Roles of collagen cross-links and osteon collagen/lamellar morphotypes in equine third metacarpals in tension and compression tests.

Many bones experience bending, placing one side in net compression and the other in net tension. Because bone mechanical properties are relatively reduced in tension compared with compression, adaptations are needed to reduce fracture risk. Several toughening mechanisms exist in bone, yet little is known of the influences of secondary osteon collagen/lamellar 'morphotypes' and potential interplay with intermolecular collagen cross-links (CCLs) in prevalent/predominant tension- and compression-loaded regions. Paired third metacarpals (MC3s) from 10 adult horses were prepared for mechanical testing. From one MC3/pair, 5 mm cubes were tested in compression at several mid-shaft locations. From contralateral bones, dumbbell-shaped specimens were tested in tension. Hence, habitual/natural tension- and compression-loaded regions were tested in both modes. Data included: elastic modulus, yield and ultimate strength, and energy absorption (toughness). Fragments of tested specimens were examined for predominant collagen fiber orientation (CFO; representing osteonal and non-osteonal bone), osteon morphotype score (MTS, representing osteonal CFO), mineralization, porosity and other histological characteristics. As a consequence of insufficient material from tension-tested specimens, CCLs were only examined in compression-tested specimens (HP, hydroxylysylpyridinoline; LP, lysylpyridinoline; PE, pentosidine). Among CCLs, only LP and HP/LP correlated significantly with mechanical parameters: LP with energy absorption, HP/LP with elastic modulus (both r=0.4). HP/LP showed a trend with energy absorption (r=-0.3, P=0.08). HP/LP more strongly correlated with osteon density and mineralization than CFO or MTS. Predominant CFO more strongly correlated with energy absorption than MTS in both testing modes. In general, CFO was found to be relatively prominent in affecting regional toughness in these equine MC3s in compression and tension.

DNA survival and physical and histological properties of heat-induced alterations in burnt bones.

During forensic casework, it is vital to be able to obtain valuable information from burnt bone fragments to ascertain the identity of the victim. Here, we report the findings of an experimental study on burnt bovine compact bone segments. Compact bones were cut to size and heated in an electric furnace at a temperature range of 100­1,100 °C with 100 °C increments. Heat-induced alterations to the bone color,weight, volume, and density were monitored using gross morphology and micro-focus X-ray computed tomography.We found that the increase in temperature caused the color of the compact bones to change in order of yellow, brown, gray,and white. In contrast to the weight reduction that occurred immediately after burning, we measured no significant reduction in volume even at 600 °C; however, volume reduced drastically once the temperature reached 700 °C. Light microscopic histological observations of burnt bone revealed heat induced alterations such as cracking and separation of the osteons at higher temperatures. In addition to these findings,we sought to examine the survival of DNA in the burnt bones using polymerase chain reaction of mitochondrial DNA. No amplification was found in the specimens burnt at 250 °C or higher, indicating the likely difficulty in testing the DNA of burnt bones from forensic casework. The results of this study will enable an estimation of the burning temperatures of burnt bones found in forensic cases and will provide an important framework with which to interpret data obtained during anthropological testing and DNA typing.

The mysteries of DNA preservation in bone: A comparative study of petrous bones and metacarpal epiphyses using ATR-FTIR spectroscopy.

A comparative analysis of 26 petrous bones and epiphyses of metacarpals from the Second World War era revealed no significant differences in DNA yield or success in STR typing. This unexpected parity in DNA preservation between the petrous bone, a renowned source of endogenous DNA in skeletal remains, and the epiphyses of metacarpals, which are porous and susceptible to taphonomic changes, is surprising. In this study, we introduced ATR-FTIR spectroscopy as an approach to unravel the correlation between bone molecular structure and DNA preservation. Metacarpals and petrous bones with same taphonomic history were sampled and prepared for DNA analyses. While one portion of the sample was used for DNA analysis, the other underwent ATR-FTIR spectroscopic examination. The normalized spectra and FTIR indices between the epiphyses of metacarpals and petrous bones were compared. Because the taphonomic history of the remains used is relatively short and stable, the ATR-FTIR spectroscopy unveiled subtle structural differences between the two bone types. Petrous bones exhibited higher mineralization, whereas epiphyses contained more organic matter. The unexpected preservation of DNA in the epiphyses of metacarpals can likely be attributed to the presence of soft tissue remnants within the trabeculae. Here observed differences in the molecular structure of bones indicate there are different mechanisms enabling DNA preservation in skeletal tissues.

