The Phenotype of Mesenchymal Stromal Cell and Articular Chondrocyte Cocultures on Highly Porous Bilayer Poly-L-Lactic Acid Scaffolds Produced by Thermally Induced Phase Separation and Supplemented with Hydroxyapatite

Abstract

Bilayer scaffolds could provide a suitable topology for osteochondral defect repair mimicking cartilage and subchondral bone architecture. Hence, they could facilitate the chondro- and osteogenic lineage commitment of multipotent mesenchymal stromal cells (MSCs) with hydroxyapatite, the major inorganic component of bone, stimulating osteogenesis. Highly porous poly-L-lactic acid (PLLA) scaffolds with two layers of different pore sizes (100 and 250 µm) and hydroxyapatite (HA) supplementation were established by thermally induced phase separation (TIPS) to study growth and osteogenesis of human (h) MSCs. The topology of the scaffold prepared via TIPS was characterized using scanning electron microscopy (SEM), a microCT scan, pycnometry and gravimetric analysis. HMSCs and porcine articular chondrocytes (pACs) were seeded on the PLLA scaffolds without/with 5% HA for 1 and 7 days, and the cell attachment, survival, morphology, proliferation and gene expression of cartilage- and bone-related markers as well as sulfated glycosaminoglycan (sGAG) synthesis were monitored. All scaffold variants were cytocompatible, and hMSCs survived for the whole culture period. Cross-sections revealed living cells that also colonized inner scaffold areas, producing an extracellular matrix (ECM) containing sGAGs. The gene expression of cartilage and bone markers could be detected. HA represents a cytocompatible supplement in PLLA composite scaffolds intended for osteochondral defects.

Keywords: chondrocytes; chondrogenesis; hydroxyapatite; mesenchymal stromal cells; osteochondral defects; osteogenesis; poly-L-lactic acid; tissue engineering.

Figure 1

Macroscopical, histological and SEM analyses of PLLA scaffolds without/with HA supplementation. Macroscopical structure of pure PLLA (unilayer: A1,A2, bilayer: C1,C2) and PLLA + HA (unilayer: B1,B2, bilayer: D1,D2) unseeded scaffolds. Hematoxylin–eosin (HE) staining of unseeded pure PLLA (unilayer: A3, bilayer: C3) and PLLA + HA (unilayer: B3, bilayer: D3) scaffolds. (C3,D3): both layers of bilayered PLLA and PLLA + HA scaffolds. Double-headed arrows (A3,B3) indicate interconnectivity. Scanning electron microscopy (SEM) of unseeded pure PLLA (unilayer: A4, bilayer: E1,E2) and PLLA + HA (unilayer: B4, bilayer: E3) scaffold. Scale bars: 1 mm (A1,B1,C1,D1), 100 µm (A3,B3), 250 µm (C3,D3), 1 mm (E1–E3), 200 µm (A4,B4). HA: hydroxyapatite, PLLA: poly-L-lactic acid. Red arrow: HA particles.

Figure 2

(A,B): Representative microCT images of bilayer scaffolds. Three-dimensional view on pure PLLA and PLLA + HA scaffolds (A1,B1) and cross-sectional views (A2,B2). MicroCT image (3D) of the isolated HA scaffold segment (B3), visualization of the HA particles in the HA segment of the bilayer scaffold (B4, red), merged view of B3 + B4 for localization of the HA particles in the HA layer (B5). HA: hydroxyapatite, PLLA: poly-L-lactic acid. Scale bars: 500 µm.

Figure 3

Cytotoxicity assay with bilayer PLLA scaffolds without/with HA supplementation. L929 fibroblasts, human mesenchymal stem cells (hMSCs) and porcine articular chondrocytes (pACs) precultured for 24 h in 96 well plates were exposed for 24 h to extracts of bilayer pure PLLA and PLLA + HA scaffolds (extraction time 48 h). Cytotoxicity was determined using MTS assay. co: control, DMSO: dimethyl sulfoxide, GM: growth medium, HA: hydroxyapatite, PLLA: poly-L-lactic acid. Mean values and standard deviations are shown. Statistical analysis was performed with One-way ANOVA and Tukey’s multiple comparison test. n = 5. The red lines mark different levels (low and high) of cytotoxicity.

