CN110478531A - A kind of platelet rich plasma tissue engineering bracket and preparation method thereof - Google Patents
A kind of platelet rich plasma tissue engineering bracket and preparation method thereof Download PDFInfo
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- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
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- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/3604—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix characterised by the human or animal origin of the biological material, e.g. hair, fascia, fish scales, silk, shellac, pericardium, pleura, renal tissue, amniotic membrane, parenchymal tissue, fetal tissue, muscle tissue, fat tissue, enamel
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- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
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- A61L2300/236—Glycosaminoglycans, e.g. heparin, hyaluronic acid, chondroitin
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- A61L2430/02—Materials or treatment for tissue regeneration for reconstruction of bones; weight-bearing implants
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Abstract
The invention belongs to biomedicine fields, are related to a kind of composite biological material and preparation method thereof of platelet rich plasma tissue.The composite biological material includes the PRP for loading icariin, its with good bioactivity, at the functional characteristic of cartilage, osteogenic and knitting, it can be used as a kind of tissue engineering bracket of novel bone tendon interface damage, and the damage for treating bone tendon interface, realize the quick healing of bone tendon interface high quality, and the composite biological material shows excellent slow release effect, it can gently be released with speed appropriate and duration, to play the drug action of icariin to a greater extent, cause side effect without regard to dosage is excessively high.
Description
Technical field
The invention belongs to biomedicine field, it is related to a kind of platelet rich plasma tissue engineering bracket and preparation method thereof.
Background technique
Tendon-bone interface is the common a kind of injury gained in sports of medicine in field of sports medicine.According to statistics, only just have about 25 every year in the U.S.
Ten thousand rotator cuff injuries and 100,000 cross ligament damages need operative treatment, and it is negative to bring heavy economy to society and family
Load.With the increase of the average life span and the extensive development of nationwide fitness programs, the incidence of tendon-bone interface damage is estimated can also be after
It is continuous to rise.
After tendon-bone interface damage, often tendon or ligament are affixed directly on bone by all kinds of surgical stapling technologies, tendon
Bone interface early stage is filled by fibrous scar tissue, and the reconstruction of characteristic fibrocartilage layer is incomplete, lacks gradient-structure again
It is raw, cannot effective dispersive stress, the tendon bone structure rebuild after restoring movement is easy avulsion again.The healing of tendon-bone interface is
Very slow and difficult, level before restoring to wound is difficult to after damage.
It is clinically usually super using non-steroid anti-inflammatory drug, cold therapy, low intensity pulse after tendon-bone interface damage at present
Sound, physiotherapy etc., although these treatments are widely applied in orthopaedics or medicine in field of sports medicine, patient's early stage motor function is still
Restore bad, healing effect is unsatisfactory, cannot be effectively facilitated patient's quickly high-quality healing.
Summary of the invention
One of the objects of the present invention is to provide a kind of composite biological materials.
One of the objects of the present invention is to provide a kind of platelet rich plasma tissue engineering brackets.
Another object of the present invention is to provide a kind of tissue engineering bracket repaired for bone tendon interface.
It is also an object of the present invention to provide a kind of preparation methods of platelet rich plasma tissue engineering bracket.
Above-mentioned purpose of the invention is realized by following technological means:
On the one hand, the present invention provides composite biological materials, and the ingredient of the composite biological material is by PRP, icariin
It is formed with calcium chloride.
As an implementation, it is 0.63%-1.49% that the content of the calcium chloride, which is mass percent,.
In some embodiments, the mass percent of the PRP is 63.6%--88.9%.
In some embodiments, the mass percent of the icariin is 21.6%-25.93%.
In some embodiments, the weight ratio of the PRP and icariin is 2.85-3.53:1.
In some embodiments, the composite biological material passes through frozen dried.
On the other hand, the present invention provides the tissue engineering brackets that the composite biological material is formed.
In some embodiments, the tissue engineering bracket is bone tendon Interface Microstructure engineering rack.
On the other hand, the application the present invention provides the composite biological material in the bone material of synthesis.
On the other hand, the application the present invention provides the composite biological material in bone implant.
On the other hand, the application the present invention provides the composite biological material in bone substitute.
On the other hand, the application the present invention provides the composite biological material in bone bracket.
In some embodiments, the bone bracket is whole or layering bone bracket.
There are also on the one hand, the present invention provides a kind of preparation methods of composite biological material, comprising the following steps:
(1) preparation includes the gel of PRP and icariin；
(2) gel in step (1) is lyophilized.
In some embodiments, the step further includes disinfection.
In some embodiments, it is carried out disinfection using ethylene oxide gas, disinfection by ultraviolet light or hydrogen peroxide fumigating system.
