improved agreement with the aged biological data.

Subject of Research: Modelling how osteocyte functional age regulates mechanical remodelling in aged mouse femur defects

Article Title: Agent-based modelling of late-stage mechanical loading of femur defects in prematurely ageing PolgA mice

Article References: Kendall, J. J., Ledoux, C., Marques, F. C., Kuhn, G. A., Schulte, F. A., & Müller, R. (2026). Agent-based modelling of late-stage mechanical loading of femur defects in prematurely ageing PolgA mice. Frontiers in Bioengineering and Biotechnology, 14, Article 1879777. https://doi.org/10.3389/fbioe.2026.1879777

Image Credits: AI Generated

DOI: 10.3389/fbioe.2026.1879777

Keywords: bone regeneration, osteocytes, agent-based modelling, mechanical loading, fracture healing, callus remodelling, cellular ageing, micro-CT, sclerostin, PolgA mice, Agent-based, modelling

Researchers recovered ML2631 from the Zimbral vertebrate microfossil assemblage near Lourinhã, where fossils occur in grey marls and silty mudstones interpreted as low-energy floodplain or lagoonal deposits. The specimen comprises a partial skull table and braincase preserved in a calcareous nodule. Its age is constrained to the upper Kimmeridgian–lower Tithonian, at the transition between those stages of the Late Jurassic.

This palaeontological study combined mechanical preparation, high-resolution micro-computed tomography, anatomical comparison, neurosensory estimation and parsimony-based phylogenetic analysis. Because matrix and dense mineral veins obscured parts of the skull, the researchers used micro-CT to segment the preserved anatomy. The scan produced 736 images with a voxel size of 0.049038 mm, although mineralisation created artefacts that complicated reconstruction.

Despite those artefacts, the team digitally reconstructed most of the skull, a nearly complete brain endocast, parts of both inner ears, several cranial nerves, part of the left internal carotid artery and most of the left paratympanic sinus system. The reconstructed brain has the sigmoidal profile reported in mesoeucrocodylians, with estimated cephalic and pontine flexure angles of approximately 150 and 145 degrees, respectively. The rhombencephalon accounts for about 55–60% of total brain length, a proportion the authors associated with neurocranial maturity.

The preserved inner ear was incomplete, although the left side was nearly complete and was combined with the anterior half of the right side in a tentative reconstruction. Its elevated anterior semicircular canal and tall, slender crus commune resembled conditions described in fully terrestrial crocodylomorphs. However, moderate canal thickness and the absence of a dorsoventrally compressed labyrinth were also compatible with eusuchians and living crocodylians. The authors therefore described the vestibular anatomy as intermediate between terrestrial and semi-aquatic forms, rather than treating it as direct proof of terrestrial locomotion.

Auditory capacity was estimated from an approximately 4.01 mm endosseous cochlear duct and an approximately 40.13 mm basicranial length, producing a logarithmic ratio of −1. This approximate result falls within the reported ranges for allodaposuchids and crocodylians and suggests adaptation for detecting low-frequency sounds. The authors noted that the cochlear region and pterygoid–palatine suture are poorly preserved, making both measurements uncertain. Olfactory capacity and the Reptile Encephalisation Quotient could not be calculated because the olfactory bulbs and body-mass proxies were unavailable.

Visual acuity was assessed by comparing reconstructed brain regions. The estimated total brain volume was 1,716.02 mm³, while the mesencephalon measured 154.2 mm³, or 8.98% of the total. This ratio is closer to the lower end of the range reported for large living crocodylians than to the higher ratios typical of smaller individuals. The authors suggested that ML2631 may therefore have been a fully grown, dwarf individual, although its ontogenetic stage could not be established securely. Comparisons with more complete atoposaurid skeletons produced a rough total-length estimate of 0.5–1 metre, probably near 0.8 metres.

The phylogenetic matrix contained 81 taxa and 321 characters. All searches recovered 30 most parsimonious trees of 1,489 steps and a strict consensus in which ML2631 was placed within Atoposauridae, between Knoetschkesuchus and Sabresuchus. Including the specimen resolved polytomies in the earlier analysis, but most internal nodes had Bremer values of 1 and bootstrap support below 50%. The Varanosuchus–Aprosuchus pairing was the exception, with a Bremer value of 6 and 83% bootstrap support.

ML2631 preserves a dorsolaterally open cranioquadrate passage, large supratemporal fenestrae with posterior fossae, a posteriorly placed septate choana and a discontinuous groove on the squamosal lobe. This combination was not matched by any known atoposaurid species and supports the possibility that the specimen represents a distinct taxon. Nevertheless, the authors retained the open designation Atoposauridae indet. because the missing teeth, much of the snout, palate and postcranial skeleton prevent adequate diagnosis and leave the species-level status and ecological interpretation uncertain.

Subject of Research: Micro-CT reconstruction and phylogenetic analysis of an indeterminate Upper Jurassic atoposaurid from Portugal

Article Title: New dwarf crocodylomorph from the Upper Jurassic of Portugal and the first neuroanatomical data for Atoposauridae

Article References: Puértolas-Pascual, E. (2025). New dwarf crocodylomorph from the Upper Jurassic of Portugal and the first neuroanatomical data for Atoposauridae. Fossil Record, 28(2), 321-346. https://doi.org/10.3897/fr.28.167846

Image Credits: AI Generated

DOI: 10.3897/fr.28.167846

Keywords: Atoposauridae, crocodylomorph, palaeoneurology, micro-CT, Lourinhã Formation, Upper Jurassic, Portugal, neurosensory reconstruction, phylogenetics, palaeontology, dwarf, Upper

Most of the genus's diversity has been described from caves explored on the Spanish, Cantabrian-Pyrenean side of its range, but only rarely from the French side of the western Pyrenees. The species I. bellesi, first described in 1973, is considered uniquely Pyrenean and differentiates morphologically from its eight known Basque-Cantabrian congeners, including I. schaufussi, I. biscaiense, I. vasconicum, I. zaldivarae, I. percostulatum, I. gittenbergeri, I. praetermissum and I. costulatum, as well as the considered fossil I. goliath. In contrast to its eastern Alpine-Dinaric sister genus Zospeum, multiple species sympatry is not unusual for Iberozospeum.

Although empty shells of I. bellesi have frequently been found in southwestern French caves, only two live populations had been recorded so far before this study. Enhanced exploration in April 2025 contributed four more to the tally, of which only one, Lezea cave, was previously known for which DNA sequence data existed from the French side of the Franco-Spanish border region. In the most current phylogenetic study of the genus, only two populations from two proximate non-contiguous cave systems, one from Lexotoa cave in Spain, roughly 100 meters eastwards from the Franco-Spanish border, and Lezea cave in Pyrénées-Atlantiques, less than 1 kilometer from Lexotoa, were assigned to the I. bellesi clade.

That clade differentiated genetically from the biscaiense-zaldivarae clade but did not differentiate from other species using the selected genetic markers, COI, 16S, H3 and 5.8 S rRNA plus ITS2, leading researchers to suggest that I. bellesi likely constitutes a young radiation. Owing to the scarcity of data from caves on the French side of the Pyrenean chain and the paucity of DNA sequence data for I. bellesi, new biospeleological expeditions were conducted in March 2024 and April 2025 in karst caves of the Sare-Aïnhoa sector of the Franco-Spanish border region and its foothills. The Sare-Aïnhoa karst region lies about fifteen kilometers from the Atlantic Ocean and spans only a few square kilometers into both France and Spain.

