micro-CT Analysis Research Articles

Injectable thermosensitive hydrogel loading erythropoietin and FK506 alleviates gingival inflammation and promotes periodontal tissue regeneration.

Background: Periodontitis is a chronic multifactorial inflammatory disease associated with dysbiotic plaque biofilms and characterized by progressive destruction of the tooth-supporting apparatus. Therefore, there is significant potential in the discovery of drugs that inhibit periodontal inflammatory responses and promote periodontal regeneration. Methods: In this study, we generated a periodontitis rat model to detect the effects of chitosan/β-sodium glycerophosphate (β-GP)/glycolic acid (GA) hydrogel carried Erythropoietin and FK506 (EPO-FK506-CS/β-GP/GA). A total of forty-eight male Wistar rats were used to establish the periodontitis model. Drug injection was administered every 3days for a total of five times over a 2-week period. After a period of 2weeks following implantation, the rats underwent anesthesia, and a section of their maxillae encompassing the maxillary first and secondmolars, along with the alveolar bone, was obtained. micro-CT scanning, histopathology, immunohistochemistry and reverse transcription-quantitative PCR (RT-qPCR) assays were performed. Meanwhile, ELISA assay was performed to detect the levels of inflammatory mediators (TNF-α, IL-6 and IL-1β). Results: The synthesis and characterization of EPO-FK506-CS/β-GP/GA revealed that the hydrogel has stability and sustained release of drugs. The application of FK506+EPO was found to significantly enhance new bone formation in the defect area, as evidenced by the results of HE staining. Additionally, the use of FK506+EPO in the treated groups led to a notable increase in the density of alveolar bone, as observed through micro-CT analysis, when compared to the Model group. EPO-FK506-CS/β-GP/GA hydrogel exhibited notable efficacy in modulating inflammatory mediators (TNF-α, IL-6 and IL-1β). Furthermore, the osteoinductive properties of the EPO-FK506-CS/β-GP/GA hydrogel were extensive, as evidenced by a significant upregulation in the expression of key markers (Collagen I, Runx2, OPN, and OCN) associated with osteoblastic differentiation. Conclusion: Taken together, EPO-FK506-CS/β-GP/GA hydrogel alleviates gingival inflammation and promotes periodontal tissue regeneration in the periodontitis.

Adaptations of bone and bone vasculature to muscular stretch training.

The magnitude of bone formation and remodeling is linked to both the magnitude of strain placed on the bone and the perfusion of bone. It was previously reported that an increase in bone perfusion and bone density occurs in the femur of old rats with moderate aerobic exercise training. This study determined the acute and chronic effects of static muscle stretching on bone blood flow and remodeling. Old male Fischer 344 rats were randomized to either a naive or stretch-trained group. Static stretching of ankle flexor muscles was achieved by placement of a dorsiflexion splint on the left ankle for 30min/d, 5d/wk for 4wk. The opposite hindlimb served as a contralateral control (nonstretched) limb. Bone blood flow was assessed during and after acute stretching in naive rats, and at rest and during exercise in stretch-trained rats. Vascular reactivity of the nutrient artery of the proximal tibia was also assessed in stretch-trained rats. MicroCT analysis was used to assess bone volume and micro-architecture of the trabecular bone of both tibias near that growth plate. In naive rats, static stretching increased blood flow to the proximal tibial metaphasis. Blood flow to the proximal tibial metaphysis during treadmill exercise was higher in the stretched limb after 4wk of daily stretching. Daily stretching also increased tibial bone weight and increased total volume in both the proximal and distal tibial metaphyses. In the trabecular bone immediately below the proximal tibial growth plate, total volume and bone volume increased, but bone volume/total volume was unchanged and trabecular connectivity decreased. In contrast, intravascular volume increased in this region of the bone. These data suggest that blood flow to the tibia increases during bouts of static stretching of the hindlimb muscles, and that 4wk of daily muscle stretching leads to bone remodeling and an increase in intravascular volume of the tibial bone.

Insulin Therapy on Bone Macroscopic, Microarchitecture, and MechanicalProperties of Tibia in Diabetic Rats.

