Establishing a minimum PMI for bone sun bleaching in a UK environment with a controlled desert-simulated comparison.

Microenvironments play a significant part in understanding the post-mortem interval in forensic taphonomy. Recently, the value of weathering factors in relation to obtaining a PMI has been investigated further. In this study, observations were made to calculate the length of time it takes for three different bone elements (femur, rib, and scapula) to bleach in a UK summer and winter. This research also investigated whether there were any physicochemical modifications to the bone caused by bleaching. Porcine femora, scapulae, and ribs were placed into open and shaded areas of an outdoor research facility located in Oxfordshire, UK, during summer (July-Sep) and winter months (Dec-Mar). The specimens were monitored at 3-week intervals using photography, and an observational scoring method was developed to quantify the extent of bleaching. As temperatures are typically much lower in the UK compared with warmer climates, a controlled indoor-simulated desert experiment was also undertaken to be used as a control. This allowed sun bleaching and changes to the bone chemistry to be monitored in a controlled, high-UV environment for comparison with the UK outdoor experiments. Fourier transform infrared spectroscopy (FTIR) was employed to analyze physicochemical modifications to both the mineral and organic components of the bone. The FTIR was used to calculate crystallinity index (CI), mineral to organic ratio, and the relative amount of carbonate concentrations. Weather data was collected and a positive correlation was found between both ultraviolet (UV) levels and accumulated degree days (ADD) when compared with observational bleaching scores. Bleaching (whitening) of the bone samples occurred in both seasons but at different rates, with the bleaching process occurring at a slower rate in winter. During summer, the initial bleaching process was evident at 6 weeks, and by 9 weeks, the bones were an off-white colour. During the winter period, whitening of the bone started at 9 weeks; however, only the scapula and rib samples displayed a similar off-white colour. This colouration was observed at 13 weeks rather than at 9 weeks. The desert simulation samples started bleaching in a similar pattern to the outdoor samples after 1 week but the bones did not fully bleach. The bone chemistry, based on physicochemical properties obtained from the FTIR, showed a significant statistical difference between the simulated desert and winter season when compared against a control sample. For the winter samples, the mineral to organic ratio was significantly higher than that in the control, suggesting a reduction in the proportion of organic. For the samples in the simulated desert environment, the crystallinity index was significantly higher than that in the control samples, suggesting an increase in crystallinity. The results of this experiment support the fact that it is possible to achieve bleaching in a UK environment and that the minimal time frame for this to occur differs in seasons.

The Neanderthal in the karst: First dating, morphometric, and paleogenetic data on the fossil skeleton from Altamura (Italy).

In 1993, a fossil hominin skeleton was discovered in the karst caves of Lamalunga, near Altamura, in southern Italy. Despite the fact that this specimen represents one of the most extraordinary hominin specimens ever found in Europe, for the last two decades our knowledge of it has been based purely on the documented on-site observations. Recently, the retrieval from the cave of a fragment of bone (part of the right scapula) allowed the first dating of the individual, the quantitative analysis of a diagnostic morphological feature, and a preliminary paleogenetic characterization of this hominin skeleton from Altamura. Overall, the results concur in indicating that it belongs to the hypodigm of Homo neanderthalensis, with some phenetic peculiarities that appear consistent with a chronology ranging from 172 ± 15 ka to 130.1 ± 1.9 ka. Thus, the skeleton from Altamura represents the most ancient Neanderthal from which endogenous DNA has ever been extracted.

Development of a quantitative PCR-based method for studying temporal DNA degradation in waterlogged bone.

While several studies have examined temporal DNA degradation in bones collected from terrestrial environments, studies on temporal DNA degradation in bones collected from aquatic environments are limited and mostly based on case studies. The objective of this study was to assess the impact of long-term submersion, aquatic environment, bone type and DNA extraction method on DNA quality and quantity. Bone samples (scapulae and ribs), collected every ~1000 ADD from a freshwater lake and river, underwent DNA extraction via ChargeSwitch® gDNA Plant Kit and organic phenol-chloroform methods, and DNA quantitation using both TaqMan and SYBR Green-based quantitative PCR (qPCR) methods. Results suggest that in both bone types, quality of recovered DNA (i.e., degradation index) declined significantly with increase in submersion time. Among two bone types, quality of recovered DNA from scapulae declined faster than rib samples. There was no significant difference in recovered DNA quantity between bone types, DNA extraction methods, or locations but various interactions between these variables showed significant difference. Overall, it can be concluded that DNA can be extracted from waterlogged bone in sufficient quantity to generate an STR profile up to 4000 ADD.

