Bone natural autofluorescence and confocal laser scanning microscopy: Preliminary results of a novel useful tool to distinguish between forensic and ancient human skeletal remains.

The fast, high-throughput distinction between palaeoanthropological/archaeological remains and recent forensic/clinical bone samples is of vital importance in the field of medico-legal science. In this paper, a novel dating method was developed using the autofluorescence of human bones and the confocal laser scanning microscope as the means to distinguish between archaeological and forensic anthropological skeletal findings. Human bones exhibit fluorescence, typically induced by natural antibiotics that are absorbed by collagen, and provide secondary, exogenous fluorophores. However, primary natural fluorescence (or autofluorescence) caused by enigmatic endogenous fluorophores is also present as a micro-phenomenon, whose nature is still obscure. Here, we show that the endogenous fluorophores are mucopolysaccharides of the Rouget-Neumann sheath and, more relevant, that the intensity of the natural fluorescence in human bone decreases in a relationship to the antiquity of the samples. These results suggest that the autofluorescence of bone is a promising technique for the assessment of skeletal remains that may be potentially of medico-legal interest. A larger study is proposed to confirm these findings and to create a predictive model between the autofluorescence intensity and the time since death.

Decision tree analysis as a supplementary tool to enhance histomorphological differentiation when distinguishing human from non-human cranial bone in both burnt and unburnt states: A feasibility study.

This feasibility study was undertaken to describe and record the histological characteristics of burnt and unburnt cranial bone fragments from human and non-human bones. Reference series of fully mineralized, transverse sections of cranial bone, from all variables and specimen states, were prepared by manual cutting and semi-automated grinding and polishing methods. A photomicrograph catalogue reflecting differences in burnt and unburnt bone from human and non-humans was recorded and qualitative analysis was performed using an established classification system based on primary bone characteristics. The histomorphology associated with human and non-human samples was, for the main part, preserved following burning at high temperature. Clearly, fibro-lamellar complex tissue subtypes, such as plexiform or laminar primary bone, were only present in non-human bones. A decision tree analysis based on histological features provided a definitive identification key for distinguishing human from non-human bone, with an accuracy of 100%. The decision tree for samples where burning was unknown was 96% accurate, and multi-step classification to taxon was possible with 100% accuracy. The results of this feasibility study strongly suggest that histology remains a viable alternative technique if fragments of cranial bone require forensic examination in both burnt and unburnt states. The decision tree analysis may provide an additional but vital tool to enhance data interpretation. Further studies are needed to assess variation in histomorphology taking into account other cranial bones, ontogeny, species and burning conditions.

Comparative histomorphometric study of bone tissue synthesized after electric and ultrasound stimulation.

The clinical use of the alternative therapies in traumatology is conditioned by the knowledge and understanding of their actions on the bone tissue. The hereby study aims at the comparative assessment of the effectiveness of the direct current and ultrasounds in treating the fractures. Thus, we have proceeded to a comparative histological study of the bone tissue in the fractured area and the biomechanical description and the three-dimensional model of the stimulated bone's behavior by using micro-CT X-rays and the finite element analysis. The findings clearly show that the bone, which has been stimulated during a period of two weeks, has regained its functions, that is 85% of the compression one and 95% of the shearing one. These values prove that 90% of the bone structure has healed.

Bone regeneration using beta-tricalcium phosphate in a calcium sulfate matrix.

