Search

DNA testing of human bones is performed for identification when there is no remaining soft tissue, which often means the samples are old or environmentally compromised. Under these circumstances, it can be difficult to obtain a STR DNA profile. It is important to recover the highest quantity and quality of DNA for STR typing. This study compared the DNA recovery and STR profiles from five anatomical locations in five femora and five tibiae. These locations include the proximal metaphysis, proximal diaphysis, mid-diaphysis, distal diaphysis, and distal metaphysis. Twenty-five femur samples and 25 tibia samples were analyzed using the Qiagen Investigator Quantiplex Pro RGQ Kit for quantitating the extracted DNA and the Qiagen Investigator 24plex QS Kit for STR DNA typing. The highest DNA recovery of the five regions tested in both the femur and the tibia was from the midshaft diaphysis. The femur samples resulted in a significantly higher DNA recovery than the tibia samples as analyzed using a Kruskal-Wallis test (P = 0.002103). The midshaft diaphysis and distal diaphysis yielded the most complete STR DNA profiles in the femora, while the distal and proximal diaphysis yielded the most complete STR DNA profiles in the tibiae. There was no correlation between the amount of DNA recovered and the completeness of the STR DNA profile produced with low template extracts in this study., (© 2020 American Academy of Forensic Sciences.)

The development of safe and practical strategies to prevent weakening of bone tissue is vital, yet attempts to achieve this have been hindered by a lack of understanding of the short-term (days-weeks) physiology of bone collagen turnover. To address this, we have developed a method to quantify bone collagen synthesis in vivo, using deuterium oxide (D 2 O) tracer incorporation techniques combined with gas chromatography pyrolysis isotope-ratio mass spectrometry (GC-pyrolysis-IRMS). Forty-six male and female rats from a selectively bred model ingested D 2 O for 3 weeks. Femur diaphyses (FEM), tibia proximal (T-PRO), and distal (T-DIS) epiphyses-metaphyses and tibia mid-shaft diaphyses (T-MID) were obtained from all rats after necropsy. After demineralisation, collagen proteins were isolated and hydrolysed and collagen fractional synthetic rates (FSRs) determined by incorporation of deuterium into protein-bound alanine via GC-pyrolysis-IRMS. The collagen FSR for the FEM (0.131 ± 0.078%/day; 95% CI [0.106-0.156]) was greater than the FSR at T-MID (0.055 ± 0.049%/day; 95% CI [0.040-0.070]; p < 0.001). The T-PRO site had the highest FSR (0.203 ± 0.123%/day; 95% CI [0.166-0.241]) and T-DIS the lowest (0.027 ± 0.015%/day; 95% CI [0.022-0.031]). The three tibial sites exhibited different FSRs (p < 0.001). Herein, we have developed a sensitive method to quantify in vivo bone collagen synthesis and identified site-specific rates of synthesis, which could be applicable to studies of human bone collagen turnover., (© 2021 The Authors. Physiological Reports published by Wiley Periodicals LLC on behalf of The Physiological Society and the American Physiological Society.)

Traditional methods for bone pulverization typically generate heat, risking stability of DNA sample. SPEX™ has developed cryogenic grinders which introduce liquid nitrogen to cool the sample and aid in the grinding process. In this study, the Freezer Mill 6970 EFM was used with two DNA extraction methods and routine downstream STR analysis procedures. DNA from as little as 0.1 g of bone powder was used to develop full STR profiles after freezer mill pulverization, and the method was reproducible. Further, no contamination was detected upon cleaning/reuse of the sample vials. There were no significant differences in DNA yield, STR alleles detected, or peak heights using the freezer mill as compared to traditional grinding, and successful DNA profiles were achieved from as low as 0.1 g of bone powder with this method. Overall, this work indicates that this cryogenic mill method may be used as a viable alternative to traditional tissue grinders., (© 2017 American Academy of Forensic Sciences.)

