CN103100112A - Preparation method and use of graft material in double membrane structure - Google Patents
Preparation method and use of graft material in double membrane structure Download PDFInfo
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- CN103100112A CN103100112A CN2012105250979A CN201210525097A CN103100112A CN 103100112 A CN103100112 A CN 103100112A CN 2012105250979 A CN2012105250979 A CN 2012105250979A CN 201210525097 A CN201210525097 A CN 201210525097A CN 103100112 A CN103100112 A CN 103100112A
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Abstract
The invention belongs to the field of tissue engineering and biomaterials and discloses a preparation method and use of a graft material in a double membrane structure. The graft material comprises stem cell membrane patch segments and platelet-rich fibrin membrane particles, wherein the stem cells are firstly expanded-cultured, and then induction-cultured by using a membrane induction fluid to obtain a stem cell membrane patch, and the stem cell membrane patch is sheared to prepare the cell membrane patch segment; a platelet-rich fibrin gel is extruded, then liquid content of the extruded platelet-rich fibrin gel is removed to obtain the platelet-rich fibrin membrane, and the platelet-rich fibrin membrane is sheared to form particles; and the cell membrane patch segments and the platelet-rich fibrin membrane particles are mixed according to an optimal proportioning relation of in-vitro screening to prepare the needed graft material. The graft material can be widely applied to tissue repair and regeneration of small-area lesion in oral activity and other parts of the body, and the tissue repair effect is improved.
Description
Technical field
The invention belongs to organizational project and technical field of biological material, relate to a kind of pair of membrane structure graft materials, particularly the Preparation method and use of the two membrane structure graft materials of the compound rich platelet fibrin film of a kind of stem cell diaphragm fragment.
Background technology
At present, utilize stem cells technology to carry out Repair of tissue defect and usually adopt stem cell suspension and the compound method of exogenous timbering material, principal mode has three kinds: simple cell suspension local transplantation; Cell suspension inoculation is in exogenous timbering material; Cell suspension inoculation is external row dimensional culture after timbering material.Yet there are many deficiencies in above-mentioned implantation method: at first, cell is in obtaining the process of suspension with trypsinization, and the structure of intercellular signal molecule and extracellular matrix is destroyed, and the activity of cell itself also has been subject to impact; Secondly, the amount of cell transplantation is difficult to control, cell concentration very little, cell is difficult to be colonizated in rapidly to be transplanted locally, cell concentration is too many, can cause again local cells to pile up, and is unfavorable for that cell stretches and nutrition supply; At last, the transplanting of timbering material and degraded may cause host toxicity reaction and immunoreation, and undegradable timbering material can cause fibers encapsulation, affects blood confession and organizational structure and rebuilds.Therefore, need to seek a kind of new method for preparing the stem cell compound support frame material, reduce the loss of stem cell, improve its biologic activity, reduce the absorption of timbering material and host's immunoreation, improve the effect of Repair of tissue defect.
Summary of the invention
In order to overcome above-mentioned the deficiencies in the prior art, the object of the present invention is to provide the graft materials and its production and use of two membrane structures of the compound PRF of a kind of CSF, CSF (cell sheet fragment) is the cell patch fragment, PRF (platelet-rich fibrin) is the rich platelet fibrin, graft materials of the present invention can be applied to the regeneration as the reparation of cartilage, soft tissue defects and dental pulp, periodontal membrane tissue, effectively improves the effect of tissue repair.
To achieve these goals, the technical solution used in the present invention is:
A kind of pair of membrane structure graft materials is characterized in that: by stem cell diaphragm fragment and rich platelet fibrin film granulometric composition.
Preferably, described stem cell is selected from mesenchymal stem cells MSCs, fat stem cell, dental pulp stem cell or periodontal ligament stem cell.
Preferably, described rich platelet fibrin diaphragm granule obtains after via centrifugal blood.
Preferably, cell patch adopts six orifice plates to be prepared, and the ratio of cell patch fragment and rich platelet fibrin film is as follows: every six orifice plate cell patch quantity: be used for the blood milliliter number of centrifugal acquisition rich platelet fibrin diaphragm=1: 3.5～4.0.
Preferably, described stem cell and rich platelet fibrin all derive from same individuality.
Preferably, stem cell must be prepared into the form of cell patch fragment.
A kind of preparation method of graft materials is characterized in that its preparation process comprises the following steps:
The first step, the cultivation of stem cell diaphragm: primary stem cell is inoculated in six orifice plates after amplification culture, merges until growth and is cultivated with the diaphragm induced liquid to 80% the time, changes liquid once in 3 days, until occurring thinking that cell patch is ripe when curling around cell patch;
Second step, prepare stem cell diaphragm fragment: the method with mechanical stripping is peeled off cell patch from culture dish, with aseptic eye scissors, it is shredded, and is prepared into cell patch fragment (big or small approximately 0.5mm * 0.5mm);
the 3rd step, extract rich platelet fibrin diaphragm granule: according to required ratio, take 10ml as unit, blood (not adding any anticoagulant) is placed in the glass centrifuge tube of aseptic glass centrifuge tube or plastic bushing, with the speed of 3000rpm/min centrifugal 10 minutes immediately, after standing 5 minutes, aseptic tweezer clamp takes out the rich platelet fibrin gel of centrifuge tube mid portion, its bottom erythrocyte end is gently dipped in to remove unnecessary erythrocyte on sterile gauze, keep complete light yellow gel part and bottom and adhere on a small quantity firmly erythrocyte, latter's tiling is placed between sterile gauze, the wherein liquid component of gently extruding out, be prepared into a faint yellow tough and tensile membrane structure, with aseptic eye scissors, it is shredded, be prepared into the rich platelet fibrin diaphragm granule of big or small approximately 0.5mm * 0.5mm * 0.5mm,
The 4th step, cell patch fragment and the rich platelet fibrin diaphragm granule of preparation are placed in sterile petri dish, proportionally manually with its mixing, guarantee both fully to mix, can obtain two membrane structure graft materials of stem cell diaphragm fragment and the compound rear formation of rich platelet fibrin film.
Preferably, the ratio of cell patch fragment and rich platelet fibrin film is as follows: every six orifice plate cell patch quantity: be used for the blood milliliter number of centrifugal acquisition rich platelet fibrin diaphragm=1: 3.5～4.0.
Graft materials of the present invention is the application in Repair of tissue defect among a small circle in oromaxillo-facial region or whole body.
Graft materials of the present invention in preparation oromaxillo-facial region or whole body among a small circle Repair of tissue defect with the purposes in graft materials.
