Tissue Fixation Research Articles

Meniscal allograft transplantation (MAT) combined with revision ACL reconstruction and lateral extra-articular tenodesis results in significantly improved outcomes, but patient-reported knee function is inferior compared to an isolated MAT.

Meniscal allograft transplantation (MAT) is a valuable option for postmeniscectomy syndrome in young and active patients, which can successfully improve symptoms, function and quality of life. This study aimed to report outcomes and complications in patients treated with isolated MAT or MAT combined with revision anterior cruciate ligament reconstruction (ACLR) and lateral extra-articular tenodesis (LET). In total, 18 patients who were treated with MAT using a soft tissue fixation technique were retrospectively analysed. Patients were allocated into two groups, the isolated MAT group and the MAT plus revision ACLR + LET group (MAT+ group). Both groups were assessed using the following Patient-Reported Outcome Measures (PROMs): Knee Injury and Osteoarthritis Outcome Score (KOOS), International Knee Documentation Committee (IKDC), Tegner Activity Scale, Lysholm score, EuroQol 5-Dimension 5-level (EQ-5D-5L) questionnaire and a patient acceptable symptom state (PASS) statement. The mean follow-up was 3.2 years and the mean age of patients was 29 (±7.6) years. Ten patients were treated with isolated MAT and eight patients were treated with MAT combined with revision ACLR and LET. No complications and no reoperations were reported. In both groups, all PROMs significantly improved postoperatively (p < 0.05), except for the Tegner score in the MAT+ group, which did not reach statistical significance (not significant [n.s.]). No significant difference was found postoperatively in PROMs between the MAT and the MAT+ group; however, differences in the KOOS Symptoms, Sports and Quality-of-life subscales reached the minimal clinically important threshold. Mean values were: IKDC 61.2 (±13.9) versus 64.7 (±23.2), KOOS Total 72.8 (±14.9) versus 68.0 (±16.7), Lysholm score 83.2 (±11) versus 84.2 (±8.7) and EQ-5D-5L 71.8 (±14.9) versus 72.2 (±22.4). Median values for the Tegner Activity Scale were 4 versus 3. PASS statement was negative in 37.5% of the MAT+ group in comparison to 20% of the MAT group (n.s.). In terms of patient-reported outcomes, MAT is an effective and safe procedure, even when combined with revision ACLR + LET. Compared to the preoperative assessment, a significant improvement of the postoperative PROMs can be expected either when MAT is performed isolated or in combination with ACLR + LET. However, clinically important differences between the two groups can be noted in favour of the isolated MAT procedure, especially regarding symptoms and sporting activity. Level III.

Diagnostic accuracy of autofluorescence-Raman microspectroscopy for surgical margin assessment during Mohs micrographic surgery of basal cell carcinoma.

Autofluorescence (AF)-Raman microspectroscopy is a technology that can detect residual basal cell carcinoma (BCC) on the resection margin of fresh, surgically excised tissue specimens. The technology does not require tissue fixation, staining, labelling or sectioning, and provides quantitative diagnosis maps of the surgical margins in 30 min. To determine the accuracy of the AF-Raman instrument in detecting incomplete BCC excisions during Mohs micrographic surgery (MMS), using histology as the reference standard. Skin layers from 130 patients undergoing MMS at the Nottingham University Hospitals NHS Trust (September 2022-July 2023) were investigated with the AF-Raman instrument. The layers were measured when fresh, immediately after excision. The AF-Raman results and the intraoperative assessment by Mohs surgeons were compared with a postoperative consensus-derived reference produced by three dermatopathologists. The sensitivity, specificity, and positive and negative predictive values were calculated. The study was registered with ClinicalTrials.gov (NCT03482622). AF-Raman analysis was successfully completed for 125 of 130 layers and, on average, covered 91% of the specimen surface area, with the lowest surface area covered being 87% for the eyelid and the highest being 94% for forehead specimens. The AF-Raman instrument identified positive margins in 24 of 36 BCC-positive cases [67% sensitivity, 95% confidence interval (CI) 49-82] and negative margins in 65 of 89 BCC-negative cases (73% specificity, 95% CI 63-82). Only one of 12 false-negative cases was caused by misclassification by the AF-Raman algorithm. The other 11 false-negatives cases were a result of no valid Raman signal being recorded at the location of the residual BCC due to either occlusion by blood or poor contact between tissue and the cassette window. The intraoperative diagnosis by Mohs surgeons identified positive margins in 31 of 36 BCC-positive cases (86% sensitivity, 95% CI 70-95) and negative margins in 79 of 89 BCC-negative cases (89% specificity, 95% CI 81-95). The AF-Raman instrument has the potential to provide intraoperative microscopic assessment of surgical margins in BCC surgery. Further improvements are required for tissue processing, to ensure complete coverage of the surgical specimens.

