Soft Tissue Samples Research Articles

Identification of novel candidate biomarkers related to immune cell infiltration in peri-implantitis.

The present study was performed to identify key biomarkers associated with immune cell infiltration in peri-implantitis through bioinformatic analyses. Six peri-implantitis soft tissue samples and six healthy gingiva samples were obtained from GSE106090, and were used to identify immune-associated differentially expressed genes (DEGs) in peri-implantitis. The candidate biomarkers associated with immune cell infiltration were examined by immunohistochemical staining. We identified 2089 upregulated and 2173 downregulated genes. Upregulated DEGs were significantly associated with immune response. Ten key candidate biomarkers were identified in the PPI network, including IL1B, TLR2, TLR4, CCL4, CXCL8, IL10, IL6, CD4, CCL3, and PTPRC. The expression level of the 10 genes increased in peri-implantitis soft tissue samples compared with healthy gingiva samples. The proportion of CD4+ T cells, iTreg, and Tfh in infiltration immune cells increased in peri-implantitis soft tissue samples and were positively correlated with the expression level of candidate biomarkers TLR4, CCL3, CXCL8, and IL1B. Immunohistochemistry showed that there were more lymphocytes in peri-implantitis soft tissue samples, with an increased expression level of TLR4, CCL3, CXCL8, and IL1B. Identification of four novel diagnostic biomarkers was helpful for revealing the molecular mechanisms and could serve as a risk predictor for the immune microenvironment in peri-implantitis.

Carbapenem resistant Enterobacterales in the United Arab Emirates: a retrospective analysis from 2010 to 2021.

Carbapenem-resistant Enterobacterales (CRE) are spreading in the United Arab Emirates (UAE) where their dissemination is facilitated by international travel, trade, and tourism. The objective of this study is to describe the longitudinal changes of CRE as reported by the national AMR surveillance system of the UAE. In this study, we retrospectively describe CRE isolated from 317 surveillance sites, including 87 hospitals and 230 centers/clinics from 2010 to 2021. The associated clinical, demographic, and microbiological characteristics are presented by relying on the UAE national AMR surveillance program. Data was analyzed using WHONET microbiology laboratory database software (http://www.whonet.org). A total of 14,593 carbapenem resistant Enterobacterales were analyzed, of which 48.1% were carbapenem resistant Klebsiella pneumoniae (CRKp), 25.1% carbapenem resistant Escherichia coli (CREc), and 26.8% represented 72 other carbapenem resistant species. Carbapenem resistant strains were mostly associated with adults and isolated from urine samples (36.9% of CRKp and 66.6% of CREc) followed by respiratory samples (26.95% for CRKp) and soft tissue samples (19.5% for CRKp). Over the studied period carbapenem resistance rates remained high, especially in K. pneumoniae, and in 2021 were equivalent to 67.6% for imipenem, 76.2% for meropenem, and 91.6% for ertapenem. Nevertheless, there was a statistically significant decreasing trend for imipenem and meropenem resistance in Klebsiella species (p < 0.01) while the decrease in ertapenem resistance was non-significant. Concerning E. coli, there was a statistically significant decreasing trend for meropenem and imipenem resistance over the 12 years, while ertapenem resistance increased significantly with 83.8% of E. coli exhibiting ertapenem resistance in 2021. Resistance rates to ceftazidime and cefotaxime remained higher than 90% (in 2021) for CRKp and cefotaxime rates increased to 90.5% in 2021 for CREc. Starting 2014, resistance to colistin and tigecycline was observed in carbapenem resistant Enterobacterales. CRE were associated with a higher mortality (RR: 6.3), admission to ICU (RR 3.9), and increased length of stay (LOS; 10 excess inpatient days per CRE case). This study supports the need to monitor CRE in the UAE and draws attention to the significant increase of ertapenem resistance in E. coli. Future surveillance analysis should include a genetic description of carbapenem resistance to provide new strategies.