In this work, three new populations sampled in March 2024 were assessed using DNA sequence data in sync with the earlier phylogenetic reconstruction, and the new sequences were added to the existing dataset. A total of seven individuals were imaged, measured and molecularly processed, with images and DNA extracts housed in the Natural History Museum Bern in Switzerland. Live specimens were preserved initially in 96 percent ethanol and later transferred to an 85 percent ethanol solution.

Before DNA extraction, every specimen was imaged under sterile conditions from frontal, lateral and apical view. Total DNA extraction used a commercial tissue kit in combination with a QIAcube extraction robot, with each specimen incubated for roughly four hours at 56 degrees Celsius. Interestingly, though successfully sequenced, the DNA content of the individuals from Burgosa cave was somehow too low, so sequences of these specimens could not be included in the phylogenetic study.

The maximum likelihood analysis revealed that the French populations cluster in the I. bellesi lineage. The only animal delivering the complete dataset, specimen NMBE_0585639 from Estérençuby cave ES102 in the Vallée d'Estérenguibel, integrated into the cladogram shown by the earlier study. Remarkably, the other six specimens with an incomplete marker set always clustered in a polytomy including the Spanish I. bellesi populations, for which reason the species identification is considered reliable.

Addition of the new dataset resulted in a new arrangement of the lineages: the I. bellesi and the I. zaldivarae plus I. biscaiense subclades are united, although only weakly supported, taking over the sister position with a support value above 92. This shows that the current state of knowledge is still rather incomplete and that more precise phylogenetic relationships can be expected when applying other methods and markers. Most specimens were scanned with an XRadia MicroXCT-200 imaging system generating 1600 projection images over 360 degrees of rotation for roughly 90 minutes at a voxel size of about 2 micrometers, while the paratype shell of I. bellesi was scanned separately using a NeoScan N80 system at a voxel size of 1.8 micrometers.

The 16-bit image datasets were cropped, histogram adjusted and converted to 8-bit, and further 3D graphical procedures were conducted with specialized volume rendering software. An initial segmentation step involving the watershed function was used to digitally remove impurities from the shells, followed by manual segmentation to discriminate external and internal shell structures. Shell measurements were made on the Micro-CT images, capturing shell height, shell width, aperture height, aperture width, height of last whorl and spire angle for each specimen.

The measurement data reveal that these shells, while minute, demonstrate considerable variation in shape and proportions. Shell height ranges narrowly from 1.17 to 1.60 millimeters and shell width from 0.85 to 1.02 millimeters, yet the ratio of shell height to shell width varies from 1.28 to 1.63, indicating the presence of both relatively squat and markedly elongate shell forms. The shell from cave ES102 demonstrates the stoutest form of the study, most comparable to the paratype material from Cueva de Aso in Huesca, Spain, while an individual from Urio gaina presents a much slenderer shell.

Aperture dimensions are consistent, with the aperture height to width ratio frequently close to 1, indicating that the aperture scales proportionally with shell size and is roughly oval to rounded in shape. The height of the last whorl consistently makes up more than half the total shell height, reflecting a compact spire dominated by the body whorl. Of all measurements, the spire angle presents the greatest variation, ranging from 51.84 to 70.93 degrees, with a low angle reflecting a tall, narrow and pointed spire and a high angle defining a short, wide and blunt spire, and an apparent negative correlation between shell slenderness and spire angle.

The Micro-CT image data reveal minimal variation in morphological characters such as teleoconch sculpture, peristome thickness, columellar configuration, protoconch and whorl configuration, and peristome rim alignment, but noticeable differentiation in aperture shape from ovate to roundish-ovate to round, with the aperture alignment either oblique to the shell axis or vertically aligned to it. The configuration of the parietal callus is quite variable in thickness and extent, and the shells vary in the configuration of the last quarter coiling of the body whorl in relationship to the umbilical zone. In the paratype shell, the end of the final whorl is very short and compactly formed, aligning straight next to a narrow umbilical depression, while one shell from Turziloa cave shows this part extending to the right side of the body whorl, forming a large umbilical depression.

Contrasting conditions are observable even between shells collected in the same cavities of the same caves, which may well reflect different generations of I. bellesi impacted by different environmental influences at different times. Ecologically, living Iberozospeum were found in areas typically characterized by moist to moderately humid walls, which occasionally exhibit signs of carbonated efflorescence. The runoff from these walls fosters the regular accumulation of organic mud where the snails were found on cave walls, in rock crevices and at the base of walls above the interface to the ground.

These sites are characterized by their relative protection from air currents, serving as microhabitats that remain unexposed and prone to continuous, consistent environmental conditions over time. The sampled caves themselves are geologically diverse, developed in Lower Cretaceous limestone, Cretaceous sedimentary layers resting on a Paleozoic base, and Carboniferous limestone, with entrances charted at altitudes ranging from 300 to 650 meters and topography ranging from simple cavities to complex networks. Turziloa cave, for example, is distinguished by an active subterranean river, with the snails found toward the end of the system at the junction of active flow and a fossil gallery, in areas exposed to spray from water flows.

The study concludes that I. bellesi demonstrates a genetically reliable but ecophenotypically variable species in multiple caves of the Pyrénées-Atlantiques, and that neither the size, the altitude nor the structure of the karst network appear to be determining factors for the presence of living Iberozospeum in the western Pyrenees, while studies are in progress to better characterize the ecological parameters that determine the distribution and habitat requirements of this troglobiont species.

Subject of Research: Integrative taxonomic study of the cave snail Iberozospeum bellesi in French Pyrenean caves

Article Title: New exploration, DNA sequence data and 3D X-ray microscopy reveal prevalence of live populations and wide variability in shells of Iberozospeum bellesi in caves of the Pyrénées-Atlantiques, France ( Gastropoda , Ellobioidea , Ellobiidae )

Article References: Jochum, A., Kneubühler, J., Neubert, E., Chauvin, J., Gansoinat, S., Wackenheim, Q., Michalik, P., & Ruthensteiner, B. (2026). New exploration, DNA sequence data and 3D X-ray microscopy reveal prevalence of live populations and wide variability in shells of Iberozospeum bellesi in caves of the Pyrénées-Atlantiques, France (Gastropoda, Ellobioidea, Ellobiidae). Subterranean Biology, 55, 161-186. https://doi.org/10.3897/subtbiol.55.188007

Image Credits: AI Generated

DOI: 10.3897/subtbiol.55.188007

Keywords: Iberozospeum bellesi, cave snails, troglobionts, Pyrénées-Atlantiques, DNA barcoding, Micro-CT, integrative taxonomy, subterranean biology, Pyrenees, shell morphology, karst caves, biodiversity

The synthesis included 275 animals receiving stem cell-based interventions and 275 controls, each assigned within the included studies. Eleven experiments used rabbits and five used rats. Cell products varied substantially, encompassing bone marrow-derived mesenchymal stem cells, adipose-derived stem cells, tendon-derived stem cells, synovium-derived mesenchymal stem cells and human umbilical cord blood-derived mesenchymal stem cells. Other interventions included mesenchymal stem cell-derived extracellular vesicles and exosomes. Delivery methods included direct injection, graft wrapping, cell sheets, fibrin glue, alginate gel, scaffold-based systems and three-dimensional bio-printed sleeves.

