Static and dynamic bone histomorphometry in children with osteogenesis imperfecta

doi:10.1016/S8756-3282(00)00269-6

Bone

Volume 26, Issue 6, June 2000, Pages 581-589

Part of this work was presented at the second joint meeting of the American Society for Bone and Mineral Research and the International Bone and Mineral Society.

https://doi.org/10.1016/S8756-3282(00)00269-6 Get rights and content

Abstract

Osteogenesis imperfecta (OI) is a genetic disorder characterized by increased bone fragility and low bone mass. Four clinical types are commonly distinguished. Schematically, type I is the mildest phenotype, type II is usually lethal, type III is the most severe form compatible with postnatal survival, and type IV is moderately severe. Although mutations affecting collagen type I are responsible for the disease in most patients, the mechanisms by which the genetic defects cause abnormal bone development have not been well characterized. Therefore, we evaluated quantitative static and dynamic histomorphometric parameters in tetracycline-labeled iliac bone biopsies from 70 children, aged 1.5 to 13.5 years, with OI types I (n = 32), III (n = 11), and IV (n = 27). Results were compared with those of 27 age-matched controls without metabolic bone disease. Biopsy core width, cortical width, and cancellous bone volume were clearly decreased in all OI types. Decreased cancellous bone volume was due to a 41%–57% reduction in trabecular number and a 15%–27% lower trabecular thickness. Regression analyses revealed that trabecular number did not vary with age in either controls or OI patients, indicating that no trabecular loss occurred. The annual increase in trabecular thickness was 5.8 μm in controls and 3.6 μm in type I OI, whereas no trabecular thickening was evident in type III and IV OI. Wall thickness, which reflects the amount of bone formed during a remodeling cycle, was decreased by 14% in a subgroup of 17 type I OI patients, but was not determined in the other OI types. The remodeling balance was less positive in type I OI than in controls, and probably close to zero in types III and IV. Surface-based parameters of bone remodeling were increased in all OI types, indicating increased recruitment of remodeling units. No defect in matrix mineralization was found. In conclusion, there was evidence of defects in all three mechanisms, which normally lead to an increase in bone mass during childhood; that is, modeling of external bone size and shape, production of secondary trabeculae by endochondral ossification, and thickening of secondary trabeculae by remodeling. Thus, OI might be regarded as a disease in which a single genetic defect in the osteoblast interferes with multiple mechanisms that normally ensure adaptation of the skeleton to the increasing mechanical needs during growth.

Introduction

Osteogenesis imperfecta (OI) is a heritable disorder that is characterized by increased bone fragility and low bone mass.30 Often, bone shape is also abnormal with metaphyseal flaring and thin diaphyses. Four clinical types are commonly distinguished.32 Type I OI comprises patients with a mild presentation and a low normal or slightly reduced height, whereas type II is usually lethal in the perinatal period. Type III OI is the most severe form in children surviving the neonatal period. These patients have a well-defined phenotype, including extremely short stature, progressive bone deformity and growth plate fractures. Patients who do not fit into one of the aforementioned categories are usually classified as having type IV OI.

In most OI patients, the disease is caused by mutations in either the procollagen type I α1 or the procollagen type I α2 gene.30 The pathophysiological effects of these mutations on the skeleton are not completely understood. The obvious direct effect is a perturbed osteoblast function, the cell type that expresses the mutated gene product in bone.30 In addition, there are indirect effects, as abnormalities in collagen production and secondary changes in the organic and inorganic bone matrix components4, 9, 18, 31, 35, 36 alter the environment for all cell types in bone. However, these indirect consequences of matrix abnormalities are not well characterized.

