Platelets and plasma stimulate sheep rotator cuff tendon tenocytes when cultured in an extracellular matrix scaffold

Abstract

The addition of platelet-rich plasma (PRP) to rotator cuff repair has not translated into improved outcomes after surgery. However, recent work stimulating ligament healing has demonstrated improved outcomes when PRP or whole blood is combined with an extracellular matrix carrier. The objective of this study was to evaluate the effect of three components of blood (plasma, platelets, and macrophages) on the in vitro activity of ovine rotator cuff cells cultured in an extracellular matrix environment. Tenocytes were obtained from six ovine infraspinatus tendons and cultured over 14 days in an extracellular matrix scaffold with the following additives: (1) plasma (PPP), (2) plasma and platelets (PAP), (3) plasma and macrophages (PPPM), (4) plasma, platelets and macrophages (PAPM), (5) phosphate buffered saline (PBS), and (6) PBS with macrophages (PBSM). Assays measuring cellular metabolism (AlamarBlue), proliferation (Quantitative DNA assay), synthesis of collagen and cytokines (SIRCOL, TNF-α and IL-10 ELISA, and MMP assay), and collagen gene expression (qPCR) were performed over the duration of the experiment, as well as histology at the conclusion. Plasma was found to stimulate cell attachment and spreading on the scaffold, as well as cellular proliferation. Platelets also stimulated cell proliferation, cellular metabolism, transition of cells to a myofibroblast phenotype, and contraction of the scaffolds. The addition of macrophages did not have any significant effect on the sheep rotator cuff cells in vitro. In vivo studies are needed to determine whether these changes in cellular function will translate into improved tendon healing.

Keywords: fibroblast; in vitro; macrophage; platelet-rich plasma; rotator cuff; sheep.

Figure 1

Proportion of AlamarBlue reduced (n=6 for all groups) over the course of the experiment. Error bars represent standard error. * = significant difference (P<0.05).

Figure 2

Amount of DNA (ng) measured by PicoGreen DNA assay at day 14. N as indicated. Error bars represent standard error. Bars capped with different letters significantly different at P<0.05.

Figure 3

Amount of collagen (μg/ml) measured by SIRCOL at day 1 (n=6 for all groups). Error bars represent standard error. Bars capped with different letters significantly different at P<0.05.

Figure 4

Minimum width of constructs on day 14. Error bars represent standard error. N as indicated. Bars capped with different letters significantly different at P<0.05.

Figure 5

Representative histology of constructs at day 14 (200x magnification). Note the number and morphology of fibroblasts (arrows), and the increased positive IHC staining (brown) for α-SMA actin in the PPP and PAP groups. Scale bars = 20μm.