The purposes of the composite that graft materials of the present invention and other biological material form in the preparation Repair of tissue defect is used graft materials.
Two membrane structure graft materials of the compound rich platelet fibrin film of a kind of stem cell diaphragm fragment are by stem cell diaphragm fragment, rich platelet fibrin film granulometric composition.The stem cell form can be different according to the type of repair tissue, comprise mesenchymal stem cells MSCs, fat stem cell, dental pulp stem cell and periodontal ligament stem cell etc., and the rich platelet fibrin film is that blood prepares after centrifugal.Externally carry out both screenings of proportioning, with the propagation of stem cell, minute be turned to evaluation index, determine that both optimum volume ratio is: six orifice plates obtain cell patches: be used for the blood of centrifugal acquisition rich platelet fibrin diaphragm=1: 3.5～4.0.Carry out the preparation of graft according to this proportioning, then it is migrated in experimental animals, carry out the checking of experiment in body, result proves, the two membrane structure grafts under this proportioning have good tissue repair and tissue regeneration effect.
The mode that graft materials two components are mixed is: stem cell is prepared into the diaphragm pieces, the rich platelet fibrin film is prepared into graininess (big or small approximately 0.5mm * 0.5mm * 0.5mm), both armstrong's patent mixing in sterile petri dish, fully mix two components.
The present invention also provides the preparation method of described stem cell diaphragm fragment and rich platelet fibrin membrane granule, comprises the following steps:
The first step, the cultivation of stem cell diaphragm: primary stem cell is inoculated in six orifice plates after amplification culture, merges until growth and is cultivated with the diaphragm induced liquid to 80% the time, changes liquid once in 3 days, until occurring thinking that cell patch is ripe when curling around cell patch;
Second step, prepare stem cell diaphragm fragment: the method with mechanical stripping is peeled off cell patch from culture dish, with aseptic eye scissors, it is shredded, and is prepared into cell patch fragment (big or small approximately 0.5mm * 0.5mm);
the 3rd step, extract rich platelet fibrin diaphragm granule: according to required ratio, take 10ml as unit, blood (not adding any anticoagulant) is placed in the glass centrifuge tube of aseptic glass centrifuge tube or plastic bushing, with the speed of 3000rpm/min centrifugal 10 minutes immediately, after standing 5 minutes, aseptic tweezer clamp takes out the rich platelet fibrin gel of centrifuge tube mid portion, its bottom erythrocyte end is gently dipped in to remove unnecessary erythrocyte on sterile gauze, keep complete light yellow gel part and bottom and adhere on a small quantity firmly erythrocyte, latter's tiling is placed between sterile gauze, the wherein liquid component of gently extruding out, be prepared into a faint yellow tough and tensile membrane structure, with aseptic eye scissors, it is shredded, be prepared into the rich platelet fibrin diaphragm granule of big or small approximately 0.5mm * 0.5mm * 0.5mm,
The 4th step, cell patch fragment and the rich platelet fibrin diaphragm granule of preparation are placed in sterile petri dish, proportionally manually with its mixing, guarantee both fully to mix, can obtain the two membrane structure graft materials of the compound rich platelet fibrin film of stem cell diaphragm fragment.
During two membrane structure graft materials of the compound rich platelet fibrin of described stem cell diaphragm fragment membrane granule can be used among a small circle cartilage and the reparation of soft tissue defects, can directly complex be placed in damaged place for operating better position, the visual field, not good or can't expose the case of defective region fully for the operation visual field, also graft materials can be placed in syringe or adopt endoscopic procedures that it is injected in damaged place.
The present invention compared with prior art has the following advantages:
The timbering material that existing tissue engineering technique uses is all almost exogenous material, seed cell is inoculated on exogenous material with the form of cell suspension, add again one or more exogenous growth factors, the weak point of this method is: ectogenic timbering material degraded needs certain hour, and has the inadequate possibility of degraded; And seed cell is inoculated with suspensions and be transplanted to tissue local after, very easily run off, make it possible to the stable cell concentration that plays a role of local field planting obviously tail off; And the somatomedin kind of exogenous interpolation is less, and fully different from multiple somatomedin eurythmy under physiological situation, the collaborative pattern that plays a role, these all can affect the effect of tissue engineering technique clinical practice.The most outstanding characteristics of the present invention are the shortcomings that fundamentally solved above-mentioned prior art, be in particular in following some:
1) the low repulsion.Two kinds of compositions in the cograft material of this law preparation, namely stem cell diaphragm fragment (CSF) and rich platelet fibrin film (PRF) source is sufficient, has splendid biologic activity, has hardly immunological rejection and toxic reaction.
2) proportioning is good.One of important feature of the present invention is the best proportioning that has filtered out stem cell population and rich platelet fibrin film.Under this conditions of mixture ratios, cell has good propagation and differentiation capability, is conducive to tissue repair and regeneration, and this proportioning obtains the milliliter number of cell patch quantity and blood as the ultimate unit of proportioning with six orifice plates, easily accurately grasp, convenient operation.
3) high efficiency.The rich platelet fibrin film comes from blood, and is simple to operate, very easily obtains; Itself contain a large amount of somatomedin, comprise transforming growth factor β-1 (TGF β-1), platelet-derived growth factor (PDGF), insulin like growth factor (IGF), above-mentioned three kinds of somatomedin can promote cell migration, propagation, induce fibrin matrix to reinvent, and the secretion of promotion collagen stroma, play an important role in initial organization healing; With the exception of this, PRF also contains epidermal growth factor (EGF) and the somatomedin relevant to wound healing and osteanagenesis such as VEGF (VEGF).PRF itself is exactly the three-dimensional rack structure that the fibrin cross-linked network forms, and this structure can be so that somatomedin wherein slowly discharges.So the tissue repair of PRF and tissue regeneration effect mainly realize by two aspects, i.e. the regulating action of somatomedin and fibrin scaffold which effect.
4) easy to operate.The cell type of cell patch fragment (CSF) can be according to the difference of the zone of repair deficiency and types of organization and difference, comprise mesenchymal stem cells MSCs (BMSCs), fat stem cell (ASCs), dental pulp stem cell (DPSCs), periodontal ligament stem cell (PDLSCs) etc., above-mentioned cell all has been proved good tissue regeneration ability, and obtain relatively easily to be easy to amplification culture in the external short time; Cell patch technology itself has stronger tissue regeneration potential owing to having kept extracellular matrix and cytoactive than suspension cell; Simultaneously, diaphragm fragment technology has changed again traditional diaphragm and has transplanted pattern, diaphragm is prepared into a certain size fragment, on the one hand cell fragment is fully mixed with timbering material, the cell rapid field planting is in timbering material surface and stretch out synapse to extend to the support gap inner, both form an integral body (Fig. 7), on the other hand can be with injection cell to the zone that is difficult to expose fully the operation visual field.
