Osteochondral repair using scaffolds with gradient pore sizes constructed with silk fibroin, chitosan, and nano-hydroxyapatite

Abstract

Background: One of the main problems associated with the development of osteochondral reparative materials is that the accurate imitation of the structure of the natural osteochondral tissue and fabrication of a suitable scaffold material for osteochondral repair are difficult. The long-term outcomes of single- or bilayered scaffolds are often unsatisfactory because of the absence of a progressive osteochondral structure. Therefore, only scaffolds with gradient pore sizes are suitable for osteochondral repair to achieve better proliferation and differentiation of the stem cells into osteochondral tissues to complete the repair of defects.

Methods: A silk fibroin (SF) solution, chitosan (CS) solution, and nano-hydroxyapatite (nHA) suspension were mixed at the same weight fraction to obtain osteochondral scaffolds with gradient pore diameters by centrifugation, freeze-drying, and chemical cross-linking.

Results: The scaffolds prepared in this study are confirmed to have a progressive structure starting from the cartilage layer to bone layer, similar to that of the normal osteochondral tissues. The prepared scaffolds are cylindrical in shape and have high internal porosity. The structure consists of regular and highly interconnected pores with a progressively increasing pore distribution as well as a progressively changing pore diameter. The scaffold strongly absorbs water, and has a suitable degradation rate, sufficient space for cell growth and proliferation, and good resistance to compression. Thus, the scaffold can provide sufficient nutrients and space for cell growth, proliferation, and migration. Further, bone marrow mesenchymal stem cells seeded onto the scaffold closely attach to the scaffold and stably grow and proliferate, indicating that the scaffold has good biocompatibility with no cytotoxicity.

Conclusion: In brief, the physical properties and biocompatibility of our scaffolds fully comply with the requirements of scaffold materials required for osteochondral tissue engineering, and they are expected to become a new type of scaffolds with gradient pore sizes for osteochondral repair.

Keywords: bioscaffolds; bone marrow mesenchymal stem cells; osteochondral defect; tissue engineering.

Figure 1

Schematic of the preparation of the cell–scaffold complex.

Figure 2

Morphology of passage 3 bone marrow mesenchymal stem cells (A, 40×; B, 100×; C, 200×).

Figure 3

Appearance of the silk fibroin/chitosan/nano-hydroxyapatite scaffolds: Scaffold-1 (A), Scaffold-2 (B), and Scaffold-3 (C).

Figure 4

Height (A), diameter (B), and weight (C) of silk fibroin/chitosan/nano-hydroxyapatite scaffolds. Notes: ^aP<0.05, vs Scaffold-1. ^bP<0.05, vs Scaffold-2.

Figure 5

Porosity (A), water swelling rate (B), and hot-water dissolution rate (C) of silk fibroin/chitosan/nano-hydroxyapatite scaffolds. Notes: ^aP<0.05, vs Scaffold-1. ^bP<0.05, vs Scaffold-2.

Figure 6

Hot-water dissolution rate as a function of dissolution time for silk fibroin/chitosan/nano-hydroxyapatite scaffolds.

Figure 7

Hot-water dissolution rate vs dissolution time of the silk fibroin/chitosan/nano-hydroxyapatite scaffolds: (A) Scaffold-1, (B) Scaffold-2, and (C) Scaffold-3.

Figure 8

Stress–strain curves of silk fibroin/chitosan/nano-hydroxyapatite scaffolds: (A) Scaffold-1, (B) Scaffold-2, and (C) Scaffold-3. The averaged curves for comparing the three scaffolds (D). The linear fit curves for comparing the elastic modulus of scaffolds (E).

Figure 9

The water swelling rate of the silk fibroin/chitosan/nano-hydroxyapatite scaffolds with gradient pore diameters before and after compression. Note: ^aP<0.05, vs water swelling rate before compression.

Figure 10

Scanning electron micrographs (×210) showing the internal structure of the silk fibroin/chitosan/nano-hydroxyapatite scaffolds. Abbreviation: L, layer.

Figure 11

Pore size of the silk fibroin/chitosan/nano-hydroxyapatite scaffolds (A and B). Notes: ^aP<0.05, vs L1. ^bP<0.05, vs L2. ^cP<0.05, vs L3. Abbreviation: L, layer.

Figure 12

Cell proliferation curves corresponding to the different layers of the cell-scaffold complex. Note: *P<0.05. Abbreviation: L, layer.

Figure 13

DAPI staining of the cell-scaffold complex after 9 days of culture (A, 100×; B, 200×). Abbreviations: DAPI, 4,6-diamidino-2-phenylindole; L, layer.

Figure 14

Scanning electron microscope observation of cell morphology and distribution of the cell-scaffold complex at 9 days of culture. Abbreviation: L, layer.