Mechanical Strain Regulates Levels Of RANKL And OPG MRNA By Primary Murine Stromal Cells In Vitro

Objective: Murine stromal cells(MSCs) play a unique role in the differentiation of osteoclasts in vivo. To elucidate the effect of mechanical strain on MSCs, we investigate receptor activator of NF-kB ligand (RANKL) and osteoprotegerin (OPG) production from the primary MSCs under mechanical tensile stress in vitro.Method: To isolate the cell targeted by strain, primary murine stromal cells were cultured from murine marrow. The murine stromal cells and culture fluid were collected after 0,1,3,6,9,12, hours mechanical tensile stress applying. Total RNA and cell protein was extracted respectively, the expression of RANKL mRNA and OPG mRNA was determined by RT-PCR.Results:. In vitro study indicated that the mechanical tensile stress significantly increased the secretion of RANKL mRNA and decreased that of OPG mRNA in MSCs in a time and force magnitude-dependent manner. The mechanical tensile stress stimulated secretion of RANKL mRNA decreased approximately 3-fold and that of OPG increased 1.5-fold.Conclusions: The results obtained suggest that the changes of amount of RANKL mRNA and OPG mRNA,the changes of amount of RANKLprotein(both membrane and secretion form) and OPG protein may be involved in bone resorption as a response to mechanical tensile stress.BackgroundMechanical loading plays an important role in the regulation of bone remodeling. Skeletal unloading occurs with bed rest or space flight, which results in net losses in bone mineral density. Measurement of markers of bone resorption show that osteoclasts are activated quickly after weightlessness (6) and indeed show continued evidence of bone resorption for at least several weeks after returning to normal gravity (7). Exercise is known to affect remodeling, causing bone formation in children (8). Adults(9,10). and animals (11,12,13). It is thus clear that the skeleton responds to its mechanical environment with a complex array of responses which include balanced remodeling, as well as remodeling that favors either formation or resorption. Despite the surmounting in-vivo evidence to indicate correlations between mechanical loading and bone homeostasis, the underlying cellular and molecular mechanisms of these events are yet to be elucidated.Both animal work and in vitro marrow cell culture have allowed biologists to define many of the processes involved in osteoclast recruitment. Although many hormones and cytokines can modulate the recruitment of osteoclasts from marrow progenitors, there are three components shown to be absolutely necessary for the basic process. The first is the marrow osteoclast progenitor itself, which are modulated by the second components, receptor activator of NFkB ligand (RANKL).which is expressed on the surface of the bone stromal cells(14,15) and in thymus, lymph nodes, and spleen(16) This tumor necrosisfactor family member, when added as a genetically engineered soluble molecule, negates the necessity for stromal support cells during osteoclast differentiation from MCSF-supported progenitor cells; and the third components is osteoprotegerin (OPG), which functions as a decoy receptor for RANKL, thereby inhibiting these processes and accelerating osteoclast apoptosis (17). Thus, the signaling and regulation of the expression of RANKL and OPG may play critical roles in bone remodeling. However, little information is available concerning the production of these modulators during bone remodeling in human subjects.It was hypothesized that mechanical strain affected the formations and functions of osteoclasts which pertinent to bone homeostasis. For this reason, a custommade laboratory setup and rat bone marrow cells were used to investigate the effects of mechanical tensile strain on the release and mRNA expression of select cytokines.Materials and MethodsBone Marrow Cell Culture.Bone marrow cells, a source of osteoclast-precursors, were harvested from the femurs of 14-day old Sprague-Dawley rats according to procedures reported in the literature (18). Briefly, the femurs were excised, the epiphyses were cut off, and the marrow cavity was flushed out with Dulbecco's modified Eagle Medium (MEM; Life Technologies, Long Island, NY), supplemented with 10% fetal bovine serum (FBS; Life Technologies). To generate primary stromal cell cultures, murine marrow cells were plated in cell culture bottle for 30 min to separate adherent macrophages from nonadherent cells containing thestromal elements; nonadherent cells were collected and plated at 1.0×10~6 cells/cm~2 and cultured on substrates(as described in the "Substrates" section). Twentyfour hours later, all nonadherent cells were discarded and the remaining stromal cells cultured for 1 wk until nearly confluent(19), These stromal cells represent 5% of the initial marrow collection(20),and were cultured under standard cell culture conditions, that is, a 37°C, humidified, 5% CO2/95% air environment.Substrates.The tissue-culture polystyrene bottles were deliberately cut into rectangle blocks(4cm×10cm cross section),each 2mm thick, using a low-speed diamond saw (Buehler, Evanston, IL). These tissue-culture polystyrene dishes were cleaned by deionized water and sterilizated by ultraviolet rays for 1 h until used as substrates for bone marrow cell cultures.Mechanical tensile strain systemThe custom-made, computer operated mechanical tensile strain system used in the present study was designed, assembled, and calibrated by university electronic science and technology of china. Briefly, a computer, with software specially written for this system, controlled and maintained a cyclic pressure environment inside a sealed, polystyrene chamber, which housed standard tissue-culture substrates loaded with cells. During experiments, the cyclic pressure system (except for the computer and electronic components)was maintained under standard cell culture conditions, i.e., a 37°C, humidified, 5% CO2/95% air environment.Mechanical tensile strain experimentsThe bone marrow cells cultured on the substrates were maintained under standard, that is static cell culture conditions for 7 days until confluent, then the substrates were put into the chamber filled with culture medium and exposed to tensile strain with 2mm deformation, at 0.5 Hz frequency, each substrate underwent tensile strain for 1 h ,3 h,6 h,9h,12 h, respectively. Control specimens were prepared from the same batch of marrow cells and maintained under static.standard cell culture conditions. During these experiments culture medium was changed following the change of the murine stromal cells substrates.RT- PCRTotal RNA was isolated by using RNAeasy mini kit (Qiagen) and treated with DNase I to remove contaminating genomic DNA. Reverse transcription was performed with 1 ug of RNA in a total volume of 20 ml per reaction. RT-PCR was performed using the iCycler instrument (Bio-Rad Laboratories, Hercules, CA). Amplification reactions were performed in 25 ml containing primers at 0.5 mMand dNTPs (0.2mMeach) in PCR buffer, and 0.03 U Taq polymerase along with SYBRgreen (Molecular Probes, Eugene, OR) at 1:150,000. Aliquots of cDNA were diluted 10-10,000-fold to generate relative standard curves to which sample cDNA was compared. For RANKL, forward and reverse primers were 5-CCT CTC GACCCGACTGCAGATC-3 and 5-AGCTGCAAGCTCTCT GTA ACC ATG AC-3, respectively, creating a product of 107 bp. A 102 bp of OPG amplicon was generated from forward primer 5-GAA TGG CAG CAC GCT ATT AAA TCC-3 and reverse primer 5-GCC GCT AGA ATT CAA AAC AGT TGG-3. Standards and samples were run in triplicate. Dilution curves showed that PCR efficiency was more than95% for all species studied. PCR products from all species were normalized for amount of 18S in the sameRT sample, which was also standardized on a dilution curve from RT sample.Resultsmechanical strain enhanced the expression of OPG genes to 73% after 9 h applying, while reduced the expression of RANKL genes to 34.4% after 6 h applying respectively.ConclusionsThe results obtained suggest that the changes of amount of RANKL mRNA and OPG mRNA.the consequencent changes of amount of RANKL protein(both membrane and secretion form) and OPG protein may be involved in bone resorption as a response to mechanical tensile stress.