Undecalcified Bone Sections Research Articles

FGF-23 and sclerostin in serum and bone of CKD patients.

Renal osteodystrophy occurs in the early stages of chronic kidney disease (CKD) and progresses during loss of kidney function. Fibroblast growth factor (FGF)-23 and sclerostin, both produced by osteocytes, are increased in blood of patients with CKD. The aim of this study was to analyze the impact of decline in kidney function on FGF-23 and sclerostin protein expression in bone and to study their relationship with their serum levels and bone histomorphometry. 108 patients aged 25-81 years (mean ± SD: 56±13 years) underwent anterior iliac crest biopsies after double-tetracycline labeling. Eleven patients were CKD-2, 16 were CKD-3, 9 were CKD-4 - 5, and 64 CKD-5D. Patients were on hemodialysis for 49±117 months. 18 age-matched patients without CKD were included as controls. Immunostaining was performed on undecalcified bone sections to quantify FGF-23 and sclerostin expression. Bone sections were also evaluated by histomorphometry for bone turnover, mineralization, and volume. FGF-23 expression in bone correlated positively with CKD stages (p<0.001) increasing from 5.3- to 7.1-fold starting at CKD-2. No difference in FGF-23 expression was seen between trabecular and cortical bone. Sclerostin expression in bone correlated positively with CKD stages (p<0.001) with an increase from 3.8- to 5.1-fold starting at CKD-2. This increase was progressive and significantly greater in cortical than cancellous bone. FGF-23 and sclerostin in blood and bone were strongly associated with bone turnover parameters. Expression of FGF-23 in cortical bone correlated positively with activation frequency (Ac.f) and bone formation rate (BFR/BS) (p<0.05), while sclerostin correlated negatively with Ac.f, BFR/BS, and osteoblast and osteoclast numbers (p<0.05). FGF-23 trabecular and cortical expressions correlated positively with cortical thickness (p<0.001). Sclerostin bone expression correlated negatively with parameters of trabecular thickness and osteoid surface (p<0.05). These data show a progressive increase in FGF-23 and sclerostin in blood and bone associated with decrease in kidney function. The observed relationships between bone turnover and sclerostin or FGF-23 should be considered when treatment modalities are developed for management of turnover abnormalities in CKD patients.

Spatial quantitation of antibiotics in bone tissue compartments by laser-capture microdissection coupled with UHPLC-tandem mass spectrometry

Bones are the site of multiple diseases requiring chemotherapy, including cancer, arthritis, osteoporosis and infections. Yet limited methodologies are available to investigate the spatial distribution and quantitation of small molecule drugs in bone compartments, due to the difficulty of sectioning undecalcified bones and the interference of decalcification methods with spatially resolved drug quantitation. To measure drug concentrations in distinct anatomical bone regions, we have developed a workflow that enables spatial quantitation of thin undecalcified bone sections by laser-capture microdissection coupled to HPLC/tandem mass spectrometry, and spatial mapping on adjacent sections by mass spectrometry imaging. The adhesive film and staining methods were optimized to facilitate histology staining on the same sections used for mass spectrometry image acquisition, revealing drug accumulation in the underlying bone tissue architecture, for the first time. Absolute spatial concentrations of rifampicin, bedaquiline, doxycycline, vancomycin and several of their active metabolites are shown for both small rodent bones and larger rabbit bones that more closely resemble human bone density. Overlaid MALDI mass spectrometry images of drugs and histology staining enabled the generation of semi-quantitative data from regions of interest within anatomical bone compartments. These data correlated with absolute drug concentrations determined by HPLC–MS/MS in laser-capture microdissection samples. Collectively, these techniques enable semi- and fully quantitative drug distribution investigations within bone tissue compartments for the first time. Our workflow can be translated to image and quantify not only drugs but also biomarkers of disease to investigate drug penetration as well as mechanisms underlying bone disorders.Graphical abstract

A quantitative analysis of bone lamellarity and bone collagen linearity induced by distinct dosing and frequencies of teriparatide administration in ovariectomized rats and monkeys.

