Medullary Side Research Articles

O-037 Revascularization of human ovarian grafts occurs equally efficient from both sides of the cortex tissue: implications for clinical practice

Abstract Study question Does revascularization of xenotransplanted human ovarian grafts occur equally efficient from both the cortical and medullary side of the cortex tissue? Summary answer Murine vessels were established equally efficient from both sides of human ovarian grafts, demonstrating that the fibrous cortical side does not hinder the neovascularization process. What is known already There is a lack of consensus regarding the surgical approach for transplanting frozen-thawed ovarian tissue and various techniques are being used clinically worldwide. Some centers attach ovarian tissue with stitches or Interceed® on top of the decorticated remaining ovary to mimic normal ovarian architecture. Other centers transplant ovarian tissue in subcortical pockets made by longitudinal or transverse incisions in the remaining intact ovary to facilitate vascularization from both sides of the graft. While other centers transplant to sub-peritoneal pelvic sites only. It is unknown which techniques and transplantation sites provide the most efficient revascularization of the ovarian grafts. Study design, size, duration Three pieces of cryopreserved ovarian cortex (approximately 5x5x1 mm in size) were donated and thawed from 12 women and two of the pieces were transplanted to subcutaneous pockets on the dorsal side of ovariectomized immunodeficient female mice. The third piece was processed as an ungrafted control. Ovarian grafts were retrieved after 8 days to analyze the spatial distribution of graft-revascularization using immunohistochemical detection of murine CD31, and after 8 weeks to evaluate follicle density (follicles/mm3). Participants/materials, setting, methods Ovarian cortex was donated from 12 women aged 27-31 years having their tissue frozen for fertility preservation. The CD31 positive vessel area and density were quantified using a custom designed application (APP) from Visiopharm®. Three regions of interest (ROIs) were defined in each tissue section; the cortical side, the center, and the medullary side. Vessels were sub-divided into three categories according to size: micro-vessels (<300 µm2), small vessels (300-1000 µm2), and large vessels (1000-3000 µm2). Main results and the role of chance After 8 days xenotransplantation, a statistical significant lower density of vessels was found in the center of the human ovarian grafts compared to the cortical and medullary sides (cortical side: 323 ± 14 vessels/mm2; center: 240 ± 12 vessels/mm2; medullary side: 301 ± 18 vessels/mm2; p < 0.001). The mean percentage of CD31 positive vessel area in the human ovarian grafts was similar in the three ROIs, but lowest in the center of the grafts (cortical side: 3.9% ± 0.2 (SEM); center: 3.5% ± 0.2; medullary side: 4.0% ± 0.3; p = 0.17). Micro-vessels comprised 89-91% of all vessels in the three ROIs, demonstrating that the vast majority of vessels were newly formed. Heatmaps were generated for each section based on the CD31 positive area to visualize the localization of vessels in the ovarian grafts and the maps showed highly variable vascularization patterns in the grafts from different patients. Overall, vascularization appeared to occur equally efficient in all peripherical areas of the ovarian grafts. The follicle density in ungrafted cortex tissue was 51.8 ± 17.3 follicles/mm3 and 14.7 ± 3.7 follicles/mm3 after 8 weeks xenografting, resulting in an average follicle survival rate of 28%. Limitations, reasons for caution The current study was not designed to directly compare clinically used transplantation techniques, which should be studied in larger animal models, like sheep, in which the structure of ovarian tissue is similar to human. Wider implications of the findings Revascularization was established equally efficient from both sides of xenotransplanted human ovarian cortex, suggesting that transplantation techniques ensuring revascularization from both sides of the ovarian graft, such as sub-cortical or peritoneal pockets, may facilitate a more efficient revascularization of the graft than techniques allowing revascularization from only the medullary side. Trial registration number not applicable

