Effects of single layer and bilayer hydroxyapatite scaffold architectures on cell activity
Estimates of over 500,000 bone-grafting procedures are performed annually in the United States, with approximately half of these procedures related to spine fusion (Greenwald AS 2001). Common therapies include autograft and allograft bone substitutes; however, these treatments carry associated risks such as donor site morbidity and disease transmission that may decrease the quality of life for any patient. This study is intended for development of a ceramic-based bone-graft substitute that can help substitute current bone-graft therapies. Currently, it is unclear as how pore gradients affect cellular activity. This study evaluated open-porous scaffolds with varying pore architecture in terms of metabolic activity, differentiation, and distribution for human embryonic palatal mesenchymal (HEPM) cells. Two hydroxyapatite (HAp) architectures consisting of different pore size configurations were evaluated. Five scaffold groups consisting of single (three uniform porous) and bilayer (two dual porous) designs were used and further evaluated through biological characterization via in vitro studies.
HAp characterization studies were evaluated for cell attachment and loading confluence to evaluate HEPM cellular response for 3-D scaffold seeding. Individual scaffold groups (n=6) were also evaluated for their surface area through BSA (Bovine Serum Albumin) protein adsorption along with porosity characterization. HEPM cells were further loaded onto each scaffold for 28 days to determine cell metabolic activity through alamarBlue and alkaline phosphatase detection. Cell distribution was evaluated via polarized light through longitudinal histological sections.
Porosity differences amongst each individual scaffold group showed no significant differences. BSA protein adsorption was highest in the 250μl scaffold group. Surface area through BSA adsorption was determined to not be significant. In addition, 28 days revealed high metabolic activity of a 200/340μm bilayer and 340μm single layer scaffold. Secreted alkaline phosphatase was measured to reveal that after 4 weeks, both bilayers (200/450μm, 200/340μm) and the 340μm single layer had the highest differentiation activity. Furthermore, cell distribution revealed that scaffolds consisting of larger pore size had an even distribution of HEPM cells throughout the construct.