WO2009119935A1

WO2009119935A1 - Method and composition for inducing regeneration of damaged cartilage using microfracture and hyaluronic acid

Info

Publication number: WO2009119935A1
Application number: PCT/KR2008/002417
Authority: WO
Inventors: Byung-Soo Kim; Sun-Woong Kang
Original assignee: Industry University Cooperation Foundation IUCF HYU; Industry University Cooperation Foundation of Sogang University
Current assignee: Industry University Cooperation Foundation IUCF HYU; Industry University Cooperation Foundation of Sogang University
Priority date: 2008-03-26
Filing date: 2008-04-29
Publication date: 2009-10-01
Anticipated expiration: 2010-09-26
Also published as: KR20090102552A

Abstract

The present invention relates to a method, a composition and use thereof for inducing regeneration of damaged cartilage. Specifically, the invention relates to a method, a composition and use thereof for inducing regeneration of damaged cartilage by applying hyaluronic acid to damaged area of cartilage after microfracture formation. The present method and composition for inducing regeneration of damaged cartilage may regenerate hyaline-like cartilage with a simpler surgery procedure. No involvement of any transplantation step, make the present method more useful.

Description

METHOD AND COMPOSITION FOR INDUCING REGENERATION OF DAMAGED CARTILAGE USING MICROFRACTURE AND HYALURONIC ACID

Technical Field

[1] The present invention relates to a method, a composition and use thereof for inducing regeneration of damaged cartilage. Specifically, the invention relates to a method, a composition and use thereof for inducing regeneration of damaged cartilage by applying hyaluronic acid to damaged area of cartilage after microfracture formation. Background Art

[2] Articular cartilage has limited self-healing potential (Buckwalter et al. 1997,

Hunziker 2002, Prakash et al. 2002). Since articular cartilage has neither vascular supply nor easy access to stem cells, repair is often unsuccessful. Partial thickness defects cannot heal spontaneously because stem cells found in the bone marrow cannot be accessed (Buckwalter 1998). Full thickness defects, however, do have access to these cells and thus can heal spontaneously (Wei et al. 1997). However, the regenerated cartilage is fibrocartilage rather than hyaline cartilage (Frisbie et al. 2003) and, being mechanically weaker than hyaline cartilage, it gradually degenerates over time (Shapiro et al. 1993).

[3] For treating damaged articular cartilage, no promising therapeutic method has been established until recently. Two major methods may be used to regenerate damaged cartilage. Those are microfracture surgery, which stimulates the subchondral bone to induce regeneration of articular cartilage, and transplantation of chondrogenic cells. Disclosure of Invention Technical Problem

[4] Microfracture used to treat articular cartilage injuries can facilitate the intrinsic healing response of stem or progenitor cells recruited by subchondral penetration to the marrow and can stimulate cartilage regeneration. Multiple drilling, arthroplasty, microfracture surgery, or the like may be used in this method. However, microfracture has been shown to result in regeneration of fibrocartilage, with inferior mechanical properties, rather than the desired hyaline cartilage. Microfracture has been failed to regenerate cartilage that maintains its functional integrity of articular cartilage.

[5] Cartilage transplantation has limitations. Various problems result at the area where the graft has been provided and the method cannot be used when the chondral lesion is too large. Furthermore, margins are generated between the original tissues and healed regions.

[6] Autologous chondrocyte transplantation, wherein chondrocytes that were harvested and in vitro cultured beforehand are implanted in a chondral lesion. This method involves two serial surgeries, chondral harvest and cell implantation, which necessitate chondrocyte-harvest site morbidity and also burdensome in terms of time, cost and medical aspects.

[7] There also have been developed methods of chondral regeneration using bone marrow- or adipose tissue-derived stem cell. In these methods, in vitro cultured stem cells are differentiated to chondrocytes and implanted in a chondral lesion. Dexa- methasone which is added to the cell culture may be toxic to cells. The possibility of contamination during in vitro cultivation and the duration of time that is required to culture cells before they can be implanted to treat a patient are also raised as main problems. Technical Solution

[8] The present inventors, surprisingly, found that application of hyaluronic acid to the area including a microfractured site for treating damaged cartilage resulted in regeneration of hyaline-like cartilage rather than fibrocartilage by the stem or progenitor cells recruited by subchondral penetration to the marrow.

[9] The present invention will be hereinafter described in detail.

[10] In an embodiment, the present invention provides a method for inducing regeneration of cartilage in a mammal comprising:

[11] forming one or more microfractures on a damaged area of cartilage;

[12] applying biomaterial containing hyaluronic acid to said damaged area of cartilage including the formed microfracture(s); and

[13] allowing said damaged area regenerate cartilage.

[14] Hyaluronic acid is a type of complex polysaccharide composed of N-acetyl glucosamine and glucuronic acid.

[15] In an embodiment, the present invention provides the present method for inducing regeneration of cartilage is provided, wherein the hyaluronic acid being in a cross-linked form or the carboxyl group or alcohol group of the hyaluronic acid being chemically modified.