Control animals underwent the same type of ACL reconstruction as the corresponding intervention group but did not receive a cellular treatment. Depending on the experiment, controls received PBS, vehicle, fibrin glue, alginate gel, a scaffold without cells or another non-cellular intervention. Sham-operated animals and animals without ACL reconstruction were excluded from the quantitative analysis. This design meant that the pooled comparisons primarily addressed the added value of stem cell-related treatments in reconstructed knees rather than differences between reconstructed and unreconstructed animals.

The researchers searched PubMed, Embase, Scopus, SPORTDiscus and the Cochrane Library from each database’s inception through December 2025. They also screened reference lists from included studies and relevant reviews. Following PRISMA 2020 guidance, two reviewers independently screened records, assessed full texts, extracted data and evaluated risk of bias with SYRCLE’s animal-study tool. The protocol was prospectively registered in PROSPERO as CRD420251137985. The search returned 4,528 records; after 2,130 duplicates were removed, 2,398 records were screened and 132 full-text reports were assessed. Sixteen studies met all inclusion criteria.

Continuous outcomes were combined in random-effects models, with standardized mean differences used when studies employed different measurement scales. Statistical heterogeneity was evaluated with the chi-square test and the I² statistic. The analysis estimated 61% to 76% heterogeneity for the principal pooled outcomes, indicating substantial variation among studies. Follow-up duration was examined in subgroup analyses. Publication-bias assessments included inspection of funnel plots and Egger’s regression tests, although the small number of studies available for each outcome limited the reliability of those tests.

Ultimate failure load showed the largest pooled biomechanical association. Fourteen studies contributed 280 animals per group, yielding a standardized mean difference of 1.34, a 95% confidence interval of 0.93 to 1.75 and P below 0.00001. Heterogeneity for this outcome was high at I² = 75%. Thirteen studies, each contributing 264 animals per group, were available for stiffness. Their pooled standardized mean difference was 0.96, with a 95% confidence interval of 0.64 to 1.27 and P below 0.00001; heterogeneity was 61%. The stiffness result was not statistically significant at two weeks, when the standardized mean difference was 0.60 and the confidence interval ran from minus 0.31 to 1.50, but was significant across the reported later follow-up period from four to 12 weeks.

Micro-computed tomography results also favored the interventions. Seven studies, with 144 animals in each group, contributed to the bone volume fraction analysis. The pooled standardized mean difference was 1.03, the 95% confidence interval was 0.75 to 1.31 and P was below 0.00001. Heterogeneity was high at I² = 76%. Histological evaluation drew on only two studies, with 36 animals in the intervention arms and 36 in the control arms. The pooled standardized mean difference for graft-bone integration was 0.86, with a 95% confidence interval of 0.59 to 1.13 and P below 0.00001. That estimate had high heterogeneity of I² = 72%, and its basis in two studies makes it less secure than the broader biomechanical and imaging results.

Timing varied across the outcomes. The ultimate failure load effect was greatest at four weeks, with a subgroup standardized mean difference of 2.09, and declined at later observations. Stiffness reached a standardized mean difference of 1.49 at six weeks, followed by estimates of 0.85 at eight weeks and 1.02 at 12 weeks or later. Bone volume fraction was highest at four weeks, at 1.61, and fell to 0.68 at eight weeks. The histological estimate decreased from 1.05 at four weeks to 0.59 at 12 weeks or later. These subgroup estimates indicate that the measured differences were most pronounced during early healing, but they do not establish why the effects varied over time.

Several biological processes could plausibly be relevant, but the pooled comparisons did not directly test them. Mesenchymal stem cells have multipotent differentiation potential and may generate bone-like and fibrocartilage-forming tissue at the graft-bone interface. Their immunomodulatory and paracrine activities may also influence inflammation, blood-vessel formation, collagen organization and mineral deposition. Extracellular vesicles and exosomes were investigated as cell-derived products, while scaffolds and bio-printed constructs may improve local cell retention and viability. Because the included studies used different cell sources, doses, graft materials, animal species and delivery systems, the review could not reliably attribute the pooled effects to a particular mechanism, cell type or implantation method.

Risk of bias was judged moderate overall. Most studies were rated low risk for sequence generation, baseline comparability, incomplete outcome data and selective outcome reporting. By contrast, allocation concealment, random housing, blinding of caregivers and random outcome assessment were frequently unclear, and only a small number of studies adequately reported blinding of outcome assessors. Small samples, inconsistent dose reporting, varied timing of administration and short follow-up further restricted the analysis. The evidence did not permit dose-response analysis or the identification of an optimal therapeutic window, and the studies could not assess long-term graft durability, joint stability or possible post-traumatic osteoarthritis.

Funnel plots for ultimate failure load, stiffness and bone volume fraction showed no obvious asymmetry, while Egger’s regression tests were not statistically significant, with all P values above 0.05. These results did not provide strong evidence of publication bias, but they remain limited by the small number of studies contributing to each outcome. Null or negative findings may also be underrepresented in the broader mesenchymal stem cell literature. The meta-analysis therefore indicates that stem cell-based strategies can improve measured tendon-bone healing in preclinical ACL reconstruction models, especially during early integration, but standardized animal protocols, longer follow-up and adequately randomized and blinded studies are needed before clinical trials can test whether those laboratory gains translate into durable patient benefit.

Subject of Research: Preclinical evidence on stem cell therapies for tendon-bone healing after ACL reconstruction

Article Title: Effect of stem cell therapies on tendon-bone healing after anterior cruciate ligament reconstruction in animal models: a systematic review and meta-analysis

Article References: Sun, X., Zhao, S., & Wang, C. (2026). Effect of stem cell therapies on tendon-bone healing after anterior cruciate ligament reconstruction in animal models: a systematic review and meta-analysis. Frontiers in Bioengineering and Biotechnology, 14, Article 1869863. https://doi.org/10.3389/fbioe.2026.1869863

Image Credits: AI Generated

DOI: 10.3389/fbioe.2026.1869863

Keywords: ACL reconstruction, animal studies, stem cell therapy, mesenchymal stem cells, tendon-bone healing, graft integration, meta-analysis, biomechanics, micro-CT, regenerative medicine, Effect, stem

The genus Plusioglyphiulus is one of the most conspicuous and species-rich millipede lineages in Southeast Asia, ranging from southern Myanmar, Thailand, and Laos in the west to Borneo in the east. These millipedes are strongly associated with limestone karst landscapes, which are recognized biodiversity hotspots under severe threat across the region. Most species show a striking degree of micro-endemism: because of limited dispersal capabilities and specific habitat requirements, populations tend to be confined to a single cave or even a solitary limestone outcrop. Despite frequently forming massive populations within caves, nearly all Plusioglyphiulus species lack the classic troglomorphic features of true cave dwellers, such as elongated appendages or complete loss of eyes, and are therefore regarded as troglophiles. Bat guano serves as the primary nutrient source sustaining these subterranean ecosystems.

Cambodia’s karst systems, which blanket large areas of the country’s western and southern parts, remain dramatically understudied compared with neighboring Thailand, which alone harbors 14 recorded Plusioglyphiulus species documented from more than 300 sampled localities. Until recently, only two Cambodian species were known: Plusioglyphiulus dubius, described in 1938 from Angkor, and Plusioglyphiulus boutini, described in 1970 from a cave near Kampong Trach in Kampot Province. A 2020 field survey added two more cavernicolous species from the southern provinces of Kampot and Kep. The paucity of records, the researchers argue, reflects a severe lack of collecting effort rather than any true biogeographical pattern of the genus.

