WO2016188123A1 - A composition for repairing chondral defects - Google Patents

A composition for repairing chondral defects Download PDF

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Publication number
WO2016188123A1
WO2016188123A1 PCT/CN2016/000274 CN2016000274W WO2016188123A1 WO 2016188123 A1 WO2016188123 A1 WO 2016188123A1 CN 2016000274 W CN2016000274 W CN 2016000274W WO 2016188123 A1 WO2016188123 A1 WO 2016188123A1
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Prior art keywords
composition
cartilage
chondral
bone marrow
defect
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PCT/CN2016/000274
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French (fr)
Inventor
Hsu-Wei Fang
Chih-Hung Chang
Chia-Chun Chen
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Biotegy Corporation
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Priority to CN201680029271.7A priority Critical patent/CN107708712B/en
Publication of WO2016188123A1 publication Critical patent/WO2016188123A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/32—Bones; Osteocytes; Osteoblasts; Tendons; Tenocytes; Teeth; Odontoblasts; Cartilage; Chondrocytes; Synovial membrane
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/3604—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix characterised by the human or animal origin of the biological material, e.g. hair, fascia, fish scales, silk, shellac, pericardium, pleura, renal tissue, amniotic membrane, parenchymal tissue, fetal tissue, muscle tissue, fat tissue, enamel
    • A61L27/3608—Bone, e.g. demineralised bone matrix [DBM], bone powder
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/3604—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix characterised by the human or animal origin of the biological material, e.g. hair, fascia, fish scales, silk, shellac, pericardium, pleura, renal tissue, amniotic membrane, parenchymal tissue, fetal tissue, muscle tissue, fat tissue, enamel
    • A61L27/3612—Cartilage, synovial fluid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/3641—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix characterised by the site of application in the body
    • A61L27/3645—Connective tissue
    • A61L27/3654—Cartilage, e.g. meniscus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease

Definitions

  • the present invention pertains to a composition for repairing chondral defects.
  • MSCs Mesenchymal stem cells
  • ECM is thought to participate significantly in guiding the differentiation process of MSCs (Quesenberry P Jet al., 1 Stem Cells 16 (Suppl 1) : 33-35 (1998) ; Philp D et al., Stem Cells 23: 288-296 (2005) ) .
  • known cell-based compositions require an effective carrier or sealant to fix onto the defect site.
  • sufficient initialmechanical stability of the graft is of utmost importance (Efe Tet al., Knee Surg Sports TraumatolArthrosc. 2012 Feb; 20 (2) : 210-5) .
  • the graft must withstand the forces in vivo duringthe early postoperative phase during joint movement. Adequate fixation is necessary to keep the graft inthe defect zone and to achieve optimal joint surface congruity.
  • a periosteal patch or collagen fleece is sutured over cartilage defect to avoid the loss of cells or carrier/sealant (Slynarski Ket al., Transplant Proc.
  • a composition comprising a bone marrow concentrate, a cartilage fragment and a synovial membrane exhibit superior properties in the repair of chondral defects. Further, it was unexpectedly found that a fibrin glue applied onto the composition of the present invention shows superior cohesiveness and does not disperse or lose over time as usually used on a chondral defect site, such that a conventional step of covering the chondral defect filled with a cell-based composition for repair with a periosteal or synthetic membrane by suturing is not required.
  • the present invention provides a composition for repairing chondral defects, which comprises a bone marrow concentrate, a cartilage fragmentor powder, and synovial membranes.
  • the composition of the present invention may be used in combination with a fibrin glue.
  • the composition of the present invention enables a novel one-stage-orientated cartilage repair surgery without the needs of a GTP laboratory for cell culturing and GMP factory for manufacturing new scaffolds. This could lower the threshold of clinical feasibility and benefit the performance from bench to clinic.
  • the invention provides a method for treating a chondral defect in a subject, comprising applying a composition of the present invention into the chondral defect, and applying a fibrin glue onto the composition to cover the chondral defect.
  • a composition of the present invention into the chondral defect
  • a fibrin glue onto the composition to cover the chondral defect.
  • the present invention provides said composition for use in a method of treating a chondral defect in a subject, the method comprising applying the composition into the chondral defect, and applying a fibrin glue onto the composition to cover the chondral defect.
  • the composition is applied by filling the chondral defect.
  • the method may not comprise a step of covering the chondral defect with a periosteal or synthetic membrane by suturing.
  • the subject is preferably a mammal.
  • the mammal includes but is not limited to humans, rodents, monkeys, pigs, dogs and cats. More preferably, the subject is human.
  • Cartilage and synovial membrane from a donor having the same or different genus from the subject to be treated may be used in the present invention.
  • Fig. 1A shows the RT-PCR results for culture of Group A1 (BMC (5 x 10 5 ) ) .
  • Fig. 1B shows the RT-PCR results for culture of Group A2 (cartilage-synovium) . ***p ⁇ 0.001.
