Introduction: Guided bone regeneration (GBR) using the principle of guided tissue regeneration (GTR) has been widely demonstrated to be a useful technique for enhancing bone healing in a variety of osseous defects including those in periodontal and peri-implant regions. Placement of a biocompatible membrane to guide desirable cells such as osteoblasts to the appropriate site is a critical step in GBR. Optimization of the process includes the prevention of collapse of the membrane with bone grafting materials that also enhance the growth and differentiation of osteoblastic cells at the desired site. The purpose of this in vitro study was to assess the ability of a nano-sized calcium sulfate product (nCS) to enhance the growth and differentiation of human osteoblastic cells on biocompatible collagen materials commonly used in GBR.