The two new species were collected by hand in August 2024 from separate cave localities in Battambang Province. Plusioglyphiulus parviserratus was found in the dark zone of Wat La-ang Phnom Takream, exclusively on bat guano deposits, where the type series comprised a holotype male together with 26 males and 43 females. Plusioglyphiulus battambangensis was discovered about 20 meters from the entrance of a cave at Asram Kompir Temple, occurring on moist limestone floor, with 17 males and 35 females joining its holotype. Live animals were photographed in their natural habitats with a macro lens, and specimens were preserved in ethanol for morphological study or in high-grade ethanol for molecular analysis, with the preservative replaced after 24 hours to prevent defensive secretions from compromising DNA quality. All type specimens are deposited in the Museum of Zoology, Chulalongkorn University in Bangkok.

Both new species are small, light brown millipedes with darker lateral sides and a narrow median line, adults ranging roughly from 18 to 41 millimeters in length depending on sex and species. Like all cambalopsids, they carry reduced but functional eyes, with each eye patch holding only 7 to 16 flat ommatidia arranged in two or three longitudinal rows. Their bodies are armored with low crests and tubercles whose arrangement, known as the carinotaxic formula, follows a precise pattern that differs between the two species and provides one of the key diagnostic characters separating them from relatives. The male genital structures, the gonopods, are the critical features for species-level identification in this group, and here the two new taxa show distinctive architectures: in P. parviserratus, the male first leg telopodite is distinctly two-segmented with a rudimentary knob-like second segment, the anterior gonopod coxosternal processes are long, slender, and regularly curved anteriorly, vividly resembling a bird’s head, and the broad flagellum process of the posterior gonopod bears a unique micro-serrate upper margin. In P. battambangensis, the second telopoditomere is significantly smaller, the paramedian coxal process is slender and elongated, and the flagellum process is narrow with a smooth margin.

The closest known relative of P. parviserratus is Plusioglyphiulus likhitrakarni, described from eastern Thailand. The two share remarkably similar general somatic characters and basic gonopod structure, but the new species is readily distinguishable by its unique carinotaxic pattern on the collum and midbody metaterga, a significantly reduced number of ommatidia, and two-segmented rather than one-segmented male first leg telopodites. Molecular data robustly support this differentiation: the uncorrected pairwise COI p-distance between the two species is 9.9 percent. The two new Cambodian species are separated from each other by an interspecific distance of 10.3 percent and show divergences of 9.6 to 9.9 percent from their closest known relatives. Although these values approach the lower threshold of the interspecific distances generally observed within the genus, which typically range between 11.4 and 18.2 percent, the substantial genetic gap coupled with clear diagnostic morphological boundaries strongly corroborates the independent evolutionary status of each newly described taxon.

Beyond the descriptions themselves, the study evaluates the resolution efficiency and practical limitations of multiple imaging modalities, offering a methodological comparison that will interest taxonomists working on any small, heavily sclerotized invertebrate. Digital stereomicroscopic photography remains indispensable for capturing true-to-life coloration and providing an overview of the body habitus, but even state-of-the-art optical systems often fail to resolve minute, densely crowded, or overlapping appendages. Traditional line drawings, prepared from focus-stacked digital renderings, allow selective emphasis of diagnostic features, yet their accuracy depends heavily on the investigator’s interpretation, particularly problematic for membranous or semi-translucent structures such as the delicate flagellum process of the posterior gonopods.

Scanning electron microscopy provides unparalleled resolution and depth of field, bypassing optical limitations associated with tissue translucency and rendering the entire specimen surface with uniform, crisp focus, making it the gold standard for illustrating micro-characters. Its drawbacks are that it reveals nothing about internal tissue density and requires preparatory procedures that demand structurally rigid specimens capable of withstanding high-vacuum conditions, with fragile elements risking distortion or artifactual displacement. Micro-computed tomography scanning, performed with a SkyScan 1273 at a voxel size of 4.0 micrometers, has emerged as a powerful, non-destructive alternative. The 3D reconstructions yield highly satisfactory visualizations of external somatic architecture, offering exceptional rotational capabilities and allowing virtual segmentation with color-coded isolation of internal and closely appressed structures in situ, removing the necessity for physical dissection that might damage a unique specimen.

Yet the researchers demonstrate that current in-situ tomographic data remain insufficient for resolving the highly condensed, tightly appressed gonopodial microscopic characters that are essential for species-level discrimination. The primary constraint lies in post-scanning segmentation, where manually delineating boundaries of densely packed structures is hampered by a lack of contrast. Subtle micro-characters, such as the exact setation pattern on the mesal surface of the first leg or the precise configuration of a vestigial second telopoditomere reduced to a mere knob, cannot be successfully discriminated. Because the anterior and posterior gonopods remain naturally tightly appressed in situ, without physical dissection prior to scanning these overlapping elements mutually obscure one another, making reconstruction of individual hidden surfaces exceedingly difficult. The team concludes that a tiered workflow, emphasizing routine photography for quick initial diagnosis while reserving advanced methods such as micro-CT and SEM for complex gonopod analysis, provides the best compromise between cost, time, and morphological resolution.

The discoveries also carry urgent conservation implications. Limestone karsts and their subterranean networks in Southeast Asia are critical hotspots of narrow-range endemism, but they are extremely fragile and increasingly threatened by commercial limestone mining for cement manufacture, agricultural development, and unmanaged cave tourism. Such activities destabilize underground microclimates and deplete essential organic resources, especially bat guano, the fundamental substrate with which many cambalopsid millipedes are intimately connected. Being micro-endemics strictly confined to isolated karst towers, both new species are highly vulnerable to habitat degradation, and any structural compromise to their type localities could result in extinction before their ecological roles are understood. The researchers provide an updated identification key to all six Cambodian Plusioglyphiulus species and stress that basic taxonomic inventories are an essential first step toward assessing the biological value of individual karsts, providing the empirical baseline needed to advocate for sustainable cave management and formal site protection. Given that Cambodia’s extensive limestone formations remain vastly under-sampled, future systematic explorations will undoubtedly reveal many more undescribed diplopods hiding in the region’s caves.

Subject of Research: Integrated taxonomy of two new cavernicolous Plusioglyphiulus millipede species from Cambodia

Article Title: Integrated taxonomy of two new species of the millipede genus Plusioglyphiulus Silvestri, 1923 from Cambodia ( Diplopoda , Spirostreptida )

Article References: Likhitrakarn, N., Golovatch, S. I., Srisonchai, R., Thi, S., Chhin, S., Lou, V., Sinovas, P., Jirapatrasilp, P., Sutcharit, C., Panha, S., & Seesamut, T. (2026). Integrated taxonomy of two new species of the millipede genus Plusioglyphiulus Silvestri, 1923 from Cambodia (Diplopoda, Spirostreptida). ZooKeys, 1289, 237-269. https://doi.org/10.3897/zookeys.1289.204580

Image Credits: AI Generated

DOI: 10.3897/zookeys.1289.204580

Keywords: millipedes, Cambodia, taxonomy, cave biology, karst conservation, COI barcoding, micro-CT, ZooKeys, Integrated, species, millipede, genus

The research team, led by Seung Hoon Lee and Tae-Hwan Kim along with colleagues, set out to address a striking gap in the experimental literature. Although clinicians have long suspected that physical trauma contributes to the onset or worsening of ankylosing spondylitis—patients frequently report that symptoms begin in regions subjected to mechanical strain—rigorous laboratory evidence for this connection has been scarce. Human studies, by their nature, struggle to separate mechanical stress from the many confounding factors that accompany it, from occupation and sport to infection and genetic background. The Korean group therefore turned to a carefully controlled animal model in which mechanical load could be manipulated with precision and its downstream consequences measured at the tissue level.

