KR100704537B1 - Compositions for healing and repair of articular cartilage - Google Patents

Abstract

The present invention provides methods and compositions for treating articular cartilage defects and diseases, including mixtures of tissues such as osteochondral implants and active growth factors. The active growth factor is preferably a composition comprising at least one bone morphogenic protein and a suitable carrier. The method results in the regeneration of the functional recovery of articular cartilage tissue.

Bone Morphogenic Protein (BMP), Joints, Cartilage, Osteochondral, Growth and Differentiation Factors, Vgr-2, BIP, Tendons, Ligaments

Description

Compositions for healing and repairing articular cartilage {Compositions for healing and repair of articular cartilage}

The present invention relates to the field of tissue repair, in particular to the restoration of stable and functional articular cartilage repair. Thus, the present invention may be useful in reconstructive surgery or other methods for the regeneration or recovery of articular cartilage.

Recovery of articular cartilage wounds remains a challenge for orthopedic surgery. Several of the existing treatment strategies are based on the transplantation of cartilage and osteochondral tissue. Autologous osteochondral transplantation provides the most suitable physiological material. However, donor tissues are limited and often require surgery at the second site to harvest tissue for implants. Thus, despite substantial efforts in the art, there is a need for effective methods of repairing articular cartilage defects and wounds that provide adequate physiological recovery without the need to take autologous tissue from the patient.

Summary of the Invention

The present invention provides methods and compositions for regenerating functional and physiologically suitable tissue repair for the repair of articular cartilage wounds and defects. In particular, the present invention includes a method of treating a patient with articular cartilage wound or defect. The methods and compositions of the present invention benefit from the use of bone morphogenic proteins (BMPs), which are known to have osteogenic and / or cartilage properties and can be produced via recombinant DNA technology, which is a potentially limitless source. Have The methods and compositions of the present invention can accelerate the regeneration of functional articular cartilage or have more ultimate efficacy and stability, and have the additional advantage that tissue formed at the defect or wound site is physiologically compatible.

Using BMP to increase the repair of articular cartilage defects and wounds may provide a better treatment for osteoarthritis, thereby unnecessary, delaying or reducing the need for artificial femur replacement and other general interventions. Preclinical evaluation has shown that rhBMP-2 improves early healing of penetrating defects of articular cartilage in rabbits.

In accordance with the present invention, methods and compositions are provided for treating patients suffering from certain types of articular cartilage wounds or defects. The wound may be the result of a sudden compression, or wound, such as from exercise or from an unexpected accident that tears, damages or otherwise injures the articular cartilage.

The present methods and compositions are advantageous for the recovery or amelioration of articular cartilage defects, in particular full surface articular cartilage defects. In particular, other defects that undergo an additional course of deterioration to the extent that the defect site reaches the underlying subchondral bone can also be treated with the methods and compositions of the present invention.

In the present invention, an active growth factor such as BMP is added to a suitable tissue source. The tissue source may be the patient's own osteochondral implant, or may include allograft or artificially prepared tissue. In a preferred embodiment, the tissue source may be a chondrocyte culture, such as a stem cell culture prepared by chondrocyte or in vitro cell culture methods, with or without additional growth factors. See, eg, US Pat. No. 5,226,914; 5,811,094; 5,053,050; 5,053,050; No. 5,486,359; 5,786,217 and 5,723,331. The technical content of all of these applications is incorporated herein by reference.

The tissue also contained cartilage from Acufex 7 (Smith and Nephew, Inc., Andover, Mass.); COR system (Innovasive Technologies, Marlborough, Mass.); Or by traditional non-cell culture based methods, using techniques such as mosaic plasty harvested using commercially available devices such as the Arthrex 7 osteochondral autograft delivery system (Arthrex, Munich, Germany). You may. The harvested tissue can be applied directly in the method of the invention or mixed with the tissue based cell culture system described above.

