Preliminary in situ identification of estrogen target cells in bone.

Although estrogens profoundly influence skeletal growth and maturation, their mechanism of action is still unclear. To identify their target cells in bone, estrogen receptors were located by immunofluorescence using the H222 monoclonal antibody in cryosections (both undecalcified and briefly decalcified) of hyperplastic mandibular condyle (persistent asymmetric mandibular growth) from a 14-year-old girl and radius and ulna from an 18-month-old female pig (epiphyseal fusion) and from a 3-month-old guinea pig (epiphyses open). Bone was removed from the animals at the peak of estrus. The most striking feature in all three species was the high proportion (approximately 50%) of receptor positive osteocytes. Although all sections contained active bone-forming surfaces, we were unable to identify clearly osteoblasts or lining cells that were estrogen receptor positive. In pig bone only, distinctive groups of receptor positive chondrocytes, with a pericellular localization of collagen type 1, were detected above the growth plate but below secondary centers of ossification. This observation suggests that osteocytes are major skeletal estrogen target cells and may be involved in coordinating the response of surface bone cells to the hormone, and further that chondrocytes may be involved in estrogen-induced epiphyseal growth plate fusion.

Evidence for an extensive collagen type III/VI proximal domain in the rat femur. I. Diminution with ovariectomy.

Collagenous proteins other than Type I have received little attention in hypogonadal bone loss. Using femora from 25 young (2.5 months) and older (11 months) control and ovariectomized adult rats killed 1-4 months postoperation, cancellous atrophy was histologically confirmed, and the immunolocalization of collagen Type III was examined. This occurred as numerous immunofluorescent Sharpey-like fibers, 5-25 microm thick, regularly associated with collagen Type VI, which ramified the femoral cortex. Sequential transverse cryosections enabled the mapping of the fibers in three-dimensions, demonstrating that they constituted an extensive subperiosteal domain which may be a lasting legacy of early skeletal development. Fiber density was greatest in the trochanters and femoral neck. The domain tapered distally and was apparently anchored into the mid-shaft by intracortical cartilaginous islands, staining for collagen Type VI (as well as Type II and fibronectin). Ovariectomy caused disconnection of the fibers and reduced the proximal domain of both young and older animals, previously positive areas of the cortex becoming negative. It is concluded that collagen Type III/VI occupies a substantial, discrete domain in the rat proximal femur as a complex extension of the periosteum. Diminution of this cortical domain with trabecular atrophy suggests that it has a proactive or reactive role in determining bone mass and strength by facilitating musculoskeletal exchange in a form that is disengaged by ovariectomy.

Effect of deproteination on bone mineral morphology: implications for biomaterials and aging.

Bone mineral morphology is altered by processing and this is rarely considered when preparing bone as a bioimplant material. To examine the degree of transformation, a commercial, coarsely particulate bone mineral biomaterial produced by prolonged deproteination, defatting, dehydration, and heating (donor material) was compared with similar particles of human bone (recipient material) prepared optimally by low-temperature milling. The two powders were freeze-substituted and embedded without thawing in Lowicryl K4M before sectioning for transmission electron microscopy (TEM) (other aliquots were processed by traditional TEM methods). To maximize resolution, electron micrographs were image-enhanced by digitization and printed as negatives using a Polaroid Sprint Scan 45. In addition to their morphology, the particles were examined for antigenicity (specific by reference to fluorescein isothiocyanate [FITC]-conjugated fibronectin, and nonspecific by reference to general FITC-conjugated immunoglobulins). Results showed that the optimally prepared human bone fragments stained discretely for fibronectin with negligible background autofluorescence. In contrast, the bioimplant fragments stained extensively with this and any other FITC-conjugated antibody and, unlike fresh bone, it also autofluoresced a uniform yellow. This difference was also expressed structurally and, although the bioimplant mineral consisted of rhomboidal plates up to 200 nm across and 10 nm thick, the optimally prepared bone mineral was composed of numerous clusters of 5-nm-wide sinuous calcified filaments of variable density and indeterminate length (which became straight needles 50 nm long and 5 nm thick following traditional chemical TEM fixation/staining). It was concluded that the inorganic phase of bone is both morphologically and immunologically transmutable and that, in biomaterials, the transformation is apparently so great that a broad indigenous antigenicity is unmasked, increasing the likelihood of resorption or rejection. This marked change may also provide preliminary insight into a more modest natural aging phenomenon with the localized lateral fusion of calcified filaments into less flexible, more immunologically reactive fenestrated plates.

