Altered type I collagen networking in osteoporotic human femoral head revealed by histomorphometric and Fourier transform infrared imaging correlated analyses.

Bone homeostasis is the equilibrium between organic and inorganic components of the extracellular matrix (ECM) and cells. Alteration of this balance has consequences on bone mass and architecture, resulting in conditions such as osteoporosis (OP). Given ECM protein mutual regulation and their effects on bone structure and mineralization, further insight into their expression is crucial to understanding bone biology under normal and pathological conditions. This study focused on Type I Collagen, which is mainly responsible for structural properties and mineralization of bone, and selected proteins implicated in matrix composition, mineral deposition, and cell-matrix interaction such as Decorin, Osteocalcin, Osteopontin, Bone Sialoprotein 2, Osteonectin and Transforming Growth Factor beta. We developed a novel multidisciplinary approach in order to assess bone matrix in healthy and OP conditions more comprehensively by exploiting the Fourier Transform Infrared Imaging (FTIRI) technique combined with histomorphometry, Sirius Red staining, immunohistochemistry, and Western Blotting. This innovatory procedure allowed for the analysis of superimposed tissue sections and revealed that the alterations in OP bone tissue architecture were associated with warped Type I Collagen structure and deposition but not with changes in the total protein amount. The detected changes in the expression and/or cooperative or antagonist role of Decorin, Osteocalcin, Osteopontin, and Bone Sialoprotein-2 indicate the deep impact of these NCPs on collagen features of OP bone. Overall, our strategy may represent a starting point for designing targeted clinical strategies aimed at bone mass preservation and sustain the FTIRI translational capability as upcoming support for traditional diagnostic methods.

Short-term clinical and radiographic outcomes of total hip arthroplasty with PMPC-grafted highly cross-linked polyethylene liners against 32-mm femoral heads.

We evaluated the clinical and radiographic outcomes of femoral head penetration and total hip arthroplasties with untreated and poly (2-methacryloyloxyethyl phosphorylcholine) (PMPC)-grafted highly cross-linked polyethylene (HXLPE) acetabular liners against 26-, 28-, and 32-mm cobalt-chromium alloy femoral heads 3 years after the index surgery. Three combinations of the articulating surfaces were evaluated in the present study: untreated or PMPC-grafted HXLPE liner against 26- or 28-mm femoral heads (n = 16, 24) [control (26 or 28 mm) and PMPC (26 or 28 mm)] and PMPC-grafted HXLPE liner against 32-mm femoral heads (n = 64) [PMPC (32 mm)]. The clinical outcomes improved at 3 years postoperatively for the groups. No periprosthetic osteolysis or acetabular component migration was detected, and no revision surgery was performed among the groups. The steady-state wear rate of the PMPC (26 or 28 mm) group (0.021 mm/year) was lower than that of the control (26 or 28 mm) group (- 0.015 mm/year); the steady-state wear was under the clinical threshold. In contrast, the steady-state wear rate of the PMPC (32 mm) group (-0.006 mm/year) showed no significant difference when compared to that of the PMPC (26 or 28 mm) group (p < 0.01). The results obtained in the present study clearly demonstrate that PMPC-grafting onto an HXLPE surface improved the wear resistance of acetabular liners, even when coupled with larger femoral heads. Although further follow-up evaluations are required, PMPC-grafted HXLPE acetabular liners may be a promising approach to extend the longevity of artificial joints.

The effect of femoral head size on edge loading in metal-on-metal hip joint replacement under dynamic separation conditions.

Edge loading that occurs in hip joint replacements due to dynamic separation of the joint bearings has been shown to cause severe wear for meal-on-metal bearings. In the present study, the multibody dynamics model for metal-on-metal (MoM) hip joints with a medial-lateral translational mismatch in the centers of rotation of the cup and head has been developed to predict the dynamic separation and contact force of edge loading under gait loading conditions. The effects of larger head diameters (28-55 mm), in combination with the translational mismatch (0-4 mm) and varied cup inclination angles (45°-65°), on edge loading of MoM bearings have been computationally investigated. For the given translational mismatch, increasing head diameters results in negligible effects on the dynamic separation, contact force and severity of edge loading. Increasing head size also leads to increased offset loading torque which has been found to reach at the level that may cause cup loosening under larger translational mismatch at 4 mm. The result highlights the importance of the cup inclination angle of 45° and a lower translational mismatch to avoid severe edge loading.

