Integration of Bioglass Into PHBV-Constructed Tissue-Engineered Cartilages to Improve Chondrogenic Properties of Cartilage Progenitor Cells

Abstract

Background: The Poly (3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) scaffold has proven to be a promising three-dimensional (3D) biodegradable and bioactive scaffold for the growth and proliferation of cartilage progenitor cells (CPCs). The addition of Bioglass into PHBV was reported to increase the bioactivity and mechanical properties of the bioactive materials. Methods: In the current study, the influence of the addition of Bioglass into PHBV 3D porous scaffolds on the characteristics of CPC-based tissue-engineered cartilages in vivo were compared. CPCs were seeded into 3D macroporous PHBV scaffolds and PHBV/10% Bioglass scaffolds. The CPC-scaffold constructs underwent 6 weeks in vitro chondrogenic induction culture and were then transplanted in vivo for another 6 weeks to evaluate the difference between the CPC-PHBV construct and CPC-PHBV/10% Bioglass construct in vivo. Results: Compared with the pure PHBV scaffold, the PHBV/10% Bioglass scaffold has better hydrophilicity and a higher percentage of adhered cells. The CPC-PHBV/10%Bioglass construct produced much more cartilage-like tissues with higher cartilage-relative gene expression and cartilage matrix protein production and better biomechanical performance than the CPC-PHBV construct. Conclusion: The addition of Bioglass into 3D PHBV macroporous scaffolds improves the characteristics of CPC-based tissue-engineered cartilages in vivo.

Keywords: Bioglass; PHBV; cartilage engineering; cartilage progenitor cells; hydrophilicity.

FIGURE 1

Optical microscopy and SEM of PHBV/10%Bioglass scaffolds and the CPC–PHBV/10%Bioglass construct. (A) The PHBV/10%Bioglass scaffolds exhibited a cylindrical shape (5 mm diameter and 2 mm thickness), with a lot of pores on the surface of the composite scaffolds. (B) PHBV/10%Bioglass composite porous 3D scaffolds had a macroporous structure with interconnected open pores of 30–300 μm in diameter. (C) Gross view of in vitro CPC–scaffold constructs after 6 weeks of in vitro culture. These engineered tissues roughly maintained their original cylindrical shape and size and exhibited an ivory-whitish appearance. (D) SEM view of CPCs-PHBV/10%Bioglass constructs after 6 weeks of in vitro culture, exhibiting abundant extracellular matrix production and good compatibility of the CPCs with the composite scaffold.

FIGURE 2

Characterization of PHBV and PHBV/10%Bioglass 3D porous scaffolds. The PHBV and PHBV/10%Bioglass 3D porous scaffolds exhibited the same volume (A) and dry weight (B) and the same porosity (C) (p > 0.05). The compressive modulus (D) of the CPC–PHBV/10%Bioglass constructs was greater than that of CPC–PHBV constructs (p < 0.05).

FIGURE 3

Contact angle, water absorptivity, and cell adhesion of the scaffolds. (A) The water contact angles of the PHBV/Bioglass composite scaffolds were significantly lower than that of the pure PHBV scaffold, indicating that there was a significant increase in surface hydrophilicity with the addition of Bioglass into PHBV (p < 0.05). (B) The water absorptivity of the PHBV/Bioglass composite scaffolds was obviously greater than that of pure PHBV (p < 0.05). (C) The percentage of adhered cells increased significantly with the addition of Bioglass (p < 0.05).

FIGURE 4

Cell Proliferation. There was an obvious difference in the cellular proliferation between the CPC–PHBV construct and CPC–PHBV/10%Bioglass construct (p < 0.05) (A). The DNA content of the CPC–PHBV/10%Bioglass construct is higher than that of the CPC–PHBV construct (p < 0.05) (B).

FIGURE 5

Gross analysis of in vivo engineered tissue cartilages. The diameter (A), thickness (B), volume (C), and wet weight (D) of the CPC–PHBV/10%Bioglass construct were higher than those of the CPC–PHBV construct in vivo (p < 0.05).

FIGURE 6

Histological and immunohistological analysis of in vivo engineered constructs. There is an obvious difference in the thickness between the CPC–PHBV/10%Bioglass construct and CPC–PHBV construct (p < 0.05). The CPC–PHBV/10%Bioglass construct produced much more cartilage-like tissues than the CPC–PHBV construct. Scale bar = 100 μm.

FIGURE 7

Collagen content, GAG content, and compression modulus. There is a significant difference in terms of collagen (A) and GAG (B) contents and the compression modulus (C) between the CPC–PHBV construct and CPC–PHBV/10%Bioglass construct (p < 0.05).

FIGURE 8

Chondrogenic differentiation of the CPC–PHBV construct and CPC–PHBV/10%Bioglass construct. RT-PCR analysis reveals the stronger expression of COL II (A), aggrecan (B), and the SOX-9 gene of the CPC–PHBV/10%Bioglass construct than the CPC–PHBV construct (p < 0.05).