Comparative in vitro Study on Immunomodulatory Effects of Nano-Hydroxpatite Powders on Human Immune Cells

Introduction: Significant advancement was recently achieved in the field of hydroxyapatite nanoparticles, contributing to their implementation in numerous biomedical applications, including bone tissue reconstruction. Nevertheless, the influence of these materials on the immune system still remains an unresolved issue. Materials: Here, we managed to reveal a direct in vitro impact of nano-hydroxyapatite particles on immunological, cellular and humoral components. Further, we analyzed the effect of nanoparticle size and shape on healthy human immune cells residing in the peripheral blood. Special attention was given to particles produced using the microwave hydrothermal synthesis method and precisely controlled size. Viability, phenotypes, activation status, cytokine production and release were assessed using flow cytometry and immunoenzymatic techniques. Results: We showed that large-sized nanoparticles caused significant induction of immune response associated with innate and acquired immunity, in size-dependent manner. The synthesized nanoparticles allowed for avoidance of the immunogenicity, with no changes in inflammatory cytokines, supporting high biocompatibility. Various effects suggest a different potential use of examined nanoparticles, whether proinflammatory or neutral conditions are required in the field of anticancer therapy or transplantology respectively. Discussion: Obtained results indicate that precise selection of nano-hydroxyapatites with specific immunomodulatory properties might be crucial for application in the clinical setting. Subsequent studies would establish most suitable therapeutic approaches where selected nano-hydroxyapatites could be implemented. Plain Language Summary: Nanoparticles are tiny materials with unique properties used in many medical applications, such as bone repair or cancer treatment. One type, called nano-hydroxyapatite (nHAP), is especially promising due to its similarity to human bone. However, we still do not fully understand how these particles affect the immune system, our natural defense network. In this study, we tested several types of nHAPs to see how they interact with healthy human immune cells. We found that some particles, especially larger ones, can harm immune cells at high concentrations. In contrast, smaller particles had no harmful effects and seemed to be well tolerated. We also discovered that certain nHAPs changed how immune cells behave. For example, Sigma particles caused monocytes, cells that participate in both inflammatory and non-inflammatory immune response, to shift toward a more inflammatory state. They also increased activity in helper T cells (acquired immunity cells) and led to the production of both protective and inflammatory signaling proteins. These findings suggest that not all nHAPs are equal. Choosing the right type and size of particle is essential depending on the intended medical use. Some nHAPs may help fight cancer by boosting the immune response, while others could be better suited for treatments requiring low immune activation, such as wound healing or organ transplants. Our research highlights the need for careful design of nanoparticle-based therapies to ensure safety and effectiveness in future clinical applications. The immunomodulatory effects of various nano-hydroxyapatite (nHAP) materials, including commercial HAP Sigma, HAP Berkley, and synthesized GoHAP types, on immune cells were examined. HAP Sigma and HAP Berkley affected cell viability, with particle size influencing apoptosis in monocytes and lymphocytes. GoHAP Type 3, which had no adverse effects on viability, showed improved biocompatibility. Phagocytic responses to the tested nanoparticles were observed in monocytes and were influenced by size and thermogravimetric properties. Exposure to nHAPs led to a shift from intermediate to non-classical monocytes. HAP Sigma significantly induced CD4+ T cell proliferation and activation, as indicated by CD69 expression. All tested nHAPs influenced cytokine secretion. HAP Sigma affected both pro- and anti-inflammatory cytokines, highlighting its potential for immune modulation.Effects of nano-hydroxyapatite materials (HAP Sigma, HAP Berkley, and GoHAP) on lymphocyte and monocyte activity, cell viability, phagocytosis, and cytokine secretion. Keywords: nanoparticles, hydroxyapatite, immunomodulation, monocytes, lymphocytes