This study evaluated tibia's macroscopic structure, mechanical properties, and bone microarchitecture in rats with type 1 diabetes mellitus (T1DM). Eighteen animals were divided into three groups (n=6): Non-diabetic (ND), diabetic (D), and diabetic+insulin (DI). T1DM was induced by streptozotocin; insulin was administered daily (4IU). The animals were euthanized 35 days after induction. The tibiae were removed and analyzed using macroscopic, micro-computed tomography (micro-CT) and three-point bending. The macroscopic analysis measured proximal-distal length (PD), antero-posterior thickness (AP) of proximal (AP-P) and distal (AP-D) epiphysis, and lateral-medial thickness (LM) of proximal (LM-P) and distal (LM-D) epiphysis. Micro-CT analysis closed porosity, tissue mineral density, and cortical thickness. The three-point bending test measured maximum strength, energy, and stiffness. The macroscopic analysis showed that D presented smaller measures of length and thickness (AP and AP-P) than ND and DI. More extensive measurements were observed of LM and AP-D thickness in DI than in D. In micro-CT, DI showed larger cortical thickness than D. Mechanical analysis showed lower strength in D than in other groups. T1DM reduces bone growth and mechanical strength. Insulin therapy in diabetic rats improved bone growth and fracture resistance, making diabetic bone similar to normoglycemic animals.

ADVANCED IN VITRO 3D TISSUE CULTURE MODEL FOR THE ASSESSMENT OF TITANIUM IMPLANT OSTEOINTEGRATION

The use of implant biomaterials for prosthetic reconstructive surgery and osteosynthesis is consolidated in the orthopaedic field, improving the quality of life of patients and allowing for healthy and better ageing. However, there is the lack of advanced innovative methods to investigate the potentialities of smart biomaterials, particularly for the study of local effects of implant and osteointegration. Despite the complex process of osseointegration is difficult to recreate in vitro, the growing challenges in developing alternative models require to set-up and validate new approaches. Aim of the present study is to evaluate an advanced in vitro tissue culture model of osteointegration of titanium implants in human trabecular bone. Cubic samples (1.5×1.5 cm) of trabecular bone were harvested as waste material from hip arthroplasty surgery (CE AVEC 829/2019/Sper/IOR); cylindrical defects (2 mm Ø, 6 mm length) were created, and tissue specimens assigned to the following groups: 1) empty defects- CTR-; 2) defects implanted with a cytotoxic copper pin (Merck cod. 326429)- CTR+; 3) defects implanted with standard titanium pins of 6 µm-rough (ZARE S.r.l) -Ti6. Tissue specimens were cultured in mini rotating bioreactors in standard conditions, weekly assessing viability. At the 8-week-timepoint, immunoenzymatic, microtomographic, histological and histomorphometric analyses were performed. The model was able to simulate the effects of implantation of the materials, showing a drop in viability in CTR+, differently from Ti6 which appears to have a trophic effect on the bone. MicroCT and histological analysis supported the results, with lower BV/TV and Tb.Th values observed in CTR- compared to CTR+ and Ti6 and signs of matrix and bone deposition at the implant site. The collected data suggest the reliability of the tested model which can recreate the osseointegration process in vitro and can therefore be used for preliminary evaluations to reduce and refine in vivo preclinical models.Acknowledgment: This work was supported by Emilia-Romagna Region for the project “Sviluppo di modelli biologici in vitro ed in silico per la valutazione e predizione dell'osteointegrazione di dispositivi medici da impianto nel tessuto osseo”

3D PRINTING OF PCL TO FABRICATE POROUS SCAFFOLDS FOR BONE TISSUE ENGINEERING APPLICATIONS

The aim of this study is to print 3D polycaprolactone (PCL) scaffolds at high and low temperature (HT/LT) combined with salt leaching to induced porosity/larger pore size and improve material degradation without compromising cellular activity of printed scaffolds. PCL solutions with sodium chloride (NaCl) particles either directly printed in LT or were casted, dried, and printed in HT followed by washing in deionized water (DI) to leach out the salt. Micro-Computed tomography (Micro-CT) and scanning electron microscope (SEM) were performed for morphological analysis. The effect of the porosity on the mechanical properties and degradation was evaluated by a tensile test and etching with NaOH, respectively. To evaluate cellular responses, human bone marrow-derived mesenchymal stem/stromal cells (hBMSCs) were cultured on the scaffolds and their viability, attachment, morphology, proliferation, and osteogenic differentiation were assessed. Micro-CT and SEM analysis showed that porosity induced by the salt leaching increased with increasing the salt content in HT, however no change was observed in LT. Structure thickness reduced with elevating NaCl content. Mass loss of scaffolds dramatically increased with elevated porosity in HT. Dog bone-shaped specimens with induced porosity exhibited higher ductility and toughness but less strength and stiffness under the tension in HT whereas they showed decrease in all mechanical properties in LT. All scaffolds showed excellent cytocompatibility. Cells were able to attach on the surface of the scaffolds and grow up to 14 days. Microscopy images of the seeded scaffolds showed substantial increase in the formation of extracellular matrix (ECM) network and elongation of the cells. The study demonstrated the ability of combining 3D printing and particulate leaching together to fabricate porous PCL scaffolds. The scaffolds were successfully printed with various salt content without negatively affecting cell responses. Printing porous thermoplastic polymer could be of great importance for temporary biocompatible implants in bone tissue engineering applications.

