Vascular biosafety of commercial hydroxyapatite particles: discrepancy between blood compatibility assays and endothelial cell behavior

Abstract

BackgroundVascular homeostasis is ensured by a dynamic interplay involving the endothelium, the platelets and the coagulation system. Thus, the vascular safety of particulate materials must address this integrated system, an approach that has been largely neglected. This work analysed the effects of commercial hydroxyapatite (HA) particles in blood compatibility and in endothelial cell behavior, due to their clinical relevance and scarcity of data on their vascular biosafety.ResultsParticles with similar chemical composition and distinct size and morphology were tested, i.e. rod-like, nano dimensions and low aspect ratio (HAp1) and needle-shape with wider size and aspect ratio (HAp2). HAp1 and HAp2, at 1 to 10 mg/mL, did not affect haemolysis, platelet adhesion, aggregation and activation, or the coagulation system (intrinsic and extrinsic pathways), although HAp2 exhibited a slight thrombogenic potential at 10 mg/mL. Notwithstanding, significantly lower levels presented dose-dependent toxicity on endothelial cells’ behavior. HAp1 and HAp2 decreased cell viability at levels ≥ 250 and ≥ 50 μg/mL, respectively. At 10 and 50 μg/mL, HAp1 did not interfere with the F-actin cytoskeleton, apoptotic index, cell cycle progression, expression of vWF, VECad and CD31, and the ability to form a network of tubular-like structures. Comparatively, HAp2 caused dose-dependent toxic effects in these parameters in the same concentration range.ConclusionThe most relevant observation is the great discrepancy of HA particles’ levels that interfere with the routine blood compatibility assays and the endothelial cell behavior. Further, this difference was also found to be dependent on the particles’ size, morphology and aspect ratio, emphasizing the need of a complementary biological characterization, taking into consideration the endothelial cells’ functionality, to establish the vascular safety of particulate HA.

<p>Comparative study of in vitro effects of different nanoparticles at non-cytotoxic concentration on the adherens junction of human vascular endothelial cells</p>

“…For example, Ag NPs induce excess ROS production of HUVECs by activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity through upregulation of NADPH oxidase 4 (NOX4) gene,43 increase the permeability of rat brain microvascular endothelial cells by inhibiting the antioxidant defence of the astrocytes that inhibits the Trx system, and decreasing Nr4a1 and Dusp1 gene 45. Hydroxyapatite NPs down-regulated VE-cadherin and CD31 gene of ECs 44…”

The comprehensive in vitro evaluation of eight different calcium phosphates: Significant parameters for cell behavior

“…Conversely, the cytoplasmic Ca and P concentrations increased in both nanoparticle groups at 24 hours. Commercially available needle‐shaped HA (Plasma Biotal Ltd) inhibited the proliferation/viability of endothelial cells at a concentration of 50 mg/mL and higher, and the rod‐like particles (nanoXIM•HAp102R) at a concentration of 250 mg/mL. The needle‐like particles exhibited slight thrombogenic potential at 10 mg/mL.…”

“…The needle‐like particles exhibited slight thrombogenic potential at 10 mg/mL. Moreover, these particles exerted an adverse impact on the F‐actin cytoskeleton, the apoptotic index, cell cycle progression, the expression of vWF, VECad, and CD31, and the ability to form a network of tubular‐like structures in a dose‐dependent manner …”

Calcium phosphate bions: towards a pathogenetic concept

“…При этом известно, что форма наночастиц гидроксиапатита является одним из основных факторов, определяющих их токсичность; в частности, наночастицы игольчатой формы значительно токсичнее сферических [70][71][72], причем данная зависимость также характерна и для других типов наночастиц, не являясь специфичной для гидроксиапатита или форм фосфата кальция в целом [73]. Токсичность кристаллов гидроксиапатита определяется в том числе другими физико-химическими параметрами, такими как размерность [69,70,74], степень кристалличности [75], площадь поверхности [76], соотношение продольного и поперечного диаметров [76], а также наличие или отсутствие белковой оболочки [59]. Интересно, что значение для цитотоксичности КФБ имеют даже на первый взгляд незначительные различия их формы; так, игольчатые КФБ токсичнее палочковидных [76].…”

“…Токсичность кристаллов гидроксиапатита определяется в том числе другими физико-химическими параметрами, такими как размерность [69,70,74], степень кристалличности [75], площадь поверхности [76], соотношение продольного и поперечного диаметров [76], а также наличие или отсутствие белковой оболочки [59]. Интересно, что значение для цитотоксичности КФБ имеют даже на первый взгляд незначительные различия их формы; так, игольчатые КФБ токсичнее палочковидных [76].…”