Hard Tissue Samples Research Articles

Dietary and homeostatic controls of Zn isotopes in rats: a controlled feeding experiment and modeling approach.

The stable isotope composition of zinc (δ66Zn), which is an essential trace metal for many biological processes in vertebrates, is increasingly used in ecological, archeological, and paleontological studies to assess diet and trophic level discrimination among vertebrates. However, the limited understanding of dietary controls and isotopic fractionation processes on Zn isotope variability in animal tissues and biofluids limits precise dietary reconstructions. The current study systematically investigates the dietary effects on Zn isotope composition in consumers using a combined controlled feeding experiment and box-modeling approach. For this purpose, 21 rats were fed one of seven distinct animal- and plant-based diets and a total of 148 samples including soft and hard tissue, biofluid, and excreta samples of these individuals were measured for δ66Zn. Relatively constant Zn isotope fractionation is observed across the different dietary groups for each tissue type, implying that diet is the main factor controlling consumer tissue δ66Zn values, independent of diet composition. Furthermore, a systematic δ66Zn diet-enamel fractionation is reported for the first time, enabling diet reconstruction based on δ66Zn values from tooth enamel. In addition, we investigated the dynamics of Zn isotope variability in the body using a box-modeling approach, providing a model of Zn isotope homeostasis and inferring residence times, while also further supporting the hypothesis that δ66Zn values of vertebrate tissues are primarily determined by that of the diet. Altogether this provides a solid foundation for refined (paleo)dietary reconstruction using Zn isotopes of vertebrate tissues.

Methods to Enable Spatial Transcriptomics of Bone Tissues.

Understanding the relationship between the cells and their location within each tissue is critical to uncover the biological processes associated with normal development and disease pathology. Spatial transcriptomics is a powerful method that enables the analysis of the whole transcriptome within tissue samples, thus providing information about the cellular gene expression and the histological context in which the cells reside. While this method has been extensively utilized for many soft tissues, its application for the analyses of hard tissues such as bone has been challenging. The major challenge resides in the inability to preserve good quality RNA and tissue morphology while processing the hard tissue samples for sectioning. Therefore, a method is described here to process freshly obtained bone tissue samples to effectively generate spatial transcriptomics data. The method allows for the decalcification of the samples, granting successful tissue sections with preserved morphological details while avoiding RNA degradation. In addition, detailed guidelines are provided for samples that were previously paraffin-embedded, without demineralization, such assamples collected from tissue banks. Using these guidelines, high-quality spatial transcriptomics data generated from tissue bank samples of primary tumor and lung metastasis of bone osteosarcoma are shown.

Applying laser induced breakdown spectroscopy (LIBS) and elemental imaging on marine shells for archaeological and environmental research.

Using LIBS for the analysis of archaeological and geological marine mollusc shells is a growing research area that relies on customised instrumentation and specific workflows that can accommodate the variety and precision of the required sampling parameters. However, the increased efficiency offered by LIBS, which enables the study of a larger quantity of shell samples for temperature variation, ecological parameters, and human consumption practices, outweighs the initial efforts required to develop customised instrumentation and workflows. In this work, we present detailed specifications and parameters for the development of a LIBS system capable of generating Mg/Ca images on marine shells that directly correlate with seasonal sea temperatures. Our main objective was to develop specifications that enable easy adaptation of LIBS systems to existing laboratories for studying hard-tissue samples. These specifications were used to develop a customised micro-LIBS system and apply it to a real-world example of an archaeological study to better understand its efficiency on the marine mollusc shells and demonstrate its potential for broader applications in interdisciplinary research. In total 101 shell specimens have been analysed within a time frame of approximately 71h of machine time, producing 234 images (100µm resolution: 100 images, 30µm resolution: 134 images). SEM analysis of the irradiated sections of the shell revealed a primary ablated area of 10-15µm in diameter, while a secondary affected area of the shell's crystal fabric extended to 30-50µm after repeated shots. Overall, this new customised system reliably and efficiently analysed marine mollusc specimens without major destructive effects, enabling additional analyses for other proxies to be carried out. This study highlights the potential of the LIBS method for interdisciplinary research, encompassing applications in paleoclimatology, marine ecology, and archaeology.

