Masses Of Various Sizes Research Articles

Successful surgical excision of vaginal leiomyoma in a labrador bitch

Neoplasia of reproductive system in bitch is more common in lower reproductive tract i.e. vagina and vulva, as compare to uterus and ovaries. Majority of the canine vaginal or vulvar neoplasms are leiomyoma, leiomyosarcoma, fibroma and transmissible venereal tumor Vaginal leiomyomas may be single or multiple, intraluminal or extraluminal and usually round or oval, well Defined and encapsulated. Minimum size of excised masses of various sizes was ranging from 0.4 x 0.2 to 2.4 x 2.3 inches and weighing 0.65 to 113 grams, respectively. A case of multiple vaginal leiomyoma that was presented to the department of Veterinary Gynecology, Veterinary College, Gadag, was successfully surgically excised andafter 10 days the bitch was completely recovered from surgical wound and reported.

Histological changes in Horabagrus brachysoma acclimated to warm temperature and exposed to critical thermal maxima and minima

Escalating concerns about global climate change necessitate investigations into its consequences for the survival of poikilothermic animals like fish. The present study describes histological alterations in gill, skin, kidney, liver, and brain tissues of the juvenile Horabagrus brachysoma specimens acclimated to 26°C (control) and 36°C for 30 days, and those exposed to dynamic temperature changes (increased or decreased at the rate of 0.3°C/min from 26°C), the critical thermal maxima (CTMax) and critical thermal minima (CTMin). The fish acclimated to 36°C and those exposed to CTMax (40.23 ± 0.12°C) and CTMin (14.15 ± 0.10°C) showed severe histological aberrations in gill, skin, liver, kidney, and brain tissues. Histological alterations in gill tissues included loss of epithelial cells in the branchial arch, thinning of the primary lamellae, and loss of the secondary lamellae. Thickening of the epithelial layer (36°C), and desquamation of epithelial cells (CTMax) were the histological alterations detected in skin tissues. Alterations in liver tissues included severe congestion with vacuolization and the cloudy appearance of cells and extensive loss of cellular contour. Extensive vacuolization with complete flattening of the tubule epithelial cells, distorted appearance of tubular lamellae, and marked loss of glomerular tuft were the changes recorded in kidney tissues. Brain tissue alterations comprised increased cellularity and vacuolization in the cerebrum (36°C), and nodular masses of various sizes in the cerebrum (CTMin). The present study showed that acclimation to warm temperature (36°C) and exposure to dynamic temperature changes (CTMax and CTMin) cause histopathological alterations in the vital organs of H. brachysoma. The findings of the present study can help in monitoring the health of H. brachysoma in the natural environment and in culture systems under a climate change scenario.

Independent particle and lot selection of increment samples in the theory of sampling

Introduction. The theory of sampling developed by Pierre Gy does not prove the compatibility and consistency of discrete and continuous selection models. Discrete (independent particle) and continuous (lot) selection models are determined by incompatible properties of increment samples, which prevented from creating the consistent theory of sampling. Research methodology. The inconsistencies are removed at assuming the idea that there are differences in both separate ore lumps or mineral-dressing products and any locally selected parts of the rock mass under test called increment samples simultaneously available in any rock mass under test. The said differences are described by independent particle dispersion and increment samples dispersion. Composite sample permissible error formed in both discrete and continuous selection is attained by selection of the number of particles collected into the increment sample or their parts and the number of increment samples. Particle dispersion, increment sample dispersion, the number of particles in an increment sample and the number of increment samples combined in one formula make up the complete formula of the fundamental sampling error. The development of the theory of sampling. Based on the complete formula, the possibility of obtaining minimum masses of various sizes has been shown. Thus, for one and the same preset permissible error and from one and the same massif under test, it is possible to collect minimum mass of 17.55 kg (individual particle selection), minimum mass of 170.3 kg (collecting with the bucket sampler), and minimum mass of 10 g under the individual particle selection of particle parts (which is fulfilled under X-ray fluorescent on-stream analysis of material). Discrete selection of increment samples without particles destruction is an especial case of continuous selection method when it is possible to accept the condition that increment samples dispersion is equal to zero. It is possible under ideal mixing of the massif under test. Discussion. Minimum mass of a sample is not a constant. It is a function of increment sample mass. Minimum mass in individual particle selection can be accepted as a reference value of the minimum mass. In lot selection it can be significantly higher than the reference one, while in case of reducing particle size it can be significantly lower than the reference one.

