No Benefit To Taking Bisphosphonates More Than Five Years – Current FDA Report
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Bone Matrix and Mineral
Raman imaging of two orthogonal planes within cortical bone
The characterization of bone material quality which essentially contributes to the mechanical properties of bone is rather difficult due to bone’s hierarchical structure. The bone material itself is a nano-composite consisting of hydroxyapatite mineral particles embedded in collagen fibres. Generally, the combination of these components (stiff and hard mineral particles with highly elastic and tough collagen fibres) provides rigidity and resistance against fracture. However, mechanical behaviour of the bone material is highly influenced by several structural aspects such as tissue organization, amount of mineral, orientation and cross-linking of collagen. Raman microspectroscopic and imaging analysis represents a rather novel method for studying bone material properties with a high spatial resolution. The principle of this technique is based on light scattering and therefore Raman spectroscopy can be used in reflection mode of polished bone samples for simultaneous information on the mineral and the organic component. However, as already described previously Raman line intensities depend strongly on both the chemical composition of the tissue and on the fibre orientation with respect to the linear polarization direction of the exciting laser beam. While n1 PO4 and amide I bands were sensitive to the orientation amide III, n2 PO4 and n4 PO4 were less influenced by orientation effects. In the present study, Raman microspectroscopic and imaging analyses were employed to study a normal human femoral midshaft bone cube-like specimen with a spatial resolution of about 1–2 μm. Identical bone lamellae in both longitudinal and transverse directions were analyzed, which made the separation of orientation and composition effects of Raman lines depending on the laser beam polarization directions possible. Consequently, information on lamellar bone orientation and variation of bone composition was available. It was shown that the n1 PO4 to amide I ratio mainly displayed lamellar bone orientation; n2 PO4 to amide III and CO3 to n2 PO4 ratios displayed variations in bone composition. Compared to osteonal bone, the n2 PO4 to amide III ratio was higher in the interstitial bone region, whereas the CO3 to n2 PO4 ratio has lower values in the same region. Fig. 1 shows additionally the importance of the polarization direction. Structures lying perpendicular to this direction in (A) are almost invisible while in (B) the same structures (now parallel to the polarization direction) are clearly visible.
The bone mineralization density distribution as a fingerprint of the mineralization process
Bone is a heterogeneous material consisting of areas of different mineral content distributed on a microscopic scale. The reason for this inhomogeneous pattern of mineral content are the kinetics of bone deposition (modelling and remodelling activities of the bone cells) and the kinetics of mineralization (the mineral apposition in the newly formed collagen). The heterogeneity of mineral content can be quantified by the bone mineralization density distribution (BMDD) which is the frequency distribution of the occurring calcium concentrations. It was shown that the BMDD is one important determinant of bone quality in view of the mechanical properties of the bone material. Experimentally, the BMDD can be accessed by quantitative backscattered electron imaging (qBEI) for instance. In the present work, computed modelling of the BMDD was used for a deeper understanding of how the kinetics of bone deposition (rate of bone turnover) and the kinetics of mineralization affect the shape of the BMDD. We demonstrate that the shape of the trabecular BMDD histogram reflects directly the mineralization kinetics. Using a mathematical model for the remodelling and the mineralization process and the experimentally obtained trabecular BMDD from healthy human adults the following main results were obtained. A two-phase mineralization process with a fast primary phase and a slow secondary phase (with the corresponding time constants differing three orders of magnitude) is necessary to receive a peaked BMDD. The obtained mineralization law describes the increase in the mineral content in a bone packet as a function of time and is shown to influence not only the initial mineralization surge but also the slow increase afterwards on the time scale of years. Additionally, the turnover rate of the remodelling process has a strong influence on the peak position and the shape of the BMDD. This theoretical work helps to separate the effects of variations in bone turnover or alterations in the kinetics of mineralization on the experimentally measured BMDD. It allows addressing whether changes in the BMDD have to be attributed to a variation in the turnover rate which consequently affects the density distribution or to a primary disorder in the mineralization process most likely caused by alterations of the organic matrix. This has an important clinical impact since it may help to understand the primary etiological defects of an altered BMDD and to find therapeutic approaches targeting these defects to correct the patient’s BMDD towards normal.
Scanning texture analysis of lamellar bone using microbeam synchrotron X-ray radiation
Cortical or compact bone is an example of a hierarchically structured biocomposite, which is built mainly of cylindrical osteons (or so called Haversian systems) which are concentric cylindrical sheets of mineralized collagen around a blood vessel. The texture (the spatial distribution of the orientation of the crystallites) of a material such as osteonal bone affects the mechanical properties essentially. For a deeper mechanical understanding it is important to characterize the osteonal bone structure at fibre composite (micrometer) level quantitatively. In the present work, we describe the novel combination of microbeam synchrotron X-ray texture analysis with thin sections of osteonal bone to measure the three-dimensional distribution of the c-axis orientation of the mineral apatite in bone with a positional resolution of 1 mm. Using lateral sample scanning with single axis rotation together with the two dimensional position-sensitive detector provides the reconstruction of the mineralized fibril orientation within the osteon. The aim of the present work was the detailed description of the technique, the data reduction procedure needed to go from the stereographic projection of X-ray intensity to the determination of the local orientation of mineralized collagen fibrils. This procedure is of interest also for studying other polycrystalline materials with micrometer scale and biologically controlled fibrillar texture such as other mineralized tissues (trabecular bone and chitin e.g.).
J Appl Cryst 40:115-120
Cell & Molecular Biology
Three-dimensional growth behaviour of osteoblasts on biomimetic hydroxylapatite scaffolds
In addition to autografts synthetic materials (such as polymers, metals or ceramics) play an important role in the development of bone replacement materials. Among those, hydroxylapatite (HA) - based bioceramics have the advantage that they are chemically similar to bone mineral. Additionally to their chemical composition, the architecture (porosity) is essential for biocompatibility. Pore size, type and interconnectivity should be within certain ranges to provide optimal cell migration and flow of body fluids. In the present work, we studied preosteoblastic cells seeded on a three-dimensional scaffold produced by rapid prototyping (allowing controlled, fully interconnected porosity). The effect of hormones on the osteogenic differentiation and how osteoblasts colonize the scaffold focusing on the formation of the cellular network were investigated. The preosteoblasts were optionally treated with the osteogenic hormones triiodo-L-thyronine (T3) and 1,25-dihydroxyvitamin-D3 (D3), and the expression of osteoblastic marker genes was investigated. Confocal laser scanning microscopy was used to investigate the three-dimensional growth behaviour (see Figure 2). Culturing cells on 3-dimensional scaffolds strongly increased the expression of, osteoprotegerin, Runx2, and receptor activator of NFkB-ligand (RANKL) compared to a standard 2-dimensional culture system. Treatment with T3 increased the expression of osteocalcin but did not change that of osteoprotegerin and Runx2. Treatment with D3 inhibited the expression of osteocalcin, Runx2, and osteoprotegerin. Both hormones had similar effects in the three-dimensional system as found in two-dimensional cultures although more accentuated, indicating that preosteoblasts behave more naturally on three-dimensional structures. Investigating kinetics of tissue formation revealed that the osteoblasts completely covered the surface of the scaffold and had the tendency to fill micorpores or cracks present in the scaffold. With ongoing time, the cells colonized the three-dimensional squared pores of scaffolds by forming a cellular network starting in the corners and forming a round central channel keeping it into the depth. These cellular networks consist of a highly organized collagenour matrix with embedded cells and look similar to bone osteons with the Haversian canal being the central channel. Our finding showed that a three-dimensional environment promoted the differentiation of pre-osteoblasts to a mature osteoblastic phenotype compared to normal two-dimensional cell culture systems. In conclusion, the 3-dimensional HA scaffolds provide an environment more similar to the in vivo situation and are therefore an excellent tool for studying growth and differentiation of these cells.
