Substitution of murine type I collagen A1 3-hydroxylation site alters matrix structure but does not recapitulate osteogenesis imperfecta bone dysplasia

Null mutations in CRTAP or P3H1, encoding cartilage-associated protein and prolyl 3-hydroxylase 1, cause the severe bone dysplasias, types VII and VIII osteogenesis imperfecta. Lack of either protein prevents formation of the ER prolyl 3-hydroxylation complex, which catalyzes 3Hyp modification of types I and II collagen and also acts as a collagen chaperone. To clarify the role of the A1 3Hyp substrate site in recessive bone dysplasia, we generated knock-in mice with an α1(I)P986A substitution that cannot be 3-hydroxylated. Mutant mice have normal survival, growth, femoral breaking strength and mean bone mineralization. However, the bone collagen HP/LP crosslink ratio is nearly doubled in mutant mice, while collagen fibril diameter and bone yield energy are decreased. Thus, 3-hydroxylation of the A1 site α1(I)P986 affects collagen crosslinking and structural organization, but its absence does not directly cause recessive bone dysplasia. Our study suggests that the functions of the modification complex as a collagen chaperone are thus distinct from its role as prolyl 3-hydroxylase.     

Penetration of cutting tool into cortical bone: Experimental and numerical investigation of anisotropic mechanical behaviour

An anisotropic mechanical behaviour of cortical bone and its intrinsic hierarchical microstructure act as protective mechanisms to prevent catastrophic failure due to natural loading conditions; however, they increase the extent of complexity of a penetration process in the case of orthopaedic surgery. Experimental results available in literature provide only limited information about processes in the vicinity of a tool–bone interaction zone. Also, available numerical models the bone-cutting process do not account for material anisotropy or the effect of damage mechanisms. In this study, both experimental and numerical studies were conducted to address these issues and to elucidate the effect of anisotropic mechanical behaviour of cortical bone tissue on penetration of a sharp cutting tool. First, a set of tool-penetration experiments was performed in directions parallel and perpendicular to bone axis. Also, these experiments included bone samples cut from four different cortices to evaluate the effect of spatial variability and material anisotropy on the penetration processes. Distinct deformation and damage mechanisms linked to different microstructure orientations were captured using a micro-lens high-speed camera. Then, a novel hybrid FE model employing a smoothed-particle-hydrodynamic domain embedded into a continuum FE one was developed based on the experimental configuration to characterise the anisotropic deformation and damage behaviour of cortical bone under a penetration process. The results of our study revealed a clear anisotropic material behaviour of the studied cortical bone tissue and the influence of the underlying microstructure. The proposed FE model reflected adequately the experimental results and demonstrated the need for the use of the anisotropic and damage material model to analyse cutting of the cortical-bone tissue.     

A comparison of the fatigue behavior of human trabecular and cortical bone tissue

The fatigue properties of trabecular bone tissue (single trabeculae) and similarly sized cortical bone specimens from human tibia were experimentally determined on a microstructural level using four-point bending cyclic tests, and they were compared based on modulus, mineral density, and microstructural characteristics. The results showed that trabecular specimens had significantly lower moduli and lower fatigue strength than cortical specimens, despite their higher mineral density values. Fracture surface and microdamage analyses illustrated different fracture and damage patterns between trabecular and cortical bone tissue, depending upon their microstructural characteristics. Based on the results from mechanical tests and qualitative observations, a possible mechanical role of the cement lines in trabecular tissue microfracture was suggested.     

Transfer of proalpha2(I) cDNA into cells of a murine model of human Osteogenesis Imperfecta restores synthesis of type I collagen comprised of alpha1(I) and alpha2(I) heterotrimers in vitro and in vivo

