HK1231785B - Fgf-18 in graft transplantation and tissue engineering procedures - Google Patents

Description

FGF-18 for transplantation and tissue engineering procedures

Technical Field

The present invention relates to regenerative medicine, in particular for the treatment of cartilage diseases (e.g., osteoarthritis, cartilage damage and osteochondral defects). More specifically, the present invention relates to FGF-18 compounds for use in tissue engineering and transplantation procedures (e.g., osteochondral or cartilage transplantation, or autologous chondrocyte transplantation (ACI)).

Background Art

Generally speaking, cartilage diseases are conditions characterized by metabolic deterioration in connective tissue, manifesting as pain, stiffness, and limited mobility in the affected body part. These diseases can be caused by pathogens (e.g., osteoarthritis (OA)) or by trauma or injury. Osteochondral defects (OCDs)—losses of the cartilage covering the ends of bones in joints—are more commonly caused by trauma or injury, but can also be caused by pathogens. OCDs can lead to OA. Mature cartilage has limited self-repair capacity, primarily because mature chondrocytes have little proliferative potential and lack blood vessels. Replacing damaged cartilage, particularly articular cartilage, caused by injury or disease presents a major challenge for physicians, and existing surgical procedures are considered unpredictable and effective only for a limited time. Consequently, most young patients do not seek treatment or are advised to delay treatment as much as possible. When treatment is necessary, the standard procedure varies by age and varies between total joint replacement, cartilage block transplantation, or bone marrow stimulation techniques such as microfracture surgery. Microfracture surgery is an economical and common procedure that involves puncturing the subchondral bone to stimulate cartilage deposition using bone marrow stem cells. However, this technique has been shown to be inadequate for repairing cartilage defects, and the newly formed cartilage is primarily fibrocartilage, leading to inadequate or altered function. Indeed, fibrocartilage does not possess the same durability and may not properly adhere to the surrounding hyaline cartilage. Consequently, the newly synthesized fibrocartilage may be more susceptible to damage (estimated duration: 5-10 years).

For patients with osteoarthritis, nonsurgical treatments primarily include physical therapy, lifestyle changes (such as reduced activity), support devices, oral or injectable medications (such as nonsteroidal anti-inflammatory drugs), and medical management (although there are no commercially available therapies that can repair cartilage damage (see Lotz, 2010)). If these treatments fail, the patient's main option is surgery, such as (partial or total) joint replacement. This option can relieve symptoms but often results in decreased joint function. Tibial or femoral osteotomy (cutting the bone to restore balance to the wear and tear of the joint) can relieve symptoms, help maintain an active lifestyle, and delay the need for total joint replacement. Total joint replacement can improve symptoms in advanced osteoarthritis but generally requires changes in the patient's lifestyle and/or activity level.

Current cartilage repair surgeries include total joint replacement, bone marrow stimulation (e.g., microfracture surgery), osteochondral allograft or autograft, and cultured cartilage implantation (e.g., autologous chondrocyte implantation (ACI)). These surgeries provide treatment options for patients with symptomatic cartilage damage. Osteochondral allograft or autograft is a common surgery for treating localized joint defects. Various factors may affect the effectiveness of the surgery, including the source of the donor cartilage, the health of the cartilage around the defect site, and the quality of integration. Unfortunately, in many cases, osteochondral transplantation surgery results in poor integration.

In general, in tissue engineering methods, cells are grown in a three-dimensional (3D) matrix, wherein each component of the matrix plays a key role in tissue regeneration. The main type of stem cell used for cartilage formation is human MSC (hMSC) (Zhang et al., 2013). However, the type, skeleton and other factors of MSC are important in tissue engineering. In addition, it is important to ensure that the regeneration of uniform hyaline cartilage-like structure is integrated into the defect with high quality. It is complex to establish and maintain the phenotype during articular cartilage tissue engineering and can be optimized by using factors that inhibit hypertrophy (Tang et al., 2012). For example, although adding TGF-beta1 promotes the expression of aggrecan, type II collagen and Sox-9 genes of hMSC, the newly synthesized cartilage is mainly composed of fibrous transient tissue rather than hyaline tissue (Zhang et al., 2013).

Another type of tissue engineering procedure is cultured cartilage implantation, such as autologous chondrocyte implantation (ACI), in which cartilage is harvested from a low-load-bearing area of the joint surface of the patient to be treated; the chondrocytes are then isolated and cultured in vitro in a monolayer or 3D culture; after a period of culture, the resulting chondrocytes or 3D structures are implanted into the defect to fill the defect. Unfortunately, it is known that the expansion of chondrocytes (particularly monolayer cultures) can induce fibroblast-like chondrocytes (Magill et al., 2011).

Fibroblast growth factor 18 (FGF-18) is a proliferator of chondrocytes and osteoblasts (Ellsworth et al., 2002; Shimoaka et al., 2002). It has been proposed to be used alone (WO2008/023063) or in combination with hyaluronic acid (WO2004/032849) for the treatment of cartilage diseases such as osteoarthritis and cartilage damage. A lyophilized formulation containing FGF-18 has shown potential for the treatment of OA or CI when injected intra-articularly.

Although cartilage repair surgery such as osteochondral transplantation and cultivating cartilage implantation (such as ACI) is promising, the rate of integration and the quality of the cartilage produced therein are still to be improved. Therefore, a kind of improved surgical method is needed so that the cartilage produced has good integration and good quality (that is, mainly hyaline cartilage). In fact, the generation of described hyaline cartilage is all valuable (Getgood etc., 2010) as therapeutic agent and as the composition two aspects of biological matrix.

Summary of the Invention

The present invention provides a method for preparing an implantable cartilage material for use in tissue engineering or osteochondral/cartilage transplantation, wherein the method comprises or consists of culturing chondrocytes, or osteochondral/cartilage explants, in a culture medium containing an FGF-18 compound, in a monolayer or 3D culture for a sufficient period of time to allow for the formation of the implantable osteochondral/cartilage material. Optionally, the FGF-18 compound may be additionally injected into the implantation site of the resulting osteochondral/cartilage material before, simultaneously with, or after transplantation.

In another embodiment, the present invention relates to a method for regenerating cartilage in a mammal at a site of joint defect (e.g., cartilage defect) caused by a cartilage disease, the method comprising or consisting of the following steps: (a) culturing chondrocytes, or culturing osteocartilage or cartilage explants, in a culture medium containing an FGF-18 compound in a monolayer culture or 3D culture, and (b) administering the cultured chondrocytes or cultured osteocartilage/cartilage explants obtained in step (a) to a mammal in need thereof. Optionally, the FGF-18 compound may be additionally injected into the site of administration of the cultured chondrocytes or osteocartilage/cartilage explants before, simultaneously with, or after administration of the cells/explants.

In another embodiment, the present invention discloses a method for regenerating cartilage in a mammal at a site of joint defect (e.g., cartilage defect) caused by cartilage disease, the method comprising or consisting of the following steps: (a) culturing chondrocytes, or culturing osteocartilage or cartilage explants, in culture medium in a monolayer or 3D culture, (b) administering the cultured chondrocytes or cultured osteocartilage/cartilage explants obtained in step (a) to a mammal in need thereof, and (c) injecting an FGF-18 compound into the site of administration of the cultured chondrocytes or osteocartilage/cartilage explants. Step (c) may be performed before, simultaneously with, or after administration of the cells/explants.

In a third embodiment, the present invention relates to an FGF-18 compound for use in a method for treating a defect in cartilage tissue of a mammal, wherein the defect is caused by a cartilage disease, the method comprising or consisting of the following steps: (a) subjecting chondrocytes or osteochondral/cartilage explants to in vitro or in vitro culturing, wherein the culturing is carried out in a cell culture medium containing the FGF-18 compound, (b) optionally repeating step (a) to obtain a graft material comprising the cultured chondrocytes or the cultured osteochondral/cartilage graft, and (c) transplanting the graft material obtained in step (b) into the defect of the mammal in need of said treatment, wherein the chondrocytes in steps (a) and (b) can be cultured in a monolayer culture or a 3D culture. Optionally, the FGF-18 compound can be additionally injected into the transplantation site before, simultaneously with, or after the transplantation.