[16] In another embodiment, the present invention provides the present method for inducing regeneration of cartilage, wherein bone marrow-derived stem cells flowing over the damaged area of cartilage as a result of the formation of microfracture(s). Preferably, when carrying out the present method for inducing regeneration of cartilage, one forms one or more microfractures and confirms that bone marrow- derived stem cells flow over the damaged area of cartilage before applying biomaterial containing hyaluronic acid.

[17] 'Biomaterial', as used herein, means material which is generally allowed to be implanted, injected or applied to human and whose biocompatibility is verified. Biomaterial may comprise pharmaceutically acceptable buffer, injection or the like including saline solution. The biomaterial may also comprise one or more carrier, vehicle, excipient for efficient delivery of an effective component.

[18] In the method of the present invention, any type of biomaterial containing hyaluronic acid may be used as long as that can be applied to a damaged area of cartilage including microfracture(s). Preferable, the biomaterial containing hyaluronic acid is gel-type.

[19] In another embodiment, the method for inducing regeneration of cartilage of the present invention does not comprise transplantation of cells including chondrocytes.

[20] The method for inducing regeneration of cartilage of the present invention may be used in combination with other method for inducing cartilage regeneration. The method that can be used with the present method include any surgical or drug administering methods known in the art.

[21] In an embodiment, the present invention provides a composition for applying to a mi- crofractured site for inducing regeneration of damaged cartilage comprising hyaluronic acid. Preferably, the molecular weight of hyaluronic acid is greater than 10,000 Da.

[22] In another embodiment, the present composition for applying to a microfractured site for inducing regeneration of damaged cartilage further comprises one or more inducers of chondrogenic differentiation. Preferable inducers of chondrogenic differentiation may be selected from the group consisting of transforming growth factor -β(TGF-β), BMPs, IGF, Wnt proteins. More preferably, the inducer of chondrogenic differentiation is TGF-β or BMP-2.

[23] In an embodiment, the present invention provides a use of hyaluronic acid for applying to a microfractured site for inducing regeneration of damaged cartilage.

Advantageous Effects

[24] The composition of the present invention may be applied to an damaged area of cartilage after microfrature formation in that area. Bone marrow-derived stem cells flowed over the damaged area of cartilage facilitate regeneration of hyaline-like cartilage, which is more natural, rather than fibrocartilage thereby improve remedial results.

[25] Furthermore, no additional surgery for harvesting bone marrow or adipose tissue is needed to obtain bone marrow- or adipose tissue-derived stem cells. This single-stage surgical procedure requires minimal surgical time, equipment and supply cost.

[26] Even further, since no cell transplantation is involved, there are no need for chondrocyte cultivation and no fear of contamination or infection, which makes the method much safe. A patient can be treated immediately and does not need to wait until cells are cultured and ready to use, which generally takes more than one month. This may be very advantageous for clinical perspectives. Brief Description of the Drawings

[27] Fig. 1 shows photographs of rabbit knee articular cartilage defects immediately after creation (A) and after microfracture surgery (B). (A) A full-thickness cartilage defect (2.5 x 5 mm, depth 1-2 mm) was created on the patellar groove. The dotted line indicates the defect. (B) Four holes of subchondral penetration (arrows) were made in the defects.

[28] Fig. 2. shows photographs of rabbit knee articular cartilage defects three months after treatments. (A) Treatment with microfracture only (group A). (B) Treatment with microfracture and hyaluronic gel (group B). (C) Treatment with microfracture and Hyaluronic gel containing TGF- β (group C). The dotted lines indicate the defects.

[29] Fig. 3 illustrates H&E-stained histological sections of defects three months after treatments. (A) Treatment with microfracture only (group A). (B) Treatment with microfracture and HA gel (group B). (C) Treatment with microfracture and HA gel containing TGF-β (group C). The arrows indicate defect edge. The scale bars indicate 1 mm.

[30] Fig. 4 illustrates Alcian blue-stained histological sections of defects three months after treatments. (A) Treatment with microfracture only (group A). (B) Treatment with microfracture and HA gel (group B). (C) Treatment with microfracture and HA gel containing TGF-β (group C). The arrows indicate defect edge. The scale bars indicate 1 mm. Mode for the Invention

[31] Certain embodiments of the invention are described in the following examples. Other embodiments within the scope of the claims herein will be apparent to one with ordinary skill in the art. It is intended that the specification including the examples be considered exemplary, with the scope of the invention only being indicated by the claims.

[32] Examples

[33] Example 1 : Preparation of Hyaluronic acid solution

[34] Hyaluronic acid (molecular weight=4,000,000 Da, LG life Sciences Ltd. Seoul,

Korea) was dissolved in saline solution or DMEM culture medium (Invitrogen, USA) containing 100 U/ml of Penicillin and 100 μg/ml of Streptomycin to make 4% hyaluronic acid solution. To test the therapeutic effect of the inducers of chondrogenic differentiation, TGF-β (5 μg/ml) were added to saline solution or DMEM medium to prepare 4% hyaluronic acid solution.