The oim mouse is a model of human Osteogenesis Imperfecta (OI) that has deficient synthesis of proalpha2(I) chains. Cells isolated from oim mice synthesize alpha1(I) collagen homotrimers that accumulate in tissues. To explore the feasibility of gene therapy for OI, a murine proalpha2(I) cDNA was inserted into an adenovirus vector and transferred into bone marrow stromal cells isolated from oim mice femurs. The murine cDNA under the control of the cytomegalovirus early promoter was expressed by the transduced cells. Analysis of the collagens synthesized by the transduced cells demonstrated that the cells synthesized stable type I collagen comprised of alpha1(I) and alpha2(I) heterotrimers in the correct ratio of 2:1. The collagen was efficiently secreted and also the cells retained the osteogenic potential as indicated by the expression of alkaline phosphatase activity when the transduced cells were treated with recombinant human bone morphogenetic protein 2. Injection of the virus carrying the murine proalpha2(I) cDNA into oim skin demonstrated synthesis of type I collagen comprised of alpha1 and alpha2 chains at the injection site. These preliminary data demonstrate that collagen genes can be transferred into bone marrow stromal cells as well as fibroblasts in vivo and that the genes are efficiently expressed. These data encourage further studies in gene replacement for some forms of OI and use of bone marrow stromal cells as vehicles to deliver therapeutic genes to bone.     

Effect of gamma sterilization on microhardness of the cortical bone tissue of bovine femur in presence of N-Acetyl-L-Cysteine free radical scavenger

Gamma sterilization is usually used to minimize the risk of infection transmission through bone allografts. However, it is believed that gamma irradiation affects the mechanical properties of allografts and free radical scavengers can be used to alleviate the radiation-induced degradation of these properties. The aim of this study was to investigate the radioprotective effects of N-Acetyl-L-Cysteine (NAC) free radical scavenger on the material properties of sterilized bovine cortical bone at microstructure level. Forty-two cortical tissue specimens were excised from three bovine femurs and irradiated to 35 and 70 kGy gamma rays in the presence of 5, 50, and 100 mM concentrations of NAC. The localized variations in microhardness were evaluated via indentation in the radial and longitudinal directions to examine different regions of the microstructures of the specimens, including the osteonal and interstitial tissues. A significant increase was observed in the hardness of osteonal, interstitial, and longitudinal combined microstructures exposed to 35 and 70 kGy radiations (P < 0.05), whereas a relative reduction of the hardness was observed in the radial direction. Furthermore, it was found that the application of 50 and 100 mM NAC during gamma irradiation significantly subsided the hardening in longitudinal combined microstructure. Moreover, the reduction of hardness in radial direction was suppressed in the presence of 100 mM of NAC. In conclusion, the results indicated that NAC free radical scavenger can protect the cortical bone against deteriorative effects of ionizing radiation and can be used to improve the material properties of sterilized allografts.     

Anatomical, physiological, and epidemiological correlates of the aging process: a confirmation of multifactorial age determination in the Libben skeletal population

Paleodemographic analyses based on estimates of skeletal age at death consistently report high levels of young adult mortality with few individuals living in excess of 50 years. Critics assert these data indicate systematic underaging of adults and justifiably remark that criteria for estimating skeletal age at death may be unreliable, age determinations are too frequently based on one or two criteria alone, and adult paleodemographic age profiles often mimic the age distribution of the modern population from which an age indicator's standards were originally derived. This study reports a series of tests based on well-documented biological aging phenomena that can be used to investigate potential effects of systematic underaging in adults, assuming the skeletal population is of sufficient size to permit such tests. These include patterns of third decade sternal clavicular epiphyseal fusion, multiple age and sex criteria associated with cortical bone dynamics, and fractures known to occur throughout the entire adult ages range. These phenomena are examined here for the Libben site skeletal population where adult age at death was determined by the multifactorial summary age technique. None of the biological criteria reported here were used in the Libben summary age analysis and thus serve as an independent test of accuracy in age determination. In addition, the summary age method has recently been applied to a series of modern skeletons of known age (Todd samples 1 and 2). Age standards for criteria employed with Libben and Todd 1 were identical. Since Todd 1 displayed underaging in older adults, a second Libben age distribution adjusted for Todd 1 bias was generated for comparison. A third Libben adult survivorship profile based on a Coale and Demeny West level 3 mortality experience, considered by some to be a more realistic model for skeletal populations, was produced for comparison. For all criteria examined, original Libben summary ages provided superior concordance with known patterns of biological aging in human populations. While Libben ages adjusted for Todd 1 bias were slightly better in the third decade, both Todd 1 adjusted and Coale and Demeny West level 3 age distributions produced unrealistic patterns of biological aging for individuals greater than 35 years. Implications of these results are discussed.     

