Changes in the osteoinductive activity of the bone matrix in ontogeny

Correlation between the animal's age, degree of mineralization of its bone tissue and the osteoinductive activity of the bone organic matrix was established in experiments with rats of the same litter at an age of 2, 4, 8, 16 weeks. The osteoinductive activity was estimated by the capacity of matrix to induce ectopic osteogenesis using biochemical methods. Bone mineralization increased and the capacity for osteoinduction decreased roughly 1,5-fold with the age of donor animals. It is suggested that the reduction of osteoinductive potencies is based on the decrease in lability of the bond between the protein osteoinducer and the collagen matrix, as shown by unequal sensitivity of bone matrix of rats of different age to the doubling of the shortest possible time of bone demineralization.     

Methods for the analysis of the composition of bone tissue, with a focus on imaging mass spectrometry (TOF-SIMS)

The review describes methods available for analyzing mineralization of bone tissue in healing of fractures and implants in bone. The recent development of imaging MS, TOF-secondary ion MS (SIMS), enabling localization of hydroxyapatite (HA) in tissue samples will be presented in some detail. We strongly believe that imaging MS has the potential of becoming an important method for the studies of bone mineralization. Formation and mineralization of bone tissue with HA is a process controlled by cells, the osteoblasts, osteocytes, and osteoclasts. Formation, de novo, of bone in embryonic tissue takes place in extracellular areas within cell clusters that regulate the environment of the mineralization zone. The process of de novo formation of bone as in embryonic tissue is reactivated in adults for example during fracture healing, with or without the presence of bone implants. Thus, bone healing is one of few examples of scar-free healing of a differentiated tissue. Much of the interest of researchers in bone mineralization stems from a desire to influence the process of bone formation towards fast and endurable bone healing. There is also a wish to understand the pathogenesis of bone diseases, for example osteogenesis imperfecta, Turner's syndrome and osteoporosis.     

Bone material properties and mineral matrix contributions to fracture risk or age in women and men

The strength of bone is related to its mass and geometry, but also to the physical properties of the tissue itself. Bone tissue is composed primarily of collagen and mineral, each of which changes with age, and each of which can be affected by pharmaceutical treatments designed to prevent or reverse the loss of bone. With age, there is a decrease in collagen content, which is associated with an increased mean tissue mineralization, but there is no difference in cross-link levels compared to younger adult bone. In osteoporosis, however, there is a decrease in the reducible collagen cross-links without an alteration in collagen concentration; this would tend to increase bone fragility. In older people, the mean tissue age (MTA) increases, causing the tissue to become more highly mineralized. The increased bone turnover following menopause may reduce global MTA, and would reduce overall tissue mineralization. Bone strength and toughness are positively correlated to bone mineral content, but when bone tissue becomes too highly mineralized, it tends to become brittle. This reduces its toughness, and makes it more prone to fracture from repeated loads and accumulated microcracking. Most approved pharmaceutical treatments for osteoporosis suppress bone turnover, increasing MTA and mineralization of the tissue. This might have either or both of two effects. It could increase bone volume from refilling of the remodeling space, reducing the risk for fracture. Alternatively, the increased MTA could increase the propensity to develop microcracks, and reduce the toughness of bone, making it more likely to fracture. There may also be changes in the morphology of the mineral crystals that could affect the homogeneity of the tissue and impact mechanical properties. These changes might have large positive or negative effects on fracture incidence, and could contribute to the paradox that both large and small increases in density have about the same effect on fracture risk. Bone mineral density measured by DXA does not discriminate between density differences caused by volume changes, and those caused by changes in mineralization. As such, it does not entirely reflect material property changes in aging or osteoporotic bone that contribute to bone's risk for fracture.     

Bone strength in hypokinesia]

Experiments (20- and 100-day) on rats showed that decrease of the animal motor activity with preserved static function of the extremities led to a marked thinning of the cortical layer of the femoral bone. However, the ability of the whole bone to bear mechanical load decreased but slightly. An increase in the firmness of the bone tissue as a result of its mineralization compensated for the cortical layer thinning.     