Differential location of NKT and MAIT cells within lymphoid tissue

Natural Killer T (NKT) cells and Mucosal-Associated Invariant T (MAIT) cells are innate-like T cells that express semi-invariant αβ T cell receptors (TCRs) through which they recognise CD1d and MR1 molecules, respectively, in complex with specific ligands. These cells play important roles in health and disease in many organs, but their precise intra-organ location is not well established. Here, using CD1d and MR1 tetramer staining techniques, we describe the precise location of NKT and MAIT cells in lymphoid and peripheral organs. Within the thymus, NKT cells were concentrated in the medullary side of the corticomedullary junction. In spleen and lymph nodes, NKT cells were mainly localised within T cell zones, although following in vivo activation with the potent NKT-cell ligand α-GalCer, they expanded throughout the spleen. MAIT cells were clearly detectable in Vα19 TCR transgenic mice and were rare but detectable in lymphoid tissue of non-transgenic mice. In contrast to NKT cells, MAIT cells were more closely associated with the B cell zone and red pulp of the spleen. Accordingly, we have provided an extensive analysis of the in situ localisation of NKT and MAIT cells and suggest differences between the intra-organ location of these two cell types.

Revascularization of human ovarian grafts is equally efficient from both sides of the cortex tissue

Research questionDoes revascularization of human ovarian grafts in a mouse model occur with equal efficiency from both sides of the cortex tissue? DesignTwenty-four frozen-thawed ovarian cortex pieces from 12 women were transplanted to immunodeficient mice, for 8 days to analyse graft revascularization using immunohistochemical detection of murine CD31, or for 8 weeks to evaluate follicle density (follicles/mm3). The CD31-positive vessel area and density were quantified using a custom-designed application. Three regions of interest (ROI) were defined in each tissue section: the cortical side, the centre and the medullary side. Vessels were subdivided into three categories according to size: microvessels (<300 µm2), small vessels (300–1000 µm2) and large vessels (>1000–3000 µm2). ResultsNo significant difference in the mean percentage of the CD31-positive vessel area was found between the three ROI (cortical side: 3.9% ± 0.2%; centre: 3.5% ± 0.2%; medullary side: 4.0% ± 0.3%; P = 0.17), but a significantly lower density of vessels was found in the centre of the human ovarian grafts compared with the cortical and medullary sides (cortical side: 323 ± 14 vessels/mm2; centre: 240 ± 12 vessels/mm2; medullary side: 301 ± 18 vessels/mm2; P < 0.001). Microvessels comprised 89–91% of all vessels in the three ROI. Follicle density in ungrafted cortex pieces was 51.8 ± 17.3 and 14.7 ± 3.7 follicles/mm3 after 8 weeks of xenografting, resulting in a follicle survival rate of 28%. ConclusionsHost revascularization was established equally efficiently from both sides of transplanted human ovarian cortex, suggesting that transplantation techniques ensuring revascularization from both sides of the ovarian graft could potentially facilitate faster graft revascularization.

Short term results of Sub trochanteric femur fractures treated by Ender’s nail and Plate: A study of 25 cases

Introduction: Fractures in sub-trochanteric regions are common in elder as well as younger population though force of injury varies. In younger age this results from high velocity trauma around hip while in elder population this may occur due to trivial trauma like simple fall. This type of fracture constitutes 10-30% of fractures around hip. Usually these type of fractures result in poor outcome because of difficult reduction due to muscle forces across fracture, comminution of fracture fragments and factors like osteoporosis. Treatment includes fixation of this fracture using various intramedullary as well as extra medullary side support implants. Proximal femoral nail is most commonly used implant as they share the load across the fracture. This type of fracture is having a very high failure rate resulting in non-union or varus mal-union with implant failure. The forces across the fracture are well countered by combining intramedullary as well side support implants. In this study we used Ender’s Nails as intramedullary implant and with 4.5 mm Dynamic compression plate and 4.5mm and 6.5 mm screws as extra medullary implant that adds the rotational stability to bone implant construct. Materials and methods: 25 patients operated from June 2015 to December 2019 were identified from the available records and included in the study. All the treatment documents of selected patients till their last follow-up were reviewed. During their last follow-up Harris Hip Score was counted and Tegner Activity scores were obtained.Results: Out of 25, 18 were Male and 7 were female patients. Mean age of patients was 43. Most common mode of injury was road traffic accident. Fracture was united in all the patients (Union rate was 100%). 2 patients developed minor complication like nail backing out at knee joint. Harris Hip Scores is excellent in 20(80%) and good in 5(20%) numbers of patients. None of the patients were having movement restriction for hip and knee joint of the affected side. Conclusion: Combination of intra medullary device like Ender’s nail and extra medullary implant like Dynamic compression plate for sub-trochanteric fracture is having good functional results and they are beneficial in terms of cost of the implants and technical ease.