Their experimental design hinged on the SKG mouse, a strain carrying a mutation in the ZAP-70 gene that predisposes it to a spondyloarthritis-like syndrome closely mirroring human ankylosing spondylitis. When these mice receive an injection of curdlan, a gel-forming polysaccharide that triggers innate immune activation through inflammasome pathways, they develop progressive peripheral arthritis as well as inflammation at the entheses—the critical junctions where tendons and ligaments anchor into bone. Because the disease unfolds in a synchronized, predictable fashion after injection, the model offers a clean experimental window in which to ask whether an added mechanical insult changes the course of pathology.

To induce physiological microtrauma, the researchers immobilized the right ankle joints of the mice for one week using surgical plaster combined with a pipette tip as a rigid splint. The logic is counterintuitive but physiologically sound: when a limb is immobilized, the contralateral limb must compensate, absorbing greater mechanical load during normal movement. In this arrangement, the splinted right ankle served as an internal control, shielded from loading, while the freely moving left ankle became the microtraumatized limb, subjected to compensatory overload. After the plaster was removed, the mice received an intraperitoneal injection of 3 milligrams of curdlan to ignite the inflammatory cascade, and the investigators then tracked both ankles over time under identical systemic conditions.

Clinical arthritis scores, assessed weekly for five weeks, told the first part of the story. The microtraumatized left ankles of the curdlan-injected mice consistently exhibited more severe arthritis than their protected right counterparts, despite both joints sharing the same bloodstream, immune system, and genetic makeup. The only meaningful difference between them was mechanical history. That asymmetry, replicated across the experimental cohort, provides some of the most direct evidence to date that sub-traumatic mechanical stress—far below the threshold of a fracture or dislocation—can amplify inflammatory disease in a susceptible host.

Under the microscope, the differences became even more pronounced. Histological analysis at six weeks revealed aggravated synovitis, the inflammation of the joint lining that drives much of the pain and destruction in spondyloarthritis, and exacerbated enthesitis, the inflammation at tendon and ligament insertion sites that is considered a hallmark lesion of the disease family. Immunohistochemistry then pinpointed the molecular fingerprint of this aggravation: the synovium and enthesis of the overloaded ankles expressed markedly higher levels of the pro-inflammatory cytokines tumor necrosis factor-alpha, interleukin-1 beta, and interleukin-6. These signaling molecules are central players in inflammatory arthritis—biologic drugs targeting tumor necrosis factor in particular have transformed the treatment of ankylosing spondylitis in humans—so their local upregulation in mechanically stressed joints provides a plausible mechanistic bridge between tissue strain and immune escalation.

The structural consequences were equally striking when viewed in three dimensions. Using micro-computed tomography, the team quantified bone changes with high resolution and found a significantly elevated volume of inflamed bone in the microtraumatized ankles, indicating that mechanical stress had not merely intensified the soft-tissue inflammation but had also driven pathological remodeling of the skeleton itself. In ankylosing spondylitis, such remodeling is doubly destructive: chronic inflammation erodes bone while abnormal repair processes produce new bone that progressively fuses joints, robbing them of mobility. Any factor that accelerates this process is therefore of substantial clinical concern.

Perhaps the most unexpected finding emerged far from the site of the original stress. The left hip joints—the contralateral hip, located on the same side as the overloaded ankle—showed proteoglycan loss and narrowing of the joint space, classic radiological signs of cartilage degeneration. This distant pathology implies that the effects of compensatory microtrauma are not confined to the stressed joint itself. Altered gait and load distribution likely propagated abnormal forces up the limb to the hip, where the combination of mechanical derangement and systemic inflammation conspired to degrade cartilage. Alternatively, inflammatory mediators generated in the traumatized ankle may have contributed to distant tissue damage. Either way, the observation underscores how a focal mechanical insult can ripple through the musculoskeletal system when the body is primed for autoinflammation.

Taken together, the results position mechanical stress as a genuine aggravating factor in ankylosing spondylitis progression rather than a passive bystander. The compensatory overload of a single ankle was enough to worsen clinical scores, intensify cytokine expression in synovium and enthesis, expand the burden of inflamed bone, and instigate cartilage breakdown in a distant joint. For patients and clinicians, the message is provocative: protecting joints from repetitive strain, particularly during active or incipient phases of inflammatory disease, might meaningfully slow structural damage. The study also raises questions that the authors’ model is well suited to explore in future work—whether the same holds true for other joints, whether timing matters, and whether interventions that redistribute mechanical load could blunt the effect. For now, the experiment offers a compelling laboratory demonstration of what rheumatologists have long suspected: in a susceptible body, the slow accumulation of small injuries can help turn an inflammatory predisposition into a disease that locks joints in place.

Subject of Research: The effect of mechanical microtrauma on joint inflammation and structural damage in a mouse model of ankylosing spondylitis

Article Title: Effects of microtrauma on joint structural changes in the Curdlan-injected SKG mouse model of ankylosing spondylitis

Article References: Lee, S. H., Kim, D., Jeon, C., Jo, H.-R., Youn, J., Jo, S., & Kim, T.-H. (2026). Effects of microtrauma on joint structural changes in the Curdlan-injected SKG mouse model of ankylosing spondylitis. PLOS One, 21(10), e0360912. https://doi.org/10.1371/journal.pone.0360912

Image Credits: AI Generated

DOI: 10.1371/journal.pone.0360912

Keywords: ankylosing spondylitis, microtrauma, SKG mouse model, curdlan, synovitis, enthesitis, TNF-alpha, micro-CT, bone remodeling, spondyloarthritis, mechanical stress, joint pathology

The discovery rests on four fossil requiem shark teeth of the genus Carcharhinus, surface-collected from two sites near Pienza in the Siena-Radicofani Basin and housed in the public collections of Gruppo AVIS Mineralogia e Paleontologia Scandicci. The teeth come from offshore mudstones of the Argille Azzurre Formation, deposited during the Zanclean age of the Early Pliocene in a semi-enclosed embayment on the outer shelf and upper slope, where low oxygen and abundant organic matter favoured the preservation of marine vertebrates. Three of the teeth are lowers and one is an upper, identified by their serrated cutting edges, narrow cusps and roots with widely separated lobes bisected by a nutrient groove, characters typical of a genus that counted at least eight Mediterranean species in Pliocene times.

What turned these ordinary-looking teeth into evidence was micro-computed tomography. Scanned at the CNR Institute of Applied Physics in Sesto Fiorentino with voxel sizes down to 18 micrometres, the teeth revealed tree-like cavities hidden inside the root dentine, invisible or barely visible from the outside as tiny subcircular holes a fraction of a millimetre across. Each cavity opens at the surface through a single aperture with no rim or platform, extends inward through a short neck and swells into a globular or irregular chamber studded with exploratory branches, some bulbous, some tapering to a point. That architecture is the unmistakable fingerprint of Osedax, whose branching root tissues, housing symbiotic bacteria, are what the worm sinks into the substrate it digests.