Selective inhibition of interleukin 6 receptor decreased inflammatory cytokines and increased proteases in an experimental model of critical calvarial defect.

Considering the lack of consensus related to the impact of selective IL-6 receptor inhibition on bone remodeling and the scarcity of reports, especially on large bone defects, this study proposed to evaluate the biological impact of the selective inhibitor of interleukin-6 receptor (tocilizumab) in an experimental model of critical calvarial defect in rats. In this preclinical and in vivo study, 24 male Wistar rats were randomly divided into two groups (n=12/group): defect treated with collagen sponge (CG) and defect treated with collagen sponge associated with 2 mg/kg tocilizumab (TCZ). The defect in the parietal bone was created using an 8-mm diameter trephine drill. After 90 days, the animals were euthanized, and tissue samples (skull caps) were evaluated through micro-CT, histological, immunohistochemistry, cytokines, and RT-qPCR analyses. Tocilizumab reduced mononuclear inflammatory infiltration (P<0.05) and tumor necrosis factor (TNF)-α levels (P<0.01) and down-regulated tissue gene expression of BMP-2 (P<0.001), RUNX-2 (P<0.05), and interleukin (IL)-6 (P<0.05). Moreover, it promoted a stronger immunostaining of cathepsin and RANKL (P<0.05). Micro-CT and histological analyses revealed no impact on general bone formation (P>0.05). The bone cells (osteoblasts, osteoclasts, and osteocytes) in the defect area were similar in both groups (P>0.05). Tocilizumab reduced inflammatory cytokines, decreased osteogenic protein, and increased proteases in a critical bone defect in rats. Ninety days after the local application of tocilizumab in the cranial defect, we did not find a significant formation of bone tissue compared with a collagen sponge.

Atomic Layer Deposition of Zirconia on Titanium Implants Improves Osseointegration in Rabbit Bones.

Atomic layer deposition (ALD) is a method that can deposit zirconia uniformly on an atomic basis. The effect of deposited zirconia on titanium implants using ALD was evaluated in vivo. Machined titanium implants (MTIs) were used as the Control. MTIs treated by sandblasting with large grit and acid etching (SA) and MTIs deposited with zirconia using ALD are referred to as Groups S and Z, respectively. Twelve implants were prepared for each group. Six rabbits were used as experimental animals. To evaluate the osteogenesis and osteocyte aspects around the implants, radiological and histological analyses were performed. The bone-to-implant contact (BIC) ratio was measured and statistically analyzed to evaluate the osseointegration capabilities. In the micro-CT analysis, more radiopaque bone tissues were observed around the implants in Groups S and Z. Histological observation found that Groups S and Z had more and denser mature bone tissues around the implants in the cortical bone area. Many new and mature bone tissues were also observed in the medullary cavity area. For the BIC ratio, Groups S and Z were significantly higher than the Control in the cortical bone area (P < 0.017), but there was no significant difference between Groups S and Z. MTIs deposited with zirconia using ALD (Group Z) radiologically and histologically showed more mature bone formation and activated osteocytes compared with MTIs (Control). Group Z also had a significantly higher BIC ratio than the Control. Within the limitations of this study, depositing zirconia on the surface of MTIs using ALD can improve osseointegration in vivo.

Nicotinamide mononucleotide enhances fracture healing by promoting skeletal stem cell proliferation.