J Biomed Mater Res 2007; 81A:40-50
Evaluation of Biocompatible Photopolymers I: Photoreactivity and Mechanical Properties of Reactive Diluents
Artificial bone substitute materials must be biocompatible (not toxic or mutagen), should reveal appropriate mechanical stability and should be osteoconductive (they should support the growth and differentiation of osteogenic cells). Bone substitutes with well-defined geometry and inner trabecular-like structure can be generated by the polymerization of specific acrylate-based components during rapid prototyping. However, further investigation is needed to find monomer formulations which can be photopolymerized and which are optimal in biocompatibility and in their mechanical behaviour. Therefore, different commercially available monomers (either mono-acrylates or multi-substituted monomers together with different cross-linkers, reactive diluents, fillers and initiators) were studied in the present work. For rapid prototyping, the short building time of the material is an important criterion which was measured by differential scanning photocalorimetry (Photo-DSC). Three-point bending tests revealed stiffness and strength of the materials. Viability of osteoblast-like cells was related to the functional groups in the monomers present, e.g., oligoethyleneglycol, urethane-, hydroxy- or carboxy groups. Considering the building times, multi-substituted monomers yielded lower double bond conversion than mono-acrylates due to the network formation. It was found that polymers obtained from acrylates with urethane units, most dialkylacrylamide and especially trimethylolpropane triacrylate gave outstanding biocompatibility. Mechanical testing proved that they have significantly better performance than many known thermoplastic biopolymers.
Journal of Macromolecular Science, Part A, 44:547-557
Evaluation of Biocompatible Photopolymers II: Further Reactive Diluents
Bone replacement materials are needed in medicine in cases of bone loss by trauma, disease or tumor resection to provide mechanical stability. Most important properties of the implanted material are its biocompatibility and bioresorbability and the support of attachment and differentiation of osteogenic cells. Up to now, autografts (bone tissue from the same subject) or allografts (bone tissue from another subject) are used for this purpose but limited availibilty and the risk of viral transmission are their disadvantages. For these reasons, artificial material has been developed during recent years by stereolithography which offers the production of materials with a well-defined highly complex geometry and inner structure. However, substances are needed which are polymerizable (for the stereolithography technique), biocompatible and which show adequate mechanical stability. Up to now, biocompatibilty was proven for solid photopolymerizable polymers which alone are not appropriate for stereolithography. In this work, we developed a new monomer formulation – consisting of a biodegradable basis monomer, reactive diluents, fillers, and an appropriate photoinitiator – for the stereolithographic fabrication of bone replacement materials. Testing of several acrylate based reactive diluents – having different functional groups – was done including photoreactivity, biocompatibility, and mechanical properties. Dynamical mechanical testing and 3-point bending tests revealed excellent strength and stiffness for polymers from di- or multiacrylated monomers which form dense networks. Considering also the two other criteria (photoreactivity and biocompatibility), polymers from tripropylene-glyco-diacrylate, propoxylated-glycerol-diacrylate and tricyclodecan-dimethanol-diacrylate showed the most promising results. All monomers tested can be used for stereolithography. Fig. 3 shows a spongiosa-like structure generated by sterolithography using 30wt% hydroxyapatite as osteoconductive filler which further improves mechanical and biocompatibility properties.
Chemical monthly 138:261-268
Novel PHEX mutation associated with hypophosphatemic rickets
X-linked hypophosphatemia (XLH) - the most prevalent heritable form of rickets - is dominantly inherited and clinically characterized by rickets or osteomalacia, short stature, bone pain, enthesopathy, poor dental development, defective bone mineralization and deformation of the lower extremities. Radiographic examination often reveals increased axial and decreased peripheral bone mass in these patients. Laboratory parameters give evidence for abnormalities in XLH including renal phosphate wasting, abnormal vitamin D and PTH metabolism. During recent years, 179 inactivating mutations in the gene encoding PHEX (phosphate regulating gene with homologies to endopeptidases on the X chromosome) have been found to be associated with XLH. There are indications that truncating mutations are associated with the more severe forms of XLH, however, no correlation between mutation and clinical phenotype has been proved so far. In the present work, we report about a 54-year-old male patient, who exhibited the typical features of XLH. This patient showed highly elevated FGF23 and PTH levels. Bone resorption serum markers (cross-laps) and osteocalcin, a maker for bone remodeling, was significantly above the normal range as well. An increased axial BMD score was measured and radiographs of the lower limbs showed multiple old fatigue fractures together with increased cortical thickness and bone diameter. Genomic DNA was extracted from whole blood samples and mutational analysis was performed. We found a 1bp-deletion in exon 2 of the PHEX gene (177delC), which has not been reported yet. This deletion results in a premature stop codon (C59X), suggesting a truncation of the PHEX protein at the beginning of the extracellular domain. For conclusion, this novel mutation in the PHEX gene was associated with a severe form of XLH. These findings support previous results and contribute to deciphering the pathogenetic pathways of XLH.
Nephron Physiol 106:8-12
New observations on bone quality in mild hyperparathyroidism as determined by quantitative backscattered electron imaging
Mild primary hyperparathyroidism (PHPT) is a relatively common endocrine disease best characterized by asymptomatic hypercalcemia, most commonly in the absence of classical signs and symptoms. The majority of PHPT patients are postmenopausal women who have normally no skeletal or renal complications. However, BMD (DXA) and histomorphometric measurements reveal differential effects on cortical and cancellous bone caused by the elevated PTH level: cortical bone is thinned while trabecular bone is preserved in PHPT. The effects of PHPT on the intrinsic bone material quality have not been studied yet. Hence, there is need to characterize this disorder with particular attention to the bone material. Bone mineralization density distribution (BMDD) of transiliacal bone from 51 patients with PHPT (16 men, 28-68 years of age; 35 women, 26-74 years of age) was analyzed by quantitative backscattered electron imaging (qBEI). Four parameters were used to characterize the shape of the BMDD: CaMEAN, the weighted mean calcium concentration; CaPEAK, the most frequent Ca concentration; CaWIDTH, the width of the distribution, a measure of the mineralization homogeneity; and CaLOW, the percentage of bone area that is mineralized below the 5th percentile in the reference range. The results were compared to normal reference BMDD data we have previously established. The greatest differences were found in CaWIDTH (+15.7%, p<0.0001) and CaLOW (+44.7%, p<0.001), both of which were significantly higher in PHPT than control. CaMEAN was significantly lower (-2.5%, p<0.0001) in PHPT compared with controls. The BE-image of the transiliacal bone biopsy together with corresponding BMDD for a typical patient can be seen in Fig. 4. These differences were reversed in seven patients who underwent parathyroidectomy. CaMEAN and CaPEAK variables were negatively, whereas CaWIDTH and CaLOW were positively correlated with mineralizing surface and bone formation rate (both dynamic variables of bone formation determined by histomorphometry: Correlation coefficient r varying from +/-0.3 to 0.8 and statistical significance p varying from 0.05 to 0.0001. These results are the first BMDD measurements in mild PHPT using qBEI. We found a reduction in the average mineralization density and an increase in the heterogeneity of the degree of mineralization. These changes were significantly correlated with the bone turnover rate revealing that the higher bone turnover rate in PHPT is leading to a reduction of mean bone tissue age. The relevance of this finding for the mechanical strength of bone in PHPT is unknown. In most cases, lower mineralization densities were associated with compromised bone strength, however, no increase of fracture risk has been reported for patients with mild PHPT so far. Further studies are needed to draw conclusions about the altered BMDD and its relation to fracture risk in this disease.