In another embodiment, the present invention discloses an FGF-18 compound for use in a method of treating a defect in cartilage tissue of a mammal, wherein the defect is caused by a cartilage disease, the method comprising or consisting of the following steps: (a) subjecting chondrocytes or osteocartilage/cartilage explants to in vitro or in vitro culture, (b) optionally repeating step (a) to obtain a graft material comprising the cultured chondrocytes or the cultured osteocartilage/cartilage explants, (c) transplanting the graft material obtained in step (b) into the defect of the mammal in need of said treatment, wherein in steps (a) and (b) the chondrocytes can be cultured in a monolayer culture or a 3D culture, and (d) injecting the FGF-18 compound into the transplantation site. Step (d) can be performed before, simultaneously with, or after the transplantation.

In a fifth embodiment, the present invention provides a composition comprising mammalian osteochondral/cartilage explants or cultured mammalian chondrocytes in a culture medium containing an FGF-18 compound for use in tissue engineering or osteochondral/cartilage transplantation in a mammal in need thereof.

In the context of the present invention as a whole, said chondrocytes or said osteocartilage/cartilage explants are preferably collected or isolated from a mammal prior to the expansion or culturing step.

In the context of the present invention as a whole, for 3D cultures of chondrocytes or osteochondral/cartilage explants, the FGF-18 compound is preferably intermittently added to the culture medium on approximately one, two, or three days per week, with the one, two, or three-day addition repeated weekly for at least two weeks of culture, at least three weeks of culture, or at least four weeks of culture. Preferably, the FGF-18 compound is intermittently added to the culture medium on one, two, or three days per week, with the one, two, or three-day addition repeated weekly for two weeks of culture, three weeks of culture, or four weeks of culture. Alternatively, the FGF-18 compound may be intermittently added to the culture medium on approximately one, two, or three days per month, with the one, two, or three-day addition repeated monthly for at least two months of culture, at least three months of culture, or at least four months of culture. Preferably, the FGF-18 compound is intermittently added to the culture medium on one, two, or three days per month, with the one, two, or three-day addition repeated monthly for two months of culture, three months of culture, or four months of culture. Alternatively, the FGF-18 compound may be permanently maintained in the culture medium. For chondrocytes cultured in a monolayer, the FGF-18 compound is preferably, but not limited to, permanently added.

According to any embodiment of the present invention, the cartilage disease is preferably osteoarthritis, cartilage injury or osteochondral defect.

In the context of the present invention as a whole, the FGF-18 compound is preferably selected from the group consisting of: a) a polypeptide comprising or consisting of the mature form of human FGF-18, said mature form of human FGF-18 comprising residues 28-207 of SEQ ID NO: 1, b) a polypeptide comprising or consisting of residues 28-196 of SEQ ID NO: 1, or c) a polypeptide comprising or consisting of SEQ ID NO: 2.

Furthermore, in the context of the present invention as a whole, the explant is preferably a cartilage explant, and the chondrocytes are preferably chondrocytes or mesenchymal stem cells derived from mature tissue. As desired, the chondrocytes or the osteocartilage/cartilage explant are collected from the mammal to be treated or a different mammal, preferably the same species as the mammal to be treated. The mammal to be treated is preferably a human, but may also be, but is not limited to, a horse, camel, sheep, dog, or a smaller mammal such as a cat, rabbit, rat, or mouse.

definition

As used herein, the terms "FGF-18 compound" or "FGF-18" refer to proteins that retain at least one biological activity of the human FGF-18 protein. FGF-18 can be native, in its mature form, in a recombinant form, or in a truncated form. Biological activities of the human FGF-18 protein include, inter alia, increased chondrocyte or osteoblast proliferation (see WO98/16644) or increased cartilage formation (see WO2008/023063). Native, or wild-type, human FGF-18 is a protein expressed by chondrocytes of articular cartilage. WO98/16644 first designated human FGF-18 as zFGF-5 and fully described it. SEQ ID NO: 1 corresponds to the amino acid sequence of native human FGF-18, whose signal peptide consists of amino acid residues 1 (Met) through 27 (Ala). The mature form of human FGF-18 corresponds to the amino acid sequence of residue 28 (Glu) to residue 207 (Ala) of SEQ ID NO: 1 (180 amino acids).

In the present invention, FGF-18 can be produced by recombinant methods, such as those taught in patent application WO2006/063362. Depending on the expression system and conditions, FGF-18 is expressed in recombinant host cells and has either an initial methionine (Met) residue or a secretion signal sequence. When expressed in a prokaryotic host (e.g., E. coli), FGF-18 contains an additional Met residue at the N-terminus of its sequence. For example, when expressed in E. coli, the amino acid sequence of human FGF-18 begins with a Met residue at the N-terminus (position 1), followed by residues 28 (Glu) to 207 (Ala) of SEQ ID NO: 1.

As used herein, the term "truncated form" of FGF-18 refers to a protein comprising or consisting of residues 28 (Glu)-196 (Lys) of SEQ ID NO:1. Preferably, the truncated form of the FGF-18 protein is a polypeptide designated "trFGF-18" (170 amino acids; also known as rhFGF18 or sprifermin), which begins with a Met residue (N-terminus) followed by amino acid residues 28 (Glu)-196 (Lys) of wild-type human FGF-18. The amino acid sequence of trFGF-18 is set forth in SEQ ID NO:2 (amino acid residues 2-170 of SEQ ID NO:2 correspond to amino acid residues 28-196 of SEQ ID NO:1). trFGF-18 is a truncated form of recombinant human FGF-18 produced in Escherichia coli (see WO2006/063362). It has been demonstrated that FGF-18 exhibits activities similar to those of mature human FGF-18, for example, it increases the proliferation of chondrocytes and the deposition of cartilage, leading to the repair and reconstruction of various cartilage tissues (see WO2008/023063).

As used herein, "cartilage disease" includes conditions resulting from damage caused by injury, such as trauma, osteochondritis, or arthritis. Such diseases result in defects, preferably cartilage defects. Examples of cartilage diseases that can be treated by administering the FGF-18 formulations described herein include, but are not limited to, arthritis (e.g., osteoarthritis), cartilage damage, and osteochondral defects. The term also includes degenerative diseases/conditions of cartilage or joints, such as chondrocalcinosis, polychondritis, relapsing polychondritis, ankylosing spondylitis, or costochondritis. The International Cartilage Repair Society has proposed an arthroscopic grading system for assessing the severity of cartilage defects: Grade 0: (normal) healthy cartilage, Grade 1: cartilage with soft spots or blisters, Grade 2: tiny tears visible in the cartilage, Grade 3: lesions with deep cracks (more than 50% of the cartilage layer), and Grade 4: cartilage tears exposing the underlying (subchondral) bone (see page 13 of the ICRS publication http://www.cartilage.org/_files/contentmanagement/ICRS_evaluation.pdf).

The term "arthritis" as used herein includes diseases such as osteoarthritis, rheumatoid arthritis, juvenile rheumatoid arthritis, infectious arthritis, psoriatic arthritis, Still's disease (a cause of juvenile rheumatoid arthritis) or osteochondritis dissecans, and preferably includes diseases or conditions in which the cartilage is damaged or separated from the underlying bone.

The term "osteoarthritis" or "OA" is used to refer to the most common form of arthritis. The term "osteoarthritis" refers to cartilage diseases, including primary osteoarthritis and secondary osteoarthritis (e.g., see The Merck Manual, 17th edition, page 449). Osteoarthritis can be caused by cartilage breakdown. Small pieces of cartilage can break off and cause pain and swelling in the joints between bones. Over time, the cartilage can completely wear away, and the bones can rub against each other. Osteoarthritis can affect any joint, but commonly involves the hands, shoulders, and weight-bearing joints such as the hips, knees, feet, and spine. In a preferred embodiment, the osteoarthritis can be knee osteoarthritis or hip osteoarthritis. The term primarily encompasses forms of osteoarthritis that can be classified as Stage 1 to Stage 4 or Grade 1 to Grade 6 according to the OARSI classification system. Those skilled in the art are familiar with the osteoarthritis classification used in the art, particularly the OARSI Assessment System (also known as OOCHAS; see, e.g., Custers et al., 2007). Osteoarthritis is one of the preferred cartilage diseases that can be treated by administering the FGF-18 compounds of the present invention.