Comparative assessment of the efficiency of various decellularization agents for bone tissue engineering.

Bone regeneration can be possible through grafts or engineered bone replacement when bone defects are larger than the critical size. Decellularized bone extracellular matrix (ECM) is an alternative that is able to accelerate tissue regeneration, while decellularization protocols influence engineered bone quality. The objective of this study was to compare the quality of decellularized bone produced through different methods. Four decellularization methods were employed using (a) sodium lauryl ether sulfate (SLES), (b) sodium dodecyl sulfate (SDS) 0.5%, (c) SDS 1% and (d) trypsin/EDTA. All samples were then washed in triton X-100. DNA quantification, hematoxylin and eosin, and Hoechst staining showed that although DNA was depleted in all scaffolds, treatment with SLES led to a significantly lower DNA content. Glycosaminoglycan quantification, Raman confocal microscopy, alcian blue and PAS staining exhibited higher carbohydrate retention in the scaffolds treated with SLES and SDS 0.5%. Raman spectra, scanning electron microscopy and trichrom Masson staining showed more collagen content in SLES and SDS-treated scaffolds compared to trypsin/EDTA-treated scaffolds. Therefore, although trypsin/EDTA could efficiently decellularize the scaffolds, it washed out the ECM contents. Also, both MTT and attachment tests showed a significantly higher cell viability in SLES-treated scaffolds. Raman spectra revealed that while the first washing procedure did not remove SLES traces in the scaffolds, excessive washing reduced ECM contents. In conclusion, SLES and, to a lesser degree, SDS 0.5% protocols could efficiently preserve ultrastructure and ECM constituents of decellularized bone tissue and can thus be suggested as nontoxic and safe protocols for bone regeneration.

The Effect of Material Heterogeneity, Element Type, and Down-Sampling on Trabecular Stiffness in Micro Finite Element Models.

Preclinical and clinical bone strength predictions can be elucidated by understanding bone mechanics at a variety of hierarchical levels. As such, down-sampled micro-CT images are often used to make comparisons across image resolutions or used to reduce computational resources in micro finite element models (µFEMs). Therefore, the objectives of this study were to compare trabecular apparent modulus among (i) hexahedral and tetrahedral µFEMs, (ii) µFEMs generated from 32, 64, and 64 µm down-sampled from 32 µm µCT scans, and (iii) µFEMs with homogeneous and heterogeneous tissue moduli. Trabecular µFEMs were generated from scans at the three spatial resolutions taken from the glenoid vault of 14 cadaveric specimens. Simulated unconstrained compression was performed and used to calculate and compare the apparent modulus of each µFEM. It was found that models derived from high-resolution images that account for material heterogeneity are nearly equivalent whether hexahedral or tetrahedral elements are used. However, translation of stiffness from down-sampled scans are not equivalent to scans performed at the down-sampled resolution, or that account for trabecular material heterogeneity. Material heterogeneity is most representative of in vivo trabecular bone and to accurately model trabecular mechanical properties, material heterogeneity should be considered in future µFEM development.

Indentation across interfaces between stiff and compliant tissues.

Bone-tendon, bone-ligament and bone-cartilage junctions are multi-tissue interfaces that connect materials that differ by two orders of magnitude in mechanical properties, via gradual variations in mineral content and matrix composition. These sites mediate load transfer between highly dissimilar materials and are consequently a primary site of injury during orthopedic failure. Given the large incidence rate and the lack of suitable surgical solutions for their regeneration or repair, characterization of their natural structure and subsequent replication through tissue engineering is important. Here, we evaluate the ability and accuracy of instrumented indentation to characterize the mechanical properties of both biological tissues and engineered scaffolds with interfaces between materials that contain significant changes in mechanical properties. In this study, finite element simulations and reference samples are developed that characterize how accurately indentation measures the modulus of a material as it varies with distance across a continuous interface between dissimilar tissues with multiple orders of magnitude difference in properties. Finite element simulations accurately predicted discrepancies between the modulus function across an interface observed by indentation and the true modulus function of the material and hence allow us to understand the limits of instrumented indentation as a technique for quantifying gradual changes in material properties. It was found that in order to accurately investigate mechanical property variations in tissues with significant modulus heterogeneity the indenter size should be less than 10 percent of the expected length scale of the modulus variations. STATEMENT OF SIGNIFICANCE: The interfaces between stiff and compliant orthopedic tissues such as bone-tendon, bone-ligament and bone-cartilage are frequent sites of failure during both acute and chronic orthopedic injury and as such their replication via tissue engineering is of importance. The characterization and understanding of these tissue interfaces on a mechanical basis is a key component of elucidating the structure-function relationships that allow them to function naturally and hence a core component of efforts to replicate them. This work uses finite element models and exeperiments to outline the ability of instrumented indentation to characterize the elastic modulus variations across tissue interfaces and provides guidelines for investigators seeking to use this method to understand any interface between dissimilar tissues.