Histo-morphology of cutaneous papillomatosis in indigenous cattle

Background: Papillomatosis is a viral disease manifested with benign cutaneous growths (skin epithelium hyperplasia) in different body parts which is encountered in cattle, goat, dog, rabbit, horse, rodent and also in human. The purpose of the present study is to detect the histo-morphological changes of cutaneous papillomatosis in indigenous cattle. Methods: Ten (10) random samples from skin lesions of warts were collected from adult indigenous cattle (10 cattle) that were diagnosed as cutaneous papillomatosis by clinical examination at Veterinary Teaching Hospital, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh. For gross observations, location of appearance of warts, color and size was taken into consideration. Immediately after collection of sample, they were preserved in Bouin’s fluid followed by dehydration, clearing and infiltration and finally embedded by paraffin wax. Then samples were sectioned using rotatory microtome and stained with Harris’s Haematoxylin and Eosin Y to facilitate microscopic study. Results: The results of present study revealed that cutaneous papillomas were mostly located on the head and neck, around eyes, under the chin and perineal region in indigenous cattle. Grossly, warts in cattle are bumpy or cauliflower-like masses of various sizes and numbers that projected from the skin having grey, rough, scaly, and dry surface. When incised, its cut surface has an external layer- keratinized epithelium and an internal core- moist white connective tissue. Histopathological changes indicate hyperplastic features of the affected skin layers of epidermis. Further, the microscopic analysis also revealed orthokeratotic hyperkeratosis, acanthosis and down word hyperplasia, fibrovascular area, keratohyalin granules and koilocytes at different layers of the affected skin. Conclusion: The results of this present study might be useful for the classification and treatment of this common malady based on its histological changes.

Advanced multicentric lymphoma in a Belgian Draft Horse mare

A 15-year old draft horse mare was presented to the University Clinic for evaluation of lethargy, anorexia, ptyalism, weight loss and ventral oedema. Clinical examination and rectal palpation revealed generalised lymphadenopathy and numerous firm subcutaneous and abdominal masses of various sizes. Transcutaneous ultrasonography revealed significant bilateral pleural fluid accumulation and a single hypoechoic structure in the abdomen lateral to the liver. Blood analysis showed several abnormalities including a marked leucocytosis with an increased number of segmented neutrophils, atypical lymphocytes and monocytosis, suggestive of leukaemic lymphoma. A significant hyperproteinaemia with a hypoalbuminemia and a monoclonal gammopathy was identified. At necropsy myriad masses presented through the whole body. Histology confirmed the suspicion of lymphoma, which was classified as a T-cell rich B-cell multicentric lymphoma. This article describes the clinical and pathologic findings of this case of leukaemic lymphoma.

Adrenal lymphoma: presentation, management and prognosis.

