CN101332313A - Bracket for regenerating decalcification cortical-bone articular cartilage with vertical drill and preparation method thereof - Google Patents
Bracket for regenerating decalcification cortical-bone articular cartilage with vertical drill and preparation method thereof Download PDFInfo
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- CN101332313A CN101332313A CNA2008100208911A CN200810020891A CN101332313A CN 101332313 A CN101332313 A CN 101332313A CN A2008100208911 A CNA2008100208911 A CN A2008100208911A CN 200810020891 A CN200810020891 A CN 200810020891A CN 101332313 A CN101332313 A CN 101332313A
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Abstract
The invention discloses a stent used for the joint cartilage regeneration of the decalcification cortex bone of longitudinal drilling and a preparation method thereof, and the stent belongs to an animal decalcification cortex bone. Holes with the diameter of 0.1 to 1 mm are drilled perpendicularly on the horizontal surface of the decalcification cortex bone; the interval among the holes is 0.5 to 5 mm, thereby forming three-dimensional porous stereo network structure; the porosity reaches 5 percent to 20 percent. The preparation method is as follows: a first step, the decalcification of the cortex bone: a cortex bone is taken out from the cattle or goat to remove the bone marrow and then soaked in decalcifying solution which is replaced everyday and the cortex bone is completely decalcified to enter into the next step; a second step, drilling: holes with the diameter of 0.1 to 1 mm are drilled on the horizontal surface of the decalcification cortex bone; the interval among the holes is supposed to be 0.5 to 5 mm, after the cortex bone is tailored into a designed dimension, then a stent used for the joint cartilage regeneration of the decalcification cortex bone of longitudinal drilling is gotten. The stent used for the joint cartilage regeneration of the decalcification cortex bone of longitudinal drilling has extensive material selection and quite good biocompatibility.
Description
Technical field
The invention belongs to the preparation field of artificial joint cartilage, particularly utilize the prior biological material to prepare decalcification cortical-bone articular cartilage bracket for regenerating of artificial vertical boring and preparation method thereof.
Background technology
The biological study of articular cartilage is verified, because the cartilaginous tissue structure is special, impassivity, blood vessel, lymphatic vessel in it, though childhood, articular cartilage can obtain part nutrition by the cartilage underpass, form a year joint cartilage and do not have nutritional channel under the cartilage, the nutrition supply of cartilage is to change by the pressure that joint motion produces, synovial fluid is exchanged between articular cavity and cartilage matrix, so cartilage self lacks the effective for repairing ability, the treatment of damage is also more difficult than fracture sometimes, and easily causes tangible dysfunction.Athletic articular cartilage damage incidence rate is very high, and regular meeting causes symptoms such as arthralgia, and the secondary osteoarthritis influences the raising of sports achievement, even LOM, so the articular cartilage reparation is a sports medical science field important subject always.
The repair of cartilage method mainly contains the method that stimulates articular cartilage self to repair at present, methods such as the plane that comprises fiber deliquescing bone is scraped, subchondral bone boring, it is not clear that shortcoming mainly is that the plane of fiber deliquescing bone is scraped late result, can cause fiber become increase the weight of, in the defective region and contiguous necrocytosis.Though and repair tissue has the appearance of hyaline cartilage in early days in the subchondral bone boring method, the articular cartilage that can not bear stress, generation in early days lacks normal structure, mechanical property and durability, finally regression can take place.Also having a kind of is that the transplanting and repairing method of tissue, cell mainly comprises the transplanting of articular cartilage, transplanting, periosteum and the perichondral transplanting of chondrocyte, shortcoming mainly is the operation technique complexity, relate to the donor operation of drawing materials, and limited from body or homogeneous allogenic bone cartilage source.And synthetic substrate influences the expression of Phenotype chondrocyte.Generation mostly is fibrous cartilage and improper hyaline cartilage and operating difficulties, needs to transplant in cultured and amplified in vitro and the body, is difficult to be widely used in clinical., fixedly difficulty etc. limited for the district.Also having a kind of at last is the organization engineered cartilage recovery technique, and shortcoming mainly contains the autogenous cell transplantation operating difficulties, and cultured and amplified in vitro and the interior transplanting of second operation body of drawing materials that need to perform the operation is difficult to be widely used in clinical.And there is immunological rejection in variant cell, and cell survival rate is low.Tissue engineering bracket employing artificial-synthetic copolymer (PLGA) has biocompatibility issues in various degree, and natural biologic material rack mechanical mechanics such as silkworm silk, collagen protein, fibrin, people's amniotic membrane are poor, the mechanical stress that produces in the time of can't resisting joint motions, cause subsiding of mend easily, also influence the growth of cell on the material simultaneously.The shortcoming of syringeability water-setting is that the controllability and the mechanical strength of gelation process is relatively poor equally.And cytokine itself is fine to the facilitation of cartilage-derived growth in the cytokine auxiliary treatment cartilage injury method, but the factor will be fixed in tissue engineering bracket material and the release of energy slow release, needs further technology exploration.