A beginner’s guide to immunohistochemistry

Using the affinity between antibodies and antigens, immunohistochemistry (IHC) is a helpful method for analysing the cellular components within tissue sections placed on glass slides. The chemistry of antibodies was not discovered until 1941, and since then, their use has increased due to the discovery of enzymes and fluorochromes conjugated to antibodies, enabling observations through optical or fluorescence microscopy (FM). Hence, immunofluorescence (IF), or IHC with fluorescent antibodies, has been associated with the advancement of FM and fluorochromes. Specifically, the discovery of three fluorochromes that emit blue, red and green led to a rise in IF application in the context of visualizing numerous components in a single cell. The proximity ligation assay (PLA) method, so termed because the antibody is conjugated with short DNA strands called PLA probes, is the most recent development in the field of antibody conjugation. Protein–protein interactions and sub-localization of proteins can both be studied using this technique. The improvement of conjugation techniques has occurred in parallel with the development of sophisticated microscopies, including confocal and slide scanners. Confocal microscopy allows for the reconstruction of three-dimensional structures while a slide scanner can digitally convert up to 100 slides' worth of high-resolution pictures. The first step in the IHC process is the collection and fixation of tissues from animal models and humans. Paraformaldehyde is the most commonly used fixative. Fixation prevents antigen blocking and creates cross-links between proteins to preserve tissue structures. Fixation is followed by cutting tissues using microtome or cryostat, then slides are treated for antigen exposition (antigen retrieval) and then included in Triton X solution, which increases membrane permeability. Subsequently, incubations with enzyme- or fluorochrome-conjugated antibodies enable observations via optical microscopy or FM, respectively. In clinical practice and research, IHC and IF are still helpful methods for identifying the antigen-proteins linked to specific diseases.

Retinoic acid modulation guides human-induced pluripotent stem cell differentiation towards left or right ventricle-like cardiomyocytes

Background: Cardiomyocytes (CMs) induced from human induced pluripotent stem cells (hiPSCs) by traditional methods are a mix of atrial and ventricular CMs and other non-cardiomyocytes. Retinoic acid (RA) plays an important role in regulation of the spatiotemporal development of the embryonic heart. Aim: Create engineered heart tissue (EHT) with left and right ventricular phenotype from hiPSCs by intervening with specific concentrations of RA during hiPSC differentiation towards CM. Methods: hiPSC were derived by reprogramming skin fibroblasts and erythroid progenitors. Different concentrations of RA (Control without RA, LowRA with 0.05 μM and HighRA with 0.1 μM) were administered during third to sixth days of the differentiation process. EHTs were generated by assembling CMs derived from hiPSC (hiPSC-CM) at high cell density in a low collagen hydrogel. EHTs were further matured and grown in a customized biomimetic tissue culture system, that provides continuous electrical stimulation, medium agitation and stretch. Finally, RNA extraction and tissue fixation were performed on CMs and EHTs for RT-qPCR and immunofluorescence staining analysis, while RNA sequencing was conducted on EHTs to examine how RA affects gene expression associated with function and structure of EHTs. Results: HighRA-hiPSC-CMs exhibited highest expression of maturity genes MYH7 and cTnT. The expression of TBX5, NKX2.5 and CORIN, which are transcription factors associated with left ventricular development, was also the highest in the HighRA-hiPSC-CM. The expression of TBX5 and NKX2.5 was also found to be highest in the HighRA-EHTs, while expression of right ventricular transcrition factors TBX20 and ISL1 was highest in control-EHTs. RNAseq of EHTs showed augmented extracellular matrix gene expression in HighRA-EHTs. The HighRA-EHTs were functionally most similar to left ventricle with the highest contraction force, the lowest beating frequency, and the highest sensitivity to hypoxia and isoprenaline. Conclusion: By interfering with the differentiation process of hiPSC with a specific concentration of RA at a specific time, we were able to successfully induce CMs and EHT with a phenotype similar to that of the left ventricle or right ventricle. Our research paves the way for future studies on in vitro left ventricular or right ventricular function, personalized drug screening, and precision medicine. German Center for Cardiovascular Research (DZHK81Z0600207 to D.M. and P.S.) China Scholarship Council (CSC202108410141) and Henan Provincial Medical Science and Technology Research Project. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Effect of tissue fixatives on the corrosion of biomedical magnesium alloys

