USE OF HYALURONIC ACID DERIVATIVES IN THE REGENERATION OF BONE AND CARTILAGE TISSUES

Abstract

The present invention relates to the use of derivatives between hyaluronic acid, heterocyclic compounds and naturally occurring amino acids in single, oligomeric or polymeric form for the treatment of skeletal diseases, in particular in the regeneration of bone and cartilage tissues.

Claims

1. A compound of hyaluronic acid and at least one heterocycle derived from purine and/or pyrimidine, said compound being further associated with at least one different organic compound selected from naturally occurring amino acids in the single, oligomeric or polymeric form, for its use in the treatment of skeletal conditions, in particular in the regeneration of hard tissues.

2. The compound for the use according to claim 1, wherein said at least one heterocycle is thymine.

3. The compound for the use according to claim 1 or 2, wherein said at least one different organic compound is lysine.

4. The compound for the use according to anyone of the preceding claims, wherein the bond between said hyaluronic acid, said heterocycle and said naturally occurring amino acid is a chemical bond of ionic type.

5. The compound for the use according to anyone of the preceding claims, wherein said compound of the hyaluronic acid is T-LysYal®, which comprises hyaluronic acid in combination with lysine and thymine.

6. The compound according to anyone of the preceding claims, for its use in the induction and stimulation of cell differentiation in the osteogenic and chondrogenic lineage of mesenchymal stem cells.

7. The compound for its use according to claim 6, wherein said mesenchymal stem cells are stem cells of the dental germ.

8. An implantable scaffold comprising a compound of hyaluronic acid and at least one heterocycle derived from purine and/or pyrimidine, said compound being further associated with at least one different organic compound selected from naturally occurring amino acids in the single, oligomeric or polymeric form.

9. The implantable scaffold according to claim 8, for its use in the treatment of skeletal conditions, in particular in the regeneration of hard tissues.

10. A pharmaceutical formulation comprising a compound of hyaluronic acid and at least one heterocycle derived from purine and/or pyrimidine, said compound being further associated with at least one different organic compound selected from naturally occurring amino acids in the single, oligomeric or polymeric form, for its use in the treatment of skeletal conditions, in particular in the regeneration of hard tissues.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0045] FIG. 1: Effect of T-Lys in the differentiation of MSCs towards the osteoblastic lineage. Sections: A) qPCR carried out on DBSCs grown with osteogenic medium and stimulated with 0.3% T-Lys and control DBSC; B) immunoblotting test for the expression of the Runx-2 and Col 1 proteins; C) histochemical assay on the ALP enzyme (purple staining).

[0046] FIG. 2: Effect of T-Lys in the deposition of mineral matrix during the osteogenic differentiation of MSCs. Deposition of mineral matrix tested by ARS (red staining) in cells treated with T-Lys, hyaluronic acid and control.

[0047] FIG. 3: Effect of T-Lys on the expression of typical markers in chondrocyte cultures. qPCR carried out on chondrocyte pellet cultures grown with chondrogenic medium and stimulated with 0.3% T-Lys and negative control group (Ctr).

[0048] FIG. 4: Effect of T-Lys on the chondrocyte proliferation and tissue growth. Sections: A) images and measurements of culture pellets of dissected chondrocyte treated with T-Lys and control groups; B) images and measurements of the deposition of cartilage matrix of dissected chondrocytes treated with T-Lys and control groups; C) theoretical reconstruction of the thickness of the culture pellets of chondrocytes treated with T-Lys and control groups.

EXPERIMENTAL SECTION

Example 1

[0049] Effect of T-Lys in the Differentiation of MSCs Towards the Osteoblastic Lineage

[0050] The stem cells of the dental germ (DBSC) were used as a source of MSC and were differentiated for 12 days in osteogenic medium. A portion of the tested cells was treated with 0.3% T-Lys (T-Lys—treatment group) added to the culture medium with each change. The fraction of cells not treated with T-Lys was used as control group (Ctr). The mRNA levels of the early markers of typical osteoblasts, Runx-2 and Collagen I (Col 1) were determined in Ctr and T-Lys samples by using real time PCR.

[0051] FIG. 1(A) shows how the expression of both markers has increased significantly in T-Lys-treated cells compared to Ctr cells, suggesting that T-Lys treatment has improved the ability of MSCs to differentiate in the lineage of the osteoblasts.

[0052] The protein expression levels of these osteoblastic markers were further evaluated in T-Lys and Ctr cells by Western Blot analysis.

[0053] FIG. 1(B) highlights how the level of the Runx-2 and Col 1 proteins is increased in T-Lys-treated cells compared to Ctr cells, thus confirming the mRNA expression trend.

[0054] A histochemical test was then carried out to explore the expression of another marker of osteoblastic cells, the alkaline phosphatase (ALP) enzyme, in response to the treatment with T-Lys. The result of this experiment, shown in FIG. 1(C), revealed that the stimulation of MSCs with T-Lys, during osteogenic differentiation, significantly increased the purple staining that identifies ALP expression.