Immunolocalisation of fibrillin microfibrils in the calf metacarpal and vertebral growth plate.

Overgrowth of limbs and spinal deformities are typical clinical manifestations of Marfan syndrome (MFS) and congenital contractural arachnodactyly (CCA), caused by mutations of the genes encoding fibrillin-1 (FBN1) and fibrillin-2 (FBN2), respectively. FBN1 mutations are also associated with acromicric (AD) and geleophysic dysplasias (GD), and with Weill-Marchesani syndrome (WMS), which is characterised by short stature. The mechanisms leading to such abnormal skeletal growth and the involvement of the fibrillins are not understood. Postnatal longitudinal bone growth mainly occurs in the epiphyseal growth plate. Here we investigated the organisation of fibrillin microfibrils in the growth plate of the long bone and vertebra immunohistochemically. Fibrillin-1 was dual-immunostained with elastin, with fibrillin-2 or with collagen X. We report that fibrillin microfibrils are distributed throughout all regions of the growth plate, and that fibrillin-1 and fibrillin-2 were differentially organised. Fibrillin-1 was more abundant in the extracellular matrix of the resting and proliferative zones of the growth plate than in the hypertrophic zone. More fibrillin-2 was found in the calcified region than in the other regions. No elastin fibres were observed in either the proliferative or hypertrophic zones. This study indicates that, as fibrillin microfibrils are involved in growth factor binding and may play a mechanical role, they could be directly involved in regulating bone growth. Hence, mutations of the fibrillins could affect their functional role in growth and lead to the growth disorders seen in patients with MFS, CCA, AD, GD and WMS.

High DNA yield from metatarsal and metacarpal bones from Slovenian Second World War skeletal remains.

DNA yield varies by anatomical region, and the selection of bone types that yield maximum recovery of DNA is important to maximize the success of human identification of skeletal remains. The goal of our study was to explore inter- and intra-individual variation in DNA content by measuring nuclear DNA quantity and quality and autosomal STR typing success to determine the most promising skeletal elements for bone sampling. To exclude the influence of taphonomic issues as much as possible, three complete male skeletons from a single Second World War mass grave were examined and all representative skeletal element types of the human body were analyzed. Forty-eight different types of bones from the head, torso, arm, leg, hand, and foot were sampled from each skeleton, 144 bones altogether. The samples were cleaned, and half a gram of bone powder was decalcified using a full demineralization extraction method. The DNA was purified in a Biorobot EZ1 (Qiagen). DNA content and rates of DNA degradation were determined with the PowerQuant (Promega), and the Investigator ESSplex SE QS (Qiagen) was used for STR typing. The highest-yielding bones mostly produced the most complete STR profiles. Among the skeletal elements containing on average the most DNA and producing the most complete profiles in all three skeletons examined were metacarpals, metatarsals, and the petrous portion of the temporal bone. Metatarsals and metacarpals can easily be sampled without using a saw, thus reducing potential DNA contamination. Skeletons from the Second World War can be used as a model for poorly preserved skeletal remains, and the results of the investigation can be applied for genetic identification of highly degraded skeletal remains in routine forensic casework. Although the research was limited to only three skeletons found in a unique mass grave, the data obtained could contribute to sampling strategies for identifying old skeletal remains. More Second World War skeletons will be analyzed in the future to investigate inter-bone variation in the preservation of DNA.

Assessment of the mineral density and mineral content of the equine third metacarpal and first phalanx bone by dual energy x-ray absorptiometry.

