Thin Tissue Research Articles

Histological Comparative Analysis of Bowman Layer Grafts Procured Using 3 Different Techniques.

The objective of this study is to perform a histological analysis of Bowman layer (BL) grafts. BL grafts were procured from 13 human cadaver corneal tissues using 3 different donor preparation techniques. Subsequently, the grafts were fixed in 10% buffered formalin phosphate and embedded in paraffin. Hematoxylin and eosin sections of BL grafts were obtained and analyzed under a light microscope. BL and full graft thickness were measured using an image-processing software. All 13 BL grafts contained residual anterior stromal tissue. BL stripping using Kelman-McPherson and Moorfield forceps (technique 3) achieved the thinnest graft thickness with a mean full graft thickness of 18.7 μm (95% confidence interval [CI], -9.8 to 47.2) at the thinnest point of the graft, whereas BL procurement using the Melles lamellar dissector (technique 2) led to the highest mean full graft thickness of 279.9 μm (95% CI, 251.4-308.5) even at the thinnest area of the graft. By contrast, BL dissection using a blunt dissector (technique 1) provided a mean full graft thickness of 70.2 μm (95% CI, 40.4-100.1) at the graft's thinnest point. Although peripheral graft tears occurred in 50%, 50%, and 100% of techniques 1, 2, and 3, respectively, intact 6.25-mm diameter BL grafts could be secured in 50%, 100%, and 80% of techniques 1, 2, and 3, respectively. None of the techniques used led to the procurement of pure BL grafts devoid of the anterior stroma. Peripheral scoring with a thin needle and tissue manipulation with Kelman-McPherson and Moorfield forceps led to the thinnest grafts in this study.

Super-resolution STED imaging in the inner and outer whole-mount mouse retina.

Since its invention, super-resolution microscopy has become a popular tool for advanced imaging of biological structures, allowing visualisation of subcellular structures at a spatial scale below the diffraction limit. Thus, it is not surprising that recently, different super-resolution techniques are being applied in neuroscience, e.g. to resolve the clustering of neurotransmitter receptors and protein complex composition in presynaptic terminals. Still, the vast majority of these experiments were carried out either in cell cultures or very thin tissue sections, while there are only a few examples of super-resolution imaging in deeper layers (30 - 50 µm) of biological samples. In that context, the mammalian whole-mount retina has rarely been studied with super-resolution microscopy. Here, we aimed at establishing a stimulated-emission-depletion (STED) microscopy protocol for imaging whole-mount retina. To this end, we developed sample preparation including horizontal slicing of retinal tissue, an immunolabeling protocol with STED-compatible fluorophores and optimised the image acquisition settings. We labelled subcellular structures in somata, dendrites, and axons of retinal ganglion cells in the inner mouse retina. By measuring the full width at half maximum of the thinnest filamentous structures in our preparation, we achieved a resolution enhancement of two or higher compared to conventional confocal images. When combined with horizontal slicing of the retina, these settings allowed visualisation of putative GABAergic horizontal cell synapses in the outer retina. Taken together, we successfully established a STED protocol for reliable super-resolution imaging in the whole-mount mouse retina at depths between 30 and 50 µm, which enables investigating, for instance, protein complex composition and cytoskeletal ultrastructure at retinal synapses in health and disease.

Influence of supracrestal tissue height on peri-implant hard and soft tissues of single implants placed in the esthetic areas using one abutment-one time approach: A pilot, prospective interventional study.

