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Alizarin red S staining of cortical bone. Sections decalcified for 0 ( a ), 1( b ), 3 ( c ), and 5 ( d ) min. a Intense staining is apparent throughout the bone matrix, although none was observed in osteoid ( arrow head ). b–d Following decalcification, a gradual reduction in intensity of staining of bone matrix is observed, which disappeared after 5 min of decalcification (b–d). BM bone marrow; CB cortical bone; M skeletal muscle. Bar =200 l m
Source publication
To examine the stability of bone matrix proteins for crystal dislocation, the immunolocalization of type I collagen, bone sialoprotein, and osteopontin was investigated during different stages of fixation and decalcification. Four-week-old rat femurs were rapidly frozen, and were sectioned without fixation or decalcification. Thereafter, following...
Citations
... Specimens were obtained from 6 mice at each time period. Each undecalcified frozen sample was sagittally sectioned with the film (Cryofilm Type IIC; Section-Lab Co. Ltd.) at a thickness of 5 µm (Kawamoto and Shimizu 2000;Hosoya et al. 2005). Some sections on the mesial root plane of the first molar were stained with 0.1% toluidine blue (pH 7.0) or 1% alizarin red. ...
The periodontal ligament (PDL) contains mesenchymal stem cells (MSCs) that can differentiate into osteoblasts, cementoblasts, and fibroblasts. Nevertheless, the distribution and characteristics of these cells remain uncertain. Gli1, an essential hedgehog signaling transcription factor, functions in undifferentiated cells during embryogenesis. Therefore, in the present study, the differentiation ability of Gli1 ⁺ cells was examined using Gli1-CreERT2/ROSA26-loxP-stop-loxP-tdTomato (iGli1/Tomato) mice. In 4-wk-old iGli1/Tomato mice, Gli1/Tomato ⁺ cells were only slightly detected in the PDL, around endomucin-expressing blood vessels. These cells had proliferated over time, localizing in the PDL as well as on the bone and cementum surfaces at day 28. However, in 8-wk-old iGli1/Tomato mice, Gli1/Tomato ⁺ cells were quiescent, as most cells were not immunoreactive for Ki-67. These cells in 8-wk-old mice exhibited high colony-forming unit fibroblast activity and were capable of osteogenic, chondrogenic, and adipogenic differentiation in vitro. In addition, after transplantation of teeth of iGli1/Tomato mice into the hypodermis of wild-type mice, Tomato fluorescence indicating the progeny of Gli1 ⁺ cells was detected in the osteoblasts and osteocytes of the regenerated bone. These results demonstrate that Gli1 ⁺ cells in the PDL were MSCs and could contribute to the alveolar bone regeneration.
... 16 However, these methods can lead to inconsistent preservation of tissue and loss of chemical integrity, in terms of the native structure and distribution of biomolecules. 17,18 Formalin fixation maintains vascular and cellular structures in the bone marrow but results in removal, crosslinking, or delocalization of molecular species. Therefore, imaging of undecalcified and unfixed tissue is ideal, but extensive preparation is required to preserve the heterogenous morphology of the cortical bone, trabecular bone, and bone marrow; all of which differ in porosity and density. ...
Bone and bone marrow are vital to mammalian structure, movement, and immunity. These tissues are also commonly subjected to pathological alterations giving rise to debilitating diseases like rheumatoid arthritis, osteoporosis, osteomyelitis, and cancer. Technologies such as matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) enable the discovery of spatially resolved chemical information in biological tissue samples to help elucidate the complex molecular processes underlying pathology. Traditionally, preparation of native osseous tissue for MALDI IMS has been difficult due to the mineralized composition and heterogenous morphology of the tissue, and compensation for these challenges with decalcification and fixation protocols can remove or delocalize molecular species. Here, sample preparation methods were advanced to enable multimodal MALDI IMS of undecalcified, fresh-frozen murine femurs allowing the distribution of endogenous lipids to be linked to specific tissue structures and cell types. Adhesive-bound bone sections were mounted onto ITO coated glass slides with a microscopy-compatible glue and freeze-dried to minimize artificial bone marrow damage. Subliming matrix does not induce further bone marrow cracks, and recrystallizing the deposited matrix improves lipid signal. High spatial resolution (10 μm) MALDI IMS was leveraged to characterize lipid distributions in fresh-frozen bone, and complementary microscopy modalities aided tissue and cell assignments. For example, various phosphatidylcholines localize to bone marrow, adipose tissue, marrow adipose tissue, and muscle. Furthermore, we discovered that [sphingomyelin(42:1) + H]+ was abundant in megakaryocytes, whereas [sphingomyelin(42:2) + H]+ was diminished in this cell type. These data reflect the vast molecular and cellular heterogeneity indicative of the bone marrow and the soft tissue surrounding the femur. Therefore, this application of multimodal MALDI IMS has the potential to advance bone-related biomedical research by offering deep molecular coverage in a preserved native bone microenvironment.