The term "cartilage injury" as used herein refers primarily to cartilage diseases or cartilage damage caused by trauma. Cartilage injury can primarily occur after traumatic mechanical damage, particularly accidents or surgery (e.g., microfracture surgery). The term "cartilage injury" also includes cartilage or osteochondral fractures and meniscal injuries. The definition also includes sports-related injuries or wear and tear of joint tissue related to sports. The term also includes microtrauma or blunt trauma, cartilage fractures, osteochondral fractures, or meniscal injuries.

The term "osteochondral defect" (OCD) refers to cartilage disorders in joints where a defect in the cartilage covering the end of a bone occurs. These defects are most often caused by trauma or injury, but can also be caused by pathogens. OCD can lead to OA. Generally, OCD means that part of the bone, not just the cartilage, is involved. If only the cartilage is involved, we prefer to use the term "chondral lesion" (see above).

The term "tissue engineering" also includes autologous chondrocyte implantation (ACI). This is also known as regenerative medicine. Cells or tissues can be grown in monolayer or 3D cultures. The goal of this procedure is to repair or replace part or all of a tissue.

The term "transplantation" refers to transplantation or implantation. This procedure is also part of regenerative medicine. It includes osteocartilage or cartilage (also referred to herein as osteocartilage/cartilage) transplantation/implantation, such as autologous or allogeneic transplantation/implantation of osteocartilage/cartilage. Within the scope of transplantation, the explant is collected from a mammal, i.e. the mammal to be treated (i.e. autologous transplantation) or preferably another mammal of the same species (allografting). It is usually taken from a healthy cartilage area or healthy osteocartilage tissue. This transplant is preferably performed at the level of the cartilage defect.

The terms "transplantable cartilage material" and "implantable material" are used interchangeably. They refer to cartilage-derived cells (e.g., chondrocytes) or osteocartilage/cartilage explants prepared for transplantation into a mammal in need thereof. Such transplantable materials are preferably transplanted/implanted at the level of the cartilage defect.

In the context of the present invention, the "effectiveness" of a treatment can be measured based on changes in cartilage thickness, such as the thickness of the articular cartilage of a joint. For example, the thickness can be measured by X-ray computed tomography, magnetic resonance imaging (MRI), or ultrasound.

The term "about" in "about 24, 48, or 72 hours" or "about one, two, or three days" includes changes in the culture medium 24, 48, or 72 hours after supplementation with the FGF-18 compound, as well as changes in the culture medium 24, 48, or 72 hours +/- several hours (e.g., +/- 1, 2, 3, or 4 hours) after supplementation with the FGF-18 compound. Similarly, the term "about" in "about 7 days," "about one week," "about four weeks," or "about one month" includes 7 days, 1 week, 4 weeks (i.e., 28 days), or one month, respectively, and also includes administrations separated by 7 days +/- 1 or 2 days, one week +/- 1 or 2 days, 4 weeks +/- several days (e.g., +/- 1, 2, 3, 4 days), or one month +/- several days (e.g., +/- 1, 2, 3, 4 days), respectively. In practice, it should be understood that, from a practical perspective, it is not always possible to change the medium or the next FGF-18 compound supplement at precise intervals, for example, exactly 24, 48, or 72 hours after the FGF-18 compound supplement, or exactly 4 weeks (28 days) after the last supplement (to the nearest day). Thus, in the context of the present invention, for example, 4 weeks can mean 28 days, but also 24, 25, 26, 27, 28, 29, 30, 31, or 32 days after the last administration. In the context of the present invention, "4 weeks" has a meaning similar to "1 month," and the two terms are used interchangeably ( FIG1 ). When referring to "days of the week," "4 weeks" is preferably used (e.g., the first supplement is on Monday, the next supplement is on Monday four weeks later), while when referring to "dates," "months" is preferably used (e.g., the first supplement is on August 1, the next supplement is on September 1).

The term "cycle" refers to the period of supplementation. In the context of the present invention, a weekly cycle (or a 7-day cycle) means that the culture medium is supplemented with an FGF-18 compound approximately one day per week (or approximately every 7 days). Thus, the cycle includes one day of culturing in supplemented culture medium and approximately six days of culturing in unsupplemented (i.e., FGF-18-free) culture medium. Similarly, a 4-week cycle means that the culture medium is supplemented with an FGF-18 compound approximately one day per 4 weeks. Thus, the cycle includes one day of culturing in supplemented culture medium and approximately four weeks of culturing in unsupplemented (i.e., FGF-18-free) culture medium. Similarly, for a monthly cycle: a monthly cycle means that the culture medium is supplemented with an FGF-18 compound approximately one day per month. Thus, the cycle includes one day of culturing in supplemented culture medium and approximately one month of culturing in unsupplemented (i.e., FGF-18-free) culture medium. The cycle can be repeated.

Detailed Description of the Invention

While cartilage repair procedures such as osteochondral transplantation and cultured cartilage implantation (e.g., ACI) are promising, the rate of integration and quality of the resulting cartilage remain to be improved. Therefore, there is a need for an improved surgical method that results in cartilage with good integration and quality (i.e., primarily hyaline cartilage) produced. The inventors have surprisingly discovered that when FGF-18 is used in regenerative medicine (e.g., tissue engineering procedures or transplantation procedures), the quality of the resulting cartilage is improved, and the cells/explants are better integrated into the defect.

The present invention provides a method for preparing a transplantable cartilage material for tissue engineering or osteochondral/cartilage transplantation, wherein the method comprises or consists of the following steps: culturing chondrocytes in a monolayer culture or 3D culture, or culturing osteochondral/cartilage explants in a culture medium containing an FGF-18 compound, for a period of time sufficient to allow the formation of the transplantable cartilage/cartilage material. The transplantable cartilage material can be used to treat cartilage diseases such as osteoarthritis, cartilage damage (including cartilage defects), or osteochondral defects. Preferably, the chondrocytes or the osteochondral/cartilage explants are collected or isolated from a mammal before the expansion or culturing step. Therefore, another object of the present invention is to provide a method for preparing an implantable cartilage material for use in tissue engineering or osteochondral/cartilage transplantation, wherein the method comprises or consists of the following steps: (a) collecting or isolating chondrocytes or osteochondral/cartilage explants from a mammal, and (b) culturing the chondrocytes, or the osteochondral/cartilage explants, in a culture medium containing an FGF-18 compound, in a monolayer or 3D culture, for a period of time sufficient to allow the formation of the implantable cartilage material. The implantable cartilage material can be used to treat cartilage diseases, such as osteoarthritis, cartilage damage, or osteochondral defects. Optionally, the implantable cartilage material or osteochondral/cartilage explant can be injected with the FGF-18 compound at the implantation site before, during, or after the implantation.

In another embodiment, the present invention relates to a method for regenerating cartilage in a mammal at a site where articular cartilage defects are caused by cartilage diseases, the method comprising or consisting of the following steps: (a) culturing chondrocytes, or culturing osteocartilage or cartilage explants, in a culture medium containing an FGF-18 compound in a monolayer culture or 3D culture, and (b) administering the cultured chondrocytes or osteocartilage/cartilage explants obtained in step (a) to a mammal in need thereof. The method for regenerating cartilage can be used to treat cartilage diseases, such as osteoarthritis, cartilage damage, or osteocartilage defects. Preferably, the chondrocytes or the osteocartilage/cartilage explants are collected or isolated from a mammal prior to the culturing step. Therefore, another object of the present invention is to provide a method for regenerating cartilage in a mammal at a site of articular cartilage defect caused by a cartilage disease, the method comprising or consisting of the following steps: (a) collecting or isolating chondrocytes or osteocartilage/cartilage explants from the mammal, (b) culturing the chondrocytes or osteocartilage/cartilage explants in a culture medium containing an FGF-18 compound in a monolayer or 3D culture, and (c) administering the cultured chondrocytes or osteocartilage/cartilage explants obtained in step (b) to a mammal in need thereof. The method for regenerating cartilage can be used to treat cartilage diseases such as osteoarthritis, cartilage damage, or osteocartilage defects. Optionally, the FGF-18 compound may be additionally injected into the site of administration of the cultured chondrocytes or osteocartilage/cartilage explants before, simultaneously with, or after administration of the cells/explants.

