Characterization of a shorter recombinant polypeptide chain of bone morphogenetic protein 2 on osteoblast behaviour

rhBMP2_108 was kindly provided by Jiuyuan biotech company (Hangzhou, China). The shortened amino acid sequence was fabricated as follows: MKKLKSSCKRHPLYVDFSDVGWNDWIVAPPGYHAFYCHGECPFPLADHLNSTNHAIVQTLVNSVNSKIPKACCVPTELSAISMLYLDENEKVVLKNYQDMVVEGCGCR for a protein chain of 108 amino acids. The commercially available BMP2 was purchased from R&D systems (Minneapolis, USA).

According to the amino acid sequence, intracellular expression of rhBMP2_108 in E. coli was constructed for bulk production of rhBMP2_108 as previously described [24]. Briefly, the sequence of BMP2 was designed according to its amino acid sequence, and then was reverse transcribed with reverse transcriptase (Gibco BRL, NY, USA) as previously described [24]. Polymerase chain reaction (PCR) was used to amplify the cDNA encoding the shortened amino acid sequence rhBMP2_108 protein. This rhBMP2_108 cDNA was then subcloned using a pRSET(A) vector (Invitrogen, UK) to yield the pRSET(A)/rhBMP2_108 expression vector. Then, pRSET(A)/rhBMP2_108 was further utilized to transform the E. coli BL21(DE3) strain. A bioreactor was then utilized to yield high cell-density cultivation of E. coli. Briefly, transformed E. coli was cultured in a 5 L fermenter, with 3 L of the defined medium (batch culture; yeast extract 1 g/L, peptone 2 g/L) inoculated with the pre-culture (10 % of the batch culture volume). The cultivation was performed with stirring at 250 rpm for 48 h at 30 °C, including the addition of a nutrient medium (glucose 33.3 g/L, peptone 10 g/L, yeast extract 5 g/L,MgSO₄1 g/L, FeSO₄8.0 g/L, CaCl₂0.048 g/L, ZnSO₄0.0176 g/L, CuSO₄0.008 g/L).

The cultured biomass was then harvested by crushing twice the cells in a French press and thereafter centrifuged. The pellet was resuspended at 25 mg wet weight/ml in a suspension buffer (20 mMTris–HCl [pH 8.5], 0.5 mMEDTA, 2 % [v/v]Triton X-100). The inclusion bodies (pellets) were resuspended in a solubilization buffer (6Mguanidine-HCl, 0.1 M Tris–HCl [pH 8.5],0.1 M DTT, 1 mM EDTA) and thereafter incubated with constant stirring at room temperature overnight. Insoluble particles were then removed from the suspension by centrifugation.

A Heparin Sepharose 6 Fast Flow column was then used to purify the active rhBMP2_108 (dimer). A continuous NaCl gradient (0.1–1.5 M) was used to elute the bound protein. Afterwards, a stepped NaCl gradient (0.15 M, 0.3 M, and 0.5 M) was used to elute and separate the active rhBMP2_108 protein. Thereafter rhBMP2_108 powder was frozen at−20 °C. When used, 0.1 % acetic acid was utilized to dissolve the BMP2 powder. Control rhBMP2 was purchased from R & D systems (Minneapolis, USA) derived from CHO cells.

MC3T3-E1 pre-osteoblast cell line was used for this study. No human or animal samples were utilized and thus no ethical consent was required. The migration assay of cells was performed with a Transwell chamber using a 24-well plate and polycarbonate filters (Transwell Costar, Corning, Acton, MA) with a pore size of 8 μm as previously described [25]. 10⁴ MC3T3-E1 cells in 50 μl DMEM were seeded in the upper compartment. rhBMP2_108 at concentrations of 0, 10, 50, 100, 200 and 500 ng/ml and control rhBMP2 at 100 ng/ml were seeded into the lower compartment. The cells were allowed to migrate for 24 h at 37 °C in a humidified 5 % CO₂ atmosphere. The filter was then removed, cells were fixed in 4 % formaldehyde for 20 min and washed in PBS, after washings, filters were incubated for 15 min at room temperature with Methylrosanilnium Chloride Solution (Wuhan Google biotechnology limited company G1014), then the filters were washed with PBS for 10 min. Samples were examined by microscopy. Non-migrated cells on the upper side were eliminated by rinsing the filter with cold PBS and scraping with a rubber wiper. The remaining migrated cells on the lower side of the filter were counted in nine random fields per filter (×100 magnification). All samples were performed in triplicate with 3 independent experiments performed.

To investigate the effect of rhBMP2_108 on the proliferation of MC3T3-E1, CCK-8 assay for each group was performed as previously described [26]. Briefly, cells were seeded in 96-well plates at a density of 5×10³ cells/well. At time points 1, 3 and 5 days, the CCK-8 assay was performed by adding 10 μL of the CCK-8 solution (Dojindo Molecular Technologies, Inc. Japan) to each well and incubating for 1.5 h according to manufacturer’s protocol. The absorbance was measured at λ = 450 nm on a plate reader. Results were demonstrated as the absorbance of each experimental well minus the optical density value of blank wells. All samples were repeated in triplicate with three independent experiments.

Alkaline phosphatase activity was analyzed colorimetrically using alkaline phosphatase assay kit (Nanjing Jiancheng Bioengineering Insitute) using a starting seeding density of 50’000 cells per 24 well dish as previously described [27, 28]. At time point of seven and 14 days, cells were washed three times with PBS and solubilized in 0.1 % Triton X-100 (buffered in 0.1 mol/L PBS, pH 7.3) at 4 °C for 1 h. After sonication and centrifugation, ALP activity in the supernatant was determined colorimetrically using readings OD405/OD562. Total protein was quantified by BCA Protein Assay Kit (Thermo Fisher Scientific Inc.). The ALP activity was normalized to the total protein. Samples were run in triplicate with three independent experiments performed.

