Enhanced study of facial soft tissues using a novel large scale histology technique.

The safety and effectiveness of facial cosmetic surgery procedures are dependent on detailed 3D understanding of the complex surgical anatomy of the face. Traditional, small sample size anatomical dissection studies have limitations in providing definitive clarification of the fascial layers of the face, and especially in their relationship with the facial nerve, and their reaction to surgical manipulation. The objective study of large tissue areas is required to effectively demonstrate the broader architecture. Conventional histology techniques were modified to handle extraordinarily large tissue samples to fulfill this requirement. Full-thickness soft tissue samples (skin to bone) of maximum length 18 cm, width 4 cm, and tissue thickness 1 cm, were harvested from 20 hemifaces of 15 fresh human cadavers (mean age at death = 81 years). After fixation, the samples were processed with an automated processor using paraffin wax for 156 h, sectioned at 30 µm, collected on gelatin-chromium-coated glass slides, stained with a Masson's Trichrome technique and photographed. Using this technique, excellent visualization was obtained of the fascial connective tissue and its relationship with the facial mimetic muscles, muscles of mastication and salivary glands in 73 large histological slides. The resulting slides improved the study of the platysma and superficial musculo-aponeurotic system (SMAS), the spaces and ligaments, the malar fat pad, and the facial nerve in relations to the deep fascia. Additionally, surgically induced changes in the soft-tissue organization were successfully visualized. This technique enables improved insight into the broad structural architecture and histomorphology of large-scale facial tissues.

Negative Pressure Neurogenesis: A Novel Approach to Accelerate Nerve Regeneration after Complete Peripheral Nerve Transection.

Various modalities to facilitate nerve regeneration have been described in the literature with limited success. We hypothesized that negative pressure applied to a sectioned peripheral nerve would enhance nerve regeneration by promoting angiogenesis and axonal lengthening. METHODS: Wistar rats' sciatic nerves were cut (creating ~7 mm nerve gap) and placed into a silicone T-tube, to which negative pressure was applied. The rats were divided into 4 groups: control (no pressure), group A (low pressure: 10 mm Hg), group B (medium pressure: 20/30 mm Hg) and group C (high pressure: 50/70 mm Hg). The nerve segments were retrieved after 7 days for gross and histological analysis. RESULTS: In total, 22 rats completed the study. The control group showed insignificant nerve growth, whereas the 3 negative pressure groups showed nerve growth and nerve gap reduction. The true nerve growth was highest in group A (median: 3.54 mm) compared to group B, C, and control (medians: 1.19 mm, 1.3 mm, and 0.35 mm); however, only group A was found to be significantly different to the control group (**P < 0.01). Similarly, angiogenesis was observed to be significantly greater in group A (**P < 0.01) in comparison to the control. CONCLUSIONS: Negative pressure stimulated nerve lengthening and angiogenesis within an in vivo rat model. Low negative pressure (10 mm Hg) provided superior results over the higher negative pressure groups and the control, favoring axonal growth. Further studies are required with greater number of rats and longer recovery time to assess the functional outcome.

Novel resin tissue array system reduces sample preparation time, labour and reagent costs in bone tissue histology.

Resin histology plays an essential role in the analysis of hard tissues, such as bone and teeth, as well as in the context of metallic implant analysis. However, the techniques of resin embedding, followed by ground sectioning, are very costly due to significantly increased reagent cost and labour time when compared to the conventional paraffin histology approach. In the present study, a novel resin array system was developed to increase the affordability of a project analysing rat femur tissues containing metallic or polymeric implants. The resin array system enabled the simultaneous embedding of the femur samples in groups of eight samples compared to the conventional resin method where samples are processed individually. The ground sections produced with the resin array system allowed uniform ROI selection, ground section thickness, staining consistency, and histological analysis with Goldner's trichrome stain, offering a substantial opportunity for reproducible immunohistochemistry which is unable to be achieved when processing samples embedded individually. The application of this novel resin array system significantly reduced resource usage when compared to doing the same analysis on individual samples. A reduction of approximately 40% was achieved for both total labour time and total reagent cost through the use of the array system compared with individual embedding. This novel resin array system has widespread applicability to many bone, hard tissue, and metallic implant studies, offering substantial conservation of research funds and increased accessibility to advanced techniques for commercial partners due to more cost-effective sample preparation and more accurate, reproducible data.

Histomorphometric Evaluation of Critical-Sized Bone Defects Using Osteomeasure and Aperio Image Analysis Systems.