Growth factor

The active growth factors used in the present invention have preferably been described as having bone formation, cartilage formation and other growth and differentiation type activity and are derived from a subset of proteins commonly known as bone morphogenic proteins (BMPs). do. Such BMPs are rhBMP-2, rhBMP-3, rhBMP-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-11, rhGDF-12, and rhGDF-14. For example, BMP-2, BMP-3, BMP-4, BMP-5, BMP-6 and BMP-7 are described in US Pat. Nos. 5,108,922, 5,013,649, 5,116,738, 5,106,748, 5,187,076 And 5,141,905; BMP-8 is described in PCT Publication No. WO91 / 18098; BMP-9 is described in PCT Publication No. WO93 / 00432, BMP-10 is described in US Pat. No. 5,637,480, and BMP-11 is described in US Pat. No. 5,639,638. BMP-12 or BMP-13 are described in US Pat. No. 5,658,882, BMP-15 is described in US Pat. No. 5,635,372, and BMP-16 is a co-pending publication. US patent application Ser. No. 08 / 715,202. Other compositions that may also be useful include Vgr-2 and PCT Publications WO94 / 15965, WO94 / 15949, WO95 / 01801, WO95 / 01802, WO94 / 21681, WO94 / 15966, WO95 / 10539, WO96 / 01845, WO96 / 02559, and others, including any growth and differentiation factors [GDF]. Also useful in the present invention are the BIPs described in PCT Publication No. WO94 / 01557, HP00269 described in Japanese Patent Publication No. 7-250688, and PCT Publication No. WO93 / 16099. ] MP52. The technical content of all of these applications is incorporated herein by reference. Also useful in the present invention are heterodimers of the above and modified proteins or partial deletion products thereof. These proteins can be used individually or as a mixture of two or more, with rhBMP-2 being preferred.

BMPs can be produced recombinantly or purified from protein compositions. BMP is a homodimer, or in combination with another BMP (e.g., a heterodimer consisting of one monomer each of BMP-2 and BMP-6) or TGF-β super, such as actibin, inhibin and TGF-β1 Heterodimers with other members of the family (eg, heterodimers consisting of one monomer of each of the related members of the BMP and TGF-β superfamily). Examples of such heterodimeric proteins are described, for example, in PCT Publication No. WO93 / 09229, the specification of which is incorporated herein by reference. The amount of osteogenic protein useful herein is an amount effective to stimulate an increase in osteogenic activity of infiltrating progenitor cells, depending on the carrier used as well as the size and nature of the defect to be treated. In general, the amount of protein to be delivered is in the range of about 0.05 to about 1.5 mg.

In a preferred embodiment, the osteogenic protein is administered with an effective amount of protein that can induce the formation of tendons or ligament-like tissues. The protein includes MP52 as well as other members of the BMP-12, BMP-13, and BMP-12 subfamily. The proteins, and their use for regeneration of tendons and ligament-like tissues, are described in US patent application Ser. No. 08 / 362,670, filed Dec. 22, 1994, the contents of which are incorporated herein by reference. Is introduced. In another preferred embodiment, one monomer unit is a bone forming protein such as BMP-2 and the other monomer subunit is a heterodimer, which is a tendon-derived protein such as BMP-12, according to the method described below to connect connective tissue and bone Induce functional adhesion formation between.

Application of Growth Factors

Growth factors can be applied to tissue sources in the form of buffers. One preferred buffer is, in addition to the active growth factor, about 1.0 to about 10.0% (w / v) glycine, sugars, preferably about 0.1 to about 5.0% (w / v) sucrose, about 1 to about glutamic acid hydrochloride 20 mM, and optionally 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 the cellulosic carrier / buffer. In some cases, salts may be added.

Other materials that may be suitable for use in applying growth factors in the methods and compositions of the invention include hyaluronic acid, surgical meshes or sutures, polyglyconates, thermosensitive polymers, desalted bones, minerals and ceramics (e.g., , Calcium phosphate, hydroxyapatite, etc.) as well as mixtures of the aforementioned materials. However, in a preferred embodiment of the present invention, no carrier is used.

The growth factors of the present invention in suitable buffers such as those described above, or in admixture with suitable carriers, can be applied directly to tissues and / or sites in need of tissue repair. For example, growth factors may be physically applied to the tissue via spraying or dipping, or using other suitable application devices such as brushes or syringes for injection. Alternatively, or in combination, the protein may be applied directly to the site in need of tissue repair.

The following examples further describe the practice of aspects of the invention using BMP-2. The examples are not intended to be limiting and may be modified in accordance with the above specification, as will be appreciated by those skilled in the art.

I. Rabbit Allograft

All procedures are carried out with the approval of IACUC. Use 12 male New Zealand white rabbits (6 months old). Two rabbits are used as donors and ten as recipients. Osteochondral implants (3.5 mm in diameter) are taken from the donor's troclear groove or medial femoral condyle and implanted into a 3.5 mm deep defect within the recipient's trocar. The implant is immersed in either rhBMP-2 (0.5 mg / ml) or buffer control prior to transplantation. Rabbits were sacrificed 4 weeks after surgery and implants and surrounding tissues were histologically as described in Sellers et al., J. Bone Joint Surg., 79-A: 1452-1463 (1997). Evaluate according to histochemical grade criteria. Computer image analysis of histological sections is also performed. Results are assessed using an unpaired Students t-test.

