Valerio Canè

Size and density of osteocyte lacunae in different regions of long bones

SummarySize and density of osteocyte lacunae were evaluated at different levels of long bones to investigate whether or not the proportion of bone tissue occupied by osteocytes changes in skeletal regions, characterized by clear-cut differences in bone turnover rates. Statistical analysis of the results shows that the mean cross-sectional area of osteocyte lacunae (C) is lowest in compact bone of diaphysis and metaphysis, highest in spongy bone of metaphysis and epiphysis. On the contrary, the mean surface of bone tissue surrounding each osteocyte (T=bidimensional osteocyte territory, indirectly calculated from the number of lacunae/mm2 of bone) is largest in compact bone of diaphysis, smallest in metaphyseal spongiosa, and shows intermediate values in the cortex of metaphysis and in epiphyseal spongiosa. The proportion of bone tissue occupied by osteocyte lacunae (%C/T) appears to follow at different levels of long bones, the same pattern recorded for the data of bone turnover rate, by the tetracycline labeling technique: it is lowest in mid-diaphyses, highest in metaphyses, and intermediate in epiphyses. On the basis of these findings, it is suggested that the action exerted by osteocytes on the surrounding calcified matrix, whatever the function of these cells, is not uniform throughout the skeleton and is to some extent correlated with the activity of the other bone cells—osteoblasts and osteoclasts.The significance of some of the data reported is also discussed in relation to investigations of periosteocytic lacunar morphometry.

The effect of pulsed electromagnetic fields on the osteointegration of hydroxyapatite implants in cancellous bone: a morphologic and microstructural in vivo study

Effects of pulsed electromagnetic fields (PEMFs, 75 Hz, 1.6 mT) were investigated in 12 rabbits after placing hydroxyapatite (HA) implants in their femoral condyles. Six animals were stimulated with PEMFs for three consecutive weeks, 6 h/day, while the remaining animals were sham‐treated (Control Group). Rabbits were sacrificed at 3 and 6 weeks (after a 3‐week non‐stimulation period) for histomorphometric analysis and microhardness testing (at 200, 500, 1000, 2000 μm from the implant) around the implants around the implants. Histomorphometric analysis did not highlight any significant changes. On the contrary, there were statistically significant differences between the effects produced by PEMFs and Control Groups (F = 149.70, p < 0.0005) on the Affinity Index results, as well as by the experimental time of 6 and 3 weeks (F = 17.12, p = 0.001) on the same results. In PEMF‐stimulated animals the microhardness (HV) values measured in trabecular bone at a distance of 200 and 500 μm from the implants, were significantly higher with respect to controls. At 6 weeks, HV values at the bone‐implant interface in PEMF‐stimulated animals were not significantly different with respect to normal bone, while they remained significantly lower in control animals. Both morphological and structural results demonstrated a positive therapeutic effect of PEMFs in accelerating HA osteointegration in trabecular bone.

Influence of Bone Tissue Density and Elasticity on Ultrasound Propagation: An In Vitro Study

Ultrasound (US) waves are mechanical vibrations that are applied to a material—bone tissue—in order to study its properties, that is, density, elasticity, and structure. In this study we evaluated in which way density and elasticity of the spongy bone influenced the transmission of 1.25 MHz US pulses. Twelve cylindrical specimens (diameter, 8 mm; height, 5 mm) excised from phalanxes of pig were decalcified with 0.5 M EDTA for different times (0, 2, and 5 days). During these periods, the samples underwent the following investigations: US transmission, density, and elasticity measurements. To assess the homogeneity of decalcification, the cross‐sections of some samples were microradiographed. A detailed analysis of the US signal received was performed using velocity, Fourier analysis, and some parameters typical of signal processing technique. A good correlation was found between US velocity and density (r2 = 0.70); a lower correlation was found between velocity and elasticity (r2 = 0.59). If density and elasticity are considered simultaneously, the correlation with the US velocity improves significantly (r2 = 0.84). Fourier analysis enabled us to observe a shift of the main frequency toward lower values as the decalcification process advanced. We also observed that in the regressions weighted for density, US velocity correlated poorly with elasticity (r2 = 0.16), whereas signal processing parameters maintain a good correlation with elasticity (ultrasound peak amplitude [UPA], r2 = 0.48; slope, r2 = 0.62). In this study, it has been observed that when using a signal processing technique to analyze US pulses, it is possible to identify some parameters that are related in different ways to density and to elastic properties of bone. Our results show the potentiality of US technique to separate information on bone density and elasticity that X‐ray‐based densitometric methods do not provide.

