Higher BMP receptor expression and BMP-2-induced osteogenic differentiation in tendon-derived stem cells compared with bone-marrow-derived mesenchymal stem cells

Abstract

Purpose: Surgical reattachment of tendon to bone often fails due to regeneration failure of the specialised tendon-bone junction (TBJ). The use of mesenchymal stem cells for TBJ regeneration has been reported with promising results. Tendon-derived stem cells (TDSCs) with high proliferative and multi-lineage differentiation potential have been isolated. As stem cells residing in tendons, TDSCs can be considered a new cell source for TBJ repair. Bone morphogenic protein 2 (BMP-2) is a potent osteogenic factor with roles in normal bone healing and pathological ectopic bone formation in soft tissues. The use of BMP-2 to promote TBJ repair has been well reported. This study aimed to compare TDSCs to the gold standard bone-marrow-derived mesenchymal stem cells (BMSCs) with respect to osteogenic response to BMP-2 in vitro.

Method: The clonogenicity and multi-differentiation potential of TDSCs and BMSCs were identified by colony-forming-unit assay, osteogenic, adipogenic and chondrogenic differentiation assays. Their osteogenic response to BMP-2 in vitro was examined by alkaline phosphatase (ALP) cytochemical staining, ALP activity assay and Alizarin red S staining of calcium nodule formation. Messenger RNA (mRNA) and BMP receptor (types IA, IB and II) protein expression were examined by quantitative real-time reverse-transcriptase polymerase chain reaction (qRT-PCR) and Western blotting.

Results: Our results showed that both TDSCs and BMSCs exhibited stem cell properties, including clonogenicity and multi-differentiation potential. TDSCs expressed higher mRNA and protein levels of BMP receptors IA, IB and II. They also exhibited higher osteogenic differentiation with and without BMP-2 stimulation compared with BMSCs.

Conclusions: TDSCs with/without BMP-2 might be an attractive source for TBJ repair compared with BMSCs.

Fig. 1

Clonogenicity and multi-lineage differentiation potential of tendon-derived and bone-marrow-derived mesenchymal stem cells (TDSCs and BMSCs). a, c Formation of cell colonies as indicated by crystal violet staining. Magnification ×1. b, d Chondrogenic differentiation of TDSCs and BMSCs: cell pellet of TDSCs (b) and BMSCs (d) expressed proteoglycan, as indicated by positive Safranin O/fast green staining. Magnification ×200. e–h Osteogenic differentiation of TDSCs and BMSCs: calcium nodules formed only when TDSCs (e, f) and BMSCs (g, h) were cultured in osteogenic medium (e, g) compared with basal medium (f, h), as indicated by Alizarin red S staining. Magnification ×100. i–l Adipogenic differentiation of TDSCs and BMSCs. Lipid droplets formed only when TDSCs (i, j) and BMSCs (k, l) were cultured in adipogenic medium (i, k) compared with basal medium (j, l), as shown by Oil red O staining. Magnification ×100

Fig. 2

Alkaline phosphatase (ALP) cytochemical staining in tendon-derived and bone-marrow-derived mesenchymal stem cells (TDSCs and BMSCs) at day 3 treated with and without bone morphogenic protein 2 (BMP-2) stimulation. TDSCs treated a–b without and e–f with BMP-2 showed higher ALP cytochemical activity staining than BMSCs treated c–d without and g–h with BMP-2. a, c, e, g magnification ×1; b, d, f, h magnification ×100. Scale bar =100 μm. i ALP activity in TDSCs and BMSCs at day 3 with and without BMP-2 induction. *p ≤ 0.050

Fig. 3

Alizarin red S staining and quantification of Alizarin red S bound to calcium nodules in tendon-derived and bone-marrow-derived mesenchymal stem cells (TDSCs and BMSCs) treated with and without bone morphogenic protein 2 (BMP-2) for 10 days. a–h Alizarin red S staining of calcium nodules. i Intensity of Alizarin red S stain bound to calcium nodules. TDCSs treated a–b without and e–f with BMP-2 showed higher Alizarin-red-S-bound calcium nodules than BMSCs treated c–d without and g–h with BMP-2. a, c, e, g magnification ×1; b, d, f, h) magnification ×100. Scale bar =100 μm. *p ≤ 0.050

Fig. 4

Messenger RNA expressions of aBMPR-IA, bBMPR-IB and cBMPR-II in tendon-derived and bone-morphogenic-derived mesenchymal stem cells ( TDSCs and BMSCs). *p ≤ 0.050

Fig. 5

a–c BMPR-IA, BMPR-IB, and BMPR-II protein expression in tendon-derived and bone-morphogenic-derived mesenchymal stem cells (TDSCs and BMSCs). d–f Semiquantitative image analysis results. *p ≤ 0.050