In the first part of this methodological study eleven metacarpi of 9 skeletally normal horses were examined from 4 directions by dual energy x-ray absorptiometry (DXA). The differences between the dorsopalmar-palmarodorsal and lateromedial-mediolateral (opposite sites) bone mineral density (BMD) values were found to be nonsignificant. In the second part of the study the precision of the Norland XR-26 densitometer was tested by measuring 34 metacarpal bones and 34 proximal phalanges, each of them three times, from a single direction. The difference between the individual measurements of the first phalanges and of the metacarpal bones originating from the right or the left side of the same horse were not significant, nor did the age or breed have a significant effect on BMD or bone mineral content (BMC). However, both BMD and BMC are greater in the metacarpal bones than in the proximal phalanges and are higher in geldings than in mares or to stallions, while the BMD or BMC values of mares and stallions did not differ from each other significantly. These data point to the necessity of further BMD studies in a higher number of patients.

Standardized total tract digestible phosphorus requirement of 24- to 130-kg pigs1,2.

A study was conducted to determine the standardized total tract digestible phosphorus (STTD P) requirement for 24- to 130-kg finishing pigs housed under commercial conditions. A total of 1,130 barrows and gilts (PIC 359 × 1050, Hendersonville, TN; initially 24.2 kg) were used, with 26 to 27 pigs per pen with 7 replicates per treatment. Pens of pigs were allotted to treatments in a randomized complete block design with body weight (BW) as the blocking factor. The dietary treatments were fed in 4 phases and were formulated to contain 80%, 90%, 100%, 115%, 130%, and 150% of the National Research Council (NRC) requirement estimate for finishing pigs within each phase. Weight ranges for each phase were: 27 to 49, 49 to 76, 76 to 90, and 90 to 130 kg. Treatments were achieved by increasing the amount of monocalcium phosphate at the expense of corn in the diet with no added phytase. All diets were formulated to contain a similar 1.14:1 to 1.16:1 total Ca:P ratio across treatments in all phases. Increasing STTD P resulted in a quadratic response (P < 0.05) in average daily gain (ADG), gain-to-feed ratio (G:F), and final BW. The greatest improvement was observed with STTD P at 130% of NRC for ADG and final BW and at 115% STTD P for G:F. Average daily feed intake increased linearly (linear, P < 0.05) with the inclusion of STTD P. Increasing STTD P resulted in an increase (quadratic, P < 0.05) in hot carcass weight (HCW) and carcass ADG with the greatest response observed with STTD P at 130% of NRC. There was a marginally significant response (quadratic, P < 0.10) in carcass G:F, with the greatest improvement with STTD P at 115% of NRC. Carcass yield decreased (linear, P < 0.05) with increasing STTD P, while there was a marginally significant (linear, P < 0.10) decrease in backfat and increase in fat-free lean. At the end of the study, a metacarpal was collected and analyzed for bone ash. Increasing STTD P resulted in an increase (linear, P < 0.05) in bone ash weight and percentage ash. For ADG and G:F, the quadratic model demonstrated the best fit. The maximum response in ADG and G:F was estimated at 122% and 116% of NRC STTD P, respectively. The broken-line linear model best fit the data for percentage bone ash, with a plateau achieved at 131% of the NRC STTD P. In conclusion, the estimated STTD P requirement of 24 to 130 kg ranged from 116% to 131% of the NRC publication (2012) requirement estimate.

(90)Sr, (210)Po and (210)Pb in lichen and reindeer in Norway.

Concentrations of (90)Sr, (210)Po and (210)Pb in lichen and reindeer were studied in central (Østre Namdal) and southern Norway (Vågå) during 2000-2003. The study focussed on potential differences in concentrations of these nuclides in reindeer of different ages. Concentrations of (90)Sr in bones of approximately 10 year old adult females were about 40% higher than those in calves' antlers ((90)Sr concentrations in antlers and bones of calves are similar), while the available data from Vågå suggest that (90)Sr concentrations in reindeer calves decreased with an effective ecological half-time of 9.03+/-0.06 years during 1988-2002. Furthermore, (90)Sr concentrations were 50-80% higher in bone of reindeer of a similar age from Vågå compared to those from Østre Namdal. Concentrations of (210)Po and (210)Pb in muscle and liver tissues were comparable to those reported for reindeer in other Nordic areas, with no significant difference in (210)Po and (210)Pb concentrations between adults and calves or between reindeer from the two different study areas.

Remineralized bone matrix as a scaffold for bone tissue engineering.