In this work, the corrosion behavior of pure Mg, Mg3Ag, Mg6Ag, and MgZnYNd alloys in different fixatives (ethyl alcohol (EA), 85 % ethyl alcohol (85 % EA), 10 % neutral buffered formalin (10 % NBF), 4 % glutaric dialdehyde (4 % GD), and 4 % paraformaldehyde (4 % PFA)) was investigated to provide a valuable reference for the selection of fixatives during the histological evaluation of Mg implants. Through the hydrogen evolution test, pH test, and corrosion morphology and product characterization, it was found that corrosion proceeded slowest in the EA and 85 % EA groups, slightly faster in 4 % GD, faster in 10 % NBF, and fastest in 4 % PFA. After corrosion, the EA group surface remained unchanged, while the 85%EA group surface developed minor cracks and warping. The 4%GD fixative formed a dense needle-like protective layer on the Mg substrate. The 10%NBF group initially grew a uniform layer, but later developed irregular pits due to accelerated corrosion. In contrast, the 4%PFA solution caused more severe corrosion attributed to chloride ions. The main corrosion products in the EA and 85%EA groups were MgO and Mg(OH)2, while the other fixatives containing diverse ions also yielded phosphates like Mg3(PO4)2 and MgHPO4. In 4 % PFA, AgCl formed on the surface of Mg6Ag alloy after corrosion. Therefore, to minimize Mg alloy corrosion without compromising staining quality, EA or 85 % EA is recommended, while 4 % PFA is not recommended due to its significant impact.

Descending perineal syndrome cure by deep transversus perinei ligament repair: surgical options.

Descending perineal syndrome (DPS) was described by Parks et al. as descent of the anus on straining, typically 3-4 cm below a line drawn from the coccyx to the lower end of the. DPS is associated with obstructed defecation, with increased bulging of the perineum with straining, although perineal descent can also be seen at rest. In their review, Chaudhry and Tarnay stated: "It is controversial whether surgical management is even an option for patients with DPS". The deep transversus perinei (DTP) ligaments are the suspensory ligaments of the perineal body (PB). DTP are approximately 4 cm long. They attach behind the upper 2/3 and lower 1/3 of the descending ramus. If, at childbirth, the PB is overstretched and displaced laterally and inferiorly, the DTP lengthens. DPS is described as descent of the anus on straining, typically 3-4 cm below a line drawn from the coccyx to the lower end of the symphysis. DPS is associated with obstructed and often, assisted defecation, with increased bulging of the perineum with straining descent of the anus on straining. The surgical methodology begins as a standard PB repair which dissects the rectum from the vagina and PB and approximates the displaced components of the PB. We added an additional step: identifying the DTPs, shortening and reinforcing them with the Tissue Fixation System (TFS) minisling or No. 2 polyester sutures. High cure rates for obstructed defecation were achieved with the TFS minisling, and initial results using No. 2 polyester sutures are favourable. The key messages from both operations is DPS is caused by stretching and elongation of DPS ligaments, and these are surgically repairable.

A comparison of native vaginal and ligament surgery for cure of pelvic organ prolapse and overactive bladder.