J Bone Miner Res 22:717-723
Prophylactic bisphosphonate treatment prevents bone fractures after liver transplantation
Patients who are referred to liver transplantation (LT) are known to have an increased fracture risk associated with lower bone mass (lower bone mineral density (BMD)) and altered bone architecture. After LT, several factors such as the administration of immunosuppressive agents and immobilization of the patients even increase fracture risk in the liver recipients. During recent years, bisphosphonates which are commonly used for the treatment of postmenopausal osteoporosis have also been described for the treatment of bone loss after organ transplantation. In this work, a randomized controlled prospective open-label single center trial was performed for studying the effect of zoledronic acid (ZOL) a potent novel bisphosphonate on fractures in LT patients. At the time of transplantation patients were randomly assigned to one of two treatment arms: The study group (n= 47 patients) received ZOL (8 infusions at 4 mg during the first 12 months after LT), calcium (1000 mg/d) and vitamin D (800 IE/d). The control group (n= 49 patients) received calcium and vitamin D at same doses (CON). Primary endpoints were the incidence of bone fractures or death; secondary endpoints included bone mineral density (BMD), serum biochemical markers of bone metabolism, parameters of trabecular bone histomorphometry and bone mineralization density distribution (BMDD). Patients were followed up for 24 months. Analysis was performed on an intention-to-treat basis. The primary endpoint fracture or death was reached in 26% of patients in the ZOL group and 46% in the CON group (p= 0.047, log rank test). BMD measures were significantly different between the groups at the femoral neck at 6 months after LT (mean+/-SD, ZOL: 0.80 +/- 0.19 g/cm2 vs. CON: 0.73 +/- 0.14 g/cm2, p= 0.036). Mixed linear models of biochemical bone markers showed less increase of osteocalcin, histomorphometrical and bone mineralization parameters indicated reduced bone turnover in the ZOL group. For conclusion, it was shown that prophylactic treatment with the bisphosphonate zoledronic acid reduces bone turnover and fractures after LT.
American Journal of Transplantation 7:1763-1769
Evidence that Treatment with Risedronate in Women with Postmenopausal Osteoporosis Affects Bone Mineralization and Bone Volume
Bisphosphonates are known to reduce bone turnover markers, to increase bone mineral density (BMD) and to decrease vertebral and non-vertebral fracture risk in postmenopausal osteoporosis. While it is generally accepted that low BMD is associated with increased fracture risk, it was shown that the fracture risk reduction after bisphosphonate therapy was poorly related to the increase in BMD. One of the difficulties is that BMD is a surrogate parameter representing the total amount of bone mineral in a volume of bone tissue, thereby including the effects of bone tissue volume and the mineral content of the bone tissue on the X-ray absorption. Additionally, BMD is also influenced by the geometry of bone at the studied site. For these reasons it is difficult to interpret the changes in BMD together with fracture risk reduction in bisphosphonate treated patients. In this work, we combine BMD results with measures of the mineral content of the bone tissue as obtained from quantitative backscattered electron imaging from the same patient before and after bisphosphonate treatment. The studied transiliacal biopsies were from postmenopausal women enrolled in the Vertebral Efficacy with Risedronate Therapy trial who received either risedronate (n=18, 5 mg/day) or placebo (n=13) for 3 years. All patients received calcium and vitamin D supplementation if deficient at baseline. The novel combination of lumbar spine BMD values (at baseline and at 3 years of treatment with risedronate) with the mineral volume fraction at the same time points for the same patients made the calculation of the relative change in trabecular bone volume with treatment possible. The results showed that changes in both matrix mineralization and bone volume contributed to the changes in BMD. After risedronate, there was a trend of an increase in trabecular bone volume for the risedronate group (+2.4%, nonsignificant) but a significant decrease (-3.7%, P < 0.05) for the placebo group. Calcium supplementation with adequate levels of vitamin D independent of risedronate increased the bone tissue mineral content about 3.3% (with the increase being larger in patients with lower baseline matrix mineralization). In conclusion, by combining BMD and bone mineralization density distribution data we could show that 3-year treatment with risedronate of postmenopausal osteoporotic patients preserves or may increase trabecular bone volume, unlike placebo.
Calcif Tissue Int 80:73-80
High-dose bisphosphonate therapy in an urgent case of spontaneous multiple fractures in a 55 year old woman
Bisphosphonates are widely used for the treatment of postmenopausal osteoporosis. They are known to increase vertebral and non-vertebral bone mineral density (BMD) and to reduce fracture risk. As previous works have shown, part of the increase in BMD is due to an increase in bone mineralization. In the present work, we report a high-dose bisphosphonate therapy in an early postmenopausal Caucasian woman aged 55 years who sustained multiple vertebral fractures after a minor trauma. Any kind of secondary osteoporosis could be excluded. Due to clinical severity we administered combined oral and cyclic intravenous bisphosphonate therapy (oral risedronate 35 mg/week, iv pamidronate 30 mg quarterly) with adequate calcium and vitamin D supplementation for 28 months. Transiliacal bone biopsies were obtained at baseline and at month 28. The paired samples were investigated by histomorphometry, by microCT-analysis for 3D structure and by quantitative backscattered electron imaging (qBEI) for bone mineralization density distribution (BMDD). At baseline, the patient had lower normal vitaminD and calcium levels and indices of bone turnover were reduced. Interestingly, the BMDD was significantly shifted to lower mineralization densities compared to normal. Bisphosphonate treatment and supplementation of calcium and vitamin D increased BMD and trabecular bone volume (BV/TV). The BMDD was found more narrow which is considered as typical change after bisphosphonate treatment. However, no increase in mineralization densities occurred. These findings indicate that the high-dose bisphosphonate therapy did not influence bone mineralization significantly in this patient but improved bone architecture and BMD, reduced pain, increased mobility and prevented further osteoporotic fractures during the time of investigation.
Wien Med Wochenschr 157:388-391
Patients with Rare Syndromes
Asymmetrical skull, ptosis, hypertelorism, high nasal bridge, clefting, umbilical anomalies, and skeletal anomalies in sibs: is Carnevale syndrome a separate entity?