In sum, more or less the shortcoming that exists biocompatibility and mechanical strength to be difficult to take into account simultaneously in the said method, and how to carry out In vitro culture, complicated operation.
Summary of the invention
The present invention is in order to solve the shortcoming that is difficult to have concurrently excellent biological compatibility and mechanical strength that prior art exists, the present invention proposes decalcification cortical-bone articular cartilage bracket for regenerating of a kind of vertical boring and preparation method thereof, the decalcification cortical-bone articular cartilage bracket for regenerating of vertical boring of this method manufacturing has excellent biological compatibility and mechanical strength, need not carry out In vitro culture and can use.
Technical scheme of the present invention is: the decalcification cortical-bone articular cartilage bracket for regenerating of this vertical boring, be a kind of animal decalcification cortical bone, be provided with the hole of 0.1~1mm diameter at horizontal surface perpendicular to described decalcification cortical bone, the hole interbody spacer is 0.5~5mm, form three-dimensional porous space network, porosity reaches 5%-20%, and thickness is greater than 2mm.The cortical bone decalcification that described decalcification cortical bone is cattle or goat makes.
The preparation method of the decalcification cortical-bone articular cartilage bracket for regenerating of described vertical boring, step is: the first step, cortical bone decalcification: the cortical bone of getting cattle or goat, remove bone marrow, be soaked in 2 ℃-10 ℃, the concentration of pH between 6-10 is in the 0.1mol/L-0.7mol/L ethylenediaminetetraacetic acid decalcifying Fluid, decalcifying Fluid is changed every day, the decalcifying Fluid of changing every day detects calcium ion concentration, Ca chelate concentration<0.5g/L in decalcifying Fluid, be complete decalcification, be complete decalcification, cortical bone taken out carry out the x line and CT detects, see whether decalcification fully, fully the cortical bone of decalcification enters next step, the cortical bone that does not have a complete decalcification again decalcification until complete decalcification;
Second step, boring: determine that according to the trial test screening aperture is 0.1-1mm, by the machine drilling technology perpendicular to the diameter punching of the horizontal surface of decalcification cortical bone with 0.1-1mm, set hole interbody spacer 0.5-5mm, be cut into the decalcification cortical-bone articular cartilage bracket for regenerating that design size is promptly vertically holed.Described decalcifying Fluid is to use the deionized water preparation, and plastic containers are contained and carried.The decalcification cortical-bone articular cartilage bracket for regenerating of described vertical boring uses cobalt 60 irradiations sterilization in 12-24 hour before use.
The present invention vertically decalcification cortical-bone articular cartilage bracket for regenerating of boring is that utilization natural biological decalcification cortical bone support prepares, adopt the organizational project recovery technique of cartilage in the time of use, hole in conjunction with subchondral bone, autologous stem cell transplantation, reaching bone morphogenetic protein BMP factor in treatment is one, has given full play to the advantage of every technology.