... Consequently, mineralised skeletal tissues are commonly subjected to 1-2 weeks of decalcification before being processed for embedding and sectioning [5,6]. However, decalcification may impair antigenicity and enzymatic activity, therefore preventing the utility of these sections for certain immunohistochemistry and enzymatic assays [5,7]. Moreover, decalcification destroys fluorescent labels (such as calcein labels) that are used to evaluate bone formation parameters [6]. ...
Background/objective:
Histology-based analyses are important tools to dissect cellular and molecular mechanisms of skeletal homeostasis, diseases, and regeneration. The success of these efforts is highly dependent on rapidly obtaining high-quality sections of mineralized skeletal tissues suitable for various analyses. However, the current techniques for preparing such sections are still far from satisfactory. This study aimed to develop a new approach for preparing high-quality undecalcified bone sections applicable to various histological analyses.
Methods:
Two important modifications were made to the conventional Cryojane Tape-Transfer System, including utilization of an optimized adhesive to prepare adhesive glass slides for improving the transfer efficiency, and a cheap conventional benchtop UV transilluminator for UV curing. Cryosections of undecalcified rodent bones were prepared using this modified tape transfer approach, and their tissue morphology and structural integrity were visually examined. A variety of histological analyses, including calcein labeling, Von kossa staining, immunofluorescence, and enzymatic activity staining as well as 5-Ethynyl-2'-deoxyuridine (EdU) and TUNEL assays, were performed on these sections.
Results:
We developed a modified version of tape transfer approach that can prepare cryosections of undecalcified rodent adult bones within 4 days at a low cost. Bone sections prepared by this approach exhibited good tissue morphology and structural integrity. Moreover, these sections were applicable to a variety of histological analyses, including calcein labeling, Von kossa staining, immunofluorescence, and enzymatic activity staining as well as EdU and TUNEL assays.
Conclusion:
The tape transfer approach we developed provides a rapid, affordable, and easy learning method for preparing high-quality undecalcified bone sections valuable for bone research.
The translational potential of this article:
Our research provides a rapid, affordable, and easy learning method for preparing high-quality undecalcified bone sections that can be potentially used for accurate diagnosis of various bone disorders and evaluation of the efficacy of different therapies in the treatment of these diseases.
... 19 Previously published studies comparing different decalcification methods for mineralized tissues mostly used teeth and mandibular bone of human and rat origin [20][21][22][23][24][25] or rat femurs. 10,26,27 Therefore, the main goal of this study was to evaluate and compare commonly used decalcification methods for mouse and rat tibiae. To investigate this, micro-computed tomography (microCT) was used to accurately measure rates of decalcification for exact demineralization end points and to determine the effects different decalcification solutions and temperatures have on the preservation of tissue morphology and antigenicity of mouse and rat bone tissue. ...
... When decalcified in EDTA, formic acid, or nitric acid, the cartilage showed weaker staining at 37C. These findings complement the results of several other studies, confirming that both fixation and decalcification methods have been associated with proteoglycan and glycosaminoglycan depletion 4,26,27,32 reducing the staining intensity of Safranin O, which is directly proportional to the proteoglycan content in normal cartilage. 37 Optimal fixation and decalcification conditions for cartilage-containing specimens should always be decided in a pilot study. ...
