CN101820895A

CN101820895A - The compositions and the method that are used for the treatment of spinal column

Info

Publication number: CN101820895A
Application number: CN200880101796A
Authority: CN
Inventors: C·E·哈特; S·E·林奇; C·S·杨; D·佩里恩
Original assignee: Biomimetic Therapeutics LLC
Current assignee: Biomimetic Therapeutics LLC
Priority date: 2007-06-04
Filing date: 2008-06-03
Publication date: 2010-09-01
Also published as: WO2008151193A1; CA2689986C; CA2689986A1; AU2008259785B2; AU2008259785A1; CN106110299A

Abstract

The present invention relates to be used for the treatment of the compositions and the method for the spine structure that comprises vertebral body.In one embodiment, being used for promoting the osteoplastic method of vertebral body to comprise provides the compositions that comprises PDGF solution and biocompatible matrix, and comprises described compositions is administered at least one vertebral body.According to some embodiment, promote that the bone formation in the vertebral body can increase bone volume, quality and/or density, cause mechanical strength increase with the vertebral body of present composition treatment.

Description

The compositions and the method that are used for the treatment of spinal column

Related application data

The application is according to PCT I chapter clause 8 and 35U.S.C. § 119 (e), be attained at the interim patent serial number of submitting on June 4th, 2,007 60/933 of the U.S. in this requirement, 202 and the interim patent serial number 61/026 of the U.S. submitted on February 7th, 2008,835 priority, the two all is hereby incorporated by reference.

Invention field

The present invention relates to be used for the treatment of the compositions and the method for the spine structure that comprises vertebral body.

Background of invention

All age group of flesh skeleton problem exists throughout and both sexes crowd.(half American is in the service of need fracturing of certain time in its all one's life for American Academy of Orthopedic Surgeons, AAOS) a large amount of disclosed article that proposes of annual meeting in 2003 according to U.S. orthopedist association.According to this report, the U.S. is annual to be spent on the inpatient relevant with fractures above 10,000,000,000 dollars.

(Vertebral compression fracture VCF) is modal osteoporotic fracture to vertebral compression fractures, and (Eastell etc., J BoneMiner Res 1991 take place in about 20% postmenopausal women; 6:207-215).Estimate annual 700, the 000 routine VCF of generation, and only 250,000 diagnosed and treat in these cases.Because these fracture do not obtain medical treatment, thereby osteoporosis may not obtain medical treatment yet and develop rapidly.The postmenopausal women continued another time of generation vertebral fracture in following 1 year risk increases by 500, and comprises that the risk of other vulnerability fracture of hip fracture increases by 2 times of (Klotzbuecher etc., J Bone Miner Res, 2000; 15:721-739).

When one of terminal plate of vertebral body or both are broken VCF takes place then, soleplate breaks and is caused by wound usually, causes the front pillar fault and vertebra is died down to the support of the health in activities of daily living.The vertebral compression fractures that is caused by osteoporosis can cause weakness backache, deformity of spine and height reduction.The two is all relevant with high incidence and mortality rate for the vertebral fracture of reveal any symptoms and asymptomatic vertebral fracture.Old people's number sharply increase at following 10 years along with expection osteoporosis risk needs accurately to identify VCF and treat and get involved to reduce the huge potential impact of this disease to patient and healthcare network.

Traditionally always with lie up, narcosis analgesic, brace (brace) and physiotherapy treat the VCF that is caused by osteoporosis.Yet lying up causes bone loss to quicken and health deadaptation (physical deconditioning), makes the patient further worsen and promote the osteoporosis problem.In addition, the use of anesthetics can make in the old people may this ubiquitous emotion and psychological problems more worsen.In addition, the wearing of brace can not be tolerated by the old people.Although the treatment (for example hormone replacement, diphosphate, calcitonin and parathyroid hormone (PTH) analog) of osteoporosis can solve secular problem at present, but except that calcitonin, in case fracture takes place, and they all can not provide benefit immediately (Kapuscinski etc., Master Med.Pol.1996 at ease pain; 28:83-86).

Developed at present the minimally-invasive treatment that is used for vertebral compression fractures already: vertebroplasty (vertebroplasty) and kyphoplasty art (kyphoplasty), to solve pain and fracture stabilization problem.Vertebroplasty is the vertebral body of filling fracture for the purpose of stabilizing bone, prevents from further to subside and eliminate acute fracture pain.Yet vertebroplasty does not attempt to recover vertebral levels and/or sagittal plain is arranged (sagittal alignment).In addition, because in skeleton, there is not the space, carry out vertebra with the less cement of viscosity and fill difficulty and control, so implant tends to seepage.

The kyphoplasty art is the Minimally Invasive Surgery method with security purpose, and it improves vertebral height and stablizes VCF.Under the radioscopic image guiding, distensible bone sacculus (bone tamp) is expanded.This makes inner spongy bone tight, because it pushes back its normal position with disruptive cortex.Can fix by filling the space then with biomaterial under the control volume and the cement that has more viscosity.Although the kyphoplasty art is considered to the treatment safely and effectively of vertebral compression fractures, the increase of biomechanics Research proof cement has produced extra pressure to adjacent segment (level).In fact, this hardness increase can make the lost efficacy limit load of (failure) of adjacent vertebrae reduce 8-30%, and cause subsequently fracture (Berlemann etc., J Bone Joint Surgery BR, 2002; 84:748-52).This paper is called " secondary vertebral compression fractures " with a place or the many places compression fracture of vertabral body of vertebroplasty or kyphoplasty postoperative.

In nearest clinical research, compare with the historical data of untreated fracture, arrive secondary vertebral compression fractures higher incidence in the kyphoplasty clinical follow.Major part in these cases occurs in adjacent segment in 2 months after described method.After these two months, only there is accidental secondary vertebral compression fractures to occur in sections at a distance.This research proved conclusively the cement that shows increase to adjacent segment produced extra pressure biomechanics Research (Fribourg etc., Incidence of subsequent vertebral fracture after kyphoplasty (incidence rate of the vertebral fracture that the kyphoplasty postoperative is concurrent), Spine, 2004; 20; 2270-76).

Increased sickness rate since be used for the treatment of the use of the Minimally Invasive Surgery technology of vertebral compression fractures, and adjacent vertebrae is easy to stand the second-compressed fracture, so for prophylactic treatment with prevent that from there are unsatisfied clinical needs in secondary VCF.

The invention summary

The invention provides the compositions and the method that are used for the treatment of the spine structure that comprises vertebral body.In certain embodiments of the invention, be provided for promoting the osteoplastic compositions in vertebral body.In other embodiments, be provided for preventing or reducing the compositions and the method for the probability of vertebral compression fractures.In another embodiment, be provided for preventing or the method and composition of the probability of the secondary vertebral compression fractures that reduction and vertebroplasty and/or kyphoplasty art link.The present composition and method can be used for treating easy ill patient (compromised patient) (patient's who for example suffers from osteoporosis, diabetes or other disease or disease) vertebral body.

In one aspect, be used for promoting that the osteoplastic compositions of vertebral body comprises solution and the biocompatible matrix that contains platelet derived growth factor (PDGF), wherein said solution is configured or is incorporated in the described biocompatible matrix.In some embodiments, PDGF is adsorbed by biocompatible matrix.In other embodiments, PDGF is adsorbed to one or more surfaces of biocompatible matrix.In the embodiment, PDGF is adsorbed by biocompatible matrix, and is adsorbed to one or more surfaces of biocompatible matrix again in the present invention.

In some embodiments, PDGF is to exist in solution between following concentration range: the about 10mg/ml of about 0.01mg/ml-, the about 5mg/ml of about 0.05mg/ml-, the about 1.0mg/ml of about 0.1mg/ml-or the about 0.4mg/ml of about 0.2mg/ml-.PDGF concentration in the solution can be in above-mentioned any concentration range.

In certain embodiments of the invention, PDGF comprises PDGF homodimer and heterodimer, comprises PDGF-AA, PDGF-BB, PDGF-AB, PDGF-CC, PDGF-DD and composition thereof and derivant.In one embodiment, PDGF comprises PDGF-BB.In another embodiment, PDGF comprises recombined human (rh) PDGF, for example recombined human PDGF-BB (rhPDGF-BB).

In certain embodiments of the invention, PDGF comprises the PDGF fragment.In one embodiment, rhPDGF-B comprises following fragment: the aminoacid sequence of the 1-31 of complete B chain, 1-32,33-108,33-109 and/or 1-108 position.At United States Patent (USP) the 5th, 516, whole aminoacid sequences (1-109) of PDGF B chain are provided among Figure 15 of No. 896.Should be appreciated that rhPDGF compositions of the present invention can comprise complete rhPDGF-B (1-109) and segmental combination thereof.Can adopt other fragment of PDGF, for example at United States Patent (USP) the 5th, 516, disclosed fragment in No. 896.In some embodiments, rhPDGF-BB comprises at least 65% of complete rhPDGF-B (1-109).

According to certain embodiments of the invention, biocompatible matrix comprises bone and replaces agent (bonesubstituting agent) (being also referred to as timbering material (scaffolding material) herein) and optional biocompatible adhesive.In some embodiments, bone replaces agent to comprise calcium phosphate, and it comprises amorphism calcium phosphate, one water one-lime phosphate (MCPM), anhydrous phosphoric acid one calcium (MCPA), dicalcium phosphate dihydrate (DCPD), anhydrous dicalcium phosphate (DCPA), OCP (OCP), type alpha tricalcium phosphate, β-TCP, hydroxyapatite (OHAp), the hydroxyapatite that crystallinity is low, tetracalcium phosphate (TTCP), ten phosphoric acid, seven calcium (heptacalcium decaphosphate), calcium metaphosphate, calcium pyrophosphate dihydrate, calcium pyrophosphate, carbon calcium phosphate, hydroxyapatite or their derivant or mixture.In some embodiments, bone replaces agent to comprise calcium sulfate or demineralized bone (deminerialized bone), for example dried Compact bone or spongy bone.

On the other hand, the invention provides the osteoplastic compositions that is used for promoting vertebral body, described compositions comprises the PDGF solution that is configured in the biocompatible matrix, and wherein biocompatible matrix comprises bone holder material and biocompatible adhesive.PDGF solution can have aforesaid PDGF concentration.In some embodiments, bone holder material comprises calcium phosphate.In one embodiment, calcium phosphate comprises β-TCP.In one aspect, biocompatible matrix can comprise calcium phosphate particles (containing or do not contain biocompatible adhesive) or bone allograft (bone allograft), lyophilization demineralized bone allogeneic thing (demineralized freezedried bone allograft for example, DFDBA), lyophilization mineralising bone allogeneic thing (mineralizedfreeze dried bone allograft, FDBA) or the demineralized bone matrix granule (particulartedemineralized bone matrix, DBM).On the other hand, biocompatible matrix can comprise bone allograft (for example DFDBA, DBM) or other bone allograft material, comprises the Compact bone of different shape (for example bulk, wedge shape, cylinder or granule) or the spongy bone granule of different shape and size.

In addition, according to certain embodiments of the invention, biocompatible adhesive comprises protein, polysaccharide, nucleic acid, carbohydrate, synthetic polymer or its mixture.In one embodiment, biocompatible adhesive comprises collagen protein.In another embodiment, biocompatible adhesive comprises hyaluronic acid.

The present invention is provided for preventing or reducing the compositions of vertebral compression fractures probability on the other hand, and described vertebral compression fractures comprises the secondary vertebral compression fractures.In some embodiments, be used to prevent or the compositions that reduces the vertebral compression fractures probability comprises solution and the biocompatible matrix that contains PDGF, wherein with described solution allocation in described biocompatible matrix.In other embodiments, be used for preventing or the compositions that reduces the vertebral compression fractures probability comprises the PDGF solution that is configured in biocompatible matrix, wherein biocompatible matrix comprises bone holder material and biocompatible adhesive.In the compositions embodiment that is used for preventing or reducing the vertebral compression fractures probability, PDGF solution can have aforesaid PDGF concentration.In addition, in some embodiments, bone holder material comprises calcium phosphate.In embodiments, calcium phosphate comprises bata-tricalcium phosphate.According to certain embodiments of the invention, biocompatible adhesive comprises protein, polysaccharide, nucleic acid, carbohydrate, synthetic polymer or its mixture.In one embodiment, biocompatible adhesive comprises collagen protein.In another embodiment, biocompatible adhesive comprises collagen protein, for example bovine collagen albumen.

