MXPA01007731A - Methods and compositions for healing and repair of articular cartilage - Google Patents

Abstract

Methods and compositions are provided for the treatment of articular cartilage defects and disease involving the combination of the tissue, such as osteochondral grafts, with active growth factor. The active growth factor is preferably a composition containing at least one bone morphogenetic protein and a suitable carrier. The method results in the regeneration of functional repair of articular cartilage tissue.

Description

METHODS AND COMPOSITIONS TO HEAL AND REPAIR ARTICULAR CARTILAGE FIELD OF THE INVENTION The present invention relates to the field of tissue repair, specifically, the regeneration of articular cartilage repair, stable and functional. In this way, the present invention can be useful in reconstructive surgery or other procedures for the regeneration or repair of articular cartilage.

BACKGROUND OF THE INVENTION The repair of injuries in articular cartilage continues to be a challenge for the current orthopedics. Several of the current therapeutic strategies are based on the grafting of chondral and osteochondral tissues. The autologous osteochondral graft provides the most appropriate physiological material. However, the donor tissue is limited and often requires surgery at a secondary site in order to collect the tissue for transplantation. Therefore, despite substantial efforts in this field, there continues to be a need for an effective method for the repair of REF .: 132040 defects and injuries in articular cartilage, which provides an appropriate physiological repair, without the need to collect autologous tissue from the patient.

BRIEF DESCRIPTION OF THE INVENTION The present invention provides methods and compositions for regenerating tissue repair, functionally and physiologically appropriate, to repair injuries and defects in articular cartilage. In particular, the present invention comprises methods for the treatment of patients with injuries and defects in articular cartilage. The methods and compositions of the present invention are advantageous because they utilize bone morphogenetic proteins (BMPs), which are known to have osteogenic and / or chondrogenic properties, and which can be produced through recombinant DNA technology, and therefore are of a potentially unlimited supply. The methods and compositions of the present invention are further advantageous because the regeneration of functional articular cartilage can be accelerated or it can be of higher endurance and stability, and the tissue formed at the site of the defect or injury is physiologically appropriate. The use of BMP to increase the repair of defects and injuries in articular cartilage, can result in better methods for the treatment of osteoarthritis, obviating, delaying or reducing the need for replacement of artificial hips and other common interventions. Preclinical evaluations indicate that rhBMP-2 improves early healing of defects throughout the thickness of articular cartilage in rabbits.

DETAILED DESCRIPTION OF THE INVENTION In accordance with the present invention, methods and compositions are provided for the treatment of patients suffering from some form of injury or defect in articular cartilage. The injury can be the result of an acute injury or strain, such as those resulting from participation in athletic games, or from accidental events that tear, destroy or otherwise injure the articular cartilage. The methods and composition are advantageous because they repair or improve articular cartilage defects, particularly articular cartilage defects throughout the thickness. Other defects can also be treated by the methods and compositions of the present invention, particularly with an additional procedure in which the defect site is further aggravated to reach the underlying subchondral bone. In the present invention, the active growth factor, such as a BMP, is added to an appropriate tissue source. The tissue source can be an osteochondral graft, either autologous to the patient, or it can comprise allograft or artificially prepared tissue. In a preferred embodiment, the tissue source may be chondrocytic cell cultures such as chondrocyte cultures or stem cells that have been prepared through cell culture methods ex vivo, with or without additional growth factors. For example, see the description of the North American Patents Numbers: 5,226,914; 5,811,094; 5,053,050; 5,486,359; 5,786,217 and 5,723,331. The descriptions of all of these applications are incorporated herein by reference. The tissue can also be collected through traditional media based on non-cell cultures, using techniques such as mosaicplasty, in which the cartilage is grown using commercially available instruments such as Acufex7 [Smith and Nephew, Inc., Andover MA]; COR System [Innovative Technologies, Marlborough MA]; o Arthrex7 Osteochondral Autograft Transfer System [Arthrex., Munich, Germany]. The collected tissue can be applied directly in the methods of the present invention, or it can be combined with the tissue-based cell culture systems, described above.