In formal terms, the traces belong to the ichnogenus Osspecus, established in 2012 for fossil borings of this exact morphology. Five well-preserved borings were reconstructed in three dimensions, one on a lower tooth from the site of Lucciola Bella and four on an upper tooth from the same locality, with apertures between 0.16 and 0.47 millimetres and chamber volumes all below half a cubic millimetre. A sixth trace, opening within the nutrient groove of a lower tooth from the site of Selvoli, could not be fully segmented because it merges with the groove and the natural vascular canals of the dentine. Based on their short tapering necks and far-from-spherical chambers, the reconstructed borings are referred to Osspecus tusciae, an ichnospecies whose holotype also comes from the Tuscan Pliocene, from the historic whale-bone locality of Orciano Pisano.

The worms were selective eaters. Every boring excavates the root dentine, while the enameloid-coated crowns remain essentially untouched, mirroring exactly how living Osedax fed on deployed shark teeth in the Monterey experiment. The preference makes biological sense: shark tooth roots are built of osteodentine, a porous, organic-rich, bone-like tissue loaded with collagen, the protein the worms’ bacterial symbionts are equipped to digest, whereas the enameloid cap is far more mineralised. Several of the borings have since collapsed into crater-like pits called pockmarks, where the thin roof of dentine over the chamber gave way, and on some teeth these craters coalesce into large damaged areas along the root lobes, a decay pattern familiar from Osspecus traces on fossil whale bones.

The finding widens the known menu of an animal already famous for eating beyond its niche. Osedax was described only in 2004 from whale bones on the deep seafloor of California and lacks a gut entirely, relying on Oceanospirillales bacteria in its root tissues to digest collagen and lipids. Deployment experiments have since shown it feeding on the bones of terrestrial mammals, seals, bony fishes, turtles, birds and alligators, and trace fossils have placed its distinctive borings on fossil birds, bony fishes, whale tooth roots and Cretaceous plesiosaurs and marine turtles. Shark teeth, however, had never yielded the trace, despite three earlier suggestions: contentious circular borings on a Cretaceous lamniform vertebra from Italy, tiny microborings on Lower Cretaceous shark teeth from Austria, and a rootless Megalodon tooth from the deep Pacific whose loss was speculatively pinned on zombie worms. The Tuscan teeth settle the question with three-dimensional morphology, the standard of proof the earlier candidates lacked.

The taphonomic stakes are considerable. Dense Osedax colonisation can pit, degrade and ultimately destroy entire whale skeletons, an influence on the cetacean fossil record so significant it has its own name, the Osedax effect. The authors argue that a parallel, selachian version of this effect likely exists, one that progressively destroys the dentine of shark teeth, including the root and the inner core of the crown, while leaving the enameloid cap pristine. Supporting evidence may already sit in museum drawers and seafloor trawls: roughly half the shark teeth dredged from the seamounts of the southern Pacific are rootless, many deep-sea tooth assemblages are similarly depleted, and some time-averaged coastal accumulations inland contain teeth missing their roots and much of the dentine core yet bearing perfectly preserved enameloid cusps, a preservation style so localised it points to a specific agent. Fungal and bacterial bioerosion are usually blamed, but zombie worms may share the blame.

There is a paradox in the traces themselves: they are self-destroying evidence. Osedax consumes its own substrate, so a boring only survives if the tooth is buried halfway through its journey to the seafloor sediment. The Italian borings are strikingly small, volumes far below one cubic millimetre, compared with the tens of cubic millimetres living species achieve on bone, and the largest boring recorded in the literature exceeds the entire root volume of one of the studied teeth. The authors conclude the traces are probably incomplete, snapshots of worms that would have grown much larger had burial not interrupted them, meaning complete destruction of small, inconspicuous tooth roots is likely common and often total. That invisibility, compounded by the fact that nobody thought to look for Osspecus on shark teeth, explains how a behaviour coexisting with sharks for at least 100 million years went unnoticed until now.

The discovery also feeds a deeper debate about where zombie worms came from. The Monterey experiment prompted the suggestion that shark tooth dentine might be the primordial food source of the genus, and the fossil record of shark-like teeth stretches back some 425 million years, offering a deep-time stage for such an origin. The authors are sceptical: molecular clock estimates and the first appearance of Osspecus in the Albian, roughly 100 million years ago, converge on a Cretaceous origin for Osedax, and both the living worms and their fossil traces concentrate overwhelmingly on the bones of marine tetrapods, whales today, marine reptiles in the Mesozoic. Shark teeth, they conclude, were unlikely to have been the ancestral substrate. Instead, the teeth probably served as benthic islands and ecological stepping stones, small peripheral colonies seeded near whale falls or reptile carcasses that connected distant, isolated food patches across the ocean floor.

One final question hangs over the end-Cretaceous mass extinction. A stratigraphic gap separates the youngest Mesozoic Osspecus, of latest Cretaceous age, from the oldest Cenozoic record, of late middle Eocene date, and how the lineage bridged it is unknown. Sea turtles, the only Mesozoic marine vertebrate group with known Osedax traces whose descendants persist, are one candidate refuge, but shark teeth are an intriguing alternative: the K-Pg event appears to have left some elasmobranch communities largely unscathed, and many ocean floors are carpeted with shed teeth. Whether a carpet of shark dentine helped zombie worms walk through the extinction that killed the dinosaurs remains open, but the Pliocene teeth of Tuscany now prove that wherever dead whales lay, the worms were also nibbling at the sharks.

Subject of Research: First fossil evidence of Osedax zombie worms boring into shark tooth dentine, from Pliocene Carcharhinus teeth in Tuscany

Article Title: Trace fossil evidence for Osedax Rouse et al., 2004 exploiting shark tooth dentine on a Pliocene seafloor: broadening our understanding of a major taphonomic agent

Article References: Collareta, A., Mezzasalma, O., Agresti, J., Barucci, A., Bosio, G., Mulè, F., Casati, S., Di Cencio, A., Bianucci, G., Pieri, A., & Nobile, F. (2025). Trace fossil evidence for Osedax Rouse et al., 2004 exploiting shark tooth dentine on a Pliocene seafloor: broadening our understanding of a major taphonomic agent. Fossil Record, 28(2), 359-375. https://doi.org/10.3897/fr.28.167615

Image Credits: AI Generated

DOI: 10.3897/fr.28.167615

Keywords: Osedax, zombie worms, Osspecus, shark teeth, trace fossils, Carcharhinus, Pliocene, Tuscany, micro-CT, taphonomy, whale fall, Osedax effect

The genus Podosphaeraster currently comprises seven species, all found on the seabed at depths below approximately 70 metres. Their peculiar spherical morphology has made their phylogenetic placement a subject of ongoing debate since they were first described. Early researchers suggested that these animals belonged to Sphaerasteridae, a family originally established for fossil species. However, subsequent detailed skeletal analyses of the fossil forms indicated that the resemblance was likely a result of convergent evolution, leading to the establishment of a separate family, Podosphaerasteridae, within the order Valvatida. The controversy has persisted, with some taxonomists re-instating the genus within Sphaerasteridae and others maintaining the separate family classification. Meanwhile, molecular analyses have revealed that Valvatida itself is likely a large polyphyletic group, meaning its members do not all share a single common ancestor, leaving the taxonomic position of Podosphaeraster still unresolved.

To address this knowledge gap, the researchers conducted a detailed micro-focus X-ray computed tomography examination of the internal skeletal structure of Podosphaeraster toyoshiomaruae, collected from the seas around Japan. One specimen came from the Oshima-shinsone Bank in south-western Japan at depths of 147 to 149 metres, while another, used for morphological observation, was collected between Okinawa-jima Island and Miyako-jima Island at depths of 95 to 100 metres. Both specimens are deposited at the National Museum of Nature and Science in Japan. Concurrently, the team performed shotgun sequencing using next-generation sequencing technology to determine key molecular markers used in previous phylogenetic studies and, remarkably, the complete mitochondrial genome sequence.