Most histological evaluations of critical-sized bone defects are limited to the analysis of a few regions of interest at a time. Manual and semiautomated histomorphometric approaches often have intra- and interobserver subjectivity, as well as variability in image analysis methods. Moreover, the production of large image data sets makes histological assessment and histomorphometric analysis labor intensive and time consuming. Herein, we tested and compared two image segmentation methods: thresholding (automated) and region-based (manual) modes, for quantifying complete image sets across entire critical-sized bone defects, using the widely used Osteomeasure system and the freely downloadable Aperio Image Scope software. A comparison of bone histomorphometric data showed strong agreement between the automated segmentation mode of the Osteomeasure software with the manual segmentation mode of Aperio Image Scope analysis (bone formation R2 = 0.9615 and fibrous tissue formation R2 = 0.8734). These results indicate that Aperio is capable of handling large histological images, with excellent speed performance in producing highly consistent histomorphometric evaluations compared with the Osteomeasure image analysis system. The statistical evaluation of these two major bone parameters demonstrated that Aperio Image Scope is as capable as Osteomeasure. This study developed a protocol to improve the quality of results and reduce analysis time, while also promoting the standardization of image analysis protocols for the histomorphometric analysis of critical-sized bone defect samples. Impact Statement Despite bone tissue engineering innovations increasing over the last decade, histomorphometric analysis of large bone defects used to study such approaches continues to pose a challenge for pathological assessment. This is due to the resulting large image data set, and the lack of a gold standard image analysis protocol to quantify histological outcomes. Herein, we present a standardized protocol for the image analysis of critical-sized bone defect samples stained with Goldner's Trichrome using the Osteomeasure and Aperio Image Scope image analysis systems. The results were critically examined to determine their reproducibility and accuracy for analyzing large bone defects.

Characterization of Normal Murine Carpal Bone Development Prompts Re-Evaluation of Pathologic Osteolysis as the Cause of Human Carpal-Tarsal Osteolysis Disorders.

Multicentric carpal-tarsal osteolysis; multicentric osteolysis, nodulosis, and arthropathy; and Winchester syndromes, skeletal dysplasias characterized by carpal/tarsal and epiphyseal abnormalities, are caused by mutations in v-maf musculoaponeurotic fibrosarcoma oncogene ortholog B (MAFB), matrix metalloproteinase (MMP) 2, and MMP14, respectively; however, the underlying pathophysiology is unclear. Osteoclast-mediated osteolysis has been regarded as the main mechanism, but does not explain the skeletal distribution. We hypothesized that MAFB, MMP-2, and MMP-14 have integral roles in carpal/tarsal and epiphyseal bone development. Normal neonatal mouse forepaws were imaged by micro-computed tomography and examined histologically. Murine forepaw ossification occurred sequentially. Subarticular regions of endochondral ossification showed morphologic and calcification patterns that were distinct from archetypical physeal endochondral ossification. This suggests that two different forms of endochondral ossification occur. The skeletal sites showing the greatest abnormality in the carpal-tarsal osteolysis syndromes are regions of subarticular ossification. Thus, abnormal bone formation in areas of subarticular ossification may explain the site-specific distribution of the carpal-tarsal osteolysis phenotype. MafB, Mmp-2, and Mmp-14 were expressed widely, and tartrate-resistant acid phosphatase staining notably was absent in the subarticular regions of the cartilage anlagen and entheses at a time point most relevant to the human osteolysis syndromes. Thus, abnormal peri-articular skeletal development and modeling, rather than excessive bone resorption, may be the underlying pathophysiology of these skeletal syndromes.

Ureaplasma parvum and Ureaplasma urealyticum are detected in semen after washing before assisted reproductive technology procedures.

OBJECTIVE: To investigate the prevalence of ureaplasmas in semen and washed semen and to explore their effect on semen andrology variables. DESIGN: Prospective study. SETTING: In vitro fertilization (IVF) unit of a private hospital. PATIENT(S): Three hundred forty-three men participating in an assisted reproductive technology (ART) treatment cycle. MAIN OUTCOME MEASURE(S): The prevalence of ureaplasmas in semen and washed semen tested by culture, polymerase chain reaction assays, and indirect immunofluorescent antibody assays. RESULT(S): Ureaplasmas were detected in 73 of 343 (22%) semen samples and 29 of 343 (8.5%) washed semen samples. Ureaplasmas adherent to the surface of spermatozoa were demonstrated by indirect immunofluorescent antibody testing. Ureplasma parvum serovar 6 (36.6%) and U. urealyticum (30%) were the most prevalent isolates in washed semen. A comparison of the semen andrology variables of washed semen ureaplasma positive and negative groups demonstrated a lower proportion of nonmotile sperm in men ureaplasma positive for washed semen. CONCLUSION(S): Ureaplasmas are not always removed from semen by a standard ART washing procedure and can remain adherent to the surface of spermatozoa.