GROWTH FACTOR The active growth factor used in the present invention is preferably of the subclass of proteins generally known as bone morphogenetic proteins (BMPs), which have been described as having osteogenic, chondrogenic and other activities of the growth and differentiation type. . These BPMs include the rhBMP-2, rhBMP-3, rh.BMP-4, (also referred to as rhBMP-2B), rhBMP-5, rhBMP-6, rhBMP-7, (rhOP-1), rhBMP-8, rhBMP-9, rhBMP-12, rhBMP-13, rhBMP-15, rhBMP-16, rhBMP-17, rhBMP-18, rhGDF-1, rhGDF-3, rhGDF-5, rhGDF-6, rhGDF- 7, rhGDF-8, rhGDF-9, rhGDF-10, rhGDF-1lf rhGDF-12, rhGDF-14. For example, BMP-2, BMP-3, BMP-4, BMP-5, BMP-6 and BMP-7, described in US Patent Numbers: 5,108,922; 5,013,649; 5,116,738; 5,106,748; 5,187,076; and 5,141,905; BMP-8, described in PCT publication O91 / 18098; and BMP-9, described in PCT publication WO93 / 00432, BMP-10, described in U.S. Patent No. 5,637,480; BMP-11, described in U.S. Patent No. 5,639,638, or BMP-12 or BMP-13, described in U.S. Patent No. 5,658,882, BMP-15, described in U.S. Patent No. 5,635,372 and BMP- 16, described in copending patent application serial number 08 / 715,202. Other compositions that may also be useful include Vgr-2, and any of the growth and differentiation factors [GDFs], including those described in PCT applications 094/15965; 094/15949; WO95 / 01801; WO95 / 01802; W094 / 21681; 094/15966; O95 / 10539; O96 / 01845; WO96 / 02559 and others. Also useful in the present invention may be the BIP, described in O94 / 01557; HP00269, described in Japanese publication No. 7-250688; and MP52, described in PCT application O93 / 16099. The descriptions of all of these applications are incorporated herein by reference. Also useful in the present invention are the heterodimers of the above proteins and of the modified proteins or partial deletion products thereof. These proteins can be used individually or in mixtures of two or more, and rhBMP-2 is preferred. The BMP can be produced recombinantly, or it can be purified from a protein composition. BMP can be homodimeric, can be heterodimeric with other BMPs (eg, a heterodimer composed of a monomer from each of BMP-2 and BMP-6) or with other members of the TGF-β superfamily, such as activins , inhibins and TGF-β1 (for example, a heterodimer composed of a monomer from each of the BMPs and a related member of the TGF-β superfamily). Examples of such heterodimeric proteins are described, for example, in PCT Patent Application WO 93/09229, the specification of which is incorporated herein by reference. The amount of osteogenic protein useful herein is that amount effective to stimulate the increased osteogenic activity of infiltrating progenitor cells, and which will depend on the size and nature of the defect being treated, as well as the carrier that is employed. Generally the amount of protein that is delivered is in a range from about 0.05 to about 1.5 milligrams. In a preferred embodiment, the osteogenic protein is administered together with an effective amount of a protein that is capable of inducing the formation of tendon-like or ligament-like tissue. These proteins include BMP-12, BMP-13, and other members of the subfamily of BMP-12, as well as MP-52. These proteins and their use for the regeneration of tendon-like and ligament-like tissue are described in the American patent application serial number; 08 / 362,670, filed on December 22, 1994, the description of which is incorporated herein by reference. In another preferred embodiment, a heterodimer in which a monomeric unit is an osteogenic protein such as BMP-2, and the other monomeric subunit is a tendon-inducing protein, such as BMP-12, is administered in accordance with the methods described later, in order to induce the formation of a functional union between connective tissue and bone.

APPLICATION OF THE GROWTH FACTOR The growth factor can be applied to the tissue source in the form of a regulatory solution.

