Sulfated Carboxymethyl Cellulose and Carboxymethyl κ-Carrageenan Immobilization on 3D-printed Polycaprolactone Scaffolds Differentially Promote Preosteoblast Proliferation and Osteogenic Activity
Sulfated Carboxymethyl Cellulose and Carboxymethyl κ-Carrageenan Immobilization on 3D-printed Polycaprolactone Scaffolds Differentially Promote Preosteoblast Proliferation and Osteogenic Activity
Sonia Abbasi-Ravasjani,1Hadi Seddiqi,2Ali Moghaddaszadeh,3Mohammad-Ehsan Ghiasvand,4Jianfeng Jin,5Jenneke Klein-Nulend,6,*
1. Department of Oral Cell Biology, Academic Centre for Dentistry Amsterdam (ACTA), University of Amsterdam and Vrije Universiteit Amsterdam, Amsterdam Movement Sciences, Amsterdam, The Netherlands 2. Department of Oral Cell Biology, Academic Centre for Dentistry Amsterdam (ACTA), University of Amsterdam and Vrije Universiteit Amsterdam, Amsterdam Movement Sciences, Amsterdam, The Netherlands 3. Department of Biomedical Engineering, Science and Research Branch, Islamic Azad University, Tehran, Iran 4. Department of Mechanical Engineering, Amir Kabir University of Technology, Tehran, Iran 5. Department of Oral Cell Biology, Academic Centre for Dentistry Amsterdam (ACTA), University of Amsterdam and Vrije Universiteit Amsterdam, Amsterdam Movement Sciences, Amsterdam, The Netherlands 6. Department of Oral Cell Biology, Academic Centre for Dentistry Amsterdam (ACTA), University of Amsterdam and Vrije Universiteit Amsterdam, Amsterdam Movement Sciences, Amsterdam, The Netherlands
Introduction: Lack of bioactivity of three-dimensional (3D)-printed polycaprolactone (PCL) scaffolds limits cell-material interactions in bone tissue engineering. This limitation can be overcome using surface-functionalization by glycosaminoglycan-like anionic polysaccharides, e.g. carboxymethyl cellulose (CMC), a plant-based carboxymethylated, unsulfated polysaccharide, and κ-carrageenan, a seaweed-derived sulfated, non-carboxymethylated polysaccharide. Sulfation of CMC and carboxymethylation of κ-carrageenan critically improve their bioactivity, but how sulfated carboxymethyl cellulose (SCMC) and carboxymethyl κ-carrageenan (CM-κ-Car) affect the osteogenic differentiation potential of preosteoblasts on 3D-scaffolds is still unknown. Here we aimed to assess the effects of surface-functionalization by SCMC or CM-κ-Car on physicochemical and mechanical properties of 3D-printed PCL scaffolds, as well as the osteogenic response of preosteoblasts. MC3T3-E1 preosteoblasts were seeded on 3D-printed PCL scaffolds either or not functionalized by CM-κ-Car (PCL/CM-κ-Car), or SCMC (PCL/SCMC), and cultured up to 28 days. The scaffolds’ physicochemical and mechanical properties and preosteoblast function were assessed experimentally and by finite element modeling.
Methods: Sulfation of CMC: CMC was allowed to react with SO3/pyridine complex to prepare SCMC.
Carboxymethylation of ĸ-carrageenan: CM-к-Car was synthesized by alkalization of к-carrageenan to form alkoxy-к-carrageenan, followed by etherification with monochloroacetic acid.
Characterization of SCMC and CM-к-Car: The chemical structure of CMC, SCMC, к-carrageenan, and CM-к-Car was studied using FTIR spectrophotometry, and NMR spectroscopy.
3D-printing of PCL scaffolds: PCL scaffold (l×w×h:10×10×10 mm; volume: 1000 mm3) strands (diameter: 0.7 mm) were printed layer-by-layer with a 0º/90º lay-down pattern.
Surface-functionalization of 3D-printed PCL scaffolds by SCMC or CM-к-Car: SCMC and CM-к-Car were immobilized on aminolysed 3D-printed PCL scaffolds using EDC/NHS as crosslinker.
Scaffold characterization: Physicochemical properties of 3D-printed PCL, PCL/SCMC, and PCL/CM-κCar scaffolds, i.e. surface elemental composition (EDS), hydrophilicity, surface topography and morphology (SEM), void size and strand diameter, surface roughness, surface charge, surface chemical composition (ATR-FTIR), total protein adsorption, and as well as mechanical properties, i.e. compression modulus, and ultimate compression strength were determined.
Finite element (FE) modeling: FE modeling was used to quantify the mechanical behavior, von Mises stress distribution and magnitude, under uniform 2% compression strain deformation.
Cell culture and scaffold bioactivity: MC3T3-E1 preosteoblasts were seeded at 5ˣ105 cells/cm3 on the scaffolds, and cultured up to 28 days. Preosteoblast seeding efficiency, cell morphology and spreading (SEM), expression of osteogenic genes (RT-PCR), proliferation, and alkaline phosphatase (ALP) activity, collagen production, and calcium deposition were determined.
Statistics: Differences in mean values were tested using one-way ANOVA. Two-way analysis of variance with pairwise comparison was used to assess differences between groups and over time. Differences were considered significant if p<0.05.
Results: Surface-functionalization by SCMC and CM-κ-Car did not change scaffold geometry and structure, but similarly increased surface roughness (PCL/CM-κ-Car: 6.62-fold), hardness, as well as decreased water contact angle and elastic modulus. Finite element modeling showed that maximal von Mises stress for 2% compression strain did not exceed yield stress for bulk material in all scaffolds. Surface-functionalization by SCMC and CM-κ-Car increased protein adsorption, and improved cell spreading, resulting in well-spread cells with a natural spindle-shaped morphology on the surface of PCL/SCMC and PCL/CM-κ-Car scaffolds. Surface-functionalization by SCMC decreased Runx2 and Dmp1 expression, while surface-functionalization by CM-κ-Car increased Cox2 expression at day 1. Surface-functionalization by SCMC most strongly enhanced preosteoblast proliferation and collagen production, while CM-κ-Car most significantly increased alkaline phosphatase activity and mineralization after 28 days.
Conclusion: In conclusion, surface-functionalization by SCMC or CM-κ-Car of 3D-printed PCL-scaffolds enhanced preosteoblast proliferation and osteogenic activity, likely due to increased surface roughness and hydrophilicity. Surface-functionalization by SCMC most strongly enhanced cell proliferation, while CM-κ-Car most significantly promoted osteogenic activity, suggesting that surface-functionalization by CM-κ-Car may be more promising, especially in the short-term, for in vivo bone formation.