The process of skeletal regeneration initiated by stem cells following injury, especially in fractures, is significantly impaired by aging and adverse factors. Nicotinamide mononucleotide (NMN), a critical endogenous precursor of nicotinamide adenine dinucleotide (NAD), has garnered extensive attention for its multifaceted regulatory functions in living organisms and its wide-ranging therapeutic potential. However, whether NMN contributes to trauma-induced skeletal regeneration remains unclear. Methods: The transverse femoral shaft fracture model was employed to evaluate the potential advantages of NMN administration for overall repair during the initial fracture stages in male mice through micro-CT analysis, histochemistry, and biomechanical testing. The pro-proliferative function of NMN on skeletal stem cells (SSCs) was investigated through flow cytometry, qRT-PCR, NAD content measurement, and cell proliferation assay. Results: In this study, we observed that the administration of NMN during the initial phase of fracture in mice led to a larger callus and corresponding improvement in micro-CT parameters. NMN enhances the cartilaginous component of the callus by elevating the NAD content, consequently accelerating subsequent endochondral ossification and the fracture healing process. Subsequent analyses elucidated that NMN was beneficial in promoting the expansion of diverse stem cells in vivo and in vitro potentially via modulation of the Notch signaling pathway. Moreover, the depletion of macrophages profoundly obstructs the proliferation of SSCs. Conclusion: Our discoveries provide a potential strategy for enhancing fracture healing through stimulation of callus SSC proliferation at an early stage, shedding light on the translational value of NMN as an enhancer for skeletal regeneration and highlighting the pivotal role of macrophage-stem cell interactions in governing the regenerative influence of NMN on stem cells.

Manufacturing seamless three-dimensional woven preforms with complex shapes based on a new weaving technology

A new weaving technology using a modified z-binder interlacement system was designed to demonstrate its potential for the effective, continuous, efficient, and rapid manufacturing of various three-dimensional (3D) woven structures. First, three representative 3D woven preforms were fabricated. Then, epoxy resin was transferred to a preform. The manufactured 3D woven textile-reinforced composites were investigated using micro-CT analysis, tensile tests, and bending tests to study the effect of the z-binder interlacing on the structure. Furthermore, a design rule was established that could seamlessly create complex 3D woven structures with non-uniform heights in the z-direction, such as boxes, bowls, and pyramids, demonstrating that the seamless 3D woven preform of the complex shape can be fabricated with structural integrity.

Comparative Evaluation of Supplementary Cleaning Techniques for Removal of Bio-C Sealer and AH-Plus from Oval Root Canals: A Micro-CT Analysis.

This study aimed to compare the effectiveness of two endodontic cleaning techniques, passive ultrasonic irrigation (PUI) and the XP-endo Finisher R (XPR) system, in removing residual filling material during endodontic retreatment procedures. Forty mandibular premolars with oval canals were divided into four groups based on the sealer used (AH-Plus or Bio-C Sealer) and the cleaning technique employed (PUI or XPR). To ensure uniformity of canal volume among groups, initial micro-CT scans were conducted. The canals were instrumented, filled, and then re-instrumented before undergoing either PUI or XPR cleaning techniques. Residual filling material volumes were assessed through micro-CT scans, and statistical analysis was performed using the Kruskal-Wallis and Mann-Whitney U tests. Following instrumentation, there was no significant difference in residual filling material volumes between AH-Plus and Bio-C Sealer groups (1.35 mm3and 1.02 mm3, respectively; P>0.05). However, after supplementary cleaning techniques, XPR-cleaned specimens exhibited significantly less residual material compared to PUI-cleaned specimens (0.01 mm3 and 0.29 mm3 for Bio-C Sealer, and 0.07 mm3 and. 0.30 mm3for AH-Plus, P<0.05). The XPR system was found to be more effective than PUI in removing residual filling material from Bio-C Sealer-filled root canals. This highlights its potential as a useful supplementary cleaning technique in endodontic retreatment procedures.

Micro-computed tomography (micro-CT) evaluation of root canal morphology in immature maxillary third molars.