A preferred buffer is a composition comprising, in addition to the active growth factor, from about 1.0 to about 10.0% (w / v) glycine. From about 0.1 to about 5.0% (w / v) of a sugar, preferably sucrose, from about 1 to about 20 mM glutamic acid hydrochloride, and optionally from about 0.01 to about 0.1% of a nonionic surfactant such as polysorbate 80. Preferred solutions are from about 1% to about 20% w / v of cellulosic carrier / buffer. If desired, a salt can be added. Other materials that may be suitable for use in the application of growth factors, in the methods and compositions of the present invention, include hyaluronic acid, surgical mesh or sutures, polygluconate, temperature-sensitive polymers, demineralized bone, minerals and ceramics such as calcium phosphate, hydroxyapatite, etc., as well as combinations of the materials described above. However, in the preferred embodiment of the present invention, no carrier is employed. The growth factor of the present invention, in an appropriate buffer solution such as that described above, or combined with an appropriate carrier, can be applied directly to the tissue and / or site in need of tissue repair. For example, the growth factor can be physically applied to the tissue by spraying or dipping, or by using a brush or other suitable applicator such as a syringe for injection. Alternatively, or jointly, the protein can be applied directly to the site in need of tissue repair. The following examples further describe the practice of the embodiments of the invention, with BMP-2. The examples are not limiting, and as will be appreciated by those skilled in the art, they may vary according to the above specification.

EXAMPLES I. Aloinjerto in Rabbit All procedures were carried out with the approval of the IACUC. Twelve white rabbits, males, New Zealanders (6 months old) were used. Two rabbits served as donors and 10 as recipients. Osteochondral grafts (with a diameter of 3.5 millimeters) were collected from the trochlear fissure or the middle femoral condyle of the donors. They were transplanted into a defect 3.5 millimeters deep in the trochlear groove of the recipient. The graft was soaked in either rhBMP-2 (0.5 milligrams / milliliters) or in control buffer solution, before implantation. The rabbits were sacrificed 4 weeks after surgery and the transplants and the surrounding tissue were evaluated by a histological-histochemical classification scale such as that described in Sellers et al., J. Bone Joint Surg., 79-A: 1452-1463 (1997). A computerized image analysis of histological sections was also performed. The results were evaluated using a Student's t-test without pairing. In a general examination, the joints showed no signs of inflammation. All the defects were filled by the repair tissue. The superficial appearance of the defects was variable but acceptable and did not correlate with the form of treatment. Osteophytes were found in 3 joints (2 in the experimental group, 1 in the control regulatory group). There was no correlation between the general and histological appearance in any of the defects. The presence of chondrocytes in the lacunar and sporadic cloning of cells in the cartilage of the donor indicated the survival of the tissue. Focal degeneration of donor cartilage was present in all control groups but only in one of the groups treated with rhBMP-2. Healing of the defect in the group treated with rhBMP-2 was significantly improved compared to that in the control group. The group treated with rh.BMP-2 had improved bone integration, indicated by smaller fibrous repair tissue in the subchondral bone compartment. Treatment with rh.BMP-2 also resulted in a greater amount of cartilage above the mark left by the original tissue, apparently consisting of tissue from both the donor and recently regenerated cartilage of the recipient. There was no significant difference in the total amount of bone observed between the two groups.

TABLE I HISTOLOGICAL SCORING AND HISTOMORFOMETRIC MEASUREMENT FOR CARTILAGE REPAIR, MEDIUM VALUE (SD) * Statistically significant difference with respect to the control (p <0.05). ** The scale system varies from 0 (normal cartilage) to 31 (no repair).

Additional histomorphometric analysis data further support the beneficial effects of rhBMP-2 on graft healing. For example, the filling percentage of the new tissue above the marker has been shown to be 81.52% in a group treated with rhBMP-2 versus 57.63% in the control. There was less degeneration in the graft cartilage in the group treated with rhBMP-2 (23.83%) than in the control group (44.52%). The integration of the newly formed graft or cartilage with the host cartilage was improved by treatment with rhBMP-2 (56.48%) compared to that of the control group (21.89%). Newer cartilage was formed under the influence of rhBMP-2, either at the edge of the graft, which eliminated the free space between the graft and the host, or in the upper part of the graft, which made the graft more congruent with the surface of the joint. The above results show that the healing of allogenic osteochondral grafts, in articular cartilage defects, was improved by the addition of rhBMP-2. The active growth factor can have an accelerated subchondral bone junction, providing support and nutrition to the articular cartilage tissue. The addition of the growth factor can also stimulate new cartilage formation of the recipient's mesenchymal stem cells, in the bone marrow, and / or the synovial tissue. These results suggest that the combination of active growth factor, particularly bone morphogenetic proteins, and osteochondral allografts, could present a powerful strategy for the treatment of articular cartilage defects, particularly defects in articular cartilage throughout the thickness.