Differential diagnosis of renal oncocytoma and chromophobe renal cell carcinoma using CT features: a central scar-matched retrospective study.

Renal oncocytoma (RO) and chromophobe renal cell carcinoma (chRCC) have a common cellular origin and different clinical management and prognosis. To explore the utility of computed tomography (CT) in the differentiation of RO and chRCC. Twenty-five patients with RO and 73 patients with chRCC presenting with the central scar were included retrospectively. Two experienced radiologists independently reviewed the CT imaging features, including location, tumor size, relative density ratio, segmental enhancement inversion (SEI), necrosis, and perirenal fascia thickening, among others. Interclass correlation coefficient (ICC, for continuous variables) or Kappa coefficient test (for categorical variables) was used to determine intra-observer and inter-observer bias between the two radiologists. The inter- and intra-reader reproducibility of the other CT imaging parameters were nearly perfect (>0.81) except for the measurements of fat (0.662). RO differed from chRCC in the cortical or medullary side (P = 0.005), relative density ratio (P = 0.020), SEI (P < 0.001), and necrosis (P = 0.045). The logistic regression model showed that location (right kidney), hypo-density on non-enhanced CT, SEI, and perirenal fascia thickening were highly predictive of RO. The combined indicators from logistic regression model were used for ROC analysis. The area under the ROC curve was 0.923 (P < 0.001). The sensitivity and specificity of the four factors combined for diagnosing RO were 88% and 86.3%, respectively. The correlation coefficient between necrosis and tumor size in all tumors including both of RO and chRCC was 0.584, indicating a positive correlation (P < 0.001). The CT imaging features of location (right kidney), hypo-density on non-enhanced CT, SEI, and perirenal fascia thickening were valuable indicators in distinguishing RO from chRCC.

Osteoinductive potential and bone-bonding ability of ProRoot MTA, MTA Plus and Biodentine in rabbit intramedullary model: Microchemical characterization and histological analysis

ObjectiveTo study the in vivo osteoinductive potential, bone-bonding ability (bioactivity) and bone biomineralization of current hydraulic calcium silicate cements used as graft materials and placed in contact with medullary bone. MethodsProRoot MTA, MTA Plus and Biodentine were used to fill surgical bone defects (2-mm diameter through the entire cortical thickness to reach the medullary bone) in the tibia of mature male rabbits. Tibiae were retrieved after 30days and submitted to histological analysis and microchemical characterization using Optical Microscopy (OM) and Environmental Scanning Electron Microscopy with Energy Dispersive X-ray analysis (ESEM-EDX). Bone neoformation and histomorphometric evaluations, degree of mineralization (by Ca/P, Ca/N and P/N ratios) and the diffusion of material elements were studied. ResultsBone neoformation was observed in response to all materials. No sign of necrosis were found on the walls of the pre-existing cortical bone. No osteoclasts and no formation of fibrous tissue were evident. Sign of angiogenesis were present.EDX (element content, line profile and element mapping) showed the increase in Ca and P and decrease in C, S and N from the mature bone towards the mineralizing interface.Ca/P, Ca/N and P/N ratios showed differences in the degree of mineralization/maturation stage of bone.MTA Plus and ProRoot MTA exhibited close contact with the pre-existing bone and good bone-bonding with neoformed bone juxtaposed on the medullary side of the materials without interposed connective tissue or resorption lacunae or gaps. The materials showed a dense appearance with 100% of residual materials and no colonization by fluids and cells. No migration of Bi or Al material elements to the newly formed bone was found.Biodentine showed newly formed trabecular bone with marrow spaces and sparse traces of residual material (≈9%). SignificanceThe in vivo osteoinductive properties with dynamic biomineralization processes around these calcium silicate materials extruded in medullary bone in appropriate animal model have been demonstrated by ESEM-EDX in association with OM. Good biocompatibility was evident as only slight inflammatory infiltrate and no sign of necrosis at the interface with the pre-existing bone were found.MTA Plus and ProRoot MTA exhibited bioactive potential as they can bond to bone directly without interposed connective tissue. Biodentine was replaced by newly formed bone. Clinical significanceThe results of the study demonstrate the capacity of calcium silicate cements to allow osteoid matrix deposition by activated osteoblasts and favour its biomineralization, and to achieve a direct bond between the (bioactive) materials surface and the mineralized bone matrix.