1. The aim of the study was to investigate the effects of caponisation on bone development of males of two native breeds in Poland. 2. The weight, length and cross-sectional area of tibiae and femora were measured, densitometric measurements and tests of strength were determined and dimensions were calculated. 3. Breed and caponisation did not influence bone weight and length. Higher mechanical strength of the femur was found in entire males, mainly in the Polbar breed. Tibial strength was reduced in capons of the Green Partridge breed. Maximum elastic strength was greater in the Polbar, irrespective of caponisation. Bone cross-sectional area was influenced by breed, while caponisation reduced femoral bone mineral density in both breeds. 4. Caponisation thus increased growth rate but had adverse effects on bone development. 5. Caponisation had fewer negative effects in the Polbar than in the Greenleg Partridge.

Natural collagen is easily available from animal tissues such as bones. Main limitations reported in the use of natural collagen are heterogeneity and loss of integrity during recovery. However, its natural complexity, functionality and bioactivity still remain to be achieved through synthetic and recombinant ways. Variability of physicochemical properties of collagen extracted from bovine bone by acetic acid was then investigated taking into account endogenous and exogenous factors. Endogenous: bovine's bones age (4 and 7 years) and anatomy (femur and tibia); exogenous: thermal treatments (spray-drying and lyophilisation). Scanning electron microscopy, spectroscopy (EDS, FTIR, UV/Vis and CD), differential scanning calorimetry (DSC), centesimal composition, mass spectrometry, amino acids and zeta-potential analysis were used for the purpose. Age correlated negatively with yield of recovery and positively with minerals and proteoglycans content. Comparing the anatomy, higher yields were found for tibias, and higher stability of tibias collagen in solution was noticed. Whatever the age and the anatomy, collagens were able to renature and to self-assemble into tri-dimensional structures. Nonetheless thermal stability and kinetics of renaturation were different. Variability of natural collagen with bone age and anatomy, and drying methodology, may be a crucial advantage to conceive tailor-made applications in either the biological or technical sector., (Copyright © 2016 Elsevier B.V. All rights reserved.)

The effect of caponization on the mechanical properties of Polbar chicken bones was the subject of investigation. The biomechanical strength of bones and the risk of their deformation or fracture were determined on the basis of their geometric, structural, material, and densitometric properties. Eight-week-old cockerels of Polbar breed were surgically castrated and then fattened until the 24th week of life. It was observed that caponization did not influence the weight and length of either of the long bones. It did however significantly reduce their ash content and the Ca and P contents in their femur, and P content in their tibia. Dual energy x-ray absorptiometry densitometry revealed that bone mineral content of the mid-diaphyseal part of both bones were reduced. Similarly, the bone tissue density of bones decreased. Caponization influenced the bone's geometric structure increasing the internal and external diameters of the bones. The bone cross-sectional area was greater in capons, and, consequently, the cross-sectional moment of inertia and the radius of gyration significantly increased. However, the relative wall thickness and cortical index were not altered. The three-point bending tests revealed the negative effect of caponization on the mechanical endurance of bones. Yield strength, Young modulus, and yield stress, characterizing the strength of the bone's material, decreased in capons. That suggests a higher risk of permanent deformation as capon bones become less elastic. Concluding, caponization negatively influenced the quality characteristics of Polbar chicken long bones., (© 2016 Poultry Science Association Inc.)

Forensic pathology often uses osteobiography, which involves biological profiles based on a determination of the age, sex, constitution, pathological states and other anomalies (paleopathology) of subjects for identification purposes. In this paper, proteins were analysed in bone remains. A total of 45 long bones from 45 different cadavers (29 males, 16 females) with a mean age of 66.31 years (S.D.=19.48, range 20-97) were used to search for pathological biomarkers which are closely related to several diseases. The bones were removed from the cement niches of a cemetery in Murcia (south-eastern Spain), where they had lain for between 18 and 45 years (mean time 25.84 years, S.D.=8.91). After a specific extraction using Tris-Urea buffer, were measured using HPLC/MS/MS. Our results show that proteins resulting from tumoral diseases and bacterial and viral pathogens can be detected and identified in the skeletal remains, making them useful pathological biomarkers for constructing biological profiles., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