Assessment of Bone Architecture with Ultrasonometry: Experimental and Clinical Experience

Over the last decade, ultrasound technology has been introduced as a method of analysing bone tissue in clinical practice, and several studies have compared various ultrasound devices with dual-energy X-ray absorptiometry (DXA). Unlike DXA, the ultrasound technique is not limited to the measurement of bone density – it also has the potential to provide information on the mechanical and architectural characteristics of bone. The first generation of ultrasound devices used the speed of sound and attenuation of the ultrasound signal to obtain information on bone mineral content. Second generation ultrasound devices, which analyse the ultrasound signal received, permit the study of bone structure and elasticity, in addition to its mineral content, in both experimental studies and clinical practice. This is accomplished by signal processing analysis, the study of backscattering and the application of Biot’s theory. This approach to the study of bone tissue represents the future for the development of ultrasound technology for use in clinical practice.

Influence of density, elasticity, and structure on ultrasound transmission through trabecular bone cylinders

The aim of this in vitro study is to evaluate the potentiality of quantitative ultrasound (QUS) to separate information on density, elasticity, and structure on specimens of trabecular bone. Fifteen cylinders of spongy bone extracted from equine vertebrae were progressively demineralized and subjected to QUS, micro computed tomography (muCT), Dual energy X-ray absorptiometry (DXA) at various mineralization levels. Eventually all cylinders underwent a compression test to calculate the Youngs modulus. Correlation analysis shows that speed of sound (SOS) is strictly associated to bone mineral density (BMD), Youngs modulus, and all muCT parameters except for degree of anisotropy (DA). Fast wave amplitude (FWA) is directly correlated with bone surface and total volume ratio (BS/TV) and trabecular separation (Tb Sp), and inversely correlated with trabecular number (Tb N). Because muCT parameters were strictly correlated to BMD and Youngs modulus data, partial correlation analysis was performed between SOS, FWA, and structural and elastic data in order to eliminate the effect of density. SOS was significantly correlated to bone volume and total volume ratio (BV/TV), BS/TV, and Youngs modulus, and FWA was significantly correlated to Tb Sp only. These results show that SOS is strongly influenced by volumetric mineral bone density and elastic modulus of the specimen, and FWA is mainly affected by trabecular separation independently on density. Therefore, SOS and FWA are able to provide different and complementary information, at least on trabecular bone samples.

Effects of essential amino acids and lactose on bony fractures and defects in rabbits: a preliminary histomorphometric study

Abstract An experimental study was performed in order to test the possibility of improving bone repair with the administration of a drug (Calciofix, Farmaceutici Damor SpA, Naples, Italy) containing essential amino acids and lactose. Fifty rabbits were submitted to an open transversal fracture of the left fibula and to a right femoral condyle defect. They were left untreated or treated daily with the drug orally and were divided into subgroups depending on the experimental time: 15, 30, 40, 50, 60 days. Histomorphometric data showed a significantly faster healing rate in treated animals compared with untreated ones. Firstly, on day 30 there was a significantly larger amount of cartilage in the control bone callus (P < 0.01). On day 50 a significant difference existed between trabecular and lacunar percentages in the two subgroups (P < 0.0005). At 60 days no significant differences were observed, but bony trabeculae had become more oriented parallel to the long axis of the bone in treated animals. Secondly, after 15 days the defect area was significantly smaller in treated animals than in the untreated ones (P < 0.01). At 30 and 40 days, respectively, significant differences existed between the two subgroups in connective tissue and mature bone percentages (P < 0.01 and P < 0.001). Our results seem to demonstrate that the drug significantly accelerates the rate of bone formation in fractures and bone defects in rabbits.