In certain embodiments of the invention, be used for promoting the osteoplastic compositions of vertebral body and be used to prevent or the compositions that reduces the vertebral compression fractures probability further comprises at least a contrast agent.According to embodiment of the present invention, contrast agent is for operating so that the material of two or more bodily tissue differences to be provided when the imaging at least in part.According to some embodiment, contrast agent comprises cation contrast agent, anion contrast agent, non-ionic contrast medium or their mixture.In some embodiments, contrast agent comprises radiopaque contrast agent (radiopaquecontrast agent).In some embodiments, the radiopaque contrast agent comprises and contains iodine compound, it comprises (S)-N, N '-two [2-hydroxyl-1-(methylol)-ethyl]-2,4,6-three iodo-5-lactoyl amido isophthaloyl amine (lactamidoisophthalamide) (iopamidol (Iopamidol)) and derivant thereof.

On the other hand, the invention provides kit, described kit is included in the biocompatible matrix in first packing and contains PDGF solution in second packing.In some embodiments, biocompatible matrix comprises timbering material, timbering material and biocompatible adhesive and/or bone allograft such as DFDBA or microgranule DBM.In one embodiment, timbering material comprises calcium phosphate such as β-TCP.In addition, in some embodiments, described solution comprises the PDGF of predetermined concentration.Can for example be scheduled to according to the predetermined PDGF concentration of surgical operation to be performed according to the probability of promotion or acceleration vertebral body osteogenesis or prevention or reduction secondary vertebral compression fractures.In addition, in some embodiments, biocompatible matrix can be present in the kit with scheduled volume.The amount of the biocompatible matrix that is provided by kit is decided by surgical operation to be performed.In some embodiments, contain second of PDGF solution and comprise syringe.Syringe can help the configuration of PDGF solution in biocompatible matrix.In some embodiments, can the compositions that obtain be placed second syringe and/or sleeve pipe and be delivered to vertebral body after the PDGF solution allocation is in the biocompatible matrix at once.

The method for compositions that the present invention also provides preparation to be used for promoting bone formation and the prevention of vertebral body or to reduce the probability of compression fracture of vertabral body (comprising the secondary vertebral compression fractures).In one embodiment, be used to prepare described method for compositions and comprise: the solution that comprises PDGF is provided, biocompatible matrix is provided and with described solution allocation in described biocompatible matrix.In some embodiments, the preparation method for compositions that is used for promoting bone formation and the prevention of vertebral body or reduces the compression fracture of vertabral body probability further comprises provides contrast agent and contrast agent is configured in the biocompatible matrix.

On the other hand, the invention provides the osteoplastic method that is used for promoting or quickening vertebral body, described method comprises: the compositions that comprises the PDGF solution that is configured in the biocompatible matrix is provided and the compositions of effective dose is administered at least one vertebral body.In some embodiments, described compositions being administered at least one vertebral body comprises compositions is expelled at least one vertebral body.

On the other hand, the invention provides the method that comprises prevention or reduce the probability of vertebral compression fractures (comprising the secondary vertebral compression fractures).According to embodiment of the present invention, prevention or reduce the vertebral compression fractures probability and comprise: the compositions that comprises the PDGF solution that is configured in the biocompatible matrix is provided and the compositions of effective dose is administered at least one vertebral body.In some embodiments, described compositions being administered at least one vertebral body comprises compositions is expelled at least one vertebral body.In one embodiment, first vertebral body being carried out vertebroplasty or kyphoplasty postoperative, compositions is administered to second vertebral body, described in some cases second vertebral body is adjacent vertebral bodies.In some embodiments, the compositions that will comprise the PDGF solution that is configured in the biocompatible matrix is administered at least one high-risk vertebral body.(high risk vertebral body HVB) refers to the vertebral body of vertebra T5 to T12 and L1 to L4, and they are in the excessive risk that stands the secondary vertebral compression fractures " high-risk vertebral body " used herein.

In some embodiment of the inventive method, biocompatible matrix comprises bone holder material.In some embodiments, biocompatible matrix comprises bone holder material and biocompatible adhesive.

In some embodiments, the method that is used for promoting bone formation and the prevention of vertebral body or reduces the probability of compression fracture of vertabral body, further comprise: except that the compositions that comprises the PDGF solution that is configured in the biocompatible matrix, at least a pharmaceutical composition also is provided, and through the part and/or whole body give described at least a pharmaceutical composition.In some embodiments, described at least a pharmaceutical composition comprises vitamin, calcium complement agent or any osteoclast inhibitor well known by persons skilled in the art, comprises diphosphate.In some embodiments, described at least a pharmaceutical composition per os gives.In such embodiments, described at least a pharmaceutical composition can be incorporated in the biocompatible matrix, or be configured in the vertebral body in addition and on every side.In other embodiments, give the patient with described at least a pharmaceutical composition whole body.For example, in one embodiment, give the patient with described at least a pharmaceutical composition per os.In another embodiment, give the patient with described at least a pharmaceutical composition intravenous.

Therefore, target of the present invention provides the osteoplastic PDGF of the containing compositions that is used for promoting vertebral body.

Another target of the present invention provide be used to reinforce vertebral body contain the PDGF compositions.

Another target of the present invention provide be used to reinforce the patients with osteoporosis vertebral body contain the PDGF compositions.

Another target of the present invention provide be used for prevention or reduce vertebral compression fractures (comprising the secondary vertebral compression fractures) probability contain the PDGF compositions.

Another target of the present invention provides and is used for containing the osteoplastic method that the PDGF compositions promotes vertebral body.

The present invention's target again provides with the method that contains prevention of PDGF compositions or reduction vertebral compression fractures (comprising the secondary vertebral compression fractures) probability.

These and other embodiment of the present invention is set forth in the following detailed Description Of The Invention in more detail.After scanning disclosed embodiment of following detailed Description Of The Invention and claim, these targets of the present invention, feature and advantage will be apparent.

The accompanying drawing summary

Fig. 1 illustrates syringe and related equipment, and its tissue that thrusts the covering vertebral body according to an embodiment of the present invention is to be delivered to vertebral body with the present composition.

Fig. 2 illustrates the X-ray photograph that according to an embodiment of the present invention compositions is injected vertebral body.

Fig. 3 illustrates the vertebra of accepting the present composition according to an embodiment of the invention.

Fig. 4 illustrates according to an embodiment of the invention, acceptance comprises the vertebral body of the compositions of the rhPDGF-BB that is configured in the 1.0mg/ml in β-TCP/ collagen matrices, the vertebral body that comprises the compositions that is configured in the 20mM sodium acetate buffer in β-TCP/ collagen matrices with acceptance is compared the variation of its volume bone mineral density percentage ratio.

Fig. 5 illustrates according to an embodiment of the invention, acceptance comprises the vertebral body of the compositions of the rhPDGF-BB that is configured in the 1.0mg/ml in β-TCP/ collagen matrices, the vertebral body that comprises the compositions that is configured in the 20mM sodium acetate buffer in β-TCP/ collagen matrices with acceptance is compared the variation of its volume bone mineral density percentage ratio.

Detailed Description Of The Invention

The invention provides the composition and the method that are used for the treatment of the spine structure that comprises centrum. According to embodiment described herein, the invention provides for the osteoplastic composition that promotes centrum with for the composition that prevents or reduce vertebral compression fractures (comprising the secondary vertebral compression fractures) possibility. In one embodiment, described composition comprises and contains PDGF solution and biocompatible matrix, wherein with described solution allocation in described biocompatible matrix. In another embodiment, described composition comprises to be configured in and contains PDGF solution in the biocompatible matrix, and wherein said biocompatible matrix comprises bone holder material and biocompatible adhesive. In one aspect, biocompatible matrix comprises calcium phosphate granules (containing or do not contain biocompatible adhesive) or bone allograft for example DFDBA or particle DBM. On the other hand, biocompatible matrix can comprise DFDBA or DBM.

Turn to now the component that can be included in the various embodiments of the present invention, the present composition comprises and contains PDGF solution.

PDGF solution

PDGF plays an important role in regulating Growth of Cells and migration. PDGF is combined with the ectodomain of receptor tyrosine kinase as other growth factor. The kinase activity in conjunction with the catalyst structure domain that activates its cytoplasm side that is positioned at film of PDGF and these transmembrane proteins. Described kinases is induced the various kinds of cell process by making the tyrosine residue phosphorylation of target proteins, comprises that Growth of Cells and extracellular matrix form.

In one aspect, composition provided by the invention comprises and contains PDGF solution and biocompatible matrix, wherein with described solution allocation or be incorporated in the described biocompatible matrix. In some embodiments, PDGF exists in solution with the concentration between the about 10mg/ml of about 0.01mg/ml-, the about 5mg/ml of about 0.05mg/ml-or the about 1.0mg/ml of about 0.1mg/ml-. PDGF can exist in solution with any concentration in described these scopes, comprises the upper and lower bound of each scope. In other embodiments, PDGF exists in solution with following any one concentration: about 0.05mg/ml; About 0.1mg/ml; About 0.15mg/ml; About 0.2mg/ml; About 0.25mg/ml; About 0.3mg/ml; About 0.35mg/ml; About 0.4mg/ml; About 0.45mg/ml; About 0.5mg/ml; About 0.55mg/ml; About 0.6mg/ml; About 0.65mg/ml; About 0.7mg/ml; About 0.75mg/ml; About 0.8mg/ml; About 0.85mg/ml; About 0.9mg/ml; About 0.95mg/ml; Or about 1.0mg/ml. In some embodiments, PDGF is to exist in solution between following concentration range: the about 2mg/ml of about 0.2mg/ml-; The about 3mg/ml of about 0.3mg/ml-; The about 4mg/ml of about 0.4mg/ml-or the about 5mg/ml of about 0.5mg/ml-. Should be appreciated that these concentration only are the example of specific embodiments, PDGF concentration can in above-mentioned any concentration range, comprise the upper and lower bound of each scope.

Various PDGF amounts can be used in the present composition. Spendable PDGF amount comprises the amount of following scope: the about 50mg of about 1ug-, the about 25mg of about 10ug-, the about 10mg of about 100ug-and the about 5mg of about 250ug-.

Can be by using such as United States Patent (USP) the 6th, 221 No. 625, the 5th, 747, No. 273 and the 5th, 290, No. 708 described enzymoimmunoassays or any other determination method for measuring PDGF concentration known in the art are measured the PDGF of embodiment of the present invention or the concentration of other growth factor. When providing in this article, the molar concentration of PDGF is based on the dimeric molecular weight of PDGF (MW) (PDGF-BB for example; MW is about 25kDa) measure.

In certain embodiments of the invention, PDGF comprises PDGF homodimer and heterodimer, comprises PDGF-AA, PDGF-BB, PDGF-AB, PDGF-CC, PDGF-DD and composition thereof and derivative. For example, in one embodiment, PDGF comprises PDGF-BB. In another embodiment, PDGF comprises rhPDGF-BB, for example rhPDGF-BB. In some embodiments, PDGF comprises the mixture of various homodimers and/or heterodimer. Embodiment of the present invention contains any combination of PDGF-AA, PDGF-BB, PDGF-AB, PDGF-CC and/or PDGF-DD.

In some embodiments, can obtain PDGF from natural origin. In other embodiments, can prepare PDGF by recombinant DNA technology. In other embodiments, the known peptide synthetic technology (for example solid-phase peptide is synthetic) of available persons skilled in the art prepares PDGF or its fragment. When PDGF obtained from natural origin, it can derive from biological fluids. According to some embodiment, biological fluids can comprise any treated or untreated liquid relevant with live organism, comprises blood.

In another embodiment, biological fluids also can comprise blood constitutent, and described blood constitutent comprises PC (PC), machine blood sampling platelet (apheresed platelet), platelet rich plasma (PRP), blood plasma, serum, FFP (FFP) and dark yellow cover layer (BC). In another embodiment, biological fluids can comprise from plasma separation and again be suspended in blood platelet in the physiology flow liquid.

In some embodiments, when preparing PDGF by recombinant DNA technology, the dna sequence dna of the single monomer of coding (for example PDGF B-chain or A-chain) can be inserted prokaryotic or the eukaryotic of the cultivation that is used for expression, to produce subsequently homodimer (for example PDGF-BB or PDGF-AA). In other embodiments, can prepare by the following method the PDGF heterodimer: the dna sequence dna of two monomeric units of the heterodimer of will encoding all is inserted in the prokaryotic or eukaryotic of cultivation, and makes cell process translated monomeric unit to produce heterodimer (for example PDGF-AB). Commercially available GMP restructuring PDGF-BB can buy from NovartisCorporation (Emeryville, CA). The rhPDGF-BB of research grade can obtain from a plurality of sources, comprises R﹠D Systems, Inc. (Minneapolis, MN), BD Biosciences (San Jose, CA) and Chemicon, International (Temecula, CA). In some embodiments, can in prokaryotic, prepare monomeric unit with denatured form, wherein again be folded into bioactive molecule with the relief denatured form.