Background: The effects of high and low jumping exercise intensities combined with honey on bone and gonadotrophins were investigated in eighty four 9 week-old female rats., Methods: The experimental groups were 20 or 80 jumps per day combined with or without honey supplementation (HJ20, HJ80, J20 and J80), honey supplementation (H), sedentary without supplementation control (C), and baseline control (C0) groups., Results: Study results showed that HJ80 elicited greatest beneficial effects on tibial and femoral mass, serum total calcium and alkaline phosphatase concentrations. There were significantly (p < 0.05) lower levels of serum follicle stimulating hormone concentrations in H, J20, J80 compared to C, with exception of HJ20 and HJ80. Serum luteinizing hormone concentrations were significantly (p < 0.05) greater in HJ20, HJ80 and J20 compared to J80., Conclusions: It appears that high intensity jumping exercise combined with honey supplementation resulted more discernable effects on bone. Meanwhile, honey may protect against the adverse effects induced by jumping exercise on gonadotropins in female rats.

Objective: To investigate the effects of soy isoflavones (Iso) and mechanical vibration treatments alone or combined on bone extracellular matrix constituents of ovariectomized rats., Methods: Forty female Wistar rats at the age of 6 months were ovariectomized (Ovx) and ten were sham-operated (sham). After 3 months, the animals were divided into five groups: GI (sham); GII (Ovx); GIII, ovariectomized and orally treated with isoflavones (200 mg/kg) for 90 consecutive days; GIV, ovariectomized and submitted to vibration for 90 days (5 days/week); GV, ovariectomized and treated with isoflavones plus vibration. After treatments, the rats were euthanized, and their femurs were removed for histological routine and biochemical study. Histological sections were stained with hematoxylin-eosin, picrosirius red and alcian blue. Shaft of femurs were submitted to biochemical assay and tibias were subjected to biophysical and biomechanical tests., Results: Treatments did not have significant effects on the trabecular bone volume, but the combined treatments showed trophic effects on the cortical bone width and area. Bone density and the content of organic material of the tibias were higher in the GIV and GV groups. The GV group showed the highest presence of mature collagen fibers and content of total glycosaminoglycans, while the highest contents of chondroitin sulfate and other sulfated glycosaminoglycans were seen in the GIV group., Conclusion: The mechanical vibration treatment is more efficient than soy isoflavones in improving bone quality by increasing the bone density, the content of sulfated glycosaminoglycans and the presence of mature collagen fibers. In addition, the combined interventions have partial trophic and synergistic effects that are bone site-specific in ovariectomized rats.

The disease- and drug-related bone disorders are rapidly increasing in the population. It is previously reported that anti-epileptic drugs (AEDs) may cause osteopenia, osteoporosis, and fractures in epilepsy patients. However, it cannot be determined whether the bone disorders in epileptic patients are due to AED therapy and/or to epilepsy and epileptic seizures. There is no study in the literature which investigates the sole effects of epilepsy and epileptic seizures on bone tissues. The current study provides the first report on determination of the possible effects of epilepsy and epileptic seizures on long bone tissues. Wistar Albino Glaxo/Rijswijk rats, which are accepted as genetic rat models for human absence epilepsy, were compared with the healthy Wistar rats to get information about the sole effects of epilepsy and epileptic seizures on bones. Cortical regions of tibia and femur bones were studied by Fourier transform infrared microspectroscopy (FTIRM). According to FTIRM parameters, variation on bone mineral and matrix composition, including decreased mineral content, decreased collagen cross-links, increased carbonate substitution, and larger crystals in epileptic group compared to the healthy one, show severe effects of epilepsy and seizures on bone tissues for the first time., (© 2013 Society of Photo-Optical Instrumentation Engineers (SPIE))