GC-MS method for metabolic profiling of mouse femoral head articular cartilage reveals distinct effects of tissue culture and development.

OBJECTIVE: The metabolic profile of cartilage is important to define as it relates to both normal and pathophysiological conditions. Our aim was to develop a precise, high-throughput method for gas/chromatography-mass/spectrometry (GC-MS) semi-targeted metabolic profiling of mouse cartilage. METHOD: Femoral head (hip) cartilage was isolated from 5- and 15-week-old male C57BL/6J mice immediately after death for in vivo analyses. In vitro conditions were evaluated in 5-week-old samples cultured ±10% fetal bovine serum (FBS). We optimized cartilage processing for GC-MS analysis and evaluated group-specific differences by multivariate and parametric statistical analyses. RESULTS: 55 metabolites were identified in pooled cartilage (4 animals per sample), with 29 metabolites shared between in vivo and in vitro conditions. Multivariate analysis of these common metabolites demonstrated that culturing explants was the strongest factor altering cartilage metabolism, followed by age and serum starvation. In vitro culture altered the relative abundance of specific metabolites; whereas, cartilage development between five and 15-weeks of age reduced the levels of 36 out of 43 metabolites >2-fold, especially in TCA cycle and alanine, aspartate, and glutamate pathways. In vitro serum starvation depleted six out of 41 metabolites. CONCLUSION: This study describes the first GC-MS method for mouse cartilage metabolite identification and quantification. We observed fundamental differences in femoral head cartilage metabolic profiles between in vivo and in vitro conditions, suggesting opportunities to optimize in vitro conditions for studying cartilage metabolism. In addition, the reductions in TCA cycle and amino acid metabolites during cartilage maturation illustrate the plasticity of chondrocyte metabolism during development.

Mechanical properties of cancellous tissue in compression test and nanoindentation.

The goal of the study was to determine material constants of cancellous tissue in a compression test taking into account stress-strain characteristics. The nineteen test samples were resected from a femoral head of a patient qualified for a hip replacement surgery. During the compression tests, load-displacement curves were evaluated and stress-strain characteristics (𝜎-𝜀) of the cancellous bone tissue were calculated. A special one-touch extensometer was used. The use of an extensometer improves the displacement measurement accuracy with respect to the measurement using standard crosshead. The obtained values of the mechanical parameters as well as the stress-strain curves are the basis for further research going into the development of bone tissue constitutive equations.

Urban and rural area differences in the interaction between oxidative process elements in human femoral bone.

Elements in the human body come from contaminated food, water, and air from the living area. Bones are a marker of long-term exposure to elements and show a relationship between them. The aim of the study was to analyze the correlation between the contents of Zn, Cu, Fe, Mo, Cr, Ni, Ba, Sr, and Pb in the proximal femoral head (cancellous bone) and femoral neck (cortical bone) in rural and urban populations. The study included 96 patients who were operated on for total hip replacement (THR), acquired in a surgical procedure with atomic absorption spectrometry, and the content of Zn, Cu, Fe, Mo, Cr, Ni, Ba, Sr, and Pb was evaluated. In rural areas, significant negative correlations were observed for Mo/Cr, Mo/Cu, and Ni/Fe, and positive correlations were observed for Fe/Zn and Pb/Zn. In urban areas, a negative correlation was found for Pb/Mo. Pb and Ni increased with age only in villagers, and Zn and Sr decreased with age in urban citizens. Ba decreased with age in people from rural areas. The correlation showed variances mainly in molybdenum, nickel, and oxidative elements between rural and urban populations.

Measuring bone stiffness using spherical indentation.