Adopt the restorative procedure key step of the decalcification cortical-bone articular cartilage bracket for regenerating of vertical boring of the present invention to be:
At first, dig in the osteochondral defect zone and scrape off except that fiber becomes, irregular pathological changes cartilage and subchondral bone.Then, carry out the subchondral bone grinding, osteochondral defect is accomplished the shape of comparison rule.The boring of carrying out subchondral bone then allows to discharge the bone marrow of self.
Then, the decalcification cortical-bone articular cartilage bracket for regenerating with vertical boring of the present invention preparation is trimmed to the shape that meets osteochondral defect, inserts damaged place.Subchondral bone can infiltrate through in the support by the aperture on the decalcification cortical-bone articular cartilage bracket for regenerating of vertical boring of the present invention owing to self the bone marrow that boring discharges, and then reparation osteochondral defect place.
Because the plane of fiber deliquescing bone is scraped, and does not discharge self bone marrow, so repair is not obvious.And the decalcification cortical-bone articular cartilage bracket for regenerating of vertical boring of the present invention digs employing subchondral bone boring on the basis of scraping at cartilage in use, discharges self bone marrow, has strengthened repair ability.Self bone marrow that simple subchondral bone boring discharges, no support can not be born stress and can't be according to the reparation of ad hoc structure standard, and the articular cartilage of generation lacks normal structure.And adopted the decalcification cortical-bone articular cartilage bracket for regenerating of vertical boring of the present invention, both increased the mechanical strength of repairing early stage mend, the bone marrow that makes release again infiltrates in the support through the duct in the support, the three-dimensional space that provides for the reparation of bone marrow.
The decalcification cortical-bone articular cartilage bracket for regenerating of vertical boring of the present invention is that the decalcification cortical bone of utilization cattle or goat prepares, and has solved from body or homogeneous allogenic bone cartilage and the limited problem of periosteum and perichondral transplantation donor source.When adopting the decalcification cortical-bone articular cartilage bracket for regenerating reparation of vertical boring of the present invention, easy and simple to handle, be easy to clinical practice.The decalcification cortical-bone articular cartilage bracket for regenerating of vertical boring of the present invention has remedied chondrocyte cell transplantation and tissue engineering bone/cartilage operating difficulties when getting seed cell needs to transplant in cultured and amplified in vitro and the body, is difficult to be widely used in clinical deficiency.And the decalcification cortical-bone articular cartilage bracket for regenerating that uses vertical boring of the present invention can solve the shortcoming of heteroplastic transplantation rejection, autologous bone marrow not only provides autologous stem cells during reparation, the microenvironment of cells survival is provided simultaneously, higher cells survival rate is arranged than simple chondrocyte or stem cell transplantation.This method uses the decalcification cortical bone support of the cattle of natural boring or goat to solve the biocompatibility issues of synthetic support.Articular cartilage regeneration by biomechanics test shows the present invention preparation more approaches cartilage with the mechanical stress and the elastic modelling quantity of decalcification cortical bone support.It is poor to have solved natural biologic material rack mechanical mechanics such as silkworm silk, collagen protein, fibrin, people's amniotic membrane, the problem of the mechanical stress that produces in the time of can't resisting joint motions.Can not cause subsiding of mend, but the also growth of cell on the support material simultaneously.And because the animal decalcification cortical bone that the decalcification cortical-bone articular cartilage bracket for regenerating of vertical boring of the present invention is to use, itself be rich in bone morphogenetic protein (BMPs), can promote intermediary stem cell of bone marrow or marrow stromal cell in the induction rack to change into chondrocyte or osteocyte, promote the secretion of stem cell secretion cartilage or bone matrix.To the startup of repair of cartilage, growth, regulation and control and mouldingly all play an important role.The decalcification cortical-bone articular cartilage bracket for regenerating of vertical boring of the present invention no longer needs during use the factor to be fixed in tissue engineering bracket material and to make its slow release discharge this complex technology.