In embodiment of the present invention, PDGF comprises the PDGF fragment. In one embodiment, rhPDGF-B comprises following fragment: the amino acid sequence of the 1-31 of complete B chain, 1-32,33-108,33-109 and/or 1-108 position. United States Patent (USP) the 5th, 516 provides whole amino acid sequences (1-109) of PDGF B chain among Figure 15 of No. 896. Should be appreciated that rhPDGF composition of the present invention can comprise the combination of complete rhPDGF-B (1-109) and fragment thereof. Can adopt other fragment of PDGF, for example United States Patent (USP) the 5th, 516, the fragment described in No. 896. According to an embodiment, rhPDGF-BB comprises at least 60% of complete rhPDGF-B (1-109). In another embodiment, rhPDGF-BB comprises at least 65%, 75%, 80%, 85%, 90%, 95% or 99% of complete rhPDGF-B (1-109).

In certain embodiments of the invention, but purifying PDGF. Before being included in and joining in the solution of the present invention, the PDGF of purifying used herein has the composition greater than the PDGF of about 95% weight. Described solution can be any medical solution. In other embodiments, purifying PDGF basically. The composition that has the PDGF of about 95% weight of about 5%-before the PDGF of basically purifying used herein is included in and joins in the solution of the present invention. In one embodiment, the PDGF of purifying is included in and joins the composition that has the PDGF of about 95% weight of about 65%-in the solution of the present invention before basically. In other embodiments, the PDGF of purifying is included in the composition that has the PDGF of following content before joining in the solution of the present invention basically: about 70%-is about 95%, about 75%-is about 95%, about 80%-is about 95%, about 85%-about 95% or about 95% weight of about 90%-. Can with the PDGF of purifying and basically the PDGF of purifying be incorporated in support and the adhesive.

In another embodiment, but partial purification PDGF. Partially purified PDGF used herein is included in the composition with PDGF in the following situation: platelet rich plasma (PRP), FFP (FFP) maybe need to gather and separate to prepare any other blood product of PDGF. Embodiment of the present invention contains any PDGF isotype provided herein, comprises homodimer and heterodimer, and they can be purified or purifying partly. The present composition that contains the PDGF mixture can contain PDGF isotype or the PDGF fragment that is the partial purification ratio. In some embodiments, can be such as the interim patent serial number 11/159,533 of the U.S. (publication No.: the 20060084602) PDGF of described preparation partial purification and purifying.

In some embodiments, contain PDGF solution by allowing PDGF be dissolved in and forming in one or more buffer solutions. The buffer solution that is applicable to PDGF solution of the present invention can include but not limited to: carbonate, phosphate (for example phosphate buffered saline (PBS)), histidine, acetate (for example sodium acetate), acidic buffer (for example acetic acid and HCl) and organic buffer liquid (for example lysine, Tris buffer solution (for example three (methylol) aminoethane), N-2-hydroxyethyl piperazine-N '-2-ethanesulfonic acid (FIEPES) and 3-(N-morpholino) propane sulfonic acid (MOPS)). Can stop undesirable protein modified buffer capacity to select buffer solution based on biocompatibility and the buffer solution with PDGF. Can be based on selecting in addition buffer solution with the compatibility of host tissue. In one embodiment, can use sodium acetate buffer. Can adopt the buffer solution of different molar concentrations, for example any molar concentration in the about 100mM of about 0.1mM-, the about 50mM of about 1mM-, the about 40mM of about 5mM-, the about 30mM of about 10mM-or the about 25mM of about 15mM-or these scopes. In some embodiments, adopt the acetate buffer of about 20mM molar concentration.

In another embodiment, contain PDGF solution by freezing PDGF being dissolved in form in the water, wherein before dissolving, allow PDGF freeze-drying from suitable buffer solution.

According to embodiment of the present invention, contain PDGF solution and can have pH between about 3.0-about 8.0. In one embodiment, contain the pH that PDGF solution has following scope: about 5.0-is about 8.0, between about 5.5-about 7.0 or the about 5.5-about 6.5 or any value in these scopes. In some embodiments, it is compatible to contain long-time stability and the effect of the BA agent that the pH of PDGF solution can need with PDGF or any other phase. PDGF is more stable in sour environment. Therefore, one embodiment of the invention comprises that the acidity of PDGF solution stores preparation. According to this embodiment, PDGF solution preferably has the pH of about 3.0-about 7.0 or about 4.0-about 6.5. Yet the BA of PDGF is best in the solution with neutral pH scope. Therefore, in yet another embodiment, the present invention includes the neutral pH preparation of PDGF solution. According to this embodiment, PDGF solution has preferably that about 5.0-is about 8.0, the pH of about 5.5-about 7.0 or about 5.5-about 6.5. According to the inventive method, acid PDGF solution is allocated as the neutral pH composition again, wherein subsequently described composition is used for the treatment of bone and promotes bone growth and/or healing. According to certain embodiments of the invention, the PDGF that uses in described solution is rh-PDGF-BB. In yet another embodiment of the invention, can change contain PDGF solution pH so that the binding kinetics optimization of PDGF and matrix substrate or attachment. If need, make pH and the adjacent material balance of described material, otherwise the PDGF of combination may change easily.

In some embodiments, the pH that contains PDGF solution can be controlled by buffer solution described herein. Different protein has the stable pH scope of different maintenances. The stability of protein is mainly reflected by the electric charge on isoelectric point and the protein. The pH scope can affect the conformational structure of protein and protein to proteolysis, hydrolysis, oxidation and cause the structure of protein and/or the sensitiveness of other process that BA changes.

In some embodiments, contain PDGF solution and can further comprise other component, for example other bioactivator. In other embodiments, contain that PDGF solution can further comprise cell culture medium, other helps stabilize proteins, antiseptic, protease inhibitors [for example ethylenediamine tetra-acetic acid (EDTA), ethylene glycol bis (beta-aminoethyl ether)-N such as albumin etc., N, N ', N '-tetraacethyl (EGTA), aprotinin, EACA (EACA) etc.] and/or other growth factor, for example fibroblast growth factor (FGF), EGF (EGF), TGF (TGF), keratinocyte growth factor (KGF), IGF (IGF), HGF (HGF), BMP (BMP) or other PDGF comprise the composition of PDGF-AA, PDGF-BB, PDGF-AB, PDGF-CC and/or PDGF-DD.

Except containing PDGF solution, the present composition also is included in the biocompatible matrix that wherein disposes PDGF solution, and also can comprise biocompatible adhesive adding or do not add under the biocompatible matrix.

Biocompatible matrix

Bone holder material

According to embodiment of the present invention, biocompatible matrix comprises bone holder material. It should be understood that term bone holder material and bone replace agent to be used interchangeably in this application. Bone holder material provides framework or support for the growth of new bone and tissue. In some embodiments, bone holder material has the hole of multi-direction and interconnected various diameters. In some embodiments, bone holder material also comprises a plurality of shrinkage pools and non-interconnected hole except interconnected hole. In some embodiments, bone holder material can permanent or temporary replacement bone. Bone holder material can be retained in the health after implanting, or it can be absorbed by health and be replaced by bone.

In some embodiments, bone holder material comprises at least a calcium phosphate.In other embodiments, bone holder material can comprise multiple calcium phosphate.In embodiment of the present invention, the calcium phosphate that is suitable for use as bone holder material has the calcium phosphorus atoms ratio between 0.5-2.0.In some embodiments, bone holder material comprises allograft, for example DFDBA, FDBA or microgranule DBM.In some embodiments, bone holder material comprises mineralising bone allograft, mineralising bone, mineralising Deproteinization xenograft or demineralized bone.

The limiting examples that is suitable for use as the calcium phosphate of bone holder material comprises: amorphous calcium phosphate, one water one-lime phosphate (MCPM), anhydrous phosphoric acid one calcium (MCPA), dicalcium phosphate dihydrate (DCPD), anhydrous dicalcium phosphate (DCPA), OCP (OCP), type alpha tricalcium phosphate, β-TCP, hydroxyapatite (OHAp), the hydroxyapatite that crystallinity is low, tetracalcium phosphate (TTCP), ten phosphoric acid, seven calcium, calcium metaphosphate, calcium pyrophosphate dihydrate, calcium pyrophosphate, carbon calcium phosphate, hydroxyapatite or their derivant or mixture.

In some embodiments, bone holder material comprises polymeric material.In some embodiments, the polymerization support comprises: collagen protein, polylactic acid, poly-(L-lactide), poly-(D, the L-lactide), polyglycolic acid, poly-(L-lactide-co-glycolide), poly-(L-lactide-be total to-D the L-lactide), polyacrylate, polymethacrylates, polymethyl methacrylate, chitosan or their combination or derivant.

In some embodiments, bone holder material comprises loose structure.Porous is the characteristic that needs institute's phase, because it promotes cell migration and be penetrated in the timbering material, so that the cell of this infiltration can be secreted born of the same parents' bone matrix outward.Porous also provides passage for vascularization.Porous also is provided for strengthening absorption again and active substance discharges and the high surface of increase cell and substrate interphase interaction.In some embodiments, bone holder material can form a certain size and shape before use.In some embodiments, bone holder material can provide with the shape that is suitable for implanting.

According to some embodiment, the porous bone holder material can comprise the hole that has between the diameter of the about 1mm of about 1 μ m-.In one embodiment, bone holder material comprises the macropore that has between about about 1mm of 1 μ m-or larger diameter.In yet another embodiment, bone holder material comprises the mesopore that has between the diameter of the about 100 μ m of about 10 μ m-.In an embodiment again, bone holder material comprises the micropore that has less than the diameter of about 10 μ m.Embodiment of the present invention contains the bone holder material that comprises macropore, mesopore and micropore or its any combination.

In one embodiment, the porous bone holder material has and is higher than about 25% or be higher than about 40% porosity (porosity).In another embodiment, the porous bone holder material have be higher than about 50%, be higher than about 60%, be higher than about 65%, be higher than about 70%, be higher than about 80% or be higher than about 85% porosity.In yet another embodiment, the porous bone holder material has and is higher than about 90% porosity.In some embodiments, the porous bone holder material comprises the porosity that the promotion cell migration is gone into timbering material.

In some embodiments, bone holder material comprises multiple granule.For example, bone holder material can comprise multiple calcium phosphate granules.In some embodiments, the bone holder material granule can show individually that this paper is provided for any aperture and the porosity of bone holder material.In other embodiments, the bone holder material granule can form association (association) and has this paper with generation and be provided for any aperture of bone holder material or the substrate of porosity.

Bone support granule can be mm, μ m or submicron (nm) size.In one embodiment, bone support granule has the average diameter between the about 5mm of about 1 μ m-.In other embodiments, granule has the average diameter between the about 2mm of about 1mm-, the about 3mm of about 1mm-or the about 750 μ m of about 250 μ m-.In another embodiment, bone support granule has the average diameter between the about 300 μ m of about 100 μ m-.In yet another embodiment, bone support granule has the average diameter between the about 300 μ m of about 75 μ m-.In other embodiments, bone support granule has less than about 25 μ m, less than about 1 μ m or less than the average diameter of about 1mm.In some embodiments, the support granule has the average diameter between about about 5mm of 100 μ m-or the about 3mm of about 100 μ m-.In other embodiments, bone support granule has the average diameter between the about 2mm of about 250 μ m-, about about 1mm of 250 μ m-or the about 3mm of about 200 μ m-.Granule also can be the about 1 μ m of about 1nm-, less than about 500nm or less than the scope of about 250nm.

According to some embodiment, can provide bone holder material with the shape (for example spheroid, cylinder or bulk) that is suitable for implanting.In other embodiments, bone holder material be moldable, can extrude and/or injectable.Moldable, can extrude and can promote effectively to be placed in the present composition in the vertebral body with injectable bone holder material and on every side.In some embodiments, bone holder material easily flows.In some embodiments, can runny bone holder material be administered to vertebral body by syringe and pin or sleeve pipe.In some embodiments, bone holder material hardening in vivo.