The lamellar structure of bone, which endows biomechanical rigidity to support the host organism, is observed in mammals, including humans. It is therefore essential to develop a quantitative analysis to evaluate the lamellarity of bone, which would especially be useful for the pharmacological evaluation of anti-osteoporotic drugs. This study applied a current system for the semi-automatic recognition of fluorescence signals to the analysis of un-decalcified bone sections from rat and monkey specimens treated with teriparatide (TPTD). Our analyses on bone formation pattern and collagen topology indicated that TPTD augmented bone lamellarity and bone collagen linearity, which were possibly associated with the recovery of collagen cross-linking, thus endowing bone rigidity.

Age-Related Increases in Marrow Fat Volumes have Regional Impacts on Bone Cell Numbers and Structure.

The increasing levels of bone marrow fat evident in aging and osteoporosis are associated with low bone mass and attributed to reduced osteoblastogenesis. Local lipotoxicity has been proposed as the primary mechanism driving this reduction in bone formation. However, no studies have examined the correlation between high levels of marrow fat volumes and changes in local cellularity. In this study, we hypothesize that areas of bone marrow with high fat volumes are associated with significant changes in cell number within a similar region of interest (ROI). Inbred albino Louvain (LOU) rats, originating from the Wistar strain, have been described as a model of healthy aging with the absence of obesity but expressing the typical features of age-related bone loss. We compared local changes in distal femur cellularity and structure in specific ROI of undecalcified bone sections from 4- and 20-month-old male and female LOU rats and Wistar controls. Our results confirmed that older LOU rats exhibited significantly higher fat volumes than Wistar rats (p < 0.001). These higher fat volume/total volume were associated with lower trabecular number (p < 0.05) and thickness (p < 0.05) and higher trabecular separation (p < 0.05). In addition, osteoblast and osteocyte numbers were reduced in the similar ROI containing high levels of adiposity, while osteoclast number was higher compared to control (p < 0.03). In summary, marrow ROIs with a high level of adiposity were associated with a lower bone mass and changes in cellularity explaining associated bone loss. Further studies assessing the levels of lipotoxicity in areas of high local marrow adiposity and identifying molecular actors involved in this phenomenon are still required.

A modified tape transfer approach for rapidly preparing high-quality cryosections of undecalcified adult rodent bones.

Background/ObjectiveHistology-based analyses are important tools to dissect cellular and molecular mechanisms of skeletal homeostasis, diseases, and regeneration. The success of these efforts is highly dependent on rapidly obtaining high-quality sections of mineralized skeletal tissues suitable for various analyses. However, the current techniques for preparing such sections are still far from satisfactory. This study aimed to develop a new approach for preparing high-quality undecalcified bone sections applicable to various histological analyses.MethodsTwo important modifications were made to the conventional Cryojane Tape-Transfer System, including utilization of an optimized adhesive to prepare adhesive glass slides for improving the transfer efficiency, and a cheap conventional benchtop UV transilluminator for UV curing. Cryosections of undecalcified rodent bones were prepared using this modified tape transfer approach, and their tissue morphology and structural integrity were visually examined. A variety of histological analyses, including calcein labeling, Von kossa staining, immunofluorescence, and enzymatic activity staining as well as 5-Ethynyl-2’-deoxyuridine (EdU) and TUNEL assays, were performed on these sections.ResultsWe developed a modified version of tape transfer approach that can prepare cryosections of undecalcified rodent adult bones within 4 days at a low cost. Bone sections prepared by this approach exhibited good tissue morphology and structural integrity. Moreover, these sections were applicable to a variety of histological analyses, including calcein labeling, Von kossa staining, immunofluorescence, and enzymatic activity staining as well as EdU and TUNEL assays.ConclusionThe tape transfer approach we developed provides a rapid, affordable, and easy learning method for preparing high-quality undecalcified bone sections valuable for bone research.The translational potential of this articleOur research provides a rapid, affordable, and easy learning method for preparing high-quality undecalcified bone sections that can be potentially used for accurate diagnosis of various bone disorders and evaluation of the efficacy of different therapies in the treatment of these diseases.

