CN105770992A - Surface pre-osteogenesis implant and preparation method thereof - Google Patents
Surface pre-osteogenesis implant and preparation method thereof Download PDFInfo
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/02—Inorganic materials
- A61L27/04—Metals or alloys
- A61L27/06—Titanium or titanium alloys
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/02—Inorganic materials
- A61L27/10—Ceramics or glasses
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/28—Materials for coating prostheses
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/3641—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix characterised by the site of application in the body
- A61L27/3645—Connective tissue
- A61L27/365—Bones
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2400/00—Materials characterised by their function or physical properties
- A61L2400/18—Modification of implant surfaces in order to improve biocompatibility, cell growth, fixation of biomolecules, e.g. plasma treatment
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2430/00—Materials or treatment for tissue regeneration
- A61L2430/12—Materials or treatment for tissue regeneration for dental implants or prostheses
Abstract
The invention discloses a surface pre-osteogenesis implant and a preparation method thereof, and belongs to the field of planting and repairing after absence of teeth. The preparation method disclosed by the invention comprises the following steps: obtaining osteosarcoma cells from fresh excised osteosarcoma tissues, performing culturing so as to form cell membrane sheets in culture mediums containing extracellular matrix secretion promoting factors, then wrapping the implant with the cell membrane sheets so as to form implant cell membrane sheet composite bodies, performing osteogenic induction on the composite bodies, performing pre-osteogenesis on the surface of the implant, after the completion of the pre-osteogenesis, removing osteosarcoma cells, and performing cleaning, disinfection and sterilization so as to obtain the surface pre-osteogenesis implant finally. The osteosarcoma cells used in the pre-osteogenesis process of the implant are extensive in sources, easy to obtain and rapid to amplify; besides, the surface pre-osteogenesis implant has good biocompatibility, after the surface pre-osteogenesis implant is planted into jaw bones, a stable synostosis effect is easy to form between the surface pre-osteogenesis implant and autogenous bones of a patient, and the surface pre-osteogenesis implant is quite suitable for patients with poor whole body conditions or exceptional bone metabolism, so that the success rate of oral implantology can be increased, the application range of the oral implantology is further extended, and more patients with absence of teeth can enjoy comfort and convenience brought by the oral implantology.
Description
Technical field
The present invention relates to stomatology absence of tooth plantation repair materials field, be specifically related to a kind of pre-skeletonization implantation body in surface and preparation method thereof.
Background technology
Tooth-planting is to be implanted in the jawbone lacking tooth patient by the artificial tooth root that titanium alloy or other materials make and make a kind of technology of artificial tooth thereon.Tooth-planting can recover the masticatory function of patient well, is increasingly subject to the welcome of absence of tooth patient.In order to promote Osseointegrated implants and improve the success rate of plantation that at present implantation body is had a lot of method of modifying.
First it is physical modification, refer mainly to the change of implant surface structure, physical modification can increase hydrophilic and the surface area of implantation body, the surface that formed is micro-coarse structured, and to be conducive to forming machinery between implant surface and osseous tissue sealed, but physical modification is limited to the facilitation of Osseointegrated implants, it is only suitable for the basis modified as implant surface.
Next to that chemical modification, refer mainly to change the chemical composition of implant surface, bone biomimetic material is applied to implant surface, implantation body is made to have good biocompatibility and then promote Osseointegrated implants preferably, but after Osseointegrated implants, chemical modification there is also the catabolite of biomimetic material may to shortcomings such as Osseointegrated implants have a negative impact.
Implantation body's biological modification of rising in recent years is more and more interested to researchers, specific cell, albumen or siRNA etc. are applied to implant surface mainly by modes such as parcel or sprayings by it, change the microenvironment of Osseointegrated implants, promote cell proliferation and differentiation and the sclerotin secretion with ossification, along with progress of research, it have also been realised that carry out biological modification to there is also the problems such as bioactive substance half-life shorter, somewhat expensive.