In this work, we present two patients, a brother and sister who both have a combination of symptoms which are found in Malpuech syndrome, Michels syndrome, Carnevale syndrome, OSA syndrome, and Mingarelli syndrome. These syndromes have in common the combination of highly arched eyebrows, ptosis, and hypertelorism, and vary in other symptoms such as asymmetry of the skull, eyelid, and anterior chamber anomalies, clefting of lip and palate, umbilical anomalies, and growth and cognitive development. We conclude that the present patients resemble most patients with Carnevale and Mingarelli syndrome, and the case reported by Guion-Almeida. Patients with one of these three syndromes share the most important manifestations and are suggested to form together most probably the same entity which we suggest to call Carnevale syndrome since this author described this combination of symptoms for the first time. Our present patients and previous reports indicate that the pattern of inheritance of Carnevale syndrome is most likely autosomal recessive. Malpuech and Michels syndromes are probably separate entities, although they could be allelic.
Am J of Med Genet Part A 143:349-354
Unusual facies, thumb hypoplasia, distinctive spinal fusions and extraspinal mobility limitation, in a pair of monozygotic twins
A combination of vertebral fusions with other extraspinal joint fusions has been described for spondylocarpotarsal synostosis syndrome and the proximal symphalangism syndrome. Non-infectious anterior vertebral fusion occurs also during the development of Forestier disease which is normally seen in the elderly. We report a pair of monozygotic twins with unusual facies and hypoplastic thumbs associated with progressive spinal fusion and joint immobility. The radiographic features were neither consistent with the multiple synostosis syndrome of Herrmann, nor with the spondylocarpotarsal synostosis syndrome. The overall spinal radiographic abnormalities seen in our patients were suggestive of an exceptionally early onset of Forestier disease (anterolateral, perivertebral, ligament ossification). However, the thumb hypoplasia and pterygium colli are not seen in Forestier disease. We suggest that the latter as observed in our patients can be considered as a novel syndrome association.
Clinical Dysmorphology 16:151-155
Vertebral hyperostosis, ankylosed vertebral fracture and atlantoaxial rotatory subluxation in an elderly patient with a history of infantile idiopathic scoliosis; a case report
Diffuse idiopathic skeletal hyperostosis (DISH) is characterized by new bone formation at the entheses leading to the narrowing of the spinal canal and is usually seen in male patients older than 45 years. DISH is normally asymptomatic but can predispose the patient to fractures, dysphagia, cervical and/or thoraric myelopathy, paraplegia and densie spinal cord injury caused by even minor trauma. While a strong correlation between osteoporosis and the development of DISH has been described, scoliosis is not considered as a major risk factor for the development of DISH. This is a case report of a 48-year-old-woman with scoliosis since early childhood. Recent radiographic spinal assessment revealed the presence of distinctive spinal abnormalities. The patient revealed spinal osteoporosis with DISH. Our observations suggest that infantile scoliosis may be a possible confounder in the relationship between spinal osteoporosis and the development of DISH.
Journal of Medical Case Reports 2007;1: 25
Progressive vertebral fusion in a girl with spinal enchondromatosis
Endochrondromas are ectopic hyaline cartilage rests which are likely displaced from the growth plate and replace normal intramedullary bone. These lesions are metabolically active; they grow and usually calcify with time. Often, patients with endochrondromas are asymptomatic, however, the lesions can cause fractures and sometimes show malignant transformations. We report on a 9-year-old girl who presented with progressive restriction of spine mobility. Standard spinal radiographs revealed enchondromatous lesions spread out along the anterior end plates of the lumbar vertebrae and MR imaging showed progressive anterior vertebral fusion. The long bones, short tubular bones and flat bones were free of enchondromata, but arcs of chondroid calcifications were observed in the proximal femoral metaphyses. The father of the patient revealed progressive ossification of the spinal ligament, however, no indices of a metabolic disorder. We suggest that father and daughter may have different manifestations of the same disorder. Considering that the clinical and the radiographic features encountered in our patient did not fit into any previous report we suggest that our patient has a novel form of endochrondomatosis.
European Journal of Radiology Extra 2007;63:125–129
Craniocervical junction malformation in a child with oromandibular-limb hypogenesis-Möbius syndrome
Occiput-atlas abnormalities in children are mainly caused by trauma. In this study, we report a male child with such abnormalities, however, associated with Oromandibular-limb hypogenesis (OMLH) – Möbius syndrome. Main features were bilateral sixth and seventh nerve palsies, limb anomalies and hypoplasia of the tongue, additionally shortness of the neck associated with torticollis. Radiographs of the cervical spine were non-contributory, but 3D computed tomography (CT) scanning of this area identified: a) congenital hypoplasia of the atlas; b) the simultaneous development of occiput-atlas malformation/developmental defect. Hazardous outcome of occiput-atlas developmental abnormalities have already been described in the literature before but the latter had never been related to a preexisting craniocervical defect. To our knowledge, this is the first clinical report assessing the cervico-cranium malformation in a child with OMLH-Möbius syndrome. In addition, our findings suggest that craniocervical junction risks should be carefully assessed and that CT imaging offers the feasibility of the evaluation of the craniocervical junction.
Orphanet Journal of Rare Diseases 2007;2:2 doi:10.1186/1750-1172-2-2
Ischiopubic and odontoid synchondrosis in a boy with progressive pseudorheumatoid chondrodysplasia
Progressive pseudorheumatoid chondrodysplasia (PPRC) is an autosomal recessive inherited chondrodysplasia characterized by the absence of inflammatory parameters. Our patient was a 14 years old boy who was initially diagnosed with juvenile rheumatoid arthritis. Further investigation revealed, however, no active inflammatory parameters. The patient was examined using 3D-computer tomography (3D-CT) scans. There was synchondrosis between the odontoid and the body of the axis and the cephalad part of the odontoid was detached. Bilateral ischiopubic ossification defects and ischiopubic and odontoid synchondroses were additional abnormalities. 3D-CT scans showed an orthotopic type of os odontoideum associated with an occult axial fracture. The persistence of an infantile odontoid, with a large pre-adulthood head in children with skeletal dysplasias, is a major risk factor for sudden death and significant morbidity. The early identification of these malformations must be followed by comprehensive orthopedic management.
Pediatric Rheumatology 2007;5:19
Distinctive spinal changes in two patients with unusual forms of autosomal dominant endosteal hyperostosis: a case series
Endosteal hyperostosis is characterized by facial dysmorphism and diaphyseal radiographic changes present at adolescence. Thickening of the endosteum of the long bones and the skull are typically seen in radiographic examinations. Rarer are the increase in the density of the posterior elements of the spine or an increased fracture incidence as described only for few patients. Our patients, a 26 years old man and his 6 years old daughter with endosteal hyperostosis presented both with fractures. Interestingly, both patients revealed also sclerosis of the posterior spinal elements and ondontoid process hyperplasia. The latter associated with both, fractures and endosteal hyperostosis, was observed for the first time.
