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.

Biomechanical evaluation of temporary epiphysiodesis at the femoral epiphysis using established devices from clinical practice.

The aim of this study is to compare biomechanical features of different devices used in clinical routine for temporary epiphysiodesis (eight-Plate® and FlexTackTM). The tested implants were divided into four different groups (eight-Plate® vs. FlexTackTM for lateral and anterior implantation) á 10 samples for testing implanted eight-Plate® vs. FlexTackTM in fresh frozen pig femora for maximum load forces (Fmax) and axial physis distance until implant failure (lmax). A servo hydraulic testing machine (858 Mini Bionix 2) was used to exert and measure reproducible forces. Statistical analyses tested for normal distribution and significant (p < 0.05) differences in primary outcome parameters. There were no significant differences between the eight-Plate® lateral group and the FlexTackTM lateral group for neither Fmax (p = 0.46) nor lmax (p = 0.65). There was a significant higher Fmax (p < 0.001) and lmax (p = 0.001) measured in the eight-Plate® group compared to the FlexTackTM group when implanted anteriorly. In anterior temporary ephiphysiodesis, eight-Plate® demonstrated superior biomechanical stability. At this stage of research, there is no clear advantage of either implant and the choice remains within the individual preference of the surgeon.

Intra-bone nuclear DNA variability in Second World War metatarsal and metacarpal bones.

DNA analysis of Second World War skeletal remains is challenging because of the limited yield of DNA that is usually recovered. Recent forensic research has focused on determining which skeletal elements are superior in their preservation of DNA, and little focus has been placed on measuring intra-bone variability. Metatarsals and metacarpals outperformed all the other bones in DNA yield when analyzing all representative skeletal elements of three Second World War victims, and intra-bone variability was not studied. Soft-tissue remnants were found to contribute to higher DNA yield in trabecular bone tissue. Because metatarsals and metacarpals are composed of trabecular epiphyses and a dense diaphysis, the goal of this study was to explore intra-bone variability in DNA content by measuring nuclear DNA quantity and quality using the PowerQuant System (Promega). A total of 193 bones from a single Second World War mass grave were examined. From each bone, DNA was extracted from the compact diaphysis and from both spongy epiphyses combined. This study confirms higher DNA quantity in epiphyses than diaphyses among all the bones analyzed, and more DNA was obtained from metacarpal epiphyses than from metatarsal epiphyses. Therefore, whenever the possibility for sampling both metacarpals and metatarsals from skeletal remains exists, collecting metacarpals is recommended. In cases in which the hands are missing, metatarsals should be sampled. In any case, epiphyses are a richer source of DNA than diaphyses.

Hydroxyapatite Crystal Thickness and Buckling Phenomenon in Bone Nanostructure During Mechanical Tests.

An investigation of bone samples taken from the left ulna of New Zealand white rabbits, with and without stresses and hysteresis loop, was undertaken using Small Angle X-ray Scattering technique. The purpose of this study is to investigate the nanostructural changes in the mean size of hydroxyapatite crystals thickness (T) during different mechanical conditions. The experiments were performed using bone samples aged 2 and 4 weeks, with and without strontium ranelate treatment, after compressive load and hysteresis loop. We did not observe any clear effects of strontium ranelate on the bones since the MANOVA test for epiphysis and diaphysis were found. On the other hand, a significant difference appears in epiphysis between 2 and 4 weeks. Furthermore, a reduction in the mean size of hydroxyapatite crystal thickness was observed when the loading pressure force increased, due to the buckling phenomenon. A return of memory points in the elastic region of the bone was observed. The significance of these results lays on the development of nanoproducts, with properties that are closer to the actual bone structure.

Intraosseous leiomyosarcoma arising in the epiphysis of the distal femur.