In some embodiments, bone holder material can absorb by biology again.In one embodiment, bone holder material can be implanted in vivo and be resorbed into few 30%, 40%, 50%, 60%, 70%, 75% or 90% in back 1 year.In another embodiment, bone holder material can implant in vivo 1,3,6,9,12 or 18 months in be resorbed into few 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75% or 90%.In some embodiments, bone holder material implant in vivo 1,3,6,9,12 or 18 months in absorb to be surpassed 90% again.Can biology again absorbability decide on following factor: the characteristic of (1) host material (being its chemical composition, physical arrangement and size); (2) the substrate intravital position of body of settling; (3) amount of employed host material; (4) patient's metabolism state (diabetes/non-diabetic, osteoporosis, smoking, old-age group, use steroid or the like); (5) degree of injury of being treated and/or type; (6) remove the use of extramatrical other material, for example other bone anabolism factor, the catabolism factor and the anti-catabolism factor.

Comprise bata-tricalcium phosphate (the bone support of β-TCP)

Bone holder material as biocompatible matrix can comprise β-TCP.According to some embodiment, β-TCP can comprise the loose structure in the hole with multi-direction and interconnected different-diameter.In some embodiments, β-TCP also comprises the shrinkage pool and the non-interconnected hole of a plurality of different-diameters except that interconnected hole.In one embodiment, the loose structure of β-TCP comprises the macropore that has between about about 1mm of 100 μ m-or larger diameter, has between the mesopore of the about 100 μ m diameters of about 10 μ m-and has micropore less than about 10 μ m diameters.The macropore of β-TCP and micropore can promote bone to induce (osteoinduction) and bone conduction (osteoconduction), and but macropore, mesopore and micropore make the nutrition of flow liquid connected sum to transport, to support everywhere that at β-TCP biocompatible matrix osteanagenesis is long.

In some embodiments, comprising under the loose structure, β-TCP can have and is higher than 25% or be higher than about 40% porosity.In other embodiments, β-TCP can have be higher than 50%, be higher than about 60%, be higher than about 65%, be higher than about 70%, be higher than about 75%, be higher than about 80% or be higher than about 85% porosity.In yet another embodiment, β-TCP can have and is higher than 90% porosity.In some embodiments, β-TCP can have the porosity that the promotion cell migration is gone into β-TCP.

In some embodiments, β-TCP bone holder material comprises β-TCP granule.In some embodiments, β-TCP granule can show individually that this paper is provided for any aperture, pore structure and the porous of timbering material.

In one embodiment, β-TCP granule has the average diameter between the about 5mm of about 1 μ m-.In other embodiments, β-TCP granule has between following average diameter: the about 2mm of about 1mm-, the about 3mm of about 1mm-, the about 5mm of about 100 μ m-, the about 3mm of about 100 μ m-, the about 2mm of about 250 μ m-, the about 750 μ m of about 250 μ m-, the about 1mm of about 250 μ m-, about about 2mm of 250 μ m-or the about 3mm of about 200 μ m-.In another embodiment, β-TCP granule has the average diameter between the about 300 μ m of about 100 μ m-.In some embodiments, β-TCP granule has the average diameter between the about 300 μ m of about 75 μ m-.In some embodiments, β-TCP granule has less than about 25 μ m, less than about 1 μ m or less than the average diameter of about 1mm.In some embodiments, β-TCP granule has the average diameter between the about 1 μ m of about 1nm-.In yet another embodiment, β-TCP granule has less than about 500nm or less than the average diameter of about 250nm.

In some embodiments, can provide bone to comprise the biocompatible matrix of β-TCP bone holder material with the shape (for example spheroid, cylinder or bulk) that is suitable for implanting.In other embodiments, that β-TCP bone holder material can be is moldable, can extrude and/or can flow, and helps thus substrate is administered to vertebral body.Can use runny substrate by syringe, pipe (tube), sleeve pipe or spatula (spatula).

In some embodiment, β-TCP bone holder material can absorb by biology again.In one embodiment, β-TCP bone holder material can be implanted in vivo and be resorbed into few 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80% or 85% in back 1 year.In another embodiment, β-TCP bone holder material can be implanted in vivo to be absorbed in back 1 year again and surpass 90%.

Bone holder material and biocompatible adhesive

In another embodiment, biocompatible matrix comprises bone holder material and biocompatible adhesive.Bone holder material in the biocompatible matrix embodiment further comprises and meets biocompatible adhesive provided above.

According to some embodiment, biocompatible adhesive can comprise can operate with adherent material between the material that helps to make up.For example, biocompatible adhesive can promote bonding between the bone holder material granule in forming the biocompatible matrix process.In some embodiments, same material can be used as timbering material.For example, in some embodiments, polymeric material described herein (for example collagen protein and chitosan) can be used as timbering material and binding agent simultaneously.

In some embodiments, biocompatible adhesive can comprise: collagen protein, polysaccharide, nucleic acid, carbohydrate, protein, polypeptide, poly-('alpha '-hydroxy acids), poly-(lactone), poly-(aminoacid), poly-(anhydride), polyurethane, poly-(ortho esters), poly-(anhydride-altogether-imines), poly-(orthocarbonic ester), poly-(Alpha-hydroxy alkanoic acid ester), poly-(dioxanone), poly-(phosphate ester), polylactic acid, gather (L-lactide) (PLLA), poly-(D, the L-lactide) (PDLLA), poly-Acetic acid, hydroxy-, bimol. cyclic ester (PGA), poly-(lactide-co-glycolide (PLGA), poly-(the L-lactide-altogether-D, the L-lactide), poly-(D, the L-lactide-altogether-carbonic acid 1, the inferior propyl ester of 3-), polyglycolic acid, poly butyric ester (PHB), poly-(6-caprolactone), poly-(δ-Wu Neizhi), poly-(gamma-butyrolacton), poly-(caprolactone), polyacrylic acid, polycarboxylic acids, poly-(allylamine hydrochloride), poly-(diallyl dimethyl ammonium chloride), poly-(aziridine), poly-fumaric acid propylene diester, polyvinyl alcohol, polyvinylpyrrolidone, polyethylene, polymethyl methacrylate, carbon fibre, poly-(ethylene glycol), poly-(oxirane), poly-(vinyl alcohol), poly-(vinylpyrrolidone), poly-(ethyl oxazoline), poly-(oxirane)-altogether-poly-(expoxy propane) block copolymer, poly-(PETP) polyamide and their copolymer and mixture.

In other embodiments, biocompatible adhesive can comprise alginic acid, Radix Acaciae senegalis, guar gum, xanthan gum, gelatin, chitin, chitosan, chitosan acetate (chitosanacetate), chitosan lactate (chitosan lactate), chondroitin sulfate, N, the O-carboxymethyl chitosan, glucosan (alpha-cyclodextrin for example, beta-schardinger dextrin-, gamma-cyclodextrin or sodium dextran sulfate), Fibrin Glue, lecithin, ovum Phosphorylcholine derivant, glycerol, hyaluronic acid, hyaluronate sodium, cellulose (methylcellulose for example, carboxymethyl cellulose, hydroxypropyl emthylcellulose or hydroxy ethyl cellulose), glycosamine, Dan Baijutang, starch (for example hetastarch or soluble starch), lactic acid, pluronic acid, sodium glycerophosphate, glycogen, keratin, fibroin (silk) and their derivant and mixture.

In some embodiments, biocompatible adhesive is water miscible.Water-soluble binder can dissolve from biocompatible matrix after it is implanted soon, thereby introduces macroporosity (macroporosity) in biocompatible matrix.Macroporosity described herein can increase the bone conductibility of embedded material by increasing passage, thereby has changed the osteoclast and the osteoblastic activity of implantation site.

In some embodiments, biocompatible adhesive can be present in the biocompatible matrix with the amount between about 5% weight-Yue 50% weight of substrate.In other embodiments, biocompatible adhesive can exist with the amount between about 10% weight-Yue 40% weight of biocompatible matrix.In another embodiment, biocompatible adhesive can exist with the amount between about 15% weight-Yue 35% weight of biocompatible matrix.In yet another embodiment, biocompatible adhesive can exist with the amount of about 20% weight of biocompatible matrix.In another embodiment, biocompatible adhesive can be to be present in the biocompatible matrix greater than about 50% weight of substrate or the amount of 60% weight.In one embodiment, biocompatible adhesive can be present in the biocompatible matrix with the amount of about 99% weight of accounting for substrate at the most.

According to some embodiment, the biocompatible matrix that comprises bone holder material and biocompatible adhesive can be can flow, moldable and/or can extrude.In described embodiment, biocompatible matrix can be in the pasty state or mud shape form.In one embodiment, in the pasty state or the biocompatible matrix of mud shape form can comprise by biocompatible adhesive and make it mutual adherent bone holder material granule.

Can with in the pasty state or the biocompatible matrix of mud shape form be compression molded into required implantation shape, maybe it can be compression molded into the profile of implantation site.In one embodiment, available syringe or sleeve pipe will be in the pasty state or the biocompatible matrix of mud shape form be expelled in the implantation site.

In some embodiments, in the pasty state or the not hardening of biocompatible matrix of mud shape form, after implantation, still easily flow and moldable.In other embodiments, stick with paste or mud implantation back solidifiable, thus the flowability and the moldable property of reduction substrate.

In some embodiments; can also provide the biocompatible matrix that comprises bone holder material and biocompatible adhesive with reservation shape; described reservation shape comprises the shape that bulk, spheroid or cylinder or any institute's phase need, for example by mould or use the shape that the site limits.

In some embodiments, the biocompatible matrix that comprises bone holder material and biocompatible adhesive is can be biological resorbent.In described embodiment, to implant in vivo in 1 year, biocompatible matrix can be absorbed again.In another embodiment, implant in vivo 1,3,6 or 9 months in, the biocompatible matrix that comprises bone holder material and biocompatible adhesive can be absorbed again.In some embodiments, implant in vivo in 1,3 or 6 year, the biocompatible matrix that comprises bone holder material and biocompatible adhesive can be absorbed again.Can biology again absorbability decide on following factor: the characteristic of (1) host material (being its chemical composition, physical arrangement and size); (2) the substrate intravital position of body of settling; (3) amount of employed host material; (4) patient's metabolism state (diabetes/non-diabetic, osteoporosis, smoking, old-age group, use steroid or the like); (5) degree of injury of being treated and/or type; (6) remove the use of extramatrical other material, for example other bone anabolism factor, the catabolism factor and the anti-catabolism factor.

The biocompatible matrix that comprises β-TCP and collagen protein

In some embodiments, biocompatible matrix can comprise β-TCP bone holder material and biocompatibility collagen protein binder.Those that β-TCP bone holder material with collagen protein bonding agent combination meets above to be provided are provided.

In some embodiments, collagen protein bonding agent can comprise the collagen protein of any kind, comprises I type, II type and III collagen type.In one embodiment, collagen protein bonding agent comprises the collagen protein mixture, for example the mixture of I type, II type and III collagen type.In other embodiments, collagen protein bonding agent is solvable under physiological condition.Can adopt the collagen protein that is present in other type in osseous tissue or the flesh skeletal tissue.The present invention can use reorganization, synthetic and naturally occurring collagen protein form.

According to some embodiment, biocompatible matrix can comprise with collagen protein bonding agent makes it adherent a plurality of β-TCP granule mutually.In some embodiments, has average diameter with the β-TCP granule of collagen protein bonding agent combination between the about 5mm of about 1 μ m-.In other embodiments, β-TCP granule has between following average diameter: the about 2mm of about 1mm-, the about 3mm of about 1mm-, the about 5mm of about 100 μ m-, the about 3mm of about 100 μ m-, the about 2mm of about 250 μ m-, the about 750 μ m of about 250 μ m-, the about 1mm of about 250 μ m-, about about 2mm of 250 μ m-or the about 3mm of about 200 μ m-.In another embodiment, β-TCP granule has the average diameter between the about 300 μ m of about 100 μ m-.In some embodiments, β-TCP granule has the average diameter between the about 300 μ m of about 75 μ m-.In some embodiments, β-TCP granule has less than about 25 μ m, less than about 1 μ m or less than the average diameter of about 1mm.In some embodiments, β-TCP granule has the average diameter between the about 1 μ m of about 1nm-.In yet another embodiment, β-TCP granule has less than about 500nm or less than the average diameter of about 250nm.

In some embodiments, β-TCP granule can make it bonded to each other by collagen protein bonding agent, so that produce the biocompatible matrix with loose structure.In some embodiments, the loose structure that comprises the biocompatible matrix of β-TCP granule and collagen protein bonding agent shows the hole of multi-direction and interconnected different-diameter.In some embodiments, biocompatible matrix also comprises the shrinkage pool and the non-interconnected hole of a plurality of different-diameters except that interconnected hole.