Figure 4

Adherence, survival, colonized surface area on unilayer PLLA scaffolds (100 µm pore size) without/with HA supplementation seeded with undifferentiated hMSCs after 2 and 8 days of statical culture. (A1–A3): PLLA, (B1–B3): PLLA + HA, (A1,B1): 2 days, (A2,A3,B2,B3): 8 days. (A3,B3): A transection through the middle of the scaffold is depicted. The scaffold surface seeded with hMSCs is shown on the upper side. (C): Scaffold surface area colonized with vital cells. (D): Cell migration into inner scaffold parts after one week. HA: hydroxyapatite, PLLA: poly-L-lactic acid. Red: dead cells, green: living cells. Scale bar: 100 µm.

Figure 5

Adherence, survival and Ca⁺⁺ deposition of non-induced and osteogenically induced hMSCs on unilayer PLLA scaffolds (A1–D1,A3–D3) or those supplemented with HA (A2–D2,A4–D4) after 7 (A1–B4), 14 (C1–C4) and 21 (D1–D4) days. A1–B4: Non-induced undifferentiated control. Red: dead cells, green: living cells. Ca⁺⁺ deposition is visualized by alizarin red staining of non-induced or osteogenically induced hMSCs on unilayer PLLA scaffolds (A3–D3) or those supplemented with HA (A4–D4). Scale bars: 100 µm.

Figure 6

Cell survival on bilayer PLLA scaffolds without/with HA supplementation and seeded with undifferentiated hMSCs/pAC cocultures after cultured dynamically for 1 (A1–B4) and 7 (C1–D4) days. (A1–D1,A2–D2): 100 µm pore size, (A3–D3,A4–D4): 250 µm pore size. PLLA: (A1–A4,C1–C4), PLLA + HA: (B1–B4,D1–D4). HA: hydroxyapatite, PLLA: poly-L-lactic acid. Life–death assay: red: dead cells, green: living cells. Scale bar: 100 µm (representative for A1–D4).

Figure 7

DNA content of hMSC/pAC cocultures and hMSC monocultures on bilayer PLLA scaffolds without/with HA supplementation after cultured dynamically for 1 and 7 days. HA: hydroxyapatite, hMSCs: human mesenchymal stem cells, L: larger pores, pAC: porcine articular chondrocytes, PLLA: poly-L-lactic acid, S: smaller pores. n = 6. Statistical analysis was performed with One-way ANOVA and Tukey’s multiple comparison test.

Figure 8

Sulfated glycosaminoglycan (sGAG) synthesis of hMSC/pAC cocultures and hMSC monocultures on bilayer PLLA scaffolds without/with HA supplementation after cultured dynamically for 1 and 7 days. HA: hydroxyapatite, hMSCs: human mesenchymal stem cells, L: larger pores, pAC: porcine articular chondrocytes, PLLA: poly-L-lactic acid, S: smaller pores. Statistical analysis was performed with One-way ANOVA and Tukey’s multiple comparison test.

Figure 9

Cytoskeletal architecture of hMSC/pAC cocultures on bilayer PLLA scaffolds without/with HA supplementation after cultured dynamically for 1 and 7 days. (A1–B4): 1 day, (C1–D4): 7 days. (A1,A2,B1,B2,C1,C2,D1,D2): 100 µm pore size, (A3,A4,B3,B4,C3,C4,D3,D4): 250 µm pore size. PLLA: (A1–A4,C1–C4), PLLA + HA: (B1–B4,D1–D4). HA: hydroxyapatite, PLLA: poly-L-lactic acid. Red: human vimentin, green: F-actin, visualized with phalloidin Alexa488. Cell nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI). Scale bar: 100 µm (representative for A1–D4).

Figure 10

Gene expression analysis of hMSC/pAC cocultures and hMSC monocultures on bilayer PLLA scaffolds without/with HA supplementation after cultured dynamically for 1 and 7 days. (A): porcine aggrecan (ACAN), (B): human and porcine collagen type 2 alpha 1 chain (COL2A1), (C): human collagen type 10 alpha1 chain (COL10A1), (D): human runt-related protein 2 gene (RUNX2). HA: hydroxyapatite, PLLA: poly-L-actic acid. n = 3–5. The red line represents the respective gene expression level of the gene in a monolayer normalized to 1. Statistical analysis was performed with One-way ANOVA and Tukey’s multiple comparison test.