This pilot, prospective interventional study aimed to analyze the influence of supracrestal tissue height (STH) when using the one abutment-one time concept (OAOT) at the time of implant placement, on peri-implant hard and soft tissue remodeling in esthetic areas. The definitive crown was placed seven days later. Facial mucosal margin position (FMMP), mesial and distal papilla levels (MPL and DPL, respectively), and mesial and distal marginal bone loss (M-MBL and D-MBL, respectively) were assessed after seven days (placement of the definitive crown), and 1, 2, 3, 6 and 12 months after implant placement. Patients were classified according to the STH in thin (STH < 3 mm) and thick (STH ≥ 3 mm). Fifteen patients fulfilled the eligibility criteria and were included in the study. Eight presented a thick STH and seven a thin STH. After 12 months, the implant success rate was 100%. The mean recession at FMMP was -0.47 ± 0.57 mm and -0.19 ± 0.41 mm in thin and thick groups, respectively (p = 0.29). The mean MPL recession was -0.19 ± 0.06 mm in the thin group and -0.01 ± 0.07 mm in the thick group (p < 0.01), and the mean DPL recession was -0.15 ± 0.09 mm in the thin group and 0.00 ± 0.15 mm in the thick group (p < 0.05). The mean bone loss was -0.21 ± 0.18 mm and -0.04 ± 0.14 mm in the thin and thick groups, respectively (p < 0.05). Single maxillary anterior implants with thin supracrestal tissue height (< 3 mm) at the time of implant placement had greater bone loss and papillary recession than implants with a thick soft tissue height (≥ 3 mm), even when using the one abutment one time concept.

Abstract 4707: LED photobleaching-based multiplex 3D microscopy of the tumor microenvironment

Abstract Multiplexing in immunofluorescence imaging is important for the spatial profiling of cells and molecules in tumor tissue samples. Cyclic immunofluorescence (IF) methods using oxidants (e.g. hydrogen peroxide) and enzymes (e.g. DNase) localize a great number of cellular makers and proteins in a tissue section while repeating a process of IF staining, imaging, and fluorescence deactivation. However, the repeated use of chemicals and enzymes might cause artifacts in tissue and cell morphologies. Furthermore, these methods are restricted to thin tissue sections (~5 μm thick) which are inappropriate to provide comprehensive structural information on tissue samples. Although reconstruction of two-dimensional (2D) images from serial tissue sections can provide a certain volumetric tissue image, it takes a huge amount of time and effort. Here we introduce a three-dimensional (3D) multiplex IF imaging method using LED photobleaching. We built high-power LED illuminators with 100W warm (emission wavelength: 480-700 nm), green (430-520 nm), and red (600- 680 nm) LED chips, which can efficiently bleach a broad or selected wavelength of fluorescence signals in tissue samples. We integrated this LED photobleaching with the Transparent Tissue Tomography (T3) protocol and created a 3D cyclic IF method involving tissue macrosectioning (400 μm), three-color IF staining, D-fructose-based tissue clearing, 3D confocal fluorescence microscopy, LED photobleaching, tissue washing, and three-color IF staining for other biomarkers, and repeating the process. By applying this method to mouse mammary tumor tissues, we could perform 8-plex fluorescence microscopy for visualizing cell nuclei (DAPI), vascular (CD31, SMA) and structural (ER-TR7) cells, immune cells (CD3, CD8, CD45), and cancer cells (CK8) in the tumor macrosections in 3D at tissue and cellular resolution. To validate the method as an evaluation tool for immunotherapy, we treated the mouse mammary tumor with a STING agonist (DMAXX) intratumorally and collected the tumor tissue 1 day after the treatment, and processed it for the 3D cyclic IF protocol. The quantitative multiplex image data showed immune-driven-cancer eradication and high tumor infiltration of a large number of CD3+CD8+CD45+ cytotoxic T cells. We also examined that Red and Green LED illumination can selectively bleach fluorophores in tissues, which would be useful for patterning fluorescence in tissue as well as studying fluorescent drug-cell interaction in a tissue. In summary, this chemical and enzyme-free 3D cyclic IF imaging method will be a powerful tissue assay tool to provide comprehensive spatial information of tissue (tumor) samples including cell types, cellular and molecular location, and their 3D organization in a tissue sample. Citation Format: Jingtian Zheng, Evan Phillips, Yi-Chien Wu, Steve Seung-Young Lee, Vytautas Bindokas. LED photobleaching-based multiplex 3D microscopy of the tumor microenvironment. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4707.