There is a need for improved biomaterials for use in treating non-healing bone defects. A number of natural and synthetic biomaterials have been used for the regeneration of bone tissue with mixed results. One approach is to modify native tissue via decellularization or other treatment for use as natural scaffolding for tissue repair. In this study, our goal was to improve on our previously published alternating solution immersion (ASI) method to fabricate a robust, biocompatible, and mechanically competent biomaterial from natural demineralized bone matrix (DBM). The improved method includes an antigen removal (AR) treatment step which improves mineralization and stiffness while removing unwanted proteins. The chemistry of the mineral in the remineralized bone matrix (RBM) was consistent with dicalcium phosphate dihydrate (brushite), a material used clinically in bone healing applications. Mass spectrometry identified proteins removed from the matrix with AR treatment to include α-2 HS-glycoprotein and osteopontin, noncollagenous proteins (NCPs) and known inhibitors of biomineralization. Additionally, the RBM supported the survival, proliferation, and differentiation of human mesenchymal stromal cells (MSCs) in vitro as well or better than other widely used biomaterials including DBM and PLG scaffolds. DNA content increased more than 10-fold on RBM compared to DBM and PLG; likewise, osteogenic gene expression was significantly increased after 1 and 2 weeks. We demonstrated that ASI remineralization has the capacity to fabricate mechanically stiff and biocompatible RBM, a suitable biomaterial for cell culture applications.

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. Under mechanical loading, immediately prior to failure, bone has been observed to visually become more opaque and has been termed stress-whitening. Stress-whitening is known to make materials tougher. The goal of this investigation was to investigate stress-whitening in the collagenous matrix of bone. Hydrogen bonds play a key role in collagen stability and we hypothesize that changes in hydrogen bonding will significantly affect matrix stiffness, toughness and stress whitening. Demineralized bone specimens were loaded in tension and stress-whitening was monitored. The effect of hydrogen bonding on mechanical properties and stress-whitening process was probed by altering the Hansen's hydrogen bonding parameter (δh) of the immersing solution. The Hansen's hydrogen bonding parameter of the immersing fluid affected the morphology, mechanical properties and stress whitening of specimens. Specimens were visually whiter in the absence of mechanical load in low δh solvents (the specimens solvent-whitened). Both the observed stress-whitening and solvent-whitening were reversible and repeatable processes. The observed solvent-whitening that occurred without the presence of load was consistent with solvent-induced optical clearing (the opposite of whitening) in skin caused by collagen fibril swelling. Stress whitening and solvent whitening can be explained by a common mechanism, collagen fibril densification and thinning, leading to an increased distinction between the collagen fibrillar phase and immersing fluid, ultimately leading to more scattering. Bones may be at a greater risk for fracture as we age because solubility of the matrix changes, thus making the collagen less hydrated (and more brittle) even in the same solvent.

Relative bioavailability of phosphorus in inorganic phosphorus sources fed to growing pigs.

The relative bioavailability of P in 5 sources of inorganic P was determined using growing pigs. The 5 sources of inorganic P were dicalcium phosphate (DCP), monocalcium phosphate (MCP) containing 50% MCP (MCP50), MCP containing 70% MCP (MCP70), MCP containing 100% MCP (MCP100), and monosodium phosphate (MSP). A total of 11 diets were formulated. The basal diet was formulated to contain 0.10% P, and 10 additional diets were formulated by adding 0.07 or 0.14% P from each of the 5 P sources to the basal diet. Growing pigs (n = 44; initial BW: 16.8 ± 4.3 kg) were individually housed and randomly allotted to the 11 experimental diets. Feed was provided on an ad libitum basis throughout the 28-d experimental period. At the conclusion of the experiment, all pigs were killed, and 4 bones (i.e., the third and fourth metacarpals on both front feet) were harvested. Bone-breaking strength, bone ash, and Ca and P concentrations were determined. The concentration of bone ash increased (P < 0.05) as MCP50, MCP70, MCP100, or MSP were added to the basal diet, and the concentration of bone P also increased (P < 0.05) as MCP70, MCP100, or MSP were added to the basal diet. The relative bioavailability of P in each of the feed phosphates was determined using slope ratio methodologies based on breaking strength, and expressed relative to MSP. The slope of the regression line for diets containing MSP or MCP100 was steeper (P < 0.05) than the slope for pigs fed the diet containing DCP, but not different (P > 0.05) from that of pigs fed diets supplemented with MCP50 or MCP70. In conclusion, P in MSP and MCP100 is more bioavailable than P in DCP, but there were no differences within MCP sources.

