Bovine osteoprogenitor isolation

The osteoprogenitors were derived from the periosteum of calf metacarpus, obtained from abattoir tissue and 1 to a maximum of 2 h after sacrifice. The cells originated from one animal. In the regular cell harvesting process, there was no sex-specific selection of animals. The periosteum was cut into 3–6 mm2 pieces and transferred into culture dishes. The osteogenic layer of the periosteum specimen were placed face downwards. Osteoprogenitors were allowed to migrate from these specimens for 3 weeks. During this time the explant specimen were cultured in High Growth Enhancement Medium (ICN Biomedicals, Germany) supplemented with 10% fetal calf serum, 250 μg/mL amphotericin B, 10,000 IU/mL penicillin, 10,000 μg/mL streptomycin, 200 mM L-glutamine (Biochrom, Germany) and 10 mM β-glycerophosphate, at 37 °C and 5% CO2 in humidified air. The cell culture medium was replaced once a week. Cells of the first passage were used for this study. The osteoblastic character of the osteoprogenitors used in this study was positively shown by immunocytochemical staining (osteopontin, osteocalcin) during cultivation. The cells were harvested by incubation with collagenase (CLS Typ II; Biochrom, Germany) and ready-to-use tyrode solution at pH 7.4, collected and pelleted by centrifugation.

Osteoprogenitor cultivation with EP – proliferation and differentiation analysis

The obtained osteoprogenitors were resuspended and seeded on the bottom of cell culture dishes (21.5 cm2) with densities of 104 cells/cm2. Cell counting was performed with CASY Cell counter and Analyser Modell T (Schärfe System, Reutlingen) as suggested by the manufacturer. For further cell cultivation experiments, conditions were chosen equal to those used for the periosteum outgrowth culture. The different groups contained eggshell extracted peptides (provided and concentration recommended by Dermaviduals USA LLC, USA) of concentrations of 0.005 g/L (EP I) and 0.5 g/L (EP II), which were added to the cell culture medium over time of cultivation. A further group contained a combination of eggshell peptides with the concentration of EP II (0.5 g/L) supplemented with hyaluronic acid (0.25%, made from Ostenil®, TRB Chemedica, Molecular weight: 1000–2000 kDa) in the cell culture medium, which is indicated as HAEP. As a negative control, reference cultures were cultivated with cell culture medium without eggshell peptides or hyaluronic acid supplementation. Cultures were examined regularly by light microscopy and the influence of different additives concerning proliferation was measured by cell counting after 5 h, 1, 2, 3, 4 and 5 days of cultivation.

Osteoblastic differentiation was analysed by cultivation until day 14. Therefore, cells were seeded at a density of 6∙104 cells/cm2 on culture plates with 56.7 cm2 under addition of cell culture medium supplemented as described before. Measurement of immunocytochemical expression patterns of the bone-specific matrix proteins collagen type I, osteonectin, osteopontin and osteocalcin was performed to prove osteoblastic differentiation. Additionally, analysis of the Richardson-staining of bovine osteoprogenitors was performed. All staining procedures were performed in the cell culture dishes. For immunocytochemistry, the cell culture medium was decanted. The specimens were washed three times with phosphate buffered saline (PBS), fixed with cold methanol at -20 °C and subsequently incubated at -20 °C. After removing and drying of residual alcohol, a blocking solution (Dako) was applied for 15 min to saturate non-specific binding sites. Specific antibodies, diluted in blocking solution, were used to detect extracellular matrix proteins by immunocytochemical staining. Therefore, anti-collagen type I (Bio-Trend Chemikalien, Germany; 1:100 in blocking solution), the antibodies anti-osteocalcin and anti-osteonectin (Takara, Japan; each 1:50 in blocking solution) and anti-osteopontin (CHEMICON International, USA; 1:50 in blocking solution) were used and analyzed with secondary antibody Dako EnVision™ + (DAKO; 1:100 in blocking solution). The stained cell cultures were additionally analyzed by light microscopy.

Richardson staining was accomplished with a blue dye (Methylen blue Azur II) as previously described [27]. After methanol-fixation of the cells and decanting the methanol, 2–3 drops of the freshly mixed warm solution were applied and cells were incubated for 2 min at 60 °C. Staining solution was removed and cell layer subsequently washed. Analyzation was performed after drying upside-down in a dark dry chamber.

Primary human osteoblasts – Biomineralisation by calcium determination

The used primary human osteoblasts belong to a primary cell line of Promocell (Promocell, Germany). Cultivation, passage and harvesting were conducted with products and protocols recommended and distributed by Promocell,

To prove the cellular driven mineral formation by primary human osteoblasts after 4 weeks, the calcium content of the cell layers was examined after dissolution with HCl. The test solution Calcium Arsenazo III consists of 90 mmol/L sodium acetate (pH 5.5) and 348 µmol/L arsenazo. For the measurement the microplate spectrophotometer µQuant-System was used. For mineral formation, 6∙104 cells/cm2 osteoblasts were cultivated in 6-well plates and the medium was supplemented with EP I, EP II, and HAEP. The blank medium was used as negative control. After incubation for 4 weeks with weekly medium change, the cells are fixed with methanol and then washed several times. The cell monolayers in the wells were covered with 0.1 M HCl and incubated for five minutes at room temperature. The HCl solution was transferred to sterile Eppendorf tubes, vortexed for 15 s and then centrifuged for three minutes. To determine the mineral content, 300 µL each of the Calcium Arsenazo III Reagent was added to 5 µL sample supernatant in the wells of a 96-well plate. The extinction was determined at a wavelength of 650 nm. Extinctions were assigned to the amount of calcium containing mineral formed by the cell.