Abstract C046: Rapid autopsy provides unique research opportunity to evaluate KRASG12C inhibitor resistance mechanisms in non-small cell lung cancer

Abstract TThe discovery of small molecule inhibitors that target mutant KRASG12C has revolutionized the treatment paradigm for KRASG12C-mutant non-small cell lung cancer (NSCLC). While the initial response rates to KRASG12C inhibitors (KRASG12Ci) are approximately 40%, the development of heterogenous resistance mechanisms remains a challenge. Understanding the heterogeneity of resistance mechanisms is critical in overcoming this obstacle. Autopsy samples provide valuable insights into therapy resistance and genomic evolution. Moffitt’s Rapid Tissue Donation (RTD) program offers patients with lung malignancies the opportunity to donate primary and metastatic tumor samples, as well as non-tumoral tissue samples and body fluids, for research purposes after their death. 16 patients with KRASG12C-mutant NSCLC enrolled into the RTD, and 15 were treated with KRASG12Ci. Postmortem samples from seven patients were collected to date, including 38 tumor samples from lung, liver, lymph nodes, soft tissue, adrenal, spleen, pancreas, brain and epicardium samples. Radiological measurements were used to evaluate therapy response in each lesion. 26/38 tumor samples were non-responding to treatment whereas the remaining 12 were stable/responding lesions. Among the seven patients, five had both non-responding and stable/responding lesions and the remaining two had only non-responding lesions. Whole exome sequencing (WES) and transcriptomic analyses (RNA-Seq) were performed in 7 tumoral samples and 1 non-tumoral liver sample from one single patient. KRASG12C mutation and PTENR55M mutations were found in all tumor lesions, irrespective of therapy response. A PIK3CAE542K mutation was present in all tumor lesions, except for one subcutaneous tumor that did not respond to treatment. In the non-responding lung tumor, as well as the stable metastatic lymph node, a MAP2K3A203S mutation was identified. Notably, the non-responding liver lesions exhibited an ERBB3A17G mutation, whereas the responding liver lesion did not. Gene expression analyses comparing non-responding and responding liver lesions revealed an upregulation of genes associated with hypoxia, epithelial mesenchymal transition, and inflammation in the non-responding lesions, while a subgroup of genes regulated by MYC was upregulated in the responding liver lesion. Additional genomic analyses of other autopsy samples are underway. Our findings reveal that therapy resistance may be driven by pathway alterations instead of genomic alterations. The collection of rapid autopsy specimens from primary and metastatic tissue sites from patients with KRASG12C-mutant NSCLC is essential to understand the heterogeneous KRASG12Ci mechanisms of resistance and explore biomarkers that may guide clinical decision-making in these patients. Citation Format: Hilal Ozakinci, Gina Nazario, Yaakov Stern, Hitendra S Solanki, Dung-Tsa Chen, Rose Trevor, Matthew Schabath, Paul Stewart, Jamie K Teer, Amer Beg, Theresa A Boyle, Eric Haura, Bruna Pellini. Rapid autopsy provides unique research opportunity to evaluate KRASG12C inhibitor resistance mechanisms in non-small cell lung cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C046.

Development of a Robotic Soft Tissue Collection Mechanism for Disease Detection

This research presents the comprehensive design, development, and testing of an innovative robotic mechanism tailored for the collection of soft tissue samples intended for disease detection applications. The proposed system integrates advanced technologies, such as hydrogels, chambers, and sensors, to enable meticulous and minimally invasive tissue manipulation while concurrently mitigating tissue damage. This paper showcases simulated tissue tests and validates the mechanism on chicken liver tissue. The integration of air sensors, strain gauge sensors, and chambers furnishes real-time feedback on applied forces, tissue interaction dynamics, and alterations in tissue dimensions. The presented work lays the foundation for further advancements in the realm of minimally invasive tissue collection, crucial for precise disease detection.