The key messages from the Shkarupa et al. native cardinal/uterosacral ligament (CL/USL) study, was that, in premenopausal women, ligament repair alone is sufficient for cure of pelvic organ prolapse (POP) and urgency, achieving cure rates of 85.7% for POP and 81.6% for urgency at 12 months. However, in postmenopausal women, the cure rates were 20.5% for POP and 33.3% for urge at 12 months. The Lancet Prospect Trial recorded 21% for native vaginal repair at 12 months. The poor POP cure rate in the Prospect Trial, and the rapid deterioration in the post-menopausal CL/USL repair group, can be explained by known biomechanics. The vagina has little structural strength. Ligaments, with a much higher breaking strain, are the main structural support of pelvic organs. Yet, even native ligament repair reported very low cure rates at 12 months. The poor results in postmenopausal women with native ligament repair can be explained by collagen breakdown after the menopause, as collagen is the key structural component of ligaments. An important question posed in the ligament repair study was, "What happens to women cured by ligament repair after the menopause when the collagen leaches out of the ligaments?". One recommendation was that collagen creating tapes be routinely applied in prolapse surgery and OAB, at least in postmenopausal women. The recommendation for routine collagen-creating ligament repair methods, especially in older women, are supported by high 5-year surgical cure rates in 70-year-old Japanese women, 91.2% for POP, at 12 months, falling to 79.0 at 60 months, using collagen creating Tissue Fixation System (TFS) minislings.

Pubourethral and uterosacral male analogues suggest parallel male/female pelvic anatomy and symptom pathogenesis.

The thesis that functional/dysfunctional male/female pelvic floor anatomy are parallel, originated from two studies: a successful retropubic perineal male sling for post-prostatectomy stress urinary incontinence (SUI) and discovery of a male uterosacral ligament (USL) analogue, we named "prostatosacral ligament" (PSL). In 25 out of the studied 27 males (92.6%), it starts on both sides of the median sulcus of the prostate the ligament passes lateral to the rectum being fused with the lateral margin of the mesorectum before leaving it as it thins out to be attached posteriorly similar to the USL. The ultrasound data during straining in men and women showed the same three oppositely acting muscle vectors contracting around analogous ligaments, puboprostatic ligament (PPL) (male), and pubourethral ligament (PUL) (female). Further parallels were pubovesical ligaments (PVLs) and arc of Gilvernet as part of the continence and micturition mechanisms. Impressive evidence for parallel anatomy came from the successful cure of 22 males with post-prostatectomy SUI using a perineal retropubic tissue fixation system (TFS) minisling applied to the PPL using a similar methodology to that used in the female for PUL midurethral sling repair for cure of SUI. Laparoscopic evidence confirmed the prostate as a male analogue of the cervix, and PSLs as analogues of USLs: PSL origin from the prostate attached laterally to the mesorectum and inserted into the sacrum. Histologically, PSLs had identical features with USLs: collagen, elastin, smooth muscle, blood vessels and nerves. Virtually identical symptoms for "chronic prostatitis" (CP) and "posterior fornix syndrome" (PFS), such as chronic pelvic pain, overactive bladder (OAB), abnormal emptying, gave birth to the hypothesis, of a common pathogenesis for "CP" and "PFS", USL (or PSL) laxity. If this could be proven by "simulated operations", "CP", at least in theory, may be potentially correctible by PSL repair.

DIY peristaltic perfusion pump and rotarod apparatuses from common and recycled materials

To reduce the high costs of research associated with specialized laboratory equipment, the current study aimed to construct and validate two devices (peristaltic perfusion pump and rotarod) critical in physiology, neuroscience, and histology laboratories using largely recycled materials and affordable electrical parts. The resulting systems are comparable to commercially available devices but at a fraction of the cost. The peristaltic perfusion pump has an adjustable flow rate suitable for the whole-body perfusion of rats and mice. Testing in rodents confirmed that the pump operated conveniently and consistently, yielding high-quality tissue perfusion and fixation. The rotarod worked flawlessly in behavioral studies with live mice, yielding speed, torque, acceleration and timing comparable to commercially-available devices. Collectively, the findings indicate that the purpose-built peristaltic perfusion pump and rotarod can be used in research laboratories as cost-effective alternatives to comparable but costly commercial devices.