Thus the synosteosis of implantation body needs to be further improved by other approach, we explore and utilize cell patch technology, use sclerotin secretion capacity and all stronger osteosarcoma cell of multiplication capacity, implantation body is carried out the biological modification of the pre-skeletonization form in surface, to expect better to promote Osseointegrated implants more easily, promote tooth-planting success rate.This is a kind of brand-new implantation body's biological modification method, and we demonstrate, by zoopery, the facilitation that surface pre-skeletonization implantation body is good to Osseointegrated implants.
Summary of the invention
It is an object of the invention to provide a kind of pre-skeletonization implantation body in surface and preparation method thereof, the method adopts the method for tissue engineering that the implantation body used by tooth-planting is carried out improvement biology, and this implantation body can expand the scope of application of tooth-planting and improve the success rate of tooth-planting.
The present invention is achieved through the following technical solutions:
The preparation method of the pre-skeletonization implantation body in a kind of surface, comprises the following steps:
1) taking fresh in vitro osteosarcoma tissue, remove macroscopic blood vessel fascia, then shred, piece of tissue being digested with collagenase is single osteosarcoma cell, after terminating digestion containing blood serum medium, and centrifuging and taking cell precipitation；
2) filter after resuspended for cell precipitation with PBS, filter liquor is centrifuged again, sucks the supernatant after being centrifuged, precipitate with DMEM complete medium re-suspended cell, it is thus achieved that osteosarcoma cell suspension；
3) being joined by osteosarcoma cell suspension in culture bottle and cultivate, the osteosarcoma cell of cultivation changes liquid once in every 2 ~ 3 days, until cell fusion reaches 80 ~ 90%, with Secondary Culture after trypsinization；
4) the 3rd generation osteosarcoma cell obtained is cultivated in the DMEM culture medium containing the short Extracellular Matrix Secretion factor, make the growth of osteosarcoma cell cladding form osteosarcoma cell diaphragm；
5) after being repaired according to implant diameter and diameter by osteosarcoma cell diaphragm, parcel implantation body, forms implantation body's cell patch complex；
6) implantation body's cell patch complex is put in osteogenic induction culture fluid carry out osteogenic induction at incubator, and regularly change liquid；
7) after having enough osteosis, from culture fluid, take out implantation body's cell patch complex, remove the osteosarcoma cell diaphragm of parcel implantation body, remove the osteosarcoma cell of remnants；
8) the pre-skeletonization implantation body in surface obtained is carried out cleaning thoroughly, sterilization, sterilizing.
Step 1) is with type i collagen enzyme at 37 DEG C, digests 60min.
Step 2) in adopt aperture be that the cell strainer of 100 μm is filtered.
Step 3) is by osteosarcoma cell suspension when 37 DEG C, 5%CO2 concentration and saturated humidity, cultivate with the DMEM complete medium containing 10%FBS.
Step 3) is with, after the trypsinization of 0.25%, going down to posterity in the ratio of 1:3.
Step 4) is the 3rd generation osteosarcoma cell is grown to degrees of fusion when being 80%, the short Extracellular Matrix Secretion factor is joined in culture medium, within every two days, changing liquid once, period often changes liquid and once rejoins the short Extracellular Matrix Secretion factor, until forming the cell patch containing multilamellar osteosarcoma cell.
Step 6) is implantation body's cell patch complex of acquisition is put into the osteogenic induction culture fluid being added with vitamin C, dexamethasone, sodium β-glycerophosphate, temperature 37 DEG C, 5%CO2 concentration, saturated humidity incubator in quiescent culture, taking out to be put in 37 DEG C of constant temperature oscillators after 1 week and be cultivated for 2 weeks when 5%CO2 concentration, saturated humidity, every about 3 days of period changed liquid once.
Step 7) is aseptically use cell scraper, distilled water ultrasonic irrigation and cell pyrolysis liquid thoroughly to be removed by the osteosarcoma cell of implant surface.
Step 8) is use Co60 irradiation thoroughly to kill antibacterial, virus cleaning, the pre-skeletonization implantation body in dried surface.
Described implantation body is implantation body in medical cranial maxillofacial bone, and material is titanium alloy or pottery, is shaped as column, tapered or foliaceous.
The invention also discloses the pre-skeletonization implantation body in surface adopting said method to prepare.