In some embodiments, the preparation step of the gel includes: concentration PRP, obtains PRP concentrate；By icariin
Dissolve to obtain icariin solution；It is mixed again with PRP concentrate after calcium chloride solution is added to icariin solution, obtains mixed liquor.
In some embodiments, the PRP concentration uses gradient centrifugation.
In some of embodiments, the gradient centrifugation is differential centrifugation.
In some embodiments, the preparation step of the PRP concentrate include: a. by 20-30mL blood sample, 250-350g from
Heart 10-15min removes red blood cell；B. then 800-1000g is centrifuged 10-15min, takes the blood sample of lower layer 2.4-3.0mL, remaining
Liquid removes to obtain PRP concentrate.
As a preferred embodiment, the preparation step of the concentrate further include: c. uniformly shakes 30-60s, i.e.,
PRP must be concentrated.In some embodiments, in step (2), icariin is dissolved using DMSO.
In some embodiments, the concentration of the icariin solution is 45-55mM.
In some embodiments, in step (3), the mass concentration of the calcium chloride solution is 5-20% (w/v).
In some embodiments, in step (3), the volume of the icariin solution, calcium chloride solution and PRP concentrate
Than for 1-2:10-15:100-110.
Compared with the prior art, the beneficial effect of the one of embodiment of the present invention is:
1, provide freeze-drying PRP load icariin tissue engineering bracket, the bracket have good bioactivity, at
The functional characteristic of cartilage, osteogenic and knitting becomes a kind of tissue engineering bracket of novel bone tendon interface damage, and
For treating the damage at bone tendon interface, the quick healing of bone tendon interface high quality is realized.
2, between each time interval of the tissue engineering bracket of freeze-drying PRP load icariin from 0h to 4h, Herba Epimedii
Glycosides burst size is in gentle incremental release, it is ensured that enables to icariin to reach effective therapeutic dose in early period；4h with
Afterwards, icariin has reached certain effective quantity, and rate of release starts to slow down；With highly uniform, gentle speed appropriate
It releases with duration, to play the drug action of icariin to a greater extent, draws without regard to dosage is excessively high
Play side effect；Show excellent slow release effect.
3, tissue engineering bracket of the invention is not necessarily to chitosan as carrier, and is able to maintain excellent sustained release and acceleration and is cured
The effect of conjunction.Its ingredient is simple, clear, does not have excessive ingredient and causes to be immunoreacted, histocompatbility is good.Meanwhile because
Its ingredient is simple, has cost advantage, is easier to commercialization production.
Detailed description of the invention
Fig. 1 is the tissue engineering bracket that freeze-drying PRP prepared by embodiment 1 loads ICA.
Fig. 2 Fig. 2A is accumulative release rate (%) curve of ICA that PRP loads ICA bracket；Fig. 2 B is that PRP loads ICA bracket
ICA real-time concentration (μM) curve.
The case where Fig. 3 is rabbit kneecap New Bone Quantity: Fig. 3 A is striograph；Fig. 3 B is statistical chart；Kneecap injury group
(CTL)；The PRP bracket group (FD-PRP) of freeze-drying；PRP load ICA group (FD-PRP/ICA) is lyophilized；
Wherein NB indicates area of new bone；RP indicates original kneecap；
Bone volume (BV), diaphysis fraction (BVF), bone density (BMD), bone trabecula thickness (Tb.Th).
The case where Fig. 4 is rabbit kneecap HE dyeing display fibrocartilage regeneration: Fig. 4 A is postoperative 8 weeks situations；Fig. 4 B is art
16 weeks situations afterwards；Wherein FD-PRP: the PRP bracket group of freeze-drying；FD-PRP/ICA: freeze-drying PRP loads ICA group；Wherein NB table
Show area of new bone；RP indicates original kneecap；RF indicates regeneration of cartilage；F indicates fibrocartilage；PT indicates kneecap kneecap tendon.
Fig. 5 is the case where rabbit kneecap Toluidine blue staining shows fibrocartilage regeneration: wherein FD-PRP: the PRP branch of freeze-drying
Frame group；FD-PRP/ICA: freeze-drying PRP loads ICA group；Wherein NB indicates area of new bone；RP indicates original kneecap；RF indicates that regeneration is soft
Bone；PT indicates kneecap kneecap tendon.
Specific embodiment
Technical solution of the present invention is further illustrated below by way of specific embodiment, and specific embodiment does not represent to this hair
The limitation of bright protection scope.Other people according to the present invention theory made it is some it is nonessential modification and adjustment still fall within this hair
Bright protection scope.
Icariin (Icariin, ICA)；No. CAS: 489-32-7；Structural formula is as follows:
In the present invention, icariin, brand: Solarbio, article No.: I8760.
