Cartilage Formation In Vivo Using High Concentration Collagen-Based Bioink with MSC and Decellularized ECM Granules

Abstract

The aim of this study was to verify the applicability of high-concentration collagen-based bioink with MSC (ADSC) and decellularized ECM granules for the formation of cartilage tissue de novo after subcutaneous implantation of the scaffolds in rats. The printability of the bioink (4% collagen, 2.5% decellularized ECM granules, derived via 280 μm sieve) was shown. Three collagen-based compositions were studied: (1) with ECM; (2) with MSC; (3) with ECM and MSC. It has been established that decellularized ECM granules are able to stimulate chondrogenesis both in cell-free and MSC-laden scaffolds. Undesirable effects have been identified: bone formation as well as cartilage formation outside of the scaffold area. The key perspectives and limitations of ECM granules (powder) application have been discussed.

Keywords: ECM; MSC; bioink; bioprinting; cartilage; collagen.

Figure 1

Study design. dECM powder was obtained from rat costal cartilage by decellularization, lyophilization and milling. Bioink containing sterile type I pig atelocollagen, dECM powder and human MSC were used to print scaffolds. The scaffolds were implanted under the skin in the withers area in 12 rats. The histological and immunohistochemical studies of material from animals was done on days 7 and 14 after implantation.

Figure 2

Costal rat cartilage. (a) native cartilage, staining with hematoxylin and eosin, objective lens ×20, scale bar—100 μm; (b) decellularized sample, staining with hematoxylin and eosin, objective lens ×10, scale bar—200 μm; (c) decellularized sample, staining with hematoxylin and eosin, objective lens ×20, scale bar—100 μm. Perichondrium cells are shown by arrows; (d) decellularized sample, staining with alcian blue, objective lens ×10, scale bar—200 μm.

Figure 3

Frequency distribution of dECM granules diameter, n = 188.

Figure 4

Input model of test printing bioink printability assessment.

Figure 5

ADSC after 48 h of cultivation. Phase contrast: (a) objective lens ×4, scale bar—800 μm; (b) objective lens ×10, scale bar—200 μm.

Figure 6

1st group (dECM only), 1 week. A connective tissue capsule surrounding the scaffold (Sc). Staining with hematoxylin and eosin, objective lens ×10, scale bar—200 μm. Cavities filled with fibrillar material are shown by arrows.

Figure 7

1st group (dECM only), 1 week. PCNA-positive multinucleated cells resorb around the perimeter of the scaffold in the connective tissue capsule (arrows). Immunohistochemical staining for PCNA, objective lens ×40, scale bar—50 μm.

Figure 8

Cartilage tissue in animals of different experimental groups, objective lens ×20, scale bar—100 μm. Explanation in the text. (A1–D1)—1st group (dECM only), 1 week; (A2–D2)—2nd group (MSC only), 2 weeks; (A3–D3)—3rd group (dECM and MSC), 1 week; (A4–D4)—3rd group (dECM and MSC), 2 weeks. (A)—staining with hematoxylin and eosin; (B)—staining for PCNA; (C)—immunohistochemical staining for type II collagen; (D)—staining with alcian blue.

Figure 9

1st group (dECM only), 1 week. (a) cartilage tissue. Staining with hematoxylin and eosin, objective lens ×20, scale bar—100 μm; (b) formation of an osteoid with randomly located osteoblasts (arrows) in muscle tissue near the scaffold. Staining with hematoxylin and eosin, objective lens ×40, scale bar—50 μm.

Figure 10

1st group (dECM only), 2 weeks. Connective tissue capsule (arrows) around the scaffold (Sc). Staining with hematoxylin and eosin. (a) objective lens ×10, scale bar—200 μm; (b) objective lens ×20, scale bar—100 μm. In the lower right corner, the remains of suture material, which marked the site of the implantation (asterics).

Figure 11

1st group (dECM only), 2 weeks. Collagen type II in the cytoplasm of multinucleated cells of foreign body resorption (arrows) in the connective tissue capsule. Diffuse background—scaffold material (Sc). Staining for type II collagen (asterisk). (a) objective lens ×20, scale bar—100 μm; (b) objective lens ×40, scale bar—50 μm.

Figure 12

1st group (dECM only), 2 weeks. Collagen type II in the cytoplasm of multinucleated cells of foreign body resorption and macrophages (arrows) in the connective tissue capsule. Diffuse background—scaffold material (Sc). Staining for type II collagen (asterisk). (a) objective lens ×20, scale bar—100 μm; (b) objective lens ×40, scale bar—50 μm.

Figure 13

1st group (dECM only), 2 weeks. Staining of white and brown (asterisk) adipose tissue for nuclear antigen of proliferating cells, objective lens ×20, scale bar—100 μm.

Figure 14

2nd group (MSC only), 1 week. Osteoid formation with randomly located osteoblasts near the implant. Staining with hematoxylin and eosin. (a) objective lens ×20, scale bar—100 μm; (b) objective lens ×40, scale bar—50 μm.

Figure 15

2nd group (MSC only), 1 week. Connective tissue capsule (arrows) around the implant. Staining with hematoxylin and eosin. Scaffold region (Sc). (a) objective lens ×2.5, scale bar—800 μm; (b) objective lens ×10, scale bar—200 μm.

Figure 16

2nd group (MSC only), 2 weeks. Eosinophilic infiltrate (arrows) in the connective tissue capsule. Staining with hematoxylin and eosin, objective lens ×40, scale bar—50 μm.

Figure 17

2nd group (MSC only), 2 weeks. Connective tissue capsule (arrows). Collagen type II staining. Macrophages with cytoplasm stained for type 2 collagen. (a) objective lens ×20, scale bar—100 μm; (b) objective lens ×40, scale bar—50 μm.

Figure 18

3rd group (dECM and MSC), 1 week. Connective tissue capsule. Staining with hematoxylin and eosin. Scaffold region (Sc) (a) objective lens ×2.5, scale bar—800 μm; (b) objective lens ×10, scale bar—200 μm.

Figure 19

3rd group (dECM and MSC), 1 week. Cartilage cells with signs of indirect osteogenesis in the muscle tissue near the scaffold. Staining with hematoxylin and eosin. Objective lens ×2.5, scale bar—800 μm.

Figure 20

3rd group (dECM and MSC), 2 weeks. Cartilage tissue in the muscles near the scaffold. Staining with hematoxylin and eosin, objective lens ×10, scale bar—200 μm.

Figure 21

3rd group (dECM and MSC), 2 weeks. Immunohistochemical staining (PCNA) of multinucleated cells of cartilage tissue resorption (arrows), objective lens ×40, scale bar—50 μm.