Designing payload and spaceflight operations for plants from extreme terrestrial environments

Terrestrial plants from the very limits of life are likely to harbor genes that confer an advantage in human space exploration. These plants are seemingly capable of performing mission critical functions in spaceflight and on extraterrestrial farms while informing directed gene manipulation in target plant species. However, their adaptations to physiologically extreme habitats may hinder the efficacy of routine laboratory techniques for model plants. We here present the development of Antarctic moss Ceratodon purpureus payload and flight operations for the ANT1 Radiation Tolerance Experiment with Moss in Orbit on the Space Station (ARTEMOSS) experiment to the International Space Station (ISS) given limited physical space and crew time. We demonstrate that the hydrophobic surface of Antarctic moss impedes chemical tissue fixation and precludes the use of RNAlater coupled with payload hardware deployed in standard plant spaceflight experiments. We show that deep-freezing the moss tissue on Petri plates provides adequate tissue fixation and allows the extraction of high-quality RNA suitable for gene expression profiling. We replaced hardware with stacks of Petri plates housing Antarctic moss and chemical fixation with deep-freezing in a cryogenic GLACIER freezer. Our design can be translated to other plant species to expand current experimentation techniques with plants from extreme terrestrial environments in order to advance human space exploration.

Histopathological Effects of Nicotine on Rats Pulmonary Cell Treated with Zinc and Vitamin D

The present study sought to determine the role of vitamin D and zinc in protecting against nicotine stress by altering the alveolar response and modulating antioxidant mechanisms. Thirty mature adults Wistar rats, used as a model for mammals, were maintained at 23 ± 2 C°, randomly divided into five equal groups, and then given therapy for 14 days. G1: Nicotine was administered at a dose of 0.0015g/kg b.w. I.P., G2: zinc was administered orally at 0.06 g/kg b.w., G3: Orally administration vitamin D at 250 µg/kg b.w., and G4: both zinc and vitamin D were administered orally at the same dose as nicotine, 0.0015g/kg b.w. I.P. Rats were anesthetized with ketamine at the end of the treatment period. All groups of rats had lung samples extracted rapidly; alveolar groups of samples were cleaned and placed in physiological used 10% of neutral buffered formalin for tissue fixation. Then lung suction were repaired for histopathological study. The results showed highly significant protective effects of zinc and vitamin D in the tissues of the lung in G4. Zinc and vitamin D group G4 decreased the effectiveness of nicotine when compared with the G1 nicotine group. Protective effect of zinc and vitamin D by decreased MNCS infiltration with thickening and slightly congested pulmonary blood vessels without emphysema and pulmonary edema

FixNCut: single-cell genomics through reversible tissue fixation and dissociation

The use of single-cell technologies for clinical applications requires disconnecting sampling from downstream processing steps. Early sample preservation can further increase robustness and reproducibility by avoiding artifacts introduced during specimen handling. We present FixNCut, a methodology for the reversible fixation of tissue followed by dissociation that overcomes current limitations. We applied FixNCut to human and mouse tissues to demonstrate the preservation of RNA integrity, sequencing library complexity, and cellular composition, while diminishing stress-related artifacts. Besides single-cell RNA sequencing, FixNCut is compatible with multiple single-cell and spatial technologies, making it a versatile tool for robust and flexible study designs.

Porous titanium alloys for medical application: Progress in preparation process and surface modification research

Excellent mechanical properties and biocompatibility are the most sought-after attributes in biomedical materials for the regeneration of damaged tissues. However, conventional dense titanium alloys possess a modulus significantly higher than that of human tissues, leading to potential stress-shielding effects. Medical porous titanium alloys can reduce the elastic modulus of the material, promote tissue fixation and vascular regeneration, and improve the suitability for human tissue properties. With the continuous development of technology, the preparation process of porous titanium alloys has undergone a series of multifaceted transformations and improvements in the aspects of powder sintering, fiber preparation, and additive manufacturing processes, and its structural characteristics and mechanical properties are constantly evolving in a controllable direction. Alongside the enhancement of the material&amp;rsquo;s mechanical properties through porous design, optimization of the properties at the implant-tissue interface also leads to improved antimicrobial and osteogenic properties of porous titanium. Due to the complex internal structure of porous titanium alloys, surface modification is mainly carried out in fluid media, which is realized by morphological modification and the introduction of functional substances. Over time, the surface modification of porous titanium alloys for medical applications has progressed from morphological modification and introduction of chemical composition to the loading of bioactive substances. This evolution aims to enhance safety and efficiency in the use of these materials. This paper reviews the preparation and surface modification processes of porous titanium alloys for medical use and summarizes the advantages, disadvantages, and influencing factors among different processes, with a view to providing new ideas for the development of porous implants for medical use.