Biomechanical effect of mineral heterogeneity in trabecular bone

Due to daily loading, trabecular bone is subjected to deformations (i.e., strain), which lead to stress in the bone tissue. When stress and/or strain deviate from the normal range, the remodeling process leads to adaptation of the bone architecture and its degree of mineralization to effectively withstand the sustained altered loading. As the apparent mechanical properties of bone are assumed to depend on the degree and distribution of mineralization, the goal of the present study was examine the influences of mineral heterogeneity on the biomechanical properties of trabecular bone in the human mandibular condyle. For this purpose nine right condyles from human dentate mandibles were scanned and evaluated with a microCT system. Cubic regional volumes of interest were defined, and each was transformed into two different types of finite element (FE) models, one homogeneous and one heterogeneous. In the heterogeneous models the element tissue moduli were scaled to the local degree of mineralization, which was determined using microCT. Compression and shear tests were simulated to determine the apparent elastic moduli in both model types. The incorporation of mineralization variation decreased the apparent Young's and shear moduli by maximally 21% in comparison to the homogeneous models. The heterogeneous model apparent moduli correlated significantly with bone volume fraction and degree of mineralization. It was concluded that disregarding mineral heterogeneity may lead to considerable overestimation of apparent elastic moduli in FE models.     

Ultrastructural observations on chondroid bone in the teleost fish Hemichromis bimaculatus

This paper presents transmission electron microscopical observations on the chondroid bone (CB) supporting the neurocraniad articulation facet of the upper pharyngeal jaws of juvenile specimens of Hemichromis bimaculatus (an acellular-boned teleost fish). Chondroid bone, a skeletal tissue morphologically intermediate between cartilage and bone, is composed of a dense mineralized collagenous matrix, resembling that of woven-fibred bone, and large chondrocyte-like cells. The latter vary considerably in their morphological features (functional cells, cells containing a large vacuole and degenerating cells). The CB is mineralized except for its upper layer. Mineralization is initiated in matrix vesicles. Clusters of apatite crystals coalesce at the mineralization front. Distally, the tissue grows by incorporation of cells which exhibit the features of osteoblasts, and which derive from less differentiated fibroblast-like cells located in the outermost layer of the tissue. Proximally, the CB is subjected to erosion by multinucleated clastic cells. The deposition of bone against the wall of lacunae which have been opened by clastic resorption may suggest a possible active involvement of the CB cells. Further studies should point out whether this bone substantially contributes to the acellular dermal dentigerous bone located below.     

Microcracks colocalize within highly mineralized regions of cortical bone tissue

While much work has been performed to quantify the extent of bone damage, its effects on the mechanical integrity of the tissue and its biological impact, the set of factors which gives forth to microdamage are nebulous, particularly the compositional properties local to microdamage. In this context, the current study tested the hypothesis that microcracks initiate within more mineralized regions of bone. Cortical bone specimens were taken from human male donors aged 31, 38, 53, 64, 71, and 84 years at the mid femoral diaphysis in a plane parallel to the osteonal orientation. The mineralization was assessed in a spatially resolved manner using Raman microspectroscopy. Arrays of measurements were taken over the entire area (i.e. global scans) of each sample followed by measurements in the vicinity of microcracks (i.e. local scans). Histograms of mineralization were constructed for global and local scans to determine whether the mineralization of damaged loci differed from the mean overall mineralization. Statistical analysis of this data revealed that the mean mineralization of damaged loci was significantly greater (P < 0.05) than the overall mineralization for each donor, indicating that there exists a highly-mineralized 'brittle volume' in bone. The presence of this damage prone 'brittle volume' has future implications for the assessment of fracture susceptibility.     

Plasticity and constraint in growth and protein mineralization of ectomycorrhizal fungi under simulated nitrogen deposition