Our aim was to investigate the effects of Maillard reaction products (MRPs) from bread crust (BC) on bone composition and its mechanical properties, determining whether any such effects are related to the molecular weight of different MRPs. For 88 days after weaning rats were fed a control diet or diets containing BC, or its soluble low molecular weight (LMW), soluble high molecular weight (HMW) or insoluble fractions. Animals' food consumption and body weights were monitored. After sacrifice, the femur, pelvic bone and tibia were removed for composition, physical and biomechanical properties analysis. It was found that body and femur weights, density, volume and organic matrix decreased, whereas pentosidine increased after consumption of experimental diets, especially in the HMW and insoluble groups (104.7 and 102.9 mmol mol(-1) collagen) vs. the control group (41.7 mmol mol(-1) collagen). Bone stiffness fell by 50% in the LMW, HMW and insoluble groups and failure load and energy to failure tended to decrease in the same animals after MRPs intake. Consumption of diets containing assayed MRPs during growth leads to lower bone size and introduces some changes in its mechanical behavior which appear to be related to an increase in the pentosidine level of bone.

Fetal and postnatal bone development in humans is traditionally viewed as a process characterized by progressively increasing mineral density. Yet, a temporary decrease in mineral density has been described in the long bones of infants in the immediate postnatal period. The mechanism that underlies this phenomenon, as well as its causes and consequences, remain unclear. Using daily μCT scans of murine femora and tibiae during perinatal development, we show that a temporary decrease in tissue mineral density (TMD) is evident in mice. By monitoring spatial and temporal structural changes during normal growth and in a mouse strain in which osteoclasts are non-functional (Src-null), we show that endosteal bone resorption is the main cause for the perinatal decrease in TMD. Mechanical testing revealed that this temporary decrease is correlated with reduced stiffness of the bones. We also show, by administration of a progestational agent to pregnant mice, that the decrease in TMD is not the result of parturition itself. This study provides a comprehensive view of perinatal long bone development in mice, and describes the process as well as the consequences of density fluctuation during this period., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Background: Osteogenesis imperfecta is a heterogeneous genetic disorder characterized by bone fragility. Previous research suggests that impaired collagen network and abnormal mineralization affect bone tissue properties, however, little data is yet available to describe bone material properties in individuals with this disorder. Bone material properties have not been characterized in individuals with the most common form of osteogenesis imperfecta, type I., Methods: Bone tissue elastic modulus and hardness were measured by nanoindentation in eleven osteotomy specimens that were harvested from children with osteogenesis imperfecta during routine surgeries. These properties were compared between osteogenesis imperfecta types I (mild, n=6) and III (severe, n=5), as well as between interstitial and osteonal microstructural regions using linear mixed model analysis., Findings: Disease severity type had a small but statistically significant effect on modulus (7%, P=0.02) and hardness (8%, P<0.01). Individuals with osteogenesis imperfecta type I had higher modulus and hardness than did those with type III. Overall, mean modulus and hardness values were 13% greater in interstitial lamellar bone regions than in osteonal regions (P<0.001)., Interpretation: The current study presents the first dataset describing bone material properties in individuals with the most common form of osteogenesis imperfecta, i.e., type I. Results indicate that intrinsic bone tissue properties are affected by phenotype. Knowledge of the material properties of bones in osteogenesis imperfecta will contribute to the ability to develop models to assist in predicting fracture risk., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

The quality and efficiency of a standard organic DNA isolation method and a silica-based method using the QIAGEN Blood Maxi Kit were compared to obtain human DNA and short tandem repeats (STRs) profiles from 39 exhumed bone samples for paternity testing. DNA samples were quantified by real-time PCR, and STR profiles were obtained using the AmpFlSTR(®) Identifiler(®) PCR amplification kit. Overall, the silica-based method recovered less DNA ranging from 0 to 147.7 ng/g (average 7.57 ng/g, median = 1.3 ng/g) than did the organic method ranging from 0 to 605 ng/g (average 44.27 ng/g, median = 5.8 ng/g). Complete profiles (16/16 loci tested) were obtained from 37/39 samples (95%) using the organic method and from 9/39 samples (23%) with the silica-based method. Compared with a standard organic DNA isolation method, our results indicate that the published silica-based method does not improve neither the quality nor the quantity of DNA for STR profiling., (© 2011 American Academy of Forensic Sciences.)