Term " ICA " is together " icariin ".
In the present invention, PRP refers to platelet rich plasma (Platelet-rich plasma).
As used herein, term " tissue engineering bracket ", which refers to, is suitable for Tenocyte cell or thin with or without adhesive
Any skeleton or bracket of born of the same parents' adherency with tissue biopsy cell combination and can be implanted into the different tissues of organism, and according to tool
The material for the function that body substitution tissue has.It by way of enumerating but does not limit, the bracket can be following form: film,
Microballoon, hair (fleece), thread (thread) or gel and/or their mixture.The bracket can be by
Prepared by any material with the required physics of implantation or mechanical attributes, as used herein, it is adapted for use as
The frame of bone bracket.It is used as frame in vivo, and wherein additional cell can be attached to the frame and existing cell and volume
Outer cell can grow with regenerate may because damage or disease lose tissue.
Composite biological material: composite biological material is a kind of factor containing multiple biological activities, and has good biology slow
Effect is released, can be used as a kind of biological support, loads some drugs/bioactive substance, realizes the good healing of bone tendinous tissue.In
Some aspects, the composite biological material specifically can be bone material, bone implant, bone graft, bone substitute or bone bracket.
Or the composite biological material specifically can be bone tendon interface graft；More preferably kneecap kneecap tendon graft.
Term " freeze-drying ", which refers to, under a high vacuum steams it from freezing state by chilled solvent (such as water) and then
It sends out to dry the material of cryogenic refrigeration.
Embodiment 1PRP loads the tissue engineering bracket of icariin and preparation method thereof (1)
1, the preparation of PRP is concentrated
It is derived from body blood 20mL by rabbit auricular vein, using gradient centrifugation (differential centrifugation), 300g first ×
10min removes red blood cell.Then 800g × 10min, takes the 2.4mL of lower layer, and remaining supernatant liquid abandons it；Shake up concussion
30s prepares the PRP of concentration.
2, PRP loads the preparation of icariin tissue engineering bracket
Icariin is dissolved in the DMSO solution (ICA-DMSO solution) for being prepared into the icariin containing 50mM in DMSO, takes 10
10% calcium chloride solution of mass concentration (PBS solution that solvent is 0.1M) and 1.0mL of μ L ICA-DMSO solution and 0.1mL are dense
The PRP of contracting is mixed, and is obtained PRP and is loaded icariin gel.After being lyophilized in vacuum freeze-drying machine and carry out ethylene oxide gas
The tissue engineering bracket of PRP load icariin is prepared in body disinfection.The morphosis of the tissue engineering bracket such as Fig. 1 institute
Show.
Comparative example 1
Such as: on the basis of embodiment 1, the amount that icariin solution (ICA-DMSO solution) is added is 50 μ L,
His condition is in the same manner as in Example 1.
The results show that because the ratio of icariin solution and calcium chloride solution, PRP concentrate goes beyond the scope, nothing
Method binds to form bracket.
Comparative example 2 (FD-PRP)
On the basis of embodiment 1, ICA-DMSO solution is not added, other conditions are in the same manner as in Example 1.
Comparative example 3 (the PRP load ICA bracket not being lyophilized)
On the basis of embodiment 1, frozen dried, other conditions and reality are not carried out to PRP load icariin gel
As applying example 1.
Icariin slow release effect is tested in 3 tissue engineering bracket of embodiment
Bracket prepared by embodiment 1 and comparative example 3 immerses the sterile PBS solution of 1mL at room temperature, does not stop in centrifuge tube
Ground gentle agitation respectively takes the incubation of 0.6mL at the time point of 0h, 1h, 2h, 4h, 8h, 16h, 1d, 2d, 3d, 4d, 5d, 6d, 7d
Liquid, and new isometric PBS liquid is added simultaneously into centrifuge tube after liquid.With efficient liquid phase mass spectrograph (high-
Performance liquid chromatography, HPLC) ICA in inspection sample to be tested correspond to peak area, and it is bent according to standard
Line computation goes out corresponding ICA concentration in sample to be tested, to quantify the In-vitro release curves of icariin, as a result such as table 1,2 and
Shown in Fig. 2.
