This paper summarizes the recent advances in image processing methods for morphological cell analysis. The topic of morphological analysis has received much attention with the increasing demands in both bioinformatics and biomedical applications. Among many factors that affect the diagnosis of a disease, morphological cell analysis and statistics have made great contributions to results and effects for a doctor. Morphological cell analysis finds the cellar shape, cellar regularity, classification, statistics, diagnosis, and so forth. In the last 20 years, about 1000 publications have reported the use of morphological cell analysis in biomedical research. Relevant solutions encompass a rather wide application area, such as cell clumps segmentation, morphological characteristics extraction, 3D reconstruction, abnormal cells identification, and statistical analysis. These reports are summarized in this paper to enable easy referral to suitable methods for practical solutions. Representative contributions and future research trends are also addressed.

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Recent Advances in Morphological Cell Image Analysis

Mechanical stress produced by orthodontic forces is a factor in the remodeling of periodontal ligaments (PDLs) and alveolar bone. It has been reported that the expression of a number of cytokines associated with osteoclastogenesis is upregulated when compressive forces act on osteoblasts and PDL cells. The present study investigated the effects of compressive forces on the formation of osteoclasts from the macrophage cell line RAW264.7. Compressive forces on osteoclasts were exerted using layers of 3, 5, 7, 9 or 14 glass cover slips on the 4th day of culture for 24 h. The number of osteoclasts was determined by counting the number of cells positive for tartrate-resistant acid phosphatase staining. Osteoclastogenesis advanced rapidly on days four and five. The number of osteoclasts with >8 nuclei peaked when the force of 7 slips was applied, which was therefore regarded as the optimal compressive force. Alterations in the expression of osteoclast-associated genes are associated with changes in the differentiation and fusion of macrophages in response to compressive forces; therefore, osteoclast-associated genes were assessed by reverse transcription quantitative polymerase chain reaction in the present study. The mRNA expression of osteoclast‑associated genes increased significantly after 3 h of optimal compression, whereas mRNA expression increased after 24 h in the control group. These findings suggested that osteoclastogenesis of macrophages was accelerated when an optimal compressive force was applied.

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Optimal compressive force accelerates osteoclastogenesis in RAW264.7 cells

Bone remodeling is an important factor in orthodontic tooth movement. During orthodontic treatment, osteoclasts are subjected to various mechanical stimuli, and this promotes or inhibits osteoclast differentiation and fusion. It has been previously reported that the release from tensile force induces osteoclast differentiation. However, little is known about how release from compressive force affects osteoclasts. The present study investigated the effects of release from compressive force on osteoclasts. The number of tartrate‑resistant acid phosphatase (TRAP)‑positive multinucleated osteoclasts derived from RAW264.7 cells was counted, and gene expression associated with osteoclast differentiation and fusion in response to release from compressive force was evaluated by reverse transcription‑quantitative polymerase chain reaction. Osteoclast number was increased by optimal compressive force application. On release from this force, osteoclast differentiation and fusion were suppressed. mRNA expression of NFATc1 was inhibited for 6 h subsequent to release from compressive force. mRNA expression of the other osteoclast‑specific genes, TRAP, RANK, matrix metalloproteinase‑9, cathepsin‑K, chloride channel 7, ATPase H+ transporting vacuolar proton pump member I, dendritic cell‑specific transmembrane protein and osteoclast stimulatory transmembrane protein (OC‑STAMP) was significantly inhibited at 3 h following release from compressive force compared with control cells. These findings suggest that release from optimal compressive force suppresses osteoclast differentiation and fusion, which may be important for developing orthodontic treatments.

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Release from optimal compressive force suppresses osteoclast differentiation.

This study investigated the effects of alendronate (ALN) on critical cell factors in osteoclasts. RAW 264.7 cells were induced by sRANKL to change to mature osteoclasts. On the sixth day of incubation, the osteoclasts were treated with ALN at various concentrations and for different incubation times. The concentration groups included 10-5 M, 10-6 M and 10-7 M ALN, respectively. The cells were incubated for 0 (control group), 2, 4, 6 and 8 h for each dose group. The mRNA and protein expression of tartrate‑resistant acid phosphatase, carbonic anhydrase II, osteoclast‑associated receptor and FAS/FASL genes in osteoclasts was analyzed. A concentration- and time‑dependent decrease in the mRNA and protein expression levels of the five genes was observed, and no significant difference between the two control groups was observed (P>0.05). Notably, significant differences between any two experimental groups were observed (P<0.05). Thus, ALN significantly decreased the expression of critical factors involved in osteoclast function.