In some embodiments, the biocompatible matrix that comprises β-TCP granule and collagen protein bonding agent can comprise the hole that has between the about 1mm diameter of about 1 μ m-.The biocompatible matrix that comprises β-TCP granule and collagen protein bonding agent can comprise the macropore that has between about about 1mm of 100 μ m-or larger diameter, have between the mesopore of about 10 μ m-100 μ m diameters and have micropore less than about 10 μ m diameters.

The biocompatible matrix that comprises β-TCP granule and collagen protein bonding agent can have and is higher than about 25% or be higher than about 40% porosity.In another embodiment, biocompatible matrix can have be higher than about 50%, be higher than about 65%, be higher than about 70%, be higher than about 75%, be higher than about 80% or be higher than about 85% porosity.In yet another embodiment, biocompatible matrix can have and is higher than about 90% porosity.In some embodiments, biocompatible matrix can have and promotes cell migration to go into the porosity in the substrate.

In some embodiments, β-TCP granule can show any aperture, pore structure and the porosity of the biocompatible matrix of the β of comprising provided herein-TCP and collagen protein bonding agent individually.

In some embodiments, comprise the particulate biocompatible matrix of β-TCP and can comprise collagen protein bonding agent with amount between about 5% weight-Yue 50% weight of substrate.In other embodiments, collagen protein bonding agent can exist with the amount between about 10% weight-Yue 40% weight of biocompatible matrix.In another embodiment, collagen protein bonding agent can exist with the amount between about 15% weight-Yue 35% weight of biocompatible matrix.In yet another embodiment, collagen protein bonding agent can exist with the amount of about 20% weight of accounting for biocompatible matrix.

According to some embodiment, the biocompatible matrix that comprises β-TCP granule and collagen protein bonding agent can be can flow, moldable and/or can extrude.In described embodiment, biocompatible matrix can be in the pasty state or mud shape form.Paste or mud can be compression molded into required implantation shape, can be the profile of implantation site with its pressing mold maybe.In one embodiment, available syringe or sleeve pipe will be in the pasty state or the biocompatible matrix that comprises β-TCP granule and collagen protein bonding agent of mud shape form be expelled in the implantation site.

In some embodiments, in the pasty state or the biocompatible matrix that comprises β-TCP granule and collagen protein bonding agent of mud shape form can when implanting, keep and easily flow and moldable form.In other embodiments, stick with paste or mud can hardening after implantation, thereby reduce the flowability and the moldable property of substrate.

In some embodiments, can provide the biocompatible matrix that comprises β-TCP granule and collagen protein bonding agent with reservation shape (for example bulk, spheroid or cylinder).

The biocompatible matrix that comprises β-TCP granule and collagen protein bonding agent can be absorbed again.In one embodiment, implanted in vivo back 1 year, the biocompatible matrix that comprises β-TCP granule and collagen protein bonding agent can be resorbed into few 75%.In another embodiment, implant 1 year in vivo after, the biocompatible matrix that comprises β-TCP granule and collagen protein bonding agent can absorb be surpassed 90% again.

According to embodiment described herein, contain PDGF solution and can be configured in the compositions that is used for the treatment of spine structure in the biocompatible matrix with generation.

In some embodiments, the compositions that comprises the PDGF solution that is configured in the biocompatible matrix that is used for promoting bone formation and the prevention of vertebral body or reduces the vertebral compression fractures probability as described herein further comprises at least a contrast agent.According to some embodiment, contrast agent comprises cation contrast agent, anion contrast agent, non-ionic contrast medium or its mixture.In some embodiments, contrast agent comprises the radiopaque contrast agent.In some embodiments, the radiopaque contrast agent comprises and contains iodine compound that it comprises (S)-N, N '-two [2-hydroxyl-1-(methylol)-ethyl]-2,4,6-three iodo-5-lactoyl amido isophthaloyl amine (iopamidol) and derivants thereof.

The configuration of PDGF solution in biocompatible matrix

The invention provides the preparation of compositions method that is used for promoting bone formation and the prevention of vertebral body or reduces compression fracture of vertabral body (comprising the secondary vertebral fracture) probability.In one embodiment, be used to prepare described method for compositions and comprise: provide to contain PDGF solution, biocompatible matrix is provided and with described solution allocation in described biocompatible matrix.The PDGF solution and the biocompatible matrix that are applicable to combination meet those PDGF solution and biocompatible matrixes mentioned above.

In some embodiments, can be by allowing biocompatible matrix soak in PDGF solution the PDGF solution allocation in biocompatible matrix.In another embodiment, can be by going into the PDGF injection of solution in the biocompatible matrix the PDGF solution allocation in biocompatible matrix.In some embodiments, injection PDGF solution can comprise with the PDGF solution allocation in syringe and with the PDGF injection of solution in the biocompatible matrix to soak into biocompatible matrix.

In some embodiments, PDGF is adsorbed in the hole of biocompatible matrix.In some embodiments, PDGF is adsorbed on one or more surfaces of biocompatible matrix, comprises the surface in the hole of biocompatible matrix.

According to some embodiment, before accepting PDGF solution, biocompatible matrix can be reservation shape, and is for example block or cylindrical.After accepting PDGF solution, biocompatible matrix can be easily and flows, can extrude and/or injectable pasty state or mud shape form.In other embodiments, before accepting to contain PDGF solution, biocompatible matrix can show runny pasty state or mud shape form.The easy of compositions that comprises the PDGF solution that is configured in the biocompatible matrix flowed, can extrude and/or injectable pasty state or mud shape form, it is favourable using in the method that the present invention uses, because available syringe and/or sleeve pipe are administered to vertebral body with it.

In some embodiments, the method for compositions that preparation is used for promoting bone formation and the prevention of vertebral body or reduces the compression fracture of vertabral body probability further comprises to be provided: at least a contrast agent, and described at least a contrast agent is configured in the biocompatible matrix.In some embodiments, at least a contrast agent is configured in the biocompatible matrix comprises: allow described at least a contrast agent combine and be injected into described biocompatible matrix with the PDGF/ contrast agent solution with PDGF solution.

In another embodiment, at least a contrast agent is configured in the biocompatible matrix comprises: allow described at least a contrast agent combine, and biocompatible matrix is soaked in the PDGF/ contrast agent solution with PDGF solution.Perhaps, in some embodiments, contrast agent is independent of PDGF solution and is configured in the biocompatible matrix.

According to certain embodiments of the invention, contrast agent helps the present composition settled or is applied in the vertebral body and on every side.According to some embodiment, contrast agent comprises: cation contrast agent, anion contrast agent, non-ionic contrast medium or its mixture.In some embodiments, contrast agent comprises the radiopaque contrast agent.In some embodiments, the radiopaque contrast agent comprises and contains iodine compound that it comprises (S)-N, N '-two [2-hydroxyl-1-(methylol)-ethyl]-2,4,6-three iodo-5-lactoyl amido isophthaloyl amine (iopamidol) and derivants thereof.

The compositions that further comprises bioactivator

According to some embodiment, the present composition also can further comprise one or more bioactivators except that PDGF.The bioactivator that can be incorporated in the present composition also can comprise except that PDGF: organic molecule, inorganic material, protein, peptide, nucleic acid (for example gene, genetic fragment, small molecule disturbance ribonucleic acid [si-RNA], gene regulation sequence, nuclear factor and antisense molecule), nucleoprotein, polysaccharide (for example heparin), glycoprotein and lipoprotein.The limiting examples that can be incorporated into the bioactive compound in the present composition for example comprises: anticarcinogen, antibiotic, analgesic, anti-inflammatory agent, immunosuppressant, enzyme inhibitor, hydryllin, hormone, muscle relaxant, prostaglandin, trophic factors, osteoinductive protein, somatomedin and vaccine, they are disclosed in U.S. Provisional Patent Application serial number 11/159,533 (publication No.: 20060084602).In some embodiments, the biologically active cpds that can be incorporated in the present composition comprises: bone stimulating factor (for example insulin like growth factor), fibroblast growth factor or other PDGF.According to other embodiment, the biologically active cpds that can be incorporated in the present composition preferably includes: the antibody antagonist (for example os osseum element (sclerostin), DKK, solubility Wnt receptor) and/or the parathyroid hormone of bone-inducing factor and bone stimulating factor (for example bone morphogenetic protein(BMP) (BMP), BMP analogies, calcitonin or calcitonin mimetic thing), inhibin, inhibin derivant, fibroblast growth factor, insulin like growth factor, growth and differentiation factor, micromolecule or Wnt blocker.In some embodiments, the factor also comprises protease inhibitor and the osteoporosis therapy that reduces bone resorption, comprises the antibody of diphosphate, teriparatide (teriparadide) and NF-kB part (RANK) part activator receptor.

The standard scheme and the plan of sending additional bioactive agents are known in the art.Can additional bioactive agents be incorporated in the present composition with the medicament that allows to send suitable dosage amount to implantation site.Under most of situation, known and be applicable to that the guilding principle of the medicament of concrete discussion determines dosage with the professional.The amount that is included in the additional bioactive agents in the present composition can be depending on following variable: the overall health of the type of disease and degree, particular patient, the dosage form of bioactivator, release dynamics and biocompatible matrix can biology absorbability again.For any specific additional bioactive agents, dosage and administration frequency are optimized in the available standards clinical trial.

According to some embodiment, the present composition can further comprise the other bone grafting material that adds with PDGF, comprises autologous bone marrow, autologous platelet extract, allograft, synthetic extracellular matrix material, xenograft and their derivant.

The method of treatment vertebral body

In some embodiments, the invention provides the osteoplastic method that is used for promoting vertebral body, this method comprises: the compositions that comprises the PDGF solution that is configured in the biocompatible matrix is provided and described compositions is administered at least one vertebral body.In some embodiments, described compositions can be administered to a plurality of vertebral bodys.In some embodiments, applying said compositions comprises with described compositions and is injected at least one vertebral body.In some embodiments, the present composition is injected in the spongy bone of vertebral body.In some embodiments, vertebral body comprises body of thoracic vertebre, lumbar vertebra body or its combination.In some embodiments, vertebral body comprises body of cervical vertebra, coccyx vertebral body, rumpbone or its combination.

On the other hand, the invention provides the method that is used to prevent or reduce the probability of the vertebral compression fractures (comprising the secondary vertebral compression fractures) that causes by the reinforcing vertebra.According to embodiment of the present invention, prevention or reduction vertebral compression fractures probability comprise: the compositions that comprises PDGF solution that is configured in the biocompatible matrix is provided, and said composition is administered at least one vertebral body.In some embodiments, described compositions being administered at least one vertebral body comprises described compositions is expelled at least one vertebral body.

In some embodiments, in that first vertebral body is implemented vertebroplasty or kyphoplasty postoperative, the present composition is administered to second vertebral body.In some embodiments, described second vertebral body is adjacent with described first vertebral body.In other embodiments, described second vertebral body is not adjacent with described first vertebral body.In yet another embodiment, in that first vertebral body is implemented vertebroplasty or kyphoplasty postoperative, the present composition is administered to the 3rd vertebral body.In some embodiments, described the 3rd vertebral body is adjacent with described first vertebral body.In other embodiments, described the 3rd vertebral body is not adjacent with described first vertebral body.Embodiment of the present invention is encompassed in addition implements vertebroplasty or kyphoplasty postoperative to first vertebral body, and the compositions that this paper provided is administered to a plurality of vertebral bodys, comprises high-risk vertebral body.It should be understood that, first, second and the 3rd vertebral body used herein are not meant any ad-hoc location of spinal column, all types of vertebral bodys be can be administered to because be used to suppress the method for vertebral compression fractures (comprising the second-compressed fracture), body of thoracic vertebre, lumbar vertebra body, body of cervical vertebra, coccyx vertebral body and rumpbone comprised.

In some embodiments, the method that is used for promoting bone formation and the prevention of vertebral body or reduces the probability of compression fracture of vertabral body further comprises: provide the other at least a pharmaceutical composition except the compositions that comprises the PDGF solution that is configured in the biocompatible matrix, and part and/or whole body give described at least a pharmaceutical composition.In some embodiments, described at least a pharmaceutical composition comprises: vitamin (for example vitamin D3), calcium complement agent or any osteoclast inhibitor well known by persons skilled in the art (comprising diphosphate).In some embodiments, the described at least a pharmaceutical composition of topical administration.In described embodiment, described at least a pharmaceutical composition can be incorporated in the biocompatible matrix, or be configured in the vertebral body in addition and on every side.In other embodiments, give the patient with described at least a pharmaceutical composition whole body.For example, in one embodiment, give the patient with described at least a pharmaceutical composition per os.In another embodiment, give the patient with described at least a pharmaceutical composition intravenous.