The Return of Organic Soft Tissue Preservation Abilities and the “Bog Body” Phenomenon in MLTT Restored Peatlands

Climate change and human manipulation in peatlands threatens peatland health, presaging the loss of instrumental peatland functions including carbon sequestration or the bog body mummification function. In response, peatland ecologists are studying restoration methods to revive degenerating bogs, including the Rochefort Moss Layer Transfer Technique (MLTT). Since the advent of these studies, questions regarding the return of lost peatland functions have arisen. This study employed a qualitative experimental study of mammal soft tissue samples in a controlled incubatory environment mimicking an MLTT restored peatland to discern if the MLTT method induced the return of the bog body mummification function. The findings indicate that the function does appear to return, which crucially implies that restorative efforts in peatland ecology have a valid prospect with promising returns, therefore this field is worthwhile to continue because this study shows that it is possible to save peatlands and their functions, further permitting the archaeological community to save the artifacts found in peatlands.

Genotyping strategies for tissues fixed with various embalming fluids for human identification, databasing, and traceability.

Within anatomical willed body programs and skeletal collections, whole bodies and their disassociated limbs and organs are identified and tracked. However, if these tracking mechanisms fail, DNA recovered from the formalin-fixed tissues/organs could provide an additional layer of quality assurance. Embalming fluids preserve biological tissues; however, they also damage, fragment, and cross-link DNA and protein molecules. This project investigated the success of STR-typing from various soft tissue and bone samples that were fixed with embalming solutions with a range of formaldehyde concentrations. Formalin-fixed samples dissected from five cadavers, including skin, muscle, bone, heart, and kidney were used in Phase 1 of this study. In Phase 2, an additional 57 tissue samples from various embalmed organs and body parts were collected to demonstrate long-term fixation and direct applicability within a body donor program. DNA was extracted from the samples using the QIAamp® FFPE Tissue Kit (QIAGEN), quantified with the Investigator® QuantiPlex® Pro RGQ qPCR Kit (QIAGEN), and amplified using the Investigator® 24plex and 26plex QS Kits and the Investigator® DIPplex Kit (QIAGEN). The results show the DNA was severely damaged, degraded, and often in low amounts (after one year post-embalming). Sampling from skin and muscle tissues embalmed with ~2.5%-5% formaldehyde solutions appears to be the best strategy for identification, while also maintaining the preservation of the tissues. The results of this project can provide informative data when determining which genotyping strategy may be best suited for the identification, re-association, and establishment of a database for the provenance of formalin-fixed human remains.

In Vitro Evaluation of the Effect of Soft Tissue Samples Produced Using Different Gingival Masks on Implant Angulations

In Vitro Evaluation of the Effect of Soft Tissue Samples Produced Using Different Gingival Masks on Implant Angulations

A Two-Dimensional K-Shell X-ray Fluorescence (2D-KXRF) Model for Soft Tissue Attenuation Corrections of Strontium Measurements in a Cortical Lamb Bone Sample

Human bones store elements such as calcium, phosphorus, and strontium, and accumulate toxic elements such as lead. In vivo measurements of elemental bone concentration can be done using X-ray fluorescence (XRF) techniques. Monte Carlo (MC) simulations of X-ray interactions were predominantly employed in this field to develop calibration methods that linked XRF measurements to concentrations. A simple and fast two-dimensional K-shell X-ray fluorescence model was developed to compute the KXRF signal of elements in bone and overlying soft tissue samples. The model is an alternative to MC methods and can guide future bone XRF studies. Contours of bone and soft tissue cross sections were elliptical and only KXRF signals from absorption of primary photons were considered. Predictions of the model were compared to Sr KXRF measurements using the bare lamb bone (LB) and the LB with overlying leather. XRF experiments used a small X-ray beam, silicon X-ray detector, and three positioning stages. Linear attenuation coefficients of the leather and LB were measured and used in the model. Measured and model-derived values of the Sr X-rays leather attenuation and Sr Kβ/Kα ratio agreed, but estimated bone Sr concentrations were likely overestimated. Results, approximations, future work directions, and applications were discussed.