Herein, we present a rare case of intraosseous leiomyosarcoma arising in the epiphysis of the distal femur and showing unusual radiographic features. A 44-year-old man presented with a pain in the left knee joint. Computed tomography revealed an intraosseous lesion with slightly increased attenuation and a thin marginal sclerotic rim in the femoral medial condyle. The signal of the lesion was hypointense on T1-weighted magnetic resonance (MR) images and hyperintense on fat-suppressed T2-weighted MR images. After gadolinium administration, the signal of the lesion was moderately and diffusely enhanced. The histological diagnosis of leiomyosarcoma was made based on a preoperative core biopsy specimen. Microscopic examination of the resected specimen revealed an ill-defined intraosseous tumor composed of proliferated atypical and mildly pleomorphic smooth muscle cells permeating among the bone trabeculae with only focal destruction of the bone trabeculae and low mitotic activity, indicating low grade leiomyosarcoma. The bone trabeculae at the periphery of the tumor were mildly thickened and anastomosed with a rim of an increased number of osteoblasts. Systemic examination showed no tumorous lesions in other anatomical sites. Leiomyosarcomas rarely present in the bone as a diffuse intertrabecular growth, even in low grade tumors.

Total water, phosphorus relaxation and inter-atomic organic to inorganic interface are new determinants of trabecular bone integrity.

Bone is the living composite biomaterial having unique structural property. Presently, there is a considerable gap in our understanding of bone structure and composition in the native state, particularly with respect to the trabecular bone, which is metabolically more active than cortical bones, and is readily lost in post-menopausal osteoporosis. We used solid-state nuclear magnetic resonance (NMR) to compare trabecular bone structure and composition in the native state between normal, bone loss and bone restoration conditions in rat. Trabecular osteopenia was induced by lactation as well as prolonged estrogen deficiency (bilateral ovariectomy, Ovx). Ovx rats with established osteopenia were administered with PTH (parathyroid hormone, trabecular restoration group), and restoration was allowed to become comparable to sham Ovx (control) group using bone mineral density (BMD) and µCT determinants. We used a technique combining (1)H NMR spectroscopy with (31)P and (13)C to measure various NMR parameters described below. Our results revealed that trabecular bones had diminished total water content, inorganic phosphorus NMR relaxation time (T1) and space between the collagen and inorganic phosphorus in the osteopenic groups compared to control, and these changes were significantly reversed in the bone restoration group. Remarkably, bound water was decreased in both osteopenic and bone restoration groups compared to control. Total water and T1 correlated strongly with trabecular bone density, volume, thickness, connectivity, spacing and resistance to compression. Bound water did not correlate with any of the microarchitectural and compression parameters. We conclude that total water, T1 and atomic space between the crystal and organic surface are altered in the trabecular bones of osteopenic rats, and PTH reverses these parameters. Furthermore, from these data, it appears that total water and T1 could serve as trabecular surrogates of micro-architecture and compression strength.

Evaluation of cytotoxic compounds in different organs of the sea bream Sarpa salpa as related to phytoplankton consumption: an in vitro study in human liver cell lines HepG2 and WRL68.

The present study was aimed to assess the cytotoxic effects of not-yet identified compounds present in organ extracts of Sarpa salpa, collected in autumn, the period with a peak in health problems. In addition, we studied the cytotoxicity of extracts of epiphytes found in the stomach content of S. salpa collected in summer and of epiphytes collected from the sea in the Sfax area at the end of spring. We tested these fractions in two human hepatic cell lines: HepG2 and WRL68. We observed a significant loss of viable cells when HepG2 cells were exposed for 72 h to acetone extracts of livers of S. salpa at a concentration of 2.5 mg/ml protein. Proteins extracted from brain or muscle did not significantly induce cell death at the studied concentrations (≤10 mg/ml). There was a significant loss of viable cells when treated with liver extract of S. salpa dissolved in DMSO. Extracts of epiphytes collected in late spring showed a cytotoxic effect in a concentration-dependent manner. Moreover, we observed a significantly decreased cell viability of HepG2 at a dilution (1/40) of epiphyte extracts from stomach contents of two fish we had collected. The cytotoxic effect of the observed epiphyte extracts confirms the transfer of toxins originating from toxic dinoflagellates which live in epiphyte on the Posidonia oceanica leaves to fish organs by grazing. Hence, the liver of this fish can cause a threat to human health and consumption should for this reason be dissuaded.

Effect of hyperlipidemia on femoral biomechanics and morphology in low-density lipoprotein receptor gene knockout mice.