Conventional bone decalcification is a time-consuming process and is therefore unsuitable for clinical applications and time-limited research projects. Consequently, we compared the effect of four different decalcification solutions applied at three different temperatures, and assessed the rate of decalcification and the implications on tissue morphology and antigenicity of mouse and rat tibiae. Bones were decalcified with 10% ethylenediaminetetraacetic acid (EDTA), 10% formic acid, 5% hydrochloric acid, and 5% nitric acid at 4C, 25C, and 37C. Decalcification in both species was fastest in nitric acid at 37C and slowest in EDTA at 4C. Histological and immunohistochemical staining confirmed that the conventional protocols of EDTA at 4C and 25C remain the best option regarding the quality of tissue preservation. Whereas formic acid at 4C is a good alternative saving about 90% of the decalcification time, hydrochloric and nitric acids should be avoided particularly in case of rat tibia. By contrast, due to their smaller size, mouse tibiae had shorter decalcification times and tolerated higher temperatures and exposure to acids much better. In conclusion, this study demonstrated that depending on the specific research question and sample size, alternative decalcification methods could be used to decrease the time of decalcification while maintaining histological accuracy.
... Microscopic characterization of calcified matrices is methodologically challenging, as two key prerequisites have to be fulfilled: chemically diverse sample constituents have to be preserved during all steps, and sample has to be prepared appropriately for imaging, which generally implies that thin sectioning is necessary. Preparation of calcified tissues for sectioning frequently involves artificial decalcification, which could impact also other components in the sample [23][24][25]. Generally, it is known that crustacean cuticle, once fully calcified, includes different calcium minerals, namely, crystalline calcium carbonate (calcite), amorphous calcium carbonate (ACC), and amorphous calcium phosphate (ACP) [16,18,19,[26][27][28][29][30]. ...
Biomineralized structures consist of an organic matrix and mineral constituents. Imaging of the mineralized biological
tissues is demanding due to specific requirements for the preservation and visualization of chemically different constituents and due to sectioning difficulties. In this study, a characterization of the cuticular matrix of the crustacean exoskeleton was performed by a combination of microscopic methods, aiming to obtain spatial information on the matrix
composition. Histochemical procedures were performed and compared in artificially decalcified and non-decalcified
samples, in paraffin and resin sections. Wheat germ agglutinin (WGA) lectin-gold conjugate and a fluorescent chitinbinding probe were used to localize chitin in paraffin and resin sections of samples prepared by different fixations.
Calcified regions of the matrix were determined by histochemical staining of aldehyde-fixed, methanol-fixed, and resinembedded samples and by scanning electron microscopy with energy dispersive X-ray spectroscopy (SEM–EDXS) of
“intact” cuticle transverse profiles that were not subjected to any surface processing. We show that the spatial distribution of cuticular organic matrix components is not homogenous, as a differential staining of exocuticular and endocuticular matrix was obtained by histochemistry. Chitin localization, performed by two different methods, shows a
localization pattern of horizontal lines of alternating intensity in the transversely cut endocuticle. Histochemical demonstration of calcified cuticular matrix in resin sections was successful in 2.5 μm thick resin sections of methanol-fixed
samples. Methanol-fixed, dried, and fractured samples displayed the characteristic elemental composition and are useful
to obtain qualitative composition data from the non-processed surface profiles of the cuticle
... To count human PKH-positive cells in bone marrow, femora harvested from the mice were immersed in 4% paraformaldehyde in a darkroom at 4 C and cryosections were prepared for analysis using the Kawamoto method 24 . ...
Acute kidney injury (AKI) is a major clinical problem that still has no established treatment. We investigated the efficacy of cultured human peripheral blood mononuclear cells (PBMNCs) for AKI. Ischemia/reperfusion injury (IRI) was used to induce AKI in male nonobese diabetic (NOD/severe combined immunodeficiency) mice aged 7 to 8 wk. PBMNCs were isolated from healthy volunteers and were subjected to quality and quantity controlled (QQc) culture for 7 d in medium containing stem cell factor, thrombopoietin, Flt-3 ligand, vascular endothelial growth factor, and interleukin 6. IRI-induced mice were divided into 3 groups and administered (1) 1 × 10⁶ PBMNCs after QQc culture (QQc PBMNCs group), (2) 1 × 10⁶ PBMNCs without QQc culture (non-QQc PBMNCs group), or (3) vehicle without PBMNCs (IRI control group). PBMNCs were injected via the tail vein 24 h after induction of IRI, followed by assessment of renal function, histological changes, and homing of injected cells. Blood urea nitrogen and serum creatinine (Cr) 72 h after induction of IRI in the QQc PBMNCs group dramatically improved compared with those in the IRI control and the non-QQc PBMNCs groups, accompanied by the improvement of tubular damages. Interstitial fibrosis 14 d after induction of IRI was also significantly improved in the QQc PBMNCs group compared with the other groups. The renoprotective effect noted in the QQc PBMNCs group was accompanied by reduction of peritubular capillary loss. The change of PBMNCs’ population (increase of CD34+ cells, CD133+ cells, and CD206+ cells) and increased endothelial progenitor cell colony-forming potential by QQc culture might be one of the beneficial mechanisms for restoring AKI. In conclusion, an injection of human QQc PBMNCs 24 h after induction of IRI dramatically improved AKI in mice.
