Characterization of cytolytic neutrophil activation in vitro by amorphous hydrated calcium phosphate as a model of biomaterial inflammation.
Calcium ions are utilized in biomolecular biomaterial design for osteomimetic scaffolds and as divalent cross-linking agents, typically for gelation of alginates, stabilisation of protein structure (e.g., fibrinogen) and enzyme activation (e.g., thrombin). Biological interactions with defined calcium phosphates (e.g., hydroxyapatite) are exploited for osteogenesis, although crystalline calcium phosphates (e.g., calcium pyrophosphate) stimulate inflammation. We found that the calcium concentration used in the manufacture of prototype dermal scaffolds made from fibrin/alginate composite was related to the inflammatory infiltration during in vivo integration. In investigating a cause for this inflammatory response, we have identified and characterized a cytolytic inflammatory effect of amorphous calcium phosphate (CaP) formed in physiological solutions, relevant to biomaterial biocompatibility. Isolated human neutrophils (Nφ) were incubated in phosphate-buffered saline with CaCl(2) ranging 2.5-20 mM total calcium. Nφ activation was assessed by morphology and integrin-β2 (CD18a) expression. Mediator release (Nφ-elastase, IL-8, and TNFα) was measured from both Nφ and whole blood cultures plus CaCl(2). CaP exposure increased CD18a expression over 1 h (maximal at 10 mM calcium/ phosphate) with concurrent phagocytosis, cytolysis, and Nφ-elastase release. CaCl(2) induced expression of IL-8 and TNFα in whole blood cultures. These results suggest that CaP formed from the resorption of calcium-containing biomaterials could induce inflammation and accelerate biomaterial degradation, driving further CaP release. This demonstrates a novel mechanism for biomaterial-induced inflammation. The in vitro system described could aid preclinical evaluation of novel biomaterial inflammatory potential.
RAFT Institute of Plastic Surgery Research, Mount Vernon Hospital, Middlesex, United Kingdom.
Felicity C
FC
Edwards
Amir
A
Taheri
Sophie C
SC
Dann
Julian F
JF
Dye
D000464:Alginates / Q000494:pharmacology; D001672:Biocompatible Materials / Q000009:adverse effects*; D002021:Buffers; D018821:CD18 Antigens / Q000378:metabolism; D002118:Calcium / Q000494:pharmacology; D002130:Calcium Phosphates / Q000494:pharmacology*; D003602:Cytotoxicity, Immunologic / Q000187:drug effects*; D020405:Dermis / Q000187:drug effects; D005337:Fibrin / Q000494:pharmacology; D020723:Glucuronic Acid / Q000494:pharmacology; D006603:Hexuronic Acids / Q000494:pharmacology; D006801:Humans; D007249:Inflammation / Q000473:pathology*; D018836:Inflammation Mediators / Q000378:metabolism; D019272:Leukocyte Elastase / Q000378:metabolism; D008274:Magnesium / Q000494:pharmacology; D008954:Models, Biological; D018375:Neutrophil Activation / Q000187:drug effects*; D009504:Neutrophils / Q000166:cytology / Q000187:drug effects / Q000276:immunology / Q000648:ultrastructure; D010587:Phagocytosis / Q000187:drug effects; D010710:Phosphates / Q000494:pharmacology; D013052:Spectrometry, X-Ray Emission; D013997:Time Factors; D054457:Tissue Scaffolds / Q000737:chemistry
D000464:Alginates; D001672:Biocompatible Materials; D002021:Buffers; D018821:CD18 Antigens; D002130:Calcium Phosphates; D006603:Hexuronic Acids; D018836:Inflammation Mediators; D010710:Phosphates; C519480:amorphous calcium phosphate; D020723:Glucuronic Acid; D005337:Fibrin; D019272:Leukocyte Elastase; D008274:Magnesium; D002118:Calcium