Combination of graphene oxide and platelet-rich plasma improves tendon-bone healing in a rabbit model of supraspinatus tendon reconstruction

Abstract

The treatment of rotator cuff tear is one of the major challenges for orthopedic surgeons. The key to treatment is the reconstruction of the tendon-bone interface (TBI). Autologous platelet-rich plasma (PRP) is used as a therapeutic agent to accelerate the healing of tendons, as it contains a variety of growth factors and is easy to prepare. Graphene oxide (GO) is known to improve the physical properties of biomaterials and promote tissue repair. In this study, PRP gels containing various concentrations of GO were prepared to promote TBI healing and supraspinatus tendon reconstruction in a rabbit model. The incorporation of GO improved the ultrastructure and mechanical properties of the PRP gels. The gels containing 0.5 mg/ml GO (0.5 GO/PRP) continuously released transforming growth factor-β1 (TGF-β1) and platelet-derived growth factor (PDGF)-AB, and the released TGF-β1 and PDGF-AB were still at high concentrations, ∼1063.451 pg/ml and ∼814.217 pg/ml, respectively, on the 14th day. In vitro assays showed that the 0.5 GO/PRP gels had good biocompatibility and promoted bone marrow mesenchymal stem cells proliferation and osteogenic and chondrogenic differentiation. After 12 weeks of implantation, the magnetic resonance imaging, micro-computed tomography and histological results indicated that the newly regenerated tendons in the 0.5 GO/PRP group had a similar structure to natural tendons. Moreover, the biomechanical results showed that the newly formed tendons in the 0.5 GO/PRP group had better biomechanical properties compared to those in the other groups, and had more stable TBI tissue. Therefore, the combination of PRP and GO has the potential to be a powerful advancement in the treatment of rotator cuff injuries.

Keywords: controlled release; graphene oxide; platelet-rich plasma; rotator cuff tear; tendon–bone interface healing.

Figure 1.

General observation of gels. (A) PRP gel. (B) 0.25 GO/PRP gel. (C) 0.5 GO/PRP gel. (D) 1.0 GO/PRP gel. (E) PRP gel at 30 s of gelification. (F) 1.0 GO/PRP gel at 30 s of gelification

Figure 2.

(A) SEM images of gels. Scale bar = 100 µm. (B) Degradation of gels. (C) Release of TGF-β1 at each time point. (D) Release of PDGF-AB at each tome point. *P < 0.05 and **P < 0.01 vs PRP

Figure 3.

(A) Cell proliferation of BMSCs cultured with gels using a CCK-8 kit. (B) Images of live–dead staining of BMSCs cultured on gels (green representing living cells and red representing dead cells, scale bar = 100 μm). *P < 0.05 and **P < 0.01 vs blank. ^#P < 0.05 and ^##P < 0.01 vs PRP

Figure 4.

(A) The expressions of genes related to osteogenic differentiation analyzed by qRT-PCR. (B) Immunofluorescence staining images of OCN expression at 14 d. (C) The expressions of genes related to chondrogenic differentiation analyzed by qRT-PCR. (D) Immunofluorescence staining images of col II expression at 21 d. Scale bar = 50 μm. *P < 0.05 and **P < 0.01 vs PRP

Figure 5.

(A) MRI images of specimens. (B) The average signal-to-noise quotient (SNQ) value. (C) μCT results of the specimens. (D–F) Correlative analysis of new bone formation in the region of interest, (D) bone volume fraction (BV/TV), (E) mean trabecular thickness (TbTh) and (F) mean trabecular spacing (TbSp). The tendon–bone interface is marked by an arrow. *P < 0.05 and **P < 0.01 vs blank. ^#P < 0.05 and ^##P < 0.01 vs PRP

Figure 6.

(A) Images of H&E staining of specimens. (B) Images of Masson’s trichrome staining of specimens. (C) Images of picrosirius red staining of specimens. Arrows or if represent tendon–bone interface, t represents tendon, and b represents bone. (D) H&E staining images of livers, lungs, hearts and kidneys at 12 weeks post surgery. (E) The load to failure and stiffness of specimens. Scale bar = 100 µm. *P < 0.05 and **P < 0.01 vs blank. ^#P < 0.05 and ^##P < 0.01 vs PRP