Bone Formation Analysis: Effect of Quantification Procedures on the Study Outcome

Abstract

Quantification of the amount of newly formed bone is an essential part of bone regeneration studies. Histomorphometry, based on histological sections of plastic-embedded specimens, is the most frequently applied technique in this assessment. Before performing image analysis, a specific region of interest (ROI) has to be determined. Based on the histological procedure, different areas within the ROI can be discriminated and assigned to relevant tissue structures. However, in literature not much attention is paid to the effect of the histological procedures on the final outcome of the histomorphometrical measurements on bone regeneration. In this study, the histomorphometrical bone formation of the intramedullary cavity of the guinea pig tibia, filled with calcium phosphate cement, was quantified in plastic-embedded and paraffin-embedded specimens and in specimens analyzed with scanning electron microscopy in the backscattering mode (SEM-BS). The data showed that the histological procedure significantly affected the measured bone amount. Therefore, it is recommended that scaffold characteristics are carefully considered in selecting a proper technique for the analysis of bone formation in bone tissue engineering studies. The results of this study identified high-resolution SEM-BS and elastic van Gieson staining of decalcified histological sections as recommendable techniques for evaluating bone formation.

Subcutaneous tissue response and osteogenic performance of calcium phosphate nanoparticle-enriched hydrogels in the tibial medullary cavity of guinea pigs

“…from the cortical endosteal surface towards the center of the medullary cavity, until reaching almost complete filling with transient trabecular mesh at the peak of the osteogenic phase. Subsequently, the bone trabeculae are subjected to osteoclastic resorption and replacement by new marrow, which is identical to the original tissue that was removed (the so-called second osteogenic phase) [35,36]. In rats, the entire process is completed within 35 days [2].…”

The performance of CPC/PLGA and Bio‐Oss® for bone regeneration in healthy and osteoporotic rats

“…Using different filters on the light microscope, pictures could be made of the fluorescent labels at the four different time points. Quantitative assessment of the pMMA specimens was performed using ImageJ computer‐based image analysis software (Java ® ImageJ 1.47, Image processing and analysis in Java) . From digitalized images of the sections (magnification: 5×), a circular region of interest (ROI) with a diameter equal to the created defects was positioned.…”

Size matters: effects of PLGA-microsphere size in injectable CPC/PLGA on bone formation

“…Examination of the PMMA sections revealed that both CPC/PLGA formulations were still present within the intramedullary cavity at the end of the 12 weeks implantation time. Due to the high density of the CPC/PLGA composite material, the relative thickness of PMMA sections and artifacts caused by staining, analysis of bone formation using PMMA embedding is inaccurate (Lopez‐Heredia et al ., ). In contrast, H&E and EVG staining of decalcified paraffin sections indicated the presence of abundant bone formation within the CPC matrix.…”