Injured Achilles Tendons Treated with Adipose-Derived Stem Cells Transplantation and GDF-5

Abstract

Tendon injuries represent a clinical challenge in regenerative medicine because their natural repair process is complex and inefficient. The high incidence of tendon injuries is frequently associated with sports practice, aging, tendinopathies, hypertension, diabetes mellitus, and the use of corticosteroids. The growing interest of scientists in using adipose-derived mesenchymal stem cells (ADMSC) in repair processes seems to be mostly due to their paracrine and immunomodulatory effects in stimulating specific cellular events. ADMSC activity can be influenced by GDF-5, which has been successfully used to drive tenogenic differentiation of ADMSC in vitro. Thus, we hypothesized that the application of ADMSC in isolation or in association with GDF-5 could improve Achilles tendon repair through the regulation of important remodeling genes expression. Lewis rats had tendons distributed in four groups: Transected (T), transected and treated with ADMSC (ASC) or GDF-5 (GDF5), or with both (ASC+GDF5). In the characterization of cells before application, ADMSC expressed the positive surface markers, CD90 (90%) and CD105 (95%), and the negative marker, CD45 (7%). ADMSC were also differentiated in chondrocytes, osteoblast, and adipocytes. On the 14th day after the tendon injury, GFP-ADMSC were observed in the transected region of tendons in the ASC and ASC+GDF5 groups, and exhibited and/or stimulated a similar genes expression profile when compared to the in vitro assay. ADMSC up-regulated Lox, Dcn, and Tgfb1 genes expression in comparison to T and ASC+GDF5 groups, which contributed to a lower proteoglycans arrangement, and to a higher collagen fiber organization and tendon biomechanics in the ASC group. The application of ADMSC in association with GDF-5 down-regulated Dcn, Gdf5, Lox, Tgfb1, Mmp2, and Timp2 genes expression, which contributed to a lower hydroxyproline concentration, lower collagen fiber organization, and to an improvement of the rats' gait 24 h after the injury. In conclusion, although the literature describes the benefic effect of GDF-5 for the tendon healing process, our results show that its application, isolated or associated with ADMSC, cannot improve the repair process of partial transected tendons, indicating the higher effectiveness of the application of ADMSC in injured Achilles tendons. Our results show that the application of ADMSC in injured Achilles tendons was more effective in relation to its association with GDF-5.

Keywords: biomechanics; collagen; extracellular matrix; gait; gene expression; repair.

Figure 1

In vitro adipogenic (A), condrogenic (B), and osteogenic (C) differentiation of adipose-derived mesenchymal stem cells (ADMSC) in 5P: Observe intracellular lipid droplets stained with Sudan IV (→), proteoglycans stained with toluidine blue (▶), and extracellular calcium stained with alizarin red S (▶). Bars = A and C: 200 μm; B: 100 μm. (D) Histograms demonstrate the x-axis fluorescence scale considered positive when the cell peak is above 10¹ (CD45) or 10² (CD105 and CD90). (E) Control for –PE and –APC (with very low fluorescence), corresponding to non-marked cells. (F) Flow cytometry of ADMSC for CD105, CD90, and CD45 surface markers.

Figure 2

Morphology of ADMSC on 5P: (A) ADMSC-GFP: Fibroblast-like morphology, with a fusiform shape. (B) Contrast by Differential Interference (DIC): The red to blue band showing the interference effect of the nucleus due to higher concentrations of material. Nucleoli presence seen in the initial of the blue band (▶). (C) ADMSC stained with AT (pH 4.3): Note the presence of nuclei stained in blue (→), and the presence of granules of various sizes highly stained in blue in some regions (▶) due to the disponibility of phosphate groups. (D) Polarization microscopy: In the detailed image, note the abnormal interference colors due to abnormal dispersion of birefringence because of the differences in the high packing of DNA. Bars = 50 μm (A), 70 μm (B,C), and 35 μm (D). (E) Number of ADMSC used for application in tendons, presenting about 80% viability after tripsinization. (F) RT-PCR array of ADMSC on 5P in vitro showing the expression profile of the genes Lox, Dcn, Timp2, Mmp2, and Tgfb1. No expression was observed for Scx, Tnmd, Mmp9, Gdf5, Tnf, and Ilb1 genes.

