Improvement of Distribution and Osteogenic Differentiation of Human Mesenchymal Stem Cells by Hyaluronic Acid and β-Tricalcium Phosphate-Coated Polymeric Scaffold In Vitro.

BioResearch Open Access Volume 4.1, 2015 DOI: 10.1089/biores.2015.0021 BioResearch OPEN ACCESS ORIGINAL RESEARCH ARTICLE Open Access Improvement of Distribution and Osteogenic Differentiation of Human Mesenchymal Stem Cells by Hyaluronic Acid and b-Tricalcium Phosphate-Coated Polymeric Scaffold In Vitro Muwan Chen,1,2,* Dang Q.S. Le,1,2 Jørgen Kjems,2 Cody Bünger,1 and Helle Lysdahl1 Abstract Bone tissue engineering requires a well-designed scaffold that can be biodegradable, biocompatible, and support the stem cells to osteogenic differentiation. Porous polycaprolactone (PCL) scaffold prepared by fused deposition modeling is an attractive biomaterial that has been used in clinic. However, PCL scaffolds lack biological function and osteoinductivity. In this study, we functionalized the PCL scaffolds by embedding them with a matrix of hyaluronic acid/b-tricalcium phosphate (HA/TCP). Human mesenchymal stem cells (MSCs) were cultured on scaffolds with and without coating to investigate proliferation and osteogenic differentiation. The DNA amount was significantly higher in the HA/TCP-coated scaffold on day 21. At the gene expression level, HA/ TCP coating significantly increased the expression of ALP and COLI on day 4. These data correlated with the ALP activity peaking on day 7 in the HA/TCP-coated scaffold. Scanning electron microscope and histological analysis revealed that the cell matrix and calcium deposition were distributed more uniformly in the coated scaffolds compared to scaffolds without coating. In conclusion, the HA/TCP coating improved cellular proliferation, osteogenic differentiation, and uniform distribution of the cellular matrix in vitro. The HA/TCP-PCL scaffold holds great promise to accommodate human bone marrow-derived MSCs for bone reconstruction purposes, which warrants future in vivo studies. Key words: bone tissue engineering; cell distribution; human mesenchymal stem cell; osteogenic differentiation; scaffold Introduction Bone tissue engineering (BTE) and reconstructive surgery have been intensively researched in the past 20 years. The key challenge for successful BTE is creating an ideal scaffold having the following properties: a porous structure, high interconnectivity, adequate mechanical properties, biodegradability, biocompatibility, osteoinduction, and osteoconduction. The scaffold should support cell attachment, migration, proliferation, and differentiation and eventually be replaced by the regenerated host tissue.1–3 No single biomaterial accomplishes all the required properties of the BTE scaffold. Composite scaffold designs can take each material’s advantage to fulfill most of the requirements and significantly improve the physical, chemical, and biological properties.4–6 Biodegradable polyesters, such as polyglycolic acid, polylactic acid, and polycaprolactone (PCL), are the most commonly used synthetic polymer materials for BTE applications. They are attractive candidates for use as scaffolds because they can be fabricated for a wide range of biodegradable biomedical applications 1 Orthopaedic Research Laboratory, Aarhus University Hospital, Aarhus, Denmark. Interdisciplinary Nanoscience Center, Aarhus University, Aarhus, Denmark. 2 *Address correspondence to: Muwan Chen, PhD, Orthopaedic Research Laboratory, Aarhus University Hospital, Nørrebrogade 44, Building 1A, Aarhus C 8000, Denmark, E-mail: ª Muwan Chen et al. 2015; Published by Mary Ann Liebert, Inc. This Open Access article is distributed under the terms of the Creative Commons License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. 363 Chen, et al.; BioResearch Open Access 2015, 4.1 http://online.liebertpub.com/doi/10.1089/biores.2015.0021 with a high-process ability, controlled degradation, adjustable mechanical properties, and with the possibility of a wide range of modifications.7 Their degradation products are tolerated by the human body and can be removed by physiological metabolic pathways. Clinically, PCL scaffolds made by fused deposition modeling (FDM) have been studied for more than 10 years and gained FDA approval in 2006.8 However, these FDM-manufactured PCL scaffolds comprise a macroporous structure, lack biological functionality in the scaffold–cell interface, and are inherently hydrophobic. To overcome such disadvantages, improvements by incorporating natural polymer and bioceramic material into the scaffolds have been performed: PCL scaffolds have been coated with natural polymers such as collagen,9,10 chitosan,11 hyaluronic acid (HA),12 and silk fibroin13 to improve cell affinity and biocompatibility. Bioceramics, such as hydroxyapatite and b-tricalcium phosphate (TCP), are calcium phosphate products, which have often been used for BTE.14,15 However, their brittle and fragile properties limit their uses as defect fillers in orthopedic surgery. Therefore, blending bioceramics with PCL polymer to reduce the ceramic materials’ intrinsic brittleness and to promote osteoconductivity of PCL material has been performed.4,6,16 We have previously shown that functionalization of PCL scaffolds with HA and TCP facilitates migration and osteogenic differentiation of human dental pulp stem cells in vitro.17 The aim of the current study was to investigate the osteogenic potential of this scaffold seeded with human bone marrow-derived mesenchymal stem cells (MSCs). We hypothesized that HA/ TCP coating would promote distribution, proliferation, as well as osteogenic differentiation of human MSCs (hMSCs) in vitro. Materials and Methods Scaffold fabrication FDM scaffolds were made from PCL with a molecular weight of 50 kDa (Perstorp) at a processing temperature of 106C with a BioScaffolder (SYS+ENG GmbH). The stainless steel extrusion needle (DL Technology) had a 200 lm opening, which produced extruded polymer strands with a width of *190 lm. A square of 36 · 36 · 2 mm mat was made first, and cylindrical scaffolds with a diameter of 4 mm were punched out using a biopsy punch (Acuderm). To increase surface hydrophilicity and, thus, improve cell attachment, the scaffolds were treated in a 5 M sodium hydroxide bath for 3 h, neutralized by washing with phosphate- 364 buffered saline (PBS) and sterile water, and disinfected using 70% ethanol. The scaffolds were rinsed in sterile water multiple times and dried. These scaffolds are hereafter referred to as PCL scaffolds. HA/TCP-PCL scaffolds were fabricated as described previously.17 Briefly, the PCL scaffolds were soaked in the HA/TCP suspension for 12 h. HA/TCP suspension was prepared by dispersing 10 wt% TCP (BABI-TCPN100, particle average size 100 nm; Berkeley Advanced Biomaterials, Inc.) into an aqueous 4 mg/mL HA solution (MW = 780 kDa; Lifecore Biomedical). Finally, these scaffolds were placed in a freeze dryer (FreeZone Triad Freeze Dry Systems) at 20C, 30 mTorr, for 4 days. Scaffold ch (...truncated)