Use of a biological reactor and platelet-rich plasma for the construction of tissue-engineered bone to repair articular cartilage defects

Abstract

Articular cartilage defects are a major clinical burden worldwide. Current methods to repair bone defects include bone autografts, allografts and external fixation. In recent years, the repair of bone defects by tissue engineering has emerged as a promising approach. The present study aimed to assess a novel method using a biological reactor with platelet-rich plasma to construct tissue-engineered bone. Beagle bone marrow mesenchymal stem cells (BMSCs) were isolated and differentiated into osteoblasts and chondroblasts using platelet-rich plasma and tricalcium phosphate scaffolds cultured in a bioreactor for 3 weeks. The cell scaffold composites were examined by scanning electron microscopy (SEM) and implanted into beagles with articular cartilage defects. The expression of osteogenic markers, alkaline phosphatase and bone γ-carboxyglutamate protein (BGLAP) were assessed using polymerase chain reaction after 3 months. Articular cartilage specimens were observed histologically. Adhesion and distribution of BMSCs on the β-tricalcium phosphate (β-TCP) scaffold were confirmed by SEM. Histological examination revealed that in vivo bone defects were largely repaired 12 weeks following implantation. The expression levels of alkaline phosphatase (ALP) and BGLAP in the experimental groups were significantly elevated compared with the negative controls. BMSCs may be optimum seed cells for tissue engineering in bone repair. Platelet-rich plasma (PRP) provides a rich source of cytokines to promote BMSC function. The β-TCP scaffold is advantageous for tissue engineering due to its biocompatibility and 3D structure that promotes cell adhesion, growth and differentiation. The tissue-engineered bone was constructed in a bioreactor using BMSCs, β-TCP scaffolds and PRP and displayed appropriate morphology and biological function. The present study provides an efficient method for the generation of tissue-engineered bone for cartilage repair, compared with previously used methods.

Keywords: bioreactor; bone regeneration; bone repair; bone tissue engineering; platelet-rich plasma.

Figure 1.

Perfusion bioreactor design used in the present study.

Figure 2.

Preparation of platelet-rich plasma.

Figure 3.

Cell-scaffold placement into the bioreactor.

Figure 4.

Bone marrow stem cells from beagle bone marrow (A) 3 days after seeding, (B) primary cells cultured for 7 days, and (C) 7 days following subculture (magnification, ×100).

Figure 5.

Type II collagen immunohistochemical staining. (A) In the chondroblast-induced group, the cells displayed brownish yellow granules in the cytoplasm following type II collagen immunohistochemical staining (magnification, ×200). (B) In the osteoblast-induced group (magnification, ×200), gray-black granules or black deposit were observed in the (C) cytoplasm (magnification, ×200) and (D) Alizarin red staining revealed a large number of mineral nodes in the osteoblast-induced group (magnification, ×400).

Figure 6.

(A) Scanning electron microscopy showed the structure of the β-tricalcium phosphate scaffold. (B) Scanning electron microscopy revealed good adhesion and distribution of beagle bone marrow mesenchymal stem cells on the β-tricalcium phosphate scaffold. (magnification, ×5,000).

Figure 7.

Histological staining revealing cartilage defects following implantation of BMSC scaffolds. The tissue samples were sectioned and stained with hematoxylin and eosin. (A) BMSCs cultured on scaffolds in the presence of PRP in a bioreactor. (B) BMSCs cultured on scaffolds without the use of the bioreactor. (C) BMSCs cultured in the bioreactor without PRP. (D) BMSCs cultured in the absence of the bioreactor or PRP. (E) Negative controls: Scaffold with no BMSCs. BMSCs, bone marrow stem cells; PRP, platelet-rich plasma.

Figure 8.

Gene expression levels of (A) ALP and (B) BGLAP2 were detected in the various experimental groups using reverse transcription-quantitative polymerase chain reaction. *P<0.05 vs. control group (group E); ^#P<0.05. Group A, BMSCs cultured with PRP in the bioreactor; Group B, BMSCs cultured with PRP without the use of the bioreactor; Group C, BMSCs cultured in the bioreactor without PRP; Group D, BMSCs cultured without PRP or use of the bioreactor; Group E, β-tricalcium phosphate scaffold only. ALP, alkaline phosphatase; BGLAP2, bone γ-carboxyglutamate protein 2; ALP, alkaline phosphatase; BGLAP2, bone γ-carboxyglutamate protein; BMSC, bone marrow stem cell; PRP, platelet-rich plasma.