Ectomycorrhizal fungi allow their host plants access to organic forms of N through enzymatic mineralization of the substrate and enhanced absorption of amino acids and mineral N. The cost to the plant is carbohydrates that support fungal growth and metabolism. Enrichment of soils with mineral N, as through atmospheric deposition, may affect the growth and function of these fungi by direct effects of increased N availability on fungi and indirect effects through reduced plant C allocation to roots. We tested the potential of N enrichment and altered carbohydrate supply to affect the growth and protein mineralization activity of 10 ectomycorrhizal fungi in sterile liquid media. Nitrogen treatments consisted of organic N only vs organic plus mineral N. Carbon treatments consisted of 5 g per liter glucose vs. no glucose added. Fungi differed widely in their growth and mineralization responses to these variables. Seven of 10 fungi had at least 20% reduced growth with reduced carbohydrates. Only 2 of 10 increased growth by 20% or more with increased mineral N. Carbohydrates affected growth more in a purely organic N environment suggesting an energy limitation to mineralization. Protein mineralization activity tended to be depressed by reductions in carbohydrates and increased by increased mineral N. The high sensitivity of fungal growth to carbohydrates suggests important indirect effects of N enrichment via altered C allocation in host trees. Principal Components analysis separated most fungal species along an axis representing a gradient from high protein mineralization efficiency to high intrinsic growth rate. Those fungi with slow growth and efficient mineralization activity corresponded closely to fungi often cited as late successional species, while fungi with high growth rates and low mineralization efficiency are often categorized as early successional. One fungus, Cenococcum geophillum, separated from others on an axis representing strong N dependence in growth. Nitrogen enrichment has the potential to alter the composition and function of the ectomycorrhizal fungus community. Physiological differences among species provide a starting point for predicting community responses and anticipating ecosystem consequences.     

The effect of stable strontium on the incorporation and metabolism of radioactive strontium in young and adult rabbit bones.

The experiments were performed on 5-, 8-, 12- and 14 months old rabbits. The effect of stable strontium enriched diet on the bone tissue formation (apposition) and on physiocochemical processes consisting in ion exchange were studied using radioactive isotopes: Sr-85 and Ra-226 and tetracycline. The results of kinetic and autoradiographic studies and micoscopical analysis of bone preparations suggest that stable strontium inhibits the mineralization of newly formed bone tissue without affecting the physico-chemical processes related to ion exchange.     

The influence of caffeine on the biomechanical properties of bone tissue during pregnancy in a population of rats

The influence of pregnancy on bone tissue metabolism is not completely understood. Caffeine also has a potentially negative influence on bones. The aim of this study was the evaluation of changes in the bones of pregnant rats under the influence of caffeine. The experiment was carried out on Wistar rats. The evaluation of rats' bone tissue quality was performed based on bone density measurements and resistance examinations. It analyzed the impact of caffeine on the degree of bone tissue mineralization and the composition of the bones. The mean value of pelvises 'wet' and 'dry' densities in a group of pregnant rats with caffeine intake was lower compared to the control group. The deformation in maximal load point of the femur shaft in the experimental group was significantly higher than in the control group. In the experimental group, the percentage of water in the bones was significantly higher, while the content of inorganic phase was significantly lower compared to the control group. The changes of biomechanical parameters in the group of pregnant rats with caffeine intake indicate its negative influence on the bone. Our results show higher plasticization of the bone shafts of the animals under the influence of caffeine. Higher deformation of bone shafts may have an effect on the statics of the skeleton. The administration of caffeine significantly affected the quantitative composition of the bone.     

Identification of an osteopontin-like protein in fish associated with mineral formation

Fish has been recently recognized as a suitable vertebrate model and represents a promising alternative to mammals for studying mechanisms of tissue mineralization and unravelling specific questions related to vertebrate bone formation. The recently developed Sparus aurata (gilthead seabream) osteoblast-like cell line VSa16 was used to construct a cDNA subtractive library aimed at the identification of genes associated with fish tissue mineralization. Suppression subtractive hybridization, combined with mirror orientation selection, identified 194 cDNA clones representing 20 different genes up-regulated during the mineralization of the VSa16 extracellular matrix. One of these genes accounted for 69% of the total number of clones obtained and was later identified as theS. aurata osteopontin-like gene. The 2138-bp full-length S. aurata osteopontin-like cDNA was shown to encode a 374 amino-acid protein containing domains and motifs characteristic of osteopontins, such as an integrin receptor-binding RGD motif, a negatively charged domain and numerous post-translational modifications (e.g. phosphorylations and glycosylations). The common origin of mammalian osteopontin and fish osteopontin-like proteins was indicated through an in silico analysis of available sequences showing similar gene and protein structures and was further demonstrated by their specific expression in mineralized tissues and cell cultures. Accordingly, and given its proven association with mineral formation and its characteristic protein domains, we propose that the fish osteopontin-like protein may play a role in hard tissue mineralization, in a manner similar to osteopontin in higher vertebrates.     

