Evaluation of early cellular influences of bone morphogenetic proteins 12 and 2 on equine superficial digital flexor tenocytes and bone marrow-derived mesenchymal stem cells in vitro

Abstract

Objective: To evaluate early cellular influences of bone morphogenetic protein (BMP)12 and BMP2 on equine superficial digital flexor tenocytes (SDFTNs) and equine bone marrow-derived mesenchymal stem cells (BMDMSCs).

Animals: 9 adult clinically normal horses.

Procedures: BMDMSCs and SDFTNs were cultured in monolayer, either untreated or transduced with adenovirus encoding green fluorescent protein, adenovirus encoding BMP12, or adenovirus encoding BMP2. Cytomorphologic, cytochemical, immunocytochemical, and reverse transcriptase-quantitative PCR (RT-qPCR) analyses were performed on days 3 and 6. Genetic profiling for effects of BMP12 was evaluated by use of an equine gene expression microarray on day 6.

Results: BMDMSCs and SDFTNs had high BMP12 gene expression and remained viable and healthy for at least 6 days. Type l collagen immunocytochemical staining for SDFTNs and tenocyte-like morphology for SDFTNs and BMDMSCs were greatest in BMP12 cells. Cartilage oligomeric matrix protein, as determined via RT-qPCR assay, and chondroitin sulfate, as determined via gene expression microarray analysis, were upregulated relative to control groups in SDFTN-BMP12 cells. The BMDMSCs and SDFTNs became mineralized with BMP2, but not BMP12. Superficial digital flexor tenocytes responded to BMP12 with upregulation of genes relevant to tendon healing and without mineralization as seen with BMP2.

Conclusions and clinical relevance: Targeted equine SDFTNs may respond to BMP12 with improved tenocyte morphology and without mineralization, as seen with BMP2. Bone marrow-derived mesenchymal stem cells may be able to serve as a cell delivery method for BMP12.

Figure 1

Photomicrographs of pluripotent equine BMDMSCs (A). The cells are undergoing mineralization, seen as alizarin red-stained nodules, in osteogenic differentiation media (B). Cytochemical stain; bars = 100 μm (A) and 20 μm (B).

Figure 2

Photomicrographs of SDFTNs (A; light microscopy; bar = 20 μm) seen as GFP-positive cells via fluorescent microscopy (B; bar = 20 μm). Quantitative assessment of transduction efficiency via flow cytometry analysis; representative scatterplots of control SDFTNs (C) and SDFTN-GFP cells (D) are illustrated. Notice > 95% transduction efficiency in SDFTN-GFP samples. P2 = Region of cells lacking detectable fluorescent activity on the FITC-A channel. P3 = Region of detectable fluorescent activity in analyzed cells. FITC-A = Designated laser used to detect fluorescent activity. Y-axis = Channel numbers detected by use of the indicated laser. X-axis = Number of cells analyzed.

Figure 3

Morphology scores on cell culture days 3 and 6 for equine BMDMSCs (A, B) and SDFTNs (C, D) transduced with adenovirus encoding GFP, BMP12, or BMP2. In A, BMDMSC-BMP12 cells are significantly (P < 0.001) more elongated, compared with that of other groups. In B, BMDMSC-BMP12 cells are significantly more elongated, compared with that of BMDMSC -GFP cells (P < 0.001) and untreated control cells (P < 0.01), respectively. In C, SDFTN-BMP12 cells are significantly (P < 0.001) more elongated, compared with that of other groups. In D, SDFTN-BMP12 cells and SDFTN-BMP2 cells are significantly (P < 0.01) more elongated, compared with that of other groups.

Figure 4

Graph of gene expression (via RT-qPCR assay) for alkaline phosphatase in equine BMDMSCs. On cell culture day 6, the value for BMDMSC-BMP2 cells was significantly (P < 0.01) greater, compared with results for other groups.

Figure 5

Photomicrographs of equine BMDMSC-BMP2 cells stained for alkaline phosphatase on cell culture day 3 (A; alkaline phosphatase stain; bar = 20 μm) and stained for focal mineral formation on day 6 (B; von Kossa stain; bar = 20 μm).

Figure 6

Type I collagen in equine SDFTNs (A, C) and BMDMSCs (B, D). On cell culture day 6, SDFTN-BMP12 and BMP2 cells had significantly (P < 0.001) greater type I collagen staining intensity, compared with that of the other groups (A). On cell culture day 6, BMDMSC-BMP12 cells had significantly (P < 0.001) greater staining intensity, compared with that of the other groups (B). C—Photomicrograph of SFTN-BMP12 cells containing type I collagen. Immunohistochemical stain; bar = 20 μm. D—Photomicrograph of BMD-MSC-BMP12 cells containing type I collagen. Immunohistochemical stain; bar = 20 μm.

Figure 7

Gene expression of BMP12 in equine BMDMSCs (A, B) and SDFTNs (C, D). A—On cell culture day 3, gene expression was significantly greater in BMDMSC-BMP12 cells than in untreated control cells, BMDSMC-BMP2 cells (P < 0.01), or BMDMSC-GFP cells (P < 0.05). B—On cell culture day 6, gene expression was significantly (P < 0.001) greater in BMDMSC-BMP12 cells than in other groups. C—On cell culture day 3, gene expression was significantly (P < 0.001) greater in SDFTN-BMP12 cells, compared with that of other groups. D—On cell culture day 6, gene expression was significantly (P < 0.01) greater in SDFTN-BMP12 cells, compared that of with other groups.

Figure 8

Gene expression of COMP in equine SDFTNs. On cell culture day 6, gene expression was significantly (P < 0.001) greater for SDFTN-BMP12 cells, compared with that of other groups.