Introduction: Synthetic osteoinductuve bone substitutes are considered the future of bone engineering in critical sized defects. Osteoinduction is referred to as the process of inducing differentiation of mesenchymal stem cells in the healing site into osteoprogenitor cells in order to form new bone[1]. Incorporating an osteoinductive factor with currently used osteoconductive bone scaffolds enhances the biological responsiveness and success rate. Self-assembling peptides (SAPs) form hydrogels composed mainly of water. These nanostructured materials have the ability to enhance the local environment for cells, induce tissue regeneration and drug delivery[2,][3]. SAPs have been shown to modify surface properties of materials and stimulate bone conduction when compared to MatrigelTm in a calvaria mice model[4],[5]. The objective of the present study was to investigate the functionalization potential of tailored EAbuK-TAMRA SAPs synthesized as C-terminal amides (where Abu refers to α-aminobutyric acid and TAMRA, 5(6)-Carboxy-tetramethyl-rhodamine respectively ) on the surface of 3 dimensional (3D) printed biphasic calcium phosphate (BCP) scaffolds composed of 15:85 hydroxyapatite/beta-tricalcium phosphate (HA/ β-TCP).
Methods: Human mesenchymal stem cells (hMSCs) were seeded on SAPs coated scaffolds and compared to non-coated scaffolds. 3D printed scaffolds were produced with high porosity and similar architecture thus minimizing the variability between scaffolds. Scaffolds were immersed in peptide solution (0.15% w/w self-assembling peptide- SAP- in water) for one hour at 37°C. Non-coated scaffolds were used as controls. Cells were micro-seeded at a density of 200,000 cells per scaffold. These were micro-seeded in 200 µL. Scaffolds containing cells were then immersed in stem cell medium without differentiating factors. The scaffolds were cultured for two time points; 14 and 42 days, media was replaced every 3 days. At each time point, scaffolds were either treated to obtain cell lysates by a freeze-thaw method or used for RNA extraction by the Trizol method. Cell growth and turnover was assessed by measuring total DNA production using the Hoechst 33258 dye. Cell differentiation of the osteoblastic phenotype was determined by measuring the alkaline phosphatase (ALP) production of hMSCs. Quantitative real-time reverse transcription– polymerase chain reaction (qPCR) measured the gene expression of runt-related transcription factor 2 (Runx2) osteonectin (ON), Osteopontin (OPN) and type IA collagen (COL1A1). The expression pattern of COL1A1 and osteocalcin (OC) were examined by immunofluorescent staining of scaffolds using a Leica multi-photon 2 confocal microscope with acousto-optical beam-splitter spectral scan head (AOBS).
Results and discussion:
DNA production was only significantly higher at day 42 in peptide-coated scaffolds in comparison to controls (Figure 1-A). Alkaline phosphatase (ALP) activity, however, was significantly higher in peptide-coated scaffolds at days 14 and 42 in comparison to controls (Figure 1-B). Gene expression for Runx2, ON, OPN and COL1A1 were all significantly enhanced in peptide-coated scaffolds in relation to controls. In addition, significant increase in gene expressions was detected at day 14 when compared to day 42 in all genes. Also, OC (green) and COL1A1 (red) staining showed more abundant OC production in peptide-coated scaffolds, whereas COL1A1 was similar (Figure 2-A and B).
Bridging of seeded cells over empty pores was observed in the peptide-functionalized scaffolds (Figure 2-C). These scaffolds induced cell proliferation and osteogenic differentiation without the addition of osteogenic stimulatory factors.
Conclusion: Scaffolds were successfully functionalized using EAbuK-TAMRA SAPs in vitro. SAPs can be used as a quick and practical functionalizing material that can be added to scaffolds prior implantation. An in vivo study using these scaffolds has been performed and scaffolds are being processed for analysis. Translating the in vitro findings in a true defect in vivo will give a broader picture of the peptides potential in osteoinduction.
King Abdulaziz University and the Saudi Ministry of Higher Education
References:
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