Cartilage product

Abstract

The present invention relates to a method for preparing a cartilage product comprising a protein hydrolysate with a degree of hydrolysis comprised between 0.5% and 3.0%, at least one glycosaminoglycan and at least one growth factor. The present invention also relates to the cartilage product obtainable through said method. Said cartilage product is useful in the treatment or prevention of wounds, ulcers, burns, psoriasis, osteoarthritis, synovitis, osteoporosis, osteopenia, diseases of the tendons and ligaments, periodontal diseases, signs of skin aging, the harmful effects of ultraviolet radiation exposure or stretch marks.

Claims

1. A cartilage product, comprising: a) between 67% and 87% by weight of protein hydrolysate with a degree of hydrolysis comprised between 0.5% and 3.0%, with respect to the weight of the anhydrous cartilage product; b) between 15% and 25% by weight of chondroitin sulfate, with respect to the weight of the anhydrous cartilage product; c) between 0.1% and 1.0% by weight of hyaluronic acid, with respect to the weight of the anhydrous cartilage product; d) between 20 pg and 200 pg of TGF-1 for every 100 mg of anhydrous cartilage product, and e) between 20 pg and 200 pg of TGF-3 for every 100 mg of anhydrous cartilage product.

2. The cartilage product of claim 1 which is an aqueous solution.

3. The cartilage product of claim 1 which is a solid product.

4. The cartilage product of claim 1, comprising: a) 77% by weight of protein hydrolysate with a degree of hydrolysis of the 1.7%, with respect to the weight of the anhydrous cartilage product; b) 19.7% by weight of chondroitin sulfate, with respect to the weight of the anhydrous cartilage product; c) 0.3% by weight of hyaluronic acid, with respect to the weight of the anhydrous cartilage product; d) 53.0 pg of TGF-1 for every 100 mg of anhydrous cartilage product, and e) 31.3 pg of TGF-3 for every 100 mg of anhydrous cartilage product.

5. The cartilage product of claim 1, wherein the protein hydrolysate includes collagen hydrolysate and the collagen hydrolysate comprises between 45% and 65% by weight of the collagen hydrolysate with a degree of hydrolysis less than 3.0%, with respect to the weight of the anhydrous cartilage product.

6. The cartilage product of claim 5, comprising 54.9% by weight of collagen hydrolysate with a degree of hydrolysis less than 3.0%, with respect to the weight of the anhydrous cartilage product.

7. The cartilage product of claim 5, wherein the collagen hydrolysate has a degree of hydrolysis of 0.1%.

8. A cartilage product, characterized by the following analytical parameters: Degree of hydrolysis of the protein: comprised between 0.5% and 3.0%; Degree of hydrolysis of the collagen hydrolysate: less than 3%; Protein hydrolysate: between 67% and 87% by weight with respect to the weight of the anhydrous cartilage product wherein the protein hydrolysate includes collagen hydrolysate; Collagen hydrolysate: between 45% and 65% by weight with respect to the weight of the anhydrous cartilage product; Chondroitin sulfate: between 15% and 25% by weight with respect to the weight of the anhydrous cartilage product; Hyaluronic acid: between 0.1% and 1.0% by weight with respect to the weight of the anhydrous cartilage product; Growth factor TGF-1: between 20 pg and 200 pg for every 100 mg of anhydrous cartilage product; Growth factor TGF-3: between 20 pg and 200 pg for every 100 mg of anhydrous cartilage product.

9. A food supplement or a functional food comprising the cartilage product according to claim 1 and at least one nutritional excipient.

10. A cosmetic composition comprising the cartilage product according to claim 1 and at least one cosmetically acceptable excipient.

11. A pharmaceutical composition comprising the cartilage product according to claim 1 and at least one pharmaceutically acceptable excipient.

12. A food supplement or a functional food comprising the cartilage product according to claim 8 and at least one nutritional excipient.

13. A cosmetic composition comprising the cartilage product according to claim 8 and at least one cosmetically acceptable excipient.

14. A pharmaceutical composition comprising the cartilage product according to claim 8 and at least one pharmaceutically acceptable excipient.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 shows the effect of the cartilage product on the percentage of human dermal fibroblast proliferation at 48 hours at three concentrations (50 g/ml, 500 g/ml and 2 mg/ml). The positive control (human dermal fibroblast culture in 10% FCS medium, in the absence of the cartilage product and in the presence of bromodeoxyuridine) and the baseline control (human dermal fibroblast culture in culture medium and in the absence of the cartilage product) are also included.