Inflammatory Gene Profile and Particle Presence in Peri-Implant Mucosa: a Pilot Study on 9 Patients.

The purpose of this pilot study is to compare gene expression in mucosa around dental implants with zirconia abutment to titanium and investigate presence of particles in mucosa samples and on implant heads. Ten patients with a single implant supported prosthesis connected to zirconia or titanium abutments were invited at the five-year control. A clinical examination and a survey on experience of function and appearance were conducted. A mucosa biopsy taken in close vicinity to the implant were analysed by real-time polymerase chain reaction (qPCR) and presence of particles in a scanning electron microscope/energy-dispersive X-ray spectroscope (SEM/EDX). Cytological smear samples were collected and analysed through inductively coupled plasma mass spectrometry (ICP-MS) to investigate presence of particles on implant heads. In total, 9 patients participated in the study, five with titanium abutments and four with zirconia abutments. All patients were satisfied with function and aesthetics. Titanium and iron particles were detected in mucosa biopsies. The ICP - MS analysis demonstrated presence of zirconia and titanium. Several proinflammatory genes were upregulated in the zirconia abutment group. Around zirconia abutments a slight increase in proinflammatory response and amount of wear particles was seen as compared to titanium. Wear particles of titanium were present in all soft tissue samples, however zirconia particles only in the samples from implants heads/mucosa with zirconia abutments.

Feasibility and safety of synchrotron-based X-ray phase contrast imaging as a technique complementary to histopathology analysis.

X-ray phase contrast imaging (X-PCI) is a powerful technique for high-resolution, three-dimensional imaging of soft tissue samples in a non-destructive manner. In this technical report, we assess the quality of standard histopathological techniques performed on formalin-fixed, paraffin-embedded (FFPE) human tissue samples that have been irradiated with different doses of X-rays in the context of an X-PCI experiment. The data from this study demonstrate that routine histochemical and immunohistochemical staining quality as well as DNA and RNA analyses arenot affected by previous X-PCI on human FFPE samples. From these data we conclude it is feasible and acceptable to perform X-PCI on FFPE human biopsies.

Comparison of Heavy Metal Distribution in Mouse Soft Tissue Samples Prepared for Serial Block Face SEM Using Different Protocols.

Comparison of Heavy Metal Distribution in Mouse Soft Tissue Samples Prepared for Serial Block Face SEM Using Different Protocols.

Potentialities of iodine-enhanced micro-CT imaging in the morphological study of adult Aedes (Stegomyia) aegypti (Linnaeus, 1762) mosquitoes

X-ray micro-computed tomography (CT) produces three-dimensional images of samples on a micrometer scale. This technique has several advantages, such as its nondestructive character and low measurement time, compared with other techniques. However, when applied to biological samples of soft tissue, the low attenuation and low effective contrast between structures pose difficulties in creating appropriate images for morphological studies. Diffusible iodine-based contrast-enhanced CT (DICE-CT), which uses iodine solutions to enhance contrast, is a viable alternative for addressing the aforementioned challenges. Given the variety of biological samples, an appropriate methodology must be adapted depending on the dimensions and morphological characteristics of the investigated object. A specimen that has not been morphologically studied by micro-CT and is of high sanitary importance is the adult Aedes aegypti mosquito. This study investigated the stage of iodine staining in the treatment of the A. aegypti mosquito to determine the most suitable staining time for the morphological study of this mosquito in adulthood. After determining the appropriate staining time, we discuss the potential of applying DICE-CT and methodology to mosquito studies. Seven A. aegypti females were treated using fixation steps with Bouin's solution, dehydration in a graded ethanol series, staining with iodine solution (1%), and washing in absolute ethanol. Only the staining step was different between samples. Each mosquito spent a varying amount of time (6–72 h) in the iodine solution (1%). For comparison, one of the mosquitoes was not stained. After treatment, the samples were scanned using the Bruker SkyScan 1172 micro-CT scanner. The reconstructed volumes and histograms were compared to determine the most suitable time. In addition, a quantitative analysis was performed based on a comparison of the attenuation profiles of the mosquito brains. Thereafter, the most suitable treatment process was selected, and two other samples were scanned after applying the selected process. Although fewer than 18 h was insufficient for an effective increase in attenuation and effective contrast, surpassing 24 h proved unnecessary and resulted in saturating the gray tones visualized through the histograms, leading to information loss. Therefore, a time of approximately 24 h was the most suitable staining time for studying adult A. aegypti. It was possible to isolate the organs of the digestive and reproductive systems of the mosquito stained for 24 h. Thus, micro-CT was confirmed to be an excellent technique in studies of individual structures of adult A. aegypti mosquitoes.