Compared with prior art, the present invention has following useful technique effect:
The preparation method of the pre-skeletonization implantation body in a kind of surface disclosed by the invention, implant surface is carried out external pre-skeletonization, biological synosteosis has been formed before plantation is implanted, and surface preformation osteoplaque is the natural secretion sclerotin that biocompatibility is splendid, it is more easy to the synosteosis stable with the autologous bone formation of patient after implantation body implants jawbone, and the catabolite that long-term synosteosis is had adverse effect will not be produced, so the pre-skeletonization implantation body in this surface is highly suitable for implantation body's implantation region poor arterial inflow, sclerotin healing ability, it is prone to the situations such as infection, the patient jawbone inner surface pre-skeletonization implantation body abnormal at these bone metabolisms also can form good synosteosis.Therefore, implant surface is carried out pre-skeletonization and processes the scope of application that can expand tooth-planting further and improve the success rate of tooth-planting, make more absence of tooth patient enjoy the benefit that tooth-planting brings.Additionally the present invention is using osteosarcoma cell as cell derived, and its sclerotin secretion capacity is strong, have can continuous passage, be prone to cultivate, grow the advantages such as rapid, so source is very extensive.Utilizing cell patch to carry out the pre-skeletonization of implant surface and also have easy and simple to handle, raw material is cheap, and equipment requirements is relatively low, conveniently carries out large-scale standardized production and the advantage such as promotes the use of.
Accompanying drawing explanation
Fig. 1 is induction osteosarcoma cell cladding growth figure.
Fig. 2 is osteosarcoma cell diaphragm figure.
Fig. 3 is the osteosarcoma cell diaphragm figure after finishing.
Fig. 4 is cell patch parcel implantation body figure.
Fig. 5 is the pre-skeletonization figure of implant surface.
Fig. 6 is the implantation body figure after the pre-skeletonization in surface.
Fig. 7 is that Micro-CT detects Osseointegrated implants figure, and wherein (A) is common species implant, and (B) is the pre-skeletonization implantation body in surface.
1. osteosarcoma cell in figure, 2. cell culture fluid, the 3. short Extracellular Matrix Secretion factor, 4. culture dish, 5. osteosarcoma cell diaphragm, 6. the osteosarcoma cell diaphragm after finishing, 7. cell scraper, 8. implantation body, 9. implantation body's cell patch complex, 10. osteogenic induction liquid, 11. surface preformation osteoplaques.
Detailed description of the invention
Below in conjunction with specific embodiment, the present invention is described further, below described in be the specific explanations to the present invention, rather than limit scope of invention.
The preparation method of the pre-skeletonization implantation body in a kind of surface disclosed by the invention, comprises the steps:
1) under aseptic condition, fresh in vitro osteosarcoma tissue is removed macroscopic blood vessel and fascia, then use PBS to rinse and remove chip；
2) osteosarcoma tissue after flushing being shredded, put in sterile centrifugation tube, adding type i collagen enzyme by piece of tissue digestion is single osteosarcoma cell, re-uses and terminates digesting containing blood serum medium；
3) after being centrifuged by postdigestive product, remove upper strata Digestive system, retain the cell precipitation at the bottom of centrifuge tube pipe；
4) PBS is used after resuspended for the cell precipitation at the bottom of centrifuge tube pipe, cell strainer will to be used to filter, filter liquor re-started centrifugal, remove supernatant liquid, retain the precipitation at the bottom of centrifuge tube pipe；
5) use DMEM complete medium by resuspended for the cell precipitation at the bottom of centrifuge tube pipe, it is thus achieved that osteosarcoma cell suspension, join in Tissue Culture Flask and cultivate；
6) osteosarcoma cell obtained changes liquid once in every 3 days, when cell fusion reaches about 80%, and Secondary Culture after trypsinization；
7) the 3rd fat subsitutes stem cell obtained is cultivated in the culture dish of 6cm, add the DMEM culture medium containing the short Extracellular Matrix Secretion factor, make the growth of osteosarcoma cell cladding form osteosarcoma cell diaphragm under the effect of the short Extracellular Matrix Secretion factor；
8) after being repaired according to implant diameter and diameter by osteosarcoma cell diaphragm, parcel implantation body, forms implantation body's cell patch complex；
9) it is configured to self-bone grafting culture fluid, implantation body's cell patch complex is put in osteogenic induction culture fluid and carries out osteogenic induction at incubator, and regularly change liquid；
10) after having enough osteosis, from culture fluid, take out implantation body's cell patch complex, remove the osteosarcoma cell diaphragm of parcel implantation body and remaining osteosarcoma cell；
11) the pre-skeletonization implantation body in surface obtained is carried out cleaning thoroughly, sterilization, sterilizing.