Assessment of a bone biopsy technique for measuring tiludronate in horses: a preliminary study.

This study assessed the feasibility of measuring tiludronate in horses using a minimally invasive bone biopsy technique. Eight horses were treated with intravenous (IV) tiludronate [1 mg/kg bodyweight (BW)], either once (n = 4) or twice, 28 d apart (n = 4). The horses that were treated once were euthanized on days 1, 43, 57, or 92 and those that were treated twice, were euthanized on days 112, 154, 194, or 364. Bone samples were taken bilaterally from each horse at 4 sites: the third metacarpal bone (MCIII), the 13th rib (R13), the tuber coxae (TC), and the cuboid bone (CB). Test samples were taken with a 5-mm diameter dental drill, while larger reference samples were taken with an osteotome. The concentrations of tiludronate were measured by high performance liquid chromatography (HPLC) with ultraviolet (UV) detection. The TC was the easiest site to sample, and no technical difficulties were encountered for extraction and measurement. Drill sampling at the MCIII was difficult. Moreover, both the extraction and measurement caused technical problems and results were unreliable in most cases (93%). Drill samples obtained from the R13 were very small and access to the CB required considerable dissection, which would not be feasible in vivo. Forty-six percent and 36% of the tiludronate measurements performed on the R13 and CB samples, respectively, were unreliable. The ratio of tiludronate concentrations ranged from 73% to 185% (median: 118%) in the TC, 65% to 208% (median: 81%) in the R13, and 26% to 110% (median: 57%) in the CB. In all but 1 horse, the highest concentrations of tiludronate were found in the TC. It was concluded that bone biopsies performed at the TC were adequate for measuring tiludronate in horses and should be considered in future for repeated measurements over time in living animals.

Intrinsic hand proportions of euarchontans and other mammals: implications for the locomotor behavior of plesiadapiforms.

Arboreal primates have distinctive intrinsic hand proportions compared with many other mammals. Within Euarchonta, platyrrhines and strepsirrhines have longer manual proximal phalanges relative to metacarpal length than colugos and terrestrial tree shrews. This trait is part of a complex of features allowing primates to grasp small-diameter arboreal substrates. In addition to many living and Eocene primates, relative elongation of proximal manual phalanges is also present in most plesiadapiforms. In order to evaluate the functional and evolutionary implications of manual similarities between crown primates and plesiadapiforms, we measured the lengths of the metacarpal, proximal phalanx, and intermediate phalanx of manual ray III for 132 extant mammal species (n=702 individuals). These data were compared with measurements of hands in six plesiadapiform species using ternary diagrams and phalangeal indices. Our analyses reveal that many arboreal mammals (including some tree shrews, rodents, marsupials, and carnivorans) have manual ray III proportions similar to those of various arboreal primates. By contrast, terrestrial tree shrews have hand proportions most similar to those of other terrestrial mammals, and colugos are highly derived in having relatively long intermediate phalanges. Phalangeal indices of arboreal species are significantly greater than those of the terrestrial species in our sample, reflecting the utility of having relatively long digits in an arboreal context. Although mammals known to be capable of prehensile grips demonstrate long digits relative to palm length, this feature is not uniquely associated with manual prehension and should be interpreted with caution in fossil taxa. Among plesiadapiforms, Carpolestes, Nannodectes, Ignacius, and Dryomomys have manual ray III proportions that are unlike those of most terrestrial species and most similar to those of various arboreal species of primates, tree shrews, and rodents. Within Euarchonta, Ignacius and Carpolestes have intrinsic hand proportions most comparable to those of living arboreal primates, while Nannodectes is very similar to the arboreal tree shrew Tupaia minor. These results provide additional evidence that plesiadapiforms were arboreal and support the hypothesis that Euarchonta originated in an arboreal milieu.