... Kawamoto allows sectioning and four-color IF-staining of bones without previous decalcification (Hosoya et al., 2005), enabling us to (co)-visualize different matrix proteins, BM-cells and vessels simultaneously. ...
Platelets, small anucleated blood cells responsible for hemostasis, interact at sights of injury with several exposed extracellular matrix (ECM) proteins through specific receptors. Ligand binding leads to activation, adhesion and aggregation of platelets. Already megakaryocytes (MKs), the immediate precursor cells in bone marrow (BM), are in constant contact to these ECM proteins (ECMP). The interaction of ECMP with MKs is, in contrast to platelets, less well understood. It is therefore important to study how MKs interact with sinusoids via the underlying ECMP. This thesis addresses three major topics to elucidate these interactions and their role in platelet biogenesis. First, we studied the topology of ECMP within BM and their impact on proplatelet formation (PPF) in vitro. By establishing a four-color immunofluorescence microscopy we localized collagens and other ECMP and determined their degree of contact towards vessels and megakaryocytes (MKs). In in vitro assays we could demonstrate that Col I mediates increased MK adhesion, but inhibits PPF by collagen receptor GPVI. By immunoblot analyses we identified that the signaling events underyling this inhibition are different from those in platelet activation at the Src family kinase level. Second, we determined the degree of MK-ECM interaction in situ using confocal laser scanning microscopy of four-color IF-stained femora and spleen sections. In transgenic mouse models lacking either of the two major collagen receptors we could show that these mice have an impaired association of MKs to collagens in the BM, while the MK count in spleen increased threefold. This might contribute to the overall unaltered platelet counts in collagen receptor-deficient mice. In a third approach, we studied how the equilibrium of ECMP within BM is altered after irradiation. Collagen type IV and laminin-α5 subunits were selectively degraded at the sinusoids, while the matrix degrading protease MMP9 was upregulated in MKs. Platelet numbers decreased and platelets became hyporesponsive towards agonists, especially those for GPVI activation. Taken together, the results indicate that MK-ECM interaction differs substantially from the well-known platelet-ECM signaling. Future work should further elucidate how ECMP can be targeted to ameliorate the platelet production and function defects, especially in patients after BM irradiation.
... Although we were unable to detect EGFP protein by immunohistochemistry, long-term decalcification by strong acid may have caused the disappearance of EGFP expression at the protein level because several articles indicate that the decalcification by strong acid reduces the protein or immunoreactivity of tissue sections. 52,53 However, decalcification and embedding paraffin of tissue are necessary to observe the clear soft and hard tissue surrounding teeth. 54 EGFP amplicons were found in RNA or DNA samples, which were extracted from the histological samples of the allogeneic group. ...