A kind of pre-skeletonization implantation body in surface specifically can obtain by the following method.
1, osteosarcoma cell is obtained
nullTake fresh in vitro osteosarcoma tissue，Macroscopic fascia is removed under aseptic condition、Blood vessel，PBS cuts tissue shear to about 1mm3 size with Sterile ophthalmic after rinsing，Put in aseptic centrifuge tube，Add 0.2% NTx enzyme of 3 times of volumes and mix，60min is digested under 37 DEG C of environment，Period shakes centrifuge tube once every 5min，Digestion is centrifugal 5min(1000r/min after terminating)，Remove upper strata Digestive system and floating tissue，Add the precipitation at the bottom of the resuspended centrifuge tube pipe of PBS，Re-use the strainer filtering re-suspension liquid of 100um，By filter liquor recentrifuge 5min(1000r/min)，Use the DMEM complete medium containing 10%FBS by resuspended for the cell precipitation at the bottom of centrifuge tube pipe specifically，Obtain osteosarcoma cell suspension，Cell suspension is joined in Tissue Culture Flask at 37 DEG C、5%CO2 concentration、Cultivate with the DMEM complete medium containing 10%FBS when saturated humidity，Liquid is changed first after 2d，When cell fusion reaches 80%，With 0.25% trypsinization，Go down to posterity in 1:3 ratio，Take the 3rd fat subsitutes stem cell to be layered in 6cm culture dish.
2, prepared by osteosarcoma cell diaphragm
When in culture dish, degrees of fusion reaches 80% to the 3rd generation osteosarcoma cell, by the short Extracellular Matrix Secretion factor, such as L-AA-2-phosphate ester joins in culture medium according to 50ug/ml concentration, within every 2 days, change liquid once, period often changes liquid and once rejoins L-AA-2-phosphate ester according to 50ug/ml concentration, until forming the cell patch containing multilamellar osteosarcoma cell, referring to Fig. 1, Fig. 2 and Fig. 3.
3, implantation body's cell patch compound system is standby
With cell scraper, osteosarcoma cell diaphragm is come together from culture dish four circumference center, the rectangle being suitable for parcel it is processed into by implant diameter and form, then closely, uniformity ground parcel implantation body root, it is thus achieved that implantation body's cell patch complex, referring to Fig. 4.
4, the pre-skeletonization of implant surface
Implantation body's cell patch complex of acquisition is put into containing 50mg/ml ascorbic acid, 10nmol/L dexamethasone, 10mmol/L sodium β-glycerophosphate osteogenic induction culture fluid in, temperature 37 DEG C, 5%CO2 concentration, saturated humidity incubator in quiescent culture.Taking out to be put in 37 DEG C of constant temperature oscillators after 1 week and be cultivated for 2 weeks when 5%CO2 concentration, saturated humidity, every 3 days of period changed liquid once, referring to Fig. 5.
5, the removal of osteosarcoma cell diaphragm
Complete after the pre-skeletonization of implant surface in osteogenic induction liquid until implantation body's cell patch complex, cell scraper is aseptically used to be struck off by osteosarcoma cell diaphragm, use the cleaning of distilled water ultrasonic irrigation, then implantation body is put into the osteosarcoma cell of 15 minutes cracking residuals in cell pyrolysis liquid, then reuse distilled water and carry out ultrasonic irrigation, repeat above step twice, thoroughly to remove the osteosarcoma cell in implantation body, obtain the pre-skeletonization implantation body in surface, referring to Fig. 6.