Degradation behavior and osseointegration of Mg-Zn-Ca screws in different bone regions of growing sheep: a pilot study.

Magnesium (Mg)-based implants are highly attractive for the orthopedic field and may replace titanium (Ti) as support for fracture healing. To determine the implant-bone interaction in different bony regions, we implanted Mg-based alloy ZX00 (Mg < 0.5 Zn < 0.5 Ca, in wt%) and Ti-screws into the distal epiphysis and distal metaphysis of sheep tibiae. The implant degradation and osseointegration were assessed in vivo and ex vivo after 4, 6 and 12 weeks, using a combination of clinical computed tomography, medium-resolution micro computed tomography (µCT) and high-resolution synchrotron radiation µCT (SRµCT). Implant volume loss, gas formation and bone growth were evaluated for both implantation sites and each bone region independently. Additionally, histological analysis of bone growth was performed on embedded hard-tissue samples. We demonstrate that in all cases, the degradation rate of ZX00-implants ranges between 0.23 and 0.75 mm/year. The highest degradation rates were found in the epiphysis. Bone-to-implant contact varied between the time points and bone types for both materials. Mostly, bone-volume-to-total-volume was higher around Ti-implants. However, we found an increased cortical thickness around the ZX00-screws when compared with the Ti-screws. Our results showed the suitability of ZX00-screws for implantation into the distal meta- and epiphysis.

Histology of tendon and enthesis - suitable techniques for specific research questions.

The musculoskeletal system consists of different components comprising a wide range of tissue types, with tendons being one part. Tendon degeneration or rupture have a high prevalence in all age groups, often with poor outcomes of surgical treatment such as chronic pain and high re-tear rates. Therefore, much effort has been directed to further develop diagnostic and therapeutic methods as well as reconstruction techniques, including using adequate placeholders or implants. Diagnostic approaches and advanced stages of preclinical studies will inevitably include histological examination of the pathologically affected tissue. The present study presents adequate tendon-related, histological techniques, including the embedding of soft- and hard-tissue samples in different media. Consideration is also given to samples containing residual implant materials or having been subjected to standard staining protocols and immunohistochemical procedures. The study further examines cells and tendon structure to detect degenerative, fibrotic or inflammatory conditions and possible foreign-body responses to implanted materials. Infraspinatus tendons from preclinical studies carried on rat and sheep samples, as well as human biceps tendon samples, have been used as example materials.

Quantitative Analysis of Growth Factors From Cancellous Bone Graft Collected With a Reamer-Irrigator-Aspirator System From Native Long Bones Versus Previously Reamed Long Bones.

Collection of bone graft with the Reamer-Irrigator-Aspirator (RIA) system has become common practice across the field of orthopaedic surgery. While RIA bone graft is typically obtained from native long bones, grafting material can likewise be harvested from long bones that have previously undergone the placement and removal of an intramedullary nail, a process termed re-reamed RIA (RRR). The purpose of this study was to evaluate the total protein and growth factor concentrations present in native-RIA (NR) compared with RRR samples. NR and RRR bone grafts were collected intraoperatively with the RIA system and processed to evaluate both the aqueous and the hard tissue components. Total protein concentration and specific growth factors were analyzed using standard bicinchoninic acid and multiplex assays, respectively. Analyte levels were then normalized to the total amount of protein detected. Total protein levels were comparable between NR and RRR samples for both the aqueous filtrate and the hard tissue samples. When normalized, while levels of bone morphogenic protein-2 and vascular endothelial growth factor were comparable in the hard tissue component, the aqueous filtrate from the RRR sample was found to have elevated levels of growth factors, with bone morphogenic protein-2 reaching statistical significance. This study demonstrates that ample protein is found within both NR and RRR samples, with comparable or elevated levels of osteogenic growth factors found within RRR samples. Future, larger, prospective studies will be required to evaluate the osteogenic potential and clinical efficacy of NR and RRR cancellous bone grafts to validate their equivalency.