The experimentation that the decalcification cortical-bone articular cartilage bracket for regenerating of vertical boring of the present invention is used for the reparation of rabbit coaster cartilage shows: behind the prothesis during 12 weeks, the generation of hyaline cartilage has appearred in rabbit knee coaster 4mm huge cartilage defect place, and the cartilage and the subchondral bone at postoperative damaged place during 24 weeks are repaired fully.
The present invention with respect to the beneficial effect of prior art is:
1. utilization cattle or sheep cortical bone are raw material, and donor source is abundant, are easy to clinical popularizing.Not the decalcification cortical-bone articular cartilage bracket for regenerating that can only prepare vertical boring among the present invention with the cortical bone of cattle and sheep.A lot of animal cortex decalcifications of bone are handled through method of the present invention can.But it is many to be with the cattle and sheep cortical bone that the advantage of raw material is that cattle, sheep are cultured quantity, get bone and do material and can turn waste into wealth, and the thickness behind the cattle, the decalcification of bone of Corii Caprae seu Ovis matter can be repaired people's osteocartilaginous and subchondralo bone injury greater than 2mm.
2. natural collagen timbering material, the problem of no immunological rejection is easy to cell absorption, good biocompatibility.
3. be rich in bone morphogenetic protein (BMPs) at decalcification cortical bone support self, can promote that intermediary stem cell of bone marrow or the marrow stromal cell in the induction rack changes into chondrocyte or osteocyte, promote the secretion of stem cell secretion, cartilage or bone matrix.To the startup of repair of cartilage, growth, regulation and control and mouldingly all play an important role.
4. easy and simple to handle, do not need second operation, saved in the art and drawn materials the time, be easy to clinical practice.The decalcification cortical-bone articular cartilage bracket for regenerating of vertical boring of the present invention has been walked around during use and the factor will be fixed in tissue engineering bracket material and make its slow release discharge this complex technology.
5. the decalcification cortical-bone articular cartilage bracket for regenerating of vertical boring of the present invention is three-dimensional porous space network, and the infiltration that is more conducive to self bone marrow is converted into chondrocyte or osteocyte.
6. preparation method is simple, need not add inducible factor more in addition.
Description of drawings
Fig. 1 is the preparation method flow chart of cartilage joint bracket for regenerating of the present invention.
Fig. 2 is the decalcification cortical-bone articular cartilage bracket for regenerating of vertical boring of goat decalcification cortical bone preparation.
The sketch map that Fig. 3 holes on support and designs, aperture 0.1-1mm, hole interbody spacer 0.5-5mm.
The Electronic Speculum of throwing Fig. 4 detects the internal structure of the cartilage joint bracket for regenerating of goat decalcification cortical bone preparation.
Fig. 5 scanning electron microscope detects the tomography structure of the cartilage joint bracket for regenerating of goat decalcification cortical bone preparation.
Fig. 6 operation process.
Fig. 7 New Zealand large ear rabbit uses cartilage joint bracket for regenerating of the present invention to carry out the nuclear magnetic resonance, NMR figure of repair of cartilage after 12 weeks.
Fig. 8 New Zealand large ear rabbit uses cartilage joint bracket for regenerating of the present invention to carry out the substantially figure of repair of cartilage after 12 weeks.
Fig. 9 New Zealand large ear rabbit uses cartilage joint bracket for regenerating of the present invention to carry out the histology figure of repair of cartilage after 12 weeks.
Figure 10 is in the observed result in 24 weeks, and cartilage defect is repaired fully, presents hyaline cartilage.
This is 24 hours decalcification cortical bone support after implanting for Figure 11, takes out from the centre and cuts, the scanning electron microscope of tangent plane.
The specific embodiment
A kind of decalcification cortical-bone articular cartilage bracket for regenerating of vertical boring, be a kind of animal decalcification cortical bone, in the hole that is provided with aperture 0.1~1mm diameter perpendicular to described decalcification cortical bone horizontal surface, can be 0.1mm, 0.5mm, 1mm, the hole interbody spacer is 0.5~5mm, can be 0.5mm, 1.2mm, 3.4mm, 5mm, forms three-dimensional porous space network, porosity reaches 5%20%, and thickness is greater than 2mm.The cortical bone decalcification that described decalcification cortical bone is cattle or goat makes.
