A histochemical comparison of methylene-blue/acid fuchsin with hematoxylin and eosin for differentiating calcification of stromal tissue

Benign and malignant connective tissue tumors consist of a fibrous component that contains varying amounts of one or more types of bone or other calcified tissue. Diagnosis of these connective tissue tumors often poses challenges for pathologists, because it is difficult to differentiate the organic matrix of osteoid from hyalinized stroma. To establish a definitive diagnosis, it sometimes is advantageous to demonstrate histologically by special staining either the type of calcification or the presence or absence of calcification. We compared the efficacy of methylene blue-acid fuchsin (MB-AF) to hematoxylin and eosin (H-E) for connective tissue tumors suspected to contain calcifications and to devise an optimal staining technique for calcification that would be specific, simple, and cost- and time-effective. We examined 50 benign and 45 malignant connective tissue tumors that were suspected to contain calcifications. Sections were stained with H-E and MB-AF and evaluated. MB-AF stained bone pink, which contrasted with blue soft tissue. After MB-AF staining, osteoid was faint pink in a blue stromal background. Osteoid was not visualized in H-E stained sections; it was stained the same shade of pink as stromal tissue. Dystrophic calcification and cementum could be identified equally well using either staining technique, but contrast was better after H-E staining. MB-AF staining of bone was comparable to H-E staining and could be used effectively to stain bone and osteoid. MB-AF is a simple, single step procedure. It also stains cementum blue with faint blue rimming and dystrophic calcification bluish-pink, but it cannot be used as a specific stain for types of calcification other than bone and osteoid.     

Further studies on the effect of tetracycline on the developing skeleton of the chick embryo

At intervals from 2 to 13 days of incubation, 2.5 mg of tetracycline hydrochloride was injected into the yolk sac of chick embryos. The femurs and mandibles were examined histologically at intervals between 10 and 17 days of embryonic age. The abnormalities which result include inhibition of mineralization of the developing osteoid trabeculae, retardation of erosion of the long bone cartilage model, and abnormal calcification of the cartilage matrix in the long bones. The major effects on cartilage maturation appear to occur after cellular hypertrophy has taken place and thus are found only in cartilage models which are being replaced by bone. While tetracycline does cause some retardation in the rate of osteoid deposition, the drug appears to affect intramembraneous bone formation in the mandible and femur primarily by retarding or temporarily inhibiting the rate of mineralization of the osteoid matrix. The results of this study indicate that the effects produced by tetracycline on developing bones are dependent upon the concentration of the drug and not upon the time of administration.     

Multiple osteosarcoma in spontaneously hypertensive rats (author's transl)]

For period from 1971 to 1975, 18 cases of osteosarcoma were experienced among 9132 spontaneously hypertensive rats. The onset was at 72 to 145 days of age and death occurred before 423 days of age. The tumors were multiple being distributed in the skull (11 of 14 cases), caudal vertebrae (8 of 14 cases), and fore- and hind-limb. All the tumors were mainly composed of sarcomatous tissue with some osteoid and cartilage. Metastasis was never observed. Serum A1-P levels of tumor bearing animals were 2 times higher than those without tumor. Bronchiectasis and abscess formation of submaxillary lymph nodes were noted in most affected animals.     

The effects of short-term fluoride ingestion on bone formation and resorption in the rat femur

Summary The femurs from rats given 120 ppm fluoride in their drinking water for 4 weeks were examined with histological, histochemical, and radiographic methods. Blood removed from the rats prior to sacrifice was analyzed for calcium, phosphorus, and alkaline phosphatase. Results of this study indicated that the ingestion of fluoride produced wide osteoid seams on the periosteal surface of the femoral diaphysis within 4 weeks. The increase in osteoid appeared to be due to an increase in the number of osteoid-producing cells (osteoblasts) along with a subsequent delay in the mineralization of this tissue. The metabolic activity of osteoblasts did not appear to be affected since the intracellular production of acid and alkaline phosphatase was not inhibited. However, due to the high concentration of fluoride ingested, abnormal collagen deposition and a change in bone mineral may have combined to cause a delay in osteoid mineralization. Mineralization was also delayed in the distal femoral epiphyseal plate resulting in an increase in the number of hypertrophied cells. Resorption of metaphyseal trabecular bone, presumably formed prior to fluoride administration, was increased causing a reduction in the amount of trabeculae extending into the shaft of the femur. Concurrent with these changes in bone, the serum levels of calcium, phosphorus, and alkaline phosphatase remained within normal ranges.     