This study aimed to identify the clinical, radiological and prognostic features of primary adrenal lymphoma (PAL) in order to diagnose the disease more accurately. A retrospective multi-centre study was conducted on the clinical, biological and radiological features as well as the treatment and overall survival outcomes in PAL. Between 1994 and 2014, 28 patients from five regions of eastern France were diagnosed with primary adrenal lymphoma. The revealing symptoms were a worsening general state (77%), weight loss (77%) and abdominal pain (42%). Biological features of PAL were almost omnipresent: increased LDH, β2 microglobulin, CRP or ferritinaemia levels. The PAL was bilateral in 20 cases (71%), adrenal insufficiency was searched for in 11 patients and found in eight (73%). CT scans showed masses of various sizes measuring up to 180 mm. On MRI, the lesions were hypointense in T1 and hyperintense in T2. When done, positron emission tomography with fluorodeoxyglucose (FDG-PET) showed locations not seen on the CT and revealed extra-adrenal locations in 70% of examinations. Adrenalectomy brought no benefit. The overall survival rate was poor (61.9% at 2 years) despite polychemotherapy. The clinical presentation of PAL comprised major general symptoms. Adrenal insufficiency was very common in patients with bilateral involvement but was not systematically tested. PET was an efficient examination to visualize extra-adrenal locations. The preliminary results of MRI to distinguish between PAL and adrenocortical carcinoma should be confirmed. Further studies are needed to establish an optimal strategy for the management of these primary adrenal lymphomas.

Multiple mesenteric well-differentiated liposarcoma complicated by purulent inflammation: A case report.

Multiple mesenteric well-differentiated (WD) liposarcoma is an extremely rare entity. The present study describes a case of multiple mesenteric WD liposarcoma, complicated by purulent inflammation, in a 59-year-old male who presented with abdominal pain and pyrexia of unknown origin. A computed tomography scan of the abdomen revealed a large, non-encapsulated mass in the abdomino-pelvic cavity, which was characterized by two components, a main portion of fatty density and a non-adipose solid portion. A re-evaluated CT scan, performed eight days later, revealed an enlargement of the non-adipose mass. A laparotomy was performed, and numerous separated fatty nodules and masses of various sizes were identified within the mesentery of the small intestine. The histological findings were consistent with an adipocytic subtype of multiple mesenteric WD liposarcoma, with the largest of the tumors complicated by purulent inflammation. The multiplicity of these tumors and the concurrent purulent inflammation in the present case make it unique.

Aortic Body Cell Tumor with Kidney Metastasis in a Dog

In this case, a male Terrier dog, 14-year-old evaluated with a history of cardiac arrhythmias, coughing, rhinorrhagia and exercise intolerance for six mounths. The dog was died upon worsening of clinical signs. At necropsy, two masses of various sizes were observed in the heart-base region. Microscopically, cells from masses were atypic and polyhedral with eosinophilic-granular cytoplasm and basophilic nucleus with round to oval shape. The neoplastic cells were divided into lobules by connective tissue forming nests. In addition to these findings, metastasis to the left kidney was observed. Immunohistochemically, the tumor cells from both primary and metastatic tissues showed immunoreactivity to monoclonal mouse anti-neuron specific enolase antibody but were negative for cytokeratin, vimentin, chromogranin A, α smooth muscle actin and S-100. Based on the clinical, histological and immunohistochemical findings, malignant aortic body tumor with left kidney metastasis was diagnosed in the present case.

Perforating abomasal ulcer caused by yolk sac tumor in a Holstein calf

A 2-month-old male Holstein calf showed clinical signs of abdominal bloating, melena, and pain and was suspected of having a perforating abomasal ulcer. Necropsy revealed a large mass located preferentially around the abomasum and a large perforating abomasal ulcer on the pyloric antrum. Milky white masses of various sizes were also found in the abdominal cavity that consisted of agglutinated nodules ranging in size from a pinhead to a golf ball and were distributed on the surfaces of the liver, reticulum, omasum, abomasum, and diaphragm. Microscopic examination revealed that the masses were composed primarily of hyaline matrices, epithelioid tumor cells, and large atypical cells with hyaline droplets and/or vacuoles. Stromal hyaline matrices and hyaline droplets of the large tumor cells stained positive with periodic acid-Schiff stain. Tumor cells showed a positive reaction to anti-human alpha-fetoprotein, which is a marker of yolk sac tumors. These findings strongly suggested that the masses found in the abdominal cavity were yolk sac tumor, a rare germ cell tumor in cattle.

