Histological tissue analyses resolving tissue distribution

3.4.1. Specimen preparation

For histological analysis, femurs were fixed in a 4% PBS/PFA solution for 24 h at 4°C. For tissue cryo-protection femurs are then transferred into ascending sucrose solutions for 24 h at 4°C each (sucrose solutions: 10%, 20% and 30%). Before progressing to the embedding procedure, all parts from the external fixator (screws, fixator bar and titanium wires) needed to be carefully removed without disrupting any of the hematoma tissue. All femurs were embedded in the same orientation (Figure 12) into a rectangle stain mold (in-house-made), where beforehand a layer of SCEM-Medium was placed on the bottom. Next, the specimens were fully covered by carefully filling up the mold with SCEM-Medium. Generation of air bubbles and changes in orientation needed to be avoided in order to obtain high quality sample cryo blocks. As a last step the specimen were frozen by immersing the molds in a beaker containing cold n-Hexan, surrounded by dry-ice to ensure constant low temperatures thus a rapid freezing process. The SCEM blocks, containing the femurs, were detached from the stain mold, labeled and stored at -80°C until further handling.

Table 4. Solutions used for specimen preparation, embedding and freezing.

Step Solution

Fixation 4% PFA/PBS

Cryo protection 10% sucrose H20 solution

20% sucrose H20 solution

30% sucrose H20 solution

Figure 13. Schematic overview of the fracture hematoma region of interest (ROI) used for histological analysis, RNA extraction, protein and metabolite analysis.

3.4.2. Slide preparation for histological staining

For all down stream investigations, the frozen cryo-embedded sample were sectioned into 5 µm thick slices using Kawamoto’s film method [210]. In this process a transparent, adhesive cyro tape is used, allowing the sectioning of calcified tissues. The C9 tape was used for all following investigations, suitable for immune fluorescent and immune-histochemical stainings. The samples were sectioned using a Leica microtome cryostat at a constant chamber temperature of -30°C. The cryo block containing the femur was placed and fastened with SCEM-medium on a specimen chuck and afterwards into the specimen holder within the microtome chamber. Next, the sectioning blade (Feather® microtome blade, N35HR) was inserted carefully into the cutting device. Specimen orientation was adjusted parallel in relation to the cutting edge and sectioning blade. To start with the sectioning process, the hand-wheel was unlocked and turned, moving the specimen along the blade and thus cutting a defined slice. Slices of 30 µm were cut until reaching the plane of interest, the transversal section in the middle of the femur, indicated by appearance of the drill holes from the stabilizing titanium wires adjacent to both sides of the osteotomy gap. Thickness was reduced to 5 µm and prior to each cut, a size-fitted C9 cryo-tape was placed over the tissue cross-section. The tape was held with pincers, while slowly cutting the tissue underneath. At the end, the section was placed with the non-adhesive side on a microscopy glass slide, that the tissue is facing upwards and fixed with sticky tape on the sides. This procedure was repeated until enough slides were obtained, the slides were stored at -80°C in special cases until further handling.

3.4.3. Histological staining

3.4.3.1. Movat pentachrome staining

Movat’s pentachrome staining was developed to distinguish between multiple connective tissues on one slide. The applied procedure of five subsequent stainings results in a yellow/orange color for mineralized bone, yellow color for collagen fibers, a greenish-blue staining for cartilage, dark red coloring for osteoids, bright red for elastic fibers and black staining for nuclei (Table 5). Prior to the staining procedure, slides were thawed for 45 minutes, then fixed for 15 min with 4% PFA/PBS. After fixation, samples were rehydrated for 5 minutes before beginning with the first staining of alcian blue for 30 minutes. Alcian blue targets acid proteoglycans structures like chondroitin sulfate by electrostatic binding at a pH of 2.5. This is followed by short immersion into 3% acetic acid and a 1 hour incubation in alkaline ethanol, stabilizing the blue-green pigment. After a 10 minutes washing step in cold tap water and immersion in aqua dest. for complete ethanol removal, slides were incubated in Weigert’s Iron hematoxylin solution for 15 minutes used for staining nuclei. Next, another washing step is applied to remove surplus hematoxylin solution, here slides are rinsed in aqua dest. and then incubated for 15 minutes in cold tap water. Cell plasma staining is done by the application of brilliant crocein acid fuchsine for 15 minutes, followed by differentiation in 0.5% acetic acid. The slides were then incubated for 20 minutes in phophotungstic acid, which binds to the fibers of connective tissues, thereby replacing anions of already binding dyes and decolorizing the tissues for the final staining step. Saffron du gatinias solution, an alcoholic extract of dried saffron pigments is used for final connective tissue staining, resulting in a bright yellow color.