Embodiment 1
The preparation method of the decalcification cortical-bone articular cartilage bracket for regenerating of vertical boring
Preparation flow figure sees Fig. 1, gets the femur backbone of cattle or goat, removes bone marrow, is soaked in 0.1M-0.7M ethylenediaminetetraacetic acid (EDTA) decalcifying Fluid in 2 ℃-10 ℃, and PH utilizes chelating to slough calcium in the cortical bone between 6-10, and decalcifying Fluid is changed every day.With deionized water preparation EDTA decalcifying Fluid, contain year (avoiding glass container) with plastic containers.The decalcifying Fluid of changing every day detects calcium ion concentration with monitoring decalcification process with atomic absorption spectrophotometer, and the Ca chelate concentration<0.5g/L in decalcifying Fluid is complete decalcification, and the specimen after the decalcification carries out the x line and CT detects, and proves complete decalcification.Because decalcification cortical bone surfaces externally and internally structure is too fine and close, is unsuitable for growing into of cell.Therefore the present invention by the machine drilling technology as shown in Figure 3, with the diameter vertical drilling (experimental rabbit animal model on clinical patient application aperture different) of decalcification cortical bone horizontal surface with 0.1-1mm, relatively hole interbody spacer 0.5-5mm (using different with clinical patient on the experimental rabbit animal model) is set in the back, then, as shown in Figure 2, the decalcification cortical bone is cut into certain size specification, refrigerated storage.Before transplanting, the decalcification cortical bone carries out cobalt 60 irradiations sterilization in 12-24 hour.
The internal structure transmission electron microscope picture of the cartilage joint bracket for regenerating of Fig. 4 decalcification cortical bone preparation.As seen collagen fiber are loose in the decalcification cortical bone is staggered, and is full of the space between collagen fiber, and the bone small structure is staggered to communicate, and helps the infiltration of the bone marrow matrix and the factor and adhering to of cell.The nature bone small structure of right side for amplifying.
The tomography structure tangent plane sem photograph of the cartilage joint bracket for regenerating of Fig. 5 decalcification cortical bone preparation.The natural lamella sample of decalcification cortical bone has some advantage as recovery support: 1. the anti-vertical of collagen structure is strong to pressure.
2. the gap of sheet interlayer helps the infiltration of the bone marrow matrix and the factor and adhering to of cell; Help the exchange of cytotrophy material.
Embodiment 2
On the rabbit animal model, repair the operation process of cartilage defect experiment
For verifying the effect of decalcification cortical-bone articular cartilage bracket for regenerating in method for repairing and mending of vertical boring of the present invention, we have carried out the sham operated experiment on the rabbit animal model.Select New Zealand's large ear rabbit of 2.5-3.0kg, anesthesia, preserved skin, sterilization, as shown in Figure 6, the otch knee joint is exposed the femur coaster, does the 4mm diameter on the femur coaster, dark damaged of 2mm, after subchondral bone is holed little fracture processing, remove the 4mm diameter at the boring decalcification cortical bone support that has disinfected, the cylinder that 2mm is high is packed into damaged.The layering sew up wound, 12 weeks of postoperative draw materials, and carry out nuclear magnetic resonance, NMR, electron microscopy, histological observation the results are shown in Figure 7~9.
Fig. 7 New Zealand large ear rabbit uses cartilage joint bracket for regenerating of the present invention to carry out the nuclear magnetic resonance, NMR figure of repair of cartilage after 12 weeks.From the nuclear magnetic resonance, NMR figure that squeezes into contrast agent as seen, the repair place surface smoothing, contrast agent does not enter the repair place, shows that the repair place does not have to subside.Subchondral bone becomes low signal, and similar cartilage signal conforms to tissue slice.Knee joint is not seen synovial hyperplasia, obviously immune inflammation reaction such as hydrops.