The objective of this study was to evaluate the effect of hyperlipidemia on the biomechanical and morphological properties of the femur of low-density lipoprotein receptor gene knockout mice (LDLr-/-) mice. Ten wild-type mice (C57BL6) and 10 LDLr-/- mice generated on a C57BL6 background were used. Male 3-month-old animals were divided into four groups (n = 5): group W (wild type) and group L (LDLr-/-) receiving low-fat commercial ration, and group WH (wild type) and group LH (LDLr-/-) receiving a high-fat diet. After 60 days, blood samples were collected for laboratory analysis of calcium, triglycerides, and cholesterol. The femur was excised for mechanical testing and morphometric analysis. LDLr-/- mice receiving the high-fat diet presented more marked alterations in the mechanical and morphological properties of femoral cortical and trabecular bone. Changes in the plasma levels of calcium, triglycerides, cholesterol, and fractions were also more pronounced in this group. The present results demonstrate that hyperlipidemia causes alterations in the structure and mechanical properties of the femur of LDLr-/- mice. These effects were more pronounced when associated with a high-fat diet.

Preliminary study on the mineral apposition rate in distal femoral epiphysis of New Zealand white rabbit at skeletal maturity.

Studies investigating the effect of different factors on the skeletal system require characterization of an appropriate animal model. Rabbits are among the most commonly studied animals for medical research, being used in about 35% of musculoskeletal research studies. The present dynamic cross-sectional histomorphometric study quantitatively determined mineral apposition rates (MARs) in the distal femoral epiphysis in four regions of interest (ROIs) in New Zealand white rabbits. ROIs included the craniolateral (CrL), caudolateral (CaL), craniomedial (CrM) and caudomedial (CaM) areas, using a reference height at different stages of skeletal maturity corresponding to experimental ages of 6, 7 and 8 months old (M6, M7 and M8). We evaluated whether a correlation exists in MARs between the times and the regions examined. Such data could be used in studies on growth of the rabbit's femur, on biomaterials for bone integration or regeneration and on growth disturbances produced by various pathologic factors. We found no interaction at the experimental times; thus, M6, M7 and M8 are considered homogeneous in terms of MARs. The velocity profiles of the MARs were statistically significantly different among the considered ROIs. For all experimental times, the CrM region had a higher MAR than the other ROIs. Both the CrM and CaM ROIs had higher MARs than the corresponding lateral ROIs. Our results indicate that bone formation is not constant within the cross-section, but is statistically different between the ROIs considered.

Breast tumors induced by N-methyl-N-nitrosourea are damaging to bone strength, structure, and mineralization in the absence of metastasis in rats.

Current theory on the influence of breast cancer on bone describes metastasis of tumor cells to bone tissue, followed by induction of osteoclasts and bone degradation. Tumor influences on bone health in pre- or nonmetastatic models are unknown. Female rats (n = 48, 52 days old) were injected with N-methyl-N-nitrosourea (MNU) to induce breast cancer. Animals were euthanized 10 weeks later, and tumors were weighed and classified histologically. Right femurs were extracted for testing of bone mineral density (BMD) by dual X-ray absorptiometry (DXA), bone mechanical strength by three-point bending and femoral neck bending tests, and structure by micro-computed tomography (µCT). Of 48 rats, 22 developed one or more tumors in response to MNU injection by 10 weeks. Presence of any tumor predicted significantly poorer bone health in 17 of 28 measures. In tumored versus nontumored animals, BMD was adversely affected by 3%, force at failure of the femoral midshaft by 4%, force at failure of the femoral neck by 12%, and various trabecular structural parameters by 6% to 27% (all p < .05). Similarly, greater tumor burden, represented by total tumor weight, adversely correlated with bone outcomes: r = -0.51 for BMD, -0.42 and -0.35 for femur midshaft force and work at failure, and between 0.36 and 0.59 (absolute values) for trabecular architecture (all p < .05). Presence of MNU-induced tumors and total tumor burden showed a negative association with bone health of the femur in rats in the absence of metastasis. Further study is required to elucidate mechanisms for this association.

In vivo microfocal computed tomography and micro-magnetic resonance imaging evaluation of antiresorptive and antiinflammatory drugs as preventive treatments of osteoarthritis in the rat.