In vitro evaluation of the effects of electromagnetic fields used for bone healing

The use of therapeutic electromagnetic fields (EMF) for bone healing has positive clinical effects but may have adverse biologic effects. For this reason, EMF exposure has been repeatedly investigated to exclude the possibility of genotoxic effects and tumour risk. This paper describes the effects of EMFs on cell cultures. We analyzed the effects of EMF (28 gauss, 75 Hz) on growth and metabolic activities in four different cell types: L929 fibro-blasts, osteoblast-like HOS/TE85 cells, human lymphocytes, and rabbit chondrocytes. We found no cytotoxic or mutagenic effects on cultures exposed to EMF compared with unexposed controls. Results of cell proliferation showed a statistically significant increase for all cultures exposed to EMF with respect to controls (L929 +45%, p = 0.002; HOS/TE85 +32%, p = 0.001; chondrocytes +40%, p = 0.0003; lymphocytes +39%, p = 0.0002). Biochemical and enzymatic tests gave different results, depending on cell types: all tested values were increased after EMF exposure, even i...

Histomorphological and chemico-physical analyses of the mineral matrix of micropetrotic human bone.

Micropetrotic areas of human bone were analyzed with reference to their distance from blood vessels and to the age of the subjects. Undecalcified bone sections were treated with various solvent, soaking and etching substances to establish the nature of the material occluding the osteocytic canalicular cavities, and were examined by means of microradiographic and microdurimetric methods and X-ray microanalysis to evaluate the degree of mineralization in the bone matrix. Since it was only after strong etching with HCl that the canalicular network became visible under light and scanning electron microscopy, it is clear that the debris filling the canalicular network consists almost entirely of mineral substance. The degree of mineralization of micropetrotic bone is high because it is always a more mature type of bone, but the mineral content of the matrix and the Ca/P ratio do not differ significantly from those of neighbouring bone where the canalicular network is fully pervious.

IN VIVO EFFECTS OF LOW-FREQUENCY LOW ENERGY PULSING ELECTROMAGNETIC FIELDS (PEMFs) ON GENE EXPRESSION DURING THE INFLAMMATION PHASE OF BONE REPAIR

It has been widely demonstrated that pulsed low-frequency electromagnetic fields (PEMFs) positively affect bone repair. The aim of this study is to highlight if PEMFs influence cell metabolic activity during the replacement of the blood clot with granulation tissue in the inflammation phase of bone repair. Four equal transcortical holes were made, at the same diaphyseal level, in both metacarpals (McIII) of five male adult horses. The left McIII were exposed to PEMFs 24 hr/day; the right untreated McIII were used as controls. Eight days after surgery, the horses were sacrificed. We investigated the effect of PEMFs on 1) histological aspects of the lesion, 2) histochemical detection of the bone marker alkaline phosphatase, and 3) molecular markers as tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and transforming growth factor-β1 (TGF-β) by reverse transcriptase-polymerase chain reaction amplification (RT-PCR). The histological analysis indicates that the blood clot, in both PEMF treated and control holes, is being replaced by granulation tissue extending from the endosteal towards the periosteal side of the lesion. TALP positive areas do not exactly correspond to the areas where fibroblasts are present, these being wider than the former. The study of the expression of the mRNA of TGF-β1 shows no differences between treated holes and control ones. The expression of TNF-α and IL-6 however, is not univocal, being sometimes more expressed, sometimes less in treated or control holes. These data show that PEMFs exposure affects the expression of inflammatory cytokines (TNF-α and IL-6) during the very early stages of bone repair. On the contrary TFG β expression and histological findings are not modified by PEMF exposure at least in this experimental condition.

PULSED ELECTROMAGNETIC FIELDS MODULATE ENZYMATIC ACTMTY DURING THE EARLY STAGES OF BONE REPAIR

The goal of this study was to investigate whether PEMFs influence enzymatic activities during the early stages of bone repair. Two transcortical holes (4.5 mm diameter) were drilled at the same diaphyseal level in the lateral margin of the right and left metacarpal bone (McIII) of six adult male horses. The left McIII were exposed to PEMFs (75 Hz; 2.8 mT, 1.3 ms impulse width) 24 h/day; the right untreated McIII were used as controls. Horses were sacrificed 8 and 15 days after the operation. The bone segments containing the holes were fixed, dehydrated in ethanol solutions, and, undecalcified, embedded in methylmethacrylate. The midlongitudinal sections of the holes were either stained with soluidine blue or processed for evaluation of the total alkaline phosphatase (TALP) and the tartrate-resistant acid phosphatase (TRAP). In PEMF-treated holes we found: (1) TALP is strongly positive with respect to the controls; (b) the newly formed bony trabeculae are more abundant than in the controls; (c) in both tre...