Highly efficient optical fiber sensor for instantaneous measurement of elevated temperature in dental hard tissues irradiated with an Nd:YaG laser.

In this in vitro experiment, the effect of 1.064 µm pulsed laser on both enamel- and dentin-dental tissues has been investigated. A total of fifty-five dental hard tissue samples were exposed to Nd:YAG laser that possesses a pulse width of 9 ns and 850 mJ of total energy. An optical fiber sensor was put behind the samples to measure the temperature instantaneously. A novel, to the best of our knowledge, fiber sensor has been proposed and used to measure the heat generated in dental hard tissues instantaneously after the application of laser irradiation on the tissue surface. This optical sensor exhibits a fast response time of about 1 ms and high sensitivity with about 1.975 nm/°C. The findings of this study in decreasing the probability of pulpal necrosis structure while handling the tooth, whether for ablation, welding, or tooth resurfacing purposes, may establish standards for dentists and laser manufacturers (healthcare professionals) that should be followed.

Evaluation and Comparison of Human Chorionic Amniotic Membrane and Platelet-Rich Fibrin in Achieving Bone Formation and Soft Tissue Healing in Extraction Sockets Indicated for Rehabilitation with Implants: A Preliminary Study.

To evaluate and compare human chorionic amniotic membrane and platelet-rich fibrin on new bone formation and soft tissue healing in extraction sockets indicated for rehabilitation with dental implants. A prospective, triple blind clinical study was conducted. The inclusion criteria were as follows: patient with two extraction sites each in the same arch, intact buccal bone and soft tissue around the socket, and recommended rehabilitation with dental implants. Postextraction, the sockets were randomly placed with human chorionic amniotic membrane in one site and platelet-rich fibrin in the other site. After 3 months, a trephine drill was used to take a biopsy of the respective sites for soft and hard tissue samples. The outcome parameters that were assessed histologically were percentage of new bone formation and lymphocyte density. After screening 80 patients, eight participants were recruited for the study. The mean percentage of new bone formation in the human chorionic amniotic membrane group was 45.71% ± 4.82%, and for the plasma-rich fibrin group, it was 41.39% ± 6.29%, showing no statistically significant difference (z = 0.99, P = .31). In the human chorionic amniotic membrane group, six out of eight sites had mild lymphocyte density, while the plasmarich fibrin group had equal numbers of mild and moderate lymphocyte density. No statistically significant difference between the groups (Fischer test value = 0.60, P = .25) was noted. Within the limitations of the study, the results showed that there is no difference in the efficiency of human chorionic amniotic membrane compared with platelet-rich fibrin in achieving new bone formation and soft tissue healing in the extraction socket.

Investigation of mammoth remains using the neutron activation analysis at the Training Reactor VR-1

At the time when the importance of the interdisciplinary research increases, the nuclear analytical techniques supported by the small research reactors represent a useful tool for investigation of human society, culture, history etc. The historical, archaeological, and palaeontological samples and objects of cultural heritage can be easily studied using the radioanalytical methods such as the neutron activation analysis. This paper deals with the detailed investigation of fragments of mammoth remains from the Mid-Upper Palaeolithic site Pavlov VI by means of the instrumental neutron activation analysis at the Training Reactor VR-1 of the Czech Technical University in Prague. Six mammoth hard tissue samples (fragments of bones, tusk, and molar) from the Institute of Archaeology of the Czech Academy of Sciences were irradiated in the dry vertical irradiation channel with thermal neutron field (φ=2×109cm−2s−1) at maximum reactor power (80 W). The activated mammoth samples were analysed employing the nuclear γ-spectrometry and semiconductor HPGe detector, and the composition of the remains was determined (qualitative and quantitative analysis). The presence of Na, Cl, K, As, Fe, Sr, Mn, Br, I, Ba, and U was revealed in studied mammoth samples. Based on obtained production rates, the concentrations of Fe, Sr, Na, K, As, and U were determined. The results presented in this paper show clearly that the low-power Training Reactor VR-1 is excellent tool for the neutron activation analysis experiments within the interdisciplinary research and can provide experimental data important for archaeologists and palaeontologists.