Abstract 1532: Improved reproducibility in multiplex immunofluorescence analysis of the tumor microenvironment using a novel quenching solution

Abstract Formalin-fixed, paraffin-embedded (FFPE) tissues are commonly used in histopathology because the fixed tissues are stable and easy to store. In the context of immunofluorescence detection, however, endogenous autofluorescence of FFPE tissues can significantly reduce or mask immunofluorescent signals. The autofluorescence of FFPE tissues is caused by a variety of factors, including the presence of endogenous fluorophores, tissue fixation, and processing. While there are several methods that can be used to quench autofluorescence in FFPE tissues, chemical quenchers, which absorb or scatter light, are most frequently used for multiplex IF. Examples of chemical quenchers commonly used in manual multiplex staining include Sudan Black B and TrueBlack; however, these are not chemically compatible for use on automated platforms such as the Leica Biosystems BOND RX. Due to the need for increased throughput and decreased hands-on time, automation has risen in popularity versus manual staining, leading to a need for a versatile quenching solution compatible with automation. Here we demonstrate that NovaPlex Quenching Solution (NPQS), a novel proprietary quenching agent, reduces the background caused by autofluorescence and enhances the signal of the Cell IDx UltraPlex multiplex immunofluorescent (mxIF) assay across tissue types. Qualitative studies showed a decrease in autofluorescent signal in FFPE mouse spleen stained with antibodies against B220/Foxp3/CD3e, suggesting the utility of NPQS in mouse models of cancer and immunity. Similarly, nonspecific background was decreased across FFPE human tissue types, including lung adenocarcinoma stained for CD8/Foxp3/CD3e/CD4 and tonsil stained for CD8/Foxp3/PD-1/CD4. Further statistical studies in FFPE human tonsil tissue stained with CD8/Foxp3/PD-1/CD4 showed there was no significant decrease in dye intensity across all four markers and channels, resulting in a significant increase in signal-to-noise ratio for both CD8 at 490nm and PD-1 at 650nm (p&amp;lt;0.05). These experiments using Cell IDx/LBS UltraPlex mxIF technology demonstrate an effective method to improve the signal-to-noise ratios of fluorophores without reducing signal intensity from any of the target proteins in these parallel multiplex immunofluorescent assays. Citation Format: Matt Levin, Jack Heath, April Schrank-Hacker, Shelby Dunn, Minh-Chau Vu, Imane Hamza, David Schwartz. Improved reproducibility in multiplex immunofluorescence analysis of the tumor microenvironment using a novel quenching solution [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1532.

Absorbable Sutures Repair Without Meniscopexy, Results in Superior Outcomes for Discoid Lateral Meniscus With Meniscocapsular Anterior Detachment in Children.

