AbstractBackgroundCalcium hydroxylapatite (CaHA; Radiesse, Merz North America) restores volume and stimulates collagen production. The aim of this research was to explore the role of dilution and diffusion in microsphere distribution and the effect of CaHA concentration on activation of fibroblasts to produce collagen.MethodsEx vivo: Tissue dispersion of CaHA was assessed in abdominal tissue segments obtained from patients which were subsequently injected with CaHA diluted to 1:1 and hyperdiluted to 1:2. In vitro: Collagen type III (COLIII) and type I (COLI) expression of fibroblasts was evaluated after 24 and 72 h of incubation with CaHA concentrations of 1.5 (high dilution), 3.0, and 4.5 mg/ml (low dilution).ResultsEx vivo: The 1:2 CaHA hyperdilution increased dispersion and decreased concentration of CaHA microspheres compared with the 1:1 dilution. In vitro: CaHA incubation resulted in an increased mean COLIII expression of 123% at 24 h. COLI synthesis did not change after 24 h but increased up to 124% at 72 h. Only fibroblasts in direct contact with CaHA increased COLIII expression. COLIII high‐expressing cells were fully activated by CaHA and resulted in the same level of COLIII expression per cell independent of the CaHA dilution.ConclusionsA 1:2 hyperdilution of CaHA increased tissue dispersion of CaHA microspheres. Direct contact of CaHA with fibroblasts was a key factor for inducing neocollagenesis. COLIII high‐expressing cells were fully activated by CaHA and resulted in the same expression level of COLIII per cell independent of the CaHA amount in each dilution. This indicates that increased collagen expression was due to the activation of more fibroblasts.