In osteoporosis, changes in tissue composition and structure reduce bone strength and expose it to fractures. The current primary diagnostic technique, i.e., dual energy X-ray absorptiometry, measures areal bone mineral density (BMD) but provides no direct information on trabecular structure or organic composition. Although still poorly characterized, ultrasound techniques may bring about information on bone composition and structure. In this study, relationships of 2.25-MHz ultrasound speed, attenuation, reflection and backscattering with composition of human trabecular bone (n=26) were characterized experimentally, as well as by using numerical analyses. We also determined composition of the trabecular sample (fat and water content, bone volume fraction) and that of the calcified matrix (mineral, proteoglycan and collagen content of trabeculae). In experimental analyses, bone volume fraction and mineral content of the calcified matrix were the only determinants of BMD. Further, bone volume fraction served as the strongest determinant of ultrasound parameters (r=0.51-0.87). In numerical simulations, density and mechanical properties of the calcified matrix systematically affected ultrasound speed, attenuation, reflection and backscattering. However, partial correlation coefficients revealed only low associations(|r|

To determine the short- to mid-term effects of ovariectomy on bone turnover, bone mass and bone strength in rats. SD rats aged 12 weeks were randomly divided into No-treatment, Sham and OVX groups. The rats were sacrificed for sample collection at week 0, week 4 and week 18 after surgical operation. Chemistries in serum and urine were measured by standard colorimetric methods and bone turnover markers were measured by ELISA kits. Bone mass and bone strength were determined using pQCT system and three-point bending tests, respectively. At week 4, OVX rats showed drastic increase in the level of urine Ca, P and DPD. At week 18, in OVX rats the levels of serum ALP, urine DPD and Ca were much higher and the level of serum Ca was much lower when comparing with Sham rats. Ovariectomy produced significant reduction in cancellous BMD, total BMD and SSI of proximal tibial metaphysis rapidly at week 4 and continuously at week 18 after surgical operation. However, no marked changes of bone mass and bone strength were found in the diaphysis of tibia and femur, respectively. The current study concluded that ovariectomy induced the uncoupling of bone turnover, and the proximal metaphysis of long bone was the sensitive site for the short- to mid-term effect of ovariectomy, demonstrated as the markers of bone mass and stress strain index.

The amelogenin protein is considered as the major molecular marker of developing ectodermal enamel. Recent data suggest other roles for amelogenin beyond structural regulation of enamel mineral crystal growth. Here we describe our novel discovery of amelogenin expression in long bone cells, in cartilage cells, in cells of the epiphyseal growth plate, and in bone marrow stromal cells., ((c) 2007 Wiley-Liss, Inc.)

High physiological prolactin induced positive calcium balance by stimulating intestinal calcium absorption, reducing renal calcium excretion, and increasing bone calcium deposition in female rats. Although prolactin-induced increase in trabecular bone calcium deposition was absent after ovariectomy, its effects on cortical bones were still controversial. The present investigation, therefore, aimed to study the effect of in vivo long-term high physiological prolactin induced by either anterior pituitary (AP) transplantation or 2.5 mg/kg prolactin injection on cortical bones in ovariectomized rats. Since the presence of prolactin receptors (PRLR) in different bones of normal adult rats has not been reported, we first determined mRNA expression of both short- and long-form PRLRs at the cortical sites (tibia and femur) and trabecular sites (calvaria and vertebrae) by using the RT-PCR. Our results showed the mRNA expression of both PRLR isoforms with predominant long form at all sites. However, high prolactin levels induced by AP transplantation in normal rats did not have any effect on the femoral bone mineral density or bone mineral content. By using (45)Ca kinetic study, 2.5 mg/kg prolactin did not alter bone formation, bone resorption, calcium deposition, and total calcium content in tibia and femur of adult ovariectomized rats. AP transplantation also had no effect on the cortical total calcium content in adult ovariectomized rats. Because previous work showed that the effects of prolactin were age dependent and could be modulated by high-calcium diet, interactions between prolactin and these two parameters were investigated. The results demonstrated that 2.0% wt/wt high-calcium diet significantly increased the tibial total calcium content in 9-wk-old young AP-grafted ovariectomized rats but decreased the tibial total calcium content in 22-wk-old adult rats. As for the vertebrae, the total calcium contents in both young and adult rats were not changed by high-calcium diet. The present results thus indicated that the adult cortical bones were potentially direct targets of prolactin. Moreover, the effects of high physiological prolactin on cortical bones were age dependent and were observed only under the modulation of high-calcium diet condition.