5) infection.The red end of PRF has concentrated a large amount of leukocyte, and these leukocyte are transferred the immunologic function of body on the other hand on the one hand at the good antiinflammatory action of part performance; Simultaneously, PRF has also enlisted the services of a large amount of immunoregulatory factor in blood in preparation process, as IL-1 β, and IL-4, IL-6 and TNF-α bring into play the anti-infective effect jointly, are beneficial to organization healing.
Therefore, the cograft material of the inventive method preparation have that the source is abundant, indication extensively, preparation is simple, without immunological rejection and toxic reaction, without dispute of ethic, the advantage such as safe and effective.The method is of universal significance, and is applicable to every patient.
Description of drawings
Fig. 1 sees (take dental pulp stem cell as example) after cell patch is cultivated maturation substantially.
Fig. 2 is the microstructural HE dyeing of cell patch (take dental pulp stem cell as example).
Fig. 3 is cell patch scanning electron microscope structure (take dental pulp stem cell as example).
Fig. 4 is that the rich platelet fibrin is seen substantially, and wherein A is sight substantially in centrifugal rear standing 5 minutes; B is the PRF gel; C is the PRF film; D is the PRF granule.
Fig. 5 is the microstructural HE dyeing of rich platelet fibrin film, and wherein A is 40 times of amplifications, and B is 100 times of amplifications.
Fig. 6 is rich platelet fibrin film scanning electron microscope structure, and wherein A is its upper end, is the fibrin tridimensional network; B is its lower end, visible a large amount of platelet, leukocyte recruitment.
Fig. 7 is the scanning electron microscope of two membrane structure grafts of forming after the compound rich platelet fibrin of dental pulp stem cell diaphragm fragment membrane granule, the visible cell secure adhesion is in the PRF surface, and stretch out synapse cell to the space of PRF tridimensional network, both strong bonded, form whole.
Fig. 8 is cell proliferation situation under different conditions of mixture ratios, and wherein A is the impact that PRF breeds dental pulp stem cell, and B is that PRF is on the impact of periodontal ligament stem cell propagation.
Fig. 9 be under different conditions of mixture ratios dental pulp stem cell to the expression of the differentiation phase DMP1 of odontoblast.
Figure 10 be under different conditions of mixture ratios periodontal ligament stem cell to the expression of periodontal ligament cell differentiation phase CP23.
Figure 11 repairs experimental result in the damaged body of rabbit condylar cartilage for the two membrane structures that adopt respectively simple mesenchymal stem cells MSCs diaphragm fragment, simple PRF granule, the compound rich platelet fibrin of mesenchymal stem cells MSCs diaphragm fragment membrane granule, and this figure is that experiment 2,4,8 all condyles are organized the gross examination of skeletal muscle result.
Figure 12 is that Figure 11 organizes capable HE coloration result.
Figure 13 is that Figure 11 organizes capable Toluidine blue staining result.
Figure 14 is the different cartogram of dying the tinctorial strength analysis of neocartilage district's toluidine blue in Figure 13, and abscissa represents group and time, and vertical coordinate represents the different average optical density value that dyes district's tinctorial strength.
Figure 15 is experimental result in the body that adopts respectively simple PRF granule, simple dental pulp stem cell diaphragm fragment, the two membrane structure row canine tooth marrow regeneration of the compound rich platelet fibrin of dental pulp stem cell diaphragm fragment membrane granule, and this figure is HE coloration result after January, February.
The specific embodiment
Below in conjunction with drawings and Examples, the present invention is carried out more detailed explanation.
The preparation method of two membrane structure graft materials of the compound rich platelet fibrin film of stem cell diaphragm fragment comprises checking two parts in external proportioning screening and body.
First: external proportioning screening
1. material and equipment
1.1 main agents
α-MEM culture medium (Hyclone company, the U.S.); 0.25% pancreatin (Hyclone company, the U.S.); Hyclone (Ilex purpurea Hassk.[I.chinensis Sims, Hangzhoupro, sky, Zhejiang bio tech ltd).
1.2 instrument and equipment
Multitube frame autobalance centrifuge (TD25-WS, Hunan, Hunan instrument Laboratory Instruments development corporation, Ltd.); Cell culture incubator (Heraeuc Hearcell, Thermo company, China); Six orifice plates (Nunc company, the U.S.); Superclean bench (BIOBASE, the U.S.).
2. operating procedure
2.1 the cultivation of stem cell:
1) with primary stem cell after amplification culture, go down to posterity according to 1: 2～1: 3 ratio;
2) get third generation stem cell and be inoculated in six orifice plates, cellar culture (α of 10%FBS-MEM culture fluid) is to cell fusion 80%.
2.2 rich platelet fibrin preparation:
1) will take 10ml as unit, blood (not adding any anticoagulant) be placed in the glass centrifuge tube of aseptic glass centrifuge tube or plastic bushing according to required ratio, with the speed of 3000rpm/min centrifugal 10 minutes immediately, standing 5 minutes;
2) aseptic tweezer clamp takes out the rich platelet fibrin gel of centrifuge tube mid portion, its bottom erythrocyte end is gently dipped in to remove unnecessary erythrocyte on sterile gauze, keep complete light yellow gel part and bottom and adhere on a small quantity firmly erythrocyte, latter's tiling is placed between sterile gauze, the wherein liquid component of gently extruding out is prepared into the faint yellow tough and tensile membrane structure of a rectangle;
3) it is tiled on sterile gauze, sterile scissors is cut off along its major axis, with its PRF that is prepared into 1/8 and 1/4, is placed in culture fluid standby.
2.3 stem cell and rich platelet fibrin film are cultivated altogether:
1) according to the ratio of 1/8,1/4,3/8,1/2 PRF, the PRF that shears is placed in the six orifice plate culture dishs of third generation stem cell, cellar culture 7 days changed liquid once in 3 days;
2) both cultivate altogether after 7 days and remove PRF, stem cell continues normal cultivation, changes liquid once in 3 days;
2.4 the detection of stem cells hyperplasia, differentiation:
1) respectively at both cultivating altogether the front detection of carrying out stem cell population in 1～7 day with being total to after cultivating, observe the impact of PRF on cell proliferation;
2) the Real-time PCR that both is total to 7,14,21 days capable stem cell related gene expressions after cultivating detects, and observes PRF to the impact of cell differentiation.