(2) FIG. 2 shows the effect of the cartilage product on the percentage of human dermal fibroblast migration at 48 hours at three concentrations (250 g/ml, 500 g/ml and 2 mg/ml). The baseline control (human dermal fibroblast culture in culture medium and in the absence of the cartilage product) and two positive controls (a human dermal fibroblast culture in 10% FCS culture medium and in the absence of the cartilage product and a human dermal fibroblast culture in the absence of the cartilage product and in the presence of EGF) are also included.

(3) FIG. 3 shows the effect of the cartilage product on the elastin synthesis percentage in a human dermal fibroblast culture at 72 hours of exposure at three concentrations (250 g/ml, 500 g/ml and 2 mg/ml). The baseline control (human dermal fibroblast culture in culture medium and in the absence of the cartilage product) and the positive control (human dermal fibroblast culture in the absence of the cartilage product and in the presence of TGF-) are also included.

(4) FIG. 4 shows the effect of the cartilage product on metalloprotease 1 (MMP-1) activity in an IL-1-stimulated human dermal fibroblast culture at three concentrations (250 g/ml, 500 g/ml and 2 mg/ml). The baseline control (human dermal fibroblast culture in culture medium and in the absence of the cartilage product and of IL-1), the IL-1-stimulated control and the positive inhibition control (human dermal fibroblast culture in the absence of the cartilage product and in the presence of IL-1 and of dexamethasone) are also included.

(5) FIG. 5 shows the effect of two compositions based on cartilage product, hydroxyapatite and vitamin D3, on cartilage degradation at a low dose (composition 1 LD) and at a high dose (composition 2 HD), using the OARSI scoring system. are also included. The blank (group of rats without induction of osteoporosis or osteoarthritis), control 1 (group of rats with induction of osteoporosis and osteoarthritis; this was the control group for treatment with the reference compound), control 2 (group of rats with induction of osteoporosis and osteoarthritis; this was the control group for treatment with the cartilage product-based compositions) and the OHC compound (reference product) are also included.

(6) FIG. 6 shows the effect of two compositions based on cartilage product, hydroxyapatite and vitamin D3, on bone volume (BV/TV %) at a low dose (composition 1 LD) and at a high dose (composition 2 HD). The blank (group of rats without induction of osteoporosis or osteoarthritis), control 1 (group of rats with induction of osteoporosis and osteoarthritis; this was the control group for treatment with the reference compound), control 2 (group of rats with induction of osteoporosis and osteoarthritis; this was the control group for treatment with the cartilage product-based compositions) and the OHC compound (reference product) are also included.

(7) FIG. 7 shows the effect of two compositions based on cartilage product, hydroxyapatite and vitamin D3, on bone surface density (BS/TV (mm.sup.1)) at a low dose (composition 1 LD) and at a high dose (composition 2 HD). The blank (group of rats without induction of osteoporosis or osteoarthritis), control 1 (group of rats with induction of osteoporosis and osteoarthritis; this was the control group for treatment with the reference compound), control 2 (group of rats with induction of osteoporosis and osteoarthritis; this was the control group for treatment with the cartilage product-based compositions) and the OHC compound (reference product) are also included.

(8) FIG. 8 shows the effect of two compositions based on cartilage product, hydroxyapatite and vitamin D3, on trabecular number (TbN (mm.sup.1)) at a low dose (composition 1 LD) and at a high dose (composition 2 HD). The blank (group of rats without induction of osteoporosis or osteoarthritis), control 1 (group of rats with induction of osteoporosis and osteoarthritis; this was the control group for treatment with the reference compound), control 2 (group of rats with induction of osteoporosis and osteoarthritis; this was the control group for treatment with the cartilage product-based compositions) and the OHC compound (reference product) are also included.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

EXAMPLES

(9) The following examples are merely illustrative and do not represent a limitation to the scope of the present invention.