Analysis of Cobalt Deposition in Periprosthetic Bone Specimens by High Resolution Synchroton XRF in Undecalcified Histological Thin Sections

Increasing numbers of implant revisions are a current clinical issue. Interactions of the endoprosthesis biomaterial with the body affect implantation time by wear processes i.e. corrosion and abrasion. Previously, cobalt-chrome implants were shown to cause high levels of cobalt ions being deposited in the bone matrix. To determine a potential functional role of these ions on bone homeostasis, we have developed a non-destructive dual analysis of highly sensitive elemental analysis by synchrotron XRF directly in undecalcified histological bone sections of 4 µm. In this study, samples from 28 hip endoprosthesis carriers (metal-on-metal bearing) with an implant life time of 17 to 1750 days were used. Results were compared to age-matched control specimens. Histological analysis identified areas of bone cell activity and assigned them for XRF measurements. Co-Cr wear particles were identified in the bone marrow. In addition, Co ions were highly enriched in the mineralized bone matrix. The cobalt deposits were not homogeneously distributed and areas of high signal intensity were identified. Co was distinctly deposited in the newly formed osteoid layer, but also within deeper layers of the bone matrix, whereby the Co concentration increased with higher degrees of bone matrix mineralization. In the current study, we determined cobalt accumulations in the bone matrix and showed for the first time via synchrotron XRF with a high spatial resolution on histological slides, that cobalt deposits in the mineralized bone matrix in a mineral-specific way that is dependent upon the implant lifetime.

Microcomputed tomography (microCT) and histology of the mandibular canal in human and laboratory animals

The inferior alveolar nerve (IAN) is a sensitive branch of the trigeminal nerve. It has an intra-bone path in the mandible, inside the mandibular canal, where it is accompanied by lymph, venous and arterial vessels. We have studied the mandibular canal in human mandibles and in some laboratory animals (mice, rats, rabbits and cats). Microcomputed tomography evidenced that the walls of the canal are made with thin plates of trabecular bone with numerous fenestrations. This aspect is evidenced in dentate subjects and become more evident in edentulous subjects with atrophy of the alveolar bone. In rats and mice, the wall of the canal is also clearly composed of trabecular plates coming from the surrounding alveolar bone of the mandible. In the rabbit, similar findings are also observed but the trajectory of the canal is more difficult to identify. In the cat, the floor of the canal is composed of the cortical bone from the basilar cortex of the mandible and the roof has a trabecular nature. Vascular injections of gelatin-barium evidenced the arterial trajectories inside the bone in rats and humans. Undecalcified bone sections in human evidenced the histological aspect of the IAN and its connective sheets. Some nervous bundles can be observed outside the epineurium. Bone remodeling is observed on the wall of the mandibular canal. These descriptive findings have a clinical relevance in dental implantology or mandibular surgery.

Cortical bone adaptation and mineral mobilization in the subterranean mammal Bathyergus suillus (Rodentia: Bathyergidae): effects of age and sex.

The patterns of bone modeling and mineral mobilization (skeletal homeostasis) among mammals other than humans and laboratory rodents are still poorly known. In this study we assessed the pattern of bone formation and bone resorption in the femur of a wild population of Cape dune molerats, Bathyergus suillus (n = 41) (Bathyergidae), a solitary subterranean mammal with a marked extended longevity among rodents, and which also lives in a naturally deficient state of vitamin D. In order to determine ontogenetic and sex effects on histomorphometric parameters of transversal undecalcified bone sections, two-way ANOVA, linear mixed-effects model and regression statistical analyses were performed. During ontogeny, B. suillus increased their cross sectional area, cortical area and cortical thickness, and most importantly, they showed scarce endosteal bone resorption which resulted in a retained medullary cavity size during ontogeny. This resulted in a positively imbalanced bone modeling, where bone formation considerably surpasses bone loss by almost 100-fold in adulthood. This differs markedly from other terrestrial mammals with relatively thin cortical walls. Regarding bone loss and remodeling, three main processes involving intracortical resorption were observed: modeling-related bone loss in early postnatal growth; secondary osteon formation occurring in both sexes; and subendosteal secondary reconstruction observed only in females. The latter is accompanied by females having six-fold more relative bone loss than males, which is evidenced by the development of enlarged resorption cavities (RCs) distributed circumferentially around the medullary cavity. Males have smaller, more circular and randomly distributed RCs. In general, our data indicate no age-related decline in mineral content in B. suillus, and provides strong support for a pattern of sexual dimorphism in skeletal homeostasis, similar to that occurring in humans and other mammals, with females losing more bone throughout aging as compared to males due to reproductive factors. Interestingly as well, despite the high mechanical loads experienced during burrow construction, bone remodeling in B. suillus is kept at very low levels throughout their lifespan, and dense Haversian tissue never forms. This study represents the first comprehensive assessment of skeletal homeostasis in a subterranean mammal, and it enables a better understanding of the complex processes governing the acquisition and maintenance of bone properties in this species with extraordinary fossorial adaptations.

