Quality Analysis of Minerals Formed by Jaw Periosteal Cells under Different Culture Conditions

Abstract

Previously, we detected a higher degree of mineralization in fetal calf serum (FCS) compared to serum-free cultured jaw periosteum derived osteoprogenitor cells (JPCs). By Raman spectroscopy, we detected an earlier formation of mineralized extracellular matrix (ECM) of higher quality under serum-free media conditions. However, mineralization potential remained too low. In the present study, we aimed to investigate the biochemical composition and subsequent biomechanical properties of the JPC-formed ECM and minerals under human platelet lysate (hPL) and FCS supplementation. JPCs were isolated (n = 4 donors) and expanded under FCS conditions and used in passage five for osteogenic induction under both, FCS and hPL media supplementation. Raman spectroscopy and Alizarin Red/von Kossa staining were employed for biochemical composition analyses and for visualization and quantification of mineralization. Osteocalcin gene expression was analyzed by quantitative PCR. Biomechanical properties were assessed by using atomic force microscopy (AFM). Raman spectroscopic measurements showed significantly higher (p < 0.001) phosphate to protein ratios and in the tendency, lower carbonate to phosphate ratios in osteogenically induced JPCs under hPL in comparison to FCS culturing. Furthermore, higher crystal sizes were detected under hPL culturing of the cells. With respect to the ECM, significantly higher ratios of the precursor protein proline to hydroxyproline were detected in hPL-cultured JPC monolayers (p < 0.001). Additionally, significantly higher levels (p < 0.001) of collagen cross-linking were calculated, indicating a higher degree of collagen maturation in hPL-cultured JPCs. By atomic force microscopy, a significant increase in ECM stiffness (p < 0.001) of FCS cultured JPC monolayers was observed. The reverse effect was measured for the JPC formed precipitates/minerals. Under hPL supplementation, JPCs formed minerals of significantly higher stiffness (p < 0.001) when compared to the FCS setting. This study demonstrates that hPL culturing of JPCs leads to the formation of an anorganic material of superior quality in terms of biochemical composition and mechanical properties.

Keywords: Raman spectroscopy; atomic force microscopy; bone mineral formation; fetal calf serum; human platelet lysate; mechanical properties; osteoprogenitor jaw periosteal cells.

Figure 1

Different consistency and macroscopic appearance of osteogenically induced JPC monolayers following FCS and hPL medium supplementation (Alizarin Red staining). Representative images of cell monolayers from two donors (#1, #2) cultured in AFM petri dishes (plastic bottom) under both supplementations. Abbreviations: FCS—fetal calf serum, hPL—human platelet lysate.

Figure 2

Detection of JPCs mineralization (Alizarin Red staining) in AFM and Raman petri dishes under FCS and hPL supplementation. Macroscopic images: JPC mineralization from three donors (#1, #2, #3) cultured in AFM (plastic bottom) and Raman (glass bottom) petri dishes was observed after osteogenic induction for 25 days under both medium supplementations (A). Quantification of JPC mineralization degrees in cell monolayers from the three donors (B). Calcium levels detected in cell monolayers cultured in AFM dishes are represented by red columns and those in Raman petri dishes by blue columns. Abbreviations: AFM—atomic force microscopy, FCS—fetal calf serum, hPL—human platelet lysate, * p < 0.05.

Figure 3

Detection of JPCs mineralization by von Kossa staining in Raman dishes under FCS and hPL supplementation. Microscopic images showing JPC mineralization from three donors (#1, #2, #3) cultured in Raman (glass bottom) dishes were observed after osteogenic induction for 25 days under both medium supplementations. Abbreviations: FCS—fetal calf serum, hPL—human platelet lysate.

Figure 4

Relative gene expression levels of human osteocalcin transcript levels by quantitative PCR. JPCs from four donors were cultured in 75 cm² culture flasks under normal (Co) and osteogenic (Ob) conditions for five and 10 days under both medium supplementations, following RNA isolation for osteocalcin gene quantification. Gene expression in untreated JPCs were set as 1 and induction indices in relation to untreated controls were calculated. Abbreviations: FCS—fetal calf serum, hPL—human platelet lysate.

Figure 5

Mean Raman spectra from hPL- and FCS-supplemented JPCs. Mean Raman spectra from four donors (#1, #2, #3, #4) generated from JPCs growing in Raman glass dishes under hPL (black spectra) and FCS (red spectra) supplementation. Abbreviations: FCS—fetal calf serum, hPL—human platelet lysate.

Figure 6

Ratios calculated from mean Raman spectra reflecting the biochemical composition of osteogenically-induced (OB) JPCs (four donors) under hPL and FCS supplementation. Phosphate to protein ratios (A) HA to phenylalanine; (B) HA to amid I; (C) HA to amid III; (D) carbonate content; (E) crystal size; (F) proline to hydroxyproline; (G) collagen cross-linking. Means (126–162 measurements per donor) ± standard deviations are depicted. * p < 0.05; *** p < 0.001. Abbreviations: FCS—fetal calf serum, hPL—human platelet lysate.

Figure 7

Analysis of Young’s modulus of JPC monolayers cultured under FCS and hPL supplementation. Boxplots (medians, minimum, maximum) of the stiffness (kPa) measured by atomic force microscopy for each analyzed group are depicted. In both, control (untreated) and osteogenic monolayers of FCS cultured monolayers analyzed ECM revealed higher stiffness when compared to hPL groups. The reversed effect was observed for the group of precipitates. *** p < 0.001 (for exact p-values, see Table 3). Abbreviations: FCS—fetal calf serum, hPL—human platelet lysate.

Figure 8

Representative microscopic images indicating the regions of interest subjected to elasticity measurements. Microscopic pictures of AFM measured JPCs—control monolayers (left pictures), osteogenic induced JPCs (middle) and calcium phosphate precipitates (right pictures) formed under hPL and FCS culturing conditions, respectively. Red arrows depict the position of the cantilever for elasticity measurements of non-mineralized regions in untreated (left) and osteogenic monolayers (middle, without precipitates) and of calcium phosphate precipitates. Images were acquired with the inverted AxioObserver D1 light microscope attached to the AFM system at a 10× magnification. Scale bar (black) represents 30 µm. Abbreviations: AFM—atomic force microscopy, JPCs—jaw periosteum derived progenitor cells, FCS—fetal calf serum, hPL—human platelet lysate.