Osteogenic and Chondrogenic Potential of the Supramolecular Aggregate T-LysYal®

Abstract

Hard tissue regeneration represents a challenge for the Regenerative Medicine and Mesenchymal stem cells (MSCs) could be a successful therapeutic strategy. T-LysYal® (T-Lys), a new derivative of Hyaluronic Acid (HA) possessing a superior stability, has already been proved efficient in repairing corneal epithelial cells damaged by dry conditions in vitro. We investigated the regenerative potential of T-Lys in the hard tissues bone and cartilage. We have previously demonstrated that cells isolated from the tooth germ, Dental Bud Stem Cells (DBSCs), differentiate into osteoblast-like cells, representing a promising source of MSCs for bone regeneration. Herewith, we show that T-Lys treatment stimulates the expression of typical osteoblastic markers, such as Runx-2, Collagen I (Col1) and Alkaline Phosphatase (ALP), determining a higher production of mineralized matrix nodules. In addition, we found that T-Lys treatment positively affects α_Vβ₃ integrin expression, key integrin in the osteoblastic commitment, leading to the formation of focal adhesions (FAs). The efficacy of T-Lys was also tested on chondrogenic differentiation starting from human articular chondrocytes (HACs) resulting in an increase of differentiation markers and cell number.

Keywords: bone; cartilage; focal adhesions; mesenchymal stem cells; supramolecular aggregate; tissue regeneration.

Figure 1

T-Lys treatment in DBSCs cultures. (A) Dental bud isolated from a third molar of a healthy patient. Scale bar: 1 cm. (B) Representative phase contrast pictures of DBSCs cultivated in osteogenic medium and stimulated with 0.3% v/v T-Lys (T-Lys) or not (Ctr). Scale bar: 20 μm.

Figure 2

Effects of T-Lys on the expression of osteoblast markers. (A) qPCR performed on DBSCs cultivated with osteogenic medium for 12 days and stimulated with 0.3% v/v T-Lys and Ctr. The graphs show that the treatment significantly increased the expression of the two osteoblast markers Runx-2 and Col1. Expression was normalized to β₂microglobulin (B2M). Each graph represents the mean ± SE of 3 independent experiments performed in triplicate. ^#P < 0.05 compared to Ctr. Statistics: unpaired Student's t-test. (B) Immunoblotting confirmed that the expression of Runx-2 and Col1 protein increased in T-Lys treated cells relative to Ctr cells. Each graph represents the mean OD ± SE of 3 independent experiments performed in triplicate. ^*P < 0.001 compared to Ctr. Statistics: Unpaired Student's t-test. Representative immunoblots were chosen for the figure. (C) ALP histochemical assay (purple staining) performed on DBSCs maintained in osteogenic conditions for 7 days and stimulated with T-Lys and Ctr. The graph represents the quantification of positive staining as percentage compared to Ctr (^§P < 0.01) and is representative for 3 independent experiments performed in quadruplicates. Data are presented as mean ± SE. Student's t-test was used for single comparisons. The wells of a representative experiment were chosen for the figure.

Figure 3

Mineral matrix deposition following T-Lys stimulation and Alizarin Red quantification. (A) Formation of calcium rich deposits in representative phase contrast pictures of DBSCs treated with T-Lys, HA or Ctr for 21 days in osteogenic conditions. Scale bar: 20 μm. (B) Mineral matrix deposition assayed by ARS (red staining). The graph shows the OD quantification of extracted dye from stained cell layers as mean percentage ± SE and is representative for 3 independent experiments performed in quadruplicates. ^§P < 0.01, ^*P < 0.001 compared to Ctr; ^§P < 0.01 compared to HA. Student's t-test was used for single comparisons. The wells of a representative experiment were chosen for the figure.

Figure 4

Subcellular localization of integrin α_Vβ_3. Midsection confocal microscopy images show the expression of integrin α_Vβ₃ (green) detected by the antibody LM609 in cells maintained for 4 days in osteogenic medium and treated with T-Lys and Ctr; picture (A) shows a single cell with integrin distributed in multiple pointed sites in Ctr, while T-Lys treatment induced a different organization of the integrin that appeared strongly augmented and more clustered and localized at the focal adhesion sites (B). Similar distribution is observable in clustered cells (C,D). Images of a representative experiment were chosen for the figure. Scale bar: 10 μm.

Figure 5

Effects of T-Lys on the expression of chondrocyte markers. (A) Representative phase contrast pictures of HACs treated with T-Lys (T-Lys) or not (Ctr) after 3 days (upper panel) and 15 days (lower panel) of culture. T-Lys treatment significantly increased matrix deposition at the expense of a dedifferentiation process after 15 days of culture (lower panel). Scale bar: 20 μm. (B) qPCR performed on chondrocyte pellet cultures cultivated 28 days with chondrogenic medium and stimulated with 0.3% v/v T-Lys and Ctr. The graphs show that the treatment significantly increased the expression of the chondrocyte markers Sox-9 and Col II, while had no effect on the expression of Aggrecan. Expression was normalized to β₂microglobulin (B2M). Each graph represents the mean ± SE of 3 independent experiments performed in triplicate. ^#P < 0.05, ^*P < 0.001 compared to Ctr. Statistics: unpaired Student's t-test.

Figure 6

Effects of T-Lys on chondrocytes proliferation and tissue growth. (A) Sectioned chondrocyte pellets were captured under optical microscope using a 20X objective lens and analyzed by using Image-J software for morphometric examination of the areas. T-Lys pellet appeared larger than Ctrs. The pictures selected are representative of three different experiments, scale bar: 75 μm. The graph represents the mean ± SE of 3 independent experiments performed in triplicate, ^*P < 0.001. (B) Deposition of cartilaginous matrix was demonstrated by Safranin O staining and chondrocyte nuclei were counterstained with hematoxylin. The pictures were captured with 40X objective lens, scale bar: 25 μm. The graph represents the mean ± SE of 3 independent experiments performed in triplicate, ^#P < 0.05. (C) The graph shows a theoretical reconstruction of the pellet thickness where the number of sections obtained was multiplied by the slice thickness (μm).