Metaplastic woven bone in bone metastases: A Fourier-transform infrared analysis and imaging of bone quality (FTIR)

Most osteolytic tumors are in fact mixed and contain an osteoblastic component associated with the predominant osteolytic areas. This metaplastic woven bone is always evidenced by histological analysis even in the absence of radiological expression. Metaplastic bone formation reflects the activation of new osteoblasts coming from the stimulation of the dormant lining cells. Twelve patients with secondary metastases of the iliac crest evidenced by hot spots on a 99Tc-MBP san were diagnosed by histomorphometry on bone biopsies. Fourier Transformed InfraRed analysis and Imaging (FTIRI) was used on 4μm thick sections of undecalcified bone. The mineralization degree, carbonate substitution, crystallinity and the cross-links ratio of collagen (1660/1690cm-1 bands) were determined. The matrix characteristics were analyzed and imaged in the pre-existing residual bone and in the metaplastic woven bone in the vicinity of the tumor cells. FTIRI provided images of the phosphate, amide and combination of peak ratio after having selected the peaks of interest. In addition, the matrix properties can be measured and compared between the old and newly-formed bones. Woven bone appeared poorly calcified with a low phosphate/amide ratio (P=0.03) crystallinity (P<0.0001) and carbonate substitution (P=0.003). Collagen was less mature as evidenced by lower cross-links (P=0.01). Woven bone associated with bone metastasis appears poorly mineralized and rapidly elaborated by osteoblasts. The collagenous phase of the bone matrix has a low level of reticulation. FTIRI is a powerful tool to measure and visualize the various components of the bone matrix in human diseases.

The use of a biphasic calcium phosphate in a maxillary sinus floor elevation procedure: a clinical, radiological, histological, and histomorphometric evaluation with 9- and 12-month healing times

BackgroundThis study evaluates the clinical, radiological, histological, and histomorphometric aspects of a fully synthetic biphasic calcium phosphate (BCP) (60% hydroxyapatite and 40% ß-tricalcium phosphate), used in a human maxillary sinus floor elevation (MSFE) procedure with 9- and 12-month healing time.MethodsA unilateral MSFE procedure, using 100% BCP, was performed in two series of five patients with healing times of 9 and 12 months respectively. Clinical and radiological parameters were measured up to 5 years postoperatively. Biopsy retrieval was carried out during dental implants placement. Histology and histomorphometry were performed on 5-μm sections of undecalcified bone biopsies.ResultsThe MSFE procedure with BCP showed uneventful healing in all cases. All dental implants appeared to be well osseointegrated after 3 months. Radiological evaluation showed less than 1 mm tissue height loss from MSFE to the 5-year follow-up examination. No signs of inflammation were detected on histological examination. Newly formed mineralized tissue was found cranially from the native bone. The BCP particles were surrounded by connective tissue, osteoid islands, and newly formed bone. Mineralized bone tissue was in intimate contact with the BCP particles. After 12 months, remnants of BCP were still present. The newly formed bone had a trabecular structure. Bone maturation was demonstrated by the presence of lamellar bone. Histomorphometric analysis showed at 9 and 12 months respectively an average vital bone volume/total volume of 35.2 and 28.2%, bone surface/total volume of 4.2 mm2/mm3 and 8.3 mm2/mm3, trabecular thickness of 224.7 and 66.7 μm, osteoid volume/bone volume of 8.8 and 3.4%, osteoid surface/bone surface (OS/BS) of 42.4 and 8.2%, and osteoid thickness of 93.9 and 13.6 μm.ConclusionsMFSE with BCP resulted in new bone formation within the augmented sinus floor and allowed the osseointegration of dental implants in both groups. From a histological and histomorphometric perspective, a 9-month healing time for this type of BCP may be the optimal time for placement of dental implants.

