Helen E. Gruber

Adaptations of Goldner's Masson Trichrome Stain for the Study of Undecalcified Plastic Embedded Bone

Specialized adaptations for application of Goldners Masson trichrome stain to plastic embedded undecalcified bone specimens are presented. This stain can be used successfully on methyl-glycol methacrylate, glycol methacrylate and Spurr embedded bones. The stain affords the advantage of good cellular staining due to the hematoxylin component with concomitant sharp discrimination of mature bone matrix which stains green, immature new bone matrix which stains red, and calcified cartilage which stains very pale green. Use of red filters during photomicrography aids in bone-osteoid discrimination in black and white photographs.

Perinatal lethal hypophosphatasia; Clinical, radiologic and morphologic findings

Clinical, radiographic and morphologic analysis of nineteen cases of perinatal (lethal) hypophosphatasia was performed. Three families each had two affected offspring. All of the patients had lethal short limb dwarfism with very soft calvaria. Other clinical findings included polyhydramnios, blue sclerae and spurs in the mid-portion of the forearms and lower legs. Considerable variability was found in the skeletal radiographs. In addition to the well known radiographic features such as generalized decrease in the size of ossified bones with some bones not ossified at all, other changes observed included: 1) marked variability in the amount of bone ossification; 2) variability between patients as to which bones were most severely affected; 3) unusually dense, round, flattened, butterfly shaped; and saggitally clefted vertebral bodies; 4) variability in femoral shape including “chromosome” like, “campomelic” like, and shortening with or without metaphyseal cupping or irregularities; 5) osteochondral projections (Bowdler spurs) of the midshaft of the fibula and ulna. Recognition of the marked clinical and radiographic variability in this autosomal recessive lethal skeletal dysplasia is important for accurate genetic counseling and prenatal diagnosis.

A large family with features of pseudoachondroplasia and multiple epiphyseal dysplasia: exclusion of seven candidate gene loci that encode proteins of the cartilage extracellular matrix.

We have identified a large family with a dominantly inherited chondrodysplasia characterized by a waddling gait, short limbs, and early onset osteoarthritis. The radiographic presentation resembles pseudoachondroplasia in childhood and multiple epiphyseal dysplasia in adults. Electron microscopic examination of cartilage reveals accumulation of material within the rough endoplasmic reticulum similar to that seen in pseudoachondroplasia and the Fairbank type of multiple epiphyseal dysplasia. By linkage analysis, we have excluded the genes for aggrecan, decorin, hexabrachion (tenascin), type II procollagen, the α1 chain of type XI procollagen, the α1 chain of type IX procollagen, and link protein, candidate genes that encode structural components of the cartilage extracellular matrix, as the disease locus for this disorder.

Bone and the Immune System

Summary There are several lines of evidence which provide support for an important relationship between immune cells and bone. Clinical studies of immunodeficiency syndromes have shown that abnormalities in bone shape are evident on x-rays, and peculiarities in the structure of the growth plate have been identified by histopathology. Studies of bone histology, and quantitation of cellular abnormalities, are scarce. Abnormalities in bone turnover, have, however, been identified in the nude mouse model. Many lines of evidence derived from in vitro bone studies have shown that lymphokines and monokines can influence bone formation and bone resorption. Some clinical studies of postmenopausal osteoporosis have indicated the possible presence of immune cell changes in this condition. Although several hypotheses have been formed regarding the exact mechanisms of the effect of immune cytokine on bone, this is clearly a very large area of study and there is a need for additional carefully controlled experiments with special emphasis on bone cells and bone matrix, especially in the human. As knowledge progresses regarding immunology and hematology, a clearer understanding of the lineages of the osteoblast and osteoclast will emerge and we will better understand how specialized bone cells interact with and react to their immune cell neighbors in the bone marrow and to immune system signals. These findings will have especially important implications for the local bone loss seen in rheumatoid arthritis, periodontal disease, and chronic osteomyelitis (38).

Improvements in Dehydration and Cement Line Staining for Methacrylate Embedded Human Bone Biopsies

Undemineralized methacrylate embedded bone biopsies and other bone specimens can be processed much more rapidly by application of acidified 2,2-dimethoxypropane (DMP) dehydration, which requires two hours, than by traditional graded ethanol dehydration, which requires at least four days. This shortened processing time is valuable when biopsy results are urgently needed to detect osteomalacia or to determine bone features prior to possible parathyroidectomy. We have processed over 200 bone specimens with DMP and have compared DMP dehydration to graded ethanol dehydration in 11 biopsies in which two plugs were available from the same patient. DMP dehydration does not compromise the following: tetracycline retention, Goldners stain, acid phosphatase localization or histochemical identification of aluminum. Cement lines, which provide a record of past remodelling, are useful in clinical interpretation of bone biopsies. We have adapted two stains, toluidine blue and methylene blue/basic fuchsin, for improved cement line identification. Five-micrometer sections individually demineralized in acetate buffer prior to cement line staining show best results with toluidine blue at pH 5.5 and with methylene blue/basic fuchsin at pH 2.5-3.5.