Despite the fact that meniscocapsular anterior detachment is the most common location of instability in children with a Discoid Lateral Meniscus (DLM), there is a lack of consensus about the type of repair that should be utilized for stabilization. The aim of this study was to determine the best fixation method for anterior detachment of DLM in children. Our hypothesis was that excessive rigidity with fixation would restrict meniscal mobility and increase the rate of failure or prevent full knee flexion. This study was a retrospective single-center study consisting of 51 consecutive knees (45 children) with menico-capsular anterior detachment that underwent stabilization and minimal saucerization of the meniscus between 2007 and 2018. We aimed to compare the need for revision surgery and knee flexion between the different types of fixations utilized; namely we compared meniscopexy using anchors on the tibia (n=30) with outside-in arthroscopic soft tissue fixation (n=21), and absorbable (n=18) and nonabsorbable sutures (n=33) were compared. The mean age at surgery was 10.4 years (3 to 17) with a mean follow-up of 52 months (18 to 148). The group of knees treated with absorbable sutures had a significantly better rate of full knee flexion (15/18) compared with the nonabsorbable group (17/33) ( P =0.03). Despite the absence of significance ( P =007), there was a lower rate of revision surgery due to suture failure in the soft tissue fixation group (0 revision operations) compared with the meniscopexy group (5 revision operations). For anterior meniscocapsular detachment of DLM, it is recommended to perform soft tissue fixation with absorbable sutures, as this technique resulted in better knee flexion and a lower rate of revision surgery when compared with meniscopexy and nonabsorbable suture fixation. Level III-retrospective case studies.

EXPLORING THE POTENTIAL OF MORINGA OLEIFERA EXTRACT AS A NATURAL TISSUE FIXATIVE FOR HISTOPATHOLOGICAL APPLICATIONS

Histopathology, crucial for disease diagnosis, heavily relies on effective tissue fixation to maintain cellular architecture. However, the carcinogenic nature of formalin has spurred interest in safer alternatives. Moringa oleifera, renowned for its medicinal properties, presents a promising natural fixative candidate. This study aimed to assess Moringa oleifera extract's viability as a fixative for histopathological applications, specifically focusing on its compatibility with subsequent histological analyses. Distilled water and normal saline solutions of Moringa extract were prepared at varying concentrations. Kidney tissue samples from euthanized rats were treated with these solutions alongside formalin. Tissue sections underwent manual processing, embedding, sectioning, and staining using Hematoxylin and Eosin (H&amp;E) technique. Microscopic examination revealed varying degrees of tissue preservation across Moringa concentrations and solvents. Higher concentrations (15%) in distilled water exhibited optimal preservation, akin to formalin-fixed tissues, while lower concentrations and normal saline solutions showed suboptimal fixation. These findings highlight Moringa oleifera's potential as a tissue fixative, particularly at higher concentrations in distilled water. Further research is recommended to isolate active components and explore alternative Moringa extracts for enhanced tissue preservation. Comparative studies would elucidate the efficacy of Moringa-derived fixatives and inform their utilization in histopathological practice.

Standardization of honey as a tissue fixative for histopathology: A morphometric study

Background: Tissue fixation is a crucial step to preserve the tissues in a life-like state with minimal disruption to its cellular and chemical composition for histopathological examination. The search for an effective alternate tissue fixative to the routinely used formaldehyde has gained interest as constant exposure to formaldehyde has proven to be toxic. Honey, an organic substance with high acidity and hygroscopic nature, exhibits tissue fixative properties and has been used in the present study. The present study aimed to standardize honey as a tissue fixative for histopathology by comparing it with formalin. Materials and Methods: In vitro study Oral tissue samples of goat were fixed in 10% honey and 10% formalin solution, respectively, for 24-48 h, followed by routine tissue processing and microscopic examination of 37 slides per group. 2200 epithelial cells (1100 per group) were selected for the computer-aided morphometric image analysis (Fiji-Image J) by three observers. Cell area (CA), cell perimeter (CP), nuclear area (NA), nuclear perimeter (NP), cytoplasmic area (Cyt A), and nuclear-cytoplasmic ratio were the parameters studied. Mann-Whitney U-test (STATA/IC version 16) for inter-group comparison was done and P &lt; 0.05 was considered statistically significant. Results: The probability of epithelial cells in the honey-fixed group to have greater NA, NP, and N/C ratio was about 50%-60%. The probability of epithelial cells in formalin-fixed tissues to have greater CA, CP, and Cyt A was about 70%. Conclusion: Honey is a better nuclear fixative than formalin. Cytoplasmic shrinkage of epithelial cells should be taken into consideration while fixing tissues with honey.

Comparison of Time-Zero Primary Stability Between a Biodegradable Magnesium Bone Staple and Metal Bone Staples for Knee Ligament Fixation: A Biomechanical Study in a Porcine Model.