Staphylococcus aureus From an Acute Fracture-related Infection Displays Important Bacteriological and Histopathologic Differences From a Chronic Equivalent in a Murine Bone Infection Model.

Staphylococcus aureus is the leading pathogen in fracture-related infection. Previous in vitro experiments, in vivo testing in wax moth larvae, and genomic analysis of clinical S. aureus isolates from fracture-related infection identified low-virulence (Lo-SA5464) and high-virulence (Hi-SA5458) strains. These findings correlated with acute fracture-related infection induced by Hi-SA5458, whereas Lo-SA5464 caused a chronic fracture-related infection in its human host. However, it remains unclear whether and to what extent the causative pathogen is attributable to these disparities in fracture-related infections. Are there differences in the course of infection when comparing these two different clinical isolates in a murine fracture-related infection model, as measured by (1) clinical observations of weight loss, (2) quantitative bacteriology, (3) immune response, and (4) radiographic and histopathologic morphology? Twenty-five (including one replacement animal) female (no sex-specific influences expected), skeletally mature C57Bl/6N inbred mice between 20 and 28 weeks old underwent femoral osteotomy stabilized by titanium locking plates. Fracture-related infection was established by inoculation of high-virulence S. aureus EDCC 5458 (Hi-SA5458) or low-virulence S. aureus EDCC 5464 (Lo-SA5464) in the fracture gap. Each of these groups consisted of 12 randomly assigned animals. Mice were euthanized 4 and 14 days postsurgery, resulting in six animals per group and timepoint. The severity and progression of infection were assessed in terms of clinical observation of weight loss, quantitative bacteriology, quantitative serum cytokine levels, qualitative analysis of postmortem radiographs, and semiquantitative histopathologic evaluation. For clinical observations of weight change, no differences were seen at Day 4 between Hi-SA5458- and Lo-SA5464-infected animals (mean -0.6 ± 0.1 grams versus -0.8 ± 0.2 grams, mean difference -0.2 grams [95% CI -0.8 to 0.5 grams]; p =0.43), while at 14 days, the Hi-SA5458 group lost more weight than the Lo-SA5464 group (mean -1.55 ± 0.2 grams versus -0.8 ± 0.3 grams; mean difference 0.7 grams [95% CI 0.2 to 1.3 grams]; p = 0.02). Quantitative bacteriological results 4 days postoperatively revealed a higher bacterial load in soft tissue samples in Hi-SA5458-infected animals than in the Lo-SA5464-infected cohort (median 6.8 x 107 colony-forming units [CFU]/g, range 2.2 x 107 to 2.1 x 109 CFU/g versus median 6.0 x 106 CFU/g, range 1.8 x 105 to 1.3 x 108 CFU/g; difference of medians 6.2 x 107 CFU/g; p = 0.03). At both timepoints, mice infected with the Hi-SA5458 strain also displayed higher proportions of bacterial dissemination into organs than Lo-SA5464-infected animals (67% [24 of 36 organs] versus 14% [five of 36 organs]; OR 12.0 [95% CI 3.7 to 36]; p < 0.001). This was accompanied by a pronounced proinflammatory response on Day 14, indicated by increased serum cytokine levels of interleukin-1β (mean 9.0 ± 2.2 pg/mL versus 5.3 ± 1.5 pg/mL; mean difference 3.6 pg/mL [95% CI 2.0 to 5.2 pg/mL]; p < 0.001), IL-6 (mean 458.6 ± 370.7 pg/mL versus 201.0 ±89.6 pg/mL; mean difference 257.6 pg/mL [95% CI 68.7 to 446.5 pg/mL]; p = 0.006), IL-10 (mean 15.9 ± 3.5 pg/mL versus 9.9 ± 1.0 pg/mL; mean