6, the sterilization of the pre-skeletonization implantation body in surface
Use sealing machine by cleaning, the encapsulation of the pre-skeletonization implantation body in dried surface, then use Co60 irradiation sterilization 60min, thoroughly to kill antibacterial, fungus and virus.
Pre-to common species implant and surface skeletonization implantation body is implanted in SD rat tibia by we by implant operation, after the synosteosis of 12 weeks, carries out the synosteosis situation of Micro-CT Scanning Detction bone regeneration around implant.Being common species implant referring to Fig. 7 (A), (B) is the pre-skeletonization implantation body in surface.Scanning result shows, in Fig. 7 (B), the pre-skeletonization in surface makes implantation body's (light areas) new bone (darker regions) around form relatively common species implant substantially to be increased, and finds after digital three-dimensional reestablishing, bone volume mark: common species implant 23.5%, the pre-skeletonization in surface makes implantation body 45.6%；Bone trabecula quantity: common species implant 2.6/mm, the pre-skeletonization in surface makes implantation body 3.9/mm, has obvious significant difference between two groups.Result proves that surface preformation bone relatively common species implant can significantly facilitate Osseointegrated implants.
In sum, osteosarcoma cell in vitro osteosarcoma tissue is cultivated by the present invention, the culture medium containing the short Extracellular Matrix Secretion factor forms cell patch, then cell patch is wrapped in bone regeneration around implant, then in Osteogenic Induction Medium, carry out the pre-skeletonization of implant surface, after having enough New bone formations, remove osteosarcoma cell, finally obtain the pre-skeletonization implantation body in surface through sterilization.Demonstrated by zoopery and implant surface is carried out pre-skeletonization process and can dramatically increase the biocompatibility of implantation body, promote the synosteosis of implantation body.
Claims (6)
1. the preparation method of the pre-skeletonization implantation body in surface, it is characterised in that comprise the following steps:
1) fresh in vitro osteosarcoma tissue is taken, with collagenase by piece of tissue digestion for after single osteosarcoma cell, centrifuging and taking cell precipitation；
2) by culture medium re-suspended cell precipitation, it is thus achieved that osteosarcoma cell suspension；
3) osteosarcoma cell suspension is cultivated, with Secondary Culture after trypsinization；
4) osteosarcoma cell obtained is cultivated in the culture medium containing the short Extracellular Matrix Secretion factor, make the growth of osteosarcoma cell cladding form osteosarcoma cell diaphragm；
5) parcel implantation body after being repaired by osteosarcoma cell diaphragm, forms implantation body's cell patch complex；
6) implantation body's cell patch complex is put in osteogenic induction culture fluid carry out osteogenic induction at incubator, and regularly change liquid；
7) after having enough osteosis, from culture fluid, take out implantation body's cell patch complex, remove the osteosarcoma cell diaphragm of parcel implantation body, remove the osteosarcoma cell of remnants；
8) the pre-skeletonization implantation body in surface obtained is carried out cleaning thoroughly, sterilization, sterilizing.
2. the preparation method of the pre-skeletonization implantation body in a kind of surface according to claim 1, it is characterised in that step 1) is to use type i collagen enzymic digestion.
3. the preparation method of the pre-skeletonization implantation body in a kind of surface according to claim 1, it is characterised in that be that osteosarcoma cell suspension is cultivated when 37 DEG C, 5%CO2 concentration and saturated humidity in step 3).
4. the preparation method of the pre-skeletonization implantation body in a kind of surface according to claim 1, it is characterised in that step 6) be implantation body's cell patch complex is put into containing dexamethasone, ascorbic acid, β-phosphoglycerol culture fluid in carry out osteogenic induction.
5. the preparation method of the pre-skeletonization implantation body in a kind of surface according to claim 1, it is characterised in that step 8) is to use the sterilizing of Co60 x ray irradiation x implantation body.
6. adopt the pre-skeletonization implantation body in surface that the method described in claim 1 ~ 5 prepares.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111808817A (en) * | 2020-07-24 | 2020-10-23 | 上海昊佰生物科技有限公司 | Culture method of osteosarcoma organs and bone tumor culture medium thereof |
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