The mitochondrial genome of Podosphaeraster toyoshiomaruae was determined to be 16,247 base pairs in length, with a nucleotide composition that is AT-rich at 65.5 percent. The start and stop codons for the 13 protein-coding genes followed patterns common amongst starfish. However, compared to the general gene arrangement known for the broader group of modern starfish, the COX2 gene was found to be translocated in the Podosphaeraster toyoshiomaruae mitogenome, while the arrangement of all other genes, including transfer RNAs, remained identical to the general pattern. This structural rearrangement represents the first evidence of a major protein-coding gene translocation within Asteroidea, the class that contains all starfish. The discovery is particularly striking because mitochondrial gene arrangements in echinoderms are generally highly conserved, although recent mitogenome sequencing has increasingly revealed lineage-specific rearrangements, particularly in brittle stars.

The genetic divergence is mirrored by significant morphological specialisation. The micro-CT scanning, performed at a tube voltage of 115 kilovolts and a tube current of 70 microamperes for 40 minutes, revealed the arrangement and morphology of the skeletal plates in extraordinary detail. The examined specimen had a horizontal diameter of 9.6 millimetres and a vertical diameter of 6.3 millimetres. The apical system was polygonal, located at the abactinal centre and composed of one central, six interradial, five radial and three additional plates. The central plate was oval and surrounded by six elongated interradial plates, while wedge-shaped radial plates were alternately positioned between them. A madreporite, the sieve-like structure through which water enters the water vascular system, was located at the centre of one of the primary interradial plates.

Perhaps the most significant morphological finding concerns the mesentery, the internal membrane that supports the digestive organs. The presence of calcified reinforcement in the mesentery had been previously implied, but the detailed structure of its skeleton is presented for the first time in this study. The micro-CT images revealed that the mesentery skeleton spans the I-plate, an unpaired actinal plate, and connects to the V-plate and five pairs of plates, including three kinds of marginal plates. This observation suggests that the marginal plates of this species may not be homologous with those of other starfish, as the mesentery skeleton in other asteroids is typically contiguous with abactinal and actinal plates and odontophores, but not with marginal plates. In other words, the structures that look like marginal plates in Podosphaeraster may have arisen independently rather than being inherited from a shared ancestor.

Notably, while the connectivity of the mesentery skeleton varies, there is a lack of significant morphological differentiation between the marginal plate series and the adjacent dorsolateral and actinal plates. One researcher has questioned the homology of marginal plates in Podosphaeraster, suggesting that the fossil family Sphaerasteridae evolved from the Mesozoic family Stauranderasteridae through the development of a spherical body and the subsequent loss of differentiated marginal plates. Although varying degrees of marginal plate reduction are shared with the order Velatida, the researchers consider this to be a case of convergent evolution, where similar features arise independently in unrelated lineages due to similar selective pressures rather than shared ancestry.

On the phylogenetic side, the team analysed mitochondrial 12S and 16S rRNA and nuclear histone H3 gene sequences from 117 operational taxonomic units, aligning a total of 345 sequences from the three gene regions. In their resulting phylogenetic tree, the order Valvatida was resolved as polyphyletic, consistent with previous molecular findings. Podosphaeraster toyoshiomaruae was positioned as the most basal lineage within Valvatida, with the family Poraniidae placed adjacent to it. However, this placement received low statistical support in the Maximum Likelihood tree, with a bootstrap value of 63 percent, and no support in the Bayesian Inference tree. The researchers attribute this instability to long-branch attraction, a phylogenetic artefact caused by the excessively rapid evolutionary rate of the sequenced genes in this species, which can make rapidly evolving lineages appear artificially closely related or basal.

The three genetic markers provided poor resolution for the deep phylogeny of starfish higher taxa, likely because their evolutionary rates are too rapid to accurately resolve divergences that occurred approximately 250 million years ago. Substitution saturation tests indicated that the dataset is generally unsaturated at the family level, but the results suggested that saturation may confound relationships amongst specific taxa. The phylogenetic instability is further complicated by ongoing controversy surrounding outgroup selection and root placement in starfish phylogenetics. The researchers provisionally utilised a Velatida clade to root their tree, following previous studies that suggested Velatida resolves basal to Valvatida in molecular analyses. Their resulting topology revealed several discrepancies with previous studies but consistently supported the polyphyly of Valvatida.

To resolve these deep relationships, future studies must employ a larger set of genetic markers with more conservative evolutionary rates. Additionally, discovering extant taxa closely related to the genus Podosphaeraster will be crucial to breaking the long branch and improving phylogenetic accuracy. The researchers also emphasise that more observations of mesenteric skeletons using micro-CT across various Podosphaeraster species will help assess the homology and diversity of skeletal ossicles amongst Podosphaerasteridae taxa. Strictly speaking, the evolutionary origins of the marginal plates remain unknown without comparative evolutionary-developmental studies. For now, this spherical starfish stands as a testament to how much remains to be discovered in the deep sea, where animals with bodies unlike any of their relatives continue to challenge our understanding of how evolution shapes life on Earth. The volumetric image data from the micro-CT reconstruction is openly available at MorphSource, and the genetic sequences are deposited in the DDBJ, EMBL and GenBank databases, ensuring that this enigmatic animal’s secrets remain accessible to researchers worldwide.

Subject of Research: Evolutionary Biology

Article Title: Phylogenetic position of the enigmatic starfish family Podosphaerasteridae ( Asteroidea , Valvatida ) with a morphological observation of the skeletal structure by micro-CT

Article References: Hiruta, S., Kobayashi, I., & Fujita, T. (2026). Phylogenetic position of the enigmatic starfish family Podosphaerasteridae (Asteroidea, Valvatida) with a morphological observation of the skeletal structure by micro-CT. Biodiversity Data Journal, 14, Article e194006. https://doi.org/10.3897/bdj.14.e194006

Image Credits: AI Generated

DOI: 10.3897/BDJ.14.e194006

Keywords: starfish, Podosphaeraster, mitochondrial genome, micro-CT, phylogenetics, convergent evolution, deep sea, Phylogenetic, position, enigmatic, family, Podosphaerasteridae

The teeth were recovered from two sections that have become famous among vertebrate palaeontologists. The Nimaigu section in Wusha Town, Xingyi City, south-western Guizhou Province, and the Yize section in Changdi Town, Luoping County, eastern Yunnan Province, both lie on the south-western part of the Yangtze Carbonate Platform. During the Triassic, these sites recorded an upward transition from shallow-water dolomite of the Yangliujing Formation to deep-water marl and nodular limestone of the Zhuganpo Member, and subsequently to the black shale and marl of the Wayao Member, which represents the maximum flooding surface. The ‘Falang’ Formation is renowned for yielding two exceptionally well-preserved marine vertebrate faunas: the latest Ladinian Xingyi Fauna and the early Carnian Guanling Biota, both famous for their marine reptiles and fishes.

Fieldwork in 2017, 2018 and 2019 produced large quantities of rock samples from the ‘Falang’ Formation at both sections. The samples were broken into fragments approximately four centimetres in size and dissolved using ten percent acetic acid, a standard technique for freeing microfossils from carbonate rock. Conodonts and chondrichthyan teeth were then manually picked from the residues under a stereomicroscope. Nine shark teeth were examined in total: specimen GMPKU-P-4268 from the Yize section and GMPKU-P-4261 to 4267 and 4269 from the Nimaigu section. All specimens are housed in the Geological Museum of Peking University in Beijing.

