Platelet Lysate: The Better Choice for Jaw Periosteal Cell Mineralization

Abstract

Previously, we demonstrated a high quality of minerals formed by serum-free cultured jaw periosteal cells (JPCs) by Raman spectroscopy but the mineralization extent was not satisfactory. In the present study, we analyzed the proliferation and mineralization potential of human platelet lysate- (hPL-) cultured JPCs in comparison to that of FCS-cultured JPCs. By cell impedance measurements, we detected significantly higher population doubling times of PL-cultured JPCs in comparison to FCS-cultured JPCs. However, this result was not based on lower proliferation activities but on diminished cell sizes which JPCs develop under PL cultivation. The measurements of the metabolic activities clearly showed significantly higher cell proliferation rates under PL culturing. Equivalent levels of the mesenchymal cell markers CD29, CD45, CD73, CD90, and CD105 were detected, but there were significantly increased MSCA-1 levels under PL cultivation. While JPCs only occasionally mineralize under FCS culture conditions, the mineralization potential was significantly stronger under PL cultivation. Moreover, in 4 of 5 analyzed patient cells, the addition of dexamethasone was proved no longer necessary for strong mineralization of PL-cultured JPCs. We conclude that in vitro cultivation of JPCs with platelet lysate is a suitable alternative to FCS culture conditions and a powerful tool for the development of high-quality TE constructs using jaw periosteal cells.

Figure 1

JPC population doubling times using the live-monitoring system xCELLigence. JPCs from 4 donors (numbers 1–4) were cultivated under FCS and human PL supplementation under normal (Co) and osteogenic (Ob) conditions for 5, 10, and 14 days in E-plates for electrical impedance measurements. Population doubling times (in hours, logarithmic scaling of the x-axis) were calculated by the device-specific RTCA software. The bars indicate statistical significances: p < 0.05.

Figure 2

Measurements of metabolic JPC activities under FCS- and PL-containing culture conditions. JPCs from 4 donors (numbers 1–4) were cultivated under FCS and human PL supplementation under normal (Co) and osteogenic (Ob) conditions for 5, 10, and 20 days in 96-well plates for colorimetric measurements of metabolic activities. Optical densities (OD) are presented in the diagrams. The bars indicate statistical significances: p < 0.05.

Figure 3

Representative microscopic images of FCS- and PL-cultured JPCs. JPCs were cultured under FCS or PL supplementation for 5 days and cell monolayers were visualized by microscopic images (scale bar: 200 μm). Subsequently, cells were counted after trypsinization. Days 7 and 10 of in vitro culturing were also taken into account and growth rates and population doubling times were calculated, as listed in Tables 1 and 2.

Figure 4

Measurements of cell size under FCS- and PL-containing culture conditions. Full length cell size measurements were performed using the CellProfiler and the LAS EZ software from 3 JPC donors (n = 10 measurements per donor, n = 30 for each culture condition). Cells were cultivated under FCS and human PL supplementation under normal (Co) and osteogenic (Ob) conditions for 5 days before performing the measurements. Statistical significances are indicated by asterisks: ^∗p < 0.001.

Figure 5

Flow cytometric analyses of MSC surface markers by JPCs cultivated under FCS and PL supplementation. Cell surface expression of CD29, CD45, CD73, CD90, CD105, and MSCA-1 was analyzed in JPCs (from 4 donors) cultivated under FCS and human PL supplementation under normal (Co) and osteogenic (Ob) conditions for 5 and 10 days by flow cytometry. Significantly higher levels (^∗p < 0.05) of MSCA-1 were detected under Co conditions after day 10 and under osteogenic conditions after day 5 and 10 of hPL cultivation.

Figure 6

Detection of JPC mineralization cultured under FCS- and PL-containing culture conditions. Representative microscopic images (scale bar: 200 μm) of alizarin red B stainings of PL- and FCS-cultured JPC monolayers (from 4 donors) after 24 days of osteogenic induction. Bright red-stained areas contain calcium phosphate precipitates.

Figure 7

Calcium quantification of PL- and FCS-cultured JPCs. Alizarin dye was dissolved in acetic acid and colorimetric quantification (calcium concentration (μM), logarithmic scale) was performed. Untreated and osteogenic induced JPCs under PL and FCS supplementation were considered. Statistical significances are indicated by asterisks: ^∗∗∗p < 0.0001.