The endodontic treatment of immature permanent teeth with necrotic pulp is a significant clinical challenge. The success of regenerative endodontic procedure is highly dependent on disinfection of the root canal and an accurate anatomical knowledge of the root canal. The aim of this study was to use micro-computed tomography (micro-CT) analysis to investigate the configuration of root canals in the upper permanent third maxillary molars with incomplete root development in their coronal, apical and middle third portions. Thirty immature third permanent maxillary molars were scanned using a micro-CT system. Then, we measured the diameters and areas of the root canal in the coronal, middle and apical third of the roots. The ratio between the long and short diameter of each root canal was then calculated and the canals were divided into several groups: round, oval, long oval, flat and irregular. The round configuration was not observed in the distobuccal and mesiobuccal roots in any of their anatomical regions. Oval and long oval canals predominated in the distobuccal root. The greatest variations were observed in the mesiobuccal root, with the ribbon-shaped canal predominating in the middle region and an irregular shape in the apical region. In the coronal region of the palatal canal, the round configuration predominated; in the middle third, we observed an almost equivalent distribution between round and oval configurations; apically, the oval shape predominated. In conclusion, we observed significant complexity and variation in the morphology and configuration of root canals in immature permanent molars, thus generating additional obstacles for the success of regenerative endodontics.

Minocycline reduces alveolar bone loss and bone damage in Wistar rats with experimental periodontitis.

This study aimed to investigate the impact of minocycline on the alveolar bone in experimental periodontitis in rats. Thirty Wistar rats were randomly assigned to three groups: control without periodontitis; experimental periodontitis induced by ligature; experimental periodontitis + intraperitoneal administration minocycline for seven days. Ligatures remained in place in both periodontitis groups for 14 days. At the end of the experiment, the animals were euthanized and one hemimandible underwent micro-computed tomography (micro-CT) analysis to assess vertical bone loss and alveolar bone quality. Histopathological analysis was performed on the other hemimandible. Statistical analysis was performed using ANOVA with Tukey's post-test (p<0.05). The results showed a significant reduction in vertical bone loss in the animals treated with minocycline compared with untreated animals. Minocycline also preserved the alveolar bone thickness, number, spacing, and bone volume to tissue volume ratio. Histopathological analysis indicated that minocycline reduced bone resorption, decreased inflammatory response, and maintained the bone collagen fibers. This study demonstrated the effectiveness of minocycline in reducing vertical bone loss and preserved bone quality in rats with experimental periodontitis. The results of this study indicate that minocycline has the potential to serve as an additional treatment option for periodontitis. However, further research is warranted to assess the efficacy and safety of minocycline use in patients with periodontitis.

High-throughput micro-CT analysis identifies sex-dependent biomarkers of erosive arthritis in TNF-Tg mice and differential response to anti-TNF therapy.

Development of reliable disease activity biomarkers is critical for diagnostics, prognostics, and novel drug development. Although computed tomography (CT) is the gold-standard for quantification of bone erosions, there are no consensus approaches or rationales for utilization of specific outcome measures of erosive arthritis in complex joints. In the case of preclinical models, such as sexually dimorphic tumor necrosis factor transgenic (TNF-Tg) mice, disease severity is routinely quantified in the ankle through manual segmentation of the talus or small regions of adjacent bones primarily due to the ease in measurement. Herein, we sought to determine the particular hindpaw bones that represent reliable biomarkers of sex-dependent disease progression to guide future investigation and analysis. Hindpaw micro-CT was performed on wild-type (n = 4 male, n = 4 female) and TNF-Tg (n = 4 male, n = 7 female) mice at monthly intervals from 2-5 (females) and 2-8-months (males) of age, since female TNF-Tg mice exhibit early mortality from cardiopulmonary disease at approximately 5-6-months. Further, 8-month-old WT (n = 4) and TNF-Tg males treated with anti-TNF monoclonal antibodies (n = 5) or IgG placebo isotype controls (n = 6) for 6-weeks were imaged with micro-CT every 3-weeks. For image analysis, we utilized our recently developed high-throughput and semi-automated segmentation strategy in Amira software. Synovial and osteoclast histology of ankle joints was quantified using Visiopharm. First, we demonstrated that the accuracy of automated segmentation, determined through analysis of ~9000 individual bones by a single user, was comparable in wild-type and TNF-Tg hindpaws before correction (79.2±8.9% vs 80.1±5.1%, p = 0.52). Compared to other bone compartments, the tarsal region demonstrated a sudden, specific, and significant bone volume reduction in female TNF-Tg mice, but not in males, by 5-months (4-months 4.3± 0.22 vs 5-months 3.4± 0.62 mm3, p<0.05). Specifically, the cuboid showed significantly reduced bone volumes at early timepoints compared to other tarsals (i.e., 4-months: Cuboid -24.1±7.2% vs Talus -9.0±5.9% of 2-month baseline). Additional bones localized to the anterolateral region of the ankle also exhibited dramatic erosions in the tarsal region of females, coinciding with increased synovitis and osteoclasts. In TNF-Tg male mice with severe arthritis, the talus and calcaneus exhibited the most sensitive response to anti-TNF therapy measured by effect size of bone volume change over treatment period. We demonstrated that sexually dimorphic changes in arthritic hindpaws of TNF-Tg mice are bone-specific, where the cuboid serves as a reliable early biomarker of erosive arthritis in female mice. Adoption of automated segmentation approaches in pre-clinical or clinical models has potential to translate quantitative biomarkers to monitor bone erosions in disease and evaluate therapeutic efficacy.