High-throughput single-molecule localization microscopy: Potential clinical applications.

High-throughput single-molecule localization microscopy: Potential clinical applications.

V09-07 HOW TO USE A MESH SLING SAFELY AFTER THE POST-VESICOVAGINAL FISTULA REPAIR REPAIR PATIENT WITH STRESS URINARY INCONTINENCE

You have accessJournal of UrologyCME1 Apr 2023V09-07 HOW TO USE A MESH SLING SAFELY AFTER THE POST-VESICOVAGINAL FISTULA REPAIR REPAIR PATIENT WITH STRESS URINARY INCONTINENCE Micheal Breen Tamatave, Madagascar, Ly Hoang Roberts, and Larry Sirls Micheal Breen Tamatave, MadagascarMicheal Breen Tamatave, Madagascar More articles by this author , Ly Hoang RobertsLy Hoang Roberts More articles by this author , and Larry SirlsLarry Sirls More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003317.07AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Patients with complicated obstetric fistulas may have stress urinary incontinence (SUI) after vesicovaginal fistula repair (VVF), especially if the urethra in involved. Various techniques to treat post-VVF SUI have low success rates or is technically challenging due to thin and scarred peri-urethral tissue. We describe a technique to place a midurethral mesh sling outside of the pre-existing vaginal mucosa. METHODS: With the patient in dorsal lithotomy under spinal anesthesia, a foley catheter is placed into the bladder and the labial majora is retracted for adequate exposure. A rectangular area of the same size as the mesh sling (3x1cm) at the midurethra is marked. A number 15 scalpel is used to incise the vaginal mucosa at the marked lines, and Metzenbaum scissors are used to dissect and mobilize the edges. The dissection is then carried out laterally to be behind the inferior pubic bone. This is done on both sides. The island of vaginal mucosa is then superficially scored using low power bovie cauterization to allow for mesh adhesion and tissue ingrowth. The mesh is sutured to the island of skin using a 3-0 Chromic or vicryl suture at the mesh edges in 4 areas. 0- prolen sutures are then placed on each lateral edge of the mesh in a running stitch. The Metzenbaum scissors are then used to enter into the endopelvic fascia behind the pubic bone to develop the retropubic space. Next, a suprapubic incision is made 1cm cranial to the pubic bone down to the rectus fascia. The Stamey needle is passed in a top-down approach approximately 2cm from midline and end delivered into the vaginal incision. To check for bladder perforation, a metal catheter is placed into the bladder and swept laterally to evaluate for any metallic noise indicative of contact. The prolene suture is passed up to the suprapubic incision and the same is done contralaterally. Bladder is backfilled with sterile saline with methylene blue by gravity and foley removed. Sutures are tied together midline with the sling tension adjusted while patient performs a Valsalva. Suprapubic incision is then closed in multiple layers. The vaginal incision is closed with mobilization of the proximal vaginal mucosa and reapproximation to the distal edge. For larger incisions, a Singapore flap can be done for adequate coverage. RESULTS: Successful managment of SUI. CONCLUSIONS: For challenging cases of SUI after a post-VVF repair, this technique can be an option. Further investigation is needed for clinical outcomes. Source of Funding: None © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e839 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Micheal Breen Tamatave, Madagascar More articles by this author Ly Hoang Roberts More articles by this author Larry Sirls More articles by this author Expand All Advertisement PDF downloadLoading ...

Effect of self-reported clinician experience level on perceptions of the relative rankings of risk factors for biological complications with dental implant therapy