Example 1

Preparation of a Porcine Tracheal Cartilage Product of the Invention

(10) Pieces of porcine trachea were subjected to mechanical cleaning to remove fat and non-cartilaginous tissues and were then chopped up. 3,100 ml of deionized water were introduced in a reactor. 1,575 g of chopped porcine tracheal cartilage were added. The mixture was heated to 50 C. and the pH was adjusted between 3.0 and 3.5 with H.sub.3PO.sub.4. Once the pH was adjusted, 5.9 g of pepsin (Biocatalysts, 1:3000 MDP) were added and the temperature was maintained at 50 C. The pH was checked every 30 minutes for the next three hours and was adjusted if needed. Ten hours after starting digestion 5.9 g of pepsin (Biocatalysts, 1:3000 MDP) were again added and the pH was checked every 30 minutes for the next three hours. Digestion was performed for a total of 24 hours, a total amount of 0.75% of pepsin by weight with respect to the weight of the porcine tracheal cartilage being used. After 24 hours, it was heated to 80 C. for one hour. Then it was filtered to clarify the product. Once filtered, 7.9 g of activated carbon were added and it was heated to 50 C. for 30 minutes. It was neutralized with calcium hydroxide. It was then filtered, obtaining an aqueous solution of cartilage product. The end product in solid form (cream-colored powder) was obtained by atomizing the aqueous solution of cartilage product.

(11) Analytical characteristics of the solid cartilage product obtained:

(12) Protein hydrolysate: 77% by weight with respect to the weight of the anhydrous cartilage product;

(13) Collagen hydrolysate: 54.9% by weight with respect to the weight of the anhydrous cartilage product;

(14) Total amino acids: 70.7% by weight with respect to the weight of the cartilage product;

(15) Free amino acids: 1.2% by weight with respect to the weight of the cartilage product;

(16) Total hydroxyproline: 6.9% by weight with respect to the weight of the cartilage product;

(17) Degree of hydrolysis of the protein: 1.7%;

(18) Degree of hydrolysis of the collagen hydrolysate: 0.1%;

(19) Chondroitin sulfate: 19.7% by weight with respect to the weight of the anhydrous cartilage product;

(20) Hyaluronic acid: 0.3% by weight with respect to the weight of the anhydrous cartilage product;

(21) Growth factor TGF-1: 53.0 pg for every 100 mg of anhydrous cartilage product;

(22) Growth factor TGF-3: 31.3 pg for every 100 mg of anhydrous cartilage product;

(23) Ash: 8.9% by weight with respect to the weight of the anhydrous cartilage product;

(24) Calcium: 0.4% by weight with respect to the weight of the anhydrous cartilage product;

(25) Phosphates: 0.3% by weight with respect to the weight of the anhydrous cartilage product.

(26) Collagen hydrolysate with a degree of hydrolysis less than 3.0% is included in 77% of the protein hydrolysate.

(27) The analytical characteristics of the cartilage product can slightly vary depending on the batch of cartilage product obtained.

(28) Determination of the Degrees of Hydrolysis:

(29) The degree of hydrolysis of the protein was determined by means of a standard method. It was expressed as the percentage of free amino acids in relation to total amino acids.

(30) The degree of hydrolysis of the collagen hydrolysate was determined by means of a standard method. It was expressed as the percentage of free hydroxyproline in relation to total hydroxyproline.

(31) Determination of the Growth Factors:

(32) The following of ELISA kits (R&D Systems) were used following the protocols recommended by the manufacturer to determine the growth factors: Quantikine porcine TGF-1 (catalog: MB100B); DuoSet Human TGF-3 (catalog: DY243).

Example 2

Tablets of Porcine Tracheal Cartilage Product, Calcium Salt and Vitamin D3

(33) The tablets were prepared following conventional methods.

(34) Per Tablet:

(35) TABLE-US-00001 Porcine tracheal cartilage product 300 mg Hydroxyapatite 400 mg Vitamin D3 100 IU

Example 3

Tablets of Porcine Tracheal Cartilage Product, Calcium Salt, Vitamin D3 and Vitamin K2

(36) Per Tablet:

(37) TABLE-US-00002 Porcine tracheal cartilage product 300 mg Hydroxyapatite 400 mg Vitamin D3 100 IU Vitamin K2 22.5 g

Biology

Example 4

Activity Stimulating Fibroblast Proliferation

(38) The wound healing process is a highly ordered and controlled process characterized by different phases: inflammation, proliferation and remodeling (R. F. Diegelmann and M. C. Evans, Front. Biosci. 9, 283-289 (2004)). The healing process requires the coordination of several cells, growth factors and cytokines. Inflammation is the initial phase. In the wound healing process, fibroblast proliferation is involved in restoring structure and function in the wound (R. A. Clark, Ann. N.Y. Acad. Sci. 936, 355-367 (2001)).

