Von Kossa stain

The acoustic micrograph in Fig. 1.5(a) came from a 5-week-old preparation. It was fixed in alcohol, and stained for alkaline phosphatase and, with von Kossa stain, for biomineral material. The biomineral material of interest here is hydroxyapatite, the principal crystalline mineral constituent of bone. The ordered structure visible within the matrix is not seen with either the light or electron microscopes. But the acoustic microscope can also work perfectly well with unfixed, unstained specimens. Figure 1.5(b) is an acoustic micrograph of matrix and cells from a 17-year-old male. In addition to the standard ingredients of culture medium, these cells were specifically stimulated with beta-glycerolphosphate and a vitamin C preparation. Because the acoustic... 

Bonewald, L. F., Harris, S. E., Rosser, J. et al. (2003) Von Kossa staining alone is not sufficient to confirm that mineralization in vitro represents bone formation. Calcif. Tissue Int. 72, 537—47. 

Renal parenchymal calcium deposition can be associated with significant renal dysfunction. When calcium deposits are encountered on renal biopsy, the calcium salt typically contains either phosphate or oxalate. The two anions are easily differentiated pathologically, as calcium oxalate is identified as refractile crystals under polarized light. In contrast, calcium phosphate is non-polarizable but gives a positive histochemical reaction to the von Kossa stain [16]. 

D. The tubular calcifications stain intensely with the von Kossa stain, consistent with calcium phosphate, (orig. magn. 400x). 

Figure 4.3 Tissue sections, (a) A 12-week-old mouse Von Kossa-stained for mineral and counterstained with toluidine blue for tissue morphology (b) IR images of cortical bone at 16 weeks the top panel shows the integrated area under the 900-1200cm phosphate envelope. The second and third panels illustrate the mineral to matrix ratios and crystallinity, respectively. Reprinted with permission from Ref [37].

Fig. 4 TNAP activity colocaiizes with the mineralization front (developing mouse Achilles tendon enthesis) [from ref. 50]. Ankles from 12-week-old C57BL6 mice were embedded, and sections were stained with von kossa staining to reveal mineralized bones in black a), (b) shows at higher magnification the insertion of the developing enthesis (bony insertion) of the Achilles tendon asterisk) in the calcaneum. (c) is a staining of TNAP activity indicating that TNAP activity colocalizes perfectly with the mineralization front. Moreover, TNAP activity is absent in already mineralized areas...

Figure 71.6 Distal femoral metaphysis and epiphysis (a) of a selenium-deficient rat 74 days old and (b) of selenium-adequate rats. The selenium-defioient rat shows a deteriorated trabecular bone architecture with fewer and thinner trabeculae in the epiphysis and metaphysis (Von Kossa stain magnifioation X11 bar = 1 mm). Reproduced with permission from Moreno-Reyes etai, (2001). The American Society for Bone and Mineral Research.

The amount of bone nodule formation was quantified from the surfaces of unmodified and modified polymers at 28 and 35 days in culture under osteogenic conditions, for each of the three different pore-sized polymers. Histomorphometric analysis shows that the surfaces of modified polymers had significantly increased areas of Von Kossa staining (Figure 7.14) compared to unmodified polymers. Increased amounts of nodule formation can be observed for each of the different pore sizes between days 28 and 35 but this is only significant for 100-yum pore size. There was no evidence that nodule area was significantly influenced by the three pore sizes evaluated in this study. 

FTIR is another surface-sensitive spectroscopic tool to analyze biomedical polymers since sample preparation is very simple for this technique. FTIR spectra should be recorded in reflection mode instead of transmission mode in order to analyze the functional groups present on polymer surfaces. ATR-FTIR has been apphed to study in vitro mineralization of porous starch scaffolds cultured in bone marrow stromal cells harvested from Wistar rats. Mineral deposition in in vitro cultures is usually followed by von Kossa stain or Alizarin red stain or by calcium uptake. These methods provide erroneous results because the scaffold matrix itself can take up some calcium from the medium. ATR-FTIR is devoid of the limitation and provides reliable information on the mineralization process. In the... 

Optical images of von Kossa-stained sections of tbe defects implanted with the four groups of scaffolds at 12 weeks are shown in Fig. 5. The total von Kossa-positive area (the dark-stained area) within the defect indicated the presence of phosphate materials due to mineralized bone and to HA due to conversion of the bioactive glass scaffolds. The 13-93B3 glass appeared to be fully converted to HA within 6 weeks (results not shown). In comparison, the 13-93 glass was only partially converted at 12 weeks. 

The site of calcium accumulation in tissues is readily identifiable on hematoxylin-eosin slides because the protein in the calcified area develops a strong affinity for hematoxylin. But sometimes special stains must be used to identify calcium. Among those techniques, the von Kossa staining method is one of the most popular. The method is based on the precipitation of silver salt by the anionic groups present in the calcium deposit. Calcium can be demonstrated directly either by microincineration or by visualization of the hydroxyapatite crystals with the aid of the electron microscope. 

Figure 21 Von Kossa staining views of osteoblasts cultured for 14 days on different surfaces (a) PLLAas-b-PEEPss (b) PLLABs-b-PEEPno ...

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