The assessment of trabecular bone parameters and cortical bone strength: A comparison of micro-CT and dental cone-beam CT

This study compared the capabilities of micro-computed tomography (micro-CT) and dental cone-beam computed tomography (CBCT) in assessing trabecular bone parameters and cortical bone strength. Micro-CT and CBCT scans were applied to 28 femurs from 14 rats to obtain independent measurements of the volumetric cancellous bone mineral density (vCanBMD) in the femoral head, volumetric cortical bone mineral density (vCtBMD) in the femoral diaphysis, cross-sectional moment of inertia (CSMI), and bone strength index (BSI) (=CSMI×vCtBMD). Five structural parameters of the trabecular bone of the femoral head were calculated from micro-CT images. A three-point bending test was then conducted to measure the fracture load of each femur. Bivariate linear Pearson analysis was conducted to calculate the correlation coefficients (r values) of the micro-CT, dental CBCT, and three-point bending measurements. The statistical analyses showed a strong correlation between vCanBMD values obtained using micro-CT and dental CBCT (r=0.830). There were strong or moderate correlation between vCanBMD measured using dental CBCT and five parameters of trabecular structure measured using micro-CT. Additionally, the results were satisfactory regardless of whether micro-CT or dental CBCT was used to measure the femoral diaphysis vCtBMD (r=0.733 and 0.680, respectively), CSMI (r=0.756 and 0.726, respectively), or BSI (r=0.846 and 0.847, respectively) to predict fracture loads. This study has yielded a new method for using dental CBCT to evaluate bone parameters and bone strength; however, further studies are necessary to validate the use of dental CBCT on humans.     

Changes in thermal stability and microunfolding pattern of collagen helix resulting from the loss of alpha2(I) chain in osteogenesis imperfecta murine

Homozygous mutations resulting in formation of alpha1(I)(3) homotrimers instead of normal type I collagen cause mild to severe osteogenesis imperfecta (OI) in humans and mice. Limited studies of changes in thermal stability of type I homotrimers were reported previously, but the results were not fully consistent. We revisited this question in more detail using purified tendon collagen from wild-type (alpha1(I)(2)alpha2(I) heterotrimers) and oim (alpha1(I)(3)) mice as well as artificial alpha1(I)(3) homotrimers obtained by refolding of rat-tail-tendon collagen. We found that at the same heating rate oim homotrimers completely denature at approximately 2.5deg.C higher temperature than wild-type heterotrimers, as determined by differential scanning calorimetry. At the same, constant temperature, homotrimers denature approximately 100 times slower than heterotrimers, as determined by circular dichroism. Detailed analysis of proteolytic cleavage at different temperatures revealed that microunfolding of oim homotrimers and wild-type heterotrimers occurs at similar rate but within a number of different sites. In particular, the weakest spot on the oim triple helix is located approximately 100 amino acid residues from the C-terminal end within the cyanogen bromide peptide CB6. The same microunfolding site is also present in wild-type collagen, but the weakest spot of the latter is located close to the N-terminal end of CB8. Amino acid analysis and differential gel electrophoresis showed virtually no posttranslational overmodification of oim mouse tendon collagen. Moreover, thermal stability and microunfolding of artificial rat-tail-tendon homotrimers were similar to oim homotrimers. Thus, the observed changes are associated with difference in the amino acid composition of alpha1(I) and alpha2(I) chains rather than posttranslational overmodification.     

Insulin-like growth factor I (IGF-I) in a growth-enhanced transgenic (GH-overexpressing) bony fish,the tilapia (<Emphasis Type="Italic">Oreochromis niloticus</Emphasis>): indication for a higher impact of autocrine/paracrine than of endocrine IGF-I