Influence of experimental constraints on micromechanical assessment of micromachined hard-tissue samples

Continuing technological advancement of mechanical characterization at the microscale has enabled the isolation of micron-sized specimens and their direct mechanical characterization. Such techniques, initially developed for engineering materials and MEMS, can also be applied on hard biological materials. Bone is a material with a complex hierarchical structure ranging from the macro- all the way down to the nanoscale. To fully understand bone tissue mechanics, knowledge of the mechanics of all structural elements i.e. at every length scale is necessary. Particularly, the mechanical properties of microstructural elements, such as bone lamellae are still largely unknown. In the last decade, testing protocols have been devised to close this gap including bending and compression of micrometer-sized bone specimens. However, the precision and accuracy of results obtained have not been discussed. In this study, we aim to do exactly this: we validate microbeam bending by testing silicon microbeams with known mechanical constants, and evaluate the precision and sources of errors in both microbeam bending and micropillar compression by means of finite element (FE) modeling. Bending of Si-microbeams reproduced the expected value for the bending modulus within 17% accuracy, although the effect of geometrical uncertainties was estimated to result in relative errors of up to 50%. The deformation of constraining bulk material had a smaller influence, with relative errors of 11%, for microbeam bending and 25% for micropillar compression. For the latter this error could be sufficiently eliminated by the Sneddon correction. The tapering of micropillars had a negligible effect on overall apparent stiffness, but induced inhomogeneous stress state within micropillars may lead to superposed structural deformation mechanisms and be responsible for failure patterns observed in past studies.

Simultaneous measurement of folate cycle intermediates in different biological matrices using liquid chromatography–tandem mass spectrometry

The folate cycle is an essential metabolic pathway in the cell, involved in nucleotide synthesis, maintenance of the redox balance in the cell, methionine metabolism and re-methylation reactions. Standardised methods for the measurement of folate cycle intermediates in different biological matrices are in great demand. Here we describe a rapid, sensitive, precise and accurate liquid chromatographic–tandem mass spectrometric (LC–MS/MS) method with a wide calibration curve range and a short run time for the simultaneous determination of folate cycle metabolites, including tetrahydrofolic acid (THF), 5‑methyl THF, 5‑formyl THF, 5,10‑methenyl THF, 5,10‑methylene THF, dihydrofolic acid (DHF) and folic acid in different biological matrices. Extraction of folate derivatives from soft and hard tissue samples as well as from adherent cells was achieved using homogenisation in buffer, while extraction from the whole blood and plasma relied on the anion exchange solid-phase extraction (SPE) method. Chromatographic separation was completed using a Waters Atlantis dC18 2.0 × 100 mm, 3-μ column with a gradient elution using formic acid in water (0.1% v/v) and acetonitrile as the mobile phases. LC gradient started with 95% of the aqueous phase which was gradually changed to 95% of the organic phase during 2.70 min in order to separate the selected metabolites. The analytes were separated with a run time of 5 min at a flow rate of 0.300 mL/min and detected using a Waters Xevo-TQS triple quadrupole mass spectrometer in the multiple reaction monitoring mode (MRM) at positive polarity. The instrument response was linear over a calibration range of 0.5 to 2500 ng/mL (r2 > 0.980). The developed bioanalytical method was thoroughly validated in terms of accuracy, precision, linearity, recovery, sensitivity and stability for tissue and blood samples. The matrix effect was compensated by using structurally similar isotope labelled internal standard (IS), 13C5‑methyl THF, for all folate metabolites. However, not all folate metabolites can be accurately quantified using this method due to their high interconversion rates especially at low pH. This applies to 5,10‑methylene THF which interconverts into THF, and 5,10‑methenyl‑THF interconverting into 5‑formyl‑THF. Using this method, we measured folate cycle intermediates in mouse bone marrow cells and plasma, in human whole blood; in mouse muscle, liver, heart and brain samples.

