Enhancement and mechanisms of MC3T3‐E1 osteoblast‐like cell adhesion to albumin through calcium exposure

Abstract

Serum albumin is the most prominent protein in blood, and it aids in bone fracture healing, though the manner through which enhanced healing occurs is not well understood. This study investigates the influence of calcium on the bioactivity of albumin due to the prevalence of calcium at bone injury sites. Bovine serum albumin (BSA) was exposed to varying concentrations of calcium, adsorbed to tissue culture polystyrene, and the subsequent BSA-coated surfaces were evaluated with calcium titration, and cell adhesion, viability, and binding inhibition studies. Calcium-modified BSA improved overall MC3T3-E1 osteoblast-like cell adhesion, although high calcium concentrations induced cell death. Inhibiting specific integrins revealed that without calcium exposure, cell binding to BSA was primarily mediated by integrins that typically bind to the GFOGER sequence of collagen. As calcium exposure increases, the primary binding interaction transitioned to integrins known to bind RGD. However, cell binding to calciummodified BSA was not completely eliminated during the inhibition studies indicating additional unidentified binding interactions occur. Overall, these results suggest that the exposure to calcium induces conformational changes that affect the cell-binding bioactivity of BSA, which may explain the beneficial impact of albumin in bone tissue.

Delivery of bioactive albumin from multi‐functional polyampholyte hydrogels

“…35 Recently, our group investigated the impact of calcium on the bioactivity of albumin, based on the hypothesis that calcium interactions may induce a bioactive conformational state of albumin. 38 In support of this hypothesis, we found an increase in osteoblast-like cell (MC3T3-E1) adhesion to calcium modified bovine serum albumin (BSA) adsorbed on tissue culture polystyrene (TCPS) surfaces, that correlated with the calcium exposure level. These cell culture results were supported by molecular dynamics simulations, which demonstrate calcium induced unfolding of BSA.…”

“…It has been suggested that BSA provides benefits to healing when provided to injury sites, [25][26][27][28][29][30][31][32][33][34] so this work is focused on the delivery of calcium modified, bioactive BSA. 38 Polyampholyte hydrogels were selected as the delivery approach due to their demonstrated multi-functional nonfouling and protein delivery capacity, 22 which may prove beneficial for future in vivo applications. Because TMA:CAA hydrogel scaffolds have previously been extensively characterized, [20][21][22] this work is instead focused on the MC3T3-E1 cellular response to calcium modified BSA delivered from this platform.…”

“…Further, it was previously shown that high localized calcium concentrations can induce cell death. 38 Therefore, the localized calcium F I G U R E 1 Representative fluorescent microscopy images of 1 mg/ml FITC BSA adsorbed to TMA:CAA hydrogels following exposure to varying calcium concentrations. In each image, the left-hand side is a 0 M control, while the right-hand side is a calcium-exposed sample.…”

“…Two different inhibitors were selected to help identify integrin-mediated binding interactions as done previously. 38 Briefly, a GRGDSP peptide was used to inhibit RGD binding integrins. A control surface for GRGDSP peptide studies was prepared by exposing 1 ml of 1 mg/ml fibrinogen in DI water to TCPS for 30 min and then rinsing thrice with tris buffer, followed by immediate use in cell tests.…”

“…To prepare this surface, collagen type I from rat tail was diluted to 50 μg/ml with 0.02 N acetic acid and then added to well plates at 5 μg/cm 2 for 1 h. The solution was carefully aspirated and then the surface was rinsed thrice with PBS and immediately used in cell tests. Cells were prepared for the inhibition studies identically as described above, except prior to cell seeding the cells were incubated for 15 min in serum free α-MEM containing 10 μM BTT 3033 38 or 0.1 mM GRGDSP peptide. 40,41 Due to solubility constraints, the BTT 3033 was initially dissolved in DMSO at 1 mM, followed by dilution with α-MEM/cell solution to give an overall 10 μM BTT 3033 seeding concentration.…”