Synthetic and naturally derived scaffold biomaterials in combination with osteogenic cells or bioactive factors have the potential to serve as bone graft substitutes. Porous poly(l-lactide-co-dl-lactide) (PLDL) scaffolds with mechanical properties comparable to trabecular bone and an oriented, interconnected porosity designed to enhance internal mass transport were recently developed. In this study, PLDL scaffolds were seeded with rat calvarial or rat stromal cells and cultured up to 8 weeks in media containing osteogenic supplements. Cell-seeded human demineralized trabecular bone matrix (DTBM) scaffolds were included for comparison. All constructs were imaged weekly from 4 to 8 weeks using microcomputed tomography (micro-CT) to nondestructively quantify the amount and distribution of mineralized matrix formation. The total mineralized matrix volume increased with time in culture for all construct groups. DTBM constructs contained significantly more mineralized matrix than PLDL constructs. However, an analysis of the acellular DTBM scaffolds exposed to osteogenic media revealed partial remineralization of the demineralized matrix whereas no mineralization was detected in acellular PLDL scaffolds. Differences in mineral distribution were also evident with cell-mediated mineralization found throughout the PLDL constructs but localized to the periphery of the DTBM constructs for both cell types. Expression of bone marker genes indicating osteoblast differentiation was demonstrated in all groups at 8 weeks using a quantitative reverse transcription polymerase chain reaction. Osteocalcin expression was significantly higher for calvarial cell constructs compared to stromal cell constructs, regardless of the type of scaffold. This study demonstrated that micro-CT imaging may be used to nondestructively and quantitatively monitor mineralization within three-dimensional scaffolds in vitro. PLDL scaffolds with an oriented microarchitecture were shown to support cell attachment, differentiation, and cell-mediated mineralization comparable to natural DTBM scaffolds., (Copyright 2004 Wiley Periodicals, Inc. J Biomed Mater Res 69A: 97-104, 2004)

The purpose of this study was to evaluate the effects of isolated alterations in mineral content on mouse bone torsional properties. The femora and tibiae from 25 eight-week-old male A/J strain mice were divided into five groups and selectively decalcified from 5% to 20%. The right femora were then tested to failure in torsion while the tibiae were ashed to determine final mineral content of the decalcified bones. Contralateral femora were serially cross-sectioned to determine geometric properties, and effective material properties were then calculated from the geometric and structural properties of each femoral pair. We found that the relationship between ash content and effective shear modulus or maximum effective shear stress could best be characterized through a power law, with an exponential factor of 6.79 (R2 = 0.85) and 4.04 (R2 = 0.67), respectively. This indicates that in a murine model, as with other species, small changes in ash content significantly influence effective material properties. Furthermore, it appears that (in adolescent A/J strain mice) effective shear modulus is more heavily affected by changes in mineralization than is maximum effective shear stress when these properties are derived from whole bone torsional tests to failure.

Atomic force microscopy (AFM) was used to obtain three-dimensional images of isolated mineralites extracted from young postnatal bovine bone. The mean mineralite size is 9 nm x 6 nm x 2.0 nm, significantly shorter and thicker than the mineralites of mature bovine bone measured by the same technique. Mineralites of the young postnatal bone can be accommodated within the hole zone regions of a quasi-hexagonally packed collagen fibril in the fashion described by Hodge [9] in which laterally adjacent hole zone regions form continuous "channels" across the diameter of a fibril for a distance of at least 10 nm. Deposition of mineralites of the size noted above in this void volume of the fibrils would result in little or no distortion of the collagen molecules or supramolecular structure of the collagen fibril. The new AFM data supporting this claim is consistent with findings obtained by electron microscopy and low-angle x-ray and neutron diffraction that mineralites formed within collagen fibrils during initial stages of calcification occur within the hole zone region. However, the deposition of additional mineralites in the intermolecular spaces between collagen molecules in the overlap region of the fibrils would significantly distort the fibrils since the space available between adjacent molecules is considerably less than even the smallest dimension of the mineralites.

