Development of new criteria for cortical bone histomorphometry in femoral neck: intra- and inter-observer reproducibility.

Histomorphometry is commonly applied to study bone remodeling. Histological definitions of cortical bone boundaries have not been consistent. In this study, new criteria for specific definition of the transitional zone between the cortical and cancellous bone in the femoral neck were developed. The intra- and inter-observer reproducibility of this method was determined by quantitative histomorphometry and areal overlapping analysis. The undecalcified histological sections of femoral neck specimens (n = 6; from men aged 17-59 years) were processed and scanned to acquire histological images of complete bone sections. Specific criteria were applied to define histological boundaries. "Absolute cortex area" consisted of pure cortical bone tissue only, and was defined mainly based on the size of composite canals and their distance to an additional "guide" boundary (so-called "preliminary cortex boundary," the clear demarcation line of density between compact cortex and sparse trabeculae). Endocortical bone area was defined by recognizing characteristic endocortical structures adjacent to the preliminary cortical boundary. The present results suggested moderate to high reproducibility for low-magnification parameters (e.g., cortical bone area). The coefficient of variation (CV %) ranged from 0.02 to 5.61 in the intra-observer study and from 0.09 to 16.41 in the inter-observer study. However, the intra-observer reproducibility of some high-magnification parameters (e.g., osteoid perimeter/endocortical perimeter) was lower (CV %, 0.33-87.9). The overlapping of three histological areas in repeated analyses revealed highest intra- and inter-observer reproducibility for the absolute cortex area. This study provides specific criteria for the definition of histological boundaries for femoral neck bone specimens, which may aid more precise cortical bone histomorphometry.

Pediatric solid organ transplantation and osteoporosis: a descriptive study on bone histomorphometric findings.

BACKGROUND: Organ transplantation may lead to secondary osteoporosis in children. This study characterized bone histomorphometric findings in pediatric solid organ transplant recipients who were assessed for suspected secondary osteoporosis. METHODS: Iliac crest biopsies were obtained from 19 children (7.6-18.8 years, 11 male) who had undergone kidney (n = 6), liver (n = 9), or heart (n = 4) transplantation a median 4.6 years (range 0.6-16.3 years) earlier. All patients had received oral glucocorticoids at the time of the biopsy. RESULTS: Of the 19 patients, 21 % had sustained peripheral fractures and 58 % vertebral compression fractures. Nine children (47 %) had a lumbar spine BMD Z-score below -2.0. Histomorphometric analyses showed low trabecular bone volume (< -1.0 SD) in 6 children (32 %) and decreased trabecular thickness in 14 children (74 %). Seven children (37 %) had high bone turnover at biopsy, and low turnover was found in 6 children (32 %), 1 of whom had adynamic bone disease. CONCLUSIONS: There was a great heterogeneity in the histological findings in different transplant groups, and the results were unpredictable using non-invasive methods. The observed changes in bone quality (i.e. abnormal turnover rate, thin trabeculae) rather than the actual loss of trabecular bone, might explain the increased fracture risk in pediatric solid organ transplant recipients.

Increased heterogeneity of bone matrix mineralization in pediatric patients prone to fractures: a biopsy study.

Idiopathic osteoporosis (IOP) in children is characterized by fragility fractures and/or low bone mineral density in otherwise healthy individuals. The aim of the present work was to measure bone mineralization density distribution (BMDD) based on quantitative backscattered electron imaging (qBEI) in children with suspected IOP. Entire cross-sectional areas of transiliac bone biopsy samples from children (n = 24, 17 boys; aged 6.7-16.6 years) with a history of fractures (n = 14 with at least one vertebral fracture) were analyzed for cancellous (Cn) and cortical (Ct) BMDD. Outcomes were compared with normal reference BMDD data and correlated with the patients' clinical characteristics and bone histomorphometry findings. The subjects had similar average degree but significantly higher heterogeneity of mineralization in both Cn and Ct bone (Cn.CaWidth +23%, Ct.CaWidth +15%, p < 0.001 and p = 0.002, respectively), together with higher percentages of low mineralized cancellous (Cn.CaLow +35%, p < 0.001) and highly mineralized cortical bone areas (Ct.CaHigh +82%, p = 0.032). Ct.CaWidth and Ct.CaLow were positively correlated with mineralizing surface per bone surface (MS/BS; a primary histomorphometric determinant of bone formation) and with serum bone turnover markers (all p < 0.05). The correlations of the mineralization heterogeneity with histomorphometric and serum bone turnover indices suggest that an enhanced variation in bone turnover/formation contributes to the increased heterogeneity of mineralization. However, it remains unclear whether the latter is cause for, or the response to the increased bone fragility in these children with suspected IOP.