Administration frequency as well as dosage of PTH are associated with development of cortical porosity in ovariectomized rats

To investigate whether the administration frequency of parathyroid hormone (PTH) is associated with the development of cortical porosity, this study established 15 dosage regimens of teriparatide [human PTH(1–34), TPTD] with four distinct concentrations and four distinct administration frequencies of TPTD to 16-week-old ovariectomized rats. Our analyses demonstrated that the bone mineral density, mechanical properties, and bone turnover were associated with the total amount of TPTD administered. Our observations further revealed that the cortical porosity was markedly developed as a result of an increased administration frequency with a lower concentration of total TPTD administration in our setting, although the highest concentration also induced cortical porosity. Deconvolution fluorescence tiling imaging on calcein-labeled undecalcified bone sections also demonstrated the development of cortical porosity to be closely associated with the bone site where periosteal bone formation took place. This site-specific cortical porosity involved intracortical bone resorption and an increased number and proximity of osteocytic lacunae, occasionally causing fused lacunae. Taken together, these findings suggested the involvement of local distinctions in the rate of bone growth that may be related to the site-specific mechanical properties in the development of cortical porosity induced by frequent and/or high doses of TPTD.

Presence of osteoclasts in middle ear cholesteatoma: a study of undecalcified bone sections

Conclusions: Osteoclasts are unlikely to be involved in bone resorption in middle ear cholesteatoma.Objective: The authors searched for osteoclasts in undecalcified bone sections in patients with middle ear cholesteatoma to determine whether and to what extent these cells are involved in this disease.Methods: Twelve patients, eight men and four women, aged 30–87 years, who underwent tympanomastoidectomy were enrolled. Six patients had primary acquired middle ear cholesteatoma (cholesteatoma group) and the other six patients had other otologic diseases including otosclerosis, non-cholesteatomatous chronic otitis media, adhesive otitis media, perilymphatic fistula and ossicular malformation (control group). The scutum bone was collected during surgery, fixed with ethanol, stained with Villanueva bone stain, and embedded in methyl methacrylate. Five-micrometer-thick sections were prepared and examined under a polarizing microscope. Images were analyzed using a semiautomatic graphics system.Results: No osteoclasts were seen in any of the samples in either group. To avoid the risk of under-estimating the presence of osteoclasts, the number of osteoclasts was considered to be <1 in each sample, and the osteoclast density was calculated. The osteoclast densities in both the cholesteatoma and control groups were significantly lower than the sex- and age-matched standard value of the normal iliac cortical bone (p = .028).

Serum phosphorus adds to value of serum parathyroid hormone for assessment of bone turnover in renal osteodystrophy.

It is well-established that parathyroid hormone (PTH) correlates with the level of bone turnover in patients with chronic kidney disease stage 5D (CKD-5D). Hyperphosphatemia is a well-established complication of end-stage renal disease and is usually attributed to dietary intake. This study evaluates the relationship between serum phosphorus levels and bone turnover in patients with CKD-5D. 93 patients with CKD-5D from the Kentucky Bone Registry who had sequentially undergone anterior iliac bone biopsies were reviewed. Undecalcified bone sections were qualitatively assessed for turnover and placed into a group with low turnover and a group with non-low (normal/high) turnover. Results of PTH and phosphorus concentrations in blood drawn at the time of biopsies were compared between the groups. PTH and phosphorus levels were significantly higher in the non-low turnover group compared to the low turnover group. Cutoff levels for PTH and phosphorus were tested for predictive power of bone turnover. Both PTH and phosphorus correlated with turnover. Adding serum phosphorus to serum PTH enhanced predictive power of PTH for low turnover. The vast majority of patients with serum phosphorus levels ≥ 6.0 mg/dL had non-low turnover, while the majority of those with low turnover had phosphorus values < 6.0 mg/dL. Classification and regression-tree analysis showed that elevated serum phosphorus (> 6.2 mg/dL) in patients with PTH < 440 pg/mL was helpful in diagnosing nonlow turnover in this range of PTH. In patients with PTH ranges of 440 - 814 pg/mL, serum phosphorus levels > 4.55 mg/dL ruled out low turnover bone disease. This suggests that not only dietary intake but also bone affects serum phosphorus levels.