Journal of Medical Case Reports 1:142
Persistent torticollis, facial asymmetry, grooved tongue, and dolicho-odontoid process in connection with atlas malformation complex in three family subjects
In this study, we describe the connection between congenital malformations of the cervico-cranium with the constellation of craniofacial features. Our patients in this study are siblings and their mother. They presented with congenital, persistent torticollis, phagiocephaly, facial asymmetry, grooved tongues and asymptomatic “dolicho-odontoid process”. 3D-scans were used to further visualize the cervico-cranial malformation complex in this family. Based on these analyses we suggest that the development of the “dolicho-odontoid process” in our patients was caused by the coexistence of three malformations concerning the atlas. These were the presence of an aplastic posterior arch of the atlas, the fusion of the residual part of the posterior arch to the posterior lip of the foramen magnum together with the clefting of the anterior arch of the atlas. All together were responsible for the progressive shortening of the anatomical distance between the axis and the foramen magnum. The “dolchio-odontoid process” might increase fracture risk resulting in neurological complications in our patients. Therefore it is important to consider the diagnosis of a congenitally malformed atlas in patients with congenital, persistent torticollis, phagiocephaly, facial asymmetry and grooved tongues.
European Spine Journal 16:S265-S270
Summary of the “highlights” published 2006
From brittle to ductile fracture of bone
Most biological materials which have a predominantly mechanical function reveal a hierarchical structure comprising a number of different hierarchical levels at different size scales. Human bone is a complex material and consists of elastic collagen fibers reinforced with nanosized mineral particles. Toughness (a measure for the energy necessary to break the bone) is crucial to the structural function of bone. Usually, the toughness of a material is not just determined by its composition, but by the ability of its microstructure to dissipate deformation energy without propagation of the crack. Polymers are often able to dissipate energy by viscoplastic flow or the formation of non-connected microcracks. In ceramics, well-known toughening mechanisms are based on crack ligament bridging and crack deflection. Interestingly, all these phenomena were identified in bone, a material which reveals both aspects – the fibrous polymer (collagen) and ceramic nanoparticles (carbonated hydroxyapatite). In our work, we used controlled crack-extension experiments in three-point bending tests to explain the influence of fibre orientation on the energy required to propagate a crack through the bone material. We found the fracture energy to change by two orders of magnitude depending on the collagen orientation, and the angle between collagen and crack propagation direction is decisive in switching between different toughening mechanisms. The pictures (FIGURE 1) show the two extreme cases with the crack perfectly aligned with the collagen fibrils (A) or perpendicular to the collagen fibrils (B). In the first case (A) and for low collagen angles with respect to the crack direction, the path of the crack is straight and the flanks appear smooth which is typical for a brittle fracture like in a ceramic. In (B) and for high collagen angles, the main crack is torn perpendicular to the propagation path characteristically to a quasi-ductile fracture. Within bone tissue, the collagen fibril angle varies which leads to an increased fracture resistance compared to a material with perfectly aligned fibrils.
Spiral twisting of fiber orientation inside bone lamellae
Compact bone consists mainly of secondary osteons which are multilayered cylindrical structures of mineralized collagen fibrils arranged around a blood vessel. On the one hand, the blood vessels represent the main transport system for metabolites and need therefore special protection in bone. On the other, from a mechanical viewpoint these vessels running through the bone material reveal micro-sized defects. It is therefore obvious that the osteon has also an important mechanical function. Functionally, the osteon must be adapted to the in vivo mechanical stresses in bone at the level of its microstructure. However, the precise mechanisms of the mechanical adaptation have not been clarified yet. In this work, we studied the structure of the osteon by the application of scanning x-ray diffraction with a micron-sized beam at the synchrotron. Quantitative texture analysis was needed to measure the local mineral crystallographic axis direction. Clearly, a combination of a small X-ray beamsize (smaller than a single fibril) together with a sample thickness of the same size, stepwise rotation of the sample for local texture measurements and the alignment of the beam position relative to the fiber orientation in the tissue made it possible to resolve the local crystallite orientation quantitatively. These experiments made the reconstruction of the three-dimensional orientation of the mineralized fibrils within a single osteon lamella (of a size of about 5 µm) possible. We found the mineralized collagen fibrils to spiral around the central axis with varying degrees of tilt (FIGURE 2). The orientation angle of the fibrils relative to the osteon axis in successive layers changes in a regular sequence of about 5°-25°. The sense of the helical winding is right-handed and the chirality is the same for all lamellae except the outermost one. This twisted orientation allows high extensibility in tension like a spring and protects the blood vessel against failure of the surrounding bone matrix. The deformation within osteons will occur mostly by means of shear between the fibrils.
Does mRNA level of microsomal carnitine palmitoyltransferase predict yield of peripheral blood stem cell apheresis?
After high-dose interveneous chemotherapy of a wide range of hematopoietic malignancies and some solid tumor entities the transplantation of autologous hematopoietic stem cells is a well established therapeutic procedure. These stem cells are usually harvested by peripheral blood apheresis after mobilization of the stem cells through cytokines or after a combination of chemotherapy and cytokine treatment. Despite advances in efficacy of the stem cell mobilization and apheresis process until now a predictive factor for the expected stem cell yield before initiation of mobilization therapy could not be identified. There are patients who cannot be mobilized by conventional mobilization strategies and who have an unpredictable low hematopoietic regenerative potential. The main objective of our study was to evaluate alterations in enzymes involved in fatty acid metabolism on the level of gene expression in mononuclear cells, as changes in relative mRNA levels of these enzymes could represent the hematopoietic regenerative potential. The alterations of different marker genes and their regulative role in hematopoiesis are on the one hand of theoretical interest, on the other hand, they are a key goal of research for the practical application by correlating gene expressions and the outcome of stem cell mobilization. Data of 23 consecutive patients with different lymphoid malignancies undergoing stem cell mobilization were analyzed. Our results show that mRNA levels of microsomal carnitine palmitoyltransferase in peripheral blood mononuclear cells quantified before application of mobilization therapy correlate positively with the amount of CD34 positive cells in peripheral blood before first apheresis, in the first apheresis product and in the total harvest outcome. The association of enzymes involved in fatty acid metabolism with hematopoiesis was further confirmed in healthy subjects on altitude-adaptation training and in proliferating or differentiating HL60 cells. This gives evidence for a possible predictive value of such analyzes though further data of a larger sample are to be collected to confirm our observations.
Regulation of adult bone mass by the zinc finger adapter protein Schnurri-3
Genetic mutations that disrupt osteoblast function can result in skeletal dysmorphogenesis or, more rarely, in increased postnatal bone formation. Here we show that Schnurri-3 (Shn3), a mammalian homolog of the Drosophila zinc finger adapter protein Shn, is an essential regulator of adult bone formation. The original aim of this study was to investigate the function of Shn3 in the immune system by generating knockout mice missing the gene encoding the Shn3 protein. The knockouts did exhibit defects in the immune system but these defects were nowhere near as profound as the skeletal phenotype. The Shn3 knockout mice reveal a striking increase in postnatal bone mass of their long, calvarial and vertebral bones. Most notable of all: The mice did not loose bone with ageing but they continued to gain bone as they aged. The knockout mice show increases in volumetric bone mineral density, as well as thicker and increased number of trabeculae and a huge amount of trabecular bone is present in places, such as the diaphysis of the femur, where normally there is no bone at all. It could be shown that osteoclast defects were not responsible for this altered phenotype. So the attention was focused on the bone-forming osteoblasts. When in vitro cultures from wild type mice and knockouts were compared, the knockout osteoblasts made more extracellular matrix as observed also in vivo. This led to the idea that Shn3 is an inhibitor of matrix mineralization by osteoblasts. In this study Shn3 was found to control protein levels of Runx2, the principal transcriptional regulator of osteoblast differentiation, by promoting its degradation through recruitment of the E3 ubiquitin ligase WWP1 to Runx2. By this means, Runx2-mediated extracellular matrix mineralization was antagonized, revealing an essential role for Shn3 as a central regulator of postnatal bone mass. If these identified mechanisms of the function of Shn3 can be targeted for therapy of osteoporosis for instance is not clarified yet. The most important issue now will be to study the quality of this increased bone mass found in the Shn3 knockout mice.