Incidence and bone biopsy findings of atypical femoral fractures.

Bisphosphonates are widely used in the treatment of osteoporosis. It has been suggested that bisphosphonate treatment may be associated with atypical femoral fractures (AFFs), severely suppressed bone turnover rate, and decreased mineralization. We studied bone properties using bone quantitative histomorphometry and Fourier transform infrared spectroscopic imaging (FTIRI) on patients with AFFs. Further, the incidence of AFFs was estimated. Patient records of Kuopio University Hospital, Finland from January 2007 to June 2009 were reviewed to identify all patients who had sustained and had been operated for AFF (n = 8). The incidence of AFFs among patients on bisphosphonates was 0.61 fractures/1,000 patients per year, compared to 0.0067/1,000 per year among untreated patients. The patients that underwent bone biopsy (n = 4) were postmenopausal women (aged 55.5-81.1 years) who had been treated with bisphosphonates for over 4 years. Histomorphometry revealed low trabecular bone volume. Bone formation and resorption parameters tended to be low. Trabecular bone single labels were detected in one patient in the region of interest. In the extended label search, trabecular bone double labels were found in two patients. Based on FTIRI results, higher phosphate-to-amide I ratio and collagen maturity were found compared to normal samples. The heterogeneity of phosphate-to-amide I ratio was low. Overall incidence of atypical femoral fractures is low. The poor fracture resistance in some patients on long-term bisphosphonate-therapy could be explained by low bone formation, and changes in bone composition, i.e., higher degree of mineralization, increased collagen maturity, and decreased heterogeneity of the degree of mineralization.

Altered bone composition in children with vertebral fracture.

Primary osteoporosis in children often leads to vertebral fractures, but it remains unknown whether these fractures associate with changes in bone composition. This study aimed to determine the differences in bone composition in fracture-prone children with and without vertebral fractures, as assessed by Fourier transform infrared spectroscopic imaging (FTIRI) and bone histomorphometry. Iliac crest bone biopsies (n = 24) were obtained from children who were suspected of primary osteoporosis based on evidence from the fracture history and/or low bone mineral density (BMD) by dual-energy X-ray absorptiometry. Vertebral morphology was determined by radiography. Bone biopsies were analyzed using histomorphometry and FTIRI. Phosphate-to-amide I, carbonate-to-phosphate, carbonate-to-amide I, and cross-link ratio (collagen maturity) were calculated. Children with (n = 14) and without (n = 10) vertebral fracture were compared. Low cancellous bone volume (BV/TV) was detected by histomorphometry in 36% of the children with vertebral fracture, and bone turnover rate was abnormal in 64% of them. Children with vertebral fractures had lower carbonate-to-phosphate ratios (p < .05) and higher collagen maturity (p < .05) than children without vertebral fracture. The children with low BV/TV in biopsy showed lower carbonate-to-amide I ratios (p < .05) than the children with normal bone volume. This study showed changes in bone composition among fracture-prone children who had sustained a vertebral fracture. The observed changes in bone composition in these children may contribute to their greater propensity to sustain vertebral fractures.

Bone biopsy findings and correlation with clinical, radiological, and biochemical parameters in children with fractures.