  • Fig. 1C shows the RT-PCR results for culture of Group A3 (BMC (5 x10 5 ) -cartilage-synovium) . ***p ⁇ 0.001
  • Fig. 1D shows the comparison of the results of Group A3 to the results of Group A1.
  • Fig. 1E shows the comparison of the results of Group A3 to the results of Group A2.
  • Fig. 1F shows the RT-PCR results for culture of Group B1 (BM-MSCs (5 x 10 5 ) -cartilage-synovium) .
  • Fig. 1G shows the RT-PCR results for culture of Group B2 (BM-MSCs (5 x 10 4 ) -cartilage-synovium) .
  • Fig. 2A is a picture showing a created chondral defect.
  • Fig. 2B is a picture showing that a composition of the present invention can remain in the defect site.
  • Fig. 2C is a picture showing that periosteum transplantation or periosteum suture is not required using a composition of the present invention for chondral defect repair.
  • Fig. 2D is a picture showingthe repair of cartilage 6 months after implantation.
  • the present invention provides a composition for repairing chondral defects, which comprises a bone marrow concentrate, a cartilage fragment or powder, and a synovial membrane.
  • composition of the present invention may be used in combination with a fibrin glue.
  • Fibrin glue has already proven to be useful in anumber of orthopedic procedures; however, it does not successfully work in the knee joint full-thickness cartilage defect to fix soft materials such as cells.
  • the present invention provides said composition for use in a method of treating a chondral defect in a subject, the method comprising applying the composition into the chondral defect, and applying a fibrin glue onto the composition to cover the chondral defect.
  • the composition is applied by filling the chondral defect.
  • the method may not comprise a step of covering the chondral defect with a periosteal or synthetic membrane by suturing.
  • the invention provides a method for treating a chondral defect in a subject, comprising applying a composition of the present invention into the chondral defect, and applying a fibrin glue onto the composition to cover the chondral defect.
  • a fibrin glue applied onto the composition of the present invention shows superior cohesiveness and does not disperse or lose over time as usually used on a chondral defect site, such that a conventional step of covering the chondral defect filled with a cell-based composition for repair with a periosteal or synthetic membrane by suturing is not required. Accordingly, in preferred embodiments of the present invention, the method does not comprise a step of covering the chondral defect with a periosteal or synthetic membrane by suturing.
  • the subject is preferably a mammal.
  • the mammal includes but is not limited to humans, rodents, monkeys, pigs, dogs and cats. More preferably, the subject is human.
  • the ′′bone marrow concentrate′′ used in the present invention is preferably in the form of cell pellet (of nucleated cells) and can be prepared from a bone marrow concentrate solution using a conventional method, for example, centrifugation.
  • the bone marrow concentrate solution can be prepared from a bone marrow aspirate by removing plasma and red blood cell fractions using conventional methods, for example, the bone marrow concentrate solution can be prepared by CellPointTM Concentrated Bone Marrow Aspirate System, ISTO Technologies, Missouri, U.S.
  • the resultant bone marrow concentratesolution is enriched with nucleated cells compared to the bone marrow aspirate.
  • nucleated cells includes but is not limited to bone marrow stem cells, neutrophils, lymphocytes and monocytes.
  • the composition may comprise the BMC in an amount such that it provides a desired number of nucleated cells.
  • the bone marrow concentrate used may comprise 10 4 -10 7 nucleated cells.
  • the composition may comprise the BMC in an amount such that it provides 10 4 -10 7 nucleated cells.
  • the bone marrow concentrate comprises 10 5 -10 6 nucleated cells.
  • the bone marrow concentrate comprises 5 x 10 5 nucleated cells.
  • the bone marrow concentrate is in the form of cell pellet and consists essentially of nucleated cells derived from a bone marrow aspirate.
  • nucleated cells constitute at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, or more, by weight of the bone marrow concentrate used in the present invention.
  • the cartilage fragment or powder may be prepared from a cartilage tissue from a joint including but not limited to knee joint, a hip joint, and anankle joint.
  • the cartilage powder is acellular cartilage powder, e.g.
  • the cartilage fragment preferably has a size ranging from 1 mm x 1 mm to 3 mm x 3 mm. In one embodiment, the cartilage fragment has a size of 1-2 mm x 2-3 mm.
  • chondral defect with a size of 1 ⁇ 3-mm in depthand about 8.5 mm in diameter (i.e., a round shaped defect)
  • 30-65 mg of cartilage fragment or powder may be used.
  • the composition may comprise 60 mg of cartilage powder.
  • the synovial membrane used in the composition of the invention may be in fragment form.
  • the synovial membrane is fragments of synovial membrane having a size ranging from 1 mm x 1 mm to 3 mm x 3 mm.
  • the fragments are of about 2 mm x 3 mm in size.
  • synovial membrane For a chondral defect with a size of 1 ⁇ 3-mm in depthand about 8.5 mm in diameter (i.e., a round shaped defect) , 30-65 mg of synovial membrane may be used.
  • the composition may comprise 60 mg of synovial membrane.
  • Autogenic bone marrow concentrate or non-autogenic bone marrow concentrate from a donor having the same or different genus from the subject to be treated can be used in the present invention.