The differential response of cortical and trabecular bone to aluminum administration in the rat

Osteomalacia has been noted following in vivo aluminum (Al) loading in the rat by some investigators but not by others. To determine whether the response of bone to Al differs as a function of the skeletal site examined, quantitative histology of cortical and trabecular bone was done in the tibiae from control (C, n = 10), Al-treated (AL, n = 9), nephrectomized control (NX-C, n = 7), and nephrectomized Al-treated (NX-AL, n = 8) rats given 2 mg/day of Al for 4 weeks. Bone Al content was determined by histochemical methods. In cortical bone, osteoid seam width, osteoid volume, and percent osteoid area were similar for all groups. In contrast, for trabecular bone, both forming surface (means +/- SD) (5.2 +/- 3.4 vs 1.8 +/- 1.1%, P less than 0.05) and osteoid volume (1.7 +/- 0.7 vs 1.0 +/- 0.4%, P less than 0.05) increased from control values in AL, although osteoid seam width did not differ. In NX-AL, trabecular forming surface (20.2 +/- 6.7 vs 6.2 +/- 2.4%, P less than 0.01), osteoid area (13.2 +/- 5.7 vs 3.5 +/- 0.8%, P less than 0.01), and osteoid width (18.7 +/- 5.7 vs 9.7 +/- 2.3 micron, P less than 0.01) all were greater than in NX-C. Deposits of Al were undetectable in C and NX-C, were minimal in cortical bone in AL and NX-AL, but were present at 40.5 +/- 11.5 and 71.1 +/ 6.5% of trabecular surfaces in AL and NX-AL, respectively. Osteoid area and osteoid surface each correlated with trabecular bone Al. Thus, (a) osteoid accumulates in trabecular, but not in cortical, bone after 4 weeks of Al loading; (b) the extent of osteoid accumulation correlates with the bone Al content; and (c) the histologic response to Al in cortical and trabecular bone is related to local differences in the uptake of Al into bone.     

Evidence for false aneuploid peaks in flow cytometric analysis of paraffin-embedded tissue

It has recently been shown that bimodal histograms with false aneuploid peaks may be obtained by DNA flow cytometry from histologically normal tissue allowed to autolyze. To investigate if such peaks can be generated from surgically excised archival tissue, 198 paraffin blocks from 179 patients containing histologically normal spleen (n = 65), liver (n = 26), thyroid (n = 32), pancreas (n = 19), salivary gland (n = 49), or lymph node tissue (n = 7), obtained from the archives of two university pathology departments, were analyzed for nuclear DNA content. The great majority (n = 160, 83.8%) of the 191 interpretable histograms had a single symmetrical G1 peak; and 8 histograms, all produced from liver tissue had a tetraploid pattern. A slight or a prominent repeatable deviation in the G1 peak outline was present in 14 (7.3%) cases. A peak resembling an aneuploid G1 peak with a DNA index (DI) ranging from 1.14 to 1.38 was repeatedly produced from 9 (4.7%) blocks containing histologically normal or inflamed splenic (n = 3), pancreatic (n = 3), liver (n = 1), thyroid (n = 1), or lymph node (n = 1) tissue. The three abnormal peaks produced from pancreatic tissue were rounded in shape and resembled closely the ones that can be obtained from autolytic pancreatic tissue, and the six remaining extra peaks were all fused with the "diploid" peak. In conclusion, false peaks, probably caused by degradation of the nuclear contents during formalin fixation or before it, may rarely be obtained from surgical paraffin-embedded samples.     