OBJECTIVE: To determine whether treatment with an antiresorptive drug in combination with an antiinflammatory drug reduces periarticular bone and soft tissue adaptations associated with the progression of posttraumatic secondary osteoarthritis (OA). METHODS: We used in vivo microfocal computed tomography (micro-CT) to map bony adaptations and in vivo micro-magnetic resonance imaging (micro-MRI) to examine joint inflammation in a rat model of surgically induced OA secondary to knee triad injury. We examined the arthroprotective effects of the bisphosphonates alendronate and risedronate and the nonsteroidal antiinflammatory drug (NSAID) meloxicam. RESULTS: Micro-CT revealed reduced levels of periarticular trabecular bone loss in animals with knee triad injury treated with the bisphosphonate drugs alendronate or risedronate, or the NSAID meloxicam, compared with untreated animals. Alendronate treatment reduced bony osteophyte development. While risedronate as a monotherapy did not positively impact osteophytogenesis, combination therapy with risedronate and meloxicam reduced osteophyte severity somewhat. Micro-MRI revealed an increased, diffuse water signal in the epiphyses of untreated rats with knee triad injury 8 weeks after surgery, suggestive of a bone marrow lesion-like stimulus. In contrast, meloxicam-treated rats showed a significant reduction in fluid signal compared with both bisphosphonate-treated groups 8 weeks after surgery. Histologic analysis qualitatively confirmed the chondroprotective effect of both bisphosphonate treatments, showing fewer degradative changes compared with untreated rats with knee triad injury. CONCLUSION: Our findings indicate that select combinations of bisphosphonate and NSAID drug therapy in the early stages of secondary OA preserve trabecular bone mass and reduce the impact of osteophytic bony adaptations and bone marrow lesion-like stimulus. Bisphosphonate and NSAID therapy may be an effective disease-modifying drug regimen if administered early after the initial injury.

Detection of acute diazepam exposure in bone and marrow: influence of tissue type and the dose-death interval on sensitivity of detection by ELISA with liquid chromatography tandem mass spectrometry confirmation.

Enzyme-linked immunosorbent assay (ELISA) and liquid chromatography tandem mass spectrometry (LC/MS/MS) were used to detect diazepam exposure in skeletal tissues of rats (n = 15) given diazepam acutely (20 mg/kg, i.p.), and killed at various times postdose. Marrow, epiphyseal, and diaphyseal bone were isolated from extracted femora. Bone was cleaned, ground, and incubated in methanol. Marrow underwent ultrasonic homogenization. Extracts and homogenates were diluted in phosphate buffer, and then underwent solid-phase extraction and ELISA. Relative sensitivity of detection was examined in terms of relative decrease in absorbance (ELISA) and binary classification sensitivity (ELISA and LC/MS/MS). Overall, the data showed differences in relative sensitivity of detection of diazepam exposure in different tissue types (marrow > epiphyseal bone > diaphyseal bone), which is suggestive of heterogenous distribution in these tissues, and a decreasing sensitivity with increasing dose-death interval. Thus, the tissue type sampled and dose-death interval may contribute to the probability of detection of diazepam exposure in skeletal tissues.

Gender differences in expression of androgen receptor in tibial growth plate and metaphyseal bone of the rat.

In this study, we investigate the expression of the androgen receptor (AR) in the tibial growth plate and metaphyseal bone of male and female rats at the mRNA and protein level. Using in situ hybridization and immunohistochemistry, AR mRNA and protein were demonstrated in proliferating and early hypertrophic chondrocytes in the growth plate of 1-, 4-, and 7-week-old male and female rats. Immunostaining for AR was observed both in the nucleus and the cytoplasm. After sexual maturation at 12 and 16 weeks of age, AR expression decreased in both genders and was confined to a small rim of prehypertrophic chondrocytes. In female rats of 40 weeks of age, this expression pattern was still visible. In most age groups there was a tendency toward an increased AR mRNA expression in male vs. female rats except in the 7-week-old animals. At the protein level, sexually maturing 7-week-old male rats demonstrated a higher staining intensity compared to their female counterparts. At this stage, AR staining in the males was mainly confined to the nucleus, whereas in females staining was predominantly found in the cytoplasm. In the tibial metaphysis, AR mRNA was detected in lining cells, osteoblasts, osteocytes, and osteoclasts at all stages of development. At the protein level, a similar expression pattern was observed, except for an absence of immunostaining in the lining cells. The staining was both nuclear and cytoplasmic. In most age groups, mRNA and protein signals were higher in males compared with females. We have demonstrated the presence of AR mRNA and protein in the tibial growth plate and the underlying metaphyseal bone during development of the rat. In male rats, the presence of higher messenger and protein staining intensities, as well as preferential nuclear staining during sexual maturation, suggests that direct actions of androgens in chondrocytes and in bone forming cells may be involved in establishing the gender differences in the skeleton.