(39) Stimulation of the degree of fibroblast proliferation is also interesting as an anti-age treatment. The number of dermal fibroblasts decreases with aging and, therefore, there is a progressive loss of dermal tissue.

(40) Materials and Methods

(41) The degree of proliferation was quantified by measuring the incorporation of bromodeoxyuridine (BrdU) into DNA of proliferating cells during the replication phase. A specific anti-BrdU antibody colorimetric immunoassay, ELISA (Enzyme Linked Immunoadsorbent Assay) detection and subsequent reading of absorbance at 450 nm were used to quantify the incorporated BrdU. The amount of BrdU detected is proportional to the number of cells that have been divided and, therefore, proportional to the growth or proliferation experienced by the culture.

(42) Human dermal fibroblasts were seeded at 5,000 cells/well in 96-well plates and after 24 hours, they were left over night with growth factor-deprived culture medium. The next day, the cells were treated at three concentrations (2 mg/ml, 500 g/ml and 50 g/ml) of a cartilage product of the present invention, specifically of the cartilage product of Example 1. The amount of bromodeoxyuridine was determined by means of specific immunoassay (technique ELISA) after 48 hours of exposure of the culture to the cartilage product.

(43) A fibroblast culture with culture medium was used as a baseline control and the fibroblasts were exposed to 10% FCS (Fetal Calf Serum) culture medium as a positive control.

(44) Results

(45) As can be observed in FIG. 1, the cartilage product at the doses of 500 g/ml and 2 mg/ml showed a statistically significant stimulating effect (p<0.05) on fibroblast proliferation when compared with the baseline control. Specifically, proliferation increased at the intermediate dose by 35.9% and by 73% at the high dose.

Example 5

Assessment of In Vitro Cell Migration Capacity

(46) This study allows evaluating the induction capacity of cell migration in primary human dermal fibroblast culture, and it is therefore useful to evaluate the potential efficacy of a product in wound healing and as an anti-age treatment. In fact, with aging there is a decrease in the degree of fibroblast proliferation and migration.

(47) Materials and Methods

(48) The Oris Cell Migration assay system by Platypus was used to evaluate the potential effect of a cartilage product of the present invention on cell migration. This system consists of a special plate which allows seeding fibroblasts and their single-layer growth, but leaving a central area of the well cell-free as a result of the so-called Oris Cell Seeding Stoppers which restrict seeding to the outer annular region of the well. The Oris Cell Seeding Stoppers are subsequently removed and this area is subsequently occupied by cells during the migration process.

(49) The human dermal fibroblasts were incubated for 48 hours in the presence of a cartilage product of the present invention, specifically of the cartilage product of Example 1 (2 mg/ml and 500 g/ml), and they were then labeled with fluorescent calcein dye. The fluorescence emitted by the migrating cells was measured by means of a fluorometer. Photographs were also taken by fluorescence microscopy which allowed seeing the occupied area compared with the baseline control.

(50) Culture medium was used as the baseline control and as positive controls were included 10% FCS (Fetal Calf Serum) culture medium and also culture medium with 5 ng EGF (Epidermal Growth Factor).

(51) Results

(52) As can be seen in FIG. 2, the cartilage product produced an important inductive effect on cell migration at 48 h of exposure for the concentrations of 500 g/ml and 2 mg/ml (42% and 75% induction, respectively). It must further be pointed out that the effect of said product exceeded that shown the positive controls (10% FCS medium and EGF at 5 ng/ml).

Example 6

Assessment of the Moisturizing Action

(53) Hyaluronic acid is an essential component health skin and is involved in hemostasis, moisturizing and repair processes. As a result of its capacity to retain water in a thousand-fold percentage equivalent, it carries out a primordial function in the skin. Young skin is rich in hyaluronic acid, however as one ages, the distribution and function of hyaluronic acid in the skin gradually change and the characteristics signs of aging such as wrinkles and expression lines appear.