Micro-computed tomography for evaluating alveolar bone resorption induced by hyperocclusion

PurposeOcclusal trauma, resulting in the destruction of alveolar bone, is a form of periodontal disease caused by excessive mechanical stress (MS) during hyperocclusion. Previously, we showed that CC chemokine ligand (CCL) 2/CCR2 receptor axis plays a crucial role in MS-dependent osteoclastogenesis. However, in the previous work, we were unable to precisely measure changes in alveolar bone profiles. In the present study, we sought to establish a precise method for evaluating alveolar bone resorption induced by hyperocclusion using micro-computed tomography. MethodsUnder anesthesia, a stainless steel wire was attached to the molars of 5-week-old C57/BL6 wild-type (WT) mice, CCL2−/− mice, and CCR2−/−mice to induce occlusal force overload. At days 0 and 7, hard tissue samples were harvested and analyzed by micro-computed tomography. ResultsIn the WT mice, bone mineral density of the alveolar bone was significantly decreased at day 7 as compared with day 0, with marked alveolar bone resorption observed. Similarly, significant alveolar bone resorption was observed in the CCL2−/− and CCR2−/− mice at day 7 as compared with day 0. ConclusionsMicro-computed tomographic images can be used to measure changes in bone mineral density in a mouse model of hyperocclusion. This method may be useful for further investigating bone changes in other periodontal disease research fields.

Three-Dimensional Observation of Microstructure of Bone Tissue Using High-Precision Machining

This study aims to verify whether the three-dimensional internal information acquisition system we have developed can be applied successfully to the microstructures of consecutively precision-machined biological samples, and to those of metallic samples. Therefore, this study mainly deals with biological hard tissue samples like bones. In this paper, we first studied the precision-machining characteristics of bones. From this, we determined that, to obtain machined surfaces sufficient for internal observations, we need to determine the maximum uncut chip thickness and the cutting speeds, taking the bone’s anisotropy into consideration. Next, we acquired three-dimensional internal information on consecutively precision-machined bone samples using the three-dimensional internal acquisition system we developed. Subsequently, we visualized the internal structures of these machined samples. Our tiling observations acquired an 18×9×3 mm segment as a 6.2×6.2×10μm resolution image. We obtained a three-dimensionally reconstructed image of complex blood vessel networks inside the bone by making the acquired images binary.

Noncontact monitoring of incision depth in laser surgery with air-coupled ultrasound transducers.

Lack of haptic feedback during laser surgery makes it difficult to control the incision depth, leading to high risk of undesired tissue damage. Here, we present a new feedback sensing method that accomplishes noncontact real-time monitoring of laser ablation procedures by detecting shock waves emanating from the ablation spot with air-coupled transducers. Experiments in soft and hard tissue samples attained high reproducibility in real-time depth estimation of the laser-induced cuts. The advantages derived from the noncontact nature of the suggested monitoring approach are expected to advance the general applicability of laser-based surgeries.

In situ characterization of resin–dentin interfaces using conventional vs. cryofocused ion-beam milling