A novel method for embedding neonatal murine calvaria in methyl methacrylate suitable for visualizing mineralization,cellular and structural detail

The study of undecalcified bone by histological methods is essential in the field of bone research. Culturing skeletal tissues such as neonatal murine calvaria provides a reliable bridge between assessment of bone formation in vitro and anabolic activity in vivo and contains most of the essential elements of bone for studying bone formation. Neonatal calvarial assay, supported by histological methods, is used to study the anabolic effects of a wide variety of factors and compounds on bone tissue. To optimize visualization and histomorphometric measurements using neonatal calvaria, we developed a method that provides high quality tissue sections suitable for routine and histochemical staining. Undecalcified neonatal mouse calvaria were processed and embedded using a low temperature methyl methacrylate procedure. Various staining methods were performed on deplastisized and floated sections to examine mineralization and to identify cells. The Von Kossa stain counterstained with a modified H & E yielded precise images of unmineralized bone including mineralization sites, and distinct osteoblasts and osteoclasts. Toluidine blue, Ladewig's trichrome, tartrate-resistant acid phosphatase, Goldner, H & E and Villanueva stains also were tested on the undecalcified neonatal calvaria sections.     

A novel method for embedding neonatal murine calvaria in methyl methacrylate suitable for visualizing mineralization, cellular and structural detail

The study of undecalcified bone by histological methods is essential in the field of bone research. Culturing skeletal tissues such as neonatal murine calvaria provides a reliable bridge between assessment of bone formation in vitro and anabolic activity in vivo and contains most of the essential elements of bone for studying bone formation. Neonatal calvarial assay, supported by histological methods, is used to study the anabolic effects of a wide variety of factors and compounds on bone tissue. To optimize visualization and histomorphometric measurements using neonatal calvaria, we developed a method that provides high quality tissue sections suitable for routine and histochemical staining. Undecalcified neonatal mouse calvaria were processed and embedded using a low temperature methyl methacrylate procedure. Various staining methods were performed on deplastisized and floated sections to examine mineralization and to identify cells. The Von Kossa stain counterstained with a modified H & E yielded precise images of unmineralized bone including mineralization sites, and distinct osteoblasts and osteoclasts. Toluidine blue, Ladewig's trichrome, tartrate-resistant acid phosphatase, Goldner, H & E and Villanueva stains also were tested on the undecalcified neonatal calvaria sections.     

A novel method for embedding neonatal murine calvaria in methyl methacrylate suitable for visualizing mineralization, cellular and structural detail.

The study of undecalcified bone by histological methods is essential in the field of bone research. Culturing skeletal tissues such as neonatal murine calvaria provides a reliable bridge between assessment of bone formation in vitro and anabolic activity in vivo and contains most of the essential elements of bone for studying bone formation. Neonatal calvarial assay, supported by histological methods, is used to study the anabolic effects of a wide variety of factors and compounds on bone tissue. To optimize visualization and histomorphometric measurements using neonatal calvaria, we developed a method that provides high quality tissue sections suitable for routine and histochemical staining. Undecalcified neonatal mouse calvaria were processed and embedded using a low temperature methyl methacrylate procedure. Various staining methods were performed on deplastisized and floated sections to examine mineralization and to identify cells. The Von Kossa stain counterstained with a modified H & E yielded precise images of unmineralized bone including mineralization sites, and distinct osteoblasts and osteoclasts. Toluidine blue, Ladewig's trichrome, tartrate-resistant acid phosphatase, Goldner, H & E and Villanueva stains also were tested on the undecalcified neonatal calvaria sections.     

Yes‐associated protein promotes tumour necrosis factor α–treated cementoblast mineralization partly by inactivating NF‐κB pathway

Cementum regeneration, as one of the most difficult challenges of periodontal regeneration, is influenced by inflammatory factors. Inflammation may hamper or promote periodontal tissue repair under different circumstances, as it is found to do in dentin‐pulp complex and bone tissue. Our team demonstrated that YAP promotes mineralization of OCCM, a cementoblast cell line. However, the effect of YAP on its mineralization under inflammatory microenvironment is unclear. In this study, cementogenesis in vitro was up‐regulated after transient TNF‐α treatment for 30 minutes. YAP expression also was increased by TNF‐α treatment. YAP overexpression promoted OCCM mineralization after the cells were transiently treated with TNF‐α because YAP overexpression inhibited NF‐κB pathway activity, while YAP knockdown elevated it. The inhibited mineralization potential and activated NF‐κB pathway activity by YAP knockdown also were partly rescued by the application of the NF‐κB inhibitor Bay 11‐7082. These results demonstrated that YAP plays a positive role in the mineralization of TNF‐α transiently treated cementoblast, partly by inhibiting the NF‐κB pathway activity.     