Several lines of growth hormone (GH)-overexpressing fish have been produced and analysed for growth and fertility parameters. However, only few data are available on the growth-promoting hormone insulin-like growth factor I (IGF-I) that mediates most effects of GH, and these are contradictory. Using quantitative real-time RT-PCR, radioimmunoassay, in situ hybridization, immunohistochemistry, and radiochromatography we investigated IGF-I and IGF binding proteins (IGFBPs) in an adult (17 months old) transgenic (GH-overexpressing) tilapia (Oreochromis niloticus). The transgenics showed an around 1.5-fold increase in length and an approximately 2.3-fold higher weight than the non-transgenics. Using radioimmunoassay, the serum IGF-I levels were lower (6.22 ± 0.75 ng/ml) in transgenic than in wild-type (15.01 ± 1.49 ng/ml) individuals (P = 0.0012). Radioimmunoassayable IGF-I in transgenic liver was 4.2-times higher than in wild-type (16.0 ± 2.21 vs. 3.83 ± 0.71 ng/g, P = 0.0017). No hepatocytes in wild-type but numerous hepatocytes in transgenic liver contained IGF-I-immunoreactivity. RT-PCR revealed a 1.4-times higher IGF-I mRNA expression in the liver of the transgenics (10.51 ± 0.82 vs. 7.3 ± 0.49 pg/μg total RNA, P = 0.0032). In correspondence, in situ hybridization showed more IGF-I mRNA containing hepatocytes in the transgenics. A twofold elevated IGF-I mRNA expression was determined in the skeletal muscle of transgenics (0.33 ± 0.02 vs. 0.16 ± 0.01 pg/μg total RNA, P < 0.0001). Both liver and serum of transgenics showed increased IGF-I binding. The increased IGFBP content in the liver may lead to retention of IGF-I, and/or the release of IGF-I into the circulation may be slower resulting in accumulation of IGF-I in the hepatocytes. Our results indicate that the enhanced growth of the transgenics likely is due to enhanced autocrine/paracrine action of IGF-I in extrahepatic sites, as shown here for skeletal muscle.     

Effect of rapamycin on bone mass and strength in the α2(I)‐G610C mouse model of osteogenesis imperfecta

Osteogenesis imperfecta (OI) is commonly caused by heterozygous type I collagen structural mutations that disturb triple helix folding and integrity. This mutant‐containing misfolded collagen accumulates in the endoplasmic reticulum (ER) and induces a form of ER stress associated with negative effects on osteoblast differentiation and maturation. Therapeutic induction of autophagy to degrade the mutant collagens could therefore be useful in ameliorating the ER stress and deleterious downstream consequences. To test this, we treated a mouse model of mild to moderate OI (α2(I) G610C) with dietary rapamycin from 3 to 8 weeks of age and effects on bone mass and mechanical properties were determined. OI bone mass and mechanics were, as previously reported, compromised compared to WT. While rapamycin treatment improved the trabecular parameters of WT and OI bones, the biomechanical deficits of OI bones were not rescued. Importantly, we show that rapamycin treatment suppressed the longitudinal and transverse growth of OI, but not WT, long bones. Our work demonstrates that dietary rapamycin offers no clinical benefit in this OI model and furthermore, the impact of rapamycin on OI bone growth could exacerbate the clinical consequences during periods of active bone growth in patients with OI caused by collagen misfolding mutations.     

A comparison of cortical elastic properties in the craniofacial skeletons of three primate species and its relevance to the study of human evolution

When a force is applied to an object, the resulting pattern of strain is a function of both the object's geometry and its elastic properties. Thus, knowledge of elastic properties in craniofacial cortical bone is indispensable for exploring the biomechanics and adaptation of primate skulls. However, elastic properties, such as density and stiffness, cannot be measured in all species, particularly extinct species known only from fossils. In order for advanced engineering techniques such as finite element analysis (FEA) to be applied to questions of primate and hominid craniofacial functional morphology, it is important to understand interspecific patterns of variation in elastic properties. We hypothesized that closely related species would have similar patterns of bone elastic properties, and that similarities with extant species should allow reasonable predictions of elastic properties in the skeletons of extinct primate species. In this study, we tested this hypothesis by measuring elastic properties in five areas of the external cortex of the baboon craniofacial skeleton using an ultrasonic technique, and by comparing the results to existing data from macaque and human crania. Results showed that cortical density, thickness, elastic and shear moduli, and anisotropy varied among areas in the baboon cranium. Similar variation had previously been found in rhesus and human crania, suggesting area-specific elastic patterns in the skulls of each species. Comparison among species showed differences, suggesting species-specific patterns. These patterns were more similar between macaques and baboons for density, maximum elastic and shear stiffness, and anisotropy than between either of these and humans. This finding demonstrates that patterns of cortical elastic properties are generally similar in closely related primate species with similar craniofacial morphology. Thus, reasonable estimates of cortical bone elastic properties should be possible for extinct species through the study of phylogenetically related and functionally similar modern forms. For example, reasonable elastic property estimates of cortical bone from fossil hominid skulls should be possible once adequate information about such properties in extant great apes is added to our current data from humans, macaques, and baboons. Such data should eventually allow FEA of craniofacial function in fossil hominids.     