OBJECTIVES: Bone material properties are a major determinant of bone health in older age, both in terms of fracture risk and implant fixation, in orthopaedics and dentistry. Bone is an anisotropic and hierarchical material so its measured material properties depend upon the scale of metric used. The scale used should reflect the clinical problem, whether it is fracture risk, a whole bone problem, or implant stability, at the millimetre-scale. Indentation, an engineering technique involving pressing a hard-tipped material into another material with a known force, may be able to assess bone stiffness at the millimetre-scale (the apparent elastic modulus). We aimed to investigate whether spherical-tip indentation could reliably measure the apparent elastic modulus of human cortical bone. MATERIALS AND METHODS: Cortical bone samples were retrieved from the femoral necks of nineteen patients undergoing total hip replacement surgery (10 females, 9 males, mean age: 69 years). The samples underwent indentation using a 1.5 mm diameter, ruby, spherical indenter tip, with sixty indentations per patient sample, across six locations on the bone surfaces, with ten repeated indentations at each of the six locations. The samples then underwent mechanical compression testing. The repeatability of indentation measurements of elastic modulus was assessed using the co-efficient of repeatability and the correlation between the bone elastic modulus measured by indentation and compression testing was analysed by least-squares regression. RESULTS: In total, 1140 indentations in total were performed. Indentation was found to be repeatable for indentations performed at the same locations on the bone samples with a mean co-efficient of repeatability of 0.4 GigaPascals (GPa), confidence interval (C.I): 0.33-0.42 GPa. There was variation in the indentation modulus results between different locations on the bone samples (mean co-efficient of repeatability: 3.1 GPa, C.I: 2.2-3.90 GPa). No clear correlation was observed between indentation and compression values of bone elastic modulus (r = 0.33, p = 0.17). The mean apparent elastic modulus obtained by spherical indentation was 9.9 GPa, the standard deviation for each indent cycle was 0.11 GPa, and the standard deviation between locations on the same sample was 1.01 GPa. The mean compression apparent elastic modulus was 4.42 GPa, standard deviation 1.02 GPa. DISCUSSION: Spherical-tip indentation was found to be a repeatable test for measuring the elastic modulus of human cortical bone, demonstrated by a low co-efficient of repeatability in this study. It could not, however, reliably predict cortical bone elastic modulus determined by platens compression testing in this study. This may be due to indentation only probing mechanical properties at the micro-scale while platens compression testing assesses millimetre length-scale properties. Improvements to the testing technique, including the use of a larger diameter spherical indenter tip, may improve the measurement of bone stiffness at the millimetre scale and should be investigated further.

Imaging of Lipids in Native Human Bone Sections Using TOF-Secondary Ion Mass Spectrometry, Atmospheric Pressure Scanning Microprobe Matrix-Assisted Laser Desorption/Ionization Orbitrap Mass Spectrometry, and Orbitrap-Secondary Ion Mass Spectrometry.

A method is described for high-resolution label-free molecular imaging of human bone tissue. To preserve the lipid content and the heterogeneous structure of osseous tissue, 4 µm thick human bone sections were prepared via cryoembedding and tape-assisted cryosectioning, circumventing the application of organic solvents and a decalcification step. A protocol for comparative mass spectrometry imaging (MSI) on the same section was established for initial analysis with time-of-flight secondary ion mass spectrometry (TOF-SIMS) at a lateral resolution of 10 µm to <500 nm, followed by atmospheric pressure scanning microprobe matrix-assisted laser desorption/ionization (AP-SMALDI) Orbitrap MSI at a lateral resolution of 10 µm. This procedure ultimately enabled MSI of lipids, providing the lateral localization of major lipid classes such as glycero-, glycerophospho-, and sphingolipids. Additionally, the applicability of the recently emerged Orbitrap-TOF-SIMS hybrid system was exemplarily examined and compared to the before-mentioned MSI methods.

Raman spectral characteristics of neck and head of femur in low-density lipoprotein receptor gene knockout mice submitted to treadmill aerobic training.