Polarized Ovaries of the Long‐tongued Bat, Glossophaga soricina: A Novel Model for Studying Ovarian Development, Folliculogenesis, and Ovulation

Glossophaga soricina is a spontaneously ovulating, monovular, polyestrous bat with a simplex uterus, exhibiting true menstruation. Studies conducted on reproductively active, captive-maintained animals established that G. soricina also has polarized ovaries, with the ovarian surface epithelium (OSE) restricted to the medial side of the ovary, and primordial follicles limited to an immediately adjacent zone. Follicles selected for further development are recruited from the medullary side of this zone, and ovulation is restricted to the portion of the ovary covered by the OSE. To further develop G. soricina as a model for studying ovarian development and physiology, ovaries were collected from fetal, neonatal, and adult females and processed for morphological and immunohistochemical analyses. The latter included staining for factor VIII-related antigen (von Willebrand factor) to assess regional differences in ovarian vascularity. The ovarian structure in fetal and neonatal animals was very similar to that in other species that do not have polarized ovaries at comparable stages of development. This indicated that polarization of the ovary does not occur during fetal development, but rather sometime between the neonatal period and adulthood. Vascular elements were abundant in areas of the ovary surrounding early growing follicles, but sparse in the zone of the ovary containing primordial follicles. The polarized nature of the ovaries in G. soricina suggests that this species might be used as a model to investigate the formation, long-term maintenance, and activation of primordial follicles, and the role of the OSE in ovulation and folliculogenesis.

The influence of cyclic compression and distraction on the healing of experimental tibial fractures

Interfragmentary displacement has a main effect on callus formation in fracture healing. To test whether compressive or distractive displacements have a more pronounced effect on new bone formation, a sheep osteotomy model was created whereby the gap tissue was subjected to constant bending displacement. A diaphyseal osteotomy with a gap of 2 mm was created in 18 sheep tibiae and stabilized with a special unilateral actuator-driven external fixator. Two experimental groups with six sheep each received either 10 or 1000 cycles evenly distributed over 24 h. The third group of six sheep served as a control group without actively induced displacement. The amount and direction of cyclic displacement was kept constant throughout the observation period, resulting in 50% compressive and 50% distractive displacement within the osteotomy gap. At sacrifice, six weeks after surgery, bending stiffness was measured and new bone formation was assessed radiologically and microradiographically. In all cycled groups, the amount of periosteal callus formation was up to 25 times greater on the compression compared to the distraction side ( p<0.001). The application of the higher number of daily cycles resulted in an up to 10-fold greater amount of periosteal new bone formation on the compression side ( p<0.012), while the difference on the distraction side was not significant. Ten cycles applied a day were sufficient to create an abundant periosteal callus on the compression side. In the 1000 cycle group, bending stiffness revealed slightly lower values but the difference was not significant. Solid periosteal bridging of the gap was observed in two sheep in the control group, whereas bridging in the cycled groups was observed exclusively at the medullary side. In conclusion, cyclic compressive displacements were found to be superior over distractive displacements. A higher number of enforced and maintained compressive displacements enhanced periosteal callus formation but did not allow bony bridging of the gap.

Immunohistochemical assessment of the neurosecretory cells of the chicken thymus using a novel monoclonal antibody against avian chromogranin A

An immunocytochemical approach to the identification of neuroendocrine cells in the thymus of the chicken was taken based on a novel monoclonal antibody against turkey chromogranin A (CgA), a classic marker protein for neuroendocrine cells. CgA-immunoreactive cells were readily observed in the thymus, and were typically confined to the medullary side of the corticomedullary junction of the thymic lobules. Reversed transcription PCR confirmed local production of CgA in the thymus. The majority of CgA + cells were small and round or oval in shape but some cells were larger and had conspicuous extensions. Immunofluorescent double staining experiments with antibodies against Neuron-specific enolase and with a neural crest marker (HNK-1) indicated no demonstrable overlap between the CgA-positive cells and either of the above cell populations, demonstrating the existence of three distinct neuronal/neuroendocrine cell populations in the avian thymus.