A comparative study of the expression patterns for vegf, vegf-b/vrf and vegf-c in the developing and adult mouse.

With the goal of better understanding the function and regulation of the different members of the VEGF family this study reports mapping of vegf, vegf-b and vegf-c mRNA expression in developing and adult mice. On embryonic day 14 (E14) there is a high expression of vegf and vegf-b, vegf-b being exceptionally high in heart and CNS. The vegf-c expression is lower with distinct signals in CNS and heart. Prior to birth (E17), vegf and vegf-b expression is moderately downregulated. Overlapping expression is present in intrascapular fat and heart. vegf dominates in thyroid and lung, while vegf-b appears to be the only VEGF member expressed at detectable levels in the CNS. In young adult mouse vegf and vegf-b show partly overlapping expression patterns particularly in kidney, heart and in the thymus, vegf displays higher levels in lung and liver, vegf-b appears to be dominating in brain, heart, testis and kidney. In brain the highest levels of vegf-b is present in the hippocampus. No vegf-c mRNA expression could be detected in the adult. Taken together, these results illustrate, in detail, the different regulations of the members of the VEGF gene family. There are at present at least three specific effectors of vascular proliferation with clear differences in their expression.

Structural studies of the mineral phase of calcifying cartilage.

The calcified cartilage of the epiphyseal growth plate of young calves has been studied by x-ray diffraction. Fourier transform infrared spectroscopy, magic angle 31P nuclear magnetic resonance spectroscopy, and chemical composition. The powdered tissue was separated by density centrifugation as a function of mineral content and thus qualitatively of the age of the calcium-phosphorus mineral phase. The individual density centrifugation fractions were examined separately. X-ray diffraction of the samples, especially of the lowest density fractions, revealed very poorly crystalline apatite. Fourier transform infrared spectroscopy and 31P nuclear magnetic resonance spectroscopy revealed the presence of significant amounts of nonapatitic phosphate ions. The concentration of such nonapatitic phosphates increases during the early stages of mineralization but then decreases as the mineral content steadily rises until full mineralization is achieved. The total concentration of carbonate ions was found to be much lower in calcified cartilage than in bone from the same organ (scapula). The carbonate ions are located in both A sites (OH-) and B sites (PO4(3-)), with a distribution similar to that found in bone mineral. However, discrepancies between infrared resolution factors of phosphate and carbonate bands are consistent with a heterogeneous distribution of carbonate ions in poorly organized domains of the solid phase of calcium phosphate. These initial studies permit one to characterize the calcium phosphate mineral phase as a very poorly crystalline, immature calcium phosphate apatite, rich in labile nonapatitic phosphate ions, with a low concentration of carbonate ions compared with bone mineral of the same animal, indeed from the bone of the same organ (scapula).

Elemental analysis of growth plate cartilage by synchrotron-radiation-induced X-ray emission (SRIXE).

The elemental composition of growth plate cartilage from calf scapula has been studied by means of SRIXE. X-ray emission spectra were obtained from the resting, hypertrophic and calcified regions of cartilage; then, each element was mapped with a lateral definition of about 10 microns x 10 microns. Evidence was found for a homogeneous distribution of the elements in resting cartilage compared to changes in local concentration of some atoms in the hypertrophic-calcified tissue. In this zone Ca, Sr, Ni, Zn, S, reach the maximal concentration at the calcification front while Cu shows a uniform distribution. A Zn distribution similar to that of the Zn-containing enzyme alkaline phosphatase, the key enzyme of calcification, is found.