In another embodiment, the present invention discloses a method for regenerating cartilage in a mammal at a site of articular cartilage defect caused by a cartilage disease, the method comprising or consisting of the following steps: (a) culturing chondrocytes in a culture medium in a monolayer culture or a 3D culture, or culturing osteochondral/cartilage explants, (b) administering the cultured chondrocytes or osteochondral/cartilage explants obtained in step (a) to a mammal in need thereof, and (c) injecting an FGF-18 compound into the site of administration of the cultured chondrocytes or osteochondral/cartilage explants. Step (c) may be performed before, simultaneously with, or after administration of the cells/explants. In another embodiment, the present invention relates to a method for regenerating cartilage in a mammal at a site of articular cartilage defect caused by a cartilage disease, the method comprising or consisting of the following steps: (a) collecting or isolating chondrocytes or osteochondral/cartilage explants from the mammal, (b) culturing the chondrocytes in culture medium in a monolayer culture or a 3D culture, or culturing the osteochondral/cartilage explants, (c) administering the cultured chondrocytes or osteochondral/cartilage explants obtained in step (b) to a mammal in need thereof, and (d) injecting an FGF-18 compound into the site of administration of the cultured chondrocytes or osteochondral/cartilage explants. Step (d) may be performed before, simultaneously with, or after administration of the cells/explants.

In a fourth embodiment, the present invention relates to an FGF-18 compound for use in a method of treating a defect in cartilage tissue of a mammal, wherein the cartilage defect is caused by a cartilage disease, the method comprising or consisting of the following steps: (a) subjecting chondrocytes or osteocartilage/cartilage explants to in vitro or in vitro culturing, wherein the culturing is carried out in a cell culture medium containing the FGF-18 compound, (b) optionally repeating step (a) to obtain a graft material comprising the cultured chondrocytes or the cultured osteocartilage/cartilage explants, and (c) transplanting the graft material obtained in step (b) into the defect of the mammal in need of said treatment, wherein in steps (a) and (b) the chondrocytes can be cultured in monolayer culture or 3D culture. Preferably, the chondrocytes or osteocartilage/cartilage explants are collected or isolated from the mammal prior to the expansion or culturing step. Thus, the present invention relates to an FGF-18 compound for use in a method for treating a defect in cartilage tissue of a mammal, wherein the cartilage defect is caused by a cartilage disease, the method comprising or consisting of the following steps: (a) isolating chondrocytes or osteochondral/cartilage explants from a mammal, (b) subjecting the chondrocytes or osteochondral/cartilage explants to in vitro or in vitro culture, wherein the culture is carried out in a cell culture medium containing the FGF-18 compound, (c) optionally repeating steps (a) and (b) to obtain a graft material comprising the cultured chondrocytes or osteochondral/cartilage explants, and (d) transplanting the graft material obtained in step (c) into the defect of the mammal in need of the treatment, wherein the chondrocytes in steps (b) and (c) can be cultured in a monolayer culture or a 3D culture. Optionally, the FGF-18 compound can be additionally injected into the transplantation site before, simultaneously with, or after the transplantation.

In another embodiment, the present invention discloses an FGF-18 compound for use in a method of treating a defect in cartilage tissue of a mammal, wherein the cartilage defect is caused by a cartilage disease, the method comprising or consisting of the following steps: (a) subjecting chondrocytes or osteocartilage/cartilage explants to in vitro or in vitro culture, (b) optionally repeating step (a) to obtain a graft material comprising the cultured chondrocytes or the cultured osteocartilage/cartilage explants, (c) transplanting the graft material obtained in step (b) into the defect of the mammal in need of said treatment, wherein the chondrocytes in steps (a) and (b) can be cultured in a monolayer culture or a 3D culture, and (d) injecting the FGF-18 compound into the transplantation site. Step (d) can be performed before, simultaneously with, or after the transplantation. In another embodiment, the present invention relates to an FGF-18 compound for use in a method of treating a defect in cartilage tissue of a mammal, wherein the cartilage defect is caused by a cartilage disease, the method comprising or consisting of the following steps: (a) isolating chondrocytes or osteochondral/cartilage explants from a mammal, (b) subjecting the chondrocytes or osteochondral/cartilage explants to in vitro or in vitro culture, wherein the culture is carried out in a cell culture medium containing the FGF-18 compound, (c) optionally repeating steps (a) and (b) to obtain a graft material comprising the cultured chondrocytes or osteochondral/cartilage explants, (d) transplanting the graft material obtained in step (c) into the defect of the mammal in need of said treatment, wherein in steps (b) and (c) the chondrocytes can be cultured in a monolayer culture or a 3D culture, and (e) injecting the FGF-18 compound into the transplantation site. Step (e) can be performed before, simultaneously with, or after transplantation.

Furthermore, the present invention relates to a method for treating a defect in cartilage tissue of a mammal, wherein the cartilage defect is caused by a cartilage disease, the method comprising or consisting of the following steps: (a) isolating chondrocytes or osteochondral/cartilage explants from a mammal, (b) subjecting the chondrocytes or osteochondral/cartilage explants to in vitro or in vitro culture, wherein the culture is carried out in a cell culture medium containing an FGF-18 compound, (c) optionally repeating steps (a) and (b) to obtain a transplant material comprising the cultured chondrocytes or osteochondral/cartilage explants, and (d) transplanting the transplant material obtained in step (c) into the defect of the mammal in need of the treatment, wherein the chondrocytes in steps (b) and (c) can be cultured in a monolayer culture or a 3D culture. Optionally, the FGF-18 compound can be additionally injected into the transplant site before, simultaneously with, or after the transplantation.

In another embodiment, the present invention discloses a method for treating a defect in cartilage tissue of a mammal, wherein the cartilage defect is caused by a cartilage disease, the method comprising or consisting of the following steps: (a) isolating chondrocytes or osteochondral/cartilage explants from a mammal, (b) subjecting the chondrocytes or osteochondral/cartilage explants to in vitro or in vitro culture, wherein the culture is carried out in a cell culture medium, (c) optionally repeating steps (a) and (b) to obtain a graft material comprising the cultured chondrocytes or osteochondral/cartilage graft, (d) transplanting the graft material obtained in step (c) into the defect of the mammal in need of the treatment, wherein the chondrocytes in steps (b) and (c) can be cultured in a monolayer culture or a 3D culture, and (e) injecting an FGF-18 compound into the transplantation site. Step (e) can be performed before, simultaneously with, or after the transplantation.

In a fifth embodiment, the present invention provides a composition comprising cultured mammalian osteochondral/cartilage explants or cultured mammalian chondrocytes in a culture medium containing an FGF-18 compound for use in regenerative medicine (e.g., tissue engineering or osteochondral/cartilage transplantation) in a mammal in need thereof. Preferably, the mammal in need of the composition suffers from a cartilage disease. Preferably, the chondrocytes or osteochondral/cartilage explants are collected or isolated from a mammal prior to the expansion or culturing step.

In another embodiment, the present invention describes an FGF-18 compound for use in treating a cartilage disease, such as osteoarthritis, cartilage damage (including cartilage defects), or osteochondral defects, wherein the FGF-18 compound is administered in culture medium within the context of a cartilage repair procedure. Furthermore, the present invention discloses a method for treating a cartilage disease, such as osteoarthritis, cartilage damage (including cartilage defects), or osteochondral defects, wherein the FGF-18 compound is administered in culture medium within the context of a cartilage repair procedure. Specifically, the cartilage repair procedure is selected from the group consisting of cartilage tissue engineering, autologous chondrocyte transplantation, or osteochondral transplantation.

It should be understood that the transplantable cartilage material obtained according to the first embodiment or the regenerated cartilage obtained according to the second embodiment is used to treat cartilage diseases.