II. Rabbit autograft Osteochondral grafts (2.7 millimeters in diameter and 3.0 millimeters in length) were collected from the trochlear groove or femoral condyle and transplanted into a donor site 2.7 millimeters wide and 3.5 millimeters long over the trochlear groove or femoral condyle of the joint. of the knee in rabbits. Half of the animals had received dripping buffer at the recipient site prior to transplantation, and the grafts were immersed in buffer for 2 minutes and placed inside the recipient site. The other half had been immersed in 5 μg of rhBMP-2 at the site of the recipient, before transplantation, and then the grafts were immersed in buffer containing 500 μg / ml of rhBMP-2 for 2 minutes and then transplanted into the site of the recipient. The animals were sacrificed 4 weeks after surgery, and the recipient sites were evaluated histologically using both the histological-histochemical classification scale [Sellers, et al., J. Bone Joint Surg., 79-A: 1452-63 (1997 )] and computational, quantitative tissue analysis. The data indicated that treatment with rhBMP-2 improved the healing of the autograft. The most dramatic effects were the reduction of graft cartilage degeneration (rhBMP-2 8.18% versus control 36.25%), and more cartilage was formed at the edge of the graft (rhBMP-2 88.23% versus control 50%).

III. Autograft in Non-Human Primate: The non-human primates used for the autograft experiments were cynomologous macaques. Osteochondral grafts (3.5 millimeters in diameter x 6 millimeters in length) were harvested from the trochlear groove of 6 cynomologous macaques and transplanted into drilled vessel sites in both the middle and lateral femoral condyle of the same animal (n = 12 transplants in total). Before transplantation, 25 μg of rhBMP-2 was dripped into 6 recipients' sites, and the grafts of these 6 transplants were immersed in a solution of 1.25 milligrams / milliliters of rhBMP-2 for 2 minutes. In the other 6 transplants only regulatory solution was dripped at the recipient sites and the grafts were submerged only in buffer for 2 minutes before transplantation. The limbs were immobilized with a mold for 2 weeks after the operation and the animals were sacrificed 9 weeks after the operation. All the animals had normal functioning of their knee joints. In a general examination the joints showed no signs of inflammation. No osteophytes were found in any joint. Although the surface of the defects appeared to be level with the surrounding cartilage, in a general examination, microscopic observation revealed subsidence of the grafts in most cases. The tissue observed in a general manner, covering the surface, was actually newly formed tissue on top of the graft. The computerized image analysis was carried out by a blind evaluator to quantify the percentage filling of the defect, the new tissue types were formed on top of the original mark, and the integration of the grafts and the surrounding cartilage. Favorable results were observed in the group treated with rhBMP-2 in all these parameters. Newer cartilage was formed between the graft and the host cartilage to eliminate free space, resulting in better integration of the graft with the surrounding cartilage (rhBMP-2 88.59% versus control 64.82%). The filling of the cartilage defect was better in the group treated with rhBMP-2 (95.02%) than in the control group (86.68%). There was more fibrous tissue in the control group (11.90% versus rhBMP-2, 2.65%), whereas more transition tissue was found in the group treated with rhBMP-2 (36.38% versus control, 20.53%). There were no significant differences in the total histochemical-histological score between the two groups. Computed tomography, quantitative, peripheral (pQCT) showed that bone density increased in donor sites, over time. At 6 weeks and 9 weeks after the operation, the tissue at the donor sites, treated with rhBMP-2, was significantly more dense and the healing process was more advanced compared to the control sites. Histologically, the sites of the donor contained bone trabeculae regenerated with fibrous tissue on the surface in all cases.

III. Retention of rhBMP-2 Ex Vivo: The retention of rh.BMP-2 in osteochondral graft, with this technique, was evaluated with the grafts of non-human primates. The graft was immersed in a mixing solution of rhBMP-2 labeled with 125 I and unlabelled rhBMP-2. The results showed that the amount of rhBMP-2 absorbed in the graft was proportional to the concentration of the protein, and to the time of soaking. Other factors that affect the retention of rhBMP-2, included the size of the graft, and the presence of elements of the marrow between the trabecular bone.