To elucidate how these direct and indirect effects of OI mutations affect bone development, more histomorphometric data are necessary. Quantitative histomorphometry is the only available method to study bone cell function within the in vivo structural context. As of yet, there is a surprising scarcity of information on the bone tissue characteristics of OI. Early qualitative studies on bone histology in OI evaluated samples obtained at sites of deformity or fracture during surgical procedures, and thus results were obscured by injury or repair reactions.29 The first quantitative studies used bone specimens from the rib and were limited to the analysis of a few parameters.1, 37

Few data have been obtained using the current standard bone histomorphometric procedure; that is, quantitative analysis of tetracycline-labeled iliac bone samples. Apart from several case reports, results from three small series of patients have been published.2, 20, 34 One of these20 examined adults, the other two included nine and four children, respectively. These studies provided valuable preliminary information, but were hampered by small sample numbers and the lack of adequate control groups.

Here we present quantitative static and dynamic histomorphometric data of tetracycline-labeled iliac bone specimens from 70 children, between 1.5 and 13.5 years of age, with types I, III, and IV OI. Results are compared with those of 27 age-matched controls without metabolic bone disease. The aims of this study are to assess the abnormalities of bone structure in OI and to analyze their age-dependency during bone development. Furthermore, we attempt to explain the observed structural abnormalities on the basis of indices reflecting bone cell function.

Section snippets

Subjects

The patient population comprised 70 children with OI, aged 1.5 to 13.5 years (Table 1). The patients were clinically classified according to the criteria established by Sillence.32 All patients had a history of frequent fractures and low bone mass, as assessed by dual-energy X-ray absorptiometry. Family history was positive for increased bone fragility in many cases, but this was not a prerequisite for the diagnosis of OI. Type I was diagnosed in patients who had a mild course, were of normal

Results

Figure 1 shows typical iliac bone sections from a control subject and from patients with OI types I, III, and IV. The decrease in biopsy size and in the amounts of cortical and cancellous bone are readily apparent in the OI samples. As seen under polarized light, a lamellar pattern is visible in the OI types presented here, but lamellae appear thinner and less smooth than in controls. Also, an increased number of osteocytes was evident in most samples, but this was not quantified in the

Discussion

In the present study we analyzed quantitative histological features in the three classical OI types that are compatible with postnatal survival. One of the most obvious abnormalities in OI is decreased bone mass, which has been reported in a number of previous histological and radiological studies.1, 2, 7, 10, 20, 28, 34, 37 Our data provide evidence that there are defects in all three mechanisms, which normally lead to an increase in bone mass during childhood. These are: (1) modeling of

Acknowledgements

The authors thank Guy Charette for the technical assistance with sample processing and Mark Lepik for the artwork. This study was supported by the Shriners of North America and by Deutsche Forschungsgemeinschaft (Grant Ra 803/1-1).

