Nanostructured and Functionalized Calcium Hydroxyapatite Enhances Collagen Production by Human Dermal Fibroblasts Compared With Microstructured Counterparts In Vitro.

Calcium hydroxyapatite (CaHA) is a well-established biocompatible material widely used to stimulate collagen formation in soft tissues; however, its clinical use is largely restricted to invasive deep dermal injection due to safety concerns associated with microstructured formulations, such as superficial nodule formation and vascular occlusion. Here, we report the development of a nanostructured, lactose-functionalized CaHA produced by nanospray drying, enabling safer and more versatile delivery strategies. Physicochemical properties were systematically characterized, and biological performance was evaluated in human dermal fibroblasts and co-culture with peripheral blood mononuclear cells (PBMCs). The resulting nanostructured CaHA (nanoCaHA) exhibited a submicron particle size (181.06 ± 23.83 nm), narrow polydispersity (PDI 0.23 ± 0.17), and a positive surface charge (+11.75 ± 3.63 mV), attributed to surface protonation during processing. NanoCaHA showed no cytotoxic, genotoxic, or irritant effects in vitro and induced a shift in fibroblast cell-cycle distribution consistent with enhanced proliferative activity. While both nano- and microCaHA increased total collagen production, nanoCaHA significantly upregulated COL1A1 and COL3A1 mRNA expression, particularly under PBMC co-culture conditions, outperforming a commercially available CaHA formulation. Moreover, nanoCaHA sustained collagen production for up to 5 days after treatment withdrawal. Scanning electron microscopy revealed persistent particulate structures closely associated with fibroblasts after washout, while energy-dispersive X-ray spectroscopy confirmed that these deposits were enriched in calcium and phosphorus, consistent with CaHA composition. Furthermore, skin permeation assays using porcine skin in Saarbrücken diffusion cells showed that the nanoCaHA formulation significantly increased calcium delivery into the skin (34.53 ± 6.64 μg/cm2) compared to endogenous control levels (23.43 ± 3.96 μg/cm2). Collectively, these findings demonstrate that nanoscale engineering of CaHA modulates cell-material interactions, supporting the development of safer superficial dermal and novel topical/transdermal delivery systems for regenerative aesthetics.