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

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/β-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.

Keywords: bone tissue engineering; cell distribution; human mesenchymal stem cell; osteogenic differentiation; scaffold.

FIG. 1.

SEM micrographs and EDA of the scaffolds without cell culture. (A) SEM picture of PCL scaffold with highly interconnected pores. (B) SEM picture of HA/TCP-PCL scaffold. (C) EDA of HA/TCP-PCL scaffold. (D) The distribution of Ca element within HA/TCP-PCL scaffold. Bar = 200 μm. EDA, energy dispersive X-ray spectrometer analysis; HA/TCP, hyaluronic acid/β-tricalcium phosphate; PCL, polycaprolactone; SEM, scanning electron microscope.

FIG. 2.

Toluidine blue stained cross sections of scaffolds in the Z-direction from top to bottom after 21 days of culture on (A) PCL scaffold and (B) HA/TCP-PCL scaffold. Cells only covered on the bottom part of the PCL scaffold, while cells were distributed uniformly on the HA/TCP-PCL scaffold. Scale bar = 900 μm. S, scaffold.

FIG. 3.

SEM images of scaffolds after 21 days of culture. (A) Both surfaces of cell seeding side and bottom side of the PCL and HA/TCP-PCL scaffolds with cell cultures. (B) Stitched cross sections of middle parts of PCL and HA/TCP-PCL scaffolds from the top to the bottom. Bar = 200 μm. ECM, extracellular matrix.

FIG. 4.

Quantification of dsDNA in cellular scaffolds by PicoGreen assay on days 2, 7, 14, and 21. The amount of DNA is expressed as mean ± SD (n = 3). Significant difference between PCL and HA/TCP-PCL scaffolds within the same culture condition and time point (*p < 0.05; **p < 0.01).

FIG. 5.

Gene expression of RUNX2, ALP, COLI, and OC after osteogenic culture of human mesenchymal stem cells on PCL and HA/TCP-PCL scaffolds up to 21 days. Vertical axes represent the BestKeeper relative gene expression relative to day 2 of the PCL scaffold for RUNX2, ALP, COLI, and OC. Horizontal axes represent the time points. Data are expressed as mean ± SD (n = 6). Significant difference between PCL and HA/TCP-PCL scaffolds at each time point (*p < 0.05; **p < 0.01).

FIG. 6.

Osteogenic differentiation of scaffold/cell constructs. (A) Activity of the ALP enzyme on days 2, 7, 14, and 21. The activity is expressed as mean ± SD (n = 3). Activity is indicated in nanomole p-nitrophenol/microgram DNA per minute (nmol/mL * μgDNA*min). (B) Calcium contents per scaffold on days 2, 7, 14, and 21. The amount of calcium is expressed as mean ± SD (n = 3). Significant difference between PCL and HA/TCP-PCL scaffolds within the same culture condition and time point (**p < 0.01). ALP, alkaline phosphatase.

FIG. 7.

Alizarin Red stained cross sections of scaffolds with 21 days cell culture on (A) PCL scaffold and (B) HA/TCP-PCL scaffold. Scale bar is 900 μm. Representative positive staining of calcium was pointed out with an arrow.