Statement of problemThe effect of clinician experience on the perception of risks for biological complications associated with dental implant therapy is unknown. PurposeThe purpose of this study was to determine whether clinician experience levels, as assessed by the number of implants placed and/or restored, as well as by type of clinical experience (restorative/prosthodontic and/or surgical), influence the perception of risk for biological complications with dental implant therapy. Material and methodsA 10-item survey instrument was developed that asked participants from a convenience sample to rank different risk factors for peri-implant complications in the areas of patient history, clinical findings, and clinician choices and postimplant placement findings. Information about participant experience (number of implants placed and restored and type of experience) was also captured. The Kruskal-Wallis and Mann-Whitney U test statistical analyses were performed to determine the degree, if any, to which the level of clinician experience influenced risk factor rankings (α=.05). ResultsClinicians with more experience viewed the use of selective serotonin reuptake inhibitor (SSRI) medications and clinician inexperience as more important risk factors than did less experienced clinicians. Clinicians with less experience viewed treated moderate or severe chronic periodontitis and thin tissue biotype as more important risk factors than did more experienced clinicians. Clinicians with predominantly surgical experience viewed a heavy plaque index as a more important risk factor than clinicians with predominantly restorative experience. ConclusionsThe data indicate that the level of clinical experience influences how risk factors associated with biological complications of dental implant therapy are viewed by clinicians.

Real-time and accurate estimation ex vivo of four basic optical properties from thin tissue based on a cascade forward neural network.

Double integrating sphere measurements obtained from thin ex vivo tissues provides more spectral information and hence allows full estimation of all basic optical properties (OPs) theoretically. However, the ill-conditioned nature of the OP determination increases excessively with the reduction in tissue thickness. Therefore, it is crucial to develop a model for thin ex vivo tissues that is robust to noise. Herein, we present a deep learning solution to precisely extract four basic OPs in real-time from thin ex vivo tissues, leveraging a dedicated cascade forward neural network (CFNN) for each OP with an additional introduced input of the refractive index of the cuvette holder. The results show that the CFNN-based model enables accurate and fast evaluation of OPs, as well as robustness to noise. Our proposed method overcomes the highly ill-conditioned restriction of OP evaluation and can distinguish the effects of slight changes in measurable quantities without any a priori knowledge.

The origin of an invasive air sac system in sauropodomorph dinosaurs.

One of the most remarkable features in sauropod dinosaurs relates to their pneumatized skeletons permeated by a bird-like air sac system. Many studies described the late evolution and diversification of this trait in mid to late Mesozoic forms but few focused on the origin of the invasive respiratory diverticula in sauropodomorphs. Fortunately, it is possible to solve this thanks to the boom of new species described in the last decade as well as the broad accessibility of new technologies. Here we analyze the unaysaurid sauropodomorph Macrocollum itaquii from the Late Triassic (early Norian) of southern Brazil using micro-computed tomography. We describe the chronologically oldest and phylogenetically earliest unambiguous evidence of an invasive air sac system in a dinosaur. Surprisingly, this species presented a unique pattern of pneumatization in non-sauropod sauropodomorphs, with pneumatic foramina in posterior cervical and anterior dorsal vertebrae. This suggests that patterns of pneumatization were not cladistically consistent prior to the arrival of Jurassic eusauropods. Additionally, we describe the protocamerae tissue, a new type of pneumatic tissue with properties of both camellae and camerae. This reverts the previous hypothesis which stated that the skeletal pneumatization first evolved into camarae, and derived into delicate trabecular arrangements. This tissue is evidence of thin camellate-like tissue developing into larger chambers. Finally, Macrocollum is an example of the gradual evolution of skeletal tissues responding to the fastly specializing Respiratory System of saurischian dinosaurs.

A Novel Multi-Observation System to Study the Effects of Anterior Ocular Inflammation in Zinn's Zonule Using One Specimen.

Zinn's zonule is a fragile and thin tissue, and little is known about its pathogenesis. The aim of this study was to develop an experimental setup for a comprehensive analysis of Zinn's zonule. Rats were divided into two groups: a control group (n = 4) and an alkali injury group (n = 4). Seven days after injury, the eyes were enucleated, the anterior eye was dissected and embedded in gelatin, and macroscopic observations were made. The gelatin specimens were then embedded in paraffin and observed in detail by low-vacuum scanning electron microscopy, immunofluorescence, and quantitative reverse transcription polymerase chain reaction (RT-qPCR). The results show qualitative changes in Zinn's zonules in both macroscopic and microscopic observations. In addition, macrophage infiltration and increased matrix metalloproteinase 2 (MMP2) expression were observed in the injured group, consistent with the RT-qPCR results. The experimental system in this study allowed us to capture the morphological and molecular biological changes of Zinn's zonule and to gain insight into its pathogenesis. In conclusion, this study presents a new experimental setup for the comprehensive analysis of the rat Zinn's zonule. The results suggest that this system can be used in the future to study and analyze a variety of paraffin-embedded tissues and specimens.