[0055] All the results described above have demonstrated that T-Lys is able to increase the ability of MSCs to differentiate into cells similar to osteoblasts.

Example 2

[0056] Effect of T-Lys in the Deposition of Mineral Matrix During the Osteogenic Differentiation of MSCs

[0057] In order to thoroughly investigate the effect of this new molecule on the osteogenic differentiation of MSCs, DBSC culture under mineralization conditions was followed for 21 days on different samples: Ctr (without any addition as negative control group); HA (with the addition of unmodified hyaluronic acid as positive control group) and T-Lys (in which cells were treated with 0.3% T-Lys as treatment group).

[0058] The effect of T-Lys on the deposition of DBSC mineral matrix was analyzed using the Alizarin Red Staining (ARS) histochemical test quantified by using a colorimetric technique. The mineralization capacity of the cells treated with 0.3% T-Lys demonstrated to be significantly higher than both Ctr and HA.

[0059] These data show how T-Lys is able to increase the osteogenic capacity of MSCs by also stimulating their ability to produce mineralized matrix.

Example 3

[0060] Effect of T-Lys on the Influence of the Sub-Cellular Distribution of Δvβ3 Integrin

[0061] Integrins are receptors for ECM molecules, important in cell adhesion but also in the mediation of proliferation and differentiation signals. In particular, the αvβ3 integrin is the receptor for the bone protein called Osteopontin, of fundamental importance to determine the differentiation of MSCs towards the osteogenic lineage. Therefore, it was assessed whether the treatment with T-Lys could influence the subcellular distribution of αvβ3 integrin.

[0062] The subcellular distribution of said integrin was analyzed by confocal microscopy in DBSCs treated with T-Lys and Ctr. The analyses were carried out, after only 4 days of osteogenic differentiation, to compensate for the fact that the cells show a rapid propensity to form a multilayer that prevents their microscopic observation. In Ctr cells the αvβ3 integrin proved to be distributed in several sites, while the T-Lys treatment induced a different organization of this receptor, more localized in the focal adhesion sites. Therefore, after 4 days of differentiation, the receptor under controlled conditions was still distributed throughout the cell, while in T-Lys cells it was present in the focal adhesions. The presence of “strings” (the typical pattern of αvβ3 integrins involved in focal adhesions) was detectable in T-Lys cells and not in Ctr cells. These results suggest that the effect of T-Lys on DBSC differentiation could be mediated by αvβ3 rearrangement.

Example 4

[0063] Effect of T-Lys on the Expression of the Typical Markers in Chondrocyte Cultures

[0064] Human joint chondrocytes collected from patients undergoing orthopedic surgery have been grown in pellet cultures in order to mimic the micro-architecture of the three-dimensional tissue and avoid the improper de-differentiation of chondrocytes that easily occurs when they are grown in two dimensions. The cell pellets were grown for 28 days under chondrogenic conditions. The control group (Ctr) was treated according to the conventional protocol while the T-Lys group was added with 0.3% T-Lys, at each change of vehicle. At the end of the culture period, the chondrocyte culture pellets were lysed and evaluated for the gene expression analysis. The mRNA levels of typical chondrogenic markers: Sox-9, Collagen II (Col II), Collagen X (Col X) and Aggrecan were determined in both groups of real time PCR samples.

[0065] FIG. 3 shows the results of these tests which demonstrate that, for three out of these four markers taken into account, their expression has definitely increased in the T-Lys groups. In particular, the expression of Sox-9, which is the main transcription factor involved in the chondrogenic differentiation, is significantly increased in T-Lys-treated cells, compared to Ctr cells. In accordance with this result, Col II and Col X, the proteins typical of the extracellular matrix of the cartilage, are also increased thanks to the treatment with T-Lys, indicating once again that this molecule supports and improves the differentiation of chondrocytes. On the other hand, the expression of Aggrecan, a proteoglycan of the cartilage, was not affected in any way.

Example 5

[0066] Effect of T-Lys on the Chondrocyte Proliferation and Tissue Growth

[0067] After 28 days of differentiation under the conditions described in Example 4, the chondrocyte pellets were fixed with 4% paraformaldehyde, and were incorporated, sectioned and histologically stained and examined. The morphometric examination by optical microscope of culture pellets of the dissected chondrocytes revealed that the T-Lys ones were larger than those in the control group. This result is visible in FIG. 4(A).

[0068] To quantify the pellet size, the samples were sectioned (5 μm thickness) and the area of each section, obtained for the two groups of cells, was measured by ImageJ software. The graph in FIG. 4(A) shows that the average surface area was significantly larger in the T-Lys-treated group than in the control group.