Hypercholesterolemia is associated with deterioration of bone tissue. Trough Raman spectroscopy, the present study aimed to assess the molecular changes in terms of mineral and organic bone-tissue remodeling in low-density lipoprotein receptor gene knockout mice (LDLr-/-) submitted to treadmill aerobic training. In order to evaluate alterations in trabecular bone and cartilage surface, neck and head femoral were assessed into four groups: (CON, n=7), the 1) animals did not perform any physical activity and had hypercholesterolemia (CON, n=7); The trained groups where animals underwent a protocols of low, moderate and high training for 8weeks and 4 times per week on a treadmill with progressive intensities: running speed (8-13m/min) and time of exercise (20-60min). These progressive intensities were needed to adjust the working load for the physical training to 2) 30-49% (LOW, n=7), 3) 50-79% (MOD, n=7), and 4) 80-100% (HIGH, n=7) of the maximum running speed. Each group was examined for molecular changes in both bone phases (mineral and organic) using principal components analysis (PCA). All exercised groups showed a significant increase (p<0.05) of bone remodeling in trabecular area and a higher content of proteins (organic content) in cartilage independently of physical activity intensity. Thus, this work demonstrated that physical training could act as a bone-protector factor in hyperlipidemic animal model as well as Raman technique associated with PCA analysis may be feasible methodology for assessment or monitoring of bone at the molecular level.

Rehydration improves the ductility of dry bone allografts.

Processing of bone allografts improves infectious safety and allows storing bone substitutes at room temperature. The aim of this study was to compare mechanical properties of the processed Osteopure™ bone with fresh frozen bone. All the samples were pieces from femoral heads retrieved during hip arthroplasty operations. The processing includes chemical decellularization, drying and irradiation with 25 kGy. Three types of samples were tested: 1. fresh frozen thawed wet, 2. dry non-rehydrated graft 3. dry rehydrated graft. In the 3-point bending test Young's modulus and stress at break yielded no significant difference among the 3 different sample groups. Rehydrating of the dry graft showed increased ductility in strain at break test compared with the other 2 groups (p = 0.003). In compression tests dry grafts had significantly higher maximum effective stress and apparent maximum deformation compared with the grafts of other groups (p < 0.05). Processed bone has almost similar mechanical properties compared with fresh frozen bone. However, rehydration of processed dry graft increases its ductility. These grafts may tolerate bending forces better before breakage.

Reconciling in vivo and in vitro kinetics of the polymorphic transformation in zirconia-toughened alumina for hip joints: I. Phenomenology.

Exploitation of the toughening effect induced by polymorphic phase transformation of zirconia in zirconia-toughened alumina (ZTA) requires the composite being properly designed and carefully manufactured. A sound algorithm for predicting phase stability along with strict control over manufacturing steps are required in order to prevent possible in vivo surface degradation or implant fracture. This paper is the first in a series of three monographs, which aim at: (i) statistically comparing the in vitro/in vivo phenomenology of surface-metastability for currently marketed ZTA femoral heads; (ii) refining pre-existing theoretical models for predicting in vivo zirconia phase metastability via the use of accelerated in vitro ageing experiments; and, (iii) providing a rationale for the mechanism(s) involved with the observed in vivo surface metastability. This initial paper of a series of three, which specifically deals with item (i), shows discrepancies between the levels of polymorphic phase transformation detected in ZTA retrievals and in vitro predictions, and attempts a phenomenological analysis of the reasons behind such discrepancies. Moreover, marked inhomogeneities are also found among as-manufactured components through different years of production. The phenomenology of retrievals' data suggests key roles for both the presence of metallic stain and the initial value of monoclinic volume fraction.

An analysis of factors affecting the mercury content in the human femoral bone.

The study was carried out to determine the content of mercury in bone tissue of the proximal femur (head and neck bone) of 95 patients undergoing total hip replacement due to osteoarthritis, using CF-AFS analytical technique. Furthermore, the investigations were aimed at assessing the impact of selected factors, such as age, gender, tobacco smoking, alcohol consumption, exposure to chemical substance at work, type of degenerative changes, clinical evaluation and radiological parameters, type of medications, on the concentration of mercury in the head and neck of the femur, resected in situ. Mercury was obtained in all samples of the head and neck of the femur (n = 190) in patients aged 25-91 years. The mean content of mercury for the whole group of patients was as follows: 37.1 ± 35.0 ng/g for the femoral neck and 24.2 ± 19.5 ng/g for the femoral head. The highest Hg contents were found in femoral neck samples, both in women and men, and they amounted to 169.6 and 176.5 ng/g, respectively. The research showed that the mercury content of bones can be associated with body mass index, differences in body anatomy, and gender. The uses of statistical analysis gave the possibility to define the influence of factors on mercury content in human femoral bones.