Previous 31P cross-polarization and differential cross-polarization magic angle spinning (CP/MAS and DCP/MAS) solid-state NMR spectroscopy studies of native bone and of the isolated crystals of the calcified matrix synthesized by osteoblasts in cell culture identified and characterized the major PO(-3)(4) phosphate components of the mineral phase. The isotropic and anisotropic chemical shift parameters of the minor HPO(-2)(4) component in bone mineral and in mineral deposited in osteoblast cell cultures were found to differ significantly from those of brushite, octacalcium phosphate, and other synthetic calcium phosphates. However, because of in vivo and in vitro evidence that phosphoproteins may play a significant role in the nucleation of the solid mineral phase of calcium phosphate in bone and other vertebrate calcified tissues, the focus of the current solid-state 31P NMR experiments was to detect the possible presence of and characterize the phosphoryl groups of phosphoproteins in bone at the very earliest stages of bone mineralization, as well as the possible presence of calcium-phosphoprotein complexes. The present study demonstrates that by far the major phosphate components identified by solid-state 31P NMR in the very earliest stages of mineralization are protein phosphoryl groups which are not complexed with calcium. However, very small amounts of calcium-complexed protein phosphoryl groups as well as even smaller, trace amounts of apatite crystals were also present at the earliest phases of mineralization. These data support the hypothesis that phosphoproteins complexed with calcium play a significant role in the initiation of bone calcification.

The influence of long-term hypodynamy on the calcium and phosphorus levels was studied in the bones of Japanese quails. The hypodynamy evoked different changes in the calcium and phosphorus content in males and females. The calcium content in the marrow of femurs was only changed in the hens, while in cockerels it was significantly decreased in the upper part and marrow of the tibia. Furthermore, changes in the phosphorus content were observed only in the tibia of cockerels.

Although C3H/HeJ (C3H) and C57BL/6J (B6) mice are similar in body size (and adult weight), and have bones of similar external size, C3H mice have higher peak bone densities than B6 mice (e.g., 53% higher peak bone density in the femora). The current studies were intended to assess the role of mechanical loading/unloading as a possible determinant of the bone density difference between these inbred strains of mice and, specifically, to assess the effect of sciatic neurectomy on histomorphometric indices of bone formation and resorption in the tibiae of female C3H and B6 mice. Groups of 10 mice of each strain were subjected to left-side sciatic neurectomy (left hindlimb immobilization) or a sham procedure. The contralateral (right) legs of each mouse were used as controls. Four weeks of immobilization produced no systemic changes in bone formation indices in either strain of mice (i.e., no change in serum alkaline phosphatase or serum osteocalcin). However, histomorphometric assessments at the tibiofibular junction showed that 4 weeks of immobilization caused a time-dependent decrease in the length of the endosteal bone forming perimeter (e.g., 14% of control single-labeled, noneroded surface at 4 weeks, p < 0.005) with a concomitant increase in the length of the endosteal bone resorbing perimeter (i.e., 424% of control eroded surface at 4 weeks, p < 0.005), in the B6 mice. These effects were associated with an increase in medullary area (132% of control, p < 0.05) at this site, in the B6 mice. The pattern of response was different in the tibiae of the C3 mice-a much smaller decrease in bone forming perimeter (88% of control at 4 weeks, p < 0.05), with no associated increase in bone resorbing perimeter, and no change in medullary area. Similar effects were seen at a second cross-sectional sampling site, in the proximal tibia. Together, these findings indicate that B6 mice are more sensitive to endosteal bone loss from hindlimb immobilization than C3H mice.