Quantitative Histology of Cartilage Cell Columns in the Human Costochondral Junction: Findings in Newborn and Pediatric Subjects

ABSTRACT: The mean number of cells per cartilage column and the proportion of hypertrophic and proliferative chondrocytes per column were determined in the costochondral junction in a population of normal subjects including 10 fetal-newborns and 15 subjects aged 0.3-16 y of age. Both the mean number of cells per column and the proportion of proliferative cells per column were significantly greater in the fetal-newborn population compared to the pediatric population (12.6 ± 1.0 (10) versus 8.4 ± 0.4 (15), p<0.001 and 39.6 ± 6.9 (10) versus 24.4 ± 2.5 (15), p=0.025, respectively) (mean ± sem [n]). The number of cells per column bore a significant negative relationship to subject age (r=—0.52, p=0.007). Significant positive correlations were found between the mean number of cells per column and age-specific growth velocity both in males (length-height velocity=[(6.3) (mean number of cells) — 44.1], r=0.72, p=0.02) and in females (length-height velocity=[(3.4 (mean number of cells) — 14.4], r=0.77, p=0.006). These data will provide normative values against which abnormalities characteristic of the skeletal dysplasias can be compared.

Application of Stains-All for Demarcation of Cement Lines in Methacrylate Embedded Bone

Cement lines provide a record of sites of past remodeling buried in the matrix of bone. A method is reported for application of Stains-all, a cationic carbocyanine dye, for demarcation of cement lines in bone. The method, which is simple, works well for both glycol methacrylate and methyl methacrylate undemineralized embedments and produces good concomitant staining of cytoplasm and nuclei of osteoblasts, osteoclasts and marrow cells.

Ultrastructural abnormalities in bone and calcifying cartilage in two siblings with a newly described recessive lethal chondrodysplasia.

Ultrastructural abnormalities in bone and calcifying cartilage are presented for a recently identified lethal chondrodysplasia. Two siblings, aged 20 and 30 weeks of gestation, showed severe short-limb dwarfism and histologically distinct, highly disorganized masses of cartilage, bone, and mesenchymal tissue in the long bones. Regions of inappropriate cartilage calcification showed unusual, electron-dense, amorphous islands of mineralization and larger, less dense, layered calcified masses that occasionally entrapped chondrocytes. Bone abnormalities included abnormal cartilage-bone transition at the growth plate, general bone matrix disorganization due to irregularly oriented bundles of collagen, mineral crystals on the osteocyte lacunar rim, and accumulations of thickened collagen fibrils, also along the osteocyte lacunar rim. These findings point to abnormal calcification and mineralization distinct from those seen in other reported skeletal dysplasias. These abnormalities are associated with an anarchic distribution of mesenchymelike tissue infiltrating the cartilage and bone.

In vitro tetracycline labelling and bone cell survival in human trabecular bone explants

Results are presented which document the feasibility and utility of human trabecular bone explants for in vitro bone research. This approach is advantageous because it allows bone cells to maintain their natural orientation on the endosteal surface, and keeps surrounding marrow intact--thus retaining the immediate local microenvironment. In addition, bone cells are not exposed to transient, but sometimes harsh, isolation procedures. Data are presented which show greater initial bone cell numbers from younger donors and the change in these numbers over culture periods up to 21 days. A technique is presented which achieves single and double tetracycline label incorporation into the mineralizing front of bone explants in vitro using a pulse-chase methodology.

Ultrastructural alterations and retention of the C-propeptide of type II collagen in human chondrocytes exposed in vitro to brefeldin A

Normal human chondrocytes grown in vitro were exposed to 10 micrograms/ml Brefeldin A (BFA) for 24 h, 1 microgram/ml for 4 h, or 0.1 microgram/ml for 4 h and evaluated for ultrastructural alterations. BFA in the amount of 0.1 microgram/ml resulted in vacuolization, disappearance of the Golgi, and moderate increases in rough endoplasmic reticulum (rER) vesicles. After 1 microgram/ml BFA exposure large interconnected cisternae were identified. BFA treatment of 10 micrograms/ml was associated with large dilated ER cisternae which contained material of variable electron densities. Immunocytochemical localization showed markedly increased type II procollagen intracellular retention in BFA-treated cells. High dose BFA-treated cells showed ultrastructural similarities to those seen in the skeletal dysplasia hypochondrogenesis. Results presented here show that in vitro culture of normal human chondrocytes results in retention of the C-propeptide of type II collagen and marked alterations in cytoplasmic ultrastructure.