[0069] The pellets were then stained with Safranin O to highlight the chondrocytes (FIG. 4(B)). The staining revealed the presence of cartilaginous matrix (orange staining), demonstrating that the cells were able to differentiate and produce CME components under these culture conditions; the nuclei were counter stained with hematoxylin (FIG. 4(B)). To check whether the T-Lys treatment also had an influence on the number of cells, the cells in a selected field (100×100 μm) were counted for each section. The graph in FIG. 4(B) depicts the number of cells for the two cultures and it can be seen that it has significantly increased in the case of the treated group compared to the control group. It is interesting to note that it was possible to obtain a larger number of sections with uniform thickness (5 μm), called “slices”, in the case of the T-Lys sample compared to the control group.

[0070] This difference is depicted in the graph of FIG. 4(C), in which the number of “slices” has been multiplied by the thickness of the section (5 μm) thus reconstructing a theoretical thickness of the culture pellet as a whole. These results demonstrate that T-Lys stimulates the proliferation of chondrocytes, their differentiation and matrix secretion.

DETAILED DESCRIPTION OF THE FIGURES

[0071] FIG. 1: Effect of T-Lys in the differentiation of MSCs towards the osteoblastic lineage

[0072] A) qPCR carried out on DBSCs grown with osteogenic medium and stimulated with 0.3% T-Lys and DBSC Ctr. Each graph depicts the average±standard error of 3 independent experiments performed in triplicate. *P<0.02 compared to the control group. The expression has been normalized to mic2microglobulin (B2M). The graphs show that the treatment with T-Lys significantly increased the expression of the two osteoblast markers Runx-2 and Coll.

[0073] B) Immunoblotting test for the expression of the proteins Runx-2 and Col 1; each graph depicts the average optical density calculated in relation to a constituent protein (0-Actin housekeeping gene)+standard error of 3 independent experiments carried out in triplicate. *P<0.001 compared to the control group. Representative immunoblot images were also depicted on the left side of the figure. The graphs show how the measured parameter is higher in T-Lys-treated cells than in the control group.

[0074] C) Histochemical assay on the ALP enzyme (purple staining) carried out on DBSCs maintained under osteogenic conditions for 7 days and stimulated with T-Lys compared to the control group. The graph depicts the quantification of positive staining in percentage with respect to the control group (*P<0.01) and derives from the analyses of 3 independent experiments carried out in quadruplicate. The data are shown as an average±standard error. Representative images of culture wells are also depicted on the left in the figure. The graph shows how T-Lys samples have a higher expression of the alkaline phosphatase enzyme.

[0075] FIG. 2: Effect of T-Lys in the deposition of mineral matrix during the osteogenic differentiation of MSCs

[0076] Deposition of mineral matrix tested by ARS (red staining) in cells treated with T-Lys, hyaluronic acid and Ctr under osteogenic conditions for 21 days. The graph shows the quantification of optical density of the dye extracted from the colored cell layers as average percentage±standard error and is representative of 3 independent experiments carried out in quadruplicates. *P<0.01, #P<0.001 versus negative control group (Ctr); @P<0.01 versus positive control group (HA). Representative images of culture wells are also depicted on the left in the figure. The graph shows how T-Lys samples have a higher deposition of mineral matrix than both the untreated sample and the sample treated with native hyaluronic acid.

[0077] FIG. 3: Effect of T-Lys on the expression of the typical markers in chondrocyte cultures

[0078] qPCR carried out on chondrocyte pellet cultures grown with chondrogenic medium and stimulated with 0.3% T-Lys and negative control group (Ctr). Each graph depicts the average±standard error of 3 independent experiments performed in triplicate. *P<0.04 for Sox-9, *P<0.001 for Col II, *P<0.01 for Col X compared to the control group. The expression has been normalized to mic2microglobulin (B2M). The graphs show that treatment with T-Lys significantly increased the expression of Sox-9, Col II and Col X chondrocyte markers, while it had no effect on the Aggrecan expression.

[0079] FIG. 4: Effect of T-Lys on the chondrocyte proliferation and tissue growth

[0080] A) The culture pellets of sectioned chondrocytes were photographed under an optical microscope by using a 20× objective lens and analyzed by using the Image-J software for morphometric examination of the areas. The selected images are representative of three different experiments, scale bar depicted in the bottom right corner of the figures: 75 μm. The graph depicts the average±standard error of 3 independent experiments performed in triplicate, *P<0.0003. The pellets treated with T-Lys appear larger than those in the control group.

[0081] B) The cartilage matrix deposition was measured by using Safranin O staining and the chondrocyte nuclei were counter stained with hematoxylin. The images were taken with a 40× lens, scale bar shown in the upper left corner of the control figure: 25 μm. The graph depicts the average±standard error of 3 independent experiments performed in triplicate, *P<0.04. The number of cells in the T-Lys sample is greater than the number in the control group.

[0082] C) The graph shows a theoretical reconstruction of the thickness of the chondrocyte culture pellet in which the number of sections obtained was multiplied by the thickness of the slice and expressed in μm. The group treated with T-Lys shows a greater thickness.