Biomechanical consequences of developmental changes in trabecular architecture and mineralization of the pig mandibular condyle

The purpose of the present study was to examine the changes in apparent mechanical properties of trabecular bone in the mandibular condyle during fetal development and to investigate the contributions of altering architecture, and degree and distribution of mineralization to this change. Three-dimensional, high-resolution micro-computed tomography (microCT) reconstructions were utilized to assess the altering architecture and mineralization during development. From the reconstructions, inhomogeneous finite element models were constructed, in which the tissue moduli were scaled to the local degree of mineralization of bone (DMB). In addition, homogeneous models were devised to study the separate influence of architectural and DMB changes on apparent mechanical properties. It was found that the bone structure became stiffer with age. Both the mechanical and structural anisotropies pointed to a rod-like structure that was predominantly oriented from anteroinferior to posterosuperior. Resistance against shear, also increasing with age, was highest in the sagittal plane. The reorganization of trabecular elements, which occurred without a change in bone volume fraction, contributed to the increase in apparent stiffness. The increase in DMB, however, contributed more dominantly. Incorporating the observed inhomogeneous distribution of mineralization decreased the apparent stiffness, but increased the mechanical anisotropy. This denotes that there might be a directional dependency of the DMB of trabecular elements, i.e. differently orientated trabecular elements might have different DMBs. In conclusion, the changes in DMB and its distribution are important to consider when studying mechanical properties during development and should be considered in other situations where differences in DMB are expected.     

Bone formation in bone marrow organ cultures

Bone tissue composed of typical bone trabeculae containing ground substance with incorporated osteogenic cells and osteoblast layer was formed in organ cultures of bone marrow obtained from adult mice. Electron microscopic properties of the bone formed in vitro were identical to those of the bone tissue in vivo. The mineralization of the bone took place only in the presence of Na-beta-glycerophosphate in the culture medium.     

Effects of bisphosphonates on matrix mineralization

Bone strength is determined not only by the volume of bone tissue and the microarchitectural organization of this bone, but also by the degree of mineralization of bone matrix. The mineralization process consists of a primary deposition of mineral substance on the calcification front, followed by a slow and progressive increase of the mineral deposition named secondary mineralization. In osteoporosis, there is a negative imbalance between bone resorption and bone formation, resulting in bone loss, and microarchitectural deterioration of the trabecular network. Therapeutic agents for osteoporosis could increase bone strength by three separate, but interrelated effects on bone tissue: 1) the prevention of bone loss and thus the preservation of bone microarchitecture, 2) an increase in the volume of bone matrix, and 3) an increase in the degree of mineralization to a level similar to that seen in healthy premenopausal women, through a prolongation of the duration of secondary mineralization. Therefore the use of antiresorptive agents that reduce bone turnover, as bisphosphonates, provide a rational approach to treatment of osteoporosis. Extensive phase III clinical trials have shown that osteoporotic women treated orally with alendronate (ALN) for 3 years or more had substantial increases in bone mineral density (BMD) of approximately 10% at the spine together with reductions of about 50% in the incidence of vertebral fractures. Since a marked reduction in activation frequency was evidenced in the transiliac biopsies taken after treatment with ALN compared to placebo (PLA), without detectable increase in cancellous bone volume, it was hypothesized that the increase in BMD and the reduction in the incidence of fragility fractures were due, in a substantial part, to an increase in the degree of mineralization of bone (DMB). The mean DMB was measured by quantitative microradiography on transiliac bone biopsies taken from 53 postmenopausal osteoporotic women who had been treated with ALN (10 mg/day) during 2 (9 patients) or 3 years (16 patients) or with PLA (15 and 13 patients, respectively). In the same patients, BMD values were obtained by dual-energy X-ray absorptiometry on lumbar spine at the beginning and end of treatment. Histomorphometric parameters and activation frequency of new remodeling units were also measured on the biopsies. After 2 years of ALN, mean DMB in compact bone was 9.3% (p=0.0035) and in cancellous bone was 7.3% (p=0.0009) higher, respectively, versus PLA. After 3 years of ALN, mean DMB in compact bone was 11.6% (p=0.0002) and in cancellous bone was 11.4% (p=0.0001) higher, respectively, versus PLA. After 2 and 3 years of ALN and compared to the corresponding PLA, the distribution of the DMB clearly showed a shift towards the highest mineralization values and a decrease of the number of bone structure units having low values of mineralization. The between group differences in mean DMB were similar to those of BMD at the lumbar spine level (+8.7% after 2 years +9.6% after 3 years, respectively), suggesting that mean DMB augmentation probably accounts for the major part of the increase in BMD seen with ALN. These results support our model that the reduction in the activation frequency caused by the antiresorptive effect of ALN is followed by a prolonged secondary mineralization which increases the percentage of bone structure units having reached a maximum degree of secondary mineralization and, through this mechanism, mean DMB. That these effects contribute to improved bone strength is demonstrated by the reduction in fracture incidence previously demonstrated in these patients. In conclusion, quantitative microradiography gives access to the mineral dimension of bone tissue which has been insufficiently taken into account until now as an important determinant of bone strength and quality of bone.     