Estrategias ALTERNAS para la Valoración histomorfológica de la edad a la muerte en restos esquELÉticos DETERIORADOS. Aplicaciones en la bioarqueología del área maya

KEY WORDS microscopic evaluation; age estimation methods; osteology; diagenesis ABSTRACT Our study focuses on the histological assignation of age-at-death ranges to diagenetically deteriorated skeletal remains. To this end, we review age-related microscopic metamorphoses in thin sections of undecalcified bone in order to generate selective counts of microstructures and a qualitative assessment of histological tissue. In this essay, we evaluate the potential of these standards in three case studies on the deteriorated skeletal remains of three Mayan dignitaries from the first millennium CE. In the first case, the macroscopic approximation is confirmed by histologic scrutiny, while in the second case contradicting results lead to a discussion of the individual’s pathological conditions that could explain the discrepancy in specific lines of evidence. In the third specimen, the combination of macroscopic and microscopic results improved chrono-vital assignation. We conclude our work with a discussion of the potential that histomorphological evaluation (and its different protocols) holds in age-range assignment in deteriorated skeletal remains from archaeological contexts.

Das Gräberfeld von Neumarkt

Anthropological examination of the early Bronze Age cemetery at Neumarkt on the Ybbs River revealed 75 individuals: 32 adults (17 men and 15 women), one juvenile, 38 children (4 months - 13 yrs.), two foetuses and two undetermined skeletal remains. The average age at death was calculated as 42.5 yrs. for men (Standard Deviation [SD] 8.6), 35.8 yrs. for women (SD 11.4), 9.3 yrs. for Infans II (SD 1.5), 3.7 yrs. for Infans I (SD 1.9) and 9-10 lunar months for foetuses. Despite the bad preservation of the skeletal remains, some diseases and peculiarities could be detected such as the loss of the right eye by one male several years before death; congenital malformation with deafness of both ears in a seven-year-old child and his or her death due to meningitis; purulent inflammation of the bone marrow with destruction of the left ankle of a man who was buried in a non-normal way; and early metastases due to cancer in a young woman. Proof was obtained by X-ray examination and macroscopic differential diagnosis, as well as the study of thin sections of undecalcified bone embedded in resin. Special histological light microscopic and microradiographic methods were applied. The evaluation was interdisciplinary and took place in cooperation with the archaeologist working on the materials.

Chemical etching in processing cortical bone specimens for scanning electron microscopy.

Transverse and longitudinal sectioning of undecalcified cortical bone is a commonly employed technique for investigating the lamellar structure of the osteons. Since a flat surface is required, the specimen has to be grinded and then polished. Whereas the smear of debris and inorganic/organic deposits left by these treatments cannot be removed by ultrasonication alone, a chemical treatment of the specimen surface with either a basic or an acid etching solution is currently employed. A further effect of the latter can be the enhancement of the lamellar bone pattern. The kind of etching solution, its pH, the concentration of etchants, and the contact time significantly affect the sectioned surface when it is observed with scanning electron microscopy (SEM). The etching procedures can severely influence the obtained images. Homogeneous cortical bone specimens were sampled from the first metatarsal of two fresh human subjects. One or two cut surfaces were exposed to different acid and basic solutions in bonded conditions. Considering the type of chemical agents, the solution pH, and the exposure time of the specimens, the effects of several etching media have been investigated and compared. Strong etching, either acid or basic produced surface decalcification and severe damage of the collagen matrix, compromising any morphological or morphometric analysis. Weak acid etching (for example citric and acetic acid), even though causing distinctive alteration of the sample, enhanced the visibility of the lamellar pattern, while the polyphosphate treatment of the surface decalcified a thin layer matrix, ensuring a good visibility of fibrils and avoiding rough distortions.