In the context of the present invention as a whole, the FGF-18 compound is preferably selected from the group consisting of: a) a polypeptide comprising or consisting of the mature form of human FGF-18 comprising residues 28-207 of SEQ ID NO: 1, b) a polypeptide comprising or consisting of residues 28-196 of SEQ ID NO: 1, or c) a polypeptide comprising or consisting of SEQ ID NO: 2. In particular, the compound is selected from human wild-type mature FGF-18 or trFGF-18.

The present invention describes an FGF-18 compound that is added to a culture medium at a concentration of 1 nanogram (ng) to 50 micrograms (μg or mcg) per milliliter (mL) of culture medium, preferably 5 ng to 5 μg, or preferably 5 ng to 1 μg, or more preferably 10 ng to 500 μg, or more preferably 10 ng to 100 ng. In a preferred embodiment, the culture medium is supplemented with the FGF-18 compound at a concentration of about 1, 5, 10, 20, 30, 40, 50, 100, 150, 200, 250, 300, 400, 500, or 1000 ng per mL of culture medium. Preferred concentrations include 10, 20, 30, 40, 50, 100, 150, or 200 ng per mL of culture medium.

In the context of the present invention as a whole, FGF-18 is added to the culture medium in which chondrocytes or osteochondrocytes/cartilage explants are cultured. Preferably, the FGF-18 compound is intermittently added to the culture medium on about one, two, or three days per week (about one week), with the one, two, or three-day addition repeated weekly for at least two weeks of culture, at least three weeks of culture, or at least four weeks of culture. Preferably, the FGF-18 compound is intermittently added to the culture medium on one, two, or three days per week, with the one, two, or three-day addition repeated weekly for two weeks of culture, three weeks of culture, or four weeks of culture. One day is preferably understood to be about 24 hours (i.e., 24 hours +/- 4 hours), two days is preferably understood to be about 48 hours (i.e., 48 hours +/- 4 hours), and three days is preferably understood to be about 72 hours (i.e., 72 hours +/- 4 hours). After one day of culture in supplemented medium, the cells are cultured for an additional 6 days in medium without the FGF-18 compound. After two days of culture in supplemented medium, the cells are cultured for an additional 5 days in medium without the FGF-18 compound. After three days of culture in supplemented medium, the cells are cultured for an additional 4 days in medium without the FGF-18 compound. This schedule corresponds to a weekly cycle. For example, for a one-day culture, if the FGF-18 compound is added to the culture medium on Tuesday, it is removed from the culture medium one day after the supplementation (i.e., Wednesday). The next supplementation would then be performed on the Tuesday following the first addition of FGF-18. The culture medium can be supplemented weekly (e.g., every Tuesday) according to the same schedule (i.e., one week after the previous supplementation). If more convenient, the FGF-18 compound can also be supplemented approximately one week after the previous supplementation (i.e., one week (or 7 days) +/- 1 or 2 days). For example, if the previous supplementation was performed on the previous Tuesday, the supplementation can be performed on Monday or Wednesday.

Alternatively, the FGF-18 compound can be intermittently added to the culture medium on approximately one, two, or three days per month, with the one, two, or three-day addition repeated monthly for at least two months of culture, at least three months of culture, or at least four months of culture. For 3D chondrocyte cultures, preferably, the FGF-18 compound is intermittently added to the culture medium on one, two, or three days per month, with the one, two, or three-day addition repeated monthly for two months of culture, three months of culture, or four months of culture. A day is preferably understood to mean approximately 24 hours (i.e., 24 hours +/- 4 hours). After one, two, or three days of culture in supplemented culture medium, culture is continued for one month in culture medium without the FGF-18 compound. This regimen corresponds to a monthly cycle. For example, with one-day addition, if the FGF-18 compound is added to the culture medium on August 1st, it is removed from the culture medium one day after the supplementation (i.e., August 2nd). The next supplementation would be on September 1st. The culture medium can be supplemented monthly according to the same schedule (i.e., one month after the last supplement). If more convenient, the FGF-18 compound can also be supplemented approximately one month after the last supplement (i.e., one month +/- 1, 2, 3, or 4 days). For example, if the last supplement was on August 1, supplementation can be performed on August 31 or September 3.

As defined above, "4 weeks" is used interchangeably with "monthly" or "monthly." Thus, according to the present invention, the FGF-18 compound can be intermittently added to the culture medium on one, two, or three days every four weeks, with the one, two, or three-day addition repeated every four weeks for at least two cycles of supplementation, at least three cycles of supplementation, or at least four cycles of supplementation. Preferably, the FGF-18 compound is intermittently added to the culture medium on one, two, or three days per month, with the one, two, or three-day addition repeated monthly for two months of culture, three months of culture, or four months of culture. A day is preferably understood to mean approximately 24 hours (i.e., 24 hours +/- 4 hours). After one, two, or three days of culture in supplemented culture medium, culture is continued for four weeks in culture medium without the FGF-18 compound. This regimen corresponds to a cycle of once every four weeks. For example, for a one-day addition, if the FGF-18 compound is added to the culture medium on Tuesday, it is removed from the culture medium one day after the supplementation (i.e., Wednesday). The next supplementation will be performed on Tuesday, four weeks after the first addition. Medium supplementation can be repeated every four weeks according to the same schedule (i.e., one month after the last supplementation). If more convenient, supplementation with FGF-18 compound can also be performed approximately four weeks after the last supplementation (i.e., four weeks +/- 1, 2, 3, or 4 days). For example, if the last supplementation was performed on Tuesday, October 1, supplementation can be performed on Monday, October 28 or Thursday, October 31.

For monolayer cultured chondrocytes or chondrogenic cells, the FGF-18 compound is preferably, but not limited to, permanently added. In contrast, when chondrogenic cells or chondrogenic cells are cultured in 3D culture, or for osteochondral/cartilage explants, the FGF-18 compound is preferably, but not limited to, intermittently added.

The FGF-compounds (e.g., trFGF-18), compositions comprising FGF-18 compounds ("FGF-18 compositions"), processes, uses, and methods described herein can be used to treat cartilage diseases. Specifically, they can be used to treat articular cartilage defects in synovial joints, such as those caused by age-related superficial ciliation, cartilage degeneration caused by osteoarthritis, and cartilage or osteochondral defects caused by injury or disease. They can also be used to treat joint diseases caused by osteochondritis dissecans and degenerative joint diseases. In the fields of reconstructive and plastic surgery, the FGF-18 compounds, compositions, processes, and methods according to the present invention can be used for autologous or allogeneic cartilage augmentation and transfer in the reconstruction of large tissue defects. The FGF-18 compositions can be combined with joint lavage, bone marrow stimulation, abrasive arthroplasty, subchondral drilling, or microfracture of the subchondral bone to repair cartilage damage.

In a preferred embodiment, the cartilage disease to be treated according to the present invention is osteoarthritis, such as knee osteoarthritis or hip osteoarthritis. The osteoarthritis to be treated includes, but is not limited to, primary osteoarthritis or secondary osteoarthritis, and osteoarthritis classified as stage 1 to stage 4 or grade 1 to grade 6 according to the OARSI classification system.

In the context of the present invention as a whole, the FGF-18 compound is added to the implant site. The addition can be performed before, at the same time as, or after the implant. When the addition is performed before or after the implant, it is preferably performed within a few hours before or after the implant (e.g., but not limited to, 1, 2, 3, or 4 hours before or after the implant). The injection can be performed within a few days before or after the implant (e.g., but not limited to, 1, 2, 3, or 4 days before or after the implant). If the addition is not performed at the same time as the implant, there is no disadvantage to the patient. In fact, the injection of the FGF-18 compound does not require surgery or any other invasive procedure.

In another preferred embodiment, the cartilage disorder to be treated according to the present invention is a cartilage injury including microfracture surgery or cartilage defects, or an osteochondral defect.