Bone staples have been shown previously to be a viable modality for cortical tendon graft fixation in ligament knee surgery. However, soft tissue reactions have been reported, making implant removal necessary. Magnesium alloys are a promising material for biodegradable orthopaedic implants, with mechanical properties closely resembling those of human bone. To compare the primary stability of a biodegradable bone staple prototype made from magnesium to bone staples made from metal in the cortical fixation of tendon grafts during knee surgery. Controlled laboratory study. Primary stability of peripheral tendon graft fixation was assessed in a porcine model of medial collateral ligament reconstruction. Two commercially available metal bone staples (Richards fixation staple with spikes [Me1] and spiked ligament staple [Me2]) were compared with a magnesium bone staple prototype for soft tissue fixation. Primary stability was assessed using a uniaxial materials testing machine. Cyclic loading at 50 and 100 N was applied for 500 cycles each, followed by load-to-failure testing. After 500 cycles at 50 N, elongation was 1.5 ± 0.5 mm in the Me1 group, 1.9 ± 0.5 mm in the Me2 group, and 1.8 ± 0.4 mm in the magnesium group. After 1000 cycles of loading (500 cycles at 50 N and 500 at 100 N), elongation was 3.6 ± 0.9 mm in the Me1 group, 3.5 ± 0.6 mm in the Me2 group, and 4.1 ± 1.0 mm in the magnesium group. No significant differences regarding elongation were found between the groups. Load to failure was 352 ± 115 N in the Me1 group, 373 ± 77 N in the Me2 group, and 449 ± 92 N in the magnesium group, with no significant difference between the groups. In this study, the magnesium bone staples provided appropriate time-zero biomechanical primary stability in comparison with metal bone staples and may therefore be a feasible alternative for cortical fixation of tendon grafts in knee surgery. The biodegradability of magnesium bone staples would eliminate the need for later implant removal.

Rapid Examination of Nonprocessed Renal Cell Carcinoma Using Nonlinear Microscopy.

Histology, the traditional method of examining surgical tissue under a microscope, is a time-consuming process involving the fixation of tissue in formalin, dehydration, embedding in paraffin, and cutting into thin sections for hematoxylin-eosin (H&E) staining. Frozen section analysis is a faster alternative used in surgery to quickly evaluate tissue, but it has limitations, such as the size of the specimens that can be analyzed and difficulties with fatty and bony tissues. To rapidly examine nonprocessed kidney tumors using nonlinear microscopy (NLM), a fluorescence microscopy technique that can rapidly visualize fresh or fixed, rapidly stained, nonprocessed tissue resembling H&E histology. This technology eliminates the need for fixation, embedding, microtome sectioning, or slide preparation. In this study, a total of 190 tissue specimens were collected from 46 patients who underwent partial or radical nephrectomy. Two genitourinary pathologists confirmed that diagnostically important features present in the H&E images could also be identified in the NLM images. The results of this study demonstrated that NLM had a high degree of correspondence with H&E staining for the classical variants of renal cell carcinoma. NLM offers several clinical benefits, such as facilitating rapid renal cell carcinoma diagnosis, assessment of targeted kidney biopsies for both tumor and medical kidney diseases, and collection of fresh renal cell carcinoma tissue for molecular studies.

Virtual histological staining of unlabeled autopsy tissue

Traditional histochemical staining of post-mortem samples often confronts inferior staining quality due to autolysis caused by delayed fixation of cadaver tissue, and such chemical staining procedures covering large tissue areas demand substantial labor, cost and time. Here, we demonstrate virtual staining of autopsy tissue using a trained neural network to rapidly transform autofluorescence images of label-free autopsy tissue sections into brightfield equivalent images, matching hematoxylin and eosin (H&E) stained versions of the same samples. The trained model can effectively accentuate nuclear, cytoplasmic and extracellular features in new autopsy tissue samples that experienced severe autolysis, such as COVID-19 samples never seen before, where the traditional histochemical staining fails to provide consistent staining quality. This virtual autopsy staining technique provides a rapid and resource-efficient solution to generate artifact-free H&E stains despite severe autolysis and cell death, also reducing labor, cost and infrastructure requirements associated with the standard histochemical staining.