Immunohistochemical and biochemical studies on the collagenous proteins of human osteosarcomas

The distribution of type I, II, III, IV, V and VI collagens in 20 cases of osteosarcoma was demonstrated immunohistochemically using monospecific antibodies to different collagen types. In addition, biochemical analysis was made on collagenous proteins synthesized by tumor cells in short-term cultures obtained from seven representative cases and compared with dermal fibroblasts. In osteoblastic areas, most of the tumor osteoid consisted exclusively of type I collagen. Type V collagen was associated in some of them. Type III and type VI collagens were mainly localized in the perivascular fibrous stroma. Cultured tumor cells from osteoblastic osteosarcomas produced type I collagen exclusively and small amount of type V collagen constantly, while the synthetic activity of type III collagen was extremely low. In contrast, fibroblastic areas were characterized by the codistribution of type I, III, VI collagens and chondroblastic areas by type I, V, VI collagens as well as type II. Furthermore, type IV collagen was demonstrated in the stroma, other than the basement membrane region of blood vessels, in fibroblastic, intramedullary well-differentiated and telangiectatic osteosarcomas. In vitro, the production of variable amounts of type IV collagen, which was not detected in cultured dermal fibroblasts, was also recognized in the osteoblastic, fibroblastic, undifferentiated and intramedullary well-differentiated osteosarcomas examined. These findings suggest that the immunohistochemical approach using monospecific antibodies to different collagen types is useful not only in identifying some specific organoid components, such as tumor osteoid, but also in disclosing the biological properties of osteosarcoma cells with diverse differentiation.     

Modified Tetrachrome Method for Osteoid and Defectively Mineralized Bone in Paraffin Sections

A new modification of the tetrachrome method for bone osteoid in paraffin sections has been designed. The modified tetrachrome method suitable for routine use in any histology laboratory retains the simplicity of the original method and gives good results on the freshly fixed, decalcified, paraffin embedded material. Osteoid tissue is stained deep blue and normally mineralized bone is stained red. Defectively mineralized bone stains pale blue or pink and the cellular population is clearly identifiable. The ability to distinguish the osteoid tissue from mineralized bone and connective tissue and cartilage makes diagnosis of osteomalacia or osteoid producing tumors or assessment of ossification process straightforward, without the need for un-decalcified sections. By displaying simultaneously irregularities in the mineralized matrix and morphology of bone cells, the method also permits the diagnosis of conditions recently described in patients with osteoporotic fractures, such as osteocytic degeneration and bone tissue defects.     

Three-dimensional visualization analysis of in vitro cultured bone fabricated by rat marrow mesenchymal stem cells

Marrow mesenchymal stem cells are well known for their differentiation into bone-forming osteoblasts and in vitro mineralized tissue formation. However, process details, including tissue structure and cellular environments, remain unclear. The present study demonstrates three-dimensional visualization of tissue fabricated by culturing MSCs in the presence of calcein, a fluorescent marker for bone mineralization. The 3D visualization was performed by computer-assisted confocal laser scanning microscopy and revealed that the in vitro tissue consisted of layers of a mineralized matrix with round cells in the matrix lacunae, an unmineralized matrix (osteoid), and osteoblastic cells on the osteoid surface. The findings show that the mineralization by cultured MSCs is an in vitro counterpart of in vivo bone formation and indicate that the novel technique of visualization without tissue fixation could be useful for continuous monitoring of tissue organization in an ongoing culture.     

Dropped head syndrome due to myogenic atrophy --- a case report of surgical treatment

Osteoid osteoma of the spine is a relatively rare bone-forming tumor. Pain that is worse at night and relieved by aspirin and muscle contracture are the most characteristic symptoms of spinal osteoid osteoma. Although radicular pain occasionally occurs in spinal osteoid osteoma, spinal cord and nerve root compression is absent in most cases. Although radicular pain appears to be associated with tumorous inflammation, there have been no presentations of histological findings of inflammation around the nerve root. We present here two rare cases of spinal osteoid osteoma causing radiculopathy and the first histological evidence of tumorous inflammation as a cause of radiculopathy in osteoid osteoma near the intervertebral foramen.     