Bone regeneration using activin A/BMP2 chimera (AB204) with collagen membrane in rats with calvarial defects.

Collagen has long been recognized as an excellent carrier for growth factors, and membrane-type collagen has been widely applied in dentistry for guided bone regeneration. This study was conducted to examine the effects of an activin A/BMP2 chimera (AB204) combined with a collagen membrane (CM) on bone repair in a rat calvarial defect model. A unilateral calvarial defect measuring 5.0 mm was surgically created in 32 Sprague-Dawley rats. The rats were then randomly assigned to 1 of 4 groups, each consisting of 8 animals: control (untreated), CM (treated with a CM only), CM/bone morphogenetic protein 2 (BMP2) (treated with a CM and 1.0 μg of BMP2), and CM/AB204 (treated with a CM and 1.0 μg of AB204). Bone regeneration was evaluated using micro-computed tomography (CT) and histological analysis at 2 and 4 weeks following surgery. Micro-CT analysis revealed that bone formation in the CM/BMP2 and CM/AB204 groups was superior to that observed in the control and CM groups at both 2 and 4 weeks postoperatively. BMP2 induced greater bone regeneration than AB204 at 2 weeks; however, AB204 resulted in a greater bone volume at 4 weeks, achieving the highest values recorded. No significant differences were found between the CM/BMP2 and CM/AB204 groups at either time point (P>0.05). On histological examination, new bone formation was evident in both CM/BMP2 and CM/AB204 groups. Within the limitations of this study, the findings indicate that AB204 may enhance osteogenic potential when used in combination with CM for bone regeneration.

Targeting FGFR3 signaling and drug repurposing for the treatment of SLC26A2-related chondrodysplasia in mouse model

BackgroundMutations in Slc26a2 cause a spectrum of autosomal-recessive chondrodysplasia with a significant and negligible influence on the quality of life. It has been reported that Slc26a2 deficiency triggers the ATF6 branch of the UPR, which may, in turn, activate the negative regulator of the FGFR3 signaling pathway. However, the correlation between the deletion of Slc26a2 and the augmentation of downstream phosphorylation of FGFR3 has not been investigated in vivo. MethodsFirst, we constructed Slc26a2 and Fgfr3 double knockout mouse lines and observed gross views of the born mice and histological staining of the tibial growth plates. The second approach was to construct tamoxifen-inducible Cre-ERT2 mouse models to replicate SLC26A2-related non-lethal dysplastic conditions. Pharmacological intervention was performed by administering the FGFR3 inhibitor NVP-BGJ398. The effect of NVP-BGJ398 on chondrocytes was assessed by Alcian blue staining, proliferation, apoptosis, and chondrocyte-specific markers and then verified by western blotting for variations in the downstream markers of FGFR3. The growth process was detected using X-rays, micro-CT examination, histomorphometry staining of growth plates, and immunofluorescence. ResultsGenetic ablation of Fgfr3 in embryonic Slc26a2-deficient chondrocytes slightly attenuated chondrodysplasia. Subsequently, in the constructed mild dysplasia model, we found that postnatal intervention with Fgfr3 gene in Slc26a2-deficient chondrocytes partially alleviated chondrodysplasia. In chondrocyte assays, NVP-BGJ398 suppressed the defective phenotype of Slc26a2-deficient chondrocytes and restored the phosphorylation downstream of FGFR3 in a concentration-dependent manner. In addition, in vivo experiments showed significant alleviation of impaired chondrocyte differentiation, and micro-CT analysis showed a clear improvement in trabecular bone microarchitectural parameters. ConclusionOur results suggested that inhibition of FGFR3 signaling pathway overactivation and NVP-BGJ398 has promising therapeutic implications for the development of SLC26A2-related skeletal diseases in humans. The translational potential of this articleOur data provide genetic and pharmacological evidence that targeting FGFR3 signaling via NVP-BGJ398 could be a route for the treatment of SLC26A2-associated skeletal disorders, which promisingly advances translational applications and therapeutic development.