Pullulan/dextran/nHA Macroporous Composite Beads for Bone Repair in a Femoral Condyle Defect in Rats

The repair of bone defects is of particular interest for orthopedic, oral, maxillofacial, and dental surgery. Bone loss requiring reconstruction is conventionally addressed through bone grafting. Depending on the size and the location of the defect, this method has limits and risks. Biomaterials can offer an alternative and have features supporting bone repair. Here, we propose to evaluate the cellular penetration and bone formation of new macroporous beads based on pullulan/dextran that has been supplemented with nanocrystalline hydroxyapatite in a rat model. Cross-linked beads of 300–500 µm diameters were used in a lateral femoral condyle defect and analyzed by magnetic resonance imaging, micro-computed tomography, and histology in comparison to the empty defects 15, 30, and 70 days after implantation. Inflammation was absent for both conditions. For empty defects, cellularisation and mineralization started from the periphery of the defect. For the defects containing beads, cellular structures filling out the spaces between the scaffolds with increasing interconnectivity and trabecular-like organization were observed over time. The analysis of calcified sections showed increased mineralization over time for both conditions, but was more pronounced for the samples containing beads. Bone Mineral Density and Bone Mineral Content were both significantly higher at day 70 for the beads in comparison to empty defects as well as compared with earlier time points. Analysis of newly formed tissue around the beads showed an increase of osteoid tissue, measured as percentage of the defect surface. This study suggests that the use of beads for the repair of small size defects in bone may be expanded on to meet the clinical need for a ready-to-use fill-up material that can favor bone formation and mineralization, as well as promote vessel ingrowth into the defect site.     

Fresh and in vitro osteodifferentiated human amniotic membrane,alone or associated with an additional scaffold,does not induce ectopic bone formation in Balb/c mice

The human amniotic membrane (hAM) has been successfully used as a natural carrier containing amniotic mesenchymal stromal cells, epithelial cells and growth factors. It has a little or no immunogenicity, and possesses useful anti-microbial, anti-inflammatory, anti-fibrotic and analgesic properties. It has been used for many years in several indications for soft tissue repair. We previously reported that hAM represents a natural and preformed sheet containing highly potent stem cells, and could thus be used for bone repair. Indeed, native hAM possesses pre-osteoblastic potential that can easily be stimulated, even as far as mineralization, by means of in vitro osteogenic culture. However, cell culture induces damage to the tissue, as well as to cell phenotype and function. The aim of this study was to evaluate new bone formation by fresh and in vitro osteodifferentiated hAM, alone or associated with an additional scaffold presenting osteoinductive properties. Moreover, we also aimed to determine the effect of in vitro hAM pre-osteodifferentiation on its in vivo biocompatibility/tissue degradation. Results showed that neither fresh nor osteodifferentiated hAM induced ectopic bone formation, whether or not it was associated with the osteoinductive scaffold. Secondly, fresh and osteodifferentiated hAM presented similar in vivo tissue degradation, suggesting that in vitro hAM pre-osteodifferentiation did not influence its in vivo biocompatibility.