Probiotic bacteria cell surface-associated protein mineralized hydroxyapatite incorporated in porous scaffold: In vitro evaluation for bone cell growth and differentiation

Highlights: • L. rhamnosus GG cell surface-associated protein (CSP) was mineralized to yield hydroxyapatite nanoparticle (HANP). • L. rhamnosus GG also rendered mineralization at the whole-cell (WC) level to support HANP synthesis. • WC-HANPs recovered by cell lysis were needle-shaped, with a particle size distribution of 70-110 nm. • Chitosan-gelatin scaffold incorporated with HANP (H-CG) rendered proliferation and differentiation of seeded MG-63 cells. There is a high demand for synthesis of biocompatible hydroxyapatite nanoparticle (HANP), which is a key component in bone tissue engineering scaffolds. The present study describes a facile route of HANP synthesis through mineralization of the cell surface-associated protein (CSP) from the human probiotic lactic acid bacteria (LAB) Lactobacillus rhamnosus GG. CSP extract from the LAB (consisting of ~66 kDa, ~47 kDa, ~40 kDa and ~25 kDa protein) was mineralized to yield spindle-shaped HANPs having an average particle length of 371 nm as evidenced in FETEM analysis. CSP-mineralized HANPs (CSP-HANPs) were characterized by FTIR and BET analysis, while XRD and SAED analysis indicated their crystalline nature. Mechanistic studies suggested the key role of ~25 kDa CSP (F4SP) in mineralization. In contrast to CSP-HANPs, F4SP-mineralized crystalline HA was plate-shaped having an average length of 1.68 μm and breadth of 0.95 μm. HANP mineralization at the whole-cell (WC) level resulted in clusters of aggregated HANPs (WC-HANPs) adhering onto L. rhamnosus GG cells as evident in FETEM, FESEM and AFM analysis. FETEM analysis revealed that the desorbed WC-HANPs recovered by cell lysis were needle-shaped, with a particle size distribution of 70–110 nm. Given that CSP-HANPs were non-toxic to cultured HEK 293 cells and osteoblast-like MG-63 cells, chitosan-gelatin (CG) scaffold incorporated with 15% w/v CSP-HANP (H-CG) was generated and tested for bone cell growth. H-CG exhibited a favorable pore size distribution (160–230 μm), overall porosity (~84%) and biodegradation profile. H-CG scaffold was conducive to osteogenesis and rendered enhanced proliferation, alkaline phosphatase (ALP) activity, calcium mineralization and heightened marker gene expression (ALP, Col I, Runx2 and OCN) in seeded MG-63 cells. CSP sourced from a safe probiotic LAB is thus a viable and effective mineralization template for synthesis of biocompatible HANPs that can be leveraged for bone tissue engineering applications.