  • Cartilage and synovial membrane from a donor having the same or different genus from the subject to be treated may be used in the present invention.
  • the composition may be prepared by evenly mixing a bone marrow concentrate, a cartilage fragment or powder, and a synovial membrane.
  • a bone marrow concentrate comprising 10 4 -10 7 nucleated cells may be mixed with 40-100 mg cartilage fragments or powder and 40-100 mg synovial membrane to form a composition for chondral defect repair.
  • the composition may be further mixed with 0.2-1 mL biocompatible carrier. The actual amount may be easily adjusted by a person of ordinary skill according to the dimension of the chondral defects to be repaired.
  • the composition is prepared by mixing a bone marrow concentrate comprisingabout 5 x 10 5 nucleated cells with 60 mg cartilage fragments and 60 mg synovial membrane fragments, further mixed with 0.2 mL fibrin glue.
  • the composition may comprise 0.1-1 wt%of BMC, 30-60 wt%of cartilage fragment or powder, and 30-60 wt%of synovial membrane.
  • the composition is not pre-mixed with biocompatible carrier.
  • the composition may be directly applied into the chondral defect, and then a suitable carrier or sealant is applied therein to or thereon to for fixation.
  • a suitable carrier or sealant is applied therein to or thereon to for fixation.
  • the carrier or sealant is a fibrin glue.
  • Porcine bone marrow concentrate was rapidly isolated by using a sucrose gradient.
  • porcine bone marrow was aspirated with a syringe from tibia. The bone marrow was then filtered through a 70- ⁇ m mesh. The filtered bone marrow (10 ml) overlaid into a 17.5%sucrose gradient (15 ml) in a 50-mL conical tube was centrifuged once for 5 minutes at 1,500 rpm. The bone marrow concentrate in the fractionated layer were then extracted by centrifugation for 5-10 minutes at 1,500 rpm. The obtained pellet was dispersed into 1 ml DPBS to calculate the total cell numbers by an automatic cell counter.
  • BM-MSCs bone marrow mesenchymal stem cells
  • the bone marrow concentrate collected from the fractionated layer were transferred into a new tube and then centrifuged for 5-10 minutes at 1,500 rpm to obtain nucleated cells. These cells were plated into culture flasks and cultured in ⁇ -MEM containing 10%FBS.
  • Cartilage tissues were harvested from porcine knee joint and washed with DPBS solution. These tissues were minced into small fragments by surgical knife. Approximate 1-2 mm x 2-3 mm sized cartilage fragments were washed with DPBS and collected by centrifugation for 5-10 minutes at 1,500 rpm.
  • Synovial membranes were harvested from porcine knee joint and washed with DPBS solution. The tissues were minced into small fragments by surgical knife. Approximate 2 mm x 3 mm sized synovial membrane fragments were washed with DPBS and collected by centrifugation for 5-10 minutes at 1,500 rpm.
  • Bone marrow concentrate suspension containing 1-10 x 10 5 cells and the tissue fragments including 30-60 mg cartilage fragments and 30-60 mg synovial membranes were mixed together. After centrifugation, the supernatant was discarded and the BMC-cartilage fragment-synovial membrane mixture was obtained. The mixture was further embedded into 0.2 mL TISSEEL solution containing fibrinogen and thrombin on 48-well plate to form a biological construct. The formed construct was transferred into 24-well plate and cultured in ⁇ -MEM containing 10%FBS. Other similar constructs without tissue fragments and without bone marrow concentrate were prepared for comparison.
  • the BM-MSCs were serially trypsinized.
  • the cell suspension containing 1-10 x 10 4 stem cells and the tissue fragments including 30-60 mg cartilage fragments and 30-60 mg synovial membranes were mixed together. After centrifugation, the supernatant was discarded and the BM-MSCs-cartilage fragment-synovial membrane mixture was obtained.
  • the mixture was further embedded into 0.2 mL TISSEEL solution containing fibrinogen and thrombin on 48-well plate to form the biological construct.
  • the formed construct was transferred into 24-well plate and cultured in ⁇ -MEM containing 10%FBS. Other similar constructs with different BM-MSC numbers were prepared for comparison.
  • RNA was isolated by using the Trizol purification system (Invitrogen) according to the manufacture’s protocol.
  • Reverse transcription (RT) of total RNA to single-stranded cDNA was completed by using the high-capacity cDNA RT kit (Applied Biosystems) .
  • Gene expression was analyzed by using an ABI SteponePlus TM real-time PCR system (Applied Biosystems) .
  • probes were purchased from Applied Biosystems including glyceraldehydes-3-phosphate dehydrogenase (Hs99999905_ml) , aggrecan (Hs00153936_ml) , SOX9 (Hs00165814_ml) , type I collagen (Hs00164004_ml) , type II collagen (Hs00156568_ml) , and type X collagen (Hs00166657_ml) . Data were evaluated as mRNA levels of the cells in the constructs. A cycle threshold value (Ct) was obtained from each sample and triplicate sample values were averaged. The 2 - ⁇ Ct method was used to calculate relative expression of each target gene.