difference 6.0 pg/mL [95% CI 3.2 to 8.7 pg/mL]; p < 0.001), and interferon-γ (mean 2.7 ± 1.9 pg/mL versus 0.8 ± 0.3 pg/mL; mean difference 1.8 pg/mL [95% CI 0.5 to 3.1 pg/mL]; p = 0.002) in Hi-SA5458-infected compared with Lo-SA5464-infected animals. The semiquantitative histopathologic assessment on Day 4 revealed higher grades of granulocyte infiltration in Hi-SA5458-infected animals (mean grade 2.5 ± 1.0) than in Lo-SA5464-infected animals (mean grade 1.8 ± 1.4; mean difference 0.7 [95% CI 0.001 to 1.4]; p = 0.0498). On Day 14, bone healing at the fracture site was present to a higher extent in Lo-SA5464-infected animals than in Hi-SA5458-infected animals (mean grade 0.2 ± 0.4 versus 1.8 ± 1.2; mean difference -1.6 [95% CI -2.8 to -0.5]; p = 0.008). Similar to septic infection in a human host, infection with Hi-SA5458 in this murine model was characterized by a higher bacterial load, more-pronounced systemic dissemination, and stronger systemic and local inflammation. Thus, there is strong support for the idea that pathogenic virulence plays a crucial role in fracture-related infections. To confirm our observations, future studies should focus on characterizing S. aureus virulence at the genomic and transcriptomic levels in more clinical isolates and patients. Comparing knockout and wildtype strains in vitro and in vivo, including the S. aureus strains studied, could confirm our findings and identify the genomic features responsible for S. aureus virulence in fracture-related infections. For translational use, virulence profiles of S. aureus may be useful in guiding treatment decisions in the future. Once specific virulence targets are identified, one approach to fracture-related infections with high-virulence strains might be the development of antivirulence agents, particularly to treat or prevent septic dissemination. For fracture-related infections with low virulence, prolonged antimicrobial therapy or exchange of an indwelling implant might be beneficial owing to slower growth and persistence capacity.

Towards virtual histology with X-ray grating interferometry

Breast cancer is the most common type of cancer worldwide. Diagnosing breast cancer relies on clinical examination, imaging and biopsy. A core-needle biopsy enables a morphological and biochemical characterization of the cancer and is considered the gold standard for breast cancer diagnosis. A histopathological examination uses high-resolution microscopes with outstanding contrast in the 2D plane, but the spatial resolution in the third, Z-direction, is reduced. In the present paper, we propose two high-resolution table-top systems for phase-contrast X-ray tomography of soft-tissue samples. The first system implements a classical Talbot–Lau interferometer and allows to perform ex-vivo imaging of human breast samples with a voxel size of 5.57 μm. The second system with a comparable voxel size relies on a Sigray MAAST X-ray source with structured anode. For the first time, we demonstrate the applicability of the latter to perform X-ray imaging of human breast specimens with ductal carcinoma in-situ. We assessed image quality of both setups and compared it to histology. We showed that both setups made it possible to target internal features of breast specimens with better resolution and contrast than previously achieved, demonstrating that grating-based phase-contrast X-ray CT could be a complementary tool for clinical histopathology.

