Cell cytoskeleton and proliferation study for the RANKL‐induced RAW264.7 differentiation

Abstract Although document studies (including ours) have been reported the achieved in vitro osteoclastic cellular model establishment from the RAW264.7 cell lineage, there was no study directly reported that American Type Culture Collection (ATCC) cell bank has various RAW264.7 cell lineages. Besides that, for our knowledge there was only one study compared the two different RAW264.7TIB‐71 and RAW264.7CRL‐2278 cell lineages for their osteoclastic differentiation, and they concluded that the RAW264.7CRL‐2278 demonstrated to generate much osteoclast than RAW264.7TIB‐71. However, on the contrary to their results we noticed the fusion of RAW264.7TIB‐71 in our previous studies was much compromising. Therefore, we try to explore the two cell lineages for their properties in osteoclastic differentiation with an in‐depth cellular cytoskeletal study. Our current study has showed that comparing to the RAW264.7CRL‐2278, RAW264.7TIB‐71 demonstrated a much higher efficacies for RANKL‐stimulated osteoclastic differentiation. Besides that, in our depth cytoskeletal studies, we found that the RANKL‐induced RAW264.7TIB‐71 cells could finally differentiate into mature osteoclasts. However, regardless the various pre‐treatment conditions, there was no mature osteoclast formed in RANKL‐induced RAW264.7CRL‐2278 cell lineage.


| Cell cultures and treatments
and RAW264.7 CRL-2278 were purchased from ATCC.

| Osteoclastic grouping and induction
For osteoclastic differentiation, we induced the RAW264.7 TIB-71 as we previously reported. 2 Besides that, we modified the induction method for RAW264.7 CRL-2278 in our current study in order to abrogate the culturing divergences with RAW264.7  . Specifically, we changed the induction from RPMI-1640 medium into α-MEM at the first induction day. Both cell lineages were inducted for 7 days in several culture group with different stimulate conditions, the grouping details are listed in Supplementary Data, and the concentrations of each stimulator were determined after our preliminary studies.

| Morphological study and TRAP staining
After initially seeding, both cell lineages were allowed to grow and attach the wall for 24 hours. After 24 hours, we starting to treat cells with cocktailed stimulators for 10 days, and we counted this initial treating day as day 0. Subsequently, cellular morphological alterations were recorded and evaluated at days 1, 2, 3, 4, 5, 6 and 7 by an inverted microscope Nikon (Nikon) and NIS-Element analysis software (Nikon) for image analysis. TRAP staining kits were purchased from Sigma-Aldrich Co. According to instructions, after PBS washing two cell lineages were fixed by paraformaldehyde (4%) lasting for 30 minutes. Subsequently, both cell lineages were washed by PBS and then stained by TRAP solution at 37°C in 1 hour, the staining results were evaluated by Nikon (Nikon) and NIS-Element analysis software (Nikon) for image analysis.

| Cytoskeleton F-actin staining and Scanning electron microscopy
We performed cytoskeletal fibrous actin (F-actin) staining for two subjects in order to observe the podosome patterns and sealing zone formation in each cell linages. Briefly, two cell lineages were washed by PBS, after washing cells were incubated in formaldehyde (4%) for 15 minutes fixing, then cells washed by PBS for three times. In order to increase permeability, before the phalloidin red incubation, the Triton X-100 (0.1%) was added for 1 minutes.

| Statistical analysis
Final data results were analysed by 'two-way ANOVA' and 'Student's t test' methods by the software GraphPad Prism (version 8). Final, the value of P < .05 was defined as statistical significant in our current study, the data presented by the mean ± standard deviation (SD) manner. noted the brand of cell lineages for each usage. Therefore, in our present study we performed a subtle compare for these two cell lineages ( Figure 1A).

| The multinucleated cells from
In our current study, we found RAW264.7 TIB-71 could be introduced as osteoclast by RANKL independently stimulation in α-MEM. Besides that, RANKL independently stimulation could also successfully induce osteoclastic differentiation in DMEM and 1640 media, but the α-MEM showed a significantly high producing efficacy for osteoclastic differentiation (data not be presented  group are significantly increased (P < .05) (Figure 1).

F I G U R E 2
Besides that, we could find these morphological details in both immunofluorescent and SEM figure results only for RAW264.7 TIB-71 cell lineages. Moreover, consist with previous report, in the SEM scan results, we could observe podosomes presented an increasingly interconnectivity in the SZ (Figure 3).

| D ISCUSS I ON
Osteoclast formation and functional activity are important for bone homeostasis, which is key study target for bone biology. 8 As we previously reported, RAW264.7 cell lineage not belongs to osteoclastic homogeneous. 9 It is prevailing in the fields of bone homeostasis study because it merits advantages for providing a much easier accessible osteoclastic cellular model. However, during our usage, we found methodologies for RAW-OCs still elusive and existing controversies. For instance, several studies reported that RAW-OCs were generated by M-CSF and RANKL costimulation,however, we previously demonstrated the RAW-OCs could be generated by RANKL or LPS independently treatment. [10][11][12][13] These existing discrepancies might lie in the unique properties of RAW264.7 cell lineages, such as keep changing in its phenotypes during passage process. 9,14,15  Osteoclasts could commonly be characterized by two criteria: the multiple nuclei (≥3) and TRAP activity. 4,12,16,17 In that, TRAP activity is normally performed to evaluate activity of osteoclastogenesis. However, TRAP could also express in other hematopoietic-originated cell lineages such as macrophages. 18 Therefore, studies have to examine the osteoclast nucleus numbers and their associated osteoclastic activities, which demonstrated a significant correlation in the nucleus number and cellular size with the depth of the subsequent bone lacunae. [19][20][21] Commonly, in bone physiological homeostasis, osteoclasts could contain 3 to 10 nuclei. However, in the bone pathological homeostasis, the numbers of nuclei and the size of osteoclast are commonly noticed, such as Paget's disease, periodontal disease and rheumatoid arthritis (RA). 22

CO N FLI C T O F I NTE R E S T
The authors declare that they have no competing interests.

E TH I C S A PPROVA L A N D CO N S E NT TO PA RTI CI PATE
None animal studies have involved in the current study.

CO N S E NT FO R PU B LI C ATI O N
The manuscript is approved by all authors for publication.

DATA AVA I L A B I L I T Y S TAT E M E N T
All data and materials were included in the manuscript available.