Refined Dual-Phase-Lag Theory for the 1D Behavior of Skin Tissue under Ramp-Type Heating

In this article, a mathematical analysis of thermoelastic skin tissue is presented based on a refined dual-phase-lag (DPL) thermal conduction theory that considers accounting for the effect of multiple time derivatives. The thin skin tissue is regarded as having mechanically clamped surfaces that are one-dimensional. Additionally, the skin tissue undergoes ramp-type heating on its outer surface, whereas its inner surface keeps the assessed temperature from vanishing. Some of the previous generalized thermoelasticity theories were obtained from the proposed model. The distributions of temperature, displacement, dilatation, and stress are attained by applying the Laplace transform and its numerical reversal approaches. The outcomes are explicitly illustrated to examine the significant influences on the distributions of the field variables. The refined DPL bioheat conduction model in this study predicts temperature, and the findings revealed that the model is located among the existing generalized thermoelastic theories. These findings offer a more thorough understanding of how skin tissue behaves when exposed to a particular boundary condition temperature distribution.

Histological study of the thin membranous dense connective tissues around the curving portion of the bilateral recurrent laryngeal nerves.

We aimed to demonstrate the anatomical relationship between the recurrent laryngeal nerves (RLNs), thin membranous dense connective tissue (TMDCT [e.g., the visceral or vascular sheaths around the esophagus]), and the lymph nodes around the esophagus at the curving portion of the RLNs for rational and efficient lymph node dissection. Transverse sections of the mediastinum at 5mm or 1mm intervals were obtained from four cadavers. Hematoxylin and eosin staining and Elastica van Gieson staining were performed. The visceral sheaths could not be clearly observed the curving portions of the bilateral RLNs, which were observed on the cranial and medial side of the great vessels (aortic arch and right subclavian artery [SCA]). The vascular sheaths could be clearly observed. The bilateral RLNs diverged from the bilateral vagus nerves, which ran along with the vascular sheaths, went up around the caudal side of the great vessels and the vascular sheath, and ran cranially on the medial side of the visceral sheath. Visceral sheaths were not observed around the region containing the left tracheobronchial lymph nodes (No. 106tbL) or the right recurrent nerve lymph nodes (No. 106recR). The regions containing the left recurrent nerve lymph nodes (No. 106recL) and the right cervical paraesophageal lymph nodes (No. 101R) were observed on the medial side of the visceral sheath, with the RLN. The recurrent nerve, which branched off from the vagus nerve descending along the vascular sheath, ascended the medial side of the visceral sheath after inversion. However, no clear visceral sheath could be identified in the inverted area. Therefore, during radical esophagectomy, the visceral sheath along No. 101R or 106recL may be recognized and available.

Acoustic Force Elastography Microscopy.