Evidence for an extensive collagen type III proximal domain in the rat femur. II. Expansion with exercise.

Exercise in youth may affect bone "quality" as well as quantity. Using the rat model, 1.5-month-old females were divided into four weight-matched groups, exercised short-term (6 weeks, E(s), n = 20) and long-term (14 weeks, E(L), n = 10) by access to monitored running wheels, and corresponding "sedentary" controls (S(S) short-term, n = 20; S(L) long-term, n = 10). Femora were either plastic-embedded or fresh-frozen. Transverse histological slices, 100 microm thick, were cut midshaft, while similar cryosections, 8 microm thick, were prepared from the same site and also coronal to the femoral neck region. An image analyser measured femoral neck and midshaft microarchitecture, while immunostaining localized collagen type III-rich fibres (CIII, an index of Sharpey fibre insertions) and osteopontin-rich osteons (OPN, an index of remodelling). Exercise increased cortical bone (proximal width +18%, midshaft area +7%). It also raised cancellous bone volume (+25%) by trabecular thickening (+30%) with more intraosseous vascularity and new trabecular interconnections (node-terminus ratio, +57%; trabecular pattern factor, -147%; marrow star volume. -48%). In the cortex a prominent discrete subperiosteal domain became wider (+50% midshaft) with exercise and contained more numerous (+15%) CIII-stained fibres. In contrast the encircled inner bone developed more numerous (+14%) OPN-rich osteons. It is concluded that short-term voluntary exercise augments both cortical and cancellous microarchitecture. It also alters protein composition, such that expanding arrays of Sharpey's fibres within a circumferential proximal domain (Part I) interconnect more powerfully with the musculature and interface more robustly with the core bone that in response becomes more vascular and biodynamic, providing further insight into how muscle mass may be skeletally translated.

Rapid preparation of fresh-frozen undecalcified bone for histological and histochemical analysis.

An increasing need to be able to relate hard tissue morphology to its histochemistry, and the lack of an adequate methodology, prompted this investigation of polyvinylpyrrolidones of varying chain length as supporting films for the cryomicrotomy of fresh undecalcified cortical and trabecular bone. We developed a method that parallels histological procedures but is biochemically inert and functions at -25 degrees C. Using an LKB heavy-duty cryomicrotome, large numbers of serial sections suitable for histochemistry, immunocytochemistry, or rapid histology can be prepared with ease. Sections from mouse skull, ox vertebra, and human iliac crest illustrate the versatility of the method, and have been examined for the preservation of general morphology and of histochemical activity of those enzymes most prominent in remodeling. The procedure does not preclude the subsequent application of standard hard-tissue embedding methods to the remaining material. Comparison with plastic-embedded bone shows not only close similarities in the structural preservation of the frozen tissue, making possible histomorphometry, but also shows some interesting staining differences. These include the absence of differentiation of the calcification front with toluidine blue stain in fresh frozen specimens, and the presence in frozen osteoid tissue of specific methylene blue metachromasia missing from plastic preparations.

Immunolocalization of collagen types I and III, tenascin, and fibronectin in intramembranous bone.

Structural components of the organic bone matrix were located by immunohistochemical techniques in fresh-frozen sections of normal and dysplastic bone. Fine and coarse birefringent fibers were identified as separate and distinctive features in the extracellular matrix by antibodies raised against human collagen Type III. The glycoprotein tenascin was located on a proportion of the fibers in a characteristic beaded pattern, which was absent in dysplastic bone. The fibers originated in the periosteum or in the fibrous stroma of the marrow cavity and were oriented with regard to both the spatial and the lamellar organization of the bone. The disposition and composition of the fibers suggests that they form a preliminary framework on which intramembranous bone modeling proceeds, and that the specific location of tenascin on the fibers in normal developing membrane bone may be important in determining the alignment of the bone tissue. Epitopes recognized by the collagen Type I and fibronectin antibodies were demonstrated throughout the mineralized matrix, but their incorporation into the collagen "Type III" fibers was evident only outside the mineralized matrix.