3. experimental result
3.1 stem cells hyperplasia
PRF has obvious facilitation to the propagation of dental pulp stem cell and periodontal ligament stem cell, and is the regular hour dependency.Two kinds of common characteristics of stem cell are: PRF concentration is from 1/8～3/8, the impact of its on cell proliferation presents obvious concentration dependent, and the facilitation with the 3/8PRF on cell proliferation is the most obvious, though the 1/2PRF on cell proliferation has a certain promotion, not as good as 3/8PRF effect obvious (as shown in Figure 8).
3.2 differentiation of stem cells
After both cultivating altogether 21 days, PRF has obvious rise effect to dental pulp stem cell to the expression of the specific proteins DMP1 of odontoblast direction differentiation, and be obvious concentration dependent, best with 1/2PRF, but the difference not statistically significant (Fig. 9) between itself and 3/8PRF; PRF also has obvious rise to the expression of periodontal ligament stem cell specific proteins CP23, with the concentration of 3/8PRF best (as shown in figure 10).
The result of comprehensive PRF on the impact of two kinds of dissimilar stem cells hyperplasia differentiation, we think that 3/8PRF all has obvious facilitation to propagation and the differentiation of stem cell, therefore select this concentration as optium concentration.Because every PRF film desired blood is 10mL, therefore final determine that both proportion relation is: every six orifice plate cell patch quantity: blood milliliter number=1: 3.75, in order to simplify the operation, we think 1: 3.5～4.0 all can.For transplantation effect in the body of verifying the graft under this conditions of mixture ratios, we carry out part experiment in body.
Second portion: transplant checking in object
The first step, the cultivation of stem cell diaphragm: primary stem cell is inoculated in six orifice plates after amplification culture, merges until growth and is cultivated with the diaphragm induced liquid to 80% the time, changes liquid once in 3 days, until occurring thinking that cell patch is ripe when curling around cell patch;
Second step, prepare stem cell diaphragm fragment: the method with mechanical stripping is peeled off cell patch from culture dish, with aseptic eye scissors, it is shredded, and is prepared into cell patch fragment (big or small approximately 0.5mm * 0.5mm);
In the 3rd step, extract rich platelet fibrin diaphragm granule: concrete grammar shreds with aseptic eye scissors the PRF film for preparing gained with the experiment in vitro part with it, be prepared into the rich platelet fibrin diaphragm granule of big or small approximately 0.5mm * 0.5mm * 0.5mm;
The 4th step, cell patch fragment and the rich platelet fibrin diaphragm granule of preparation are placed in sterile petri dish, proportionally manually with its mixing, guarantee both fully to mix, can obtain the two membrane structure graft materials of the compound rich platelet fibrin film of stem cell diaphragm fragment.
For operating position preferably, the visual field, the two membrane structure graft materials that prepare directly can be placed in damaged place during use, not good or can't expose the case of defective region fully for the operation visual field, also graft materials can be placed in syringe or adopt endoscopic procedures that it is injected in damaged place.
Effect for two membrane structure graft materials of verifying the compound rich platelet fibrin film of stem cell diaphragm fragment of the present invention, we have carried out respectively the two membrane structures of the compound rich platelet fibrin film of variety classes stem cell cell patch and have carried out the experiment of Repair of tissue defect, now as example, carry out following test take " it is damaged that the two membrane structures of the compound rich platelet fibrin film of bone marrow mesenchymal stem cells diaphragm fragment are repaired the rabbit condylar cartilage " and " the two membrane structures of the compound rich platelet fibrin film of canine tooth marrow stem cell diaphragm fragment are used for the dental pulp regenerative therapy " respectively:
Experiment one: it is damaged that the two membrane structures of the compound rich platelet fibrin film of bone marrow mesenchymal stem cells diaphragm fragment are repaired the rabbit condylar cartilage
1. material and equipment
1.1 main agents
α-MEM culture medium (Hyclone company, the U.S.); 0.25% pancreatin (Hyclone company, the U.S.); Hyclone (Ilex purpurea Hassk.[I.chinensis Sims, Hangzhoupro, sky, Zhejiang bio tech ltd); Vitamin C (Sigma company, the U.S.); Pentobarbital sodium (Germany, the packing of sky biological engineering Co., Ltd of section).
1.2 instrument and equipment
Multitube frame autobalance centrifuge (TD25-WS, Hunan, Hunan instrument Laboratory Instruments development corporation, Ltd.); Cell culture incubator (Heraeuc Hearcell, Thermo company, China); Six orifice plates (Nunc company, the U.S.); Superclean bench (BIOBASE, the U.S.); Nikon microscopy imaging system (XTJ30, Japan), analytical electron balance (FA1004, China), scanning electron microscope (S-4800 of Hitachi, Japan).
2. operating procedure
2.1 the cultivation of bone marrow mesenchymal stem cells (BMSCs)
The primary mesenchymal stem cells MSCs of rabbit adopts the α that contains 10% hyclone-MEM culture medium cellar culture, and the ratio according to 1: 2～1: 3 when cell fusion to 80% is carried out amplification culture, changes liquid once in every 2～3 days;
2.2BMSCs the preparation of diaphragm
When 1) BMSCs is cultured to the third generation, be passaged to 6 orifice plates; Change diaphragm culture fluid (containing 10% hyclone, the ascorbic α of 20mg/ml-MEM culture medium), the conventional continuation cultivated for two weeks, and the diaphragm culture fluid was changed once in every 2～3 days, noted lucifuge when changing liquid;
2) grow to peripheral cell patch when slightly curling until cell patch, think that namely cell patch is ripe, the cell patch of maturation is peeled off (as Fig. 1 from an end with ophthalmic tweezers, Fig. 2, shown in Figure 3), be placed in sterile petri dish, adopt aseptic eye scissors that it is cut into the fragment of big or small approximately 0.5mm * 0.5mm, standby.