Identification of the Vitamin D Receptor in Osteoblasts and Chondrocytes But Not Osteoclasts in Mouse Bone

Bone is clearly a target of vitamin D and as expected, the vitamin D receptor (VDR) is expressed in osteoblasts. However, the presence of VDR in other cells such as osteocytes, osteoclasts, chondroclasts, and chondrocytes is uncertain. Because of difficulties in obtaining sections of undecalcified adult bone, identification of the site of VDR expression in adult bone tissue has been problematic. In addition, the antibodies to VDR used in previous studies lacked specificity, a property crucial for unambiguous conclusions. In the present study, VDR in the various cells from neonatal and adult mouse bone tissues was identified by a highly specific and sensitive immunohistochemistry method following bone decalcification with EGTA. For accurate evaluation of weak immunosignals, samples from Demay VDR knockout mice were used as negative control. Molecular markers were used to identify cell types. Our results showed that EGTA-decalcification of bone tissue had no detectable effect on the immunoreactivity of VDR. VDR was found in osteoblasts and hypertrophic chondrocytes but not in the multinucleated osteoclasts, chondroclasts, and bone marrow stromal cells. Of interest is the finding that immature osteoblasts contain large amounts of VDR, whereas the levels are low or undetectable in mature osteoblasts including bone lining cells and osteocytes. Proliferating chondrocytes appear devoid of VDR, although low levels were found in the hypertrophic chondrocytes. These data demonstrate that osteoblasts and chondrocytes are major targets of 1α,25-dihydroxyvitamin D, but osteoclasts and chondroclasts are minor targets or not at all. A high level of VDR was found in the immature osteoblasts located in the cancellous bone, indicating that they are major targets of 1α,25-dihydroxyvitamin D. Thus, the immature osteoblasts are perhaps responsible for the vitamin D hormone signaling resulting in calcium mobilization and in osteogenesis.

