Image pro plus 6.0 imaging software

Protein extraction and was determination based on the manufacturer's instructions (C500005 and C503051, Sangon Biotech). Subsequently, the obtained proteins (~30 μg) were analyzed by SDS–PAGE and transferred onto PVDF membranes (ImmobilonP). Thereafter, the membrane was blocked for 1 h at normal temperature with nonfatty milk (3%), and the membranes were incubated at 4°C overnight with the following primary antibodies: rabbit anti‐human antibodies against DGKH (1:1000; 13,873‐1‐AP, Proteintech), PDE4D (1:3000; 67,062‐1‐Ig, Proteintech), PYCR1 (1:1000; 66,510‐1‐Ig, Proteintech), TKT (1:3000; 66,016‐1‐Ig, Proteintech), or β‐actin (1:5000; 66,009‐1‐Ig, Proteintech). Then, the membranes were washed three times at 10‐min intervals, with PBS containing 0.1% Tween‐20. Secondary antibodies were incubated for 1 h at normal temperature (1:5000, SA00001‐2, Proteintech). Finally, special bands were determined by a commercial kit (12,630, Cell Signaling Technology, Inc.). Image‐Pro Plus 6.0 imaging software (Media Cybernetics, Inc.) was used to quantify protein expression and followed statistics by GraphPad Prism 9 (GraphPad Software).

Each slide stained with HE (n = 5, one slide per animal) was observed under an optical microscope (OLYMPUS BX43, Tokyo, Japan), and six photomicrographs corresponding to the regions surrounding the implanted biomaterial were captured by scanning without overlapping, using a high-resolution digital camera (OLYMPUS SC100, Tokyo, Japan). The magnification used in the light microscope was 40× for histomorphometric analysis. For general histological evaluation, magnifications if 10× and 40× were also used to identify the defect area, inflammatory cell infiltration patterns, reminiscent biomaterial, and newly formed bone. Histomorphometric analysis was done using the Image-Pro Plus^® 6.0 imaging software (Media Cybernetics, Silver Spring, MD, USA). Through this program, a grid of 133 points was superimposed on the area under analysis, which allowed the determination of the volume density of the newly formed bone, of the connective tissue, and of the residual biomaterial. The 133 points superimposed on each photomicrograph were considered as 100%, so each point was classified, and the percentage of each parameter was obtained (Adapted from [36 (link)]). Quantitative values were stored in the Microsoft Excel^® database and were transferred to the Prism^® 8.0 software (GraphPad Software, Inc., Irvine, CA, USA) for statistical analysis.

For histomorphometric analysis, the photomicrographs were captured from HE-stained slides, with 20× magnification, in a bright field light microscope (OLYMPUS^® BX43, Tokyo, Japan) coupled in a high-resolution digital camera (OLYMPUS^® SC100, Tokyo, Japan) in CELLSENS^® 1.9 software (Digital Image, Olympus, Tokyo, Japan). In each histological section, 6 fields, with no overlapping areas, were captured by scanning corresponding to the interest area into the dental socket.
The photomicrographs were transferred to ImagePro-Plus^® 6.0 imaging software (Media Cybernetics, Silver Spring, MD, USA), and a grid of 200 points was superimposed on histological images. Each point of the grid was classified according to the corresponding variable: newly formed bone, biomaterial, and connective tissue, thus allowing the determination of the density of each structure. The number of points for each variable was transformed into a percentage. The average of the fields of each animal was obtained for each variable that was used in the statistical analysis.