Embryonal carcinoma in the abdominal cavity of a male calf

An embryonal carcinoma was diagnosed in the abdominal cavity of a 55-day-old male calf. Macroscopically, a large volume of ascitic fluid was observed along with white to yellowish-white masses of various sizes densely located on the abdominal wall and the surface of abdominal organs. There was an absence of testes in the scrotum. Histologically, the tumor cells were polygonal, and the tumor was mostly composed of sheets of densely packed solid patterns with occasional papillary and tubular structures. Cell nuclei were variable in size, and cellular mitotic rate was high. Immunohistochemically, tumor cells were positive for alpha-fetoprotein, placental alkaline phosphatase, cytokeratin, and carcinoembryonic antigen. Ultrastructurally, the tumor cells had large nuclei, extensive rough endoplasmic reticulum, and small numbers of mitochondria. Microvillus-like structures and desmosomes were occasionally observed. From lectin histochemical examination, the tumor cells were positive for concanavalin A, wheat germ agglutinin, peanut agglutinin, Dolichos biflorus agglutinin, soybean agglutinin, Griffonia simplicifolia I, and Bauhinia purpurea, and negative for Ulex europaeus agglutinin I. Results of histopathological, immunohistochemical, and ultrastructural examinations of the tumor were similar to those obtained for human embryonal carcinoma.

Our strategy in complication management of augmentation mammaplasty with polyacrylamide hydrogel injection in 235 patients

Polyacrylamide hydrogel (PAAG) was once widely used in breast augmentation in China. Although it had been banned for augmentation mammaplasty in 2006, a large number of patients whose breasts were augmented with the gel injection have continued to seek medical advice because of its complications. The clinical management of these complications has never been standardised. The data of a total of 235 patients with complications following PAAG-injected breast augmentation have been summarised and the types and causations analysed. Magnetic resonance imaging (MRI) was undertaken in 228 patients with palpable masses, breast pain or tenderness, asymmetry or deformity and functional or psychological problems to eliminate neoplasm, infection and to delineate the diseases. The surgical gel evacuation via periareolar incisions was performed for all patients and immediate silicone breast prostheses were implanted in 108 patients and were delayed in 28 cases by 6 months. Most patients (214/235) of the group were satisfied with the treatment, and symptoms disappeared after removal of the gel. The gel distribution and involved tissue were well defined and neoplasm was ruled out by MRI. Postoperative MRI in 68 patients revealed that no obvious PAAG remaining. In either immediate or delayed reconstructive patients with silicone breast implants, good breast contour presented. A diagnosis and management strategy for these complications is proposed. In conclusion, the breast masses, pain and deformity are major complications after PAAG-injected breast augmentation. Psychological problems should be paid attention to. MRI is a sensitive and accurate method for diagnosis and treatment evaluation. Surgical removal of injected gel is the preferred method for complication management. The implantation of silicone prostheses for breast contour restoration after PAAG evacuation is effective for patients under strict selection. Our strategy for treating PAAG-related complications proved useful.

Giant congenital melanocytic naevus associated with Dandy-Walker malformation, lipomatosis and hemihypertrophy of the leg

A 30-year-old woman presented with congenital hyperpigmented plaques and multiple tumoral lesions. Her left leg was hypertrophic, although the bones were normal. Dermatological examination revealed hyperpigmented macules and plaques with hair on the legs, abdominal and lumbar areas (covering > 60% of the total body surface) and multiple naevi on the face, arms, back and chest. Multiple soft masses of various sizes, some of which colocalized with hyperpigmented plaques, were seen on the trunk and legs. Malignant melanoma was excluded by histopathological examinations of multiple biopsy specimens obtained from the pigmented lesions. Histopathological examination of one of the soft masses showed that it was a lipoma. Cranial magnetic resonance imaging and computed tomography scans showed an enlarged fourth ventricule and vermis hypoplasia (Dandy-Walker malformation; DWM). Neurocutaneous melanosis is a rare combined abnormality of the skin and central nervous system. A few case reports relating to the coexistence of giant congenital melanocytic naevus, lipomatosis and hemihypertrophy have been published in the literature. We report this very rare case of giant melanocytic naevus with lipomatosis, hemihypertrophy of the leg, and DWM.