Craniovertebral malformation complex in a child with Weismann-Netter-Stuhl syndrome
The magnitude of the problem of congenital anomalies becomes evident when considering the fact that they cause the death of approximately one quarter of children either before or shortly after birth, and that they handicap an appreciable proportion of the survivors throughout their lives. Further, a significant percentage of infants judged to be normal at birth are found to suffer from "disguised" anomalies of the skeleton and soft tissues in later life. Though the study of genetic factors leading to congenital defects has attracted great attention during the last few decades, the importance of environmental causes of human malformations has received relatively less emphasis. Congenital malformations that are in most instances genetically determined, such bow legs, knock knees, scoliosis, kyphosis, clubfoot etc…. can manifest itself shortly after birth at any age. Bowing of the legs is usually thrown into the basket of vitamin D deficiency rickets; therefore, a significant number of affected children can be misdiagnosed and improperly managed. In this work, we observed the unusual radiographic interpretation. As part of our strategy in analysing these patients the “clinico-radiographic” phenotype is almost always our baseline. Of prime importance is the early recognition of these deformities and a proper management plan should take place. Radiographic study of his cranial base signified abnormal development of the first and second cervical spine respectively. Overlooking this sort of abnormality can lead to catastrophic complications. This case illustrates how the careful clinical and radiological assessment can lead to the adequate understanding of its etiology. We report a sporadic case of a 2-year-old male child of normal intelligence who presented with radiological features that were compatible with Weismann-Netter-Stuhl syndrome. In addition, we observed a craniovertebral malformation complex. To our knowledge, the combination of Weismann-Netter-Stuhl syndrome and presence of a hypoplastic occipitalized atlas and further C2-C3 fusion has not been reported before. Classically, Weismann-Netter-Stuhl syndrome is characterized by short stature, mental retardation (in some individuals), dural calcification, and anterior bowing of the tibiae. However, we believe that careful clinical and radiological examinations can reveal more striking data which might positively reflect on the whole process of management. We postulate that the congenital limitations in neck movements in our patient developed because of the marked fusion of the hypoplastic and occipitalized atlas and simultaneous C2-C3 fusion. Therefore, if this form of malformation is disregarded, there may be involvement of the atlantoaxial structure, and this can possibly lead to serious neurological and even life-threatening complications. The use of CT scanning for the detection of such abnormalities can be remarkably important.
- Progressive congenital torticollis in VATER association syndrome
- In this project, we describe a case of the VATER association syndrome which is characterized by a combination of vertebral anomalies, anal stenosis, tracheo-esophageal fistula, and radial anomalies and needs a multidisciplinary approach with a major input from orthopaedic surgeons. Torticollis in this condition has not been reported before. Detailed family history and radiologic study using plain radiographs and three-dimensional-reconstruction were used in this study. We found bony abnormalities at the base of the skull and upper cervical vertebrae. The child (a girl) manifested different abnormalities at different age periods. At birth oesophageal atresia and rectal stenosis were dealt. At the age of 4 years the congenital involvement of the spinal cord (a large intramedullary lipoma was the reason for her delayed walking, club foot and loss of sphincter control) this was dealt with surgically and the child improved dramatically. At age of preadolescence, progressive neck tilting was diagnosed by means of 3DCT scan and a progressive congenital fusion of the clivus with the first cervical spine was recognised. Stabilisation through occipito-cervical fixation was done until the age of post adulthood. In the latter a final assessment can be done whether to surgically intervene or keeping the child under observation. In conclusion, we could show that the bony abnormalities were the underlying cause of the neurologic problem.
Effects of 3- and 5-year treatment with risedronate on bone mineralization density distribution in triple biopsies of the iliac crest in postmenopausal women
Bisphosphonates are essential therapeutic agents in the treatment of osteoporosis and other bone diseases. They inhibit osteoclastic bone resorption, reduce bone turnover, increase bone mineral density (BMD) and reduce the risk of vertebral and non-vertebral fractures significantly. Exact mechanisms for the antifracture benefit of the bisphosphonates are not fully understood. Clinical trials show that the increase in BMD accounts less than 20% for the reduction in fracture risk. Therefore other factors determining the bone quality (such as bone geometry, bone mass distribution, microarchitecture and bone mineral and tissue properties) have to be considered for the explanation of the benefit of bisphosphonate therapy. While the antifracture efficacy of bisphosphonates is clinically proven, there is a debate about the long-term effects and safety issues. In this double-blinded study, we evaluated the long-term effects of risedronate (a nitrogen containing bisphosphonate) on the mineralization pattern of the bone material from triple transiliac crest biopsies of osteoporotic women. These osteoporotic women enrolled in the VERT-NA trial received either risedronate (5 mg/day, orally) or placebo for up to 5 years and received calcium and vitamin D supplementation if deficient at baseline. Triple iliac crest biopsies were collected from a subset of these subjects at baseline and 3 and 5 years treatment. We measured the bone mineralization density (BMDD) in these biopsies using quantitative backscattered electron imaging (qBEI) and related the results to the duration of the treatment and compared these data from osteoporotic patients also with a normal reference group (FIGURE 3). At baseline, both risedronate and placebo groups had a lower degree and a greater heterogeneity of mineralization and also an increase in low mineralized bone regions compared with the normal reference group. Three and 5 years treatment with risedronate or placebo increased the degree of mineralization significantly compared with baseline. However, the degree of mineralization did not exceed that of normal. After 3 years with risedronate the homogeneity of mineralization was significantly increased and low mineralized bone areas were slightly decreased compared with placebo. Surprisingly, the BMDD was broader after 5 years compared to 3 years of treatment. This increase in heterogeneity might indicate an increase in newly formed bone after 5 years with risedronate. We conclude that long-term treatment with risedronate affects the homogeneity and degree of mineralization without inducing hypermineralization of the bone matrix. These changes at the material level of the bone matrix may contribute to the antifracture efficacy of risedronate in osteoporotic patients.
Bone material properties in trabecular bone from human iliac crest biopsies after 3- and 5-year treatment with risedronate
As described in the project above, the bisphosphonates are essential therapeutic agents in the treatment of osteoporosis. In a study of long-term risedronate treatment changes of the mineralization pattern of iliac crest biopsies from postmenopausal osteoporotic patients could be observed compared to before treatment and compared to placebo treated patients (see project-description above). In this work, we describe the effect of risedronate on the mineral maturity/crystallinity and on the properties of collagen cross-links which are both important contributors to the mechanical quality of bone. Fourier-transform infrared imaging was used to study the bone material of triple iliac crest biopsies from patients of the VERT-NA trial (description see project above). We found that patients who received placebo had a higher mineral maturity/crystallinity and collagen cross-link ratio after 3 and 5 years compared to baseline. On the contrary, patients that received risedronate retained baseline values in both bone material indices throughout. In a more spatially detailed analysis we could show that this was achieved mainly through beneficial effects on actively bone forming areas. Surprisingly, patients that received risedronate achieved premenopausal values at bone forming sites in both indices after 5 years of treatment (FIGURE 4). These results indicate that long-term treatment with risedronate affects bone material properties (mineral maturity/crystallinity and collagen cross-link ratio). Risedronate treatment arrests the tissue ageing apparent in untreated osteoporosis and these changes at the bone material level might contribute to the rapid and sustained anti-fracture efficacy of this bisphosphonate in osteoporotic patients.