  • Ct cycle threshold value
  • Group A3 shows more increasesin type II collagen expression over time of culture (up to 6 weeks) , as compared to Groups A1 (BMCs alone) and A2 (cartilage-synovium alone) (see Figs. 1A-1E) .
  • BMCs alone BMCs alone
  • A2 cartilage-synovium alone
  • type II collagen expression did not increase significantly over time of culture.
  • Type II collagen represents 90%to 95%of the collagen in the extracellular matrix (ECM) of articular cartilage (or hyaline cartilage) , and is thus considered crucial for successful repair of chondral defects.
  • Porcine bone marrow concentrate was rapidly isolated by using a sucrose gradient.
  • 10 ml porcine bone marrow was aspirated with a syringe from tibia.
  • the bone marrow (10 ml) overlaid into a 17.5%sucrose gradient (15 ml) in a 50-mL conical tube was centrifuged once for 5 minutes at 1,500 rpm.
  • the bone marrow concentrate in the fractionated layer were then extracted by centrifugation for 5-10 minutes at 1,500 rpm.
  • Pellet was dispersed into 1 ml DPBS to calculate the total cell numbers by an automatic cell counter.
  • Cartilage tissues were harvested from porcine knee joint and washed with DPBS solution, and then minced into small fragments by surgical knife. Approximate 1-2 mm x 2-3 mm sized cartilage fragments were washed with DPBS and collected by centrifugation for 5-10 minutes at 1,500 rpm.
  • Synovial membranes were harvested from porcine knee joint and washed with DPBS solution. The tissues were then minced into small fragments using surgical knife. Approximate 2 mm x 3 mm sized synovial membrane fragments were washed with DPBS and collected by centrifugation for 5-10 minutes at 1,500 rpm.
  • Miniature pigs were used in this study. All pigs were sexually mature at the timeof surgery. All operations and interventions were performed under general anesthesia. Full-thickness defects were created in medial condyles of both knee joints (see Fig. 2A) , whereinone of which received acomposition containing bone marrow concentrate, 60 mg cartilage fragments and 60 mg synovial membranes, and then aTisseel solution (afibrin glue) (0.2-0.4 ml) for fixation; and the other received micro-fracture treatmentas a control group. The diameter of the defect was 8 mm with 1 mm depth.
  • the fibrin glue shows high cohesiveness, and the fibrin glue as well as the composition can remain in the defect site without flow around despite haemorrhages from the defect site, and thus periosteum transplantation or periosteum suture in order to hold the composition in place was not necessary.
  • known composition would flow out of the defect site and require a periosteum suture (or other membrane suture) in the surgery. See Figs. 2B and 2C.

Abstract

A composition for repairing chondral defects is provided. The composition comprises a bone marrow concentrate, a cartilage fragment or powder, and a synovial membrane. Also provided is a method for treating a chondral defect in a subject. Said method comprises a step of applying a composition of the present invention into the chondral defect, and applying a fibrin glue onto the composition to cover the chondral defect.

Description

A COMPOSITION FOR REPAIRING CHONDRAL DEFECTS FIELD OF THE INVENTION
The present invention pertains to a composition for repairing chondral defects.
BACKGROUND OF THE INVENTION
Tissue engineering using autologous chondrocytes has emerged as a promising method for cartilage regeneration. However， concerns associated with using autologous chondrocytes include injuries at the harvested site and their intrinsic tendency to lose phenotype during monolayer expansion. Mesenchymal stem cells (MSCs) are considered the cell type of choice for their ability to differentiate into chondrocytes in a single-step procedure without needing a biopsy/culture phase and staged re-implantation surgery (Fang HW， Biomed Eng 21： 149-155 (2009) ) . A key for successful cartilage regeneration from MSCs is to correctly modulate cells to differentiate into the chondrogenic lineage and express normal cartilage extracellular matrix (ECM) . Moreover， ECM is thought to participate significantly in guiding the differentiation process of MSCs (Quesenberry P Jet al.， 1 Stem Cells 16 (Suppl 1) ： 33-35 (1998) ； Philp D et al.， Stem Cells 23： 288-296 (2005) ) .
Recently， the cartilage repair products related toautologous chondrocyte implantation (ACI) have been actively promoted to various countries for creating more businesses. They offer commercial service for culturing and expansion of chondrocytes from a small cartilage biopsy tissue in the Good Tissue Practice (GTP) laboratory. However， it needs two surgeries and still has some limitations (Buda Ret al.， IntOrthop. 2015 May； 39 (5) ： 893-900； Pestka JMet al.， Am J Sports Med. 2014 Jan； 42 (1) ： 208-15) .