Table 5. Staining result of slides for Movat pentachrome staining.

Tissue Staining result

Mineralized bone Yellow/orange

Collagen fibers Yellow

Cartilage Greenish-blue

Osteoids Dark red

Elastic fibers Bright red

Nuclei Black

3.4.4. Immunehistochemistry

To visualize specific proteins the process of immunohistochemistry is utilized, where the antigens of interest are targeted with selective antibodies on a tissue slide. The most common practice to visualize the antigen-antibody complex, is to have an enzyme coupled antibody, e.g. with a peroxidase or fluorochrome that is able to catalyze a visible color reaction.

3.4.4.1.

α-smooth muscle actin staining

Vessel staining for vascularization analysis was done using an immunohistochemical approach. The target antigen used is alpha-smooth muscle actin (ɑ-SMA) which is highly expressed in vessel smooth muscle cells. Positive staining results in a bright pink color, using an alkaline phosphatase reduction of the applied chromogen. Prior to staining procedure, slides were thawed for 45 minutes. The area to be stained, was encircled with a hydrophobic pen, fixed with 4% PFA/PBS for 15 minutes and rehydrated with 1x PBS twice for 5 min. All subsequent staining steps were carried out in a humid chamber, at room temperature, unless states otherwise. To block against unspecific background binding, samples were incubated for 30 minutes with 2% normal horse serum before applying the primary antibody solution, 1:400 (ɑ-SMA, Dako M0851) over night at 4°C. The next day, slides were washed twice with 1xPBS for 5 minutes each, followed by incubation for 30 minutes with the secondary antibody, 2% in 2% PBS/BSA (anti-mouse biotinylated, rat-adsorbed). During the incubation time, the avidin-biotin complex (ABC-AP Vectastain Kit – SP 5000) should be prepared, according to manufactures instructions. After two washing steps, the AB- complex is applied to the tissue section for 50 min. Avidin has a high affinity for biotinylated sites, therefore form a complex with a biotinylated enzyme of choice (here: alkaline phosphatase) and further bind to the biotinylated secondary antibody. Afterwards slides were incubated twice (à 5 minutes) with chromogen buffer, before visualizing the α-SMA positive vessels by adding alkaline phosphatase substrate (Red AP Substrate Kit, Vector – SK 5100) to the chromogen buffer, which was catalyzed into a bright pink color at the sites of the target structures. Color development was observed under the microscope and ended by immersing the slides in 1xPBS. Nuclei were counter stained using Mayers Hämalaun for 1.5 minutes, followed by 5 minutes incubation in tap water and appeared blue/purple in color.

3.4.5. Image capturing and parameter quantifications

Pictures of the stained samples were taken with Zeiss Axioscope 40 Microscope, 10x objective (plus condenser) and the corresponding Imaging AxioVision LE Software (Carl Zeiss). White balance and focus were adjusted for each sample. A tile scan was used to image the fracture gap region of interest, which was set to be 4 mm in width and spanning the whole callus region in height. The 4 mm width included 2 mm osteotomy region and 1 mm on both, proximal and distal sides. A schematic overview of the determined region of interest can be seen in Figure 13.

Vessel quantification for α-SMA stainings was done with ImageJ (Version 1.44p; http://rsbweb.nih.gov/ij/) using a semi-automated method on blinded sections. Vessels were counted manually in a blinded approach. Inclusion criteria were a clear endothelial cell border, a visible lumen and non-muscle association. Our region of interest (ROI) included the osteotomy gap and 1 mm proximal and distal (Figure 13) from it and was analyzed for all time points and experimental groups.

Tissue quantification for Movat Pentachrome stainings was done using ImageJ. A semi-automated macro (IOCT.Sass.Dienelt.1.03), developed in-house by Mario Thiele, was applied to each picture, creating masks for the various tissue types by manual annotation of tissue areas and color thresholds. At the beginning, for each picture the ROI was defined and a length calibration done. The threshold color was defined for all white fields in the picture, making up the void area. Cortical bone was manually selected and mineralized tissue defined by a color threshold, here yellow. Areas of cartilaginous tissue, appearing blueish-green, were manually selected, as well as the hematoma area. At the end, the macro calculated the determined areas for each tissue type specified beforehand, in relation to the ROI and scale bar set at the beginning of the macro. All values obtained from the different histological analysis mentioned above were sorted for each experimental group and condition and statistical testing by one-way-analysis of variance (ANOVA) was performed using GraphPad Prism Version 8.