Following examples are used for further illustrating the present invention, yet it does not constitute any limitation of the invention simultaneously.In contrast, should be expressly understood that after having read this paper explanation, those skilled in the art can own proposition depart from different embodiments, modification and their equivalents of spirit of the present invention.

Embodiment 1

The preparation of compositions that comprises PDGF solution and biocompatible matrix

Preparation comprises the compositions of PDGF solution and biocompatible matrix in accordance with the following methods.

Obtain the biocompatible matrix of a collection of preweighted β of comprising-TCP and collagen protein.Described β-TCP comprises the β-TCP granule that has between the about 300 μ m average diameters of about 100 μ m-.Solubility cattle type i collagen protein binder with about 20% percentage by weight is allocated described β-TCP granule.Described β-TCP/ collagen protein biocompatible matrix can available from Kensey Nash (Exton, Pennsylvania).

Acquisition comprises the solution of rhPDGF-BB.Concentration is that rhPDGF-BB liquid storage (being lot number QA2217) in the sodium acetate buffer of 10mg/ml is available from Novartis Corporation.Described rhPDGF-BB is produced in yeast expression system by Chiron Corporation, and is used for product REGRANEX (Johnson ﹠amp; Johnson) and the rhPDGF-BB among the GEM 21S (BioMimeticTherapeutics) derive from same factory, REGRANEX and GEM 21S been have have been checked and approved by FDA (Food and Drug Adminstration) and have been used for the people.This rhPDGF-BB is is also checked and approved by European Union and Canada and is used for the people.Described rhPDGF-BB solution is diluted to 0.3mg/ml in acetate buffer.According to embodiment of the present invention, rhPDGF-BB solution can be diluted to the concentration of any needs, comprises 1.0mg/ml.

Use the rhPDGF-BB solution of about 3ml and the β-TCP/ collagen protein biocompatible matrix ratio of about 1g dry weight to prepare compositions.In preparation compositions process, with on the rhPDGF-BB injection of solution biocompatible matrix, allow the compositions that obtains be mixed into pasty state with syringe, place syringe to be used for being expelled to subsequently vertebral body.

Embodiment 2

The method that suppresses the secondary vertebral compression fractures

Experimental design and general introduction

This expection at random, contrast, the clinical trial of single center be the effect that is used for assessing the second-compressed fracture of the high-risk vertebral body (FFVB) when comprising the compositions that is configured in PDGF solution in the tricalcium phosphate substrate and being used for being suppressed at vertebral compression fractures kyphoplasty art.Between with the vertebral body of bata-tricalcium phosphate+rhPDGF-BB combination treatment and untreated vertebral body, compare.This research is preliminary clinical trial (pilot clinical trial), to support β-TCP+rh-PDGF-BB by improving evidence or the principle that the HVB bone formation prevented or reduced secondary vertebral compression fractures probability.

This research is carried out in amounting to 10 patients that need prophylactic treatment HVB during in the kyphoplasty art.Screen potential patient to determine whether they satisfy into group standard and exclusion standard.If reached all group standards of going into, then invite this potential patient to participate in clinical trial.On the examination log record all be considered and enroll the patient of this research, and the reason got rid of of record.

All patients accepted the kyphoplasty art, and did not have and the adjacent VCF symptom of two vertebral bodys of being treated in this research.If the surgeon determines that when operation fracture does not satisfy fracture and goes into the group standard or have other fracture of not treating in this therapeutic scheme, then be subjected to treatment target can not be incorporated into this research.

In this research, register and treat 10 patients altogether.With the 3.0ml β that presses this paper embodiment 1 preparation-injection of TCP+0.3mg/ml rhPDGF-BB compositions and the first adjacent vertebral body of each patient's kyphoplasty art vertebral body.Do not treat second vertebral body adjacent also in contrast with kyphoplasty art vertebral body.The vertebral body of being treated can be positioned at head or the afterbody of kyphoplasty art vertebral body, and it is determined at random.

Press standard scheme treatment patient, and follow up a case by regular visits to its kyphoplasty art/vertebroplasty situation.By the surgeon at 7-14 days with 6,12,24 and 52 weeks each patient was being carried out clinical, x-ray imaging and QCT art (QCT) is checked.Write down the use of all nonprescription drugss and prescription drugs.By not knowing that the independently radiotherapy Shi Jinhang QCT that the patient treatment grouping is arranged analyzes with the evaluation bone density.Record is also analyzed these measurement results.

All post-operative complication all are recorded in the appropriate cases report form with the adverse events (device-related adverseevent) relevant with apparatus.If the curee suffers from VCF subsequently during studying, or accept other operation because of serious adverse events, or remove research apparatus (investigational device), the safety of then monitoring this curee is up to the research end.Again the curee who undergos surgery and/or remove fracture fixation apparatus (fracture fixation hardware) is required to check explant with meaning histology's purpose.Duration of test at 12 months is monitored all patients, and any requirement is withdrawed from research or the person of being studied requires the curee withdraw from research to require to provide the reason that gives up the study of.Table 1 provides the timetable summary of this research.

The investigation of table 1-search time table

In the 1st the prescription on individual diagnosis examination prescription on individual diagnosis ↓ operation 21 days	In the 2nd the prescription on individual diagnosis operation prescription on individual diagnosis ↓ examination 21 days	The 3rd prescription on individual diagnosis follow up a case by regular visits to behind Tx ↓	The 4th goes to a doctor and to follow up a case by regular visits to behind Tx ↓	The 5th goes to a doctor and to follow up a case by regular visits to behind Tx ↓	The 6th prescription on individual diagnosis follow up a case by regular visits to behind Tx ↓	The 7th prescription on individual diagnosis follow up a case by regular visits to behind Tx ↓
								The 0th day	7-14 days	The 6th week ± 3 day	The 12nd week ± 7 day	The 24th week ± 7 day	The 52nd week ± 14 day

First terminal point be after operation 12 weeks by the bone density of QCT scanning survey.Second terminal point comprises curee's pain and appraisal of life quality.

Operation plan

After the patient who satisfies group and exclusion standard is incorporated into research, it is carried out following operation plan.

With standard mode the patient is sent into operating room (OR), use standard method to implement the vertebral body that the kyphoplasty art strengthens fracture with the acrylic acid methyl ester. cement.To strengthen the vertebral body shooting standard x radiograph for the treatment of with the kyphoplasty art with preventative bone.

After the treatment of kyphoplasty art, research worker defines and determines that preventative bone strengthens two sections of treatment.If be defined as according to two (2) vertebral bodys determining in when operation and be not suitable for treatment, then this patient is construed to the examination failure and does not enroll in this research.

In case identified two HVB, research worker is just asked to open and is selected code (randomization code) to determine the research treatment that gives at random.Select the treatment with β-TCP+rhPDGF compositions of code appointment at random with respect to sections near-end for the treatment of or far-end with the kyphoplasty art.Other HVB does not still treat.

Mix β-TCP+rhPDGF compositions according to the method that embodiment 1 provides.The syringe of at once pastel being packed into after mixing is used for the aseptic technique injection.After β-TCP+rhPDGF compositions is mixed, after waiting for about 10 minutes, the clinician begins to implant.The each mixing used new aseptic mixing apparatus (spatula).Research worker commander implements the cumulative amount of the compositions that blended assistant writes down implantation and the residual quantity of the compositions do not implanted.Amount and record with qualitative relative measurement value (1/3,2/3, whole) calculation composition.

Will be from Cardinal Health of Dublin, the 8-16 rule that Ohio obtains Pin is inserted into by the outer approach (extrapedicular approach) of pedicle of vertebral arch needs preventative-therapeutic vertebral body.Tinsel passes

Pin, and

Pin passes the stylet (stylet) on the tinsel.Suitable mix preparation is expelled in curee's the vertebral body.Should careful operation so that to leak into the outer pastel of vertebral body minimum.

According to embodiment of the present invention, contrast agent can help to identify the pastel that leaks into outside the vertebral body.Fig. 1 illustrates syringe and related equipment, and they penetrate into the tissue that covers the vertebral body outside and arrive vertebral body to send the present composition.Fig. 2 illustrates the X-ray photograph that the present composition is expelled to the vertebral body of L3 vertebra according to an embodiment.

Remove apparatus.Use cleaning down and standard wound suture technology.

Follow up a case by regular visits to evaluation

After operation, 7-14 days and the 6th (± 3 days), 12 (± 7 days), 24 (± 7 days) and 52 (± 14 days) week observe the patient and carry out post-operative evaluation.In follow-up period, implement conventional the evaluation and program, shown in the research flow chart in the following table 2.

Table 2-studies flow chart and follows up a case by regular visits to assessment

1. before any research specific program, must carry out

2. implement quantitative CT (QCT) to obtain the BMD data according to standard scheme, measure by specified flesh skeleton radiotherapy Shi Jinhang.

Efficacy assessment

Collect about deriving from the result data of the result of study that X-ray photograph, QCT and direct function check from this research.The timetable of these measurement results provides in table 3.

Table 3-X radiograph and functional assessment frequency

Compare with untreated vertebral body, with vertebral body expection demonstration bone mineral density (BMD) increase of β-TCP+rhPDGF compositions injection.The increase of bone mineral density can make vertebral body to by the inductive fracture in kyphoplasty art/vertebroplasty operation back (comprising the secondary fracture) susceptible more not in the vertebral body.

Embodiment 3

The method that suppresses the vertebral compression fractures in the osteoporosis individuality

The method that suppresses the vertebral compression fractures in the osteoporosis individuality comprises: by promoting bone formation in the vertebral body with comprising the combination treatment that is configured in the PDGF solution in the biocompatible matrix (for example bata-tricalcium phosphate)

Mix the present composition according to the method that embodiment 1 provides.PDGF concentration in PDGF solution is between 0.3mg/ml-1.0mg/ml.Immediately compositions is encased in after the mixing and is used for the aseptic technique injection in the syringe.The surgeon implants after waiting for about 10 minutes.The each mixing used new aseptic mixing apparatus (spatula).

Will

Pin is inserted into by the outer approach of pedicle of vertebral arch needs preventative-therapeutic vertebral body.In some embodiments, need preventative-therapeutic vertebral body to comprise to contain the high-risk vertebral body of vertebral body T5 to T12 and L1 to L4.Tinsel passes

Pin, and

Pin passes the stylet on the tinsel.The compositions that mixes is expelled in curee's the vertebral body.Careful operation is so that the pastel that leaks into outside the vertebral body is minimum.Treat a plurality of vertebral bodys according to present embodiment.The sufferers of osteoporosis face of accepting this treatment has lower vertebral compression fractures incidence rate than untreated sufferers of osteoporosis face.

Embodiment 4

The long-term safety evaluation of rh-PDGF-BB associating collagen protein/bata-tricalcium phosphate substrate in the other planting model of rabbit spinal column

Experimental design and general introduction

The safety that this research evaluation is implanted injectable rhPDGF-BB/ collagen protein/β-TCP material in the other intramuscular of the spinal column adjacent with rabbit spine site.Observe the neurotoxicity symptom situation of animal, implantation site carried out histological examination together with adjacent vertebral body and spinal cord, with record organization to specific reaction that described material was risen.

Research approach and animal feeding are ratified through local IACUC, and carry out according to the AAALAC guide.The female albino new zealand rabbit that 12 (12) of weight 〉=2.5kg are used to test first is divided into 4 groups: 0.3mg/ml PDGF; 1.0mg/ml PDGF; Rubber; Or acetate buffer.The rabbit of PDGF treatment is accepted the rhPDGF-BB implant in the suitable spissated substrate of 0.2cc, this implant is injected in the other muscle bag of right spinal column (pocket) of the 1cm adjacent with the L4-L5 vertebral body, and the while is implanted high density polyethylene (HDPE) (HDPE) at the other intramuscular of the left spinal column of the close L2-L3 of same animal with similar otch.Rabbit in the sodium acetate buffer group accepts to replace the sodium acetate buffer of PDGF+ substrate implant, and the rabbit of rubber group is only accepted rubber at the other muscle of right spinal column.After operation, put to death a rabbit in each group in 29,90 and 180 days.