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Combination of osteogenesis imperfecta and hypophosphatasia in three children with multiple fractures, low bone mass and severe osteomalacia, a challenge for therapeutic management
2023, European Journal of Medical Genetics
Osteogenesis imperfecta (OI) and hypophosphatasia (HPP) are rare skeletal disorders caused by mutations in the genes encoding collagen type I (COL1A, COL1A2) and tissue-non-specific isoenzyme of alkaline phosphatase (ALPL), respectively. Both conditions result in skeletal deformities and bone fragility although bone tissue abnormalities differ considerably. Children with OI have low bone mass and hypermineralized matrix, whereas HPP children develop rickets and osteomalacia.
We report a family, father and three children, affected with growth retardation, low bone mass and recurrent fractures. None of them had rickets, blue sclera or dentinogenesis imperfecta. ALP serum levels were low and genetics revealed in the four probands heterozygous pathogenic mutations in COL1A2 c.838G > A (p.Gly280Ser) and in ALPL c.1333T > C (p.Ser445Pro). After multidisciplinary meeting, a diagnostic transiliac bone biopsy was indicated for each sibling for therapeutic decision.
Bone histology and histomorphometry, as compared to reference values of children with OI type I as well as, to a control pediatric patient harboring the same COL1A2 mutation, revealed similarly decreased trabecular bone volume, increased osteocyte lacunae, but additionally severe osteomalacia. Quantitative backscattered electron imaging demonstrated that bone matrix mineralization was not as decreased as expected for osteomalacia.
In summary, we observed within each biopsy samples classical features of OI and classical features of HPP. The apparent nearly normal bone mineralization density distribution results presumably from divergent effects of OI and HPP on matrix mineralization. A combination therapy was initiated with ALP enzyme-replacement and one month later with bisphosphonates. The ongoing treatment led to improved skeletal growth, increased BMD and markedly reduced fracture incidence.
Cell differentiation and matrix organization are differentially affected during bone formation in osteogenesis imperfecta zebrafish models with different genetic defects impacting collagen type I structure
2023, Matrix Biology
Osteogenesis imperfecta (OI) is a family of rare heritable skeletal disorders associated with dominant mutations in the collagen type I encoding genes and recessive defects in proteins involved in collagen type I synthesis and processing and in osteoblast differentiation and activity. Historically, it was believed that the OI bone phenotype was only caused by abnormal collagen type I fibrils in the extracellular matrix, but more recently it became clear that the altered bone cell homeostasis, due to mutant collagen retention, plays a relevant role in modulating disease severity in most of the OI forms and it is correlated to impaired bone cell differentiation. Despite in vitro evidence, in vivo data are missing. To better understand the physiopathology of OI, we used two zebrafish models: Chihuahua (Chi/+), carrying a dominant p.G736D substitution in the α1 chain of collagen type I, and the recessive p3h1^−/−, lacking prolyl 3-hydroxylase (P3h1) enzyme. Both models share the delay of collagen type I folding, resulting in its overmodification and partial intracellular retention. The regeneration of the bony caudal fin of Chi/+ and p3h1^−/− was employed to investigate the impact of abnormal collagen synthesis on bone cell differentiation. Reduced regenerative ability was evident in both models, but it was associated to impaired osteoblast differentiation and osteoblastogenesis/adipogenesis switch only in Chi/+. On the contrary, reduced osteoclast number and activity were found in both models during regeneration. The dominant OI model showed a more detrimental effect in the extracellular matrix organization. Interestingly, the chemical chaperone 4-phenylbutyrate (4-PBA), known to reduce cellular stress and increase collagen secretion, improved bone formation only in p3h1^−/− by favoring caudal fin growth without affecting bone cell markers expression. Taken together, our in vivo data proved the negative impact of structurally abnormal collagen type I on bone formation but revealed a gene mutation-specific effect on bone cell differentiation and matrix organization in OI. These, together with the distinct ability to respond to the chaperone treatment, underline the need for precision medicine approaches to properly treat the disease.
Osteocyte lacunae in transiliac bone biopsy samples across life span
2023, Acta Biomaterialia
Osteocytes act as bone mechanosensors, regulators of osteoblast/osteoclast activity and mineral homeostasis, however, knowledge about their functional/morphological changes throughout life is limited.
We used quantitative backscattered electron imaging (qBEI) to investigate osteocyte lacunae sections (OLS) as a 2D-surrogate characterizing the osteocytes. OLS characteristics, the density of mineralized osteocyte lacunae (i.e., micropetrotic osteocytes, md.OLS-Density in nb/mm²) and the average degree of mineralization (Ca_Mean in weight% calcium) of cortex and spongiosa were analyzed in transiliac biopsy samples from healthy individuals under 30 (n=59) and over 30 years (n=50) (i.e., before and after the age of peak bone mass, respectively).
We found several differences in OLS-characteristics:
1). Inter-individually between the age groups: OLS-Density and OLS-Porosity were reduced by about 20% in older individuals in spongiosa and in cortex versus younger probands (both, p < 0.001).
2). Intra-individually between bone compartments: OLS-Density was higher in the cortex, +18.4%, p < 0.001 for younger and +7.6%, p < 0.05 for older individuals.
Strikingly, the most frequent OLS nearest-neighbor distance was about 30 µm in both age groups and at both bone sites revealing a preferential organization of osteocytes in clusters. OLS-Density was negatively correlated with Ca_Mean in both spongiosa and cortex (both, p < 0.001). Few mineralized OLS were found in young individuals along with an increase of md.OLS-Density with age.