Hydrogel scaffolds have attracted attention to develop cellular therapy and tissue engineering platforms for regenerative medicine applications. Among factors, local mechanical properties of scaffolds drive the functionalities of cell niche. Dynamic mechanical analysis (DMA), the standard method to characterize mechanical properties of hydrogels, restricts development in tissue engineering because the measurement provides a single elasticity value for the sample, requires direct contact, and represents a destructive evaluation preventing longitudinal studies on the same sample. We propose a novel technique, acoustic force elastography microscopy (AFEM), to evaluate elastic properties of tissue engineering scaffolds. AFEM can resolve localized and two-dimensional (2D) elastic properties of both transparent and opaque materials with advantages of being non-contact and non-destructive. Gelatin hydrogels, neat synthetic oligo[poly(ethylene glycol)fumarate] (OPF) scaffolds, OPF hydroxyapatite nanocomposite scaffolds and ex vivo biological tissue were examined with AFEM to evaluate the elastic modulus. These measurements of Young's modulus range from approximately 2 kPa to over 100 kPa were evaluated and are in good agreement with finite element simulations, surface wave measurements, and DMA tests. The AFEM can resolve localized and 2D elastic properties of hydrogels, scaffolds and thin biological tissues. These materials can either be transparent or non-transparent and their evaluation can be done in a non-contact and non-destructive manner, thereby facilitating longitudinal evaluation. AFEM is a promising technique to quantify elastic properties of scaffolds for tissue engineering and will be applied to provide new insights for exploring elastic changes of cell-laden scaffolds for tissue engineering and material science.

Chitin nanofibrils modulate mechanical response in tympanic membrane replacements

The tympanic membrane (TM), is a thin tissue lying at the intersection of the outer and the middle ear. TM perforations caused by traumas and infections often result in a conductive hearing loss. Tissue engineering has emerged as a promising approach for reconstructing the damaged TM by replicating the native material characteristics. In this regard, chitin nanofibrils (CN), a polysaccharide-derived nanomaterial, is known to exhibit excellent biocompatibility, immunomodulation and antimicrobial activity, thereby imparting essential qualities for an optimal TM regeneration. This work investigates the application of CN as a nanofiller for poly(ethylene oxide terephthalate)/poly(butylene terephthalate) (PEOT/PBT) copolymer to manufacture clinically suitable TM scaffolds using electrospinning and fused deposition modelling. The inclusion of CN within the PEOT/PBT matrix showed a three-fold reduction in the corresponding electrospun fiber diameters and demonstrated a significant improvement in the mechanical properties required for TM repair. Furthermore, in vitro biodegradation assay highlighted a favorable influence of CN in accelerating the scaffold degradation over a period of one year. Finally, the oto- and cytocompatibility response of the nanocomposite substrates corroborated their biological relevance for the reconstruction of perforated eardrums.

Outcomes of operative intervention for ulcers over hemodialysis arteriovenous access.

The native arteriovenous fistula (AVF) is the gold standard for long-term hemodialysis access. When native vein options are exhausted, arteriovenous graft (AVG) becomes the next choice. An ulcer over an AVF or AVG is a serious condition with the potential for life-threatening hemorrhage. This study aims to present our experience with surgical management of ulcers over AVFs or AVGs. Electronic records of 26 patients who underwent 27 consecutive surgical procedures for ulcers over AVFs or AVGs from December 31, 2016 to December 31, 2021 at the Royal Liverpool University Hospital were retrospectively analyzed. The majority were males (14/26, 53.8%) and the median age was 64.5 years. Operative repair was required for 25 ulcers over 24 AVFs and 2 ulcers over 2 AVGs. Ten patients (37%) presented with bleeding. Seventeen (63%) had impending bleeding suggested by a thin soft tissue covering or a false aneurysm at the site of the ulcer. Previous endovascular intervention for fistula outflow stenosis was a significant predictor for presenting with bleeding (p=0.031). All ulcers (27/27, 100%) underwent excision and primary skin closure. Fistula wall defects were directly repaired in 18/27 (66.7%). Four/26 (14.8%) fistulas had to be ligated and 2 (7.4%) had end-to-end re-anastomosis after excision of damaged segments. Three/26 (11.53%) fistulas thrombosed immediately after the intervention. The overall fistula salvage rate was 73.08% (19/26). There was 1 (3.7%) patient mortality due to bleeding from wound breakdown after surgery. An acceptable fistula salvage rate can be expected following surgical repair of ulcers over AVFs and AVGs. A history of previous endovascular interventions for fistula outflow stenosis was a predictor of bleeding from these ulcers.