Clinical, Microbiological, and Microcomputed Tomography Evaluation of Silver Diamine Fluoride in Controlling Root Carious Lesions: An in Vivo Study.

Silver diamine fluoride (SDF) application without removing necrotic tissue is an applicable non-invasive measure to primary care practice and may reduce the burden of untreated root caries. This study aims to examine clinical feature change, root caries-related bacteria, and silver penetration of SDF in arresting root caries. Ten study participants with 16 root carious teeth were included in this study. The clinical characteristics of root caries lesions (plaque deposit, color, hardness, and sensitivity symptom) were recorded. Then root caries samples were collected using a spoon excavator before and 2 weeks after treated with 38% SDF. The amounts of Streptococcus mutans (S.mutans), Actinomyces naeslundii (A. naeslundii), and Lactobacillus casei (L. casei) were determined using real-time PCR. Any tooth sample scheduled for extraction was further analyzed using micro-CT, stereoscopic microscope, and FE-SEM/ EDX to determine the silver penetration. Most treated samples were darker in color, predominantly turning black (n =15, 93.8%), had increased surface hardness (n =11, 68.8%), were non-sensitive teeth (n=14, 87.5%), and were negative to air blowing (n =12, 75%). Only S.mutans had a significantly lower number of bacteria after 2 weeks (p-value = 0.041). The micro-CT analysis revealed that the silver increased the root carious lesion's density in proportion to its depth. According to a stereoscopic microscope study, silver penetration caused dark bands, appearing along the dentinal tubule toward the dental pulp. An FE-SEM analysis showed that silver was found to be densely deposited on the surface of the lesions and penetrated through the dentinal tubule into the dental pulp direction. EDX mapping confirmed that the increased density was related to silver. Based on clinical and microbiological profiles, this investigation indicated that SDF is beneficial for controlling root caries, particularly S.mutans reduction. Silver can also penetrate deep into the lesion. Key words:Microbiology, Root caries, Silver diamine fluoride, Silver ion, Streptococcus mutans, Actinomyces naeslundii, Lactobacillus casei.

Skeletal dysmorphology and mineralization defects in Fgf20 KO mice.

Fibroblast growth factor 20 (Fgf20), a member of the Fgf9 subfamily, was identified as an important regulator of bone differentiation and homeostasis processes. However, the role of Fgf20 in bone physiology has not been approached yet. Here we present a comprehensive bone phenotype analysis of mice with functional ablation of Fgf20. The study conducts an extensive analysis of Fgf20 knockout mice compared to controls, incorporating microCT scanning, volumetric analysis, Fgf9 subfamily expression and stimulation experiment and histological evaluation. The bone phenotype could be detected especially in the area of​ the lumbar and caudal part of the spine and in fingers. Regarding the spine, Fgf20-/- mice exhibited adhesions of the transverse process of the sixth lumbar vertebra to the pelvis as well as malformations in the distal part of their tails. Preaxial polydactyly and polysyndactyly in varying degrees of severity were also detected. High resolution microCT analysis of distal femurs and the fourth lumbar vertebra showed significant differences in structure and mineralization in both cortical and trabecular bone. These findings were histologically validated and may be associated with the expression of Fgf20 in chondrocytes and their progenitors. Moreover, histological sections demonstrated increased bone tissue formation, disruption of Fgf20-/- femur cartilage, and cellular-level alterations, particularly in osteoclasts. We also observed molar dysmorphology, including root taurodontism, and described variations in mineralization and dentin thickness. Our analysis provides evidence that Fgf20, together with other members of the Fgf9 subfamily, plays a crucial regulatory role in skeletal development and bone homeostasis.

Effects of plasminogen activator inhibitor-1 deficiency on bone disorders and sarcopenia caused by adenine-induced renal dysfunction in mice.

