Whole blood and vessel interactions to biomaterials; leukocyte filtration and angiogenesis stimulation effect of calcium phosphate biomaterials

Background: When a material is implanted in the body, invariably, the first fluid it comes into contact with is blood. Although leukocytes are a small cell population in blood, they are the most crucial blood components to protect the body against foreign substances and infectious factors. Little is known about their interaction with biomaterials beyond initial attachment following activation. Similarly, while there are thousands of reports examining biomaterials and endothelial cells, there are barely 100 investigating the interaction of the tunica adventitia of blood vessels with biomaterials, even though it is more probable that an implanted material will contact the exterior of the blood vessel system than its interior lumen. In this study, the interaction of blood and blood vessels with calcium phosphate biomaterial, as the most common material used in bone repair and tissue engineering, was investigated. This project's main focus was on the leuko-reduction effect of hydroxyapatite biomaterial, and the attachment and survival of immune cells on this material, besides investigating vein tissue cell attachment and proliferation on cylindrical calcium phosphate biomaterial.Methods: Blood samples and vein tissues for this study were harvested from healthy male rats. Hydroxyapatite (HAp) interaction with leukocytes in whole blood and its leukoreduction effect were tested after incubating two types of hydroxyapatite (HAp-1 and HAp-2) and cotton with blood samples for 1h, 2h, and 4h. Leukocyte manual counting, differential counting using Giemsa staining, and MTT assay were accomplished to examine the leukocyte number in whole blood after incubation with HAp materials. Live and Dead cell assay and cell detachment were performed to determine cell viability and identify cells attached to the biomaterials. Blood vessel interaction with calcium-phosphate biomaterial was tested by detecting cells growing on the biomaterial using Live and Dead assay and Scanning Electron Microscope (SEM) imaging.Results: The number of leukocytes in whole blood after incubation with HAp-1 reduced significantly after 4h by increasing the biomaterial mass (p<0.05). Approximately 30% of leukocytes in whole blood attached to HAp-1, which in comparison to HAp-2 showed more leukoreduction effect. As the most common material in leukocyte filters, cotton showed more than 50% of leukoreduction effect from the first hour of incubation. According to the Live and Dead assay, more cells were attached to the biomaterial ( HAp-1, HAp-2, cotton, and commercial filter) after 4h rather than 1h. Lymphocytes and monocytes could be identified after cell detachment on hydroxyapatite biomaterial.Conclusion:According to obtained data, hydroxyapatite materials' leukoreduction effect in whole blood is mass-dependent and time-dependent. Leukocytes can stay attached and survive on HAp biomaterials for 5 days, and their identification showed that lymphocytes and monocytes attached more. Besides these, vein tissue can well-attached to cylindrical microporous calcium phosphate biomaterial, and according to SEM and fluorescence imaging, cells were growing through the biomaterial, and extracellular matrix was penetrating the pores.In conclusion, calcium phosphate biomaterial showed good affinity to leukocytes; furthermore, blood cells and vein tissue cells can be well-attached and survive, proliferate and differentiate