Periodontitis is a chronic inflammatory disease that induces the destruction of tooth-supporting tissues, followed by tooth loss. Although several approaches have been applied to periodontal regeneration, complete periodontal regeneration has not been accomplished. Tissue engineering using a combination of cells and scaffolds is considered to be a viable alternative strategy. We have shown that autologous transplantation of periodontal ligament-derived multipotent mesenchymal stromal cell (PDL-MSC) sheets regenerates periodontal tissue in canine models. However, the indications for autologous cell transplantation in clinical situations are limited. Therefore, this study evaluated the safety and efficacy of allogeneic transplantation of PDL-MSC sheets using a canine horizontal periodontal defect model. Canine PDL-MSCs were labeled with enhanced green fluorescent protein (EGFP) and were cultured on temperature-responsive dishes. Three-layered cell sheets were transplanted around denuded root surfaces either autologously or allogeneically. A mixture of β-tricalcium phosphate and collagen gel was placed on the bone defects. Eight weeks after transplantation, dogs were euthanized and subjected to microcomputed tomography and histological analyses. RNA and DNA were extracted from the paraffin sections to verify the presence of EGFP at the transplantation site. Inflammatory markers from peripheral blood sera were quantified using an enzyme-linked immunosorbent assay. Periodontal regeneration was observed in both the autologous and the allogeneic transplantation groups. The allogeneic transplantation group showed particularly significant regeneration of newly formed cementum, which is critical for the periodontal regeneration. Serum levels of inflammatory markers from peripheral blood sera showed little difference between the autologous and allogeneic groups. EGFP amplicons were detectable in the paraffin sections of the allogeneic group. These results suggest that allogeneic PDL-MSC sheets promoted periodontal tissue regeneration without side effects. Therefore, allogeneic transplantation of PDL-MSC sheets has a potential to become an alternative strategy for periodontal regeneration.
... The histological detection of OPN and OC in osteoid, bone marrow, cement line and cells in bone has been reported by several authors [31,37,38]. Our results are consistent with those already reported for OPN, however for OC in semi-quantitative analysis there was no significant detection in cement line. ...
Confocal immunofluorescence is a valuable technique for the detection of relevant molecules in the pathogenesis of arthritis in rat models; however, it requires efficient processing of tissues including bone decalcification. The decalcification process must ensure the complete removal of calcium and also a proper preservation of cellular structures and, specially, the antigenicity of the tissue to allow the immunodetection of the molecules of interest. In the present study, we evaluated the effect of four different decalcifying solutions: the Morse´s solution, 10% EDTA (pH 7.4), 7% HCl/2% EDTA and 5% Nitric acid, as well as four different treatments of the tissues (including microwave irradiation) in the processes of decalcification for large pieces of adult rat bones (hind paw, fore paw, knee and column). We assessed the time of decalcification, the easiness of slicing, the morphological preservation and finally, the antigenicity of two different bone proteins (Osteopontin (OPN) and Osteocalcin (OC)) measured by its immunofluorescence intensity under controlled confocal microscopy conditions. Our results showed that the specimen size and the presence of skin are critical factors for the rate of decalcification, and no significant benefit was found if microwave irradiation is applied to the tissue. The comprehensive statistical analysis showed that the optimal solution for the detection of OPN and OC by confocal immunofluorescence is the 5% Nitric Acid, and followed by 10% EDTA (pH 7.4), Ana Morse solution and 7% HCl/2% EDTA.
... Evaluation of bone tissue is still challenging due to its mineralization. The chosen chelating agent was EDTA, as it is suitable for immunohistochemical staining, because it better preserves bone matrix proteins and genetic material for future analysis [17]. Hematoxylin and eosin staining played an important role in confirming that the decalcification protocol used did not affect the presence of osteocytes. ...
: The superantigenic properties of Staphylococcus aureus have been implicated in increasing the inflammatory process in airway diseases. Local formation of IgE antibodies against staphylococcal enterotoxins by secondary lymphoid tissue in nasal polyps has been demonstrated. Staphylococcus aureus is known to colonize the nasal mucosa, and has been found invading the nasal submucosa and intracellularly.
To evaluate the limits of Staphylococcus aureus invasion in the upper airway.
Inferior turbinate samples from 3 patients without sinus disease, 6 ethmoid samples from patients with chronic rhinosinusitis with nasal polyposis, and 6 ethmoid samples from patients with chronic rhinosinusitis without nasal polyposis were studied. A fluorescein-labeled PNA probe against Staphylococcus aureus was used to test for the presence of the bacterium in bone (after decalcification) and mucosa.
We found Staphylococcus aureus invading the nasal submucosa in patients with nasal polyposis, but no cases of Staphylococcus aureus positivity in bone. In conclusion, we cannot support the hypothesis of nasal bone as a reservoir for Staphylococcus aureus, releasing massive amounts of staphylococcal enterotoxins and eliciting an inflammatory reaction, as occurs with the nasal mucosa.
