BMP12 and BMP13 gene transfer induce ligamentogenic differentiation in mesenchymal progenitor and anterior cruciate ligament cells

Abstract

Background aims: To date there are only very few data available on the ligamentogenic differentiation capacity of mesenchymal stromal/progenitor cells (MSC) and anterior cruciate ligament (ACL) fibroblasts.

Methods: We describe the in vitro potential of MSC and ACL cells to undergo ligamentogenic differentiation upon transduction with adenoviral vectors encoding the human cDNA for bone morphogenetic protein (BMP) 12 and BMP13, also known as growth and differentiation factors (GDF) 6 and 7, respectively.

Results: Transgene expression for at least 14 days was confirmed by Western blot analyzes. After 21 days of cell culture within collagen type I hydrogels, histochemical (hematoxylin/eosin (H&E), Azan and van Gieson), immunohistochemical and polymerase chain reaction (PCR) analyzes of the genetically modified constructs of both cell types revealed elongated, viable fibroblast-like cells embedded in a ligament-like matrix rich in collagens, vimentin, fibronectin, decorin, elastin, scleraxis, tenascin, and tenomodulin.

Conclusions: It appears that both MSC and ACL fibroblasts are capable of ligamentogenic differentiation with these factors. This information may aid in the development of biologic approaches to repair and restore ACL after injury.

Figure 1

Cell proliferation and viability in BMP12- and BMP13-modified ligament constructs. (A) Western blot analyzes to detect BMP12 and BMP13 in Ad.BMP12- and Ad.BMP13-transduced MSC at MOI 100 after 3, 7 and 14 days. (B) Time–course of cell proliferation by MSC and ACL fibroblasts following transduction with adenovirus encoding BMP12 and BMP13. Analyzes of cell vitality and apoptosis in MSC (C–H) and ACL fibroblasts (I–N) 3 days after transduction with Ad.BMP12 (D,G) and Ad.BMP13 (E,H) compared with the control (C,F). The cells were double-stained with 6-CFDA and Annexin V-Cy3, allowing for discrimination between living cells stained green with 6-CFDA, necrotic cells stained red with Annexin V-Cy3, and apoptotic cells stained for both. Bar = 20 μm. Original magnifications ×100.

Figure 2

Histochemical analyzes of BMP12- and BMP13-modified MSC and ACL hydrogel constructs. (A) H&E staining, (B) Azan staining and (C) Van Gieson staining. Bar = 100 μm. Original magnifications ×200.

Figure 3

Immunohistochemical analyzes of BMP12- and BMP13-modified MSC and ACL hydrogel constructs. Staining for (A) COL III, (B) FN, (C) TN and (D) VIM. Positive immunostainings are indicated by the brown-colored network in the vicinity of the blue-colored cells (stained with hematoxylin). Bar = 100 μm. Original magnifications ×200.

Figure 4

RT-PCR analyzes of ligament-specific marker gene expression of BMP12- and BMP13-modified MSC (left) and ACL fibroblast (right) collagen hydrogel constructs. Densitometry of PCR products of Ad.BMP12- (A, B) and Ad.BMP13- (C, D) transduced MSC (A, C) and ACL fibroblast (B, D) collagen hydrogel constructs. Fold changes of BGN, COL III, COL V, DCN, ELA, FN, SCL, TN-C, TNMD and VIM are shown. Gene expression was normalized to the housekeeping gene EF1α and related to controls. Mean values ± SD are derived from three patients. Differences compared with the control were considered significant for P < 0.05 (*).