The aim of the study was the histomorphometric comparison of the osteogenic potential of beta-tricalcium phosphate (beta-TCP) alone or in a calcium sulfate matrix. Three round defects, 10 mm (diameter) x 5 mm (depth), were created on each iliac crest of 4 dogs. The defects were divided into 3 groups. Ten defects were filled with beta-TCP in a calcium sulfate (CS) matrix (Fortoss Vital; group A), 10 defects were filled with beta-TCP alone (Fortoss Resorb; group B), and 4 defects were left ungrafted to heal spontaneously (group C). All defects were left to heal for 4 months without the use of a barrier membrane. Histologic evaluation and morphometric analysis of undecalcified slides was performed using the areas of regenerated bone and graft remnants. All sites exhibited uneventful healing. In group A sites (beta-TCP/CS), complete bone formation was observed in all specimens, graft granules dominated the area, and a thin bridge of cortical bone was covering the defect. Group B (beta-TCP) defects were partially filled with new bone, the graft particles still dominated the area, while the outer cortex was not restored. In the ungrafted sites (group C), incomplete new bone formation was observed. The outer dense cortical layer was restored in a lower level, near the base of the defect. The statistical analysis revealed that the mean percentage of new bone regeneration in group A was higher than in group B (49.38% and 40.31%, respectively). A statistically significant difference existed between the 2 groups. The beta-TCP/CS group exhibited significantly higher new bone regeneration according to a marginal probability value (P = .004 < .05). The use of beta-TCP in a CS matrix produced significantly more vital new bone fill and preserved bone dimensions compared with the use of beta-TCP alone.

Onlay silicone and hydroxyapatite-tricalciumphosphate composite (HAP-TCP) blocks interfere with nasal bone growth in rabbits.

We investigated how a new type of synthetic porous hydroxyapatite ceramic (HAP-TCP) acts when it is implanted in growing membranous bone. Seventy-six New Zealand White infant male rabbits (4-week-olds) were used. Rabbits received HAP-TCP block or silicone block implantation in their right nasal bone. The left nasal bone was used for a sham. Serial or cross-sectional examinations by morphometry, radiology, blood biochemistry, and histology were carried out. Both the HAP-TCP and silicone groups exhibited no systemic growth disturbance in terms of morphometry and blood biochemistry. Dual energy x-ray absorptiometry (DEXA) revealed, however, a decrease in the bone mineral content (BMC) of the right nasal bone in the silicone group. Histology revealed a superior affinity of HAP-TCP to bone tissue than that of silicone. When a HAP-TCP block was implanted under the periosteum it bonded directly to bone tissue. However, sinking of the implants into the bone tissue were noted both in the HAP-TCP and the silicone groups in longitudinal observation. These results suggest that although HAP-TCP has superior affinity to bone tissue, this by itself is not enough sufficient reason to believe that HAP-TCP can be effectively applied during the growth period.

Structure of the osseous callus formed under electric stimulation. An experimental histological study.

The study tried to evidence histologically, histometrically and mechanically the comparative qualities of the osseous callus experimentally formed in dogs and rabbits under the influence of an electric stimulation. The materials were decalcified or polished, and the collagen network and the Haversian systems analysed in the callus. The tearing strength in kg-power on the surface unity of the osseous tissue was measured using the EDZ 20 apparatus. The results indicated that the osseous tissue formed under electric stimulation had a structure and a tearing strength similar to that of controls, and that the consolidation time of fractures is reducing.

Impaired osteoclastic function and linear bone erosion rate in secondary hyperparathyroidism associated with chronic renal failure.

Osteoclastic function, as estimated by the speed with which the haversian cutting cones advance through the compacta of the rib (linear erosion) was found significantly decreased (27.97-SE 0.8 mu/day versus normal 43.61-SE 0.7 mu/day) in dogs rendered chronically uremic (BUN between 36-89 mg/100 ml). The cause of the impairment of osteoclasts' function was tentatively ascribed to the chronic excess of the circulating parathyroid hormone. A decrease in the linear erosion rate, such as observed in this study, will produce predictable morphological changes. As the time necessary to erode a cavity increases, so does the lifespan of cavities, their number and consequently the bone surface area (or in the bone section the perimeter length) covered by Howship's lacunae. Thus, the increased total bone erosion perimeter in secondary hyperparathyroidism associated with moderate uremia does not necessarily reflect an accelerated tissue level erosion rate or even bone loss. As the remodeling sites in both adult man and dog have a similar standard configuration and dimensions, these observations may be extrapolated to disease in man.