Pamidronate does not adversely affect bone intrinsic material properties in children with osteogenesis imperfecta.
Bisphosphonates, such as pamidronate, are usually used in the treatment of osteoporosis. However, these therapeutic agents could also increase bone mass and decrease fracture incidence in patients with osteogenesis imperfecta (OI), a heritable disease characterized by low bone mass, high fracture risk and an altered, hypermineralized bone material. While the bisphosphonates are known to increase the degree and the homogeneity of the bone material in patients with postmenopausal osteoporosis, it is unknown how bisphosphonates influence the bone material in OI patients which reveal a hypermineralized bone matrix prior to therapy. Thus, a possible influence of pamidronate treatment on bone quality at the material level might negate the beneficial effects of the gain in bone mass and lead to bone fragility in the long term. In the present study, we used transiliac bone biopsy samples and assessed the intrinsic material properties of the bone tissue at the micron-level by combined backscattered electron imaging and nanoindentation (FIGURE 5). Paired iliac bone samples from 14 patients (age 3 to 17 years) with severe OI before and after cyclical intravenous pamidronate treatment (average duration of treatment 2.5 years) as well as age-matched controls were examined. Bone histomorphometry was performed in all samples and confirmed an increase of bone mass in treated patients. Quantitative backscattered electron imaging was used to measure the weighted mean and the most frequently occurring calcium content, the variation in mineralization and the amount of lowly mineralized areas (that correspond to sites of primary mineralization). Nanoindentation was performed in a subgroup of 6 patients and 6 controls to determine hardness and elastic modulus. When OI patients were compared to controls, we observed that untreated OI patients had a significantly higher degree of bone matrix mineralization (+7%, P < 0.001) and a strong reduction of the percentage of lowly mineralized regions (-38%, P < 0.001) despite enhanced bone formation, as well as increased hardness (+21%, P < 0.01) and elastic modulus (+13%, P < 0.01). This shows that OI bone is stiffer and more mineralized and that, despite the enhanced bone formation rate in these patients, areas of primary mineralization are hardly visible. However, none of these parameters was significantly altered by the subsequent pamidronate treatment. We conclude that pamidronate treatment in children with OI does not have an adverse effect on the intrinsic material properties of bone and, as a consequence, that a long-term administration of the drug might not increase brittleness and fragility of the bone matrix. The antifracture effectiveness of pamidronate treatment in OI, as shown in previous clinical studies, has to be explained by the increase of mainly cortical bone volume.
Photopolymers for Rapid Prototyping of Soluble Mold Materials and Molding of Cellular Biomaterials
Synthetic bone replacement materials are often required after bone fracture or tumor surgery. These replacement materials need specific mechanical and chemical properties as they should serve as a support until the original bone structure has regenerated. Rapid prototyping was shown to be a suitable technique to produce such materials with well defined structures and mechanical properties. The molds which are generated by rapid prototyping are filled by thermosetting biopolymers. After curing, the molds are removed. Commercial resins used for rapid prototyping of the molds lead to highly cross-linked polymers which can only be removed by burning at higher temperatures. To substitute these cross-linked photopolymers and therefore extend the range of materials which can be casted, organo-soluble photopolymers were developed. Branched bisalkylacrylamides were suitable as base component for such formulations, due to their high reactivity, good mechanical properties, and excellent solubility of the formed polymers. These molding materials were used to prepare cellular biocompatible materials which could be used as bone replacement materials. Biocompatible crosslinkers based on methacrylates from hydrolyzed gelatine or lactic acid ethyleneglycol blockcopolymers and commercially available reactive diluents are the base components of such a formulation. Biocompatibility was investigated by osteoblast-like cells. First 3-dimensional cellular structures were prepared by molding as shown in FIGURE 6: First an organo-soluble sacrificial mold was prepared by rapid prototyping (1) which was then filled with a biocompatible monomer formulation (2) and cured at 65°C. Subsequently, the sacrificial mold was then removed (3) and the resulting cellular biopolymer can be seen in (4).
Lead accumulation in the tidemark of articular cartilage
Exposure to lead (Pb) causes increased risk of chronic diseases in the nervous, hematopoietic, skeletal, renal and endocrine system. Pb is one of the toxic bone seeking elements which is incorporated to bone during mineralization. During states of high bone turnover, such as osteoporosis, pregnancy or hyperthyroidism, this incorporated Pb is mobilized from the skeleton due to bone resorption. The aim of this study was to determine the spatial distribution of the toxic element lead (Pb) and other trace elements in normal articular cartilage and subchondral bone from adult humans with no history of work-related exposure to Pb. For this purpose, four macroscopically normal femoral heads and three patellae were harvested from randomly selected forensic autopsies. All subjects died of acute illnesses, had no history of work-related exposure to Pb and had no metabolic bone disease. Maps of the elemental distribution of lead (Pb) together with zinc (Zn), strontium (Sr) and calcium (Ca) in the chondral and subchondral region were measured using high resolution synchrotron radiation induced micro X-ray fluorescence (SR m-XRF) analysis. SR m-XRF line scans in conventional and SR m-XRF area scans in confocal geometry were correlated to backscattered electron (BE) images visualizing the mineralized tissue. For the first time, we reconstructed three-dimensional elemental distributions in articular cartilage and subchondral bone by data from confoncal SR m-XRF from various depth of the sample. In all samples, a highly specific accumulation of Pb in the tidemark (the transition zone between calcified and non-calcified articular cartilage) could be found (FIGURE 7). Pb fluorescence intensities in the tidemark, which is thought to be a metabolically active mineralization front, were 13-fold higher when compared to subchondral bone. Pb intensities in the subchondral region were strongly correlated with Zn, but were distinctly different from Ca and Sr. The finding of the highly specific accumulation of lead in the tidemark of human articular cartilage is novel. However, the exact mechanisms of the local Pb accumulation are not clarified yet. Cation exchange processes presumably displace Ca2+ by lead. This could alter the hydroxyapatite crystal structure and further the material properties. However, little is known how Pb could effect cartilage metabolism. The tidemark has great clinical importance in view of the development of osteoarthritis, the clinical syndrome of joint pain and dysfunction caused by joint degeneration. If the accumulation of Pb in the tidemark is related to the development of osteoarthritis is unknown yet.