(54) Materials and Methods

(55) To evaluate the inductive efficacy of skin moisturizing by a cartilage product of the present invention, specifically by the cartilage product of Example 1, a study was performed to quantify glycosaminoglycans, mainly hyaluronic acid synthesized by human dermal fibroblasts after incubation with the cartilage product (2 mg/ml, 500 g/ml and 250 g/ml) for 24 hours. To that end, the method of incorporating .sup.3H-glucosamine into newly-synthesized glycosaminoglycans was used.

(56) The bound radioactivity was analyzed in a liquid scintillation counter. The CPM (counts per minute) values obtained are proportional to the amount of hyaluronic acid synthesized in 90%.

(57) The fibroblasts were exposed to TGF-1 (transforming growth factor beta 1), known as an extracellular matrix protein production inducer, as the positive control. The baseline control consisted only of fibroblasts in culture medium.

(58) Results

(59) The cartilage product showed a mild moisturizing effect at 24 hours of exposure to the concentration of 500 g/ml. Specifically, it stimulated hyaluronic acid synthesis by 12% when compared with the baseline control.

Example 7

Assessment of the Inductive Capacity of Elastin Production

(60) In human skin, intrinsic aging is characterized by atrophy of the dermis due to collagen loss, degeneration of the elastin fiber network and loss of moisturize. Elastin is the protein conferring elastic properties to the skin.

(61) Materials and Methods

(62) The inductive capacity of skin elasticity by a cartilage product of the present invention, specifically of the cartilage product of Example 1, was evaluated from the quantification of elastin production in fibroblasts.

(63) Human dermal fibroblasts were seeded in 96-well culture plates and were kept growing until reaching confluence. Then they were exposed for 72 hours to concentrations of 2 mg/ml, 500 g/ml and 250 g/ml. After this treatment period ended, the cells were washed and fixed for ELISA processing. A primary anti-elastin antibody (Monoclonal Anti-Elastin antibody produced in mouse, Sigma), followed by a secondary antibody conjugated to the horseradish peroxidase (HRP) enzyme and developed with o-phenylenediamine (HRP substrate) and urea-H.sub.2O.sub.2, were used in this process. Absorbance was read in an ELISA reader at 492 nm.

(64) The value of elastin produced was pondered with that of total protein in each experimental condition, resulting in the values of the elastin/total protein production index.

(65) The fibroblasts were exposed to TGF-1 (transforming growth factor beta 1) as the positive control. The fibroblasts were cultured in culture medium as the baseline control.

(66) Results

(67) As it can be seen in FIG. 3, the cartilage product showed an important inductive effect on elastin production at the highest concentration studied (2 mg/ml). It should be mentioned that close to 50% of the value shown by the TGF- control, a potent inductor of elastin and extracellular matrix protein synthesis, was achieved.

Example 8

Determination of the Inhibitor Potential of MMP-1 Activity

(68) There is a loss of extracellular matrix, an increase in metalloproteases (MMPs) degrading collagen type I responsible for firmness of the skin, as well as a loss of fibroblasts and of the vascular network with aging. It is estimated that collagen of the dermis decreases 1% per year over the adult lifetime and as age increases, metalloprotease levels also increase, which progressively increases the loss of collagen. The presence of high levels of some metalloproteases has been associated with cell destruction in a wide variety of pathological and aging processes (K. C. N. Chang et al., Mol. Endocrinol. 22(11), 2407-2419 (2008); G. J. Fisher et al., Am. J. Pathol. 174(1), 101-114 (2009); A. L. Clutterbuck et al., Curr. Drug Targets 10(2), 1245-1254 (2009); H. Nagase et al., Cardiovascular Research 69, 562-573 (2006))

(69) Materials and Methods

(70) To evaluate the effect of a cartilage product of the present invention, specifically of the cartilage product of Example 1, on metalloprotease regulation, MMP-1 activity in a primary human dermal fibroblast culture exposed to the cartilage product and induced with IL1- during 48 hours was determined.