Chronic kidney disease (CKD) is a significant global health issue and often involves CKD-mineral and bone disorder (MBD) and sarcopenia. Plasminogen activator inhibitor-1 (PAI-1) is an inhibitor of fibrinolysis. PAI-1 has been implicated in the pathogenesis of osteoporosis and muscle wasting induced by inflammatory conditions. However, the roles of PAI-1 in CKD-MBD and sarcopenia remain unknown. Therefore, the present study investigated the roles of PAI-1 in bone loss and muscle wasting induced by adenine in PAI-1-deficient mice. CKD was induced in PAI-1+/+ and PAI-1-/- mice by administration of adenine for ten weeks. Muscle wasting was assessed by grip strength test, quantitative computed tomography (CT) analysis and muscle weight measurement. Osteoporosis was assessed by micro-CT analysis of femoral microstructural parameters. PAI-1 deficiency did not affect adenine-induced decreases in body weight and food intake or renal dysfunction in male or female mice. PAI-1 deficiency also did not affect adenine-induced decreases in grip strength, muscle mass in the lower limbs, or the tissue weights of the gastrocnemius, soleus, and tibialis anterior muscles in male or female mice. PAI-1 deficiency aggravated trabecular bone loss in CKD-induced male mice, but significantly increased trabecular bone in CKD-induced female mice. On the other hand, PAI-1 deficiency did not affect cortical bone loss in CKD-induced mice. In conclusion, PAI-1 is not critical for the pathophysiology of CKD-MBD or CKD-induced sarcopenia in mice. However, PAI-1 may be partly related to bone metabolism in trabecular bone in the CKD state with sex differences.

Reassessment of Archaeooides based on new material from the Fortunian (early Cambrian) of China infers algal affinity

Archaeooides originally refers to a group of early Cambrian globular microfossils with regularly arranged nodular surface ornaments, and of a diameter ranging from less than 400 μm to more than 2 mm. The traditional taxonomy assigns all Archaeooides-like microfossils to a single morphotaxon, Archaeooides granulatus, irrespective of their differences in size and surface ornamentation. Archaeooides has been interpreted as a possible diapause embryo because of its inferred palintomical cell division and thick wall with vesicular structures. However, this interpretation needs further testing based on an updated taxonomy and differentiation of taphonomic artifacts from biological structures. Here, we report new, three-dimensionally phosphatized, Archaeooides-like microfossils from the Fortunian of China. In addition to A. granulatus with nodular ornaments, we recognized three new taxa, i.e., Shaanisphaera spinosa gen. et sp. nov. with spine-like ornaments, Qinlingisphaera conica gen. et sp. nov. with cone-like ornaments, and Dahisphaera plana gen. et sp. nov. with oblate ornaments. Each taxon has a wide size interval, ranging continuously from several hundred micrometers to one or two millimeters, which is different from animal eggs that generally have a narrow and conservative size interval. Micro-CT analyses and thin sections reveal that the vesicular structures may be taphonomic in nature, and that the wall thickness is specimen-dependent. Collectively, the evidence at hand does not support an embryo interpretation for Archaeooides. Instead, the wide size interval probably represents ontogenetic variations that are common in many fossil and modern algae, and thus further suggests an algal affinity for Archaeooides as well as the other Archaeooides-like microfossils.

Quantifying the immediate post-implantation strain field of cadaveric tibiae implanted with cementless tibial trays: A time-elapsed micro-CT and digital volume correlation analysis during stair descent

Primary stability, the mechanical fixation between implant and bone prior to osseointegration, is crucial for the long-term success of cementless tibial trays. However, little is known about the mechanical interplay between the implant and bone internally, as experimental studies quantifying internal strain are limited. This study employed digital volume correlation (DVC) to quantify the immediate post-implantation strain field of five cadaveric tibiae implanted with a commercially available cementless titanium tibial tray (Attune, DePuy Synthes). The tibiae were subjected to a five-step loading sequence (0–2.5 bodyweight, BW) replicating stair descent, with concomitant time-elapsed micro-CT imaging. With progressive loads, increased compression of trabecular bone was quantified, with the highest strains directly under the posterior region of the tibial tray implant, dissipating with increasing distance from the bone-implant interface. After load removal of the last load step (2.5BW), residual strains were observed in all of the five tibiae, with residual strains confined within 3.14 mm from the bone-implant interface. The residual strain is reflective of the observed initial migration of cementless tibial trays reported in clinical studies. The presence of strains above the yield strain of bone accepted in literature suggests that inelastic properties should be included within finite element models of the initial mechanical environment. This study provides a means to experimentally quantify the internal strain distribution of human tibia with cementless trays, increasing the understanding of the mechanical interaction between bone and implant.