2.3PRF preparation
1) according to required ratio, take 10ml as unit, rabbit blood (not adding any anticoagulant) is placed in the glass centrifuge tube of aseptic glass centrifuge tube or plastic bushing, with the speed of 3000rpm/min centrifugal 10 minutes immediately, standing 5 minutes;
2) in visible centrifuge tube this moment, blood is divided into three layers: lower floor is the erythrocyte layer, and the upper strata is the faint yellow clear liquid of minute quantity, and the intermediate layer is faint yellow translucent rich platelet fibrin gel (as shown in Fig. 4 A);
3) with aseptic ophthalmic tweezers with the complete taking-up of mid portion gel, its bottom erythrocyte end is gently dipped in to remove unnecessary erythrocyte on sterile gauze, keep complete light yellow gel part and bottom and adhere on a small quantity firmly erythrocyte (as shown in Figure 4 B), latter's tiling is placed between sterile gauze, and the wherein liquid component of gently extruding out is prepared into a faint yellow tough and tensile membrane structure, be the rich platelet fibrin film, be PRF film (as Fig. 4 C, Fig. 5, shown in Figure 6);
4) with aseptic eye scissors, it is shredded, be prepared into the rich platelet fibrin diaphragm granule of big or small approximately 0.5mm * 0.5mm * 0.5mm, standby (as shown in Fig. 4 D).
2.4BMSCs/PRF the preparation of two membrane structures
BMSCs cell patch fragment and the PRF diaphragm granule of preparation are placed in sterile petri dish, obtain cell patches according to six orifice plates: the ratio that is used for the blood of centrifugal acquisition rich platelet fibrin diaphragm=1: 3.5～4.0, manually with its mixing, guarantee both fully to mix, can obtain two membrane structure graft materials of the compound rich platelet fibrin of stem cell diaphragm fragment membrane granule, standby (as shown in Figure 7).
2.5 the foundation of rabbit Temporomandibular Joint Cartilage defect model
1) anesthesia: under aseptic condition, the anesthesia of 3% pentobarbital sodium (1mL/kg) auricular vein separately imposes local anaesthesia with 1% lignocaine in the modeling side;
2) prepare before art: art side remporomandibular joint art district preserved skin, iodophor disinfection, drape;
3) make the approximately long skin incision of 2cm in 5mm place, the outer canthus outside to external auditory meatus direction;
4) separate subcutaneous tissue, cut joint capsule;
5) expose condyle prominent joint, drill through the hole of 1 diameter 3mm with electronic condyle the dash forward front bevel central authorities of being drilled in;
6) go embolia, sew up joint capsule, the skin suture layer.
2.6 experiment grouping
According to experimental design, the graft at condylar cartilage to be implanted damaged place is divided into 4 groups: 1. blank group: condylar cartilage damaged place is implantation graft not; 2. simple PRF groups of grains; 3. simple BMSCs cell patch slice groups; 4. two membrane structure groups of BMSCs/PRF.
3. experimental result
Draw materials after using pentobarbital sodium anesthesia when 2w, the 4w after implanting respectively at each group, 8w, 4% paraformaldehyde is fixed.Carry out gross examination of skeletal muscle after the 15%EDTA decalcification, and organize microstructural observation after HE, Toluidine blue staining, result is as follows:
1) gross examination of skeletal muscle as seen: the two membrane structure group reparation situations of BMSCs/PRF are better than other group (as shown in figure 11) with time point;
2) HE coloration result: the blank group has been showed no the filling of cartilage sample material in each time point defective region depression; Separately PRF group and separate cell diaphragm group be along with the prolongation defect repair district of time has neocartilage to generate gradually, but cartilage level arrangement disorder; Two membrane structure groups, the 8th when week damage zone substantially flush, repair that cartilage layers is thinner, level is more clear, with contiguous each layer of normal cartilage continuous (as shown in figure 12);
3) Toluidine blue staining result: each is organized the neocartilage zone and is heterochromaty (as shown in figure 13), adopts IPP 6.0 softwares to carry out the tinctorial strength analysis to each group Toluidine blue staining picture, calculates average optical density value.Result draws: except the blank group, all the other 3 groups after surgery 2,4,8 week average OD values be and increase gradually trend (P＜0.05), and be significantly higher than the blank group (P＜0.05) with time point; Separately PRF group and separate cell diaphragm group are in the average OD value of each time point there are no significant difference.Two membrane structure groups 4,8 all average OD values are significantly higher than other group (P＜0.05) (as shown in figure 14) with time point.
Experiment two: the two membrane structures of the compound rich platelet fibrin film of canine tooth marrow stem cell diaphragm fragment are used for the dental pulp regenerative therapy
1. material and equipment
1.1 main agents
α-MEM culture medium (Hyclone company, the U.S.); Type i collagen enzyme (GIBCO company, the U.S.); 0.25% pancreatin (Hyclone company, the U.S.); Hyclone (Ilex purpurea Hassk.[I.chinensis Sims, Hangzhoupro, sky, Zhejiang bio tech ltd); Vitamin C (Sigma company, the U.S.); Pentobarbital sodium (Germany, the packing of sky biological engineering Co., Ltd of section).
1.2 instrument and equipment
Multitube frame autobalance centrifuge (TD25-WS, Hunan, Hunan instrument Laboratory Instruments development corporation, Ltd.); Cell culture incubator (Heraeuc Hearcell, Thermo company, China); Six orifice plates (Nunc company, the U.S.); Superclean bench (BIOBASE, the U.S.); Nikon microscopy imaging system (XTJ30, Japan), analytical electron balance (FA1004, China), scanning electron microscope (S-4800 of Hitachi, Japan).
2. operating procedure
2.1 separation and the cultivation of canine tooth marrow stem cell (DPSCs)
The primary dental pulp stem cell of dog adopts the α that contains 10%FBS-MEM cellar culture, cultivates a plurality of clone cell of the appearance group after 3～5 days, when cloning cluster interior detail intracellular growth to 80%, carries out amplification culture with the ratio of 1: 2～1: 3, changes liquid once in every 2～3 days;
2.2DPSCs the preparation of diaphragm
With experiment one.
2.3PRF preparation
With experiment one.
2.4 the preparation of the two membrane structure graft materials of the compound rich platelet fibrin film of canine tooth marrow stem cell diaphragm fragment
With experiment one.
2.5 the foundation of canine tooth marrow regenerating model
1) the conventional anesthesia of dog (approximately 6 monthly ages), method is the same;
2) prepare before art: in the dog mouth, the capable gum of all premolarss employing periodontal scaling apparatuses is manually scraped up and down and is controlled, and removes tartar and soft dirt, the inside and outside povidone iodine strict sterilization in oral cavity, drape;
3) after randomized grouping, upper mandibular premolar (except frist premolar) is opened marrow, takes off marrow and pushes up, the Exposed Pulp tissue, barbed broach is complete pull out dental pulp after, after adopting 30#K to determine Open canal system, enlarge apical foramen of tooth and file to 70#K, make tip of a root open zone diameter reach 0.7mm;
4) a large amount of physiological saline solution of disposable syringe absorption carry out root canal irrigation, determine in the root pipe residual without pulp tissue and tissue of tooth chip;
5) according to grouping in 2.6, graft is placed in the root pipe, adopts auger conveyor that graft is delivered in whole pipe, reach the root canal orifice place; Calcium hydroxide (Dycal, Deng Shibai company) is gently put in the graft upper end, and thickness is 1mm approximately, and after the calcium hydroxide knot was solid, finishing hole wall adopted glass ion rebasing, and thickness is 1mm approximately, and damaged place adopts the light-cured resin filling;
2.6 experiment grouping
According to experimental design, the graft of pulp cavity to be implanted is divided into 4 groups: 1. blank group: do not implant any graft in pulp cavity; 2. simple PRF groups of grains; 3. simple DPSCs cell patch slice groups; 4. two membrane structure groups of DPSCs/PRF.