(71) Human dermal fibroblasts were seeded in 24-well culture plates. After reaching confluence and deprivation serum-free medium for 16 hours, the cartilage product was applied (2 mg/ml, 500 g/ml and 250 g/ml). After 24 hours, IL-1 was applied as a stimulator of MMP production and the culture was maintained for another 24 hours. At the end of treatment, the supernatant was pooled and the active metalloprotease was quantified by means of a specific immunofluorometric assay after enzyme activation with APMA (p-AminoPhenylMercuric Acetate).

(72) The values of active MMP-1 were weighted by the values of total protein, previously determined by the method of bicinchoninic acid, BCA (Pierce BCA Protein Assay Kit).

(73) The baseline control consisted of culture medium. An IL-1-stimulated control group was also included in the study. Dexamethasone at 5 M (dexa control), a strong anti-inflammatory glucocorticoid, was used as the positive inhibition control.

(74) Results

(75) The cartilage product showed a strong inhibitory effect (p<0.05) on IL-1-induced metalloprotease 1 activity at all the studied doses and in a dose-dependent manner, with a reduction of 61%-70% with respect to the IL-1-stimulated control.

Example 9

Efficacy of the Porcine Tracheal Cartilage Product in the Healing Quality of Wounds

(76) Materials and Methods

(77) The study was performed in an excision wound model a Yucatan-type miniature pig. The wounds were full-thickness with a diameter of 3 cm. Two female pigs were used in the study and eight wounds were made per animal. Four wounds were treated with the cartilage product of Example 1 and the other four were not treated and were considered the control group. The product was applied daily for 14 days in the form of a gel formed by 1 g of cartilage product resuspended in 1 ml of sterile saline. Healing was monitored visually with photographs and granulation, epithelialization, moisturizing and general observations for each wound were evaluated on days 1, 2, 4, 8 and 15.

(78) Results

(79) The visual evaluation demonstrated that wounds treated with the cartilage product of the present invention presented less hypertrophic scarring than those corresponding to the non-treated control.

Example 10

Effect of the Porcine Tracheal Cartilage Product on Muscular Atrophy

(80) Measurement of the size of myotubes is a suitable approach for studying compounds that can prevent or treat skeletal muscle atrophy or induce hypertrophy. The C2Cl2 cell line has been widely used as an in vitro model for muscle atrophy and hypertrophy studies. C2Cl2 cells proliferating in the form of mononucleated myoblasts which subsequently fused to and differentiated into polynucleated myotubes.

(81) Materials and Methods

(82) C2Cl2 myoblasts were seeded in culture plates with growth medium (DMEM/10%, fetal bovine serum, 2 mM L-glutamine) and were left to grow to confluence. The cells were then treated with a medium (MEM/2% bovine serum, 2 mM L-glutamine) to induce differentiation of myoblasts into myotubes. After 5 days, the myotubes were incubated for 48 hours with serum-free medium (DMEM/2 mM L-glutamine) with/without the cartilage product of Example 1 (0.3, 1 and 1.5 mg/ml). After treatment, the myotubes were fixed with formaldehyde and treated with a myosin heavy chain antibody. The size of the myotubes was determined by means of fluorescence microscopy.

(83) Results

(84) The elimination of the differentiation serum (0% control) resulted in an approximate reduction of 20% of the size of the myotubes compared with the myotubes that were maintained in differentiation conditions (2% control).

(85) Treatment with the cartilage product of the present invention was capable of counteracting in a statistically significant manner (p<0.05) myotube atrophy at all the studied concentrations.

Example 11

Effect of the Cartilage Product in an In Vitro Model of Psoriasis

Release of IL-17 cytokine by CD4+T Lymphocytes Stimulated with a Mixture of Anti-CD3 and Anti-CD28 Antibodies

(86) Psoriasis is an immunological disease mediated by T lymphocytes which induce specific physiopathological responses in keratinocytes by means of the release of cytokines. Cytokines signal and induce the expression of specific genes and the development of the pathology. IL-17 is a crucial cytokine in psoriasis pathogenesis. In fact, drugs attacking said cytokine are effective in the treatment of this disease (A. M. Lin et al., J. Immunol. 187 (1), 490-500 (2011)). An active product for this disease could act both at the lymphocyte level (inhibiting the activation/infiltration or the release of cytokines) and at the keratinocyte level (inhibiting cytokine signaling).