An intron mutation in the human alpha 1(I) collagen gene alters the efficiency of pre-mRNA splicing and is associated with osteogenesis imperfecta type II

This study describes a homozygous, G----A transition at the moderately conserved +5 position within the splice donor site of intron 14 in the human alpha 1(I) collagen gene. The mutation reduced the efficiency of normal splice-site selection since the exon upstream of the mutation was spliced alternatively. Moreover, the extent of alternative splicing was sensitive to the temperature at which the mutant cells were grown, suggesting that the mutation directly affected spliceosome assembly. To achieve exon skipping, this effect must be propagated so as to disrupt the selection of a second splice site in the adjacent intron.     

Possible effect between the molecular packing of collagen and the composition of bony tissues

The weight fractions of the organic, mineral and water components of bone have been shown to be uniquely related to the wet bone density, except for a small variation possibly due to structure, for the range of bone densities from 1.7 g/cm³ for deer antler to 2.7 g/cm³ for porpoise petrosal. In this report the mathematical expression for the organic weight fraction is shown to depend on three factors, each a function of bone density. The first factor can be ralated to the mineral fraction, the second to the volume fraction of the organic component and the third to the density of the organic component. The influence of these factors is not obvious, since the change in the organic weight fraction could be due to an absolute loss of organic matter alone, or to a combination of increased mineral concentration together with some loss of organic matter. The mathematical development is based on the generalized packing model for collagen. It is demonstrated that the mineralization process requires a decrease of the organic component as well as a compaction of the collagen fibrils and these vary with the bone density.     

Spatial heterogeneity in the strength of selection against deleterious alleles and the mutation load

According to current estimates of genomic deleterious mutation rates (which are often of the order 0.1-1) the mutation load (defined as a reduction in the average fitness of a population due to the presence of deleterious alleles) may be important in many populations. In this paper, I use multilocus simulations to explore the effect of spatial heterogeneity in the strength of selection against deleterious alleles on the mutation load (for example, it has been suggested that stressful environments may increase the strength of selection). These simulations show contrasted results: in some situations, spatial heterogeneity may greatly reduce the mutation load, due to the fact that migrants coming from demes under stronger selection carry relatively few deleterious alleles, and benefit from a strong advantage within demes under weaker selection (where individuals carry many more deleterious alleles); in other situations, however, deleterious alleles accumulate within demes under stronger selection, due to migration pressure from demes under weaker selection, leading to fitness erosion within those demes. This second situation is more frequent when the productivity of the different demes is proportional to their mean fitness. The effect of spatial heterogeneity is greatly reduced, however, when the response to environmental differences is inconsistent across loci.     

Do stress-whitening and optical clearing of collagenous tissue occur by the same mechanism?

Bone is biphasic with an organic matrix and an inorganic mineral component. As we age bone's susceptibility to fracture increases. It has been shown that there is no change in mean mineralization with aging, but bone nevertheless becomes less tough. This aging effect is therefore likely related to the organic phase.     

