IADR Abstract Archives
Bioinspired manipulation of environmental pH to induce rapid in vitro bone-like mineralization
Objectives: The objectives of this study were to investigate the environmental factors that primes initial mineralization in vivo, and then apply the obtained knowledge for rapid in vitro synthesis of bone-like tissue.
Methods: New-born Balb/c mice from post-natal day 2 (P2) to P7 were used in the experiments according to the Guidelines for Animal Research of Okayama University. Muscles and epiphyses at P3 and P6 were harvested and immediately placed onto a fluorescence pH sensor sheet (SF-HP5R VisiSens, PreSens Precision Sensing GmbH, Regensburg, Germany), which has a precision of ± 0.01 pH at pH = 7, according to the manufacturer’s protocol. Cell membrane nanofragments were collected from cultured cell lines. pH was titrated with NaOH. Mineralization was determined by Alizarin Red staining. For in vitro mineral precipitation assay, PBS solutions were titrated with HCl or NaOH to obtain solutions with different pHs (6.5, 7.5, 8.5 and 9.5), and CaCl2 solutions (0.5 mM, 1 mM and 2 mM) were prepared, and slowly added into PBS solutions of different pHs, at room temperature, without pH control.
Results: First, direct and robust measurement of pH showed that femur epiphysis is alkaline (pH=8.5) at the initial mineral stage at post-natal day 6. We showed that the alkaline milieu is decisive not only for alkaline phosphatase activity, which precedes mineral formation at P6, but also for determining initial mineral precipitation and its spherical morphology. Next, engineering approaches were used to synthesize bone-like tissue based on alkaline condition and chondrocyte membrane nanofragments, previously shown to be nucleation site for mineral formation (Hara et al., ACS Biomater Sci Eng, 2018). Interestingly, mineralization using artificial cell membrane nanofragments in an alkaline condition was achieved within just 1 day.
Conclusions: These findings demonstrate that alkaline milieu can be used as an important factor for enhancing methods for in vitro synthesis of bone tissue.
Methods: New-born Balb/c mice from post-natal day 2 (P2) to P7 were used in the experiments according to the Guidelines for Animal Research of Okayama University. Muscles and epiphyses at P3 and P6 were harvested and immediately placed onto a fluorescence pH sensor sheet (SF-HP5R VisiSens, PreSens Precision Sensing GmbH, Regensburg, Germany), which has a precision of ± 0.01 pH at pH = 7, according to the manufacturer’s protocol. Cell membrane nanofragments were collected from cultured cell lines. pH was titrated with NaOH. Mineralization was determined by Alizarin Red staining. For in vitro mineral precipitation assay, PBS solutions were titrated with HCl or NaOH to obtain solutions with different pHs (6.5, 7.5, 8.5 and 9.5), and CaCl2 solutions (0.5 mM, 1 mM and 2 mM) were prepared, and slowly added into PBS solutions of different pHs, at room temperature, without pH control.
Results: First, direct and robust measurement of pH showed that femur epiphysis is alkaline (pH=8.5) at the initial mineral stage at post-natal day 6. We showed that the alkaline milieu is decisive not only for alkaline phosphatase activity, which precedes mineral formation at P6, but also for determining initial mineral precipitation and its spherical morphology. Next, engineering approaches were used to synthesize bone-like tissue based on alkaline condition and chondrocyte membrane nanofragments, previously shown to be nucleation site for mineral formation (Hara et al., ACS Biomater Sci Eng, 2018). Interestingly, mineralization using artificial cell membrane nanofragments in an alkaline condition was achieved within just 1 day.
Conclusions: These findings demonstrate that alkaline milieu can be used as an important factor for enhancing methods for in vitro synthesis of bone tissue.