Enhanced osteogenic activity by MC3T3-E1 pre-osteoblasts on chemically surface-modified poly( ε -caprolactone) 3D-printed scaffolds compared to RGD immobilized scaffolds

Abstract

In bone tissue engineering, the intrinsic hydrophobicity and surface smoothness of three-dimensional (3D)-printed poly(ε-caprolactone) scaffolds hamper cell attachment, proliferation and differentiation. This intrinsic hydrophobicity of poly(ε-caprolactone) can be overcome by surface modifications, such as surface chemical modification or immobilization of biologically active molecules on the surface. Moreover, surface chemical modification may alter surface smoothness. Whether surface chemical modification or immobilization of a biologically active molecule on the surface is more effective to enhance pre-osteoblast proliferation and differentiation is currently unknown. Therefore, we aimed to investigate the osteogenic response of MC3T3-E1 pre-osteoblasts to chemically surface-modified and RGD-immobilized 3D-printed poly(ε-caprolactone) scaffolds. Poly(ε-caprolactone) scaffolds were 3D-printed consisting of strands deposited layer by layer with alternating 0°/90° lay-down pattern. 3D-printed poly(ε-caprolactone) scaffolds were surface-modified by either chemical modification using 3 M sodium hydroxide (NaOH) for 24 or 72 h, or by RGD-immobilization. Strands were visualized by scanning electron microscopy. MC3T3-E1 pre-osteoblasts were seeded onto the scaffolds and cultured up to 14 d. The strands of the unmodified poly(ε-caprolactone) scaffold had a smooth surface. NaOH treatment changed the scaffold surface topography from smooth to a honeycomb-like surface pattern, while RGD immobilization did not alter the surface topography. MC3T3-E1 pre-osteoblast seeding efficiency was similar (44%–54%) on all scaffolds after 12 h. Cell proliferation increased from day 1 to day 14 in unmodified controls (1.9-fold), 24 h NaOH-treated scaffolds (3-fold), 72 h NaOH-treated scaffolds (2.2-fold), and RGD-immobilized scaffolds (4.5-fold). At day 14, increased collagenous matrix deposition was achieved only on 24 h NaOH-treated (1.8-fold) and RGD-immobilized (2.2-fold) scaffolds compared to unmodified controls. Moreover, 24 h, but not 72 h, NaOH-treated scaffolds, increased alkaline phosphatase activity by 5-fold, while the increase by RGD immobilization was only 2.5-fold. Only 24 h NaOH-treated scaffolds enhanced mineralization (2.0-fold) compared to unmodified controls. In conclusion, RGD immobilization (0.011 μg mg−1 scaffold) on the surface and 24 h NaOH treatment of the surface of 3D-printed PCL scaffold both enhance pre-osteoblast proliferation and matrix deposition while only 24 h NaOH treatment results in increased osteogenic activity, making it the treatment of choice to promote bone formation by osteogenic cells.

Effect of the nano/microscale structure of biomaterial scaffolds on bone regeneration

“…Similarly, Yu et al demonstrated that the −NH 2 modified surface exhibited improved biocompatibility and osteoconductivity/osteoinductivity with increased cell adhesion and proliferation capabilities. 124 In addition, Zamani et al described 3D-printed PCL scaffolds that were surface-modified by alkaline treatment with 1 mol•L −1 and 3 mol•L −1 sodium hydroxide (NaOH) for 24 h. 125 Their investigation showed that the NaOH-treated scaffolds had a honeycomb-like surface pattern, and that the increased number of hydroxyl and carboxyl groups on the surface increased hydrophilicity via scission of PCL ester bonds by NaOH. Furthermore, the scaffold post-treated by NaOH displayed increased calcium deposition.…”

Plant Tissues as 3D Natural Scaffolds for Adipose, Bone and Tendon Tissue Regeneration

“…In vitro direct cytocompatibility tests were conducted using a pre-osteoblast cell line (MC3T3-E1), by inducing osteogenic differentiation after cell seeding (Zamani et al, 2019). Metabolic activity resulted stable up to 7 days after seeding (Figure 6B).…”

Mechanical stimulation improves osteogenesis and the mechanical properties of osteoblast‐laden RGD‐functionalized polycaprolactone/hydroxyapatite scaffolds

“…Although natural polymer‐based scaffolds (Damaraju, Matyas, Rancourt, & Duncan, 2014; Salifu, Lekakou, & Labeed, 2017a; Salifu, Lekakou, & Labeed, 2017b) have been developed for bone regeneration, synthetic polymers such as polycaprolactone (PCL) (Dettin et al, 2015; Drevelle et al, 2010; Zamani et al, 2018; Zhang & Hollister, 2009; Zhang, Lin, & Hollister, 2009), polylactic acid (PLA) (Wurm et al, 2017), and polylactic‐co‐glycolic acid (PLGA) (Li et al, 2013) have also been investigated due to their superior processability, good mechanical and controllable degradation properties (Dettin et al, 2015; Drevelle et al, 2010; Li et al, 2013; Wurm et al, 2017; Zamani et al, 2018; Zhang et al, 2009; Zhang & Hollister, 2009). Synthetic polymer scaffolds have also been combined with osteoconductive ceramics, such as hydroxyapatite (HA), into PCL/HA (Kim et al, 2017; Zhang et al, 2014), PLA/HA (Zhang et al, 2017), and PLGA/HA (Li et al, 2013) scaffolds to improve their bioactive and mechanical properties for bone regeneration (Kim et al, 2017; Li et al, 2013; Zhang et al, 2014).…”

“…Adhesion peptides, such as RGD peptides, when immobilized onto scaffolds, can promote integrin‐mediated osteogenic cell adhesion and activate cell–biomaterial interactions to promote bone formation (Dettin et al, 2015; Drevelle et al, 2010; Zhang et al, 2009; Zhang & Hollister, 2009). Prior studies have revealed that RGD‐functionalized PCL scaffolds enhance osteogenic cell adhesion (Drevelle et al, 2010; Zhang et al, 2009), proliferation and spreading (Dettin et al, 2015; Drevelle et al, 2010; Zamani et al, 2018; Zhang et al, 2009), differentiation (Dettin et al, 2015), extracellular matrix (ECM) production (Zamani et al, 2018), and mineralization (Dettin et al, 2015), compared to PCL scaffolds alone. Several studies have also shown that PCL/HA scaffolds promote osteoblast proliferation and osteogenesis (Kim et al, 2017; Zhang et al, 2014), compared to PCL alone.…”

“…, polylactic acid (PLA) (Wurm et al, 2017), and polylactic-co-glycolic acid (PLGA) (Li et al, 2013) have also been investigated due to their superior processability, good mechanical and controllable degradation properties (Dettin et al, 2015;Drevelle et al, 2010;Li et al, 2013;Wurm et al, 2017;Zamani et al, 2018;. Synthetic polymer scaffolds have also been combined with osteoconductive ceramics, such as hydroxyapatite (HA), into PCL/HA (Kim et al, 2017;Zhang et al, 2014), PLA/HA (Zhang et al, 2017), and PLGA/HA (Li et al, 2013) scaffolds to improve their bioactive and mechanical properties for bone regeneration (Kim et al, 2017;Li et al, 2013;Zhang et al, 2014).…”