Fig. 8 New Zealand large ear rabbit uses cartilage joint bracket for regenerating of the present invention to carry out the substantially figure of repair of cartilage after 12 weeks.The repair place of visible arrow indication on the cardinal principle specimen has presented the cartilage sample and changed, and is very similar with normal surrounding tissue, not obvious with the intersection of normal cartilage, the slit do not occur.Do not see obvious immune inflammation reaction.
Fig. 9 New Zealand large ear rabbit uses cartilage joint bracket for regenerating of the present invention to carry out the histology figure of repair of cartilage after 12 weeks.As seen the decalcification cortical bone support of the repair place of arrow indication is substituted by new life's hyaline cartilage fully, and subsiding does not appear in the repair place, with the intersection fluid-tight engagement of the normal cartilage on figure right side, blur boundary.Because the early stage subchondral bone of observing is not also substituted by bone, presents chondroid tissue.Do not see inflammatory cell infiltrations such as macrophage, do not see obvious immune inflammation reaction.
Figure 10 is in the observed result in 24 weeks, and cartilage defect is repaired fully, presents hyaline cartilage.Cartilage and subchondral bone are repaired fully.Do not see inflammatory cell infiltrations such as macrophage, do not see obvious immune inflammation reaction, the biocompatibility of cartilage joint bracket for regenerating of the present invention as can be known is good.
This is 24 hours decalcification cortical bone support after implanting for Figure 11, takes out from the centre and cuts, the scanning electron microscope of tangent plane.As seen have on each layer collagen gap of many cell adhesions and support, can prove that the structure of the decalcification cortical-bone articular cartilage bracket for regenerating of vertical boring of the present invention is easy to cell absorption very much.
Claims (5)
1, a kind of decalcification cortical-bone articular cartilage bracket for regenerating of vertical boring, be a kind of animal decalcification cortical bone, be provided with the hole of 0.1~1mm diameter at horizontal surface perpendicular to described decalcification cortical bone, the hole interbody spacer is 0.5~5mm, form three-dimensional porous space network, porosity reaches 5%-20%.
2, the decalcification cortical-bone articular cartilage bracket for regenerating of vertical boring as claimed in claim 1 is characterized in that the cortical bone decalcification that described decalcification cortical bone is cattle or goat makes.
3, the preparation method of the decalcification cortical-bone articular cartilage bracket for regenerating of a kind of claim 1 or 2 described vertical borings is characterized in that step is:
The first step, cortical bone decalcification: the cortical bone of getting cattle or goat, remove bone marrow, be soaked in 2 ℃-10 ℃, the concentration of pH between 6-10 is in the 0.1mol/L-0.7mol/L ethylenediaminetetraacetic acid decalcifying Fluid, decalcifying Fluid is changed every day, the decalcifying Fluid of changing every day detects calcium ion concentration, Ca chelate concentration<0.5g/L in decalcifying Fluid, be complete decalcification, cortical bone taken out carry out the x line and CT detects, see whether decalcification fully, fully the cortical bone of decalcification enters next step, the cortical bone that does not have a complete decalcification again decalcification until complete decalcification;
Second step, boring: perpendicular to the diameter punching of the horizontal surface of decalcification cortical bone, set hole interbody spacer 0.5-5mm, be cut into the decalcification cortical-bone articular cartilage bracket for regenerating that design size is promptly vertically holed with 0.1-1mm.
4, the preparation method of the decalcification cortical-bone articular cartilage bracket for regenerating of vertical boring as claimed in claim 3 is characterized in that described decalcifying Fluid is to use the deionized water preparation, and plastic containers are contained and carried.
5, the preparation method of the decalcification cortical-bone articular cartilage bracket for regenerating of vertical boring as claimed in claim 3 is characterized in that the decalcification cortical-bone articular cartilage bracket for regenerating of described vertical boring uses cobalt 60 irradiations sterilization in 12-24 hour before use.
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