Comparing x-ray phase-contrast imaging using a Talbot array illuminator to propagation-based imaging for non-homogeneous biomedical samples

Phase-contrast computed tomography can visualize soft tissue samples with high contrast. At coherent sources, propagation-based imaging (PBI) techniques are among the most common, as they are easy to implement and produce high-resolution images. Their downside is a low degree of quantitative data due to simplifying assumptions of the sample properties in the reconstruction. These assumptions can be avoided, by using quantitative phase-contrast techniques as an alternative. However, these often compromise spatial resolution and require complicated setups. In order to overcome this limitation, we designed and constructed a new imaging setup using a 2D Talbot array illuminator as a wavefront marker and speckle-based imaging phase-retrieval techniques. We developed a post-processing chain that can compensate for wavefront marker drifts and that improves the overall sensitivity. By comparing two measurements of biomedical samples, we demonstrate that the spatial resolution of our setup is comparable to the one of PBI scans while being able to successfully image a sample that breaks the typical homogeneity assumption used in PBI.

Femtosecond laser preparation of resin embedded samples for correlative microscopy workflows in life sciences.

Correlative multimodal imaging is a useful approach to investigate complex structural relations in life sciences across multiple scales. For these experiments, sample preparation workflows that are compatible with multiple imaging techniques must be established. In one such implementation, a fluorescently labeled region of interest in a biological soft tissue sample can be imaged with light microscopy before staining the specimen with heavy metals, enabling follow-up higher resolution structural imaging at the targeted location, bringing context where it is required. Alternatively, or in addition to fluorescence imaging, other microscopy methods, such as synchrotron x-ray computed tomography with propagation-based phase contrast or serial blockface scanning electron microscopy, might also be applied. When combining imaging techniques across scales, it is common that a volumetric region of interest (ROI) needs to be carved from the total sample volume before high resolution imaging with a subsequent technique can be performed. In these situations, the overall success of the correlative workflow depends on the precise targeting of the ROI and the trimming of the sample down to a suitable dimension and geometry for downstream imaging. Here, we showcase the utility of a femtosecond laser (fs laser) device to prepare microscopic samples (1) of an optimized geometry for synchrotron x-ray tomography as well as (2) for volume electron microscopy applications and compatible with correlative multimodal imaging workflows that link both imaging modalities.

Histologic and Histomorphometric Analyses of Free Gingival Grafts After Mechanical Expansion: A Study in Dogs.

This study assessed the histologic and histomorphometric changes of free gingival grafts in a canine model after mechanical expansion. A total of eight epithelialized tissue samples were obtained from the palate of eight Beagle dogs. Samples were cut in half and separated into two groups: the test group, in which a device was used to expand the grafts, and the control group, without expansion. After histologic processing, samples were evaluated by qualitative histology and histomorphometry. Histologic analysis revealed some differences in epithelial cell morphology and keratin layer integrity in the test group compared to the control group. Differences in histomorphometric parameters for the expanded and nonexpanded groups, including the thickness of the keratin layer (15.4 ± 13.4 μm and 32.3 ± 18.1 μm, respectively), thickness of the epithelium (398.0 ± 168.0 μm and 368.4 ± 142.8 μm, respectively), and the area occupied by collagen fibers in the connective tissue (62.0% ± 11.0% and 55.8% ± 7.6%, respectively), were not statistically significant (P < .05). Despite some changes in qualitative histology, free gingival grafts maintained their histomorphometric characteristics after mechanical expansion. These data provide a scientific basis for the use of mechanical expansion as a possible procedure to reduce the morbidity of autogenous grafts because a single soft tissue sample can be expanded before grafting. Int J Periodontics Restorative Dent 2023;43:e89-e97. doi: 10.11607/prd.5752.