Geometric characters of the radius and tibia in <Emphasis Type="Italic">Macaca mulatta</Emphasis> and <Emphasis Type="Italic">Macaca fascicularis</Emphasis>

We performed comparative analyses of four cross-sections of the distal radius and tibia in two species of macaque to clarify the relationships between bone morphology and locomotor type. The lengths of bones and five bone geometric properties in each section were examined and compared separately in both female and male Macaca mulatta and Macaca fascicularis. In M. mulatta, there were no significant gender-specific differences in either the radius or the tibia. In contrast, the radius and tibia of male M. fascicularis had greater geometric parameters in the 20% and 40% positions relative to the 5% and 10% positions from the distal end than those of their female counterparts. The radius and tibia of M. mulatta were relatively longer than those of M. fascicularis, and the sectional parameters of the tibia of M. mulatta were relatively larger than those of M. fascicularis. Standardization of the log-transformed bone length between the species revealed larger radial cortical bone areas in M. fascicularis. In contrast, there were minimal differences in the tibial cortical bone areas between the two species. This study suggests that the observed distinctions in bone geometry in female and male M. fascicularis may be due to gender-specific differences in the muscle weights of the forearm and calf, which may underlie the divergence in the leaping abilities of females and males of this species. Taken together, these results of interspecies comparisons may be related to the fact that arboreal primates such as M. fascicularis undergo compressive mechanical stress due to the forelimb lead that occurs as the animal descends a sloping trunk or bridges a tree gap downward, while terrestrial primates such as M. mulatta move on nearly flat substrates. Differences in fore- and hind-limb bone properties between the two species are discussed with regard to functional morphology and locomotor type.     

Type I collagen triplet duplication mutation in lethal osteogenesis imperfecta shifts register of alpha chains throughout the helix and disrupts incorporation of mutant helices into fibrils and extracellular matrix

The majority of collagen mutations causing osteogenesis imperfecta (OI) are glycine substitutions that disrupt formation of the triple helix. A rare type of collagen mutation consists of a duplication or deletion of one or two Gly-X-Y triplets. These mutations shift the register of collagen chains with respect to each other in the helix but do not interrupt the triplet sequence, yet they have severe clinical consequences. We investigated the effect of shifting the register of the collagen helix by a single Gly-X-Y triplet on collagen assembly, stability, and incorporation into fibrils and matrix. These studies utilized a triplet duplication in COL1A1 exon 44 that occurred in the cDNA and gDNA of two siblings with lethal OI. The normal allele encodes three identical Gly-Ala-Hyp triplets at aa 868-876, whereas the mutant allele encodes four. The register shift delays helix formation, causing overmodification. Differential scanning calorimetry yielded a decrease in T(m) of 2 degrees C for helices with one mutant chain and a 6 degrees C decrease in helices with two mutant chains. An in vitro binary co-processing assay of N-proteinase cleavage demonstrated that procollagen with the triplet duplication has slower N-propeptide cleavage than in normal controls or procollagen with proalpha1(I) G832S, G898S, or G997S substitutions, showing that the register shift persists through the entire helix. The register shift disrupts incorporation of mutant collagen into fibrils and matrix. Proband fibrils formed inefficiently in vitro and contained only normal helices and helices with a single mutant chain. Helices with two mutant chains and a significant portion of helices with one mutant chain did not form fibrils. In matrix deposited by proband fibroblasts, mutant chains were abundant in the immaturely cross-linked fraction but constituted a minor fraction of maturely cross-linked chains. The profound effects of shifting the collagen triplet register on chain interactions in the helix and on fibril formation correlate with the severe clinical consequences.     

A simplified method for quantitation of the relative amounts of type I and type III collagen in small tissue samples

A method is described for the quantitation of the relative amounts of types I and III collagens in rabbit lung tissue. This involved (i) repeated homogenization in the presence of 2% sodium dodecyl sulfate and the production of an acetone dried powder, (ii) reaction with cyanogen bromide, (iii) polyacrylamide gel electrophoresis, and (iv) densitometric scanning of proteins stained by Coomassie blue R-250. Several features of this procedure were shown to offer advantages over methods previously employed. First, the sodium dodecyl sulfate solution was shown to remove the bulk of noncollagen proteins leaving an insoluble residue which could then be reacted with cyanogen bromide without further purification. Second, cyanogen bromide was shown to solubilize essentially all of the collagen in the residue leaving an insoluble pellet with an amino acid analysis similar to elastin. Finally, to facilitate accurate quantitation, types I and III collagen standards were included with each gel so that a standard curve of protein versus staining density could be constructed. This method is assessed to be simpler and more accurate than those employed previously for the quantitation of collagens and can be applied to small tissue samples (<100 mg) such as would be obtained by lung biopsy.