ObjectiveThe introduction of focused ion beam (FIB) milling has facilitated preparation of hard tissue samples for transmission electron microscope (TEM). However, this technique generates high temperature that may alter or damage morphological features in biological tissue. Therefore, the aim of this study was to determine the effects of cryogenic cooling on the morphological features of dentin interfaces with dental restorative materials in samples prepared by FIB for TEM examination. MethodsAfter preparation of a cylindrical-shaped cavities in extracted, non-carious premolar teeth, the specimens were restored with dental adhesive/composite and categorized into two restorative materials groups; (PB) a combination of Clearfil Protect Bond (Kuraray Noritake Dental, Japan)/Estelite Sigma Quick composite (Tokuyama Dental, Japan), and (SB) Filtek Silorane restorative system (3M ESPE, USA). The specimens were subjected to interfacial cross-sectioning, followed by observation and area selection using confocal laser microscopy. Later, ultrathin sections were prepared using FIB with cryogenic cooling (PB-C) and (SB-C), or without cooling (PB-NC) and (SB-NC) that all were examined under TEM. ResultsResulting TEM images of the ultra-morphological features at the resin–dentin nano-interaction zone were improved when FIB preparation was conducted in the cryogenic condition and no sign of artifacts were detected. SignificanceConducting ion beam milling with cryogenic cooling was advantageous in minimizing the elevation in specimen temperature. This led to preservation of dentin microstructures that revealed additional information about substrates that are necessary for advanced characterization of tooth–biomaterial interactions.

Effects of gamma radiation on hard dental tissues of albino rats: investigation by light microscopy

The present work aims at studying the effect of gamma radiation on the hard dental tissues. Eighty adult male albino rats with weights of about 250g were used. The rats were irradiated at 0.2, 0.5, 1.0, 2.0, 4.0 and 6.0Gy whole-body gamma doses. The effects on hard dental tissue samples were investigated after 48h in histological and ground sections using light microscopy. Areas of acid phosphatase activity were detected using tartrate-resistant acid phosphatase (TRAP) stains. Observation of histological sections revealed disturbance in predentin thickness and odontoblastic layer as the irradiation dose increased. In cementum, widened cementocytes lacunae were occasionally detected even with low irradiated doses. On the other hand, relatively homogenous enamel was detected with darkened areas in enamel surface at doses over than 0.5Gy. TRAP-positive cells were detected on the surface of the dentin of irradiated groups as well as cementum surface. Minimal detectable changes were observed in ground sections.

Local expression of inflammatory cytokines in the facial tissue of children with a cleft lip and palate

The cleft lip and/or palate are among the most common congenital anomalies that occur in early development. Cytokines play an important role in the proliferation, growth, differentiation, survival and the functional activity of many cells and the upregulation of cytokines might be involved in the pathological processes of the maxillofacial region. The purpose of our study was to evaluate the expression of pro-inflammatory and anti-inflammatory cytokines in the facial tissue of children with the cleft lip and palate.The study involved 14 patients with the unilateral cleft lip and palate at the age of three months to 10 years and five months. Soft and hard palate tissue samples were collected during the primary cleft operation if the parents had given their informed written consent. All the tissue samples were stained with hematoxylin and eosin and by immunohistochemistry for IL-1α, IL-6, IL-8, IL-10 and TNF-α. The intensity of immunostaining was graded semiquantitatively.IL-1α containing structures were not detected in any of soft, cartilage and bone tissue samples, meanwhile IL-6, IL-8, IL-10 and TNF-α showed explicit expression. The expression of IL 6 was observed in the tissues of all the patients. IL-6 positive cells were found in the range from no positive to moderate of positive structures in the visual field. IL-8 positive osteocytes were found in the range from few positive to moderate, but IL-8 positive chondrocytes were found in abundance. IL-10 was richly secreted by osteocytes in bone and by chondrocytes in cartilage obtained from all the cases of plastic surgery. In all the cases we also observed numerous IL-10 positive gingival epithelial cells. Despite these data obtained, moderate to numerous macrophages and neutrophils expressed TNF-α.The sporadical and scarce expression of IL-6 indicates its insignificant role in the cleft lip and palate affected tissue. Rich cytokine IL-10 expression proves the compensated local anti-inflammatory effects of the cleft affected soft and hard tissue.