On the other hand， known cell-based compositions require an effective carrier or sealant to fix onto the defect site. For successful cartilage repair， sufficient initialmechanical stability of the graft is of utmost importance (Efe Tet al.， Knee Surg Sports TraumatolArthrosc. 2012 Feb； 20 (2) ： 210-5) . The graft must withstand the forces in vivo duringthe early postoperative phase during joint movement. Adequate fixation is necessary to keep the graft inthe defect zone and to achieve optimal joint surface congruity. Generally， a periosteal patch or collagen fleece is sutured over cartilage defect to avoid the loss of cells or carrier/sealant (Slynarski Ket al.， Transplant Proc. 2006 Jan-Feb； 38 (1) ： 318-9； Stone KRet al.， Arthroscopy. 2006 Mar； 22 (3) ： 291-9； and Christoph Erggeletet al.， Arch Orthop Trauma Surg， DOI 10.1007/s00402-009-0957-y (2009) ) . This might increase the load  of surgeon and cause some problems such as periostea hypertrophy (Kreuzet al.， Osteoarthritis Cartilage. 2007 Dec； 15 (12) ： 1339-47； Henderson Iet al.， Arthroscopy. 2006 Dec； 22 (12) ： 1318-1324) .
In view of the foregoing， there is still a need for an improvedcomposition for repairing chondral defects.
BRIEF SUMMARY OF THE INVENTION
It was unexpectedly found in the present invention that a composition comprising a bone marrow concentrate， a cartilage fragment anda synovial membrane exhibit superior properties in the repair of chondral defects. Further， it was unexpectedly found that a fibrin glue applied onto the composition of the present invention shows superior cohesiveness and does not disperse or lose over time as usually used on a chondral defect site， such that a conventional step of covering the chondral defect filled with a cell-based composition for repair with a periosteal or synthetic membrane by suturing is not required.
Accordingly， in one aspect， the present invention provides a composition for repairing chondral defects， which comprises a bone marrow concentrate， a cartilage fragmentor powder， and synovial membranes. The composition of the present invention may be used in combination with a fibrin glue. The composition of the present inventionenables a novel one-stage-orientated cartilage repair surgery without the needs of a GTP laboratory for cell culturing and GMP factory for manufacturing new scaffolds. This could lower the threshold of clinical feasibility and benefit the performance from bench to clinic.
In another aspect， the invention provides a method for treating a chondral defect in a subject， comprising applying a composition of the present invention into the chondral defect， and applying a fibrin glue onto the composition to cover the chondral defect. One characteristic of the method of the present invention is that it does not require a step of periosteum or membrane suture after the application of the composition to hold the composition in place.
In a further aspect， the present invention provides said composition for use in a method of treating a chondral defect in a subject， the method comprising applying the composition into the chondral defect， and applying a fibrin glue onto the composition to cover the chondral defect. In one embodiment of the present invention， the composition is applied by filling the chondral defect.
According to the present invention， the method may not comprise a step of covering the chondral defect with a periosteal or synthetic membrane by suturing.
According to the present invention， the subject is preferably a mammal. The mammal includes but is not limited to humans， rodents， monkeys， pigs， dogs and cats. More preferably， the subject is human.
Cartilage and synovial membrane from a donor having the same or different genus from the subject to be treated may be used in the present invention.
It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention.
BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS
The foregoing summary， as well as the following detailed description of the invention， will be better understood when read in conjunction with the appended drawings. For the purpose of illustrating the invention， there are shown in the drawings embodiments which are presently preferred.
In the drawings：
Fig. 1A shows the RT-PCR results for culture of Group A1 (BMC (5 x 105) ) .
Fig. 1B shows the RT-PCR results for culture of Group A2 (cartilage-synovium) . ***p ＜ 0.001.
Fig. 1C shows the RT-PCR results for culture of Group A3 (BMC (5 x105) -cartilage-synovium) . ***p ＜ 0.001
Fig. 1D shows the comparison of the results of Group A3 to the results of Group A1.
Fig. 1E shows the comparison of the results of Group A3 to the results of Group A2.
Fig. 1F shows the RT-PCR results for culture of Group B1 (BM-MSCs (5 x 105) -cartilage-synovium) .
Fig. 1Gshows the RT-PCR results for culture of Group B2 (BM-MSCs (5 x 104) -cartilage-synovium) .
Fig. 2A is a picture showing a created chondral defect.
Fig. 2B is a picture showing that a composition of the present invention can remain in the defect site.
Fig. 2C is a picture showing that periosteum transplantation or periosteum suture is not required using a composition of the present invention for chondral defect repair.
Fig. 2D is a picture showingthe repair of cartilage 6 months after implantation.
DETAILED DESCRIPTION OF THE INVENTION
In one aspect， the present invention provides a composition for repairing chondral defects， which comprises a bone marrow concentrate， a cartilage fragment or powder， anda synovial membrane.
The composition of the present invention may be used in combination with a fibrin glue. Fibrin glue has already proven to be useful in anumber of orthopedic procedures； however， it does not successfully work in the knee joint full-thickness cartilage defect to fix soft materials such as cells.
In another aspect， the present invention provides said composition for use in a method of treating a chondral defect in a subject， the method comprising applying the composition into the chondral defect， and applying a fibrin glue onto the composition to cover the chondral defect. In one embodiment of the present invention， the composition is applied by filling the chondral defect. According to the present invention， the method may not comprise a step of covering the chondral defect with a periosteal or synthetic membrane by suturing.
