Donor age effects on in vitro chondrogenic and osteogenic differentiation performance of equine bone marrow- and adipose tissue-derived mesenchymal stromal cells

Abstract

Background: Bone marrow (BM)- and adipose tissue (AT)-derived mesenchymal stromal cells (MSCs) have shown potential as cell-based therapies for cartilage and bone injuries and are used increasingly in human and veterinary practice to facilitate the treatment of orthopedic conditions. However, human and rodent studies have documented a sharp decline in chondrogenic and osteogenic differentiation potential with increasing donor age, which may be problematic for the important demographic of older orthopedic patients. The aim of this study was to identify the effect of donor age on the chondrogenic and osteogenic differentiation performance of equine BM- and AT-MSCs in vitro. BM- and AT-MSCs and dermal fibroblasts (biological negative control) were harvested from horses in five different age groups (n = 4, N = 60); newborn (0 days), yearling (15-17 months), adult (5-8 years), middle-aged (12-18 years), and geriatric (≥ 22 years). Chondrogenic differentiation performance was assessed quantitatively by measuring pellet size, matrix proteoglycan levels, and gene expression of articular cartilage biomarkers. Osteogenic differentiation performance was assessed quantitatively by measuring alkaline phosphatase activity, calcium deposition, and gene expression of bone biomarkers.

Results: Chondrogenic and osteogenic differentiation performance of equine BM- and AT-MSCs declined with increasing donor age. BM-MSCs had a higher chondrogenic differentiation performance. AT-MSCs showed minimal chondrogenic differentiation performance in all age groups. For osteogenesis, alkaline phosphatase activity was also higher in BM-MSCs, but BM-MSCs calcium deposition was affected by donor age earlier than AT-MSCs. Chondrogenic and osteogenic differentiation performance of BM-MSCs exhibited a decline as early as between the newborn and yearling samples. Steady state levels of mRNA encoding growth factors, chondrogenic, and osteogenic biomarkers were lower with increasing donor age in both MSC types.

Conclusions: The data showed that chondrogenic and osteogenic differentiation performance of equine BM-MSCs declined already in yearlings, and that AT-MSCs showed minimal chondrogenic potential, but were affected later by donor age with regards to osteogenesis (calcium deposition). The results highlight the importance of donor age considerations and MSC selection for cell-based treatment of orthopedic injuries and will help inform clinicians on when to implement or potentially cryopreserve cells. Moreover, the study provides molecular targets affected by donor age.

Keywords: Aging; Bone; Cartilage; Donor age; Horse; Mesenchymal stromal cells.

Fig. 1

Pellet size of chondrogenic induced cells. Box plot showing pellet size (µm) of chondrogenic induced bone marrow (BM)- and adipose tissue (AT)-derived mesenchymal stromal cells and dermal fibroblasts (DF) in five different age groups (n = 4, N = 60). All pellets were generated from 5 × 10⁵ cells and cultured in chondrogenic induction medium for 21 days. Cell types within the same age group not labeled with the same letter are significantly different from each other (p < 0.05)

Fig. 2

Safranin-O images of chondrogenic induced cells. Panel showing representative Safranin-O stained pellets of bone marrow (BM)- and adipose tissue (AT)-derived mesenchymal stromal cells (MSCs), and dermal fibroblasts (DF) from horses in five different age groups after 21 days of culture in chondrogenic induction medium. All images are taken under 5 × magnifications. The size-bar on the images is equivalent to 250 µm

Fig. 3

Proteoglycan Redness data of chondrogenic induced cells. Box plot showing proteoglycan Redness value of Safranin-O stained pellets of bone marrow (BM)- and adipose tissue (AT)- derived mesenchymal stromal cells and dermal fibroblasts (DF) (biological negative control) in five different age groups after normalization to articular cartilage (n = 4, N = 60). All pellets were cultured in chondrogenic induction medium for 21 days. Cell types within the same age group not labeled with the same letter are significantly different from each other (p < 0.05)

Fig. 4

Alkaline phosphatase activity and Alizarin Red S concentration of osteogenic induced and control cells. Bar plot showing alkaline phosphatase (ALP) activity (A) and Alizarin Red S concentration (B) as a measurement of calcium deposits of equine bone marrow (BM)- and adipose tissue (AT)-derived mesenchymal stromal cells (MSC) and dermal fibroblasts (DF) (biological negative control) in five different age groups after respectively 7 or 21 days of culture in either osteogenic induction medium or expansion medium (non-induced control) (n = 4 horses per cell type per age group per medium). Age groups within the same osteogenic induced cell type not labeled with the same letter are significantly different from each other with regards to calcium deposition (p < 0.05). Error bars indicate standard deviation. ^†N: Newborn, Y: Yearling, A: Adult, M: Middle-aged, G: Geriatric

Fig. 5

Gene expression of selected chondrogenic biomarkers. Bar plots with standard deviation illustrating mean relative gene expression of SRY-box transcription factor 9 (SOX9) (A), cartilage oligomeric matric protein (COMP) (B), collagen type 2 alpha 1 (COL2A1) (C), aggrecan core protein (ACAN) (D), and cartilage-derived retinoic acid-sensitive protein (MIA) (E) in chondrogenic induced pellets and non-induced monolayer controls from bone marrow (BM)- and adipose tissue (AT)-derived mesenchymal stromal cells (MSCs) and dermal fibroblasts (DF) from horses in five different age groups (n = 4 horses per age group per cell type per medium). Age groups within chondrogenic induced BM-MSCs not marked with the same letter are significantly different from each other (p < 0.05). No significant donor age effect was seen for SOX9 or COL2A1. Note different y-axes. ^†N: Newborn, Y: Yearling, A: Adult, M: Middle-aged, G: Geriatric

Fig. 6

Gene expression of selected osteogenic biomarkers. Bar plots with standard deviation illustrating mean relative gene expression levels of runt-related transcription factor 2 (RUNX2) (A), alkaline phosphatase (ALPL) (B), bone morphogenic factor 4 (BMP4) (C), osterix (SP7) (D), and osteocalcin (LOC100146589) (E) in osteogenic induced cells after 21 days of induction and non-induced controls at day 0 and day 21. The cells consist of bone marrow (BM)- and adipose tissue (AT)-derived mesenchymal stromal cells (MSCs) and dermal fibroblasts (DF) from horses in five different age groups (n = 4 horses per age group per cell type per medium). Age groups within the same osteogenic induced cell type not marked with the same letter are significantly different from each other (p < 0.05). Note different y-axes. ^†N: Newborn, Y: Yearling, A: Adult, M: Middle-aged, G: Geriatric