Osteogenesis Imperfecta, an Ever-Expanding Conundrum

Osteogenesis imperfecta (OI), or brittle bone disease, a rare heritable connective tissue disorder, was described more than a century ago. Its hallmark, bone fragility, may cause frequent fractures, spine and limb deformities, and chronic pain. Another characteristic of the OI syndrome is its wide spectrum of clinical severity, from the very mild to the lethal. Over the years, there have been several attempts at classifying the various forms of OI either by number or by degrees of severity (reviewed in Glorieux and Rowe). All such efforts have only been partially successful because, except for the extremes of the spectrum, clinical types are overlapping. Furthermore, it has been oftentimes observed that within the same family, affected subjects show a wide variation in clinical presentations. This variability in phenotype severity is matched by an ever‐ expanding genotypic variability. OI is in most cases associated with mutations in COL1A1 and COL1A2, the genes encoding type I collagen, the main component of the bone matrix. Detailed analysis of the large number of identified mutations allowed for two main groups to be delineated. The first one corresponds to mutations leading to haploinsufficiency and overall to a mild phenotype. The second group includes mutations that cause structural abnormalities in the collagen molecule compromising the organization of the bone matrix. Associated phenotypes are usually moderate to severe or, rarely, lethal. All such mutations are transmitted as autosomal dominant traits. However, in a small number (less than 10%) of OI cases, no such association could be established, and inmost of them there was evidence for autosomal recessive inheritance. In the first reported cases from a population isolate, mutations were identified in CRTAP, the gene encoding a cartilage associated protein part of the prolyl 3‐hydroxylation complex involved in the processing of the type I collagen triple helix. Subsequently, mutations were found that affected the other two subunits of the complex, prolyl 3‐hydroxylase 1 and cyclophylin B, as well as chaperones encoded by SERPINH1 and FKBP10 (reviewed in Forlino and colleagues). Thus, up to that point all abnormalities could be linked to the synthesis, secretion, processing, and matrix integration of the type I collagen molecule. Recently, other forms, where no abnormalities related to collagen type I could be elicited, were identified by detailed analysis of undecalcified bone sections obtained from iliac crest biopsies. Pathophysiologic aspects of two such entities are discussed in this issue. The first one, type V OI, was described 13 years ago. Unlike the other “new” forms of OI, it is transmitted as an autosomal dominant trait. Besides bone fragility, its main characteristics include short stature, ossification of the interosseous membrane of the forearm often leading to dislocation/subluxation of the proximal radial head, presence of radiodense bands under the growth plates, and the frequent development of hyperplastic calluses after fractures or corrective surgery. Its unique defining feature is, however, found on bone sections as a loss of the birefringent aspect of the matrix under polarized light illumination. It rather exhibits ameshlike appearance not observed in any other bone disease. Last year, two groups simultaneously reported the association of type V OI with a single identical mutation (c.‐14C> T) in the untranslated region of IFITM5. It introduces a new initiation codon that adds five residues at the N‐terminus of the gene product, an osteoblast‐specific transmembrane protein, BRIL. The findings were rapidly corroborated in a third publication, which added 42 patients to the 21 from the initial reports. Remarkably, all subjects carry the exact same mutation despite a wide variability in its phenotypic expression. Shapiro and colleagues further document this fascinating observation in an additional 17 patients. BRIL was discovered as part of a high‐throughput screen for cDNAs encoding secreted and membrane proteins in osteoblastic cells. BRIL is part of an evolutionary conserved family of so‐ called interferon‐inducible transmembrane (IFITM) proteins for which there are at least four closely related members in the human (IFITM1, 2, 3, and 10) and two others in the mouse (IFITM6 and 7). The inclusion of BRIL as part of this family is based on genomic and protein structure considerations. BRIL, IFITM1, 2, and 3 are all clustered within 25 kb on chromosome 11; they all possess a similar gene architecture comprising two small coding

Visualisation of methacrylate‐embedded human bone sections by infrared nanoscopy

A recently developed ultra-resolving near-field infrared nanoscope is applied to investigate methyl methacrylate embedded, un-decalcified human bone sections. Results show detail at a resolution of 30 nm. Specific contrasting of mineral components is enabled by choosing an appropriate infrared wavelength, here 9.47 μm, in the phosphate vibrational band. The method is surface-sensitive, probing to a depth of about 30 nm into the surface. The obtained infrared images are presented in direct comparison with optical and electron micrographs of the identical specimen. Lamellar bone organization, peri-cellular mineral deposition, and regional differences in mineral content are clearly detectable. Individual fibrils are resolved. - Infrared nanoscopy requires just standard hard tissue preparation techniques combined with section surface polishing. It can be integrated into existing laboratory environments without impeding subsequent routine staining and evaluation methods.

Section plane selection influences the results of histomorphometric studies: the example of dental implants.

This study was designed to determine and statistically analyze bone-to-implant contact (BIC) values for human specimens segmented in at least two different locations. Samples of human bone with fractured osseointegrated implants were obtained from six patients. Sections were prepared, dehydrated, and resin infiltrated. Undecalcified bone sections were produced using the thin-section technique according to Donath, ultimately obtaining a section thickness of approximately 20 μm. Fifteen specimens were available for histomorphometry. The bone sections were digitized and analyzed. The bone-to-metal contact (BMC) parameter was determined histomorphometrically. The BMC was returned in terms of the visibly bone-covered implant surfaces as a percentage of the total implant surface shown. The values obtained for the six implants were arranged as six maximum-distance pairs and tested for significance using the t-test for dependent samples. The mean difference in BIC was 11.69±9.79%. The two-sided test showed a significant difference (p=0.033). The accidental or deliberate choice of section plane for a bone-implant block has an influence on the BIC value. To make BIC values more comparable, a standardization of section planes is desirable.