In summary, this transiliac bone sample analysis of 200000 OLS from 109 healthy individuals throughout lifespan reveals several age-related differences in OLS characteristics. Moreover, our study provides reference data from healthy individuals for different ages to be used for diagnosis of bone abnormalities in diseases.
Osteocytes are bone cells embedded in lacunae within the mineralized bone matrix and have a key role in the bone metabolism and the mineral homeostasis. Not easily accessible, we used quantitative backscattered electron imaging to determine precisely number and shape descriptors of the osteocyte lacunae in 2D. We analyzed transiliac biopsy samples from 109 individuals with age distributed from 2 to 95 years. Compact cortical bone showed constantly higher lacunar density than cancellous bone but the lacunar density in both bone tissue decreased with age before the peak bone mass age at 30 years and stabilized or even increased after this age. This extensive study provides osteocyte lacunae reference data from healthy individuals usable for bone pathology diagnosis.
Accelerated mineralization kinetics in children with osteogenesis imperfecta type 1
2023, Bone
Not much is known about the time course of mineralization in newly formed bone from healthy individuals nor in patients with bone disease. To investigate the early phase of mineral accumulation in human bone, we measured the mean mineralization content between double fluorescence labels (CaYoung) with quantitative backscattered electron imaging (qBEI) in human transiliac biopsy samples. Fluorescent labels for histomorphometric evaluation were administered over two 2-day periods separated by a 10-day-free interval, 4–5 days before biopsy procedure. We compared n = 19 children with osteogenesis imperfecta type 1 (OI, 6 girls/13 boys, age-range 2.2–14.1 years) with both a reference group of n = 38 healthy children (REF, 24 girls/14 boys, age-range 1.5–20.9 years) and an age-matched subgroup (n = 17) of the latter (CON, 5 girls/12 boys, age-range 2.0–14.7 years).
We observed significantly higher levels of CaYoung in OI type 1 compared to CON and REF in both cortical bone (Ct.CaYoung, +8.3 % and +7.0 %, respectively) and cancellous bone (Cn.CaYoung, +6.5 % and +4.9 %, all p < 0.001). When comparing cortical and cancellous compartments intra-individually, we found a significantly higher Cn.CaYoung than Ct.CaYoung in REF (2.3 ± 2.9 %, p < 0.001), but not in the OI group (0.6 ± 1.6 %, not significant). In the REF group, n = 7 samples also contained double labels in primary woven bone (which is deposited at the external cortex during growth/lateral drift of the iliac crest). This primary woven bone in REF had a higher CaYoung than secondary osteonal bone (4.9 ± 2.7 %).
Our findings suggest that bone in OI has an accelerated mineral accumulation compared to healthy bone. This is reflecting the overall increased bone mineralization in OI as reported previously, and indicates that the higher levels of mineralization than those seen in healthy individuals are already achieved in OI type 1 early after onset of mineralization.
Alterations of bone material properties in growing Ifitm5/BRIL p.S42 knock-in mice, a new model for atypical type VI osteogenesis imperfecta
2022, Bone
Osteogenesis imperfecta (OI) is a heterogenous group of heritable connective tissue disorders characterized by high bone fragility due to low bone mass and impaired bone material properties. Atypical type VI OI is an extremely rare and severe form of bone dysplasia resulting from a loss-of-function mutation (p.S40L) in IFITM5/BRIL,the causative gene of OI type V and decreased osteoblast secretion of pigment epithelium-derived factor (PEDF), as in OI type VI. It is not yet known which alterations at the material level might lead to such a severe phenotype. We therefore characterized bone tissue at the micrometer level in a novel heterozygous Ifitm5/BRIL p.S42L knock-in murine model at 4 and 8 weeks of age.
We evaluated in female mice, total body size, femoral and lumbar bone mineral density (BMD) by dual-energy X-ray absorptiometry. In the femoral bone we examined osteoid deposition by light microscopy, assessed bone histomorphometry and mineralization density distribution by quantitative backscattered electron imaging (qBEI). Osteocyte lacunae were examined by qBEI and the osteocyte lacuno-canalicular network by confocal laser scanning microscopy. Vasculature was examined indirectly by qBEI as 2D porosity in cortex, and as 3D porosity by micro-CT in third trochanter. Collagen orientation was examined by second harmonic generation microscopy. Two-way ANOVA was used to discriminate the effect of age and genotype.
Ifitm5/BRIL p.S42L female mice are viable, do not differ in body size, fat and lean mass from wild type (WT) littermates but have lower whole-body, lumbar and femoral BMD and multiple fractures.
The average and most frequent calcium concentration, CaMean and CaPeak, increased with age in metaphyseal and cortical bone in both genotypes and were always higher in Ifitm5/BRIL p.S42L than in WT, except CaMean in metaphysis at 4 weeks of age. The fraction of highly mineralized bone area, CaHigh, was also increased in Ifitm5/BRIL p.S42L metaphyseal bone at 8 weeks of age and at both ages in cortical bone. The fraction of lowly mineralized bone area, CaLow, decreased with age and was not higher in Ifitm5/BRIL p.S42L, consistent with lack of hyperosteoidosis on histological sections by visual exam. Osteocyte lacunae density was higher in Ifitm5/BRIL p.S42L than WT, whereas canalicular density was decreased. Indirect measurements of vascularity revealed a higher pore density at 4 weeks in cortical bone of Ifitm5/BRIL p.S42L than in WT and at both ages in the third trochanter. Importantly, the proportion of bone area with disordered collagen fibrils was highly increased in Ifitm5/BRIL p.S42L at both ages.
Despite normal skeletal growth and the lack of a collagen gene mutation, the Ifitm5/BRIL p.S42L mouse shows major OI-related bone tissue alterations such as hypermineralization of the matrix and elevated osteocyte porosity. Together with the disordered lacuno-canalicular network and the disordered collagen fibril orientation, these abnormalities likely contribute to overall bone fragility.
Educational Case: Osteogenesis imperfecta
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ORIGINAL ARTICLESStatic and dynamic bone histomorphometry in children with osteogenesis imperfecta