In addition， the invention provides a method for treating a chondral defect in a subject， comprising applying a composition of the present invention into the chondral defect， and applying a fibrin glue onto the composition to cover the chondral defect.
It was unexpectedly found that a fibrin glue applied onto the composition of the present invention shows superior cohesiveness and does not disperse or lose over time as usually used on a chondral defect site， such that a conventional step of covering the chondral defect filled with a cell-based composition for repair with a periosteal or synthetic membrane by suturing is not required. Accordingly， in preferred embodiments of the present invention， the method does not comprise a step of covering the chondral defect with a periosteal or synthetic membrane by suturing.
According to the present invention， the subject is preferably a mammal. The mammal includes but is not limited to humans， rodents， monkeys， pigs， dogs and cats. More preferably， the subject is human.
The ″bone marrow concentrate″ used in the present invention is preferably in the form of cell pellet (of nucleated cells) and can be prepared from a bone marrow concentrate solution using a  conventional method， for example， centrifugation. The bone marrow concentrate solutioncan be prepared from a bone marrow aspirate by removing plasma and red blood cell fractions using conventional methods， for example， the bone marrow concentrate solution can be prepared by CellPointTM Concentrated Bone Marrow Aspirate System， ISTO Technologies， Missouri， U.S. The resultant bone marrow concentratesolution is enriched with nucleated cells compared to the bone marrow aspirate.
As used herein， the term ″nucleated cells″ includes but is not limited to bone marrow stem cells， neutrophils， lymphocytes and monocytes.
According to the present invention， the composition may comprise the BMC in an amount such that it provides a desired number of nucleated cells.
For example， for a chondral defect with a size of 1～3-mm in depthand about 8.5 mm in diameter (i.e.， a round shaped defect) ， the bone marrow concentrate used may comprise 104-107 nucleated cells. Namely， the composition may comprise the BMC in an amount such that it provides 104-107 nucleated cells. Preferably， the bone marrow concentrate comprises 105-106 nucleated cells. In one embodiment of the invention， the bone marrow concentrate comprises 5 x 105 nucleated cells.
In certain embodiments of the present invention， the bone marrow concentrateis in the form of cell pellet and consists essentially of nucleated cells derived from a bone marrow aspirate. For example， nucleated cellsconstitute at least 90％， at least 91％， at least 92％， at least 93％， at least 94％， at least 95％， at least 96％， at least 97％， at least 98％， or at least 99％， or more， by weight of the bone marrow concentrate used in the present invention.
The cartilage fragment or powder may be prepared from a cartilage tissue from a joint including but not limited to knee joint， a hip joint， and anankle joint. In certain embodiments， the cartilage powder is acellular cartilage powder， e.g. 
Figure PCTCN2016000274-appb-000001
 According to the present invention， the cartilage fragment preferably has a size ranging from 1 mm x 1 mm to 3 mm x 3 mm. In one embodiment， the cartilage fragment has a size of 1-2 mm x 2-3 mm.
For a chondral defect with a size of 1～3-mm in depthand about 8.5 mm in diameter (i.e.， a round shaped defect) ， 30-65 mg of cartilage fragment or powder may be used. For example， the composition may comprise 60 mg of cartilage powder.
The synovial membrane used in the composition of the invention may be in fragment form. Preferably， the synovial membrane is fragments of synovial membrane having a size ranging from 1 mm x 1 mm to 3 mm x 3 mm. In one embodiment of the present invention， the fragments are of about 2 mm x 3 mm in size.
For a chondral defect with a size of 1～3-mm in depthand about 8.5 mm in diameter (i.e.， a round shaped defect) ， 30-65 mg of synovial membrane may be used. For example， the composition may comprise 60 mg of synovial membrane.
Autogenic bone marrow concentrate or non-autogenic bone marrow concentrate from a donor having the same or different genus from the subject to be treated can be used in the present invention.
Cartilage and synovial membrane from a donor having the same or different genus from the subject to be treated may be used in the present invention.
According to the present invention， the composition may be prepared by evenly mixing a bone marrow concentrate， a cartilage fragment or powder， anda synovial membrane.
For example， a bone marrow concentrate comprising 104-107 nucleated cells may be mixed with 40-100 mg cartilage fragments or powder and 40-100 mg synovial membrane to form a composition for chondral defect repair. The composition may be further mixed with 0.2-1 mL biocompatible carrier. The actual amount may be easily adjusted by a person of ordinary skill according to the dimension of the chondral defects to be repaired. In one embodiment of the present invention， the composition is prepared by mixing a bone marrow concentrate comprisingabout 5 x 105 nucleated cells with 60 mg cartilage fragments and 60 mg synovial membrane fragments， further mixed with 0.2 mL fibrin glue.
According to the present invention， the composition may comprise 0.1-1 wt％of BMC， 30-60 wt％of cartilage fragment or powder， and 30-60 wt％of synovial membrane.