Cellular and molecular properties associated with osteosarcoma cells.

Osteosarcoma cells are recognized by abnormal function that causes a primary bone tumor. Osteosarcoma cells U(2)OS and SAOS-2 were analyzed for the expression of cell surface markers. High expression was quantified for hyaloronidase receptor (CD-44) > moderate for integrins (CD-51 and -61), > and lower for selectins (CD-62). High mitotic capacity were demonstrated by gene expression (measured by RT-PCR) and the protein level (measured by FACS) for cFOS, cMYC, and cJUN. The basic definition of osteosarcoma is excessive production of pathological osteoid. Expression of mRNA for matrix genes osteocalcin, osteonectin, and biglycan was studied. Osteocalcin and osteonectin were detected in RNA from primary cultured marrow stromal, trabecular bone cells, and osteosarcoma cell lines (U(2)OS, SAOS-2). mRNA for biglycan was detected only in primary cells and MG-63 cell line and was undetectable in RNA from U(2)OS, SAOS-2 osteosarcoma cell lines and by RNA extracted from bone biopsies of osteosarcoma patients. The absence of biglycan message observed in osteosarcoma samples provides evidence for the alterations in the extra cellular matrix which result with non-mineralized osteoid produced by the osteosarcoma cells.     

Uterine adenocarcinoma in a captive sika deer

A uterine adenocarcinoma with widespread visceral implantation and lymphatic metastases is described in a 4-yr-old captive nulliparous sika deer (Cervus nippon). The tumor was histologically characterized by proliferation of irregularly shaped acini and tubules lined by single- or multi-layered pleomorphic epithelial cells, which were sometimes multinucleated and contained PAS positive mucin. The neoplastic tissue was invasive and showed a marked desmoplastic reaction. A relationship between the tumor and a hormonal imbalance could not be ruled out. This is the first report of a tumor of the reproductive tract in a cervid.     

Fine needle aspiration cytology of clear cell sarcoma. Report of a case with immunocytochemical, immunohistochemical and ultrastructural studies.

Cytologic findings of clear cell sarcoma obtained by fine needle aspiration (FNA) of a tumor are described. The tumor probably originated in the retroperitoneal tissue, and the diagnosis was confirmed histologically by open biopsy. Percutaneous needle aspirates of the intraabdominal tumor and touch preparations obtained from the open biopsy specimen revealed numerous atypical cells with an extremely hyperchromatic nucleus, prominent nucleoli and clear cytoplasm. The cytoplasm was rich in glycogen. The immunocytochemical technique demonstrated S-100 protein and neuron-specific enolase in the cytoplasm, both of which were exhibited also in the histologic specimen. Clear cell sarcoma is a rare tumor of soft tissue, and to our knowledge, detailed cytologic appearances of this tumor obtained by FNA have not been reported. In addition, the present tumor was unique in location. It is possible to diagnose clear cell sarcoma accurately on an FNA cytologic specimen if the periodic acid-Schiff stain and immunocytochemical technique are utilized in addition to the routine Papanicolaou method.     

Development of the dermal skeleton in Alligator mississippiensis (Archosauria, Crocodylia) with comments on the homology of osteoderms