Role of exchanged ions in the integration of ionomeric (glass polyalkenoate) bone substitutes.

Ionomeric (glass polyalkenoate) implants are synthetic materials which can be used for repairing bone defects. It has been suggested that ions are leached from these implants during healing and that they influence cellular activity in the surrounding tissues. Morphological, immunohistochemical and microanalytical techniques were used to compare the osteogenic capacity of implants which eluted aluminium ions with implants which did not elute aluminium ions. The extracellular matrix molecules fibronectin and tenascin were located upon the surface of both implanted materials. Thick seams of lamellar bone were apposed to implants containing labile aluminium ions, but the bone was poorly mineralized. At the same time, transient increases were apparent in osteoblast activity on periosteal and endosteal surfaces and in chondrocyte activity in the growth plate and articular cartilages. In contrast, small amounts of mineralized lamellar bone were apposed to substituted implants (without aluminium) and the growth plate and articular cartilages remained normal in thickness and morphology. These results suggest that exchanged ions can influence the amount and quality of bone apposed to the implant. They also suggest that the effect of the ions depends upon their concentration and the state of differentiation of osteogenic cells.

Trabecular generation de novo. A morphological and immunohistochemical study of primary ossification in the human femoral anlagen.

An understanding of trabecular formation in early skeletal development may provide insight into the problem of trabecular replacement in the aging skeleton. In an optical and scanning electron microscope study of the processes of de novo trabecular generation, the immunohistochemical distribution of collagen Types I, II and III, together with the matrix organising proteins fibronectin and tenascin, has been examined in the ossifying human femoral anlage. In the region of the developing spongiosa, the primary osseous trabeculae that arose by endochondral ossification were assembled around calcified cartilage remnants, consisting almost entirely of aggregates of mineralised microspheres. These structures were specifically recognised by antibodies raised against collagen Type II and fibronectin. In contrast, the primary osseous trabeculae that arose by subperiosteal intramembranous processes, were assembled around a framework of prominent coarse fibres that were recognised by antibodies raised against collagen Type III and tenascin. Irrespective of their origin, all the new trabeculae were similar in their general staining character for collagen Type I and fibronectin. However, throughout the developmental stages examined here endochondral trabeculae were separated from intramembranous trabeculae by a discrete boundary of compressed cells and mineralised cartilage.

The fibrous architecture of the rat periodontal ligament in cryosections examined by scanning electron microscopy.

Cryosections through the incisor and molar teeth of the rat mandible were examined, with and without hyaluronidase pretreatment, in the scanning electron microscope. In the fully erupted molar teeth the fibres of the periodontal ligament were organized into bundles which crossed the space from the alveolus to the cementum and inserted into the associated mineralized tissues. In the erupting incisor teeth, three distinct zones were evident. The outer alveolar and cemental zones were composed of coarse fibre bundles which inserted into the adjacent mineralized tissues, while the middle zone was composed of collagenous laminates running along the axis of the tooth. These observations confirm a proposed model for the structure of the erupting periodontal ligament and suggest that the method used will provide further information about the role of the ligament in tooth support and eruption.

Patterns of integrin expression in a human mandibular explant model of osteoblast differentiation.