In children the diagnosis of osteoporosis is based on fracture history and DXA-derived BMD. Bone biopsy is an invasive but accurate method for studying bone characteristics. In this study we evaluated bone biopsy findings and their correlation with noninvasive measures of bone health. Transiliac bone biopsy was performed on 24 consecutive children (17 boys, median age 12 years, range 6 to 16 years) evaluated for suspected primary osteoporosis. Biopsy findings were compared with normative data and correlated with clinical, radiological, biochemical, and densitometric findings. The patients had sustained altogether 64 nonvertebral fractures (median 2.5) from low- or moderate-energy traumas, and 14 patients (58%) had vertebral fractures. The median lumbar spine BMD Z-score was -1.2 (range -3.1 to +1.0). Hypovitaminosis D was present in 58%. Histomorphometry showed low bone volume in 7 patients and normal bone volume in 17. Bone turnover was high in 7, low in 7, and normal in 10 patients. Histomorphometric findings correlated poorly with fracture history, serum bone turnover markers, and DXA findings. Vitamin D deficiency and low lumbar BMD were associated with high bone turnover in the biopsy. These findings underscore the difficulties in diagnosing pediatric osteoporosis. Bone histomorphometry gives additional information and may be useful when considering bisphosphonate treatment in children with suspected primary osteoporosis.

Reproducibility and agreement of micro-CT and histomorphometry in human trabecular bone with different metabolic status.

The use of micro-computed tomography (micro-CT) to study bone microstructure is continuously increasing. Thus, it is important to ensure that micro-CT can differentiate healthy and pathological bone. This study aimed to determine whether the reproducibility of bone histomorphometry and micro-CT, and agreement between the techniques, vary in bone samples with different metabolic status. Iliac crest biopsies (n = 36) were obtained from healthy subjects (n = 10) and from patients with osteoporosis (OP) (n = 15) or renal osteodystrophy (ROD) (n = 11). Micro-CT and histomorphometry analyses were repeated twice. Results were analyzed in separate groups and after pooling the data. Bone histomorphometry detected generally known differences between the diseases, whereas micro-CT did not detect differences between normal and ROD samples as effectively. Repeated measurements for BV/TV, Tb.Th, Tb.N, and Tb.Sp exhibited linear correlation coefficients (ρ) of 0.87-0.92 [coefficients of variations (CV), 8.3-27.2%] for histomorphometry and of 0.66-0.94 (CV, 4.4-23.4%) for micro-CT. There were no significant differences in reproducibility among samples from different study groups. Correlations between BV/TV (micro-CT) and mineralized bone volume (Md.V/TV, histomorphometry) were weaker than between BV/TV (micro-CT) and BV/TV (histomorphometry). When comparing the techniques, BV/TV, Tb.Th, and Tb.N displayed moderate correlations (ρ = 0.39-0.62, P < 0.05), and the agreement for BV/TV was highest in OP samples. The agreement between the techniques using clinical bone samples was moderate. Especially, micro-CT was less effective than bone histomorphometry in differentiating ROD from normal samples. The reproducibility was not affected by the health status of bone. Histomorphometry is still needed in clinical practice to study the remodeling balance in bone, and the methods are complementary.

Infrared spectroscopy indicates altered bone turnover and remodeling activity in renal osteodystrophy.

Renal osteodystrophy alters metabolic activity and remodeling rate of bone and also may lead to different bone composition. The objective of this study was to characterize the composition of bone in high-turnover renal osteodystrophy patients by means of Fourier transform infrared spectroscopic imaging (FTIRI). Iliac crest biopsies from healthy bone (n = 11) and patients with renal osteodystrophy (ROD, n = 11) were used in this study. The ROD samples were from patients with hyperparathyroid disease. By using FTIRI, phosphate-to-amide I ratio (mineral-to-matrix ratio), carbonate-to-phosphate ratio, and carbonate-to-amide I ratio (turnover rate/remodeling activity), as well as the collagen cross-link ratio (collagen maturity), were quantified. Histomorphometric analyses were conducted for comparison. The ROD samples showed significantly lower carbonate-to-phosphate (p < .01) and carbonate-to-amide I (p < .001) ratios. The spatial variation across the trabeculae highlighted a significantly lower degree of mineralization (p < .05) at the edges of the trabeculae in the ROD samples than in normal bone. Statistically significant linear correlations were found between histomorphometric parameters related to bone-remodeling activity and number of bone cells and FTIRI-calculated parameters based on carbonate-to-phosphate and carbonate-to-amide I ratios. Hence the results suggested that FTIRI parameters related to carbonate may be indicative of turnover and remodeling rate of bone.