The dermal skeleton (=exoskeleton) has long been recognized as a major determinant of vertebrate morphology. Until recently however, details of tissue development and diversity, particularly among amniotes, have been lacking. This investigation explores the development of the dermatocranium, gastralia, and osteoderms in the American alligator, Alligator mississippiensis. With the exception of osteoderms, elements of the dermal skeleton develop early during skeletogenesis, with most initiating ossification prior to mineralization of the endoskeleton. Characteristically, circumoral elements of the dermatocranium, including the pterygoid and dentigerous elements, are among the first to form. Unlike other axially arranged bones, gastralia develop in a caudolateral to craniomedial sequence. Osteoderms demonstrate a delayed onset of development compared with the rest of the skeleton, not appearing until well after hatching. Osteoderm development is asynchronous across the body, first forming dorsally adjacent to the cervical vertebrae; the majority of successive elements appear in caudal and lateral positions. Exclusive of osteoderms, the dermal skeleton initiates osteogenesis via intramembranous ossification. Following the establishment of skeletal condensations, some preossified spicules become engorged with many closely packed clusters of chondrocyte-like cells in a bone-like matrix. This combination of features is characteristic of chondroid bone, a tissue otherwise unreported among nonavian reptiles. No secondary cartilage was identified in any of the specimens examined. With continued growth, dermal bone (including chondroid bone) and osteoid are resorbed by multinucleated osteoclasts. However, there is no evidence that these cells contribute to the rugose pattern of bony ornamentation characteristic of the crocodylian dermatocranium. Instead, ornamentation develops as a result of localized concentrations of bone deposited by osteoblasts. Osteoderms develop in the absence of osteoblastic cells, osteoid, and periosteum; bone develops via the direct transformation of the preexisting dense irregular connective tissue. This mode of bone formation is identified as metaplasia. Importantly, it is also demonstrated that osteoderms are not histologically uniform but involve a range of tissues including calcified and uncalcified dense irregular connective tissue. Between taxa, not all osteoderms develop by homologous processes. However, it is concluded that all osteoderms may share a deep homology, connected by the structural and skeletogenic properties of the dermis.     

TUMOR ANGIOGENESIS : Rapid Induction of Endothelial Mitoses Demonstrated by Autoradiography

Walker ascites tumor cells and an extract derived from such cells (tumor angiogenesis factor, TAF) were injected into the subcutaneous tissue of rats by using a dorsal air sac technique. At intervals thereafter, thymidine-³H was injected into the air sac and the tissues were examined by autoradiography and electron microscopy. Autoradiographs of 1µ thick Epon sections showed thymidine-³H labeling in endothelial cells of small vessels 1–3 mm from the site of implantation, as early as 6–8 hr after exposure to live tumor cells At this time interval endothelial cells appeared histologically normal. DNA synthesis by endothelium subsequently increased and within 48 hr new blood vessel formation was detected. The presence of thymidine-³H-labeled endothelial nuclei, endothelial mitoses, and regenerating-type endothelium was confirmed by electron microscopy. TAF also induced neovascularization and endothelial cell DNA synthesis after 48 hr. A similar response was not evoked in saline controls. Formic acid, which elicited a more intense inflammatory response, was associated with less endothelial labeling and neovascularization at the times studied. Pericytes and other connective tissue cells were also stimulated by live tumor cells and TAF. The mechanism of new blood vessel formation induced by tumors is still unknown but our findings argue against cytoplasmic contact or nonspecific inflammation as prerequisites for tumor angiogenesis.     

Impact de l’imagerie hybride TEMP/TDM dans le diagnostic et la prise en charge de l’ostéome ostéoïde

IntroductionThe osteoid osteoma is a major bone benign tumors in children. It is more common in boys. The femoral and tibial locations account for 50% of cases. Clinically, it is revealed by nocturnal pain relieved by aspirin. The pain may precede by several months the radiographic abnormality. The purpose of our work is to elucidate the contribution of SPECT/CT in addition to the planar bone scintigraphy in the diagnosis and treatment of osteoid osteoma about two cases.Case reportClinical case No. 1: a 10-year-old child who presented a limp nocturnal pain in the left hip evolving for 4 months. The radiograph of the pelvis showed bone condensation subtrochanteric left femur. SPECT/CT showed an image for an osteoid osteoma. Surgical resection of the home revealed in the histological study an osteoid osteoma of the left femoral neck. The evolution was marked by an immediate pain relief and full recovery of the left lower limb mobility. Clinical case No. 2: an 11-year-old child who had a limp nocturnal pain at the upper end of the left femur evolving for 2 months. The pain was paroxysmal, relieved by salicylates. The X-ray of the pelvis showed a metaphyseal image with peripheral condensation and thickening of the cortex. SPECT/CT showed an image for an osteoid osteoma. The intervention was a tumor excision resection. Histopathological examination revealed a small nidus consistent with an osteoid osteoma. The evolution was marked by an immediate pain relief and normalization of the mobility of the left lower limb.DiscussionThe SPECT/CT can increase the sensitivity and specificity of planar bone scintigraphy. It confirms the location of osteoid osteoma and defines its anatomical relationships in order to optimize surgical management.ConclusionThe SPECT/CT contributes significantly to the diagnosis of osteoid osteoma when radiological images are atypical or unusual clinical expression.     