[35] Example 2: Microfracture surgery on animal model for cartilage defects

[36] Three-month-old, skeletally mature, male New Zealand white rabbits (3.5 ± 0.5 kg,

Halim Animal Laboratory Co. Seoul, Korea) were anesthetized with an intramuscular injection of 250 mg ketamine hydrochloride (Yuhan Co., Seoul, Korea), 35 mg xylazine hydrochloride (Bayer, Seoul, Korea), and 5 mg of acepromizine (Yuhan Co., Seoul, Korea) Guidelines for the care and use of laboratory animals published by National Institute of Health (NIH publication No. 85-23, revised 1985)were observed during the animal experiments. A longitudinal skin incision was made over the anterior aspect of the knee joint, and a medial parapatellar arthrotomy was performed. The patella was reflected laterally and the entire distal femur was exposed. A full-thickness cartilage defect (2.5 x 5 mm, depth 1-2 mm) was created on the patellar groove by controlled drilling without violation of the subchondral bone (Figure IA). Using a fine 0.03-inch Kirschner wire (Sanatmetal Co., EGER, Hungary), meticulous subchondral penetration (microfracture method) was introduced to the defects (Figure IB). Four holes were made in each defect without violation of neighboring holes. The debris was completely removed and hemostasis was achieved.

[37] Twenty-four rabbit knee joints were divided into three groups: A, B, and C. The cartilage defects in the knee joints of group A (n=8) were not treated with HA(hyaluronic acid) and served as controls. The cartilage defects in the knee joints of group B (n=8) were filled with commercially available, medical grade 4% (w/v) HA in phosphate buffered saline (50 μl/site, Hyruan plus, LG Pharmacological Co., Chunbuk, Korea). The cartilage defects in the knee joints of group C (n=8) were filled with 4% HA gel mixed with TGF- β (5 μg/ml, 250 ng/site). After confirmation of the stability of HA in the cartilage defects, the surgical wound was closed in layers. Antibiotics (gentamycin) were given for 3 days, postoperatively. All rabbits were sacrificed at three months for analysis.

[38] Example 3: eve observation

[39] Three months after treatment, all the experimental animals were sacrificed and the joint and synovium from each were examined grossly. The entire distal femur was exposed and resected without any soft tissue attachments. Femoral and tibial condyles from each knee were examined macroscopically and photographed.

[40] The articular surfaces appeared normal in all groups with no evidence of synovial hypertrophy. The defects treated with microfracture and HA with or without TGF- β (groups B and C) appeared to be filled to a greater extent than the lesions without HA (group A). The defects treated without HA are partially filled and the surface was less smooth (Fig. X).

[41] Example 4: histological analysis [42] For histological analyses, specimens were fixed in 10 % (v/v) buffered formalin, decalcified with decalcifying agent (Decal Rapid™, National Diagnostics, Georgia, USA), dehydrated with a series of graded alcohol, and embedded in paraffin. Tissue sections (4 μm thick) were stained with hematoxylin and eosin (H&E) for morphologic analysis and with alcian blue staining for glucosaminoglycan production.

[43] Histological analyses showed that the quality of the regenerated tissues differed depending on group. H&E staining showed that cartilaginous tissues were regenerated in the defects in all groups (Figure 3) but that the thickness of the regenerated cartilaginous tissue in groups B and C was much thicker than that in group A. Furthermore, alcian blue staining appeared to be more intense in the regenerated cartilaginous tissues in groups B and C than in group A. This indicates the presence of greater amounts of glucosaminoglycan in groups B and C and suggests regeneration of a more hyaline-like cartilage (Figure 4).

Claims

[I] A method for inducing regeneration of cartilage in a mammal comprising:

(a) forming one or more microfractures on a damaged area of cartilage;

(b) applying biomaterial containing hyaluronic acid to said damaged area of cartilage including the formed microfracture(s); and

(c) allowing said damaged area regenerate cartilage.

[2] The method according to claim 1, wherein as a result of step (a), bone marrow- derived stem cells flow over the damaged area of cartilage.

[3] The method according to claim 1 or 2, wherein the biomaterial being gel-type.

[4] The method according to claim 1 or 2, wherein the hyaluronic acid being in a cross-linked form or the carboxyl group or alcohol group of the hyaluronic acid being chemically modified.

[5] The method according to claim 1 or 2, wherein the method does not comprise transplantation of cells including chondrocytes.

[6] A composition for applying to a microfractured site for inducing regeneration of damaged cartilage comprising hyaluronic acid.

[7] The composition of claim 6, wherein the molecular weight of hyaluronic acid is greater than 10,000 Da.

[8] The composition of claim 6 or 7, wherein the composition further comprises one or more inducers of chondrogenic differentiation selected from the group consisting of transforming growth factor -β(TGF-β), BMPs, IGF, Wnt proteins.

[9] The composition of claim 8, wherein the inducer of chondrogenic differentiation is TGF-β or BMP-2.

[10] The composition of claim 6 or 7, wherein the hyaluronic acid being in a cross- linked form or the carboxyl group or alcohol group of the hyaluronic acid being chemically modified.

[I I] Use of hyaluronic acid for applying to a microfractured site for inducing regeneration of damaged cartilage.