The teeth were photographed with a JEOL JCM-6000 Plus scanning electron microscope under an acceleration voltage of 10 kV at the School of Earth and Space Sciences, Peking University. Two specimens, GMPKU-P-4263, assigned to Euselachii gen. et sp. indet., and GMPKU-P-4266, assigned to Neoselachii gen. et sp. indet. 1, were scanned with a SkyScan 1172 micro-CT device at the State Key Laboratory of Systematic and Evolutionary Botany, Institute of Botany, the Chinese Academy of Sciences. The scans used a 180-degree rotation, a rotation step of 0.2 degrees and a pixel size of 0.56 micrometres, with the source voltage and current set to 48 kV and 200 microamperes. The teeth and their vascular systems were then reconstructed in three dimensions using AVIZO 2022.2, revealing internal structures that no external examination could ever expose.

Euselachii gen. et sp. indet. is represented by five isolated teeth from the late Ladinian layers of the Nimaigu section. The teeth are elongated mesiodistally and narrow labiolingually, measuring 0.21 to 0.37 millimetres in height and 0.58 to 1.10 millimetres mesio-distally. A gradient monognathic heterodonty is evident: anterolateral teeth have an arched root and relatively separated cusps, whereas posterolateral teeth display a straight root and fused cusps. The crown carries a robust pyramidal main cusp flanked by two to four lateral cusplets on each side, with one or two significantly smaller intermediate cusplets between the main cusp and the first pair of lateral cusplets. All cusps are inclined distally and curved lingually, and adjacent cusps are separated by V-shaped notches on anterolateral teeth but fused at their base on posterolateral teeth.

Micro-CT scanning of specimen GMPKU-P-4263 revealed an orthodont histotype. The crown consists of enameloid and a compact layer of underlying orthodentine, which enfolds a large longitudinal vascular canal at the crown-root junction, but no ascending pulp cavity can be seen. Osteodentine is restricted to the root. The vascular system comprises a cylindrical longitudinal vascular canal with a diameter of 30 to 90 micrometres extending along the crown-root junction, a single-layered vascular plexus of interconnected canals ranging from 8 to 40 micrometres in diameter forming numerous circular grids, short connective vascular canals linking the plexus to the overlying longitudinal canal, and ascending vascular canals. At the distal extremity, the longitudinal canal branches into three thinner canals. This longitudinal canal, unaccompanied by an ascending pulp cavity, also occurs in other non-neoselachian euselachians with very different crown shapes, from the flat-crowned hybodont Palaeobates angustissimus to the multicuspid Lamarodus triangulus, suggesting that its presence may be independent of tooth morphology.

Neoselachii gen. et sp. indet. 1 is represented by a single broken tooth, GMPKU-P-4266, from layers 145 to 146 of the Nimaigu section, dated to the late Ladinian. The tooth measures 0.75 millimetres in height and 1.43 millimetres mesio-distally, with a robust biconvex main cusp curved lingually and slightly inclined distally, flanked by one lateral cusplet on the mesial side and two on the distal side. The labial face of the main cusp is devoid of ornamentation, while the lingual face bears seven faint vertical ridges. The root is low, kidney-shaped in basal view, and protrudes lingually. Its vascularisation may represent a transitional state between anaulacorhize and polyhemiaulacorhize types.

The internal architecture of this neoselachian proved even more remarkable. Micro-CT reconstruction showed an orthodont tooth in which a reduced pulp cavity ascends into the main cusp, while porous osteodentine is confined to the root. The vascular system contains two longitudinal vascular canals, each 130 to 150 micrometres in diameter and nearly as long as the tooth itself, interconnected by short vertical or oblique connective canals. Four parallel main transverse vascular canals, 52 to 93 micrometres in diameter, run labiolingually through the middle of the root, accompanied by shorter and narrower secondary transverse canals. The vascularisation resembles that of Synechodus sp. from the Early Cretaceous of northern France, which also shows a longitudinal vascular canal, ascending pulp cavity and transverse canals, but the Chinese specimen differs by possessing two large longitudinal canals rather than one significantly narrower canal, and by lacking the meshed secondary vascular canals of Synechodus.

The remaining two taxa are known from single or paired teeth. Neoselachii gen. et sp. indet. 2 is represented by one anterior tooth, GMPKU-P-4267, from layer 186 of the Nimaigu section, bearing a slender main cusp without lateral cusplets, two sharp lateral cutting edges and a low bilobate root with a lingually unroofed median canal, a vascularisation reminiscent of the hemiaulacorhize type. The specimen recalls an undetermined neoselachian from the Ladinian–Carnian interval of Guanling, Guizhou, and the two could belong to the same species, although the Guanling tooth is significantly larger. Hexanchidae? gen. et sp. indet. is represented by two broken teeth, GMPKU-P-4268 and 4269, with labiolingually compressed crowns bearing a triangular main cusp flanked by three pairs of smaller lateral cusplets that decrease in height laterally. Because the root is missing, the affiliation remains tentative, but the crown morphology points to the cow shark family Hexanchidae, comparable with material from the late Permian of Japan and the middle Jurassic of England.

Stratigraphically, Hexanchidae? gen. et sp. indet. is the only taxon that may range across the Ladinian–Carnian boundary, with one tooth from late Ladinian layers at Nimaigu and another from early Carnian strata at Yize, above the first occurrences of the conodonts Quadralella polygnathiformis and Quadralella intermedia. The remaining three taxa are confined to the late Ladinian at Nimaigu. Palaeoecologically, the four taxa display non-durophagous feeding behaviour: Euselachii gen. et sp. indet. exhibits a grasping-swallowing dentition with cladodont-like crowns, Neoselachii gen. et sp. indet. 1 may exhibit a grasping type tooth, Neoselachii gen. et sp. indet. 2 displays a tearing type tooth with a slender main cusp and sharp cutting edges, and Hexanchidae? gen. et sp. indet. bears cutting type teeth. These findings confirm that the Ladinian–Carnian shark assemblage at Nimaigu lacks durophagous forms, making it different from other contemporaneous shark faunas worldwide that were predominantly represented by durophagous sharks. With the new material, at least nine chondrichthyan taxa have now been found at the Nimaigu section, and all four newly described chondrichthyans appear to be endemic, unknown outside South China, underscoring how much of the early evolution of euselachian sharks remains hidden in the world’s Triassic rocks.

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Subject of Research: A taxonomic and micro-CT study of nine euselachian shark teeth from the Ladinian–Carnian Zhuganpo Member in Guizhou and Yunnan Provinces, South China, describing four taxa and revealing Triassic dental vascularisation in 3D.

Article Title: New euselachian teeth from the Ladinian–Carnian interval of Guizhou and Yunnan Provinces, South China

Article References: Zhao, S., Li, J., Cuny, G., & Sun, Z. (2026). New euselachian teeth from the Ladinian–Carnian interval of Guizhou and Yunnan Provinces, South China. Swiss Journal of Palaeontology, 145, 313-326. https://doi.org/10.3897/sjp.145.185785

Image Credits: AI Generated

DOI: 10.3897/sjp.145.185785

Keywords: Triassic, chondrichthyans, palaeontology, micro-CT, South China, shark teeth, euseleachii, neoselachians, hexanchids, dental histology, euselachian, teeth