V. Retention time of the r BMP-2 In Vivo: The retention time of rhBMP-2 in osteochondral graft was evaluated in rabbits. A mixture solution of rhBMP-2 labeled with 125 I and unlabelled rhBMP-2, containing 5 μg of rhBMP-2 and 20 μCi 125 I, was loaded onto the graft before implantation. The animals were explored with a camera? during the follow-up time for 22 days after the operation. Compared to the time of the collagen sponge, as a carrier, the mean time of rhBMP-2 in osteochondral graft was increased from 1 day to 3 days. The radioactivity of 10% of the initial point was maintained from 11 days of the collagen sponge to 22 days of the graft.

SAW. Allografts in non-human primates Donor sites (3.5 millimeters wide x 6 millimeters long) were removed from the trochlear grooves of 12 adult cynomolgus monkeys and transplanted into 3.5 x 6 millimeter recipient sites in the middle and lateral femoral condyles of unrelated individuals. Half of the transplants were rinsed in 1.25 mg / ml of rhBMP-2 for 2 minutes before transplantation, and half were rinsed in buffer. The identical procedure was carried out at the other extremity 7 weeks after the first surgery. The limb was immobilized with a mold for 2 weeks after the operation and after each surgery, and the animals were sacrificed 9 weeks after the second surgery for histological analysis. These results suggest that the combination of active growth factor, particularly bone morphogenetic proteins, and osteochondral autografts, could present a powerful strategy for the treatment of articular cartilage defects, particularly defects in joint cartilages throughout the thickness. In other embodiments, BMP-2 can also be applied to a frozen osteochondral allograft for the treatment of the focal defect in articular cartilage.

The foregoing descriptions detail the preferred embodiments of the present invention. It is expected that those skilled in the art will come up with numerous modifications and variations of the practice of the same, when considering these descriptions. It is believed that these modifications and variations should be included in the claims appended thereto.

It is noted that in relation to this date, the best method known by the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention.

Claims (14)

CLAIMS Having described the invention as above, the content of the following claims is claimed as property:

1. A method for the regeneration of articular cartilage, characterized in that it comprises administering to an area that needs the regeneration of the articular cartilage, at least one bone morphogenetic protein (BMP), purified.

2. A method for the regeneration of articular cartilage, characterized in that it comprises administering to an area in need of the regeneration of that articular cartilage, an appropriate tissue source, in combination with at least one bone morphogenetic protein (BMP), purified .

3. The method according to claim 1, characterized in that the BMP is BMP-2.

4. The method according to claim 2, characterized in that the BMP is BMP-2.

5. A method for the regeneration of articular cartilage, characterized in that it comprises administering to an area that needs the regeneration of that articular cartilage, at least one purified protein selected from the group consisting of Vgr-2, growth factors and differentiation ( GDFs), and BIP.

6. The method according to claim 1, characterized in that it also comprises a protein that induces the formation of tendon-like tissue or ligament. The method according to claim 6, characterized in that the protein that induces tendon or ligament tissue formation is selected from the group consisting of BMP-12, BMP-13 members of the subfamily of BMP-12 and MP52. 8. A composition for the regeneration of articular cartilage, the composition is characterized in that it comprises at least one bone morphogenetic protein (BMP), purified. 9. A composition for the regeneration of articular cartilage, the composition is characterized in that it comprises an appropriate tissue source, in combination with at least one bone morphogenetic protein (BMP), purified. 10. The composition according to claim 8, characterized in that the BMP is BMP-2. 11. The composition according to claim 9, characterized in that the BMP is BMP-2. 12. A composition for the regeneration of articular cartilage, the composition is characterized in that it comprises at least one purified protein selected from the group consisting of Vgr-2, growth and differentiation factors (GDFs), and BIP. 13. The composition according to claim 8, characterized in that it also comprises a protein that induces the formation of a tendon or ligament type of tissue. The composition according to claim 13, characterized in that the protein that induces the formation of tendon-like tissue or ligament, is selected from the group consisting of BMP-12, BMP-13 members of the subfamily of BMP-12 and MP52.