Calcium Phosphate Nanoclusters Disrupt Collagenous Structures and Alter Fibroblast Behavior

Objectives: The regulation of collagenous structure development is desirable for modulating tissue repair. Polymer-induced liquid precursors (PILP), a biomaterial which emulates key regulators of biomineralization, have shown promise in influencing collagen formation. Our group previously discovered that a high concentration formulation of PILP (HC-PILP) impairs type-I collagen remodeling in the periodontal ligament and minimizes orthodontic relapse in vivo. However, the underlying mechanism remains unclear. We hypothesize that HC-PILP disrupts collagen fibrillogenesis and fibroblast collagen production. This study aims to investigate these potential effects to advance our understanding of HC-PILP as a modulator of collagen formation and cellular behavior.
Methods: HC-PILP nanoclusters were synthesized by combining CaCl₂, Na₂PO₄, polyacrylic acid (PAA), and polyaspartic acid (PASP). A control solution containing PAA, PASP, and water (PAA/PASP) was also prepared. Collagen type-I was incubated with either HC-PILP or PAA/PASP and analyzed using a turbidity assay to monitor fibrillogenesis. To determine the effect of HC-PILP on fibroblast viability and behavior, NIH-3T3 fibroblasts grown in each condition were assayed with PrestoBlue cell viability reagent, immunofluorescence microscopy, and reverse transcription quantitative polymerase chain reaction, respectively.
Results: HC-PILP and PAA/PASP each decreased collagen fibril formation and reduced fibroblast viability compared to non-treated controls. Immunofluorescence imaging revealed diminished cell spreading in fibroblasts treated with either solution compared to non-treated fibroblasts. Furthermore, HC-PILP upregulated Col3A1 and downregulated Col1A1 gene expression in fibroblasts.
Conclusions: Here, we show that HC-PILP impairs type-1 collagen fibrillogenesis and reduces fibroblast cell spreading, underscoring its potential to modulate tissue structure and fibroblast behavior. These interferences may contribute to the observed reduction in fibroblast viability. Additionally, HC-PILP’s regulation of fibroblast gene expression suggests a shift in the Col-III/Col-I secretion ratio that may hinder normal tissue remodeling processes. Taken together, these findings enhance our understanding of HC-PILP as a biomaterial with distinct effects on collagen remodeling.