Preparation of large biological samples for high-resolution, hierarchical, synchrotron phase-contrast tomography with multimodal imaging compatibility.

Imaging across different scales is essential for understanding healthy organ morphology and pathophysiological changes. The macro- and microscale three-dimensional morphology of large samples, including intact human organs, is possible with X-ray microtomography (using laboratory or synchrotron sources). Preparation of large samples for high-resolution imaging, however, is challenging due to limitations such as sample shrinkage, insufficient contrast, movement of the sample and bubble formation during mounting or scanning. Here, we describe the preparation, stabilization, dehydration and mounting of large soft-tissue samples for X-ray microtomography. We detail the protocol applied to whole human organs and hierarchical phase-contrast tomography at the European Synchrotron Radiation Facility, yet it is applicable to a range of biological samples, including complete organisms. The protocol enhances the contrast when using X-ray imaging, while preventing sample motion during the scan, even with different sample orientations. Bubbles trapped during mounting and those formed during scanning (in the case of synchrotron X-ray imaging) are mitigated by multiple degassing steps. The sample preparation is also compatible with magnetic resonance imaging, computed tomography and histological observation. The sample preparation and mounting require 24-36 d for a large organ such as a whole human brain or heart. The preparation time varies depending on the composition, size and fragility of the tissue. Use of the protocol enables scanning of intact organs with a diameter of 150 mm with a local voxel size of 1 μm. The protocol requires users with expertise in handling human or animal organs, laboratory operation and X-ray imaging.

Methodology for estimation of undeformed thickness of arterial tissues

Soft tissue sample thickness measurement is one of the major sources of differences between mechanical responses published by different groups. New method for the estimation of unloaded sample thickness of soft tissues is proposed in this study. Ten 30 × 30 mm and ten 20 × 20 mm samples of porcine anterior thoracic aortas were loaded by gradually increased radial force. Their deformed thickness was then recorded in order to generate a pressure-thickness response. Next, the limit pressure to which the response can be considered linear was estimated. Line was fitted to the linear part of the curve and extrapolated towards zero pressure to estimate unloaded thickness (7 kPa fit). For comparison, data near zero pressure were fitted separately and extrapolated towards zero (Near Zero fit). The limit pressure for the linearity of the response was around 7 kPa. The Unloaded thickness for 30 × 30 mm samples was 2.68 ± 0.31 mm and 2.68 ± 0.3 mm for Near Zero fit and 7 kPa fit, respectively. The Unloaded thickness for 20 × 20 mm samples was 2.60 ± 0.35 mm and 2.59 ± 0.35 mm for Near Zero fit and 7 kPa fit, respectively. The median of thickness difference between smaller and larger samples was not found statistically different. Proposed method can estimate unloaded undeformed sample thickness quickly and reliably.

Optical force estimation for interactions between tool and soft tissues

Robotic assistance in minimally invasive surgery offers numerous advantages for both patient and surgeon. However, the lack of force feedback in robotic surgery is a major limitation, and accurately estimating tool-tissue interaction forces remains a challenge. Image-based force estimation offers a promising solution without the need to integrate sensors into surgical tools. In this indirect approach, interaction forces are derived from the observed deformation, with learning-based methods improving accuracy and real-time capability. However, the relationship between deformation and force is determined by the stiffness of the tissue. Consequently, both deformation and local tissue properties must be observed for an approach applicable to heterogeneous tissue. In this work, we use optical coherence tomography, which can combine the detection of tissue deformation with shear wave elastography in a single modality. We present a multi-input deep learning network for processing of local elasticity estimates and volumetric image data. Our results demonstrate that accounting for elastic properties is critical for accurate image-based force estimation across different tissue types and properties. Joint processing of local elasticity information yields the best performance throughout our phantom study. Furthermore, we test our approach on soft tissue samples that were not present during training and show that generalization to other tissue properties is possible.