3. experimental result
The HE coloration result: Normal group visible pulp tissue, odontoblast, predentin and ripe dentin ordered arrangement, odontoblast is high column ordered arrangement; The more shallow predentin of the equal visible vessels of each experimental group and dyeing forms, but there are differences between each group of blood vessel quantity, dentin thickness and cell arrangement situation: the two membrane structure group cells of DPSCs/PRF are the loose multiple layer of StarNet's shape and arrange, and cellular morphology is by changing to growing near Dentinal vertical polarization gradually away from Dentinal Parallel Growth, the similar odontoblast of form sees in pulp cavity that more new vessels forms; The two membrane structure groups of simple DPSCs cell patch group pulp cells are arranged loose, unordered, and dentin layer is thinner, but also visible more vascularization in pulp cavity; Simple PRF groups of grains cell is mostly rounded, without obviously stretching, arranges unordered and dentin wall is thin, but also as seen vascularization (as shown in figure 15) on a small quantity in pulp cavity.
4. Analysis on Mechanism
4.1 the effect of stem cell diaphragm fragment (CSF)
Stem cell (stem cells, SC) be the cell that a class has the of self-replication capacity and Multidirectional Differentiation ability, under certain condition, it can be divided into the several functions cell, potential function with the various histoorgans of regeneration and human body is therefore be widely used in organizational project as seed cell at present.
The cell patch technology is to impel seed cell to produce at short notice a large amount of extracellular matrixs (extracellular matrix, ECM) by external evoked mode, and cell is closely linked becomes a kind of laminated structure.ECM not only for cell be connected the support that connection, nutrition supply and mechanical property are provided, what is more important can be regulated intercellular communication, to the omnibearing dynamic adjustments effect of basic vital movement performance of cell.ECM has the multiformity of 26S Proteasome Structure and Function, the variation of its structure and composition will affect the structure of cytoskeleton, thereby determine shape and the activity of cell, affect the existence of cell, participate in and control differentiation and the migration of cell, generation, the growth of histoorgan, regenerate and keep the physiological activity of 26S Proteasome Structure and Function of histoorgan in all have extremely crucial effect.This unique advantage that can preserve iuntercellular autocrine signaling molecule performance of cell patch technology can reduce the quantity of invalid transplanted cells, the reconstruction that is conducive to organize or regeneration.And diaphragm fragment technology has changed traditional diaphragm transplanting pattern, complete cell patch is prepared as a certain size fragment, the activity of having preserved seed cell on the one hand, and at utmost preserved the performance of ECM and iuntercellular autocrine signaling molecule, cell can farthest evenly be mixed with support, be convenient to cell and be colonizated in rapidly rack surface, make the graft that both forms become the integral body with good biological function.
4.2 the effect of rich platelet fibrin film (PRF)
Rich platelet fibrin (PRF) fibrous reticular structure that to be whole blood form after once centrifugal fast.the main feature of PRF is to be rich in a large amount of ratios near the somatomedin of physiological situation, as transforming growth factor (transforming growth factor β, TGF β), platelet-derived growth factor (platelet-derivedgrowth factors, PDGF), insulin like growth factor (insulin-like growth factors, IGF), above-mentioned three kinds of somatomedin play an important role in initial organization healing, they can promote cell migration and proliferation, induce fibrin matrix to reinvent, and promote the secretion of collagen stroma, with the exception of this, also contain the multiple somatomedin relevant to wound healing and osteanagenesis in PRF, comprise epidermal growth factor (epidermal growth factor, EGF) and VEGF (vascular endothelial growthfactor, VEGF) etc.
As the second filial generation product of platelet concentrate, compare with first generation product platelet rich plasma (platelet-richplasma, PRP), PRF has following characteristics:
1) preparation is simple.The preparation of PRP need to be added anticoagulant, in case Hemostatic Oral Liquid solidifies; Its preparation needs two times centrifugal, needs the supernatant after centrifugal is shifted, and complex operation, and there is no at present centrifugal speed and the time of standard, different researchers is held different viewpoints to the method for its preparation.The preparation of PRF is very simple, extracts after whole blood immediately with the speed of 3000rprm/min centrifugal 10 minutes, standing 5 minutes, can obtain the PRF gel.
2) safety is good.At first PRP needs to add anticoagulant in the preparation, next needs to add thrombin of beef after two times centrifugal finishes and calcium chloride is activated, just gel can be formed in order to transplant in body, and PRP only can make just platelet alpha chain wherein take off the particle release somatomedin after activating; Yet, adds exogenous material and might cause host's immunoreation, and too much operating procedure has also increased the probability that pollutes.The preparation of PRF does not need to add any exogenous material, and single stepping completes, and safety is good.
3) growth factor slow-release.PRP is that formation is gelatinous rapidly after adding more thrombin to carry out artificial the activation.A large amount of ectogenic thrombins make the rapid polymerization of fibrin, form two-way fine and close syndeton, this just makes the somatomedin that platelet discharges not enlisted the services of wherein, but remains in suspension outside fibrous reticular structure, thereby presents fast, a large amount of characteristics that discharge.And PRF is the polymerization methods slowly that produces under self thrombin action, and this gradual polymerization methods is enlisted the services of somatomedin free in peripheral blood to the tridimensional network of PRF to a greater extent.The distribution of somatomedin in this three dimensional structure in free somatomedin and platelet will inevitably cause its slowly releasing effect, because they only can just be released when reconstruction occurs its place fibre structure, and the fibril in PRF and fibrin form tetragonal connected mode, and this elastomeric matrices structure also more is conducive to cell migration and shla molecule and keeps.
4) tridimensional network can be used as timbering material.What the fibrin in PRP formed is two-way fine and close syndeton, and this structure is unfavorable for the migration of cell and somatomedin.Fibrin in PRF forms tridimensional network, and this structure not only is beneficial to the migration of cell and somatomedin, and the place of proliferation and differentiation also is provided for the tissue repair relevant cell, has brought into play important support effect.