Lyophilized allogeneic bone tissue as an antibiotic carrier.

The rising number of primary joint replacements worldwide causes an increase of revision surgery of endoprostheses due bacterial infection. Revision surgery using non-cemented implants seems beneficial for the long-term outcome and the use of antibiotic-impregnated bone grafts might control the infection and give a good support for the implant. In this study we evaluated the release of antibiotics from fresh-frozen and lyophilized allogeneic bone grafts. Lyophilized bone chips and fresh frozen bone chips were mixed with gentamicin sulphate, gentamicin palmitate, vancomycin, calcium carbonate/calcium sulphate impregnated with gentamicin sulphate, and calcium carbonate/calcium sulphate bone substitute material impregnated with vancomycin. The efficacy of each preparation was measured by drug release tests and bacterial susceptibility using B. subtilis, S. aureus and methicillin-resistant Staphylococcus aureus. The release of gentamicin from lyophilized bone was similar to the release rate from fresh frozen bone during all the experimental time. That fact might be related to the similar porosity and microstructure of the bone chips. The release of gentamicin from lyophilized and fresh frozen bone was high in the first and second day, decreasing and keeping a low rate until the end of the second week. Depending on the surgical strategy either polymethylmethacrylate or allogeneic bone are able to deliver sufficient concentrations of gentamicin to achieve bacterial inhibition within two weeks after surgery. In case of uncemented revision of joint replacements allogeneic bone is able to deliver therapeutic doses of gentamicin and peak levels immediately after implantation during a fortnight. The use of lyophilized and fresh frozen bone allografts as antibiotic carriers is recommended for prophylaxis of bone infection.

Three Year RSA Evaluation of Vitamin E Diffused Highly Cross-linked Polyethylene Liners and Cup Stability.

Vitamin E diffusion into highly cross-linked polyethylene (E-XLPE) is a method for enhancing oxidative stability of acetabular liners. The purpose of this study was to evaluate in vivo penetration of E-XLPE using radiostereometric analysis (RSA). Eighty-four hips were recruited into a prospective 10-year RSA. This is the first evaluation of the multicenter cohort after 3-years. All patients received E-XLPE liners (E1, Biomet) and porous-titanium coated cups (Regenerex, Biomet). There was no difference (P=0.450) in median femoral head penetration into the E-XLPE liners at 3-years comparing cobalt-chrome heads (-0.028mm; inter-quartile range (IQR) - 0.065 to 0.047) with ceramic heads (-0.043mm, IQR - 0.143to0.042). The 3-year follow-up indicates minimal E-XLPE liner penetration regardless of head material and minimal early cup movement.

Experimental study of diffusion coefficients of water through the collagen: apatite porosity in human trabecular bone tissue.

We firstly measured the swelling of single trabeculae from human femur heads during water imbibition. Since the swelling is caused by water diffusing from external surfaces to the core of the sample, by measuring the sample swelling over time, we obtained direct information about the transport of fluids through the intimate constituents of bone, where the mineralization process takes place. We developed an apparatus to measure the free expansion of the tissue during the imbibition. In particular, we measured the swelling along three natural axes (length L, width W, and thickness T) of plate-like trabeculae. For this aim, we developed a 3D analytical model of the water uptake by the sample that was performed according to Fickian transport mechanism. The results were then utilized to predict the swelling over time along the three sample directions (L, W, T) and the apparent diffusion coefficients D T, D W, and D L.

Effect of storage temperature and antibiotic impregnation on the quantity of bone morphogenetic protein seven in human bone grafts.

PURPOSE: The aim of this study was to quantify the amount of bone morphogenic protein 7 (BMP-7) in bone samples in different storage and treatment conditions used in bone banks and thereby evaluate the benefit of this test as a routine measure before bone grafting. METHODS: Fresh as well as frozen bone chips, each with and without antibiotic impregnation, were screened for their BMP-7 content. Human bone chips were produced from femoral heads of two female donors who had undergone total hip replacement surgery. The amount of BMP-7 was detected using a commercially available enzyme-linked immunosorbent assay (ELISA) test. RESULTS: There were no significant differences between groups in samples obtained from the first femoral head. Bone-chip samples derived from the second femoral head showed significant differences between groups. The actual amount of these differences was small and most likely biologically irrelevant. It is important to note that there was a significant difference between groups when comparing both femoral heads, reflecting donor-to-donor variability. CONCLUSION: ELISA testing for BMP-7 as a qualitative measurement of bone grafts should be considered a routine quality-control test for bone banks.