In the context of the present invention as a whole, the explant is preferably a cartilage explant and the chondrocyte is preferably a chondrocyte or mesenchymal stem cell derived from mature tissue. As needed, the chondrocyte or osteocartilage/cartilage explant is collected from a mammal to be treated (i.e., in need of the treatment) or a different mammal (preferably of the same species). The mammal is preferably a human, but may also be, but not limited to, a horse, camel, dog, or smaller mammal such as a cat, rabbit, rat, or mouse.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1: Preparation of cartilage defect model: (A) 8 mm cartilage plug, (B) formation of a central 4 mm defect, (C) insertion of cartilage into the defect, and (D) long-term culture of the repair construct. OD denotes outer diameter, and ID denotes inner diameter.

Figure 2: (A-C) 3D μCT-reconstructed cross-sections of the different treatments. (D) Fusion strength of the repaired defect, showing increasing intensity from the control group to the 1+30 treatment group to the 1+6 treatment group. (E) Experimental setup of the extrapolation test rig. Error bars are SEM.

Figure 3: μCT scans of cartilage-to-cartilage repair structures. Left: Representative single μCT scan slice from the sample. Center: 3D reconstruction. Right: Reconstructed cross-section. μCT scans show enhanced fusion from the control group to the 1+30 treatment group to the 1+6 treatment group.

Figure 4: Treatment of CTA with rhFGF18.

Figure 5: Estimated cell mass/CTA based on DNA content/CTA after 4 weeks of treatment with no rhFGF18 (CTR), continuous rhFGF18 (perm), rhFGF18 only in the first week (1w), or rhFGF18 once weekly (1d/w). rhFGF18 was used at concentrations of 10 or 100 ng/mL. N = 4. */*** indicates significant differences from the control group, p < 0.05 and 0.001, respectively.

Figure 6: GAG and HPLo/CTA content after 4 weeks of treatment with no rhFGF18 (CTR), continuous rhFGF18 (perm), rhFGF18 only in the first week (1w), or rhFGF18 once per week (1d/w). rhFGF18 was used at concentrations of 10 or 100 ng/mL. N = 4. */**/*** indicate significant differences from the control group, with p < 0.05, 0.01, and 0.001, respectively.

Figure 7: Evaluation of collagen type I, type II, and Sox9 expression, as well as the collagen II/I ratio, in CTA after 4 weeks of culture with no rhFGF18 (CTR), continuous rhFGF18 treatment (perm), rhFGF18 treatment only during the first week (1w), or rhFGF18 treatment once weekly (1d/w). rhFGF18 was used at concentrations of 10 or 100 ng/mL. N = 4. */**/*** indicate significant differences from the control group, with p < 0.05, 0.01, and 0.001, respectively.

Figure 8: Bovine primary chondrocytes were cultured in monolayers in culture medium without rhFGF18 (CTR) or continuously containing 100 ng/mL rhFGF18 for one or two weeks. Cell concentration was determined, N = 6. Collagen type I, type II, and Sox9 expression was measured by quantitative real-time PCR, N = 4.

Figure 9: Estimated cell content/CTA based on DNA content/CTA after 4-week treatment with no rhFGF18 (CTR), continuous rhFGF18 (perm), and 1 day per week rhFGF18 (1 d/w). * indicates significant difference from the control group, p < 0.05.

Figure 10: GAG content after 4-week treatment with no rhFGF18 (CTR), continuous rhFGF18 (perm), and 1 day per week (1 d/w). * indicates significant difference from the control group, p < 0.05.

Figure 11: Evaluation of collagen type I, type II, and Sox9 expression, as well as the collagen II/I ratio, in CTA after 4 weeks of treatment with no rhFGF18 (CTR), continuous rhFGF18 (perm), and 1 day per week (1 d/w). *Indicates significant difference from the control group, p < 0.05.

Sequence Description :

SEQ ID NO. 1: Amino acid sequence of native human FGF-18.

SEQ ID NO. 2: Amino acid sequence of recombinant truncated FGF-18 (trFGF-18).

Example

Material

The recombinant truncated FGF-18 (rhrFGF-18) in the following examples was produced by expression in E. coli according to the technology described in patent application WO2006/063362. In the following examples, trFGF-18, FGF-18 and sprifermin are used interchangeably.

Example 1

method:

Fresh hyaline cartilage was collected from the trochlear groove of the knee of young cattle (3-6 months old). 8 mm cylindrical explants were removed via biopsy punch (Figure 1A) and cultured overnight in complete culture medium (DMEM 4.5 g/L D-glucose and L-glutamine, 10% FBS, 1% PSF, 1% amphotericin B (Fungizone), 1% MEM vitamins, 25 mM HEPES, and 50 μg/ml vitamin C). The specimens were then debonded and a defect (4 mm diameter) was constructed to form a core-ring repair construct (Figure 1B). The inner core and outer ring were cultured separately for 24 hours, and then the defect was filled with the original core. The specimens were then cultured in complete culture medium or treated with Sprifermin (rhFGF18, 100 ng/ml). Treatment consisted of administering rhFGF18 for 24 hours, weekly (and repeated weekly) (days 1+6), or culturing in complete culture medium for 1 month after 24 hours of treatment (days 1+30). Samples were collected after 4 weeks of culture. Extrapolated mechanical testing (n = 4-6) was performed using a custom test stand (Figure 2E, [3]) (Instron 5848, Instron, Norwood, MA). The integration strength was calculated by dividing the peak force by the integration area. For 3D visualization, samples (n = 6) were immersed in modified Lugol's solution (2.5% _I2 and 5% KI in _dH2O ) for 24 hours and scanned by μCT at an energy level of 55 kV and an intensity of 145 μA, with voxel sizes of 6 μm and 10.5 μm (μCT 35 and vivaCT 40, SCANCOMedical, Wayne, PA). Scans were analyzed and reconstructed using the manufacturer's software, and cross-sections were used to assess defect integration. Additional samples (n = 3) were fixed overnight in 4% PFA, and histological analysis of cell and matrix deposition at the interface was performed.

result:

The fusion strength of the control specimen (Figure 2D) was the lowest (2.5 ± 1.4 kPa), with progressive improvement after treatments of 1+30 (monthly cycles) (5.0 ± 2.4 kPa) and 1+6 (weekly cycles) (10.2 ± 3.7 kPa). Although the results were significant when the control group was compared with the treated groups, they did not reach statistical significance due to the number of replicates in this study. μCT analysis of the control structure (Figure 3, upper left) showed distinct dark circles, indicating separation between the outer ring and the inner core, i.e., poor fusion. The 1+30 treatment group (Figure 3, middle left) showed less distinct circles, indicating smaller gaps and better fusion, while the 1+6 treatment group (Figure 3, lower left) showed a very uniform μCT signal across the interface, indicating the highest degree of fusion. Evidence of this improved fusion was evident in both vertical and transverse sections of all specimens.

Successful cartilage repair requires that the repair material (either engineered or natural) integrates well into the surrounding cartilage to ensure continuous load transfer across the interface (and avoid stress accumulation). In this study, we investigated the potential use of sprifermin to promote cartilage fusion in a well-established in vitro (explant) cartilage repair model. Sprifermin is known to promote chondrocyte proliferation (Elthworth et al., 2002), with brief (24-hour) exposure to this biological agent triggering the most pronounced response. Our results clearly demonstrate that sprifermin improves fusion strength and matrix deposition at the interface (contrast-enhanced μCT reveals more uniform thinning due to an increase in GAG-containing proteoglycans). In this study, weekly 24-hour administration for four weeks resulted in overall better results than monthly 24-hour treatment. While not as effective as the weekly treatment regimen, the latter treatment regimen is also useful, as it unexpectedly improved compared to the control construct (i.e., no sprifermin treatment). This study demonstrates for the first time that a biological agent, specifically sprifermin, improves fusion of cartilage surfaces in a clinically relevant repair model.

in conclusion:

This study demonstrated that sprifermin improved fusion of cartilage surfaces in a cartilage repair model. These results suggest that sprifermin may be used in surgical procedures such as OATS and tissue engineering approaches, where cartilage-based biomaterials must successfully integrate with native cartilage for clinical success.

Example 2

method:

Native osteoarthritis chondrocytes were isolated from patients undergoing total joint replacement. Prior to treatment, cells were cultured in monolayers for several days and then in scaffold-free 3D cultures for one week. Treatment consisted of incubation with rhFGF-18 (100 ng/mL) continuously or once a week for four weeks. Results were compared with control culture media without sprifermin. Three-dimensional architecture was characterized using biochemical assays, quantitative PCR (qPCR), and histology.