5) contain a large amount of leukocyte.PRF obtains via whole blood is centrifugal, and in centrifugal process, in blood, most leukocyte all are distributed in the PRF lower end, i.e. red end (as shown in B figure in Fig. 6); Therefore, when two membrane structures migrated in body, concentrated leukocyte in the good antiinflammatory action of part performance, was transferred the immune system of body on the other hand on the one hand, brings into play good anti-inflammatory properties and immunoloregulation function; Simultaneously, PRF has also enlisted the services of in blood a large amount of immunoregulatory factor in preparation process, as IL-1 β, and IL-4, IL-6 and TNF-α etc., these factors protect from infection, promote the aspect such as healing all to play an important role after Repair of tissue defect.
4.3 the analysis of two membrane structure zoografting effects of the compound rich platelet fibrin film of stem cell diaphragm fragment
First of the present invention uses experiment in vitro the proportion relation of stem cell cell patch and rich platelet fibrin film is studied, obtained both between best proportion relation.Second portion according to the two membrane structure grafts of this proportion relation preparation, is implanted it in experimental animals, probes into two membrane structure grafts to the impact of tissue repair.
4.3.1 test in one, rich platelet fibrin film PRF granule is as cytoskeleton and somatomedin donor, carries out compoundly with mesenchymal stem cells MSCs diaphragm fragment, it mixed according to a certain percentage being placed on the tissue defect place, carries out the reparation of cartilage defect.The PRF diaphragm has toughness, and shredding becomes very easily operation after graininess, can be placed in the tissue defect of any shape; After mesenchymal stem cells MSCs is made into patching, due to the connection that has extracellular matrix, the shortcoming of having avoided suspension cell in the past easily to run off.Our experimental result shows, compare with natural recovering group, simple PRF and simple cell patch group, it is best that two membrane structure groups are repaired the effect of cartilage defects, and damage zone flushes substantially when the 8th week, the reparation cartilage layers is thinner, level is more clear, and is more continuous with contiguous each layer of normal cartilage.This has just proved our guess, that is: mesenchymal stem cells MSCs has been brought into play the effect of seed cell in the part, can become specific cell type by proliferation and differentiation in local microenvironment, and PRF diaphragm granule provides a large amount of collagen stroma compositions on the one hand, for cell provides supporting structure, the platelet that wherein contains on the other hand constantly is activated along with the reconstruction of matrix components, discharge a large amount of ratios near the somatomedin of physiological situation, for the propagation of stem cell and the reparation of differentiation and tissue provide nutritional support.
4.3.2 test in two, with the PRF diaphragm as cytoskeleton and somatomedin donor, with dental pulp stem cell diaphragm fragment carry out compound, with its mix according to a certain percentage be placed on that dental pulp lacks as the root tube chamber in, carry out the research of dental pulp regeneration.Found that, two membrane structure groups are than simple cell diaphragm and simple PRF group favorable regeneration effect.Visible dental pulp, odontoblast, predentin and ripe dentin ordered arrangement are observed in Normal group canine tooth longitudinal section HE dyeing, Yihong collagen stroma of having a liking for that dentin takes on a red color after demineralization is processed dyes, predentin near pulp cavity partly dyes more shallow, arrange along dentin layer, monolayer odontoblast is right after predentin and is high columnar arrangement.The two membrane structure group HE dyeing of DPSCs/PRF are found, cell is the multiple layer of loose StarNet shape and arranges, and cellular morphology by away from Dentinal Parallel Growth gradually to growth changes near Dentinal vertical polarization, more vascularization is seen by form similar odontoblast in pulp cavity.Simple cell diaphragm group cell is the multiple layer of loose StarNet shape and arranges, but it is loose, unordered that two membrane structure groups are arranged, and do not show the characteristics that significantly nearly dentin vertical polarization growth changes, and dentin layer is thinner, but cellular morphology stretches obviously, also visible more vascularization in pulp cavity.Simple PRF groups of grains cell is mostly rounded, without obviously stretching, arranges unorderedly, changes without obvious vertical polarization growth, and dentin wall is thin, but also as seen vascularization on a small quantity in pulp cavity.The above results explanation, in the dental pulp regenerative process, dental pulp stem cell is desirable seed cell, be made into the form of cell patch fragment, kept to the full extent extracellular matrix on the one hand, make the iuntercellular related signaling molecules be able to complete preservation, on the other hand, broken away from the constraint of cell suspension, the form of diaphragm is convenient operation more, guarantees that there is a q.s pulp cavity part and is difficult for the seed cell that runs off.After Open canal system, guaranteed to have in pulp cavity enough blood fortune, made seed cell that certain nutritional support be arranged, like this, provide a large amount of somatomedin and support effects even without PRF, pulp tissue's regeneration has also been arranged in pulp cavity.On the other hand, after Open canal system, blood can be full of pulp cavity, and itself there is undifferentiated mescenchymal stem cell in circulating, stem cell homing is to pulp cavity, and under the dual function of the effect of a large amount of somatomedin of PRF particle release and pulp cavity local microenvironment, the stem cell that goes back to the nest is bred, breaks up in the pulp cavity part, reach the purpose of dental pulp regeneration, this may be namely also can see regenerate the preferably reason of dental pulp of arrangement in simple PRF groups of grains.Provide somatomedin and collagen scaffold and lacked PRF in pulp cavity, even the stem cell that goes back to the nest is arranged, also may not necessarily form pulp tissue preferably.And the graft materials of the two membrane structures of the compound rich platelet fibrin of dental pulp stem cell diaphragm fragment membrane granule is placed in pulp cavity, namely satisfied simultaneously the requirement of seed cell, three-dimensional rack and somatomedin, therefore can obtain comparatively desirable dental pulp regeneration, this conforms to this experimental result.
In sum, experimental result confirms Analysis on Mechanism, and the novel biomaterial of the compound rich platelet fibrin of stem cell diaphragm fragment diaphragm shown in the present can significantly promote reparation and the tissue regeneration effect of tissue defect.The biological implantation material mixture ratio of the inventive method preparation is suitable, and the source is abundant, has the general suitability, is applicable to every individual patients.Biological implantation material by the method prepares can be widely used in the reparation of dissimilar tissue defect, also has good tissue regeneration effect, for the expansion of organizational project related experiment and clinical repair tissue defect and tissue regeneration provide new approaches.