In certain other embodiments of the present invention， the composition is not pre-mixed with biocompatible carrier. The composition may be directly applied into the chondral defect， and then a suitable carrier or sealant is applied therein to or thereon to for fixation. Preferably， the carrier or sealant is a fibrin glue.
The present invention is further illustrated by the following examples， which are provided for the purpose of demonstration rather than limitation.
Example 1. In-vitro Studies
1. Preparation of bone marrow concentrate (BMC)
Porcine bone marrow concentrate was rapidly isolated by using a sucrose gradient. In brief， porcine bone marrow was aspirated with a syringe from tibia. The bone marrow was then filtered through a 70-μm mesh. The filtered bone marrow (10 ml) overlaid into a 17.5％sucrose gradient (15 ml) in a 50-mL conical tube was centrifuged once for 5 minutes at 1,500 rpm. The bone marrow concentrate in the fractionated layer were then extracted by centrifugation for 5-10 minutes at 1,500 rpm. The obtained pellet was dispersed into 1 ml DPBS to calculate the total cell numbers by an automatic cell counter.
2. Preparation of bone marrow mesenchymal stem cells (BM-MSCs)
The bone marrow concentrate collected from the fractionated layer were transferred into a new tube and then centrifuged for 5-10 minutes at 1,500 rpm to obtain nucleated cells. These cells were plated into culture flasks and cultured in α-MEM containing 10％FBS.
3. Preparation of cartilage tissue fragments
Cartilage tissues were harvested from porcine knee joint and washed with DPBS solution. These tissues were minced into small fragments by surgical knife. Approximate 1-2 mm x 2-3 mm sized cartilage fragments were washed with DPBS and collected by centrifugation for 5-10 minutes at 1,500 rpm.
4. Preparation of synovial membrane fragments
Synovial membranes were harvested from porcine knee joint and washed with DPBS solution. The tissues were minced into small fragments by surgical knife. Approximate 2 mm x 3 mm sized synovial membrane fragments were washed with DPBS and collected by centrifugation for 5-10 minutes at 1,500 rpm.
5. Preparation of a composition for repairing chondral defects
(1) BMC-cartilage fragment-synovial membrane composition
Bone marrow concentrate suspension containing 1-10 x 105 cells and the tissue fragments including 30-60 mg cartilage fragments and 30-60 mg synovial membranes were mixed together. After centrifugation， the supernatant was discarded and the BMC-cartilage fragment-synovial membrane mixture was obtained. The mixture was further embedded into 0.2 mL TISSEEL solution containing fibrinogen and thrombin on 48-well plate to form a biological construct. The formed construct was transferred into 24-well plate and cultured in α-MEM containing 10％FBS. Other similar constructs without tissue fragments and without bone marrow concentrate were prepared for comparison.
(2) BM-MSCs-cartilage fragment-synovial membrane composition (for comparison) 
In brief， the BM-MSCs were serially trypsinized. The cell suspension containing 1-10 x 104 stem cells and the tissue fragments including 30-60 mg cartilage fragments and 30-60 mg synovial membranes were mixed together. After centrifugation， the supernatant was discarded and the BM-MSCs-cartilage fragment-synovial membrane mixture was obtained. The mixture was further embedded into 0.2 mL TISSEEL solution containing fibrinogen and thrombin on 48-well plate to form the biological construct. The formed construct was transferred into 24-well plate and cultured in α-MEM containing 10％FBS. Other similar constructs with different BM-MSC numbers were prepared for comparison.
6. RT-PCR analysis on gene expression
Total RNA was isolated by using the Trizol purification system (Invitrogen) according to the manufacture’s protocol. Reverse transcription (RT) of total RNA to single-stranded cDNA was completed by using the high-capacity cDNA RT kit (Applied Biosystems) . Gene expression was analyzed by using an ABI SteponePlusTMreal-time PCR system (Applied Biosystems) . 
Figure PCTCN2016000274-appb-000002
 probes were purchased from Applied Biosystems including glyceraldehydes-3-phosphate dehydrogenase (Hs99999905_ml) ， aggrecan (Hs00153936_ml) ， SOX9 (Hs00165814_ml) ， type I collagen (Hs00164004_ml) ， type II collagen (Hs00156568_ml) ， and type X collagen  (Hs00166657_ml) . Data were evaluated as mRNA levels of the cells in the constructs. A cycle threshold value (Ct) was obtained from each sample and triplicate sample values were averaged. The 2-ΔΔCt method was used to calculate relative expression of each target gene.
7. Results
Cultures of the following Groups were subject to RT-PCR analysis on gene expressionof type II collagen and the results are shown in the Figures： (A1) BMC (5 x 105) (Fig. 1A) ； (A2) cartilage-synovium (Fig. 1B) ； (A3) BMC (5 x105) -cartilage-synovium (Fig. 1C) ； (B1) BM-MSCs (5 x 105) -cartilage-synovium (Fig. 1F) ； and (B2) BM-MSCs (5 x 104) -cartilage-synovium (Fig. 1G) . Fig. 1D shows the comparison of the results of Group A3 to the results of Group A1， and Fig. 1Eshows the comparison of the results of Group A3 to the results of Group A2.