To analyze the interrelationships between the structure, composition, and mechanical properties of articular cartilage, canine knee (n = 10) femoral and tibial cartilages were used as experimental tissues. The biomechanical properties, instant shear modulus (IM), and equilibrium shear modulus (EM) of articular cartilage were investigated using an in situ indentation creep technique. The local variations in the concentration of glycosaminoglycans (GAGs) in the cartilage were measured with a microspectrophotometer after safranin-O staining of histological sections. Using a computer-based quantitative polarized light microscopy method, area-specific measurements of the optical path difference were performed to quantitate collagen-related optical retardation (gamma) of cartilage zones. The IM and EM were 131.3 and 51.2% higher (p < 0.001) in the femoral cartilage than in the tibial cartilage, respectively. The mean thickness of the superficial zone and the relative proportion of the superficial zone from the total uncalcified cartilage was 107.1 and 155.3% higher (p < 0.001) at the femoral test points than in the tibial ones, respectively. The mean thickness of the tibial uncalcified cartilage was 21.1% higher (p < 0.001) than the thickness of the femoral cartilage. The GAG concentration of the tibial cartilage was higher (14.8%, p < 0.001) than that of the femoral cartilage, especially in the superficial zone (50.0%, p < 0.05), whereas the gamma of the collagen network in the superficial zone of the femoral cartilage was 64.7% higher (p < 0.001) than in the tibial cartilage. The percent relative thickness and retardation gamma of the superficial zone correlated positively with the indentation stiffness of the canine knee articular cartilage. These observations indicate that cartilage is structurally inhomogenous and layered tissue and the local organization of collagen and GAG concentration of the articular cartilage regulate the biomechanical properties of the tissue. The structure and composition of the superficial articular cartilage significantly affects the indentation response of the canine knee articular cartilage.

Based on the hypothesis that bone calcification is promoted by loading physical pressure, changes in the microstructure of the bone under hyperbaric conditions were analyzed by imaging technology. Hyperbaric exposure was carried out for two weeks at 2 atm (equal to the pressure at a depth of water of 10 m) which was achieved using a mixed gas of helium and oxygen (He:O2 88%:12%) in which the oxygen partial pressure was maintained at constant (PO2: 0.21 bar). In image technological analysis, the growth and development of the bone were evaluated at different stages using Digital Magnification Radiography (DMR) images and based on changes in the X-ray absorption ratio. DMR images after hyperbaric exposure showed calcification in the heads of long bones (humeri, femora, and tibiae) in mice. There were also significant changes in the X-ray absorption ratio in the heads. The accumulation of 99mTc-MDP was higher in all long-bone heads after hyperbaric exposure than before exposure. These results suggest that the hyperbaric environment promotes bone calcification.

Histological examinations and estimations of the contents of ash, phosphorus and calcium were used to investigate the femora and tibiotarsi from lame and normal 35-day-old broilers from Holland, Northern Ireland and Scotland. The prevalence of different pathologies varied with the source of the broilers and there were correlations between histological and bone ash values. The most common condition causing lameness was bacterial infection within the physis and cartilaginous epiphysis (bacterial chondronecrosis) of the proximal tibiotarsus, and there was a possible link between rickets attributable to a relative phosphorus deficiency and this condition. There were wide variations between birds in the cortical bone quality as assessed histologically and by estimates of the bone ash content and phosphorus to calcium ratios. Theses variations may be related to different probabilities of bone fracture.

Although a number of properties of bisphosphonates have been recognized which influence the metabolic process in bones, particularly those concerning the remodelling processes, the influence of this new group of drugs on the mechanical properties of bones remains an open issue. In order to clarify this problem, the present study concentrated on the influence of a new generation bisphosphonate, i.e. pamidronate upon the mechanical properties, growth, and morphological changes in the femoral and tibial bones in rats. The experiments carried out concerned pamidronate administration to male Wistar rats in doses of 3 mg/kg of body mass subcutaneously, for the period of 3 or 6 weeks. The total changes in the osseous tissue after pamindronate administration indicate the drug to foster the development of osteopetrosis in rats, the prominent sings of the disease being mainly deformations of epiphysis, decreased bone growth, increased thickness of epiphysial cartilage and bone trabeculae, as well as lowered resistance to fractures and decreased susceptibility to deformations.