ラットを用いた術後縫合糸肉芽腫に関する実験病理学的研究

After a skin incision, spay and castration for rats, histopathological changes were compared against six different sutures widely used in the clinic. Spayed and castrated rats wereeuthanized at 30 days and 60 days after surgery, respectively. At necropsy, cutaneous fibrosis and yellowish masses of various sizes were observed in the spayed and castrated parts. Histopathologically, the lesions were characterized by various stages of foreign granuloma containing the suture materials. The inflammatory reaction wa more significant in spayed and castrated sites than in the sites of skin incision. Silk and absorbabl multifilamentsutures have produced the strongest tissue reaction among the tested suture materials. Theresults of the present study suggest that foreign granulomas occur with any suture, that tissue reactions continue as long as suture materials exist, and that the inflammatory response differs by suture materials and surgical site.

Massive intraperitoneal stromal tumour: Two-year follow-up

Gastrointestinal stromal tumours (GISTs) are rare. Few cases of such tumours are reported in the literature. Until recently, they were only treated surgically but, nowadays, for high grade tumours, we can consider wide excision, not only as a palliative measure, but also to enable subsequent treatment with Imatinib mesylate. We describe the case of a patient with a high-grade GIST, who, two years after undergoing surgery and medical treatment, is still in remission. Computerised axial tomography (CT) of the abdomen detected the presence of multiple heterogeneous solid masses of various sizes but no localised retroperitoneal adenopathies. Median laparotomy was performed and revealed multiple intraperitoneal tumours. The larger masses were excised and the smaller residual implants are currently being treated with Imatinib mesylate. The patient had visceral, parietal and peritoneal dissemination. One of them, located in the supramesocolic area, had a maximal diameter of 20cm. Another one, at the level of the retro-gastric space, had a diameter of 22cm. The other tumours were smaller and spread over the parietal and visceral peritoneum, mainly between the loops of the small intestine and pelvis. Two years after cytoreduction surgery, the patient is well and free of obvious disease and has shown a good tolerance to pharmacological treatment. Imatinib mesylate has revolutionised the treatment of GIST and offers good palliation and prolongation of overall survival.

Histoid leprosy – unusual presentation

A 39-year-old man presented with a chronic nonhealing toe ulcer and multiple skin-colored papules on the back, arms, and knees. From an initial small erosion, the toe lesion ulcerated over a 1.5-year period, while the papules progressed over a 6-month period, first appearing on the back and then spreading to the arms and knees. The past medical and family history were non-contributory. Pertinent findings included the aforementioned well-defined, asymmetric, shiny papules (Fig. 1). There were multiple, discrete, erythematous, pedunculated, tumor-like masses of various sizes over the right thigh (Fig. 2) and a well-defined 4 cm x 3 cm ulcer on the second toe of the left foot, the floor of which was covered by necrotic slough. There was distal loss of sensation to temperature, touch, and pain. The greater auricular, ulnar, radial cutaneous and common peroneal nerves were thickened bilaterally, but non-tender. A clinical diagnosis of histoid leprosy was made. The differential diagnosis for the tumor-like thigh masses included dermatofibroma, neurofibroma, and Kaposi's sarcoma. The hemogram, liver/renal function tests, chest X-ray, and abdominal ultrasound were normal. Human immunodeficiency virus enzyme-linked immunosorbent assay (HIV ELISA) was negative. The ear lobe smear (ELS) for acid-fast bacilli showed a bacterial index (BI) of 6+[> 1000 organisms/oil immersion field (oif)] and a morphological index (MI) of 50%. The skin-colored papules on the back and the pedunculated masses showed a BI of 5+ (100-1,000 organisms/oif) and an MI of 50%. The normal skin showed a BI of 4+ (10-100 organisms/oif) and an MI of 5%. Biopsy of the papules and pedunculated tumors showed a well-circumscribed area of the dermis packed with many acid-fast organisms and foamy macrophages, consistent with histoid leprosy (Fig. 3). Fite-Faraco stain demonstrated cells packed with lepra bacilli. A final diagnosis of lepromatous leprosy, histoid variant, was made. Dapsone, clofazimine, rifampicin, and, later, ofloxacin were started.