Elbow Soft Tissue Coverage after Trauma

Traumatic injuries involving the elbow can result in soft tissue defects with exposed critical structures including bone, joint, nerve, tendons or implants.1 Additionally, wounds about the elbow are subject to significant motion and tension, limiting the success of local wound care.2 In these clinical settings, soft tissue reconstruction is often indicated. The goal of reconstruction is elbow wound coverage with pliable, thin, and durable tissue that allows for repetitive motion without limiting range of motion.3 In the post-traumatic setting, optimal function is achieved if the soft tissue reconstruction allows for early return to active motion of the elbow joint.4

The applicability of Fourier transform infrared microspectroscopy for correction against matrix effects in X-ray fluorescence microimaging of tissues

X-ray fluorescence (XRF) and Fourier transform infrared (FTIR) microscopy techniques are now considered popular for rapid and label-free complementary spectrochemical analysis of chemical elements and molecular systems in biological specimens. The morphological heterogeneity but also the inhomogeneities associated with the thickness/density of biological samples demonstrate challenges for the quantitative XRF microimaging. Therefore, in the present work, we proposed for the first time the application of the total absorbance under the FTIR spectra as a mass surface correction procedure for two-dimensional (2D) XRF microimaging of tissues. We also evaluated the equivalence of the developed correction method based on total absorbance of FTIR spectra with the proposed approaches based on incoherent scattering of primary X-rays as well as on the membrane Si–Kα transmission signal, on the example of selected rat organ tissues. Thin cryo-sections taken from various organs of Wistar rats were deposited on silicon nitride membranes (Si3N4). The FTIR microscopy studies were performed to collect infrared absorption spectra, used then for the determination of total absorbance values in the selected areas of tissue samples. In turn, hard X-ray imaging based on synchrotron radiation allowed the determination of characteristic radiation intensities of the elements detectable from the tissue, as well as the characteristic radiation of the membrane Si and incoherently scattered X-ray photons (Compton scattering). The latter served as correction factors for the surface mass of the sample alongside the FTIR total absorbance. The qualitative and quantitative analyses showed a high agreement between the results of elemental surface mass correction using total absorbance under FTIR spectra of tissues with those obtained using surface mass correction factors determined directly from XRF spectra. Therefore, the proposed procedure is a good alternative in cases where the surface mass effect of the sample cannot be eliminated based on the information provided directly by the XRF spectrum, as in the case of using polymer films as sample support. We have also proposed a procedure for synchronizing SRXRF and FTIR images, not limited to visual inspection of imaging/mapping data, but also enabling quantitative analysis. We found that the total absorbance determined from FTIR spectra can be successfully used as a correction factor for eliminating the surface mass effect in XRF microimaging of thin freeze-fried tissues and therefore to obtain the surface mass-independent elemental quantities. The proposed approach for 2D-FTIR-XRF analysis can also be a powerful and versatile tool for fostering a correlation and co-localization analysis to search for common distribution patterns between molecular arrangements and chemical elements.

Better phasor calibration in turbid media using Monte Carlo simulations.

Phasor analysis has gained acceptance as a simpler yet quantitative alternative to nonlinear fitting approaches for in vitro fluorescence lifetime imaging microscopy (FLIM) and is incorporated into many commercial and research analysis software. FLIM typically involves cell monolayers, thin tissue slices or transparent spheroids, which do not introduce significant light scattering or absorption effects. As a result, the recorded fluorescence decay is a mere convolution product of the instrument response function and the emitted decay. However, the situation is more complex when in tissue phantoms or live animals observed with macroscopic fluorescence lifetime imaging (MFLI) systems, where those effects become noticeable. In particular, when using the phasor approach to analyze MFLI data, they result in an inadequacy to properly calibrate the phasor of measured decays using the simple approach used in FLIM. Namely, a discrepancy between observed calibrated phasor locations and those expected is generally observed in the simple cases where single-exponential decays are known to occur, no matter what calibration approach is used. This calls for a way to take these effects into account to obtain more quantitative results in this general situation of fluorescence in turbid media.