Results (data not shown):

To ensure phenotypic maintenance, the effect of sprifermin on hOA chondrocytes was tested using a 3D scaffold-free culture medium. In this setting, rhFGF-18 (one day per week) demonstrated beneficial effects on cell content and significantly increased the size and matrix content (GAG and HPG content) of the 3D structures. Furthermore, rhFGF18 was found to reduce collagen I expression compared to untreated cells.

in conclusion:

As observed in previous studies with bovine and porcine chondrocytes, sprifermin exhibits anabolic activity in hOA chondrocytes. These results suggest that sprifermin may be useful in tissue engineering approaches where cartilage-based biomaterials must successfully integrate with native cartilage to achieve clinical success.

Example 3

method:

Porcine chondrocytes were isolated from the femoral head cartilage of the pig hip. After joint dissection, the cartilage was collected and digested with collagenase 0.25% for 45 minutes. The loose cells were discarded and the cartilage was further digested with collagenase 0.1% overnight to extract the chondrocytes. The first week of suspension culture of porcine chondrocytes as CTA (cartilage tissue analog) was not treated, followed by one of the following treatments: 1) cultured in a culture medium containing 10 or 100 ng/mL rhFGF18 for four weeks, 2) cultured in a culture medium containing 10 or 100 ng/mL rhFGF18 for one week, followed by culture in a culture medium without rhFGF18 for three weeks, 3) cultured in a culture medium supplemented with 10 or 100 ng/mL rhFGF18 (i.e., contacted for 24 hours, followed by culture in a culture medium without rhFGF18 for 6 days) for three weeks, or 4) cultured in a culture medium without rhFGF18 for four weeks, as a control group (Figure 4). At the end of the culture period, CTA were harvested and analyzed for GAG, hydroxyproline, and cellular content. Gene expression of collagen I, II, and Sox9 was assessed, and histological analysis of Safranin O, collagen types I, and II was performed.

Results - Effects of continuous or intermittent exposure to rhFGF18 on cell growth in CTA

The CTA obtained under each culture condition was cracked and DNA content was assessed to calculate cell number/CTA (Fig. 5). In the control culture group (not containing rhFGF18), no proliferation was seen because cell number (1.2 million) was close to the seeding density (1 million cells/CTA). However, as expected, the continued presence of rhFGF18 increased the proliferation of chondrocytes (rhFGF18 of 10 and 100 ng/mL resulted in 2.2 million and 2.49 million cells/CTA, respectively). When only rhFGF18 was used for one week and chondrocytes were cultured in the culture medium without rhFGF19 for 3 weeks (1 w), compared with the control group, no proliferation increased. On the contrary, when rhFGF18 was used weekly for one day (1 d/w), compared with the control group and compared with continuous contact, rhFGF18 stimulated proliferation. One day per week of rhFGF18 administration at 100 ng/mL resulted in a cell count/CTA of 4 million cells/CTA, compared to 1.2 million without rhFGF18 and 2.49 million with continuous rhFGF18 administration.

Results - Effects of continuous or intermittent exposure to rhFGF18 on matrix production in CTA

The CTA obtained under each culture condition was digested with proteinase K and assessed for GAG and hydroxyproline content (Figure 6). GAG reflects the proteoglycan content, while hydroxyproline reflects the collagen content of the CTA. As previously observed, the continued presence of rhFGF18 reduced the GAG content per CTA (GAG decreased 2.6-fold compared to the control group) and the hydroxyproline content per CTA (hydroxyproline decreased 2.1-fold compared to the control group). Conversely, when rhFGF18 was administered intermittently (1 week or 1 day per week), both GAG and hydroxyproline content increased. For example, when 100 ng/mL of rhFGF18 was administered once a week, GAG content increased 2.67-fold and hydroxyproline content increased 2.13-fold compared to the control group.

Results - Effects of continuous or intermittent exposure to rhFGF18 on chondrocyte phenotype in CTA

RNA was isolated from the CTA obtained under each culture condition and analyzed by quantitative PCR for the expression of collagen type I, II, X and Sox9 (Figure 7). High Sox9 and collagen type II expression are markers of chondrocyte phenotype, while collagen type I is a marker of dedifferentiation and collagen type X is a marker of chondrocyte hypertrophy. The collagen II/I ratio was also calculated in addition. This ratio is generally used to show whether the phenotype of chondrocytes in culture is maintained (high ratio) or lost (low ratio). Under all culture conditions containing rhFGF18, whether it was continuous or intermittent contact, 10ng/mL or 100ng/mL, the expression of collagen type I was reduced. This reduction was most intense when rhFGF18 was applied at a concentration of 100ng/mL for one day per week. For example, compared to the control group, continuous administration of 100 ng/mL of rhFGF18 reduced the expression of collagen type I by 4 times, while weekly administration of 100 ng/mL of rhFGF18 reduced the expression of collagen type I by 123 times. In the continuous presence of rhFGF18, collagen type II decreased, but remained essentially unchanged when rhFGF18 was administered for one week (1 week) or one day per week (1 day/w). There was no significant change in the expression of Sox9, with a significant increase (x2.2) only when 10 ng/mL of rhFGF18 was administered for one week (1 week). Finally, continuous administration of rhFGF18 had no effect on the collagen II/I ratio, but when 10 ng/mL and 100 ng/mL of rhFGF18 were administered for one day per week, the ratio increased by 19 and 138 times, respectively, compared to the control group. Collagen type X was also evaluated as a marker of chondrocyte hypertrophy and was found to be unaffected by rhFGF18 in the culture conditions.

Results - Effects of continuous or intermittent exposure to rhFGF18 on the morphology and collagen II and I content of CTA (data not shown)

Histological analysis of CTAs following four weeks of rhFGF18 exposure at different schedules revealed that CTAs with continuous rhFGF18 treatment were thinner and stained lighter with Safranin O than those in the other conditions. Furthermore, a periphery of the structures with continuous rhFGF18 treatment exhibited a hyperplastic zone with a higher cell density and a lack of extracellular matrix. Furthermore, intermittent rhFGF18 exposure resulted in thickening of the structures compared to the control group. Collagen type I was not detected in all conditions (not shown), while all CTAs stained strongly positive for collagen type II.

in conclusion:

Continuous exposure to rhFGF18 stimulated cartilage proliferation but reduced the matrix content of CTA (reduced GAGs and hydroxyproline). Similarly, the expression of both collagen types I and II was reduced compared to the control group. Continuous exposure to 10 ng/mL or 100 ng/mL rhFGF18 for 4 weeks did not significantly affect Sox9 expression. Histological analysis revealed that CTAs were smaller and ECM-free proliferative zones appeared at their periphery. These results suggest that continuous administration of rhFGF18 favors proliferation while inhibiting matrix production.

When CTAs were cultured with 10 ng/mL or 100 ng/mL rhFGF18 for one week and then in rhFGF18-free medium for three weeks, in contrast to continuous exposure to rhFGF18, no stimulation of proliferation was observed. However, GAG and hydroxyproline content increased compared to the control group. Collagen I expression decreased compared to the control group, while collagen type II expression remained unchanged or even slightly increased (rhFGF18 10 ng/mL). Thus, the collagen II/I ratio increased, indicating better phenotypic maintenance. Similarly, Sox9 expression was slightly increased compared to the control group (significant only with rhFGF18 10 ng/mL). Histological analysis revealed that, similar to control CTAs, the CTAs were composed of a matrix positive for Safranin O and collagen type II. These CTAs were also thicker compared to the control group, consistent with the higher GAG and hydroxyproline content.

The best results for proliferation and matrix content were achieved when 100 ng/mL of rhFGF18 was administered for one day weekly. Collagen type I expression was also lowest under this condition, while the collagen II/I ratio was highest. However, collagen type II and Sox9 expression remained unchanged compared to the control group. CTA was positive for Safranin O and collagen type II. This was also observed in the one-week treatment group, where CTA was thicker compared to the control group, consistent with higher GAG and hydroxyproline content.