Detection of Simulated Lesions on Data-compressed Digital Mammograms

To evaluate retrospectively the effect of a wavelet-based compression method on the detection of simulated masses of various sizes and clustered microcalcifications on data-compressed digital mammograms. The images used in this study were acquired with institutional review board approval and patient informed consent, both of which allowed subsequent image data analysis. Patient identification was removed from images, and the study complied with requirements of the Health Insurance Portability and Accountability Act. Masses 3, 6, and 8 mm in diameter were analytically simulated and added to clinical mammographic backgrounds. In addition, microcalcifications were extracted from a clinical mammogram and hybridized with simulated microcalcifications for use in this study. Image compression conditions of 1:1, 15:1, and 30:1 were investigated. Observer responses were recorded with a six-point rating scale, and receiver operating characteristic (ROC) analysis was performed. In addition, two well-established numeric observer models were used to study the effect of image compression under the same compression conditions as were used with human observers. Analysis of variance was performed after observer adjustment to compare the mean values for area under the ROC curve (A(z)) across the three compression levels for the masses and microcalcification clusters. The results of the study indicated no significant differences in the A(z) values for masses with the compression conditions investigated. For images of microcalcifications, there were significant differences in A(z) values between compression ratios of 1:1 and 30:1 (P = .0005) and of 15:1 and 30:1 (P = .004); the difference between compression ratios of 1:1 and 15:1 was nonsignificant (P = .053). The observer models and human observers exhibited similar trends in detection of the masses investigated in this study. Detection of simulated masses was not affected by the compression method with the conditions used in this study, while the detection of microcalcifications was significantly reduced with a compression ratio of more than 15:1.

Unusual Metastasis of Malignant Aortic Body Tumor to <i>Multiple</i> Bones in a Dog

Unusual metastasis of malignant aortic body tumor to multiple bones was detected in a 5-year-old female English Setter dog. Radiographs exhibited an abnormal mass in the base of heart and osteolytic lesions in the bodies of T11 and L2 vertebrates, body of right femur, right proximal humoral epiphysis and infraspinous fossa near to the neck of right scapula. At necropsy, multiple tumor masses of various sizes were observed also in the bones as well as the heart base and tracheobronchial lymph node. Tumor masses of L2 and T11 protruded into the vertebral canal and compressed corresponding sites of spinal cord, leading to paraplegia. Histopathologically, the tumor cells, arranged in sheets or nests, were polyhedral, lightly eosinophilic, finely granular cytoplasm with mostly round to oval nucleus and had scattered bizarre giant cells. Ultrastructural study revealed the characteristic findings that tumor cells contained a large number of small, electron-dense, membrane-limited secretory granules in cytoplasm. This is thought to be an extremely rare case having multiple bone metastases of a malignant aortic body tumor.

Computed tomography of hereditary multifocal renal cystadenocarcinomas in German shepherd dogs.

The purpose of the study was to characterize the renal lesions of hereditary renal cystadenocarcinomas in the German shepherd dog using computed tomography (CT). Fourteen dogs with renal cystadenocarcinomas and nodular dermatofibrosis, and two unaffected dogs were studied. There were nine dogs with spontaneous disease and seven dogs from a test mating. The characteristic renal CT findings of renal cystadenocarcinomas were bilateral multiple cysts and tumor masses of various sizes. The earliest changes were detected between 4 and 5 years of age and the smallest cysts measured 2-3 mm in diameter. Abdominal CT examination gives a wide field view and excellent anatomic images of the kidneys. It is easy to differentiate between cysts and solid tumors. CT examination is useful for the early detection of renal cystadenocarcinomas and for screening suspected carrier dogs before breeding.

