A bilayer scaffold of collagen and nanohydroxyapatite promotes osteochondral defect in rabbit knee joints

Abstract

Aims: A large number of surgical operations are available to treat osteochondral defects of the knee. However, the knee joint arthroplasty materials cannot completely mimic the articular cartilage and subchondral bone, which may bring some obvious side effects. Thus, this study proposed a biocompatible osteochondral repair material prepared from a double-layer scaffold of collagen and nanohydroxyapatite (CHA), consisting of collagen hydrogel as the upper layer of the scaffold, and the composite of CHA as the lower layer of the scaffold.

Methods: The CHA scaffold was prepared, and properties including morphology, internal structure, and mechanical strength of the CHA scaffold were measured by scanning electron microscopy (SEM) and a MTS electronic universal testing machine. Then, biocompatibility and repair capability of the CHA scaffold were further evaluated using a rabbit knee cartilage defect model.

Results: The CHA scaffold was well suited for the repair of articular cartilage and subchondral bone; the in vitro results showed that the CHA scaffold had good cytocompatibility. In vivo experiments demonstrated that the material had high biocompatibility and effectively induced cartilage and subchondral bone regeneration.

Conclusion: The CHA scaffold has a high potential for commercialization and could be used as an effective knee repair material in clinical applications.

Fig. 1

Preparation diagram of collagen nanohydroxyapatite (CHA) scaffold. The collagen obtained was mixed with hydroxyapatite (HA) and gradient frozen overnight, then collagen solution was added to the frozen material, which continued de-gradient freezing and was dried in a vacuum freeze dryer.

Fig. 2

Characterization of collagen nano-hydroxyapatite (CHA) scaffolds. a) Morphological observation of double-layer scaffold of CHA. b) Mechanical property of CHA scaffolds. c) and d) Water contact angle of collagen, hydroxyapatite (HA), and CHA scaffold. e) Scanning electron microscopy (SEM) images of CHA scaffolds. f) Fourier-transform infrared spectroscopy (FITR) of collagen, HA, and CHA scaffold.

Fig. 3

Biocompatibility of collagen (Col) nano-hydroxyapatite (CHA) scaffolds. a) Neutral red staining of bone marrow mesenchymal stem cells (BMSCs) and chondrocytes in the medium containing collagen scaffolds or CHA scaffold. b) and c) Cell Counting Kit-8 (CCK-8) assay was used to test the effect of collagen scaffold and CHA scaffold on cell proliferation, containing chondrocytes and BMSCs. d) Observation of cell migration in the collagen scaffold and CHA scaffold medium. e) and f) Quantification of migration area rate on BMSCs and chondrocytes cultured in the medium containing collagen scaffold or CHA scaffold. OD, optical density.

Fig. 4

Evaluation of collagen nano-hydroxyapatite (CHA) scaffold on chondral injury. a) Model of knee joint cartilage defect. b) Gross observation of the repaired cartilage tissues two months postoperatively. c) Micro-CT of new regenerated bone. d) Quantification of bone volume/total volume (BV/TV).

Fig. 5

Histological staining of bone tissues. CHA, collagen-nanohydroxyapatite scaffold; HE, haemotoxylin and eosin; Masson, Masson’s trichrome staining; S-O, safranin O; TB, Toluidine blue.

Fig. 6

Histological staining of bone tissues. a) Immunofluorescence staining of new bone tissue with collagen II and collagen I. Quantitative analysis of b) collagen II and c) collagen I positive area. CHA, collagen-nanohydroxyapatite scaffold.