Per 2 all measuring body are heavy before operation and after the operation during studying.Before operation, after the operation immediately and face and take X-ray photograph before putting to death.When intra-operative and research end, surgical site is carried out digital photographing.The clinical observation result of weekly record implantation site, observed content is erythema, edema and inflammation symptom and neurotoxicity symptom, for example walking changes.When necropsy, gather in the crops each implantation site together together with adjacent vertebral body and spinal cord, use formalin fixed, and preparation is used for the credit of decalcification paraffin-embedded tissue and analyses.

Material

The rhPDGF-BB dosage of test is included in 0.3mg/ml and the 1.0mg/ml rhPDGF-BB in the 20mM sodium acetate buffer (pH 6.0+/-0.5) in this research.Host material is made up of β-TCP (KenseyNash Corporation) that 20% refrigerated cattle type i collagen albumen and 80% contains 100-300 μ m particle diameter.The negative control material is made up of high density polyethylene (HDPE) (HDPE), and the positive control material is made up of black rubber.Liquid/agglomerate ratio with 3: 1 before facing operation allows rhPDGF-BB and contrast solution mix with host material.

In brief, allow PDGF solution soak into described material about 2 minutes, about 3 minutes of manual mixing is to produce pasty consistency then.By from the close sample eluting PDGF of quality then by ELISA (R﹠amp; D Systems) quantitative assay PDGF confirms to make rhPDGF-BB uniform distribution situation in whole composite material with this hybrid technology.

The result

After hand mix 0.3mg/ml rhPDGF-BB and collagen protein/β-TCP substrate, confirm the inhomogeneity error of rhPDGF-BB between each sample in whole composite material+/-4% in.

All animals all recover from operation, and all clinical observation result all are reported as normally in recording process, in surgical site impassivity toxicity or unusual wound healing symptom.Two kinds of animals with sodium acetate buffer and substrate contrast treatment have been tied little crust at the surgical wound place of healing fully.Animal of accepting 0.3mg/ml rhPDGF-BB showed slight erythema at the surgical site place in 3-4 days after operation, recover normal appearance subsequently.The back 29 days histologic analysis at trier implantation site place of performing the operation show that an amount of tissue growth is in the test material of implanting and slight inflammatory reaction arranged.Do not observe the formation of dystopy bone or unusual bone at the vertebral body place adjacent with implantation site.These discoveries are summarized in the table 4, and compare with the grading of negative control HDPE implantation site.

Table 4-back 29 days implantation site place histological examination results general introduction of performing the operation

??[PDGF-BB]??(mg/ml)	Macrophage	??MGC	Tissue growth	The dystopy bone	Exostosis
??[PDGF-BB]??(mg/ml)	Macrophage	??MGC	Tissue growth	The dystopy bone	Exostosis	??0.3	??3，1(NC)	??2，0(NC)	??2，0(NC)	??0，0(NC)	??0，0(NC)
??1.0	??2，2(NC)	??2，0(NC)	??2，0(NC)	??0，0(NC)	??0，0(NC)	??0.3	??3，1(NC)	??2，0(NC)	??2，0(NC)	??0，0(NC)	??0，0(NC)

The NC=negative control; MGC=multinuclear macrophage; The biological reactivity grade: 0=does not have, 1=trace/slight, and the 2=appropriateness, 3=is medium, and 4=is significantly/seriously

The primary evidence prompting that from this research, obtains based on clinical observation result, collagen protein/bata-tricalcium phosphate and 1.0mg/ml, 0.3mg/ml rhPDGF-BB or any acute or chronic neurotoxic effect of sodium acetate buffer associating not causing.The back 29 days Histological assessments to implantation site that perform the operation show, have normal and expect an amount of tissue growth in the material of implanting, and slight inflammatory reaction is arranged.Do not observe dystopy bone formation, exostosis or unusual bone resorption at any implantation site.Based on the observed result to the animal of treatment in this research, collagen protein/bata-tricalcium phosphate associating 1.0mg/ml, 0.3mg/ml rhPDGF-BB are to use safe when injecting near the spinal column place.

Embodiment 5

PDGF-BB associating cattle type i collagen albumen/β-TCP substrate is to the safety evaluatio of vertebra treatment

This research evaluation is used for the enhanced safety of compositions after being expelled to the baboon vertebral body that comprises the rhPDGF-BB that unites with biocompatible matrix (comprising bata-tricalcium phosphate and type i collagen albumen) of bone.

Experimental design

Studied the female baboon (hunting refreshing baboon (Papio anubis)) in 6 18-21 years altogether, every baboon has been assigned in 2 treatment groups that table 5 provides.Give animal imaging and analysis with electrography, quantitative CT (QCT), nuclear magnetic resonance (MRI) technology, end flap (terminal histology) and the little calculating body section radiography of non-GLP (micro-CT) during the research.

Four vertebral levels (T12, L2, L4 and L6) in every animal, have been studied.1.0mg/ml rhPDGF-BB+ collagen protein/β-TCP (substrate) compositions of every about 0.5cc of animals received injection of I group is in each of T12, L2 and L4 vertebral body.1.0mg/mlrhPDGF-BB+ collagen protein/β-TCP (substrate) compositions prepares shown in the embodiment 1 as mentioned.Sodium acetate buffer+collagen protein/β-TCP (substrate) compositions of every about 0.5cc of animals received injection of II group is in each of T12, L2 and L4 vertebral body.Each accepts the sodium acetate buffer of the about 0.5cc of injection in addition in the L6 vertebral body I group and II treated animal.Therefore, every animal has four (4) individual vertebral bodys and has accepted injection.Fig. 3 summarizes the injection strategy of this research.The treatment situation of every animal is recorded in the research form.

The method that instructs with the percutaneous cryptoscopy undergos surgery.Except injecting injectable 1.0mg/ml rhPDGF-BB+ collagen protein/β-TCP substrate or suitable contrast treatment, this program and vertebroplasty similarly carry out.RhPDGF-BB+ collagen protein/β-TCP material, control material or the buffer of about 0.5cc are expelled to each vertebral body as mentioned above.Intra-operative provides anesthesia for every animal.

Table 5-injects the treatment general introduction of rhPDGF-BB+ collagen protein/β-TCP substrate in the baboon vertebral body

Group	Dosage	Time point	Analyze
Group	Dosage	Time point	Analyze	I	1.0mg/ml PDGF+collagen protein/β-TCP substrate.L6 only accepts sodium acetate buffer	Perioperatively and operation back 1,3,6 and 9 months	QCT, MRI, clinical observation, serum chemistry and hematology (perioperatively and operation back 1,3,6 and 9 months), body weight, electrography, histology, the micro-CT of non-GLP
II	The 20mM sodium acetate is slow	Perioperatively	QCT, MRI, clinical sight	I		Perioperatively and operation back 1,3,6 and 9 months
II	The 20mM sodium acetate is slow	Perioperatively	QCT, MRI, clinical sight		Towards liquid (pH 6.0)+collagen protein/β-TCP substrate.Only L6 accepts sodium acetate buffer	With operation back 1,3,6 and 9 months	Examine, serum chemistry and hematology (perioperatively and operation back 1,3,6 and 9 months), body weight, electrography, histology, the micro-CT of non-GLP

A. distribute the administration group

By handbook plan three animals are assigned to the treatment group to reach close group average weight through design.

B. distribute date of surgery

Animal is assigned to one of two date of surgery (I date or II date).Each animal is distributed in the I date by toss a coin decision organizes that still the II date is organized.Continue this grouping up to each on date row expired 3 animals.Record size of animal, its administration grouping and date of surgery.Research supervisor and research director know the treatment of animals group.The treatment group is not known by radiotherapy teacher and histopathologist.

C. observe in the body and measure

Clinical observation

Observation every day is in the animal in its cage during whole research.Opening entry cage limit observed result after determining pre-selection criteria is until research finishes.Overall appearance and the behavior of observing every animal change, and comprise that walking changes.During whole research, observe the menstrual cycle sign of every animal.Carry out the cycle evaluation (cycling reading) of non-GLP and be recorded in U.S. southwest biological medicine WARF that (Southwest Foundation for Biomedical Research is SFBR) in the animal data storehouse.

Handle animal according to SFBR standard operating procedure (SOP), this rule of operation meets the animal welfare method (9CFR of United States Department of Agriculture (USDA), 1st, regulations and the experimental animal feeding and guide for use (the ILAR publication of general introduction 2 and 3 parts), 1996, National Academy Press) specified condition in.The observational study animal also write down at least once disease or puzzlement symptom (comprising walking change) every day, and any such observed result is reported to reliable veterinary and studies the director.

Body weight

Before the physical examination of beginning, operation and follow up a case by regular visits to the electrography before measurement and weigh sb..Before its calmness, stop feeding, measure body weight subsequently.

Food consumption

Except fasting in research process, the quality (as the part of cage limit observed result) that every animal food of evaluation every day consumes, beginning at least 7 days before operation.(at non-sedating in the date) for every animal provides abundant food supply, writes down consumption by SFBR SOP once a day.In the calm date, feed once every day when animal recovers from anesthesia.

D. cryptoscopy, photography, electrography, MRI and QCT imaging

Before operation, after the operation immediately with postoperative 1,3,6 and injection site taken non-GLP digital photograph in 9 months.

Before treatment and operation back and postoperative about 1,3,6 and took anteroposterior position and side X-ray photograph in 9 months.For the anteroposterior position X-ray photograph, allow animal back up, support its extremity.For the side X-ray photograph, allow the animal left surface keep down, support its extremity.Record is provided with energy (kV) and the shading value (mA) of each position and animal.

Before trial target and reference substance are expelled to the animal vertebra, during and intra-operative afterwards take non-GLP photofluorogram.Photofluorogram can not be assessed as the result of this research, but can make the surgeon accurately guide needle is inserted into vertebral body at intra-operative.

Carry out nuclear magnetic resonance (MRI) with before operation and the spine imaging of giving every animal in 4-10 days of operation back.Can be in addition after operation about 1,3,6 and carry out the spine imaging that MRI gives every animal 9 months the time.MRI section (session) is made up of T1 and T2 weighted scanning.

Can carry out quantitative CT (QCT) with before operation and the spine imaging of giving every animal in 4-10 days of operation back.Can be in addition after operation about 1,3,6 and carry out the spine imaging that QCT gives every animal 9 months the time.Scanning is made up of to the cross section thin slice of a series of vicinities of the trunk of the cranium soleplate of rumpbone the afterbody soleplate from the Section 11 thoracic.

Also 1 week (before the operation) and operation back obtain injected vertebral body and the intermediary thick cross sectional image of 3mm of not treating vertebral body of every baboon during 1,4 and 12 weeks with QCT before operation.Each vertebral body perfect imaging is needed 5-8 thin slice altogether.Change is by DICOM (digital imaging and communications in medicine) format-pattern that QCT produces, and uses that (Bassersdorf, Switzerland) Kai Fa software is converted to the file format of three-D volumes analysis by Scanco AG.By selecting manually that in each thin slice the volume bone mineral density (vBMD) of the cup (anterior compartment) of each vertebral body is measured in cortical bone shell (cortical shell) excluded target area (ROI).Assessment software is created the z-stack of each section and ROI, and the root of cube ROI and calculating subsequently is from the bulk density of the arbitrary unit of gray scale in the image, and the percentage ratio that calculates with respect to baseline scan changes.Use is checked the one way replicated measures ANOVA of (Tukey ' s post-hoc test) with Tukey afterwards, measures I group and the II treated animal situation that exists from any significance,statistical variation of preceding or operation 1 week of back vBMD when research finishes that performs the operation.

Assess X-ray photograph, MRI and QCT image by qualified clinical radiotherapy teacher and a competent cooperation people, so that the concordance assessment about neuro pathology, osteopathology and the pathology result of surrounding soft tissue who is produced by described vertebra treatment to be provided.This estimate by to unusual about bone, nervous tissue is unusual and the qualitative examination of each image that adjacent surrounding soft tissue is unusual is formed.Radiotherapy teacher follows the radiotherapy evaluation scheme with assessment radiology data.

E. clinical pathology evaluation

Serum chemistry

Before operation and will about 3ml whole blood collection after the operation in the container of no anticoagulant.In addition, after operation about 1,3,6 and 9 months will about 3ml whole blood collection in the container of no anticoagulant.Be used for allowing before the blood of serum chemistry the animal overnight fast in collection.According to the parameter shown in the table 6 serum is analyzed.