MC3T3-E1 cells were seeded at a density of 2×10⁵ and cultured for 14 days as previously described [26, 28, 29]. The effects of rhBMP2_108 at various concentrations were investigated on four osteogenic-related gene expression including alkaline phosphatase (ALP), collagen I (COL I), runt-related transcription factor two (Runx 2) and osteocalcin (OCN). Total RNA was extracted from MC3T3-E1 cells by Trizol reagent (TriPure Isolation Reagent, Roche Applied Science, Germany) according to the manufacturer’s instructions. The concentration and quality of the total RNA samples was analyzed by Nanodrop (Thermo Fisher Scientific Inc.). Complementary DNA was synthesized from 2 μg of total RNA using RevertAidTm First Strand cDNA Synthesis Kit (Fermentas) following the manufacturer’s protocol. RT-qPCR was performed in 20 μL reactions containing 10 μl SYBR Green Master Mix (Roche Applied Science, Germany), 0.6 μL (10 μM) of each forward and reverse primer for each gene of interest, 2 μL of cDNA template and 6.8 μL water. The primer sequences (shown in Table 1) have designed specifically according to the reference. Glyceraldehyde-3-phosphate-dehydrogenase (GAPDH), a reference gene, was used as a control. The reaction was analyzed using an ABI Prism 7000 Sequence Detection System (Applied Biosystems), and the PCR amplification run for 40 cycles. To validate specific amplicon amplification without genomic DNA contamination, melting curve analysis was performed and the single apex appeared around the annealing temperature. Relative expression levels for each desired gene were normalized against the Ct value of GAPDH and determined by using the delta Ct method. All samples were determined in triplicate for three independent studies.

Alizarin red staining was performed to determine the presence of extracellular matrix mineralization after 14 days. MC3T3-E1 cells were seeded at a density of 50,000 cells per 24 well culture dish containing the various concentrations of rhBMP2_108. After 14 days, cells were fixed in 96 % ethanol for 15 min and stained with 2 % alizarin red solution in water (pH 4.2) at room temperature for 1 h and visualized under light microscopy. Thereafter, alizarin red quantification was dissolved using 200 μl of 1 % cetylpyridinium chloride (dissolved with double distilled water) at room temperature for 4 h. Then 100 μl solution was transferred to 96-well plate to text OD 560.

In order to investigate the effects of rhBMP2_108 on cell recruitment, a transwell chamber was utilised at five different concentrations of rhBMP2_108 (Fig. 1). It was found that after a 24 h period, all concentrations of rhBMP2_108 and control rhBMP2 were able to significantly increase cell recruitment of MC3T3-E1 cells when compared to control samples. Little variability between samples was observed demonstrating a strong potential for all concentrations of rhBMP2_108 to induce cell recruitment (Fig. 1).

The effect of rhBMP2_108 was investigated at five concentrations and control rhBMP2 for their ability to stimulate cell proliferation in vitro (Fig. 2). It was found that MC3T3-E1 cells demonstrated very similar cell number 1 day post-seeding irrespective of cell culture media concentration of rhBMP2_108 (Fig. 2). A slight increase in cell number was observed at 3 days, however, no significant difference could be observed between the various treatment modalities (Fig. 2). By 5 days post seeding, a significant increase in cell number was observed for MC3T3-E1 cells with all concentrations of rhBMP2_108 used in this study (Fig. 2). Interestingly, no advantages could be found for higher concentrations of rhBMP2_108 when compared to their lower counterparts (Fig. 2).

RhBMP2_108 was then assessed for its ability to speed up osteoblast differentiation at various concentrations (Fig. 3, Fig. 4, Fig. 5, Fig. 6). First it was found that rhBMP2_108 was able to significantly increase ALP activity at seven and 14 days post seeding for all concentrations of rhBMP2_108 (Fig. 3). Interestingly, it was observed at 7 days that ALP activity was significantly higher at rhBMP2_108 concentrations of 50, 100 and 200 ng/ml when compared to control samples (Fig. 3). At 14 days, all treatment modalities receiving rhBMP2_108 at various concentrations demonstrated a marked 16-fold increase in ALP activity when compared to control samples void of rhBMP2_108 (Fig. 3).

Then MC3T3-E1 cells were assessed for mRNA levels by real-time PCR for genes encoding ALP, OCN, COL-1 and Runx2 at various concentrations of rhBMP2_108 (Fig. 4). For gene encoding Runx2, up to a five fold increase was observed at a concentration of 500 ng/ml when compared to control samples with a significant increase at 100 and 200 ng/ml compared to lower concentrations (Fig. 4a). Similarly, COL-1 also demonstrated concentration dependent increase in gene expression (Fig. 4b). Here however, only up to a two fold increase was observed relative to control samples (Fig. 4b). It was observed that mRNA levels of ALP were over five fold increased at 14 days at concentrations of ten and 50 ng/ml rhBMP2_108 whereas at a concentration of 100 and 200 ng/ml 20 fold increase in ALP activity (Fig. 4c). The highest concentration of 500 ng/ml demonstrated a 40 fold increase in ALP activity when compared to culture media without rhBMP2 (Fig. 4c). The greatest fold change was observed in OCN levels where up to a 300 fold increase was observed when compared to their respective control without rhBMP2 (Fig. 4d).

Lastly, alizarin red staining was used to visualize in vitro mineralization (Fig. 5). It was observed that without rhBMP2, MC3T3-E1 pre-osteoblasts were unable to generate any visual mineralization as observed by alizarin red staining (Fig. 5). The effects of rhBMP2_108 demonstrated a significant concentration dependent increase in alizarin red staining at 14 days post seeding (Fig. 6).