Abstract

Introduction

Section snippets

Subjects

Results

Discussion

Acknowledgements

Orthop Clin Am

Bone

Am J Clin Nutr

Bone

Bone

Matrix Biol

Osteogenesis imperfecta tarda. The morphology of rib biopsies

Clin Orthop

Increased bone turnover with decreased bone formation by osteoblasts in children with osteogenesis imperfecta tarda

Pediatr Res

Anatomy and ultrastructure of bone

The mineralization density of iliac crest bone from children with osteogenesis imperfecta

Calcif Tissue Int

Serum concentrations of procollagen I C-terminal propeptide, osteocalcin and insulin-like growth factor-I in patients with non-lethal osteogenesis imperfecta

Acta Paediatr

Bone resorption assessed by immunoassay of urinary cross-linked collagen peptides in patients with osteogenesis imperfecta

J Bone Miner Res

Osteogenesis imperfectaA histometric analysis

J Bone Jt Surg [Am]

Quantitative analysis of trabecular morphogenesis in the human costochondral junction during the postnatal period in normal subjects

Anat Rec

Extracellular matrix stoichiometry in osteoblasts from patients with osteogenesis imperfecta

J Bone Miner Res

Computed tomographic assessment of vertebral bone mineral in childhood

Skel Radiol

Osteogenesis imperfecta. The set point proposal (a possible causative mechanism)

Clin Orthop

ORIGINAL ARTICLES
Static and dynamic bone histomorphometry in children with osteogenesis imperfecta