Primary human osteoblasts – Molecular biological analysis by microarray and qRT-PCR

For a detailed analysis of the influence of the pure higher concentrated putamen ovi proteins (EPII), a gene expression analyses of primary human osteoblasts (Promocell, Germany) was used. After a short incubation time, the cells were seeded into 75 cm2 cell culture bottles with a cell density of 104 cells/cm2. After three days, the cells were passaged, and transferred into the wells of 6-well plates with a cell density of 2∙104 cells/cm2. In order to evaluate the effect of the isolated eggshell peptides (EP) on the primary human osteoblasts, the peptides were added to the cell culture medium with a concentration of 0.5 g/L and the cells were incubated at 37 °C and 5% CO2 for five days. Similar cell cultures from the same batch and passage, provided with untreated culture medium served as the negative control. After reaching confluence (at day 5), the cells were detached from the cell culture dish and centrifuged for three minutes. The supernatant was discarded and the cells were stored in 700 μL of Qiazol (Qiagen, Germany) in cryotubes at -80 °C.

The RNA was isolated from the cells using the miRNeasy kit (Qiagen, Germany) according to the manufacturer's instructions. The quality and quantity of RNA obtained was determined by photometric measurements (NanoPhotometer™, Implen, Germany) and by the use of a bioanalyzer (RNA Nano 6000 Lab-Chip® Kit, Agilent Technologies, USA).

The cDNA was synthesized using a cDNA Synthesis Kit (Roche, Germany), according to the manufacturer´s data. 1 μg of RNA was used as starting material for cDNA synthesis. Subsequently, the NimbleGen One-Color DNA Labeling Kit (Roche, Germany) was used to label the cDNA with Cy3. The concentration of the labeled cDNA was determined using the NanoPhotometer. Subsequently, the hybridization (NimbleGen Hybridization Kit) and the required washing (NimbleGen Wash Buffer Kit) were carried out according to the manufacturer's instructions. The arrays used for the analysis were scanned with the NimbleGen MS 200 Microarray scanner (pre-scan: 40 μm, scan: 2 μm). The results of the measurements were analyzed, normalized and then statistically evaluated.

The results of the cells treated with EP were compared with those of the untreated control cells. The analysis was performed to identify the top 100 up- and down-regulated genes. The result is a matrix holding the gene name, the p-value, and the x-fold change. The ranking of this matrix is according to the x-fold change. In this analysis the p-value cutoff is at 5%. Furthermore, the heat map shows a two-dimensional clustering of the top one hundred genes, where intensities are represented by the colors red and green, for high and low intensities, respectively.

For further validation of individual genes, a qRT-PCR analysis (quantitative real-time polymerase chain reaction) was performed. Therefore, RNA of the samples was isolated with the miRNeasy Mini Kit (Qiagen, Germany). DNA digestion was performed to eliminate genomic DNA. The samples were treated in accordance with the Machery-Nagel protocol for rDNase digestion in solution with the rDNase set (Machery-Nagel, Germany). The samples were incubated for 10 min at 37 °C and the RNA was precipitated according to the protocol of the manufacturer with ethanol and finally taken up in nuclease-free water. Subsequently, the isolated RNA was transcribed into cDNA using the transcriptor High Fidelity cDNA Synthesis Kit (Roche, Germany). In parallel to the analyzed samples, a negative control without reverse transcriptase is additionally prepared in order to check the success of the reverse transcription. The reaction batches were incubated for 30 min at 55 °C in a thermocycler (GeneAmp PCR System 9700, Applied Biosystems, USA). Subsequently, the transcriptor High Fidelity Reverse Transcriptase was inactivated at 85 °C for a period of 5 min. Until further use the cDNA was stored at -20 °C.

The LightCycler 480 Instrument II, 96-well (Roche, Germany) and the LightCycler® 480 software version 1.5 (Roche, Germany) were used to quantify the expression levels. The samples were applied to 96-well plates (PCR 96-well TW-MT plate, white low profile, semi-skirted, DNase-, RNase-free, for LC 480 I and II (Biozym, Germany)) and quantified by LightCycler 480 SYBR Green I Master (Roche, Germany)) according to the manufacturer's instructions in a reaction volume of 20 μL. The LightCycler 480 Control Kit was used to check the successful quantification. The gene expression was validated using triplicates of all samples, whereby 1:20 dilutions of the samples were quantified. The primers required for the qRT-PCR were designed according to the usual criteria and synthesized by MWG (Eurofins, Germany). In this study, established housekeeping genes of human osteoblasts GAPDH (HKG 1) and HPRT were used.

Statistical analysis

All samples were treated and measured as triplicates and given as mean ± standard deviation. A one way analysis of variance (ANOVA) with Bonferroni correction was applied for statistical analysis with the aid of SigmaPlot®12 (Systat Software, Germany). P values < 0.05 were considered as significant and indicated by one asterisk (*). Furthermore, P < 0.01 is indicated by ** and P < 0.001 by ***. The validity and applicability of the different groups and the corresponding values for the statistical analysis were automatically demonstrated by Sigma Plot®12.