Bone histomorphometry: observations in cases of renal osteodystrophy

The AA report a histomorphometric study performed by measuring square areas and volumes of bone tissue. Furthermore, in 7 patients with osteodystrophy secondary to chronic renal failure, the AA investigated the amount of osteoid tissue and the presence of osteoblasts and osteoclasts. They noted signs of secondary hyperparathyroidism with increase of osteoclastic activity and fibrous metaplasia of the bone marrow associated with osteomalacia. The same patients were examined after treatment with, Vit D3 active preparations separately administered: and the AA especially noted decrease of the osteoclastic reabsorption and of the volume of osteoid tissue after the treatment with 1,25 (OH)2 D3 more than with 25 OH D3.     

Effect of aspartate and glutamate on experimental myocardial infarction in rats

Cardiac necrosis was produced in rats by administering isoproterenol sulphate (85 mg/kg, sc for 4 days). The myocardial damage was proved by observing the elevated levels of serum aspartate amino-transferase, lactate dehydrogenase and creatine phosphokinase and the changes were confirmed by histopathology of the tissue. Both aspartate and glutamate (100 mg/kg, ip) significantly reduced the elevated levels of these enzymes. The average degree of cardiac necrosis produced in these rats when observed macroscopically and histologically was also found to be significantly reduced on pretreatment with aspartate and glutamate.     

Quantitative analysis of heterogeneity of estrogen binding in human breast tumor specimens

A quantitative assessment was made of the heterogeneity of estrogen binding among histologically distinct regions and among nuclei within histologically uniform regions in ten human breast tumors. An autoradiographic method was used that employed sections from fixed tissues and required exposure times of one to two weeks. Grain counting of autoradiograms probably indicated both type I and type II estrogen binding. There were significant decreases in estrogen binding in all specimens after competition with excess diethylstilbestrol, suggesting that a component of binding was specific for estrogen. Significant differences in amount and distribution (nuclear vs cytoplasmic) of estrogen binding were seen not only among tissues with different histologies but also within histologically identical regions of a tissue. In histologically uniform regions, wide ranges of values for nuclear estrogen binding were seen. This method allows for the rapid evaluation of estrogen-binding profiles of tumor specimens and has potential for analyzing the process of neoplastic transformation in mammary epithelia as it affects, or is affected by estrogen binding.     

小鼠体内转染CH50多肽真核表达载体趋化免疫细胞在肿瘤治疗中的作用

目的 研究体内转染表达CH50多肽对免疫细胞的趋化,聚集作用,探讨趋化聚集的免疫细胞增强抗肿瘤以及化疗后抗肿瘤的免疫力。方法 分别在小鼠后肢肌肉,接种肿瘤组织或化疗的肿瘤组织内注射pCH510质粒进行转染,取转染部位组织制备石蜡切片,观察免疫细胞的聚集情况,并观察转染pCH510及联合化疗对肿瘤的治疗作用。结果 正常小鼠后肢肌肉转染pCH510后,注射部位有大量免疫细胞聚集,其中主要是巨噬细胞,淋巴细胞,中性粒细胞,聚集的免疫细胞至少持续存在一周,肿瘤部位转染后,趋化聚集的免疫细胞分布于肿瘤组织周围,抑制了肿瘤细胞的生长,化疗后转染,仍有趋化聚集免疫细胞的作用。免疫细胞聚集数量相对只转染pCH510虽有所减少,但对肿瘤的抑制作用明显增强。结论 本文结果提示,在小鼠体内转染pCH510表达的CH50多肽不仅能够趋化免疫细胞,提高局部抗肿瘤的免疫力,而且与化疗联合作用在肿瘤治疗中具有更大的研究潜力。