In summary, intermittent exposure enhances the effects of rhFGF18, leading to increased proliferation and ECM production and favoring the maintenance of a chondrocyte phenotype in culture, with the effect being 1 day per week > 1 week > control > continuous exposure. These results support cyclic administration of rhFGF18 for the treatment of OA.

Example 4

method:

Bovine chondrocytes were obtained using the same method as in Examples 2 and 3. Culture medium containing 100 ng/mL rhFGF18 was used for 1 or 2 weeks (FGF18), or cultured in a medium without FGF18 as a control group (CTR). At the end of the culture period, cells were collected and counted, or lysed to isolate RNA and analyze gene expression. The expression of Sox9, collagen I, and II was assessed by quantitative PCR.

result:

After two weeks of culture in a medium continuously containing FGF18, the cell density was higher than that of the control group. In the presence of rhFGF18, the expression of collagen type I was severely inhibited, while the expression of collagen type II and Sox9 was increased (Figure 8).

in conclusion:

When chondrocytes were cultured as monolayers, continuous exposure to 100 ng/mL of rhFGF18 resulted in increased cell proliferation and better maintenance of phenotype (increased expression of collagen II and Sox9 and decreased expression of collagen I).

Example 5

method:

Cartilage from two OA patients who underwent total joint replacement was used. Chondrocytes were isolated using the method described in Example 3 and first cultured in a high-density monolayer for 3-4 days. Then, the chondrocytes were collected and seeded in 96-well plates at 1x10 ⁶ cells/200 μL and allowed to accumulate for one week without any treatment to form CTA. They were then cultured in a medium containing or without 100 ng/mL of rhFGF18 for four weeks according to the following treatment regimen: 1) cultured in a medium containing no rhFGF18 for 4 weeks (control group), 2) cultured in a medium containing rhFGF18 for four weeks (perm), and 3) cultured for four weeks with rhFGF18 administered one day per week (i.e., contact with rhFGF18 for 24 hours, followed by culture in a medium containing no rhFGF18 for 6 days) (1 d/w) (see Figure 4). At the end of the culture period, CTA were collected and analyzed for GAG and cell content. Computed tomography (CTA) from patient 2 was evaluated for gene expression of collagen types I, II, and Sox9, and histological analysis of safranin O and collagen types I and II was performed.

Results - Cell Proliferation :

100 ng/mL of rhFGF18 increased the proliferation of human osteoarthritis chondrocytes in 3D culture (see Figure 9). For chondrocytes from Patient 1, the number of cells per CTA in the control group was lower than the starting cell number (seeding density of 1 million cells/CTA), indicating that many cells had died. However, in the groups that received either continuous or weekly rhFGF18 administration, 1.5 million cells/CTA were observed, indicating that these cells did not die but instead proliferated. For chondrocytes from Patient 2, a slight increase in cell number was observed in the untreated CTA (from 1 to 1.3 million cells/CTA), which further increased in the group that received continuous rhFGF18 administration (from 1 to 1.9 million cells/CTA).

Results - Matrix Generation :

100 ng/mL rhFGF18 increased GAG production in human osteoarthritis chondrocytes in 3D culture (see Figure 10). GAGs in CTA were significantly increased in patient 1 who received continuous rhFGF18 and rhFGF18 once a week, as well as in patient 2 who received continuous FGF18.

Results - Gene Expression :

The chondrocyte phenotype is characterized by low or no collagen type I expression, and increased expression of Sox9 and collagen II. This expression pattern is changed in osteoarthritis chondrocytes (see Figure 11). In fact, the expression of collagen type I in untreated CTA is higher than the expression of collagen type II (relative abundance is 0.67 and 0.04, respectively). rhFGF18 can reduce the expression of collagen I and increase the expression of collagen II. Therefore, in the presence of rhFGF18, the collagen II/I ratio increased by 11 to 13 times. In addition, rhFGF18 increased the expression of Sox9, which is a marker of the chondrocyte phenotype.

Results - Histology (data not shown) :

Compared to the control group, CTAs cultured with rhFGF18 for one day per week or continuously cultured with rhFGF18 showed enhanced Safranin O staining, indicating that these CTAs contained an increase in GAGs. This is consistent with the results shown in Figure 10. Collagen I staining was reduced in cells treated with rhFGF18, which also corresponds well to the gene expression results in Figure 11. Collagen II staining was not seen in the control group, indicating that these human osteoarthritis (hOA) chondrocytes are unable to produce cartilage-like matrix. However, weekly administration of rhFGF18 for one day or continuously administered rhFGF18 was able to restore the ability of hOA chondrocytes to produce collagen II.

in conclusion:

Results from chondrocytes isolated from human osteoarthritic cartilage showed that rhFGF18 promoted cell growth, increased hyaline cartilage matrix production, and favored the chondrocyte phenotype. In this study, continuous administration of rhFGF18 and weekly administration of rhFGF18 performed similarly across several parameters. However, weekly administration of rhFGF18 was slightly superior in terms of matrix production (increased GAG accumulation in patient 1 and increased collagen II expression in patient 2).

References

1.Magill et al., 2011, J. orthopedic Surg. 19(1):93-98

2.Zhang et al., 2013, Expert Opin.Biol.Ther.13(4):527-540

3.Tang et al., 2012, Expert Opin. Biol. Ther., 12(10):1361-1382

4. Ellsworth et al., 2002, Osteoarthritis and Cartilage, 10: 308-320

5. Shimoaka et al., 2002, JBC 277(9):7493-7500

6.WO2008023063

7.WO2004032849

8.WO9816644

9.WO2006063362

10.Custers et al., 2007, Osteoarthritis and Cartilage, 15:1241-1248

11. Lotz, 2010, Arthritis research therapy, 12: 211

12. The Merck manual, ^17th edition, 1999

13. Getgood et al., 2010, P116, ICRS Meeting 2010, Barcelona.

14.ICRS Publications:

http://www.cartilage.org/_files/contentmanagement/ICRS_ evaluation.pdf , page 13

15. Bian et al., 2010, Am. J. Sports. Med, 38(1):78-85.

16. Gennero et al., 2013, Cell Biochem Funct 31(3)214-227

17.Mauck et al., 2006, Osteoarthritis and Cartilage, 14(2):179-189

18. Bian et al., 2008 J. Biomech., 41(6): 1153-1159

Claims (10)

1. A method for preparing a transplantable cartilage material for tissue engineering, wherein the method comprises the following steps: intermittently culturing chondrocytes in a medium containing an FGF-18 compound in a monolayer or 3D culture manner, the cells being cultured in the medium for 24 hours, 48 hours, or 72 hours per week in the presence of the FGF-18 compound, and for the remaining time per week in the absence of the FGF-18 compound, repeating the weekly culture for at least 2 weeks. 2. The method of claim 1, wherein the weekly culture is repeated for at least 3 weeks. 3. The method of claim 1, wherein the weekly culture is repeated for at least 4 weeks. 4. The method according to any one of claims 1-3, wherein the chondrocytes are chondrocytes or mesenchymal stem cells derived from mature tissues. 5. The method according to any one of claims 1-3, wherein the FGF-18 compound is selected from the group consisting of: a) A polypeptide comprising or composed of the mature form of human FGF-18, wherein the mature form of human FGF-18 comprises residues 28-207 of SEQ ID NO:1. b) including residues 28-196 of SEQ ID NO:1 or a polypeptide consisting of residues 28-196 of SEQ ID NO:1, or c) Includes SEQ ID NO:2 or a polypeptide consisting of SEQ ID NO:2. 6. The method according to any one of claims 1-3, wherein the chondrocytes are collected or isolated from mammals prior to expansion or culture. 7. The method of claim 6, wherein the chondrocytes are collected or isolated from the mammal to be treated or from different mammals. 8. The method of claim 6, wherein the mammal is a human. 9. The use of the transplantable cartilage material obtained by the method according to any one of claims 1 to 8 in the preparation of a medicament for treating cartilage diseases. 10. The application according to claim 9, wherein the cartilage disease is osteoarthritis, cartilage damage, or osteochondral defect.