On visual inspection, the joint did not show any inflammatory symptoms. All defects were filled by the recovery tissue. The surface appearance of the defects varies but is acceptable and unrelated to the type of treatment. Osteophytes were identified in three joints (two in the experimental group and one in the buffer control group).

There was no correlation between gross and histological appearance in all defects. The presence of chondrocytes in the ossein and sporadic cell proliferation in the donor cartilage indicate tissue survival. Lesional degeneration of the donor cartilage is present in all control groups, but only in one of the rhBMP-2 treated groups. The healing of defects in the rhBMP-2 treated group was significantly improved compared to the control group. The rhBMP-2 treated group shows improved bone integration as indicated by less fibrous repair tissue in the subchondral bone compartment. rhBMP-2 treatment also produces more cartilage above the original tidemark, apparently consisting of both donor tissue and newly regenerated recipient cartilage. There was no significant difference in the total amount of bone observed between the two groups.

Histological scoring and histomorphologic measurements of cartilage recovery, mean value (SD) variable rhBMP-2 Control Average score ^** 10.0 (5.42) ^*  20.6 (5.18)  % Goal below  73.26 (13.28)  62.88 (18.07)  Fibrous tissue below peak (%)  2.19 (2.04)  15.81 (9.88)  Cartilage above peak (%) 74.70 (41.08) ^*  18.17 (26.70)  Defective Charge Rate (%) 96.53 (4.86) ^*  88.79 (8.04)   * Statistically significant difference from control (p <0.05) ** grading system ranging from 0 (normal cartilage) to 31 (no recovery)

Additional histomorphologic analytical data further supports the beneficial effect of rhBMP-2 in healing of the implant. For example, the percent fill of new tissue above the peak is 81.52% for the rhBMP-2 treated group versus 57.63% for the control. Less graft cartilage was denatured in the rhBMP-2 treated group (23.83%) compared to the control (44.52%). Integration of implants or newly formed cartilage with host cartilage was improved with rhBMP-2 treatment (56.48%) compared to control (21.89%). More new cartilage was formed under the influence of rhBMP-2 at the edge of the implant (which eliminates the gap between the implant and the host) or at the top of the implant (which makes the implant better match the joint surface). .

The results demonstrate that the healing of allogeneic osteochondral grafts in articular cartilage defects is improved due to the addition of rhBMP-2. Active growth factors can promote subchondral bone incorporation while providing support and nutrients to articular cartilage tissue. The addition of growth factors may also promote new cartilage formation from recipient mesenchymal stem cells in bone marrow and / or synovial tissue. These results suggest that the incorporation of active growth factors, especially bone morphogenetic proteins and osteochondral allografts, may provide an effective strategy for treating articular cartilage defects, in particular pectoral cartilage defects.

II. Rabbit self transplant

Osteochondral implants (2.7 mm in diameter and 3.0 mm in length) are taken from the trochlear orbital and implanted at the donor site with a width of 2.7 mm and a length of 3.5 mm on the trochlear oral cavity of the rabbit knee joint. Half of the animals are dipped into the recipient site prior to transplantation, and then the implant is immersed in the buffer for two minutes and placed at the recipient site. The other half was inoculated with 5 μg of rhBMP-2 into the recipient site prior to transplantation, then the implant was immersed in a buffer containing 3500 μg rhBMP-2 per mL for 2 minutes into the recipient site. The animals were sacrificed 4 weeks after surgery and the recipient sites were histologically- histochemically rated (Sellers, et al., J. Bone Joint Surg., 79-A: 1452-63 (1997)) and tissues. Assessment is histologically using quantitative computer image analysis. The data indicate that rhBMP-2 treatment improves the healing of autografts. The most dramatic effects are that the degeneration of transplanted cartilage is reduced (rhBMP-2 8.18% vs. control 36.25%) and that more cartilage is formed at the edge of the implant (rhBMP-2 88.23% vs. control 50%).

III. Primate self-transplant, not human                 

A non-human primate used in autotransplantation experiments is the cynomologous macaque. Osteochondral implants (3.5 mm in diameter x 6 mm in length) were harvested from the trochlear neurospheres of six cynomolgus monkeys and implanted into recipient sites perforated into both the inner and outer femoral bones of the same animal (n = 12 implants total). Prior to transplantation, 25 μg of rhBMP-2 is dispensed into 6 recipient sites and the implants from these 6 implants are immersed in a solution of rhBMP-2 1.25 mg per ml for 2 minutes. In the other six implants, buffer alone is dispensed into the recipient site and the implant is immersed in buffer alone for 2 minutes prior to implantation. The limbs are fixed with a cast bandage for 2 weeks after surgery and the animals are sacrificed 9 weeks after surgery.