NADPH diaphorase-positive dendritic profiles in rat thymus are discrete from autofluorescent cells, immunoreactive for inducible nitric oxide synthase, and show strain-specific abundance differences.

Predisposition to autoimmune disorder in Lewis rats has been associated with abnormal hypothalamic regulation of circulating steroids, leading to inadequate suppression of T helper 1 (Th1) cell-mediated inflammatory reactions. In addition, autoimmune syndromes can be triggered within formerly resistant animals, following damage to the negative selection process of the thymus. A contribution to the autoimmune-susceptible phenotype may therefore derive from the status of thymic tolerance. One mechanism of intrathymic negative selection may involve nitric oxide. Because inducible nitric oxide synthase (iNOS) is known to be inhibitable by steroids, its expression might be different within strains having neuroendocrine disturbance. We report on a study to compare intrathymic iNOS expression in autoimmune-prone Lewis rats with other resistant strains. Interdigitating cells (IDC), darkly stained for diaphorase, were confirmed as immunoreactive for iNOS. They were located towards the medullary side of an accumulation of unstained, but autofluorescent cells (presumed to be macrophages) that circumscribes the corticomedullary zone. The role of iNOS+ IDC in the apoptotic deletion of T cells has been suggested by other studies. Despite the blunted steroidal condition reported for Lewis, nitrergic cell abundance was shown, by quantitative analysis of histochemical stain, to be on average approximately twofold lower compared with resistant strains (Fischer and Sprague-Dawley). This trend was evident in males and females, and confirmed by independent observers. We hypothesize that an intrathymic, iNOS-dependent mechanism may be important for the suppression of potentially autoreactive T-cell clones.

リンパ濾胞内抗原捕捉域の変遷に関する三次元的研究

In lymph follicles of rat popliteal lymph nodes (PLNs), the transition of the localization of PAP (peroxidase-anti-peroxidase complex) trapping area with development of germinal centers was tridimensionally studied to analyze the differentiation of follicular dendritic cells (FDCs). Rats 5 weeks old were injected sheep red blood cells (SRBCs) into foot pads. The PLNs were extirpated at different intervals, at one day before extirpations PAP were injected into foot pads. Detection of PAP in lymph follicles and detail observations were performed in serial paraffin sections and in electron microscopy by immunoperoxidase technique. After the injection of SRBCs, lymph follicles and germinal centers increased in number. In early primary lymph follicles, the PAP trapping area occupied large space, however the ratio of the PAP trapping area occupying in the follicles was decreased with development of germinal centers. The trapping of PAP became weak in which centroblasts had been just appeared in the primary follicles, and then the medullary side of the germinal centers revealed no PAP trapping with further development of germinal centers. In electron microscopical observation, the trapping of PAP in secondary follicles could be found on the surface of cytoplasmic membranes of FDSs, which formed complicated labyrinth structures, in corona and light zone. On long time interval after injection of SRBCs, the germinal centers were atrophic, while cytoplasmic processes of FDCs remained complicated. The differentiation of FDCs is discussed.

The fibromuscular basket (Sporta perimedullaris musculosa) in renculi of kidneys from the ringed seal, Phoca hispida (Pinnipedia).

The medulla of renculi from kidneys of Ringed seals (Phoca hispida) is completely enclosed by cortex except at the hilum. Within the renculus, the fibromuscular coat of the calyx separates from the transitional epithelium at the level of the corticomedullary junction, where the intrarencular arteries also diverge into the parenchyma. Flat ribbons of this stromal tissue form an arborized framework near the medullary side of the intrarencular arteries and the larger of the arcuate arteries derived from them. The ribbons, which are clearly distinct from periarterial connective tissue, are composed of coarse collagenous fibers, elastic fibers, and smooth muscle cells, all oriented in the direction of the long axes of the ribbons, and myofibroblasts. The proportion of smooth muscle cells decreases and that of myofibroblasts increases with increasing distance from the calyx. At the base of the medullary pyramid, the elements of the framework diminish in width and ultimately blend with the surrounding interstitial tissue. The stromal framework, or basket, is homologous with the Sporta perimedullaris musculosa of cetacean kidneys.