Hard tissue samples as markers of occupational exposure in a phosphate fertiliser plant

This paper describes the results of study on testing of hair and nails samples as possible markers of occupational exposure in a phosphate fertiliser plant. The key objectives were to verify if: (1) elements originating from fertiliser production may accumulate in hard tissue and hence allow preliminary occupational exposure risk assessment; (2) linear discriminant function analysis (LDA) may be applied as a diversification tool in preliminary assessment of occupational exposure risk based on hard tissue samples analysis; and (3) both hairs and nails can be used successfully as an efficient biological sample in biomonitoring studies and as markers of occupational exposure in various types of plant. The complex data matrix (2025 observations) obtained by the determination of 25 elements by neutron activation analysis in hard tissue samples was treated by LDA. The obtained results indicated the presence of two discriminant functions (DFs). The data variance explained by the first DF is 78%, and that by the second DF is 22%. The first DF is highly related to S, W, Cu, K and Mg concentrations and separates hair from nails samples, and the second DF, being related to phosphate fertiliser precursors, separates the control group from the employees. Sm, Al, Mo and As accumulate in nails and hairs of fertiliser plant employees, whereas Cl and Ti are removed from hard tissues. The mean concentrations of Na, In, I, Au, Dy, Ca, La, U, Sb, V, Zn, Mn and Co in hard tissue samples do not discriminate employees and control group members and hence prove their ineffectiveness in occupational exposure assessment in phosphate fertiliser plants.

Elemental composition in mud crab Scylla serrata from Mahanadi estuary, India: In situ irradiation analysis by external PIXE

During the present study concentration of nine elements (K, Ca, Mn, Fe, Cu, Zn, Se, Br and Pb) in different tissues of mud crab Scylla serrata from Mahanadi estuary, India were determined by the external PIXE set up at Institute of Physics, Bhubaneswar, India. The study demonstrates the effectiveness of the technique in analyzing both soft and hard tissue samples from marine organisms and opens the door for non-destructive, multi-elemental analysis of tissue samples with a very little sample preparation by direct irradiation. This technique can be well utilized for analyzing the tissue samples for environmental, toxicological and nutritional purposes. The study also demonstrates the elemental concentrations from tissue samples of any crustaceans from Mahanadi estuary for the first time. Sex based difference in the elemental concentration of the mud crabs were marked, which may be related to the growth rate and other biological activities. No significant difference in the concentration of elements (Ca, Mn, Fe, Cu and Zn) in the tissues of juveniles and adults of respective sex is observed, which indicated the active regulation of these elements by S. serrata.

Rapid and conservative ablation and modification of enamel, dentin, and alveolar bone using a high repetition rate transverse excited atmospheric pressure CO[sub 2] laser operating at λ=9.3 μm

Transverse excited atmospheric pressure (TEA) CO(2) lasers tuned to the strong mineral absorption of hydroxyapatite near lambda=9 microm are well suited for the efficient ablation of dental hard tissues if the laser pulse is stretched to greater than 5 to 10 micros to avoid plasma shielding phenomena. Such CO(2) lasers are capable of operating at high repetition rates for the rapid removal of dental hard tissues. The purpose of this study was to test the hypothesis that stretched lambda=9.3-microA CO(2) laser pulses can produce lateral incisions in enamel, dentin, and alveolar bone for dental restorations and implants at repetition rates as high as 400 Hz without peripheral thermal damage. The single pulse ablation rates through enamel, dentin, and bone were determined for incident fluence ranging from (1 to 160 J/m(2)) for laser pulses from 5 to 18 mus in duration. Lateral incisions were produced in hard tissue samples using a computer-controlled scanning stage and water spray, and the crater morphology and chemical composition were measured using optical microscopy and high-resolution synchrotron radiation infrared spectromicroscopy. The residual energy remaining in tooth samples was measured to be 30 to 40% for enamel and 20 to 30% for dentin without water cooling, under optimum irradiation intensities, significantly lower than for longer CO(2) laser pulses. The transmission through 2-m length 300-, 500-, 750-, and 1000-microm silica hollow waveguides was measured and 80% transmission was achieved with 40 mJ per pulse. These results suggest that high repetition rate TEA CO(2) laser systems operating at lambda=9.3 microm with pulse durations of 10 to 20 micros are well suited for dental applications.