The measurement and analysis of surface geometric structure of ceramic femoral heads.

The surface functionality of interacting components is determined by evaluating geometric surface structure. Hence, the ceramic femoral heads originated from precision machining were subject to measurement and analysis with regard to roughness, surface damages and deviation from roundness. A variety of measurement techniques were applied in order to thoroughly examine the product quality. The obtained results proved in accordance with the specification: Ra was not greater than 0.05 µm, whereas Δ was less than 10 µm (5 µm) when ceramic balls were used in conjunction with a polyethylene (ceramic) socket. Additionally, the surface damages which can affect the life of a friction pair were analyzed.

Histological and molecular characterization of the femoral attachment of the human ligamentum capitis femoris.

The ligamentum capitis femoris (LCF) has increased in clinical significance through the development of hip arthroscopy. The histological pathologies and molecular composition of the femoral attachment of the LCF and the degeneration caused by LCF disruption were investigated in the human hip joint. Twenty-four LCFs were retrieved at surgery for femoral neck fracture (age range: 63-87 years). In the "intact" (i.e., intact throughout its length, n = 12) group, the attachment consisted of rich fibrocartilage. Fibrocartilage cells were present in the midsubstance. In contrast, the construction of the attachment in the "disrupted" (i.e., ligament no longer attached to the femoral head, n = 12) group had disappeared. The attachment in the disrupted group was not labeled for type II collagen or aggrecan, while that in the intact group was labeled for types I, II and III collagen, chondroitin 4-sulfate, chondroitin 6-sulfate, aggrecan, and versican. The percentage of single-stranded DNA-positive chondrocytes was significantly higher in the disrupted group than in the intact group. We conclude that the femoral attachment of the LCF has a characteristic fibrocartilaginous structure that is likely to adjust to the mechanical load, and suggest that its degeneration is advanced by disruption and should be regarded as a clinical pathology.

Cartilage collagen damage in hip osteoarthritis similar to that seen in knee osteoarthritis; a case-control study of relationship between collagen, glycosaminoglycan and cartilage swelling.

BACKGROUND: It remains to be shown whether OA shares molecular similarities between different joints in humans. This study provides evidence for similarities in cartilage molecular damage in osteoarthritic (OA) joints. METHODS: Articular cartilage from osteoarthritic hip joints were analysed and compared to non-OA controls regarding collagen, glycosaminoglycan and water content. Femoral heads from 16 osteoarthritic (OA) and 20 reference patients were obtained from hip replacement surgery due to OA and femoral neck fracture, respectively. Cartilage histological changes were assessed by Mankin grading and denatured collagen type II immunostaining and cartilage was extracted by α-chymotrypsin. Hydroxyproline and Alcian blue binding assays were used to measure collagen and glycosaminoglycan (GAG) content, respectively. RESULTS: Mankin and immunohistology scores were significantly higher in hip OA samples than in reference samples. Cartilage water content was 6% higher in OA samples than in references. 2.5 times more collagen was extracted from OA than from reference samples. There was a positive association between water content and percentage of extractable collagen pool (ECP) in both groups. The amounts of collagen per wet and dry weights did not differ statistically between OA and reference cartilage. % Extractable collagen was not related to collagen per dry weight in either group. However when collagen was expressed by wet weight there was a negative correlation between % extractable and collagen in OA cartilage. The amount of GAG per wet weight was similar in both groups but the amount of GAG per dry weight was higher in OA samples compared to reference samples, which suggests a capacity for GAG biosynthesis in hip OA cartilage. Neither of the studied parameters was related to age in either group. CONCLUSIONS: Increased collagen extractability and water content in human hip cartilage is associated with OA pathology and can be observed at early stages of the degenerative hip OA process. Our results suggest a common degradative pathway of collagen in articular cartilage of different joints. Furthermore, the study suggests that biochemical changes precede more overt OA changes and that chondrocytes may have a capability to compensate molecular loss in the early phase of OA.