Figure 3

ADMSC-GFP Migration to TR on the 3rd and 14th days after injury: Observe the presence of ADMSC-GFP (A,D,G,J) in the ASC and ASC+GDF5 groups. Visible higher numbers of cells can be observed on the 14th day in both groups. DAPI: Nuclei marking (B,E,H,K). Merged images of ADMSC-GFP with nuclei marked with DAPI (C,F,I,L). Bar = 50 μm.

Figure 4

RT-PCR array for expression analysis of 12 genes in transected tendons: 50% of the genes analyzed were altered. The ADMSC increased expression of Lox, Dcn, and Tgfb1 when compared to the other groups. Compared only to the ASC+GDF5 group, ADMSC increased the expression of Mmp2, Timp2, and Gdf5. The same letter between the groups corresponds to a significant difference between them.

Figure 5

Tendons hydroxyproline concentration: Observe the lower value in the ASC+GDF5 group in relation to the other groups. The same letter among groups corresponds to a significant difference between them (p ≤ 0.05).

Figure 6

Images of tendons using polarization microscopy. (A) group N: Birefringence of the collagen fibers in the proximal region of the calanear tendon. The variation in gray levels is due to the crimp and the degree of aggregation of the collagen fibers (); observe the crimp (B) by positioning the largest axis of the tendon parallel to one of the polarizers: The same region observed in (A). (C) Panoramic image of the transected tendon for identification of the transection region (TR) and the proximal and distal transition region (T1). Groups T (D–F), ASC (G–I), GDF5 (J–L), and ASC+GDF5 (M–O). Observe the complete disorganization of collagen fibers in TR. The TR from different groups (D,G,J,M): Observe freshly formed collagen fibrils and an overlapping (↘) of this region with the thicker fibers present in T1. T1 (E,H,K,N): Collagen fibers with a greater organization in relation to TR, however, with fragmentation presence (◢) mainly in groups T (E), GDF5 (K), and ASC+GDF5 (N). Crimp (F,I,L,O) from the collagen fibers observed on T1: Observe similar undulation patterns of the collagen fibers between the groups, represented by light and dark regions. The largest axis of the tendon was positioned at 45° in relation to the crossed polarizers as parallel to one of the polarizers (B,F,I,L,O). (P) TR birefringence measurements in T1: Same letter represents significant differences between groups (p ≤ 0.05). (Q) Histogram of the frequency and birefringence values showing differences in the distribution of values in the different groups. Bars = 100 μm and 200 μm (a).

Figure 7

Linear dichroism (DL) of AT-stained tendon sections and analyzed under polarization microscopy. The largest axis of the tendon was placed in the parallel (B,D,F,H) and perpendicular (C,E,G,I) position relative to the polarized light plane. Observed DL is typically more intense in the cuts in the perpendicular position. Group N (A) is observed under common light microscopy due to the low amount of PG. (J) Dichroic index (ID) calculated through linear dichroism measurements (absorbance) performed on tendons TR: Greatest value observed in group T. Same letter between groups corresponds to a significant difference between them (p ≤ 0.05). Bar = 100 μm.

Figure 8

(A) Maximum contact intensity of the paw of the animals during walking, obtained through the CatWalk system. The measurements were taken three days before the injury to obtain the normal walk pattern, and on the 1st, 3rd, 5th, 7th, 9th, 11th, and 13th days after the tendon transection. Observe the marked decrease in contact pressure of the animals’ paw on the day after surgery, with a higher value of the ASC+GDF5 group compared to the T group. Except for the GDF5 group, observe the complete recovery of the normal walk pattern of the animals in the other groups on the 13th day. (B) Comparisons between groups with significant differences observed between the 1st and 13th days.

Figure 9

(A) Biomechanical properties of the tendons: Significant differences can be observed between the groups for the parameters of maximum load, displacement, and strain. The same letter between the groups corresponds to a significant difference between them (p ≤ 0.001). (B) Stress-strain curve: Tendons treated with ADMSC presented lower deformation at higher stress in comparison to the other groups. Standard deviations are represented by vertical bars.