Soft-Tissue Augmentation around Dental Implants with a Connective Tissue Graft (CTG) and Xenogeneic Collagen Matrix (CMX)-5-Year Follow-Up.

Proper horizontal and vertical thickness of the gingival connective tissue has been proven to be one of the success criteria in dental implant and reconstructive surgery. When thin tissue is found, gingiva augmentation methods can be used to increase the quality and volume of the tissue. Many methods have been described, among them pedicle soft-tissue flaps or autogenic tissue grafts. As an alternative to patients' own tissue, xenogenic materials can be used for grafting. The fundamental issue is to choose a material that will ensure the maximum therapeutic effect, while also minimizing the negative influence on the patient's health. The aim of this study was to compare gingival augmentation procedures using a palatal connective tissue graft (CTG) and an xenogenic soft-tissue substitute, Geistlich Mucograft (xenogeneic collagen matrix; CMX), and assess whether the timing of the graft surgery influences the clinical outcomes. The original study was a randomized control trial with a total of 75 implants placed. The patients received the soft-tissue thickening 3 months before the implant placement or 3 months after the implant placement (depending on the group). A connective tissue graft (CTG) or Geistlich Mucograft were used (depending on the group). For both the CTG and Geistlich Mucograft, better clinical outcomes were observed for maintaining the alveolar bone level and the thickness of the attached gingiva compared to the control group with no gingival augmentation. The Geistlich Mucograft showed good clinical performance in comparison to the control. Soft-tissue augmentation with the CTG before the implant placement was found to be most efficient method in terms of a stable increase of the tissue thickness since, throughout the entire observation period, the greatest increase of 1.035 mm (SD = 0.73 mm) in thickness was observed. Statistically important differences in the tissue thickness baseline compared after 5 years were observed for groups G1 vs. G2b (no augmentation vs. CTG before), G1 vs. G3b (no augmentation vs. CTG after) and for groups G2b vs. G3a (CTG before vs. CMX after).

The equine patellar ligaments and the infrapatellar fat pad – a microanatomical study

BackgroundInterpretation of patellar ligament (PL) ultrasonography may be difficult, as hypoechoic or heterogenous echogenicity are common findings. Verifying suspected disease of equine PLs by histopathology is also problematic as descriptions of normal PL vascularity and histology are scarce. The current study describes the PL and infrapatellar fat pad (IFP) vascular pattern from computed tomography scans of barium perfused normal equine specimens (n = 8; age 10 days to 18 years), as well as routine histology to serve as a reference for future investigations into PL pathology and IFP disease.ResultsThe PLs received a bipolar blood supply. Vascular architecture consisted of numerous distinct longitudinal vessels with several horizontal connections, which branched into extensive latticeworks of smaller vessels throughout the ligaments. Several vascular connections between the PLs and the IFP were identified. One distinct longitudinal vessel was seen entering each of the IFP lobes at the distocranial aspect, branching extensively into lobar vascular networks which anastomosed by several horizontal branches at the mid portion of the IFP where the two lobes merge. Histologically, there were large variations in PL interfascicular endotenon thickness, vascularity and fatty infiltration; these parameters increased with age for the intermediate and medial PL. Areas of metaplastic tenocytes / chondroid metaplasia were identified in all investigated adult medial PLs; in 2/7 in the intermediate PL and in 4/7 in the lateral PL. The adult IFP consisted of white unilocular adipose tissue, organized in lobules separated by thin connective tissue septa increasing in thickness towards the periphery and the distocentral aspect.ConclusionsThe equine PLs and IFP are highly vascularized structures with ample vascular connections suggestive of crosstalk. This, together with the large variation in PL endotenon thickness, vascularity and fatty infiltration, should be taken into consideration when assessing potential PL histopathology as these changes increase with age and are found in horses without clinical signs of stifle disease. Metaplastic tenocytes / chondroid metaplasia should be considered a normal finding throughout the medial PL and is not age dependent. The role of the equine IFP in stifle disease has yet to be elucidated.