Cell-matrix interaction is crucial in regulating osteoblast differentiation and function. These interactions are themselves regulated, at least in part, by integrins. Although there are some data from mammalian models, few studies have compared integrin expression at different stages of the osteoblast lineage. Here, primary human mandibular osteoblast cultures were grown in the presence of epidermal growth factor (EGF), giving a proliferative, less differentiated phenotype, or of vitamin D(3) and hydrocortisone (D+Hc), giving a more differentiated phenotype. These cultures were compared with those of cells prepared in the absence of EGF or D+Hc by fluorescence-activated cell sorter using a panel of monoclonal antibodies to specific integrin heterodimers. To provide in vivo correlation, the same panel of antibodies was used to stain fresh-frozen, undemineralised sections of human mandibular bone. Under baseline conditions the alpha(3), alpha(5), alpha(v), alpha(v)beta(3), beta(3) and beta(1) integrin subunits were expressed strongly by the cells, with low-level expression of the alpha(1), alpha(2) and alpha(4) subunits. In the presence of EGF there was increased alpha(2) expression. With D+Hc, alpha(3) and alpha(5) expression was elevated. Immunohistochemical analysis demonstrated alpha(2), alpha(3), alpha(5), alpha(v)beta(3), beta(1) and beta(3) subunits in cells of the osteoblast lineage; alpha(2) staining was restricted to cells close to the bone surface whilst alpha(v)beta(3) and beta(3) were most frequently localised in the osteocytes. The results provide evidence that cells at successive stages of the osteoblast lineage show different patterns of integrin expression. These integrins may be important in cell-matrix interactions leading to osteoblast differentiation.

Histopathological and scintigraphic features of condylar hyperplasia.

This investigation was undertaken to correlate the scintigraphic and histological features of condylar hyperplasia to identify consistent diagnostic findings. A series of 34 surgically excised condyles were examined from a 5 year period. Of these, 20 were diagnosed clinically and histologically as condylar hyperplasia. In 18 of these the presentation was one of increasing facial asymmetry. In all 20 cases there was an increased uptake of Technetium 99 as determined by gamma scintigraphy. The thickness of the fibrous articular layer, undifferentiated germinal mesenchyme layer and the hyperplastic cartilage layer were measured using an eyepiece graticule and the presence and frequency of islands of cartilage in the subchondral bone were noted. 7 patients received tetracycline hydrochloride 14 and 4 days pre-operatively in an attempt to quantify the calcification rate. An uninterrupted layer of undifferentiated germinal mesenchyme is a consistent feature of condylar hyperplasia. An increased uptake on scintigraphy is proportionally related to the thickness of the hypertrophic cartilage and not only to the presence but also the frequency of cartilage islands in the subchondral bone.

Re-expression of procollagen type IIA in oral squamous cell carcinoma.

There are two forms of procollagen type II (IIA and IIB), both of which are expressed during chondrogenesis. Procollagen type IIA also is present at sites of developmental epithelial-mesenchymal interaction. Malignant transformation is associated with disturbed epithelial-mesenchymal interaction and the reappearance of fetal characteristics. This study aims to determine whether or not procollagen type IIA is re-expressed in oral squamous cell carcinoma (OSCC). Immunoperoxidase techniques were applied to frozen and paraffin sections of OSCC (n = 30) and normal oral mucosa (n = 5). In the carcinoma group, strong cytoplasmic staining for collagen type II was present (25/30). Staining was weak or absent in the stroma, and absent from the normal oral mucosa. Frozen sections from 10 of the carcinoma cases which showed positive staining were incubated with antibodies specific for procollagen type IIA and visualised using immunofluorescence. Staining was evident in each case and was particularly strong in the region of the basement membrane. Slot-blot analysis of collagen extracts from OSCC supported the immunohistochemical findings. We conclude, therefore, that procollagen type IIA is re-expressed in OSCC.

A survey of oral implantology teaching in the university dental hospitals and schools of the United Kingdom and Eire.

AIM: To provide an overview of the currently available academic teaching and clinical training in oral implantology at the university dental schools and hospitals of the United Kingdom and Eire. METHOD: A questionnaire was sent to the dean or director of dental studies and forwarded to the respective units involved in the academic teaching and clinical training of oral implantology. The setting was the university dental hospitals, and dental schools of the UK and Eire. Information was collected between July 1997 and March 1999. The main outcome measures were course availability, duration and emphasis for undergraduate and postgraduate study in the clinical discipline of oral implantology. The units or departments responsible for training and teaching were identified and formal degree courses were distinguished from non-degree courses. RESULTS: All institutions replied to the survey. All university dental schools provide undergraduate training in oral implantology in accordance with the guidelines provided by the General Dental Council. However, the courses vary with regard to the departments involved and the level of student participation. Thirteen centres provide informal postgraduate training with the duration ranging from one to eighteen days. Just eight centres provide formal academic graduate training based on oral implantology leading to recognised degrees. CONCLUSION: All university dental schools provide undergraduate teaching in oral implantology. Most centres also provide informal postgraduate training based on oral implantology. However, opportunities for academic graduate training, leading to recognised qualifications in this subject, appear limited at present.