Ultrastructural characterization and immunolocalization of osteopontin in rat calvarial osteoblast primary cultures

As part of ongoing studies aimed at clarifying the early events of bone matrix deposition and mineralization, we have characterized primary osteoblast cultures using ultrastructural and immunocytochemical methods. Osteogenic cells were isolated by sequential enzymatic digestion of newborn rat (2–4-day-old) calvariae and grown for periods of 7 to 28 days on polystyrene, Thermanox plastic, or sputtered titanium. Bone-like nodules, showing a stratified organization of cells and collagen, were examined by scanning and transmission electron microscopy, and further characterized for mineral by backscattered electron imaging and X-ray microanalysis. Colloidal gold immunocytochemistry was used to examine the distribution of osteopontin in these nodules. Cells at the surface of the nodules were rounded, while those within the nodules generally appeared more flattened. Both cell types, particularly at early culture intervals, exhibited well-developed protein synthetic organelles. Collagen fibrils were present between the cell layers and some individual fibrils appeared mineralized. Aggregates of needle-shaped crystallites were sometimes apposed to the cell surface, frequently within invaginated regions of the cell membrane, while other mineralized masses of various sizes were present within the collagenous scaffolding. The periphery of the mineralized masses was often delimited by an electron-dense, lamina limitans-like layer. Focal accumulations and/or a more complete layer of afibrillar, mineralized organic matrix were sometimes observed at the interface between the cells and the surface of the culture dish. Osteopontin was immunodetected over the afibrillar and collagenous mineralized matrix throughout the cultures and, in some cases, labeling was concentrated over the peripheral, electron-dense material delimiting the mineralized masses. In conclusion, these data indicate that calvaria-derived osteoblasts produce an extracellular matrix with structural and compositional similarities to bone. Although not a regular observation, the accumulation of osteopontin on the surface of the culture substrate and at the periphery of masses of mineralized matrix may be analogous to what takes place in vivo at naturally occurring bone interfaces. © 1996 Wiley-Liss, Inc.

Ultrastructural characterization and immunolocalization of osteopontin in rat calvarial osteoblast primary cultures.

As part of ongoing studies aimed at clarifying the early events of bone matrix deposition and mineralization, we have characterized primary osteoblast cultures using ultrastructural and immunocytochemical methods. Osteogenic cells were isolated by sequential enzymatic digestion of newborn rat (2-4-day-old) calvariae and grown for periods of 7 to 28 days on polystyrene, Thermanox plastic, or sputtered titanium. Bone-like nodules, showing a stratified organization of cells and collagen, were examined by scanning and transmission electron microscopy, and further characterized for mineral by backscattered electron imaging and X-ray microanalysis. Colloidal gold immunocytochemistry was used to examine the distribution of osteopontin in these nodules. Cells at the surface of the nodules were rounded, while those within the nodules generally appeared more flattened. Both cell types, particularly at early culture intervals, exhibited well-developed protein synthetic organelles. Collagen fibrils were present between the cell layers and some individual fibrils appeared mineralized. Aggregates of needle-shaped crystallites were sometimes apposed to the cell surface, frequently within invaginated regions of the cell membrane, while other mineralized masses of various sizes were present within the collagenous scaffolding. The periphery of the mineralized masses was often delimited by an electron-dense, lamina limitans-like layer. Focal accumulations and/or a more complete layer of afibrillar, mineralized organic matrix were sometimes observed at the interface between the cells and the surface of the culture dish. Osteopontin was immunodetected over the afibrillar and collagenous mineralized matrix throughout the cultures and, in some cases, labeling was concentrated over the peripheral, electron-dense material delimiting the mineralized masses. In conclusion, these data indicate that calvaria-derived osteoblasts produce an extracellular matrix with structural and compositional similarities to bone. Although not a regular observation, the accumulation of osteopontin on the surface of the culture substrate and at the periphery of masses of mineralized matrix may be analogous to what takes place in vivo at naturally occurring bone interfaces.