(87) Materials and Methods

(88) The CD4.sup.+T cells were isolated and pre-incubated for 24 hours in culture medium with or without the cartilage product of Example 1 (0.25, 0.5, 1 mg/ml) or reference cyclosporin A. In parallel, the 96-well plate was covered with anti-CD3 antibody. The cells were transferred to the plate with culture medium with anti-CD28 antibody and with or without the cartilage product or the reference. A no-stimulated control was included, incubating the cells in wells not covered with antibody and with the absence of CD28 antibody. The cells were then incubated for 24 hours and the supernatants of the culture were pooled to quantify IL-17 levels by means of an ELISA assay (R&D Systems, ref. DY317).

(89) Results

(90) The activation of CD4.sup.+T cells with anti-CD3 and anti-CD28 antibodies resulted in a significant release of IL-17. The reference compound, cyclosporin A, inhibited the release of IL-17 by 100%.

(91) The cartilage product of the present invention at 1 mg/ml inhibited IL-17 levels in a statistically significant manner: 42% inhibition.

Example 12

Effect of the Cartilage Product on an Animal Model of Osteoporosis and Osteoarthritis

(92) Materials and Methods

(93) A model of ovariectomy-induced osteoporosis and anterior cruciate ligament transection (ACLT)-induced osteoarthritis in rats was used.

(94) The study included the following treatment groups: Blank: Group without induction of osteoporosis or osteoarthritis. Treated with 1 ml of water. Control 1: Group with induction of osteoporosis and osteoarthritis treated with 1 ml of water. It is the control group for treatment with the ossein-hydroxyapatite compound (OHC; see patent EP255565B1). OHC compound: Reference product used at a dose of 290.5 mg/kg/day in rats. Control 2: Group with induction of osteoporosis and osteoarthritis treated with 1 ml of water. It is the control group for treatment with the composition based on the cartilage product of the present invention. Composition 1 at a low dose (composition 1 LD): Formulation consisting of 600 mg of the cartilage product of Example 1, 800 mg of hydroxyapatite and 200 IU of vitamin D3. The administered dose in rats was 163.5 mg/kg/day of this formulation. Composition 2 at a high dose (composition 2 HD): Formulation consisting of 900 mg of the cartilage product of the Example 1, 1,200 mg of hydroxyapatite and 200 IU of vitamin D3. The administered dose in rats was 245 mg/kg/day of this formulation.

(95) The amount of cartilage product and hydroxyapatite of compositions 1 and 2 used in the study was less than the amount of ossein and hydroxyapatite of the reference product (OHC compound).

(96) The number of rats per treatment group was 15. The compounds were administered daily for 12 weeks by means of intragastric probe, resuspended in 1 ml of water.

(97) Cartilage degradation was evaluated by means of the scale recommended by OARSI (K. P. H. Pritzker et al, Osteoarthritis and Cartilage 14, 13-29 (2006)). The effects on mineral density and on bone microarchitecture bone were evaluated by means of micro-CT technique (M. L. Bouxsein et al., J. Bone Miner. Res. 25 (7), 1468-86 (2010))

(98) Results in Osteoarthritis:

(99) Treatments with composition 2 at a high dose and also with composition 1 at a low dose have shown to be highly effective in reducing cartilage degradation (p<0.05). The compound OHC showed no significant effect (see FIG. 5).

(100) Results in Osteoporosis:

(101) As can be seen in FIG. 6, treatments with the OHC compound and composition 1 at a low dose caused a considerable increase in the bone volume percentage. Nevertheless, treatment with composition 2 at a high dose was the only treatment that entailed a statistically significant increase in bone volume percentage (p<0.05).

(102) As can be seen in FIG. 7, treatment with the OHC compound caused only a mild effect on bone surface density (comparing the OHC compound with control 1). Treatments with composition 1 at a low dose and with composition 2 at a high dose caused a considerable increase in bone surface density, the effect of composition 2 at a high dose being statistically significant (p<0.01).

(103) In addition, FIG. 8 shows that treatment with composition 1 at a low dose caused a non-statistically significant increase in the trabecular number (45% increase). The OHC compound caused an 88% increase which is, statistically significant (p<0.05). Composition 2 at a high dose was the treatment that showed better results, with an increase in the trabecular number of 180% (p<0.01).