Group A3 (a composition according to the present invention) shows more increasesin type II collagen expression over time of culture (up to 6 weeks) ， as compared to Groups A1 (BMCs alone) and A2 (cartilage-synovium alone) (see Figs. 1A-1E) . As for Groups B 1 and B2 using BM-MSCs instead of BMC， type II collagen expression did not increase significantly over time of culture.
Type II collagen represents 90％to 95％of the collagen in the extracellular matrix (ECM) of articular cartilage (or hyaline cartilage) ， and is thus considered crucial for successful repair of chondral defects.
Example 2. Animal Studies
1. Preparation of bone marrow concentrate (BMC)
Porcine bone marrow concentrate was rapidly isolated by using a sucrose gradient. In brief， 10 ml porcine bone marrow was aspirated with a syringe from tibia. The bone marrow (10 ml) overlaid into a 17.5％sucrose gradient (15 ml) in a 50-mL conical tube was centrifuged once for 5 minutes at 1,500 rpm. The bone marrow concentrate in the fractionated layer were then extracted by centrifugation for 5-10 minutes at 1,500 rpm. Pellet was dispersed into 1 ml DPBS to calculate the total cell numbers by an automatic cell counter.
2. Preparation of cartilage tissue fragments
Cartilage tissues were harvested from porcine knee joint and washed with DPBS solution， and then minced into small fragments by surgical knife. Approximate 1-2 mm x 2-3 mm sized cartilage fragments were washed with DPBS and collected by centrifugation for 5-10 minutes at 1,500 rpm.
3. Preparation of synovial membrane fragments
Synovial membranes were harvested from porcine knee joint and washed with DPBS solution. The tissues were then minced into small fragments using surgical knife. Approximate 2 mm x 3 mm sized synovial membrane fragments were washed with DPBS and collected by centrifugation for 5-10 minutes at 1,500 rpm.
4. In-vivo animal model
Miniature pigs were used in this study. All pigs were sexually mature at the timeof surgery. All operations and interventions were performed under general anesthesia. Full-thickness defects were created in medial condyles of both knee joints (see Fig. 2A) ， whereinone of which received acomposition containing bone marrow concentrate， 60 mg cartilage fragments and 60 mg synovial membranes， and then aTisseel solution (afibrin glue) (0.2-0.4 ml) for fixation； and the other received micro-fracture treatmentas a control group. The diameter of the defect was 8 mm with 1 mm depth.
5. Results
It was observed that， after application onto the composition， the fibrin glue shows high cohesiveness， and the fibrin glue as well as the composition can remain in the defect site without flow around despite haemorrhages from the defect site， and thus periosteum transplantation or periosteum suture in order to hold the composition in place was not necessary. On the contrary， known composition would flow out of the defect site and require a periosteum suture (or other membrane suture) in the surgery. See Figs. 2B and 2C.
Animals were allowed free movement upon recovery from surgery. Immediately after surgery， the pigs walked slowly and did not stand for long except when eating. They lay on the floor to rest. This type of behavior lasted for several days， after which time the pigs’behavior returned to normal. Fine repair of the chondral defect was observed 6 months after the implantation (Fig. 2D) .
It will be appreciated by those skilled in the art that changes could be made to the embodiments described above without departing from the broad inventive concept thereof. It is understood， therefore， that this invention is not limited to the particular embodiments disclosed， but it is intended to cover modifications within the spirit and scope of the present invention as defined by the appended claims.

Claims (10)

  1. A composition for repairing chondral defects comprising a bone marrow concentrate， a cartilage fragment or powder， and a synovial membrane.
  2. The composition of claim 1， comprising 0.1-1 wt％ of BMC， 30-60 wt％ of cartilage fragment or powder， and 30-60 wt％ of synovial membrane.
  3. The composition of claim 1， wherein the synovial membrane is in the form of fragments.
  4. The composition of claim 1， which is prepared by evenly mixing a bone marrow concentrate， a cartilage fragment or powder， and a synovial membrane.
  5. The composition of any of claims 1-4 for use in a method of treating a chondral defect in a subject， the method comprising applying the composition into the chondral defect， and applying a fibrin glue onto the composition to cover the chondral defect.
  6. The composition for use in claim 5， wherein the composition is applied by filling the chondral defect.
  7. The composition for use in claim 5， wherein the method does not comprise a step of covering the chondral defect with a periosteal or synthetic membrane by suturing.
  8. A method oftreating a chondral defect in a subject， comprising applying the composition of any of claims 1-3 into the chondral defect， andapplying a fibrin glue onto the composition to cover the chondral defect.
  9. The method of claim 85whichdoes not comprise a step of covering the chondral defect with a periosteal or synthetic membrane by suturing.
  10. A kit for treating chondral defect comprising： (i) the composition of any of claims 1-4； and (ii) a fibrin glue.
PCT/CN2016/000274 2015-05-22 2016-05-23 A composition for repairing chondral defects WO2016188123A1 (en)

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