Knee joint function was normal in all animals. On visual inspection, the joint did not show any inflammatory symptoms. Osteoblasts were not identified in any joints. Visual inspection showed that the defect surface appears flat with the surrounding cartilage, but most of the time, microscopic observations revealed the depression of the implant. Observed with the naked eye, the surface covering tissue is substantially the newly formed tissue on top of the implant. Computer image analysis is performed by a blinded evaluator to quantify the percentage of defect filling, new tissue types formed above the initial peak, and integration of the implant and surrounding cartilage. For all these variables, favorable results were observed in the rhBMP-2 treatment group. The more new cartilage formed to fill the gap between the implant and host cartilage, the better the implant and surrounding cartilage integrate (rhBMP-2 88.59% vs. control 64.82%). The filling of cartilage defects was better than in the rhBMP-2 treated group (95.02%) than in the control group (86.68%). More fibrous tissue (11.90% vs. rhBMP-2 5.65%) was present in the control group, whereas more transitional tissue (36.38% vs. 20.53%) was identified in the rhBMP-2 treated group. The overall histological-histochemical score between the two groups is not significantly different. Peripheral quantitative computed tomography (pQCT) indicates that bone density increases at the donor site over time. Six and nine weeks after surgery, the tissue is significantly denser at the rhBMP-2 treated donor site and the healing process is more accelerated compared to the control site. Histologically, the donor site in all cases contained bone trabeculae regenerated with fibrous tissue at the surface.

IV. RhBMP-2 retention in vitro

Using this technique, retention of rhBMP-2 in osteochondral implants is assessed using implants from non-human primates. The implant is immersed in a mixed solution of ¹²⁵ I labeled rhBMP-2 and unlabeled rhBMP-2. The results indicate that the amount of rhBMP-2 absorbed in the implant is proportional to the protein concentration and soak time. Other factors affecting the retention of rhBMP-2 include the size of the implant, and the presence of bone marrow components between the bone marrow.

V. rhBMP-2 Retention Over Time in Vivo

RhBMP-2 retention over time in osteochondral implants is evaluated in rabbits. A mixed solution of ¹²⁵ I labeled rhBMP-2 and unlabeled rhBMP-2, containing 5 μg rhBMP-2 and ¹²⁵ I 20 uCi, is added to the implant prior to transplantation. Animals are scanned with a γ-camera during the follow-up of 22 days after surgery. Compared to the time course of the collagen sponge as a carrier, the half-life of rhBMP-2 in osteochondral implants is increased by 1 to 3 days. 10% of the radioactivity at the starting point is maintained for 11 days for the collagen sponge compared to 22 days for the implant.

VI. Primate allograft, not human

The donor site (3.5 mm wide x 6 mm wide) is removed from the trochlear neurospheres of 12 adult cynomolgus monkeys and implanted into the 3.5 x 6 mm recipient site in the medial and lateral femoral bones of an unrelated individual. Half of the implant is immersed in 1.25 mg of rhBMP-2 per ml for 2 minutes prior to transplantation, and the other half is immersed in buffer. The same procedure is performed on the other limb 7 weeks after the first surgery. The limbs are fixed with a cast bandage for 2 weeks after each surgery and the animals are sacrificed for histological analysis 9 weeks after the second surgery.

These results suggest that a mixture of active growth factors, in particular bone morphogenetic proteins and osteochondral autografts, may provide an effective strategy for the treatment of articular cartilage defects, in particular full surface articular cartilage defects. In another embodiment, BMP-2 can also be applied to frozen osteochondral allografts for the treatment of lesioned articular cartilage defects.

The foregoing descriptions detail preferred embodiments of the present invention. It is foreseen that one of ordinary skill in the art would be able to contemplate its numerous modifications and variations in view of the above techniques. Such modifications and variations are considered to be included within the claims appended hereto.

Claims (17)

delete delete delete delete delete delete delete delete A composition for treating osteoarthritis, comprising osteochondral implants with one or more purified bone morphogenic proteins (BMP). delete The composition of claim 9 wherein the BMP is BMP-2. The composition of claim 9, further comprising at least one purified protein selected from the group consisting of Vgr-2, growth and differentiation factors (GDFs), and BIP. The composition of claim 9, further comprising a protein that induces the formation of tendons or ligament-like tissues. The composition of claim 13, wherein the protein that induces the formation of tendon or ligament-like tissue is selected from the group consisting of members of BMP-12, BMP-13, BMP-12 subfamily and MP52. The composition of claim 9, wherein the implant is allograft. The composition of claim 9, wherein the implant is an autograft. 10. The composition of claim 9, wherein the implant is obtained by mosaicplasty.