Histomorphometry of fresh frozen iliac crest bone biopsies.

Although methods are well established for the rapid histological preparation of fresh frozen soft tissues, they remain inadequate for the preservation of hard tissues. A recent technique for sectioning fresh frozen bone, in which sections can be prepared within an hour, has been applied to undecalcified human iliac crest bone biopsies. Quantitative analysis has shown that the static remodeling variables deduced from frozen sections closely resemble those derived from plastic-embedded sections prepared for routine use by established laboratory procedures. It is concluded that the rapid method is reliable for immediate diagnostic and histomorphometric purposes.

Cancellous bone resorption in the proximal ilium of the ovariectomized rat.

Bone histomorphometry was performed on the proximal ilium of mature Sprague-Dawley rats following ovariectomy, and these rats were compared with sham-operated controls. Bone volume per unit tissue volume (BV/TV), osteoid surface, and the depth and extent of eroded cavities were measured in animals killed at intervals after operation. The rate of bone loss and the mean osteoid surface in the proximal ilium of the ovariectomized rats was significantly greater than that of the control rats over a 210-day postoperative period. The eroded surface and mean trabecular thickness in the proximal ilium of the ovariectomized rats were not significantly different from that of the control rats, and therefore failed to explain the difference in the rate of bone loss. The distribution of the depths of trabecular eroded cavities in the ilium of ovariectomized rats was different from that in the control rats. The loss of trabecular bone mass in the proximal ilium of ovariectomized, mature rats appeared due to increased activity of individual osteoclasts, rather than to increased osteoclast numbers and thinning of trabeculae.

The ultrastructure of slam-frozen bone mineral.

Bone salt may be altered by preparative procedures. 'Slam' freezing can usefully be applied to bone mineral because it minimizes preparation and preserves the tissue chemistry. The structure and composition of the mineral in 'slam'-frozen neonatal mouse calvaria (which require neither previous slicing nor manipulation) was examined by transmission electron microscopy and X-ray microanalysis in unstained sections, 0.25 micron thick (i.e. unusually thick). Comparison was made with fresh intact calvaria and with 'snap'-frozen histological sections of mature rat femora. Under the optical microscope, calcified microspheres, up to 1 micron in diameter, were evident within 'young' osteocytes and within the extracellular matrix of both immature and mature, unstained and von Kossa-stained bone. In the electron microscope, microspheres of similar dimension and distribution were observed after 'slam' freezing and were divided into two groups. One group, found inside and outside cells, had a substructure of closely packed, electron-dense, rounded bodies 30-40 nm in diameter; despite their unusual stability in EDTA, X-ray microanalysis indicated high levels of both calcium and phosphorus. The other group was found at the calcification front and, although similar to the first group in size and chemical composition, these microspheres had a substructure of clusters of 5-nm-thick electron-dense filaments containing mineral that was characteristically EDTA labile. The 30- to 40-nm dense bodies did not appear to be mitochondrial and were absent from customary fixed and resin-embedded, ultrathin, stained preparations. They were not observed singly and their aggregation into arrangements of microspheres, sometimes linked by bridges, may be an important preliminary step in the development of the filamentous clusters. Needle-shaped and plate-like crystals of bone mineral were absent. It was concluded that 'slam' freezing preserves both intracellular and extracellular bone salt in the form of microspheres within which the mineral may modulate from dense bodies into filamentous arrays of variable density with maturity.

The cryomicrotomy of rat dental tissues: a technique for histological and immunohistochemical analysis.

We have applied a method developed for the cryomicrotomy of non-decalcified bone to the histological preparation of the tooth and related dental tissues. Cryosections of rat mandible have been cut on a heavy-duty freezing microtome. Both cellular and extracellular structure was well preserved and the sections of tooth and bone appeared to be suitable for optical and scanning electron microscopy and for immunohistochemical analysis. However, there was an overall strong non-specific binding of immunohistochemical reagents to enamel which was not evident in the other mineralized tissues of the mandible. This may relate to important differences in the nature of this tissue.

