<p>Abnormally shaped red blood cells (RBCs), called poikilocytes, can cause anemia. At present, the biochemical abnormalities in poikilocytes are not well understood. Normal RBCs and poikilocytes were analyzed using whole-blood and single-cell methods. Poikilocytes were induced in rat blood by intragastrically administering titanium dioxide (TiO<sub>2</sub>) nanoparticles. Complete blood count and inductively coupled plasma mass spectrometry analyses were performed on whole-blood to measure average RBC morphology, blood hemoglobin (HGB), iron content, and other blood parameters. Follow-up confocal Raman spectroscopy was performed on single RBCs to analyze cell-type-specific HGB content. Two types of poikilocytes, acanthocytes and echinocytes, were observed in TiO<sub>2</sub> blood samples, along with normal RBCs. Acanthocytes (diameter 7.7 ± 0.5 μm) and echinocytes (7.6 ± 0.6 μm) were microscopically larger (<italic>p</italic> < 0.05) than normal RBCs (6.6 ± 0.4 μm) found in control blood samples (no TiO<sub>2</sub> administration). Similarly, mean corpuscular volume was higher (<italic>p</italic> < 0.05) in TiO<sub>2</sub> whole-blood (70.70 ± 1.97 fl) than in control whole-blood (67.42 ± 2.03 fl). Poikilocytes also had higher HGB content. Mean corpuscular hemoglobin was higher (<italic>p</italic> < 0.05) in TiO<sub>2</sub> whole-blood (21.84 ± 0.75 pg) than in control whole-blood (20.8 ± 0.32 pg). Iron content was higher (<italic>p</italic> < 0.001) in TiO<sub>2</sub> whole-blood (697.0 ± 24.5 mg / l) than in control whole-blood (503.4 ± 38.5 mg / l), which supports elevated HGB as iron is found in HGB. HGB-associated Raman bands at 1637, 1585, and 1372 cm<sup> − 1</sup> had higher (<italic>p</italic> < 0.001) amplitudes in acanthocytes and echinocytes than in RBCs from control blood and normal RBCs from TiO<sub>2</sub> blood. Further, the 1585-cm<sup> − 1</sup> band had a lower (<italic>p</italic> < 0.05) amplitude in normal RBCs from TiO<sub>2</sub> versus control RBCs. This represents biochemical abnormalities in normal appearing RBCs. Overall, poikilocytes, especially acanthocytes, have elevated HGB.</p>
Bone healing involves compositional changes and understanding these is potentially important for prevention and treatment of bone diseases like osteoporosis. The health and proper function of bone tissues depends crucially major elements like calcium and phosphorus, but also on trace elements like iron, zinc, and strontium. This work employs energy dispersive X-ray fluorescence spectroscopy (μ-EDXRF) for trace element analysis, particularly of iron, and Raman spectroscopy for analysis of collagen, during healing of subcritical calvarial defects. In vivo defects were created on the calvaria of Sprague-Dawley rats (n=16) using a 1.4 mm burr drill. Subjects were sacrificed and additional control defects were similarly created after 7 (n=8) and 14 days (n=8) of healing. The two spectroscopy methods analyzed the bone surface at both time points and defect types without mechanical perturbation. Compared to control defects, calcium was found significantly decreased while zinc and iron increased in in vivo defects. Iron increased 6.7fold after 7 days, and this increased reduced approximately 50% after 14 days. Raman showed decreased collagen alignment after 7 days, which became insignificant after 14 days. We deduce that new collagen formation during healing, as revealed by Raman spectroscopy, scanning electron and optical microscopy and surface profiling, resulted in trace element changes. Our results show the need for studying the concentrations of major and trace elements, with iron in particular playing a crucial role in healing.
Bone healing is a complex process involving molecular changes. Bone matrix consists of collagen proteins that serve as the framework and minerals, calcium and phosphate, are deposited into the matrix accordingly. Raman spectroscopy is a promising technique to study bone mineral and matrix environments simultaneously. We studied the bone composition using 785 nm excitation during healing of subcritical calvarial defects without disrupting the fracture. Calvarial defects (in vivo) were created using 1 mm burr drill on the parietal bones of Sprague-Dawley rats (n=8). After 7 days, subjects were sacrificed and an additional defect (control) was created. Principal component analysis was utilized for the analysis of Raman spectra and helped in classifying normal and healing bone. Principal component 1 (PC1) shows that the major variation between in vivo and control defects and normal bone surface is at 958 cm<sup>-1</sup> (ʋ1 phosphate band). PC2 shows a major variation at 1448 cm<sup>-1</sup> (CH<sub>2</sub> deformation). PC2 score distinguishes in vivo defects from normal surface and control defects. The decrease in crystallinity and mineral to matrix ratio at the healing site as revealed by Raman confirms the new bone formation. Scanning electron and optical microscopy show the formation of newly generated matrix by means of bony bridges of collagens. The surface roughness increases by 23% from control to in vivo defects, as revealed by optical profiler. Overall, the new collagen formation shows the scaffolding of the bone is growing during healing.
Abnormally shaped red blood cells (poikilocytes) can cause serious health problems such as anemia and increase the risk of death. At present, the biochemical abnormalities in poikilocytes is not well understood, especially at the single cell level. In this study, confocal Raman spectroscopy revealed biochemical differences between single normal red blood cells (RBCs) and poikilocytes. Intragastric administration of nanoparticulate titanium dioxide (TiO<sub>2</sub>) was used to produce poikilocytes. Adult rats were administered by gavage 200mg/kg body weight TiO<sub>2</sub> every other day for 20 days (low-dose, N=5) or 250mg/kg every day for 60 days (high-dose, N=5). Low and high-dose controls (N=5 each) were administered distilled water for equal durations. Raman spectroscopy was performed on individual RBCs of low-dose subjects using 514nm excitation and a confocal setup. Whole blood from high-dose subjects underwent a Complete Blood Count (CBC) and inductively coupled plasma mass spectrometry (ICP-MS). Acanthocytes and echinocytes, two types of poikilocytes, were observed from TiO<sub>2</sub> subjects. RBCs were grouped into four types: normal RBCs from controls and normal-looking RBCs, Acanthocytes, and Echinocytes from TiO<sub>2</sub> subjects. The intensities of Raman bands at 1637, 1585, 1559, 1372 and 1228cm<sup>-1</sup> are larger in acanthocytes than normal and normal-looking RBCs. The 1342cm<sup>-1</sup> band is larger in normal RBCs, acanthocytes and echinocytes than in normal-looking RBCs. Also, the 975cm<sup>-1</sup> band is larger in acanthocytes than normal-looking RBCs. These bands are associated with oxygenated RBCs. Overall, poikilocytes, especially acanthocytes, carry more oxygen and haemoglobin and this is corroborated by CBC and ICPMS.
Subcritical calvarial defects are important to study bone regeneration during healing. In this study 1mm calvarial defects were created using trephine in the parietal bones of Sprague-Dawley rats (n=7) that served as in vivo defects. Subjects were sacrificed after 7 days and the additional defects were created on the harvested skull with the same method to serve as control defects. Raman spectroscopy is established to investigate mineral/matrix ratio, carbonate/phosphate ratio and crystallinity of three different surfaces; in vivo defects, control defects and normal surface. Results show 21% and 23% decrease in mineral/matrix after 7 days of healing from surface to in vivo and control to in vivo defects, respectively. Carbonate to phosphate ratio was found to be increased by 39% while crystallinity decreased by 26% in both surface to in vivo and control to in vivo defects. This model allows to study the regenerated bone without mechanically perturbing healing surface.