Mechanical modulation at the lamellar level in osteonal bone
Compact (or cortical) bone as found in the shelves of long bones for instance consists mainly of secondary osteons embedded in the higher mineralized matrix of interstitial bone. These osteons are fundamental building blocks of cortical bone at the tissue level and consist of ~ 5-7 μm thick, cylindrically arranged lamellae of mineralized collagen fibrils arranged around blood vessels (Haversian systems) which are running through the bone material. The structural and mechanical properties are crucial to the whole compact bone tissue. In the project described above, we studied the structure of the mineralized fibrils in the single lamellae and were able to reconstruct the orientation of the mineralized fibrils of an osteon by X-Ray diffraction. In this work, we were interested in the local mechanical properties within such secondary osteons. Using scanning nanoindentation and quantitative backscattered electron imaging we measured the indentation modulus at specific sites of known mineral content within the osteons from a human femoral midshaft. We found the lamellar structure within the osteons reflected by a periodic modulation of the stiffness (indentation modulus). Within a single lamella, the stiffness varied between ~ 24 GPa and ~ 27 GPa. The average lamellar value remained nearly constant across the osteon (no significant mechanical variation between the lamellae could be observed) and increased abruptly to more than 30 GPa at the interstitial bone interface (FIGURE 8). The local mineral content, determined from quantitative backscattered electron imaging at the indented locations, showed also a lamellar level modulation. Mineral content was positively correlated with the indentation modulus at the same tissue position: stiffer regions within the osteon have higher mineral content. From the mechanical viewpoint, the osteon can be considered as a mechanically modulated laminate of mineralized collagen fibril layers. We propose that such a composite structure with stiffer and softer bands of mineralized matrix alternating is an effective structure for stopping cracks. The variation of the stiffness within a single lamella may serve as a trap for the crack preventing the crack to run from the interstitial bone to the Haversian canal.
Expression and functional significance of osteocalcin splicing in disease progression of hematological malignancies
Osteocalcin (OCN) represents most abundant non-collagenous molecule in bone. It is a small molecule (MW 6.9kDa) which is tightly packed and charged and coordinates Ca2+ ions at the surface of the hydroxyapatite lattice of bone mineral crystals. OCN is thought to play a crucial role for human life and development since no OCN deficient persons have been found to date. However, the exact mechanisms of action of OCN have not been clarified yet. Originally, OCN has been found to be a marker for mature osteoblasts (bone forming cells) but OCN may also function as a matrix signal for the recruitment and the differentiation of osteoclasts (bone resorbing cells) derived from hematopoietic stem cells. And there are indications that OCN may also influence the differentiation of this stem cell pool. In bone, OCN has been shown to increase bone turnover, a process that is dependent on number and activity of both osteoclasts and osteoblasts. In addition to bone-related tissues OCN synthesis was also found in activated hematopoietic stem cells (HSC) from hematologic malignancies as characterized by KIT. The aim of this study was to investigate the expression of OCN splicing variants in hematological malignancies. We analysed bone marrow obtained from two patients with chronic myeloid leukemia (CML), seven patients with other myeloproliferative diseases (MPD) and four patients with acute myeloid leukemia (AML). RT-PCR analyses were performed in order to assess and quantify spliced (OCNs) and unspliced (OCNu) mRNA, the associated transcription factors (AML1 and AML3) as well as c-KIT which is a marker for activated stem cells. Our data confirm the presence of OCN in activated stem cells from hematologic malignancies. We found OCNs mRNA and OCN protein expressed in c-KIT positive neoplastic stem cells in hematological malignancies. These data suggest that leukemic blasts may express markers of activated hematopoietic stem cells as well as markers of osteoblasts. The definition of other osteomimetic properties of these cells which may support their proliferation within the bone microenvironment awaits further study. Further studies are also needed to find out if OCN could be used to characterize leukemic cells in general.
Recursive causality in evolution: A model for epigenetic mechanisms in cancer development
Interactions between adaptative and selective processes are illustrated in the model of recursive causality as defined in Rupert Riedl's systems theory of evolution. One of the main features of this theory also termed as theory of evolving complexity is the centrality of the notion of 'recursive' or 'feedback' causality - 'the idea that every biological effect in living systems, in some way, feeds back to its own cause'. Our hypothesis is that "recursive" or "feedback" causality provides a model for explaining the consequences of interacting genetic and epigenetic mechanisms which are known to play a key role in development of cancer. Epigenetics includes any process that alters gene activity without changes of the DNA sequence. The most important epigenetic mechanisms are DNA-methylation and chromatin remodeling. Hypomethylation of so-called oncogenes and hypermethylation of tumor suppressor genes appear to be critical determinants of cancer. Folic acid, vitamin B12 and other nutrients influence the function of enzymes that participate in various methylation processes by affecting the supply of methyl groups into a variety of molecules which may be directly or indirectly associated with cancerogenesis. We present an example from our own studies by showing that vitamin D3 has the potential to de-methylate the osteocalcin-promoter in human MG63 osteosarcoma cells. Consequently, a stimulation of osteocalcin synthesis can be observed (FIGURE 9). The above mentioned enzymes also play a role in development and differentiation of cells and organisms and thus illustrate the close association between evolutionary and developmental mechanisms. This enabled new ways to understand the interaction between the genome and environment and may improve biomedical concepts including environmental health aspects where epigenetic and genetic modifications are closely associated. Recent observations showed that methylated nucleotides in the gene promoter may serve as a target for solar UV-induced mutations of the p53 tumor suppressor gene. This illustrates the close interaction of genetic and epigenetic mechanisms in cancerogenesis resulting from changes in transcriptional regulation and its contribution to a phenotype at the micro- or macroevolutionary level. Above-mentioned interactions of genetic and epigenetic mechanisms in oncogenesis defy explanation by plain linear causality, things like the continuing adaptability of complex systems. They can be explained by the concept of recursive causality and has introduced molecular biology into the realm of cognition science and systems theory: based on the notion of so-called feedback- or recursive causality a model for epigenetic mechanisms with relevance for oncology and biomedicine is provided.
MR-imaging of anterior tibiotalar impingement syndrome: agreement, sensitivity and specificity of MR-imaging and indirect MR-arthrography
The objective of this study was to clarify the role of MR-imaging in the diagnosis of anterior ankle impingement syndromes. We prospectively examined 51 consecutive patients with chronic ankle pain by MR-imaging. Arthroscopy was performed in 29 patients who previously underwent non-enhanced MR-imaging; in 11 patients, indirect MR-arthrography additionally was performed. MR-examinations were correlated with clinical findings; MR and arthroscopy scores were statistically compared, agreement was measured. Arthroscopy demonstrated granulation tissue in the lateral gutter (38%) and anterior recess (31%), lesions of the anterior tibiofibular (31%) and the anterior talofibular ligament (21%) as well as intraarticular bodies (10%). Stenosing tenosynovitis and a ganglionic cyst were revealed as extraarticular causes for chronic ankle pain by MR-examination (17%). Agreement of MR-imaging and arthroscopy was fair for the anterior talofibular ligament and the anterior joint cavity (kappa 0.40). Major discrepancy was found for non-enhanced MR scans (kappa 0.49) when compared with indirect MR-arthrography (kappa 0.03) in the anterior cavity. The sensitivity for lesions of the anterior talofibular and calcaneofibular ligament and the anterior cavity (0.91-0.87) detected by MR-imaging was superior in comparison to lesions of the anterior tibiofibular ligament and anteromedial cavity (0.50-0.24). We conclude that MR-imaging provides additional information about the mechanics of chronic ankle impingement rather than an accurate diagnosis of this clinical entity. The method is helpful in differentiating extra- from intra-articular causes of ankle impingement. Indirect MR-arthrography has little or no additional value in patients with ankle impingement syndrome.