A survey of clinical members of the Association of Dental Implantology in the United Kingdom. Part III. The use of augmentation techniques in dental implant surgery.

The aims of the survey were to: (1) determine the use of the staged and simultaneous augmentation techniques; (2) determine trends in the use of barrier membranes; (3) establish the perceived reliability of techniques used to monitor implants that have undergone simultaneous augmentation; and (4) assess the use of biopsy techniques to confirm the histologic outcome of bone augmentation. One hundred seventy-two respondents replied to this section of the survey and indicated that the "staged" and "simultaneous" augmentation techniques were used in roughly equal numbers during 1997, and a wide range of complications was reported with the latter. The majority used barrier membranes to correct defects of between 5 and 10 mm3, and resorbable membranes were preferred. With regard to clinical techniques used to monitor augmented implants, these were mainly considered to be "adequate" or "poor." Tissue biopsy was recognized as an important tool for determining the outcome of augmentation procedures but was rarely used. The use of resorbable membranes is likely to increase. The diagnostic tools currently used to monitor augmented implants are considered to have limited reliability, and they should be evaluated by prospective, comparative studies. More widespread use of biopsy techniques might help establish an evidence base for the histologic outcome of augmentation materials and techniques.

Survey of clinical members of the association of dental implantology in the United Kingdom: Part I. Levels of activity and experience in oral implantology.

The aims of this survey were to 1) determine recruitment rates of active oral implantologists, 2) establish the proportion of participants who carry out the surgical aspects of implantology, 3) quantify levels of surgical activity, 4) determine the type of qualifications held by this sample, and 5) identify the location of implant activity of clinical members of the Association of Dental Implantology (UK). Questionnaires were mailed to the 408 members of the ADI registered as clinical members of the ADI; data were collected between July 1998 and May 1999. A response rate of 66.9% was achieved. Active members increased markedly from 1985 to 1995. Surgical activity and clinical experience varied widely: 32.9% had placed 100 to 499 implants, 29.8% had inserted 1 to 49 implants, and 4.3% had inserted > or = 2,000 implants. The total number of implants inserted by this sample could only be estimated (between 51,000 and 90,000). The majority of this sample possessed postgraduate qualifications, although only 2.6% possessed a degree in oral implantology. The data from this sample indicated that the recruitment rate to the ADI (UK) increased markedly between 1985 to 1995, after which it seems to have slowed down. Most of the respondents were involved in the surgical aspects of implantology, although the level of surgical involvement varied widely. The low incidence of postgraduate degrees in implantology might reflect the relatively limited opportunities currently available for such training in the UK.

Microbial analysis of bone collected during implant surgery: a clinical and laboratory study.

Dental implant surgery produces bone debris which can be used to correct bone defects in the "simultaneous-augmentation" technique. However, this debris is potentially contaminated with oral bacteria. Therefore, this study examined bone debris collected during dental implant surgery in order 1) to identify the microbial contaminants and 2) to compare the effects of two different aspiration protocols on the levels of microbial contamination. Twenty-four partially dentate patients were randomly allocated into two equal groups and underwent bone collection using the Frios Bone Collector during surgery to insert two endosseous dental implants. In group S (using a stringent aspiration protocol), bone collection occurred within the surgical site only. In group NS (utilizing a non-stringent aspiration protocol), bone collection and tissue fluid control was achieved using the same suction tip. Bone samples were immediately transported for microbial analysis. Colonial and microscopic morphology, gaseous requirements and identification kits were utilized for identification of the isolated microbes. Twenty-eight species were identified including a number associated with disease, in particular, Enterococcus faecalis and Staphylococcus epidermidis as well as the anaerobes Actinomyces odontolyticus, Eubacterium sp., Prevotella intermedia, Propionibacterium propionicum and Peptostreptococcus asaccharolyticus. In group S (stringent aspiration protocol), significantly fewer organisms were found than in group NS, the non-stringent aspiration protocol (P=0.001). Gram-positive cocci dominated the isolates from both groups. It is concluded that if bone debris is collected for implantation around dental implants, it should be collected with a stringent aspiration protocol (within the surgical site only) to minimize bacterial contaminants.