https://www.spandidos-publications.com/10.3892/mmr.2013.1630/download

The effect of alendronate on the expression of important cell factors in osteoclasts

This study was designed to explore the effects of rotative stress on carbonic anhydrase II (CAII), TNF receptor superfamily member 6 (FAS), FAS ligand (FASL), osteoclast-associated receptor (OSCAR), and tartrate-resistant acid phosphatase (TRAP) gene expression in osteoclasts. Osteoclasts were induced from RAW264.7 cells cultured in medium containing recombinant murine soluble receptor activator of NF-Kβ ligand (sRANKL). The mRNA and protein expression of CAII, FAS, FASL, OSCAR, and TRAP genes in osteoclasts was detected by RT-PCR and Western blot, respectively, after osteoclasts were loaded at various rotative stress strengths and times. No significant differences in mRNA and protein expression were observed between any of the control groups (P > 0.05). Importantly, rotative stress had a significant effect on the mRNA and protein expression of these genes (P < 0.05). We found a negative relationship between rotative stress strength and prolonged loading time and the expression of FAS/FASL genes in osteoclasts. In addition, there was a positive relationship between rotative stress strength and prolonged loading time and the expression of CAII, OSCAR, or TRAP genes in osteoclasts. Based on these results, rotative stress has a significant effect on CAII, FAS, FASL, OSCAR, and TRAP gene expression in osteoclasts.

The effect of rotative stress on CAII, FAS, FASL, OSCAR, and TRAP gene expression in osteoclasts.

This study was designed to investigate the effect of various strengths and action times of flow stress on mRNA expression of H+-ATPase in osteoclasts. Osteoclasts were obtained through a classical mechanical-anatomical technique. They were identified by their morphology, tartrate-resistant acid phosphatase (TRAP) staining, and by a test of their ability to form resorption lacunae. Osteoclasts were mechanically loaded by flow stress using a cell-loading system. The stress-loading experiments were divided into various strength groups and action time groups. The morphological changes of osteoclasts after application of loading stress were analyzed using an image analysis system and Image-Pro Plus software. Expression of H+-ATPase mRNA in osteoclasts was detected by real-time fluorescent quantitative polymerase chain reaction. The existence of significant differences between experimental groups was analyzed using SPSS 12.0 software. The cytoplasm of osteoclasts with positive TRAP staining appeared with a characteristic claret-red color. Cells were able to form resorption pits in the surface of dentine slices. Morphological changes of osteoclasts with applied stress assumed an early increasing tendency before reaching a peak value and following a decreasing tendency. A significant difference of H+-ATPase mRNA expression of osteoclasts was seen between any two groups (P < 0.05). H+-ATPase mRNA expression in osteoclasts had a tendency to first increase with increasing stress and was observed to then decrease in one action time group. In this present study, a close relationship between the stress and mRNA expression of H+-ATPase in osteoclasts was observed.

Effect of stress on mRNA expression of H+-ATPase in osteoclasts

The objective of the present study was to investigate chronobiological rhythms of H+-ATPase and carbonic anhydrase II mRNA expression in rat osteoclasts. Rat osteoclasts were obtained through anatomical dissociation technique from bones. Experimental groups included 3 h, 6 h, 12 h, 24 h and 48 h cell cultures. mRNA expression of Atp6a1 and Atp6i of H+-ATPase and carbonic anhydrase II in osteoclasts were detected via fluorescent probe q-PCR. The data were analyzed using ANOVA with SPSS 12.0. mRNA expression of H+-ATPase and carbonic anhydrase II increased when the cell culture time was prolonged, and reached a peak value at 24 h; however later cultures resulted in a significant decrease in mRNA expression. Furthermore, mRNA expression of H+-ATPase and carbonic anhydrase II in osteoclasts varied with time of culture.

Zhang Qing Hong

Papers

The effect of rotative stress on CAII, FAS, FASL, OSCAR, and TRAP gene expression in osteoclasts.

Effect of stress on mRNA expression of H+-ATPase in osteoclasts

mRNA expression in osteoclasts: a chronobiological approach