The mast cell: III. Distribution and maturation in various organs of the young rat

The changing population-density of mast cells in various organs was plotted for rats aged 1/2 to 90 days and correlated with histochemical studies on the stage of maturation of cells. Mast cells are present at birth in liver and spleen and are particularly associated with foci of haemopoiesis. In bone marrow mast cells are absent at birth but become progressively more numerous with increasing age of the animal. The association of mast cells firstly with foci of extramedullary haemopoiesis and secondly with medullary foci coupled with their detection in peripheral blood strongly suggests that mastopoiesis may be analogous with granulopoiesis. In thymus mast cells are usually associated with interlobular connective tissue stroma, but in the parenchyma they are found toward the medullary side of the cortico-medullary junction. Such a distribution suggests the possible origin of mast cells from lymphocyte or thymocyte. In other tissues examined mast cells are frequently associated with connective tissue stroma and blood vessels.

Localization of anti-rat kidney antibodies in new-born rats.

SummaryRabbit antibodies against adult rat kidney were found to be fixed in the kidneys of new-born rats. Since new-born rats do not develop clinical disease when injected with anti-rat kidney antibodies, this suggests either that the antigens giving rise to cytotoxic antibodies are not present at this early date or the new-born rats are deficient in complement. Six weeks after new-born rats are so injected they still have the rabbit globulin (antibody) in the glomeruli. However, rabbit globulin was demonstrated in the glomeruli on the medullary side of the cortex and not in those of the periphery indicating that new glomeruli in the peripheral region become functional as the animal matures. The increase in functional glomeruli may be the reason for the observation that new-born rats injected with anti-rat kidney serum do not develop clinical disease.

Eosinophilic Granuloma of Bone

A 25-year-old white soldier stationed in the South Pacific was in good health until the latter part of May 1944. At that time, upon lifting a heavy airplane engine part, he first experienced sharp, stabbing pain in the right axilla and beneath the right scapula. Motion of the right arm and shoulder, coughing, sneezing, and deep inspiration reproduced the pain. On occasions he was awakened at night by a heavy, dull aching sensation high in the right axilla. Physiotherapy produced only moderate relief, and on June 3, 1944, the patient was admitted to a numbered station hospital. The only finding of significance on physical examination was sharp pain at the medial and inferior border of the right scapula when the arm was abducted beyond 90°. Roentgen examination of the right shoulder disclosed what was described as a localized osteolytic lesion of the 2nd right rib, containing remnants of normal bone. The remaining long bones, skull, and vertebrae showed no similar lesions. Urinalysis gave normal findings, and tests for Bence-Jones protein were negative. Blood studies showed: hemoglobin 17.5 gm., white cells 10,650, with a normal differential count (3 per cent eosinophils), blood calcium 13.5 mg. and 13.0 mg. on two occasions, blood phosphorus 2.9 mg., sedimentation rate 8 mm. per hour (Cutler). A smear for malaria was negative, as was the Kahn reaction. The patient was evacuated to the continental United States and admitted to a General Hospital on July 21, 1944. Physical examination was entirely negative at this time; range of motion of the right shoulder was normal, and there was neither pain nor tenderness in the axilla or shoulder girdle. Laboratory findings were essentially the same as previously reported, except that the blood calcium was now 12.3 and 12.6 mg. on two occasions, the blood phosphorus was 3.9 and 4.7 mg., and phosphatase 9.2 units (Bodansky). Serum albumin was 4.88 gm. and serum globulin 2.42 gm. The patient was referred to the roentgenological service for examination of the right shoulder, and the following opinion was given on July 27, 1944: “The 2nd right rib at the mid point of its shaft in the mid-axillary line presents an eccentric, oval-shaped area of infiltrative destruction with cortical erosion on the medullary side and moderate subperiosteal new bone formation. The lesion has grown in the axial plane of the rib, and though there is subperiosteal new bone formation, there is no soft-tissue mass surrounding the rib to give it a spherical shape. The area of destruction is fairly sharply demarcated, but at the margins the destructive process has infiltrated between the bony trabeculae, leaving small islands of normal bone between areas of destruction. Similarly, in the center of the lesion, where destruction has been most severe, a coarse reticulum of bone remains, giving a faintly honeycombed appearance.