Table 6-serum analysis

Sodium	Phosphorus
Sodium	Phosphorus	Potassium	Glucose
Chloride	Blood urea nitrogen (BUN)	Potassium	Glucose
Chloride	Blood urea nitrogen (BUN)	Total CO 2 (bicarbonate)	Kreatinin
Total bilirubin	Total protein	Total CO 2 (bicarbonate)	Kreatinin
Total bilirubin	Total protein	Alkali phosphatase (AP)	Albumin
Lactic acid dehydrogenase (LDH)	Globulin	Alkali phosphatase (AP)	Albumin
Lactic acid dehydrogenase (LDH)	Globulin	Aspartate transaminase (AST)	The albumins/globulins ratio
Alanine aminotransferase (ALT)	Cholesterol	Aspartate transaminase (AST)	The albumins/globulins ratio
Alanine aminotransferase (ALT)	Cholesterol	Gamma glutamyltransferase (GGT)	Triacylglycerol
Calcium	The BUNICREAT ratio	Gamma glutamyltransferase (GGT)	Triacylglycerol
Calcium	The BUNICREAT ratio	Aninon gap	Bilirubin direct
??CPK		Aninon gap	Bilirubin direct

The hematology

Before operation and will about 3ml blood collecting after the operation in the test tube that contains EDTA.In addition, after operation about 1,3,6 and 9 months will about 3ml blood collecting in the test tube that contains EDTA.According to the parameter shown in the table 7 whole blood sample is analyzed.

Table 7-hemanalysis

Erythrocyte (RBC) counting	The average hemoglobin content of hemocyte (MCH)
Erythrocyte (RBC) counting	The average hemoglobin content of hemocyte (MCH)	Leukocyte (WBC) be (total and differentiation ^*)	Hemocyte mean corpuscular hemoglobin concentration (MCHC)

Erythrocyte (RBC) counting	The average hemoglobin content of hemocyte (MCH)
Erythrocyte (RBC) counting	The average hemoglobin content of hemocyte (MCH)	Hemoglobin concentration	Average blood cell volume (MCV)
Hematocrit	Platelet count (Plt)	Hemoglobin concentration	Average blood cell volume (MCV)
Hematocrit	Platelet count (Plt)	??RDW	The abnormal blood cell form

* comprise multi-region section neutrophil cell (polysegmented neutrophil), band form nucleus neutrophil(e) cell, lymphocyte, mononuclear cell, basophilic granulocyte, eosinophilic granulocyte.

Gather the serum that is used to analyze by presider or SFBR

Each gathers once about 14ml blood to the test tube of additive-free (i.e. " blood coagulation ") from all animals before operation and after the operation.In addition, after operation about 1,3,6 and gathered about 14ml blood to the test tube of additive-free (i.e. " blood coagulation ") from all animals in 9 months.Centrifugal blood is obtained serum and is divided into two equal portions.Serum is stored in-70 ℃ or low temperature more.

Anatomical pathology

When research finishes, all animal humanity are put to death.Every animal of putting to death under moribund condition or the disease state is carried out rough necropsy, to determine the cause and/or the characteristic of dying or disease state.

Necropsy

The animal of putting to death under to moribund condition or disease state under research pathologist's supervision during the research carries out comprehensive necropsy, to determine the cause and/or the characteristic of dying or disease state.The standard necropsy comprises: check outer surface and mouth, extremity, body cavity and internal/tissue.Collect all vertebras, and check abnormal conditions through treatment.The visible unusual morphology summary of all naked eyes of record in independent necropsy form.

Tissue sampling and preservation

To put to death animal with undertissue and organ, be kept in 10% the neutral buffered formalin (except that eyes) for optimal fixation is kept at eyes in the Bouin liquid.In order to preserve purpose each tissue or organ samples are embedded in the paraffin then, are archived in the place that the presider agrees, or be used to help determine the cause of death.

For all baboons, gather in the crops individually all through the treatment with adjacent untreated vertebra (T12 is to L6), comprise spinal cord and spinal canal, and carry out suitable evaluation according to the treatment of being accepted.Differentiate the T12 vertebral body by staying the minimum rib that is connected with bone of 2cm.Place the formalin fixed preparation to be used for the plastic cement embedding in all bone samples.

Be embedded in the paraffin each soft tissue or organ samples and file in order to preserve purpose.General introduction to the tissue sample of collecting is provided in the table 8.

The general introduction of the tissue sample that table 8-collects when necropsy

Cardiovascular	Genitourinary system
Cardiovascular	Genitourinary system	Large artery trunks	Kidney
Heart	Bladder	Large artery trunks	Kidney
Heart	Bladder	Digestive system	Ovary

Cardiovascular	Genitourinary system
Cardiovascular	Genitourinary system	Salivary gland (salivary gland of mandible)	The uterus
Tongue	Sub-neck	Salivary gland (salivary gland of mandible)	The uterus
Tongue	Sub-neck	Esophagus	Vagina
Stomach	Skin/flesh skeleton	Esophagus	Vagina
Stomach	Skin/flesh skeleton	Small intestinal	Skin/mammary gland (male and female)
Duodenum	Bone (femoral head)	Small intestinal	Skin/mammary gland (male and female)
Duodenum	Bone (femoral head)	Jejunum	Bone (the 7th rib)
Ileum	Skeletal muscle (thigh)	Jejunum	Bone (the 7th rib)
Ileum	Skeletal muscle (thigh)	Large intestine	Knee joint
Caecum	Shoulder joint	Large intestine	Knee joint
Caecum	Shoulder joint	Colon	Mandible
Rectum	Right crus of diaphragm	Colon	Mandible
Rectum	Right crus of diaphragm	Pancreas	Left side ankle
Liver	The right hand	Pancreas	Left side ankle
Liver	The right hand	Gallbladder	Left side wrist
Respiratory system	From T12 to L7 spine/rumpbone-separately	Gallbladder	Left side wrist
Respiratory system	From T12 to L7 spine/rumpbone-separately	Trachea	Nervous system/special sense organ
Lung (comprising bronchus)	The eyes that contain optic nerve	Trachea	Nervous system/special sense organ
Lung (comprising bronchus)	The eyes that contain optic nerve	Lymph/hemopoietic system	Sciatic nerve
Bone marrow (breastbone)	Brain	Lymph/hemopoietic system	Sciatic nerve
Bone marrow (breastbone)	Brain	Thymus	Optic chiasma
Spleen	Brain	Thymus	Optic chiasma

Cardiovascular	Genitourinary system
Cardiovascular	Genitourinary system	Lymph node	Cerebellum
Lymph/the hemopoietic system of axillary fossa	Medullary substance	Lymph node	Cerebellum
Lymph/the hemopoietic system of axillary fossa	Medullary substance	Lymph/the hemopoietic system of lower jaw	Pons
Mesenteric mesaraic lymph/hemopoietic system	Spinal cord (breast)	Lymph/the hemopoietic system of lower jaw	Pons
Mesenteric mesaraic lymph/hemopoietic system	Spinal cord (breast)	Hormonal system	Other
The adrenal gland	The number of animals (Animal Number Tattoo) that cicatrix is arranged	Hormonal system	Other
The adrenal gland		Hypophysis	Gross lesion
Thyroid/parathyroid gland ^*	Lachrymal gland	Hypophysis	Gross lesion

* in the conventional organization section, there is not parathyroid gland will need not to cut into slices again once in a while.

With the vertebral body that comprises the compositions injection that is configured in the rhPDGF-BB solution in β-TCP/ collagen matrices, demonstration forms normal bone and does not have deleterious neurotoxicity.In addition, comprise the soft tissue of the vertebral body that is configured in the rhPDGF-BB liquid composite in β-TCP/ collagen matrices not because of giving rh-PDGF/ base composition display abnormality adjacent to accepting.

In addition, the vertebral body with comprising the compositions injection that is configured in the rhPDGF-BB solution in β-TCP/ collagen matrices also shows the volume bone mineral density that increases.The percentage ratio that Fig. 4 illustrates the volume bone mineral density (vBMD) of I group and II treated animal vertebral body changes.The meansigma methods of all vertebral bodys of being treated in each each group of numerical point representative among Fig. 4.For example, first numerical point of I group is the meansigma methods of 9 vertebral bodys (T12, L2 and the L4 of per three animals in the I group) of measuring among Fig. 4 after the rhPDGF-BB base composition is expelled to vertebral body.Similarly, 2 groups first numerical point is the meansigma methods at 9 vertebral bodys of will measure after collagen protein/B-TCP substrate is expelled to vertebral body (T12, L2 and the L4 of per three animals in the II group) among Fig. 4.

As shown in Figure 4, with the vertebral body that comprises the combination treatment that is configured in the rhPDGF-BB solution in β-TCP/ collagen matrices (I group), being presented at vBMD stable in the research process increases, and the trimestral level in operation back that is increased in of vBMD is [contrast 5.93%+/-1.33 (the 12nd week), 2.64%+/-1.16 (the 1st week) significant on the statistics with respect to the level of operation back 1 time-of-week; P=0.023].VBMD continued to increase by 6th month up to research, reached maintenance level subsequently at 9th month.Yet,, do not show that during studying vBMD significantly increases with the vertebral body that comprises the combination treatment that is configured in the 20mM sodium acetate buffer in β-TCP/ collagen matrices (group II).

In addition, the vBMD percentage ratio that Fig. 5 illustrates I group and II treated animal vertebral body changes, wherein β-TCP/ the collagen matrices of subduction injection from volume bone mineral density analysis.The meansigma methods of Fig. 5 interior all vertebral bodys of being treated of each each group of numerical point representative as Fig. 4.

As shown in Figure 5, the vertebral body with rhPDGF-BB base composition (I group) treatment shows that vBMD increases.Clearly illustrate that from volume bone mineral density analysis subduction β-TCP/ collagen matrices, compare that the vBMD of the All Ranges of I group vertebral body all increases with the regional area of rhPDGF-BB base composition injection site.

All patents cited above, publication and digest are all incorporated by reference with its integral body at this.Should be appreciated that the aforementioned preferred embodiment of the invention that only relates to wherein can be carried out numerous modifications or change under the spirit and scope of the invention that does not depart from above accessory claim and limited.

Claims

1. prevent or suppress the method for vertebral compression fractures, described method comprises:

The compositions that comprises the PDGF solution that is configured in the biocompatible matrix is provided; With described compositions is administered at least one vertebral body.

2. the process of claim 1 wherein to use and comprise described compositions is injected to described at least one vertebral body.

3. the process of claim 1 wherein that described at least one vertebral body comprises high-risk vertebral body.

4. be used for promoting or accelerating the osteoplastic method of vertebral body, described method comprises:

The compositions that comprises the PDGF solution that is configured in the biocompatible matrix is provided; With

Applying said compositions is at least one vertebral body.

5. the method for claim 4, described method further comprises:

At least a pharmaceutical composition is provided; With

With described pharmaceutical composition topical administration patient.

6. the method for claim 5, wherein the described pharmaceutical composition of topical administration comprises described pharmaceutical composition is placed in described at least one vertebral body or around it.

7. the method for claim 4, described method further comprises:

At least a pharmaceutical composition is provided; With

Give the patient with described pharmaceutical composition whole body.

8. the method for claim 7, wherein whole body gives described pharmaceutical composition and comprises that per os gives, intravenous gives or its combination.

9. the method for claim 5, wherein said patient is an individuality of suffering from osteoporosis.

10. the method for claim 5, wherein said patient is for having accepted the individuality of kyphoplasty art or vertebroplasty.

11. be used for promoting the osteoplastic compositions of vertebral body, described compositions comprises:

Comprise the solution that derives from hematoblastic somatomedin (PDGF);

Biocompatible matrix; With

At least a contrast agent,

Wherein described solution and described contrast agent are configured in the biocompatible matrix.

12. the compositions of claim 11, wherein said at least a contrast agent comprises: cation contrast agent, anion contrast agent, non-ionic contrast medium or its combination.

13. comprising, the compositions of claim 11, wherein said contrast agent contain iodine compound.

14. the compositions of claim 13, the wherein said iodine compound that contains comprises (S)-N, N '-two [2-hydroxyl-1-(methylol)-ethyl]-2,4,6-three iodo-5-lactoyl amido isophthaloyl amine or derivatives thereofs.

15. each compositions is used for promoting purposes in the osteoplastic medicine of at least one vertebral body in preparation among the claim 11-14.

16. the compositions that comprises the solution that contains PDGF and biocompatible matrix is used for promoting purposes in the osteoplastic medicine of at least one vertebral body in preparation, wherein said solution is configured in the biocompatible matrix.