Claims (11)
1. a two membrane structure graft materials, is characterized in that: by stem cell cell patch fragment and rich platelet fibrin film granulometric composition.
2. graft materials as claimed in claim 1, it is characterized in that: described stem cell is selected from mesenchymal stem cells MSCs, fat stem cell, dental pulp stem cell or periodontal ligament stem cell.
3. graft materials as claimed in claim 1 is characterized in that: described rich platelet fibrin diaphragm granule obtains after via centrifugal blood.
4. graft materials as claimed in claim 1, it is characterized in that: cell patch adopts six orifice plates to be prepared, and the ratio of cell diaphragm fragment and rich platelet fibrin film is as follows: every six orifice plate cell patch quantity: be used for the blood milliliter number of centrifugal acquisition rich platelet fibrin diaphragm=1: 3.5～4.0.
5. graft materials as claimed in claim 1, it is characterized in that: described stem cell and rich platelet fibrin all derive from same individuality.
6. graft materials as claimed in claim 1, it is characterized in that: stem cell must be prepared into the form of cell patch fragment.
7. the preparation method of the described graft materials of claim 1 is characterized in that its preparation process comprises the following steps:
The first step, the cultivation of stem cell diaphragm: primary stem cell is inoculated in six orifice plates after amplification culture, merges until growth and is cultivated with the diaphragm induced liquid to 80% the time, changes liquid once in 3 days, until occurring thinking that cell patch is ripe when curling around cell patch;
Second step, prepare stem cell diaphragm fragment: the method with mechanical stripping is peeled off cell patch from culture dish, with aseptic eye scissors, it is shredded, and is prepared into cell patch fragment (big or small approximately 0.5mm * 0.5mm);
the 3rd step, extract rich platelet fibrin diaphragm granule: according to required ratio, take 10ml as unit, blood (not adding any anticoagulant) is placed in the glass centrifuge tube of aseptic glass centrifuge tube or plastic bushing, with the speed of 3000rpm/min centrifugal 10 minutes immediately, after standing 5 minutes, aseptic tweezer clamp takes out the rich platelet fibrin gel of centrifuge tube mid portion, its bottom erythrocyte end is gently dipped in to remove unnecessary erythrocyte on sterile gauze, keep complete light yellow gel part and bottom and adhere on a small quantity firmly erythrocyte, the latter is tiled between sterile gauze, the wherein liquid component of gently extruding out, be prepared into a faint yellow tough and tensile membrane structure, with aseptic eye scissors, it is shredded, be prepared into the rich platelet fibrin diaphragm granule of big or small approximately 0.5mm * 0.5mm * 0.5mm,
The 4th step, cell patch fragment and the rich platelet fibrin diaphragm granule of preparation are placed in sterile petri dish, proportionally manually with its mixing, guarantee both fully to mix, can obtain the two membrane structure graft materials of the compound rich platelet fibrin film of stem cell diaphragm fragment.
8. preparation method as claimed in claim 6, it is characterized in that: the ratio of cell diaphragm fragment and rich platelet fibrin film is as follows: every six orifice plate cell patch quantity: be used for the blood milliliter number of centrifugal acquisition rich platelet fibrin diaphragm=1: 3.5～4.0.
9. the described graft materials of claim 1-5 any one application in Repair of tissue defect among a small circle in oromaxillo-facial region or whole body.
The described graft materials of claim 1-5 any one in preparation oromaxillo-facial region or whole body among a small circle Repair of tissue defect with the purposes in graft materials.
11. the purposes of the composite that the described graft materials of claim 1-5 any one and other biological material form in the preparation Repair of tissue defect is used graft materials.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1857126A1 (en) * | 2005-02-28 | 2007-11-21 | Cellseed Inc. | Cultured cell sheet, production method thereof, and application method thereof |
| JP2012044970A (en) * | 2010-08-30 | 2012-03-08 | Tokyo Univ Of Agriculture & Technology | Method for producing cell sheet for transplantation, cell sheet for transplantation, and method for treating using cell sheet for transplantation |
| WO2012036225A1 (en) * | 2010-09-14 | 2012-03-22 | 学校法人 東京女子医科大学 | Method for manufacturing multilayered cell sheet, multilayered cell sheet having vascular network obtained thereby, method of use thereof |
| CN102614546A (en) * | 2011-01-26 | 2012-08-01 | 香港中文大学 | Cell sheet for tissue repair and bio-artificial tissue engineering, method of producing the same and method of using the same |
-
2012
- 2012-12-07 CN CN201210525097.9A patent/CN103100112B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1857126A1 (en) * | 2005-02-28 | 2007-11-21 | Cellseed Inc. | Cultured cell sheet, production method thereof, and application method thereof |
| JP2012044970A (en) * | 2010-08-30 | 2012-03-08 | Tokyo Univ Of Agriculture & Technology | Method for producing cell sheet for transplantation, cell sheet for transplantation, and method for treating using cell sheet for transplantation |
| WO2012036225A1 (en) * | 2010-09-14 | 2012-03-22 | 学校法人 東京女子医科大学 | Method for manufacturing multilayered cell sheet, multilayered cell sheet having vascular network obtained thereby, method of use thereof |
| CN102614546A (en) * | 2011-01-26 | 2012-08-01 | 香港中文大学 | Cell sheet for tissue repair and bio-artificial tissue engineering, method of producing the same and method of using the same |
Cited By (17)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103690998A (en) * | 2013-12-10 | 2014-04-02 | 北京大学口腔医学院 | Bone grafting material by embedding bone material gel in PRF (platelet-rich fibrin) |
| CN103690999B (en) * | 2013-12-10 | 2015-08-05 | 北京大学口腔医学院 | The liquid-solid embedded material turning to gel film of a kind of PRF precursor |
| CN103690998B (en) * | 2013-12-10 | 2015-12-09 | 北京大学口腔医学院 | The bone-grafting material of bone material gel is embedded in a kind of PRF |
| CN103690999A (en) * | 2013-12-10 | 2014-04-02 | 北京大学口腔医学院 | Implant material with PRF precursor liquid solidified into gel mask |
| CN105708858A (en) * | 2014-12-05 | 2016-06-29 | 国玺干细胞应用技术股份有限公司 | Preparation method of growth-factor-platelet-rich fibrin and releasate |
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| CN106267349A (en) * | 2016-09-21 | 2017-01-04 | 吉林大学 | A kind of preparation method of rich platelet fibrin film |
| WO2018067532A1 (en) * | 2016-10-06 | 2018-04-12 | 3M Innovative Properties Company | Methods of making fibrin compositions and articles |
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