Hydroxyapatite Particles Induced Modulation of Collagen Expression and Secretion in Primary Human Dermal Fibroblasts

Int J Nanomedicine. 2020 Jul 13;15:4943-4956. doi: 10.2147/IJN.S245500. eCollection 2020.

Abstract

Background: Hydroxyapatite (HA) [Ca5(PO4)3(OH)] is a naturally occurring calcium phosphate which makes up 60-70% of the dry weight of human bones. Nano-scale HA particles are increasingly being used as carriers for controlled and targeted delivery of bioactive agents like drugs, proteins, and nucleic acids due to their high porosity, negative charge, and biodegradability.

Purpose: Although much effort has been devoted to understanding the delivery kinetics and effects of the payloads in such carriers, a thorough understanding of the influence of the carriers themselves is lacking.

Methods: HA particles (300 µg/mL) were administered to primary human dermal fibroblasts (HDFs). The uptake and intracellular localization of the particles were determined by flow cytometry, confocal imaging, and transmission electron microscopy (TEM). Immunological assays and PCR were performed to determine the levels of pro-inflammatory cytokines and collagens in cell lysates and media supernatant.

Results: The current study explores the effects of poly-dispersed HA particles on primary HDFs as a model system. The majority of the particles were determined to range between 150 and 200 nm in diameter. Upon exposure to HA suspensions, primary HDFs internalized the particles by endocytosis within 6 hours of exposure, showing maximum uptake at 72 hours following which the particles were exocytosed by 168 hours. This correlated to reduced secretion of various pro-inflammatory and pro-collagenic cytokines. Biochemical analysis further revealed a reduction in Type I collagen expression and secretion.

Conclusion: HA particles have an immune-modulatory effect on dermal fibroblasts and reduce collagen production, which may impact the integrity of the extracellular matrix (ECM). This study demonstrates the need to consider the secondary effects of particulate carriers like HA, beyond basic cytotoxicity, in the specific tissue environment where the intended function is to be realized.

Keywords: carrier particles; collagen expression; extracellular matrix; time-weighted average exposure.

MeSH terms

  • Collagen / metabolism*
  • Collagen Type I / metabolism
  • Cytokines / metabolism
  • Durapatite / chemistry
  • Durapatite / pharmacology*
  • Extracellular Matrix / drug effects
  • Extracellular Matrix / metabolism
  • Fibroblasts / cytology
  • Fibroblasts / drug effects*
  • Fibroblasts / metabolism*
  • Gene Expression Regulation / drug effects*
  • Humans
  • Skin / cytology*

Substances

  • Collagen Type I
  • Cytokines
  • Collagen
  • Durapatite

Grant support

Funding support: SingHealth-NTU-Research Collaborative Grant awarded to T.T.W. and K.W.N. (SHS-NTU/028/2016).