[Physiological and pharmacological characteristics of epiphyseal neuropeptides]. / Fiziologicheskie i farmakologicheskie osobennosti épifizarnykh neiropeptidov.

Various epiphyseal neuropeptides, participating in regulation of the secretory processes, are capable of inducing systemic changes. This circumstance determines both pharmacological and therapeutic properties. The drug elitalamine, representing a polypeptide extract, ensures effective treatment of the immune system and brain disorders and tumors and allows a prophylaxis of tissue aging.

Normal skeletal development of mice lacking matrilin 1: redundant function of matrilins in cartilage?

Matrilin 1, or cartilage matrix protein, is a member of a novel family of extracellular matrix proteins. To date, four members of the family have been identified, but their biological role is unknown. Matrilin 1 and matrilin 3 are expressed in cartilage, while matrilin 2 and matrilin 4 are present in many tissues. Here we describe the generation and analysis of mice carrying a null mutation in the Crtm gene encoding matrilin 1. Anatomical and histological studies demonstrated normal development of homozygous mutant mice. Northern blot and biochemical analyses show no compensatory up-regulation of matrilin 2 or 3 in the cartilage of knockout mice. Although matrilin 1 interacts with the collagen II and aggrecan networks of cartilage, suggesting that it may play a role in cartilage tissue organization, studies of collagen extractability indicated that collagen fibril maturation and covalent cross-linking were unaffected by the absence of matrilin 1. Ultrastructural analysis did not reveal any abnormalities of matrix organization. These data suggest that matrilin 1 is not critically required for cartilage structure and function and that matrilin 1 and matrilin 3 may have functionally redundant roles.

Paleodietary analysis on the prehistoric population of El Hierro (Canary Islands).

In order to deepen our knowledge of the dietary habits of the prehispanic inhabitants of El Hierro, we have determined bone strontium (Sr), manganese (Mn), magnesium (Mg), copper (Cu), zinc (Zn), and calcium (Ca) in 52 human tibiae (23 belonging to males and 20 to females individuals) buried in a single burial cave; in 21 modern individuals who served as controls; and in 11 bones of herbivores found at archeological sites of the Canary Islands. Results suggest that females consumed a more vegetarian diet, although site-corrected Sr/Ca ratio of both males and females speaks for a mixed-diet consumption.

Immunolocalization of osteonectin in avian tibial dyschondroplastic cartilage.

Osteonectin is an acidic calcium-binding protein found in cartilage, bone matrix, vascular endothelium, and areas of tissue repair. Using immunocytochemistry, osteonectin has been localized in all zones of the normal avian epiphyseal growth plate with notably high amounts in the hypertrophic zone. In the proximal portion of this zone the staining was intracellular, while in the distal calcifying portion of the hypertrophic zone staining was both intracellular and extracellular. Osteonectin was also detected in the growth plate associated with lesions of chickens with tibial dyschondroplasia (TD). Intense intracellular staining was observed in hypertrophic chondrocytes proximal to the lesion; staining was markedly diminished in the TD lesion; extracellular matrix was devoid of staining. Staining intensity was high along the peripheral edges of the lesion that were undergoing vascularization and resorption. This was the only area in the dysplastic cartilage where staining was observed in the extracellular matrix as well as intracellularly. Similar patterns were viewed in all TD lesions examined, whether they were spontaneous or induced by dietary treatments or genetic selection.