Abstract
The bone-biomaterial interface has been characterized by layers of afibrillar extracellular matrix (ECM) enriched in non collagenous proteins, including osteopontin (OPN), a multifunctional protein that in bone controls cell adhesion and ECM mineralization. Physical and chemical aspects of biomaterial surfaces have been demonstrated to affect cell-ECM-substrate interactions. The present paper described the ability of oxidative nanopatterning of titanium (Ti) surfaces to control extracellular OPN deposition in vitro. Ti discs were chemically treated by a mixture of H2SO4/H2O2 for either 30 min [Nano(30') Ti] or 4 h [Nano(4h) Ti]. Non-etched Ti discs were used as control. Primary osteogenic cells derived from newborn rat calvarial bone were plated on control and etched Ti and grown under osteogenic conditions up to 7 days. High resolution scanning electron microscopy revealed that treated Ti discs exhibited a nanoporous surface and that areas of larger nanopits were noticed only for Nano(4h) Ti. Large extracellular OPN accumulation were detectable only for Nano(4h) Ti, which was associated with OPN-positive cells with typical aspects of migrating cells. At day 3, quantitative results in terms of areas of OPN labeling were as follows: Nano(4h) Ti > Nano(30') Ti > Control Ti. In conclusion, chemically nanostructured Ti surfaces may support the enhancement of endogenous extracellular OPN deposition by osteogenic cells in vitro depending on the etching time, a finding that should be taken into consideration in strategies to biofunctionalize implant surfaces with molecules with cell adhesion capacity.
Highlights
Osteopontin (OPN) is a multifunctional, matricellular protein of the SIBLING family, which is expressed by many tissues and cell types, either under physiological or pathological conditions
Considering that changes in nanoscale surface features may have an impact in protein adsorption [7,8,18] and that chemically produced nanotopography can be tuned by altering the relative proportions of acid/base and oxidant, temperature and time of etching [19], the present paper aimed to evaluate the effects of two different Ti surface nanotopographies on the early expression of OPN by osteogenic cells in vitro
Pure, grade 2 Ti discs, 13 mm in diameter and 2 mm thick, were polished with silicon carbide abrasive papers (320 and 600 grit), cleaned by sonication, rinsed with toluene, and treated with a mixture consisting of equal volumes of 10 N H2SO4 and 30% aqueous H2O2 for either 30 min or 4 h [hereafter referred to as Nano(30’) Ti and Nano(4h) Ti, respectively] at room temperature (RT) under continuous agitation
Summary
Osteopontin (OPN) is a multifunctional, matricellular protein of the SIBLING (small integrinbinding ligand, N-linked glycoprotein) family, which is expressed by many tissues and cell types, either under physiological or pathological conditions (reviewed in 1-3). OPN is expressed by osteoblastic cells and osteoclasts and have been associated with the control of extracellular matrix (ECM) mineralization/ mineral crystal growth, osteoblastic cell adhesion and proliferation, and osteoclast function [4,5]. The unique conserved regions of the OPN molecule, i.e. RGD and non-RGD domains, thrombin cleavage site, polyaspartic acid sequence, and serine/threonine phosphorylation sites, can account for the diverse functions attributed to this protein (reviewed in 3). Ultrastructural immunolabeling has revealed that in addition to be distributed throughout bone mineralized matrix, OPN preferentially accumulates at bone interfaces, including cement lines, laminae limitantes, margins of surgically created bony defects, and at bone-biomaterial interface [6].
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