1 PRP of table loads the preparation (%) of ICA in ICA bracket
| Bracket classification | 0h | 1h | 2h | 4h | 8h | 16h | 1d | 2d | 3d | 4d | 5d | 6d | 7d |
| Freeze-drying | 0 | 2.94 | 9.15 | 24.57 | 36.95 | 47.15 | 56.55 | 64.68 | 70.76 | 75.71 | 79.52 | 81.69 | 82.54 |
| It is not lyophilized | 0 | 9.17 | 26.61 | 50.31 | 65.67 | 76.59 | 82.79 | 87.44 | 89.79 | 91.35 | 91.91 | 92.20 | 92.35 |
2 PRP of table loads the real-time release concentration (μM) of ICA in ICA bracket
| Bracket classification | 0h | 1h | 2h | 4h | 8h | 16h | 1d | 2d | 3d | 4d | 5d | 6d | 7d |
| Freeze-drying | 0 | 24.48 | 51.73 | 128.46 | 103.15 | 84.92 | 78.29 | 67.77 | 50.67 | 41.17 | 31.75 | 18.11 | 14.65 |
| It is not lyophilized | 0 | 76.35 | 145.32 | 197.39 | 127.99 | 90.95 | 51.62 | 38.73 | 19.56 | 13.05 | 4.63 | 2.42 | 1.24 |
The results show that the PRP of freeze-drying shows more stable and excellent ICA slow release effect, toxic side effect not will cause.
The test of 5 tissue engineering bracket therapeutic effect of embodiment
1, the foundation and grouping of animal model
Healthy adult New Zealand White Rabbit 48 are chosen, male and female are unlimited, weight (3.0 ± 0.5) kg, and rabbit normal diet is fed
It supports, circulation light shines for 24 hours, freely ingests, drinks water, all animals are without field of operation wound, infection and deformity, also without other whole body diseases
Disease, all operation sequences abide by zoopery criterion.
48 white rabbits are often divided into 3 groups with group technology random (random digits table) according to zoopery scientific research and design, often
Group 16.Respectively kneecap injury group (control group-CLT)；The PRP bracket group (FD-PRP) of freeze-drying；PRP is lyophilized and loads ICA branch
Frame group (FD-PRP/ICA).
Carry out animals iv anesthesia with Su Mian Xin, disinfection, paving are single, take right knee kneecap tendon inner incision, isolate semitendinosus and
Gracilis retains distal end stop, cuts off from tendon and belly of muscle intersection, it is spare to be fabricated to sub-thread tendon graft.Cut knee joint
Capsule, kneecap of dislocating look into drawer test, the Lachman test positive by ACL complete resection at stop.According to trial test as a result,
Rabbit semitendinosus tendon diameter is about 1.5~2.0mm, and 2.0mm drill bit is selected to go up quadrate bone from tibial tubercle towards former ligamentaum cruciatum
Side is asked to condyle of femur condyle up and out, backward again by the tunnel in the tunnel of lower dead center drill straight diameter about 2.0mm, close to ACL
Osseous tunnel is bored at stop, the transplanting tendon made is passed through into shin bone, femoral bone tunnel with steel wire, in 30 ° of tensions of bending knee, bends and stretches knee
Outboard end transplanting tendon and femoral bone tunnel collar extension surrounding soft tissue are met conjunction by joint 20 times.
CLT group: the rabbit kneecap kneecap tendon injury group of any substance is not additionally incorporated；
FD-PRP group: the PRP bracket of freeze-drying prepared by comparative example 2 is filled in femoral bone tunnel, compacting, will be moved with surgical thread
Tendon is planted to be fixed together with bone matrix.
FD-PRP/ICA group: freeze-drying PRP load ICA bracket prepared by embodiment 1 is filled in femoral bone tunnel, is compacted, uses
Surgical thread will transplant tendon and be fixed together with bone matrix.
2, post surgery treatment
Postoperative, new zealand white rabbit divides equally cage nursing, parallel Incisional Dressing Change processing.Postoperative 3d penicillin injection liquid 800,000 is single
Position intramuscular injection.
3, ostosis situation
Postoperative 8 weeks and 16 weeks collection rabbit patellar tendon-shin bone complex samples, synchronize radiation microscopic CT scanning.
As a result as shown in figure 3, from figure 3, it can be seen that postoperative 8 weeks and micro-CT display in 16 weeks, freeze-drying PRP load ICA
Rabbit kneecap kneecap tendon New Bone Quantity in bracket group increased significantly.
4, fibrocartilage regenerates situation
Sample after iconography detects carries out histology.
(1) HE staining procedure:
1) it organizes after fixing, conventional decalcification, embedding, slicing treatment；
2) 5 μ m-thick paraffin piece 30min are toasted at 60 DEG C, at room temperature rewarming 10min；
3) dimethylbenzene dewaxing 10min × 2 time；
4) each 3min of 95%, 80%, 70%, 30% alcohol, distilled water 2min, gradient enter water, aquation tissue；
5) haematoxylin disseminates 5min, and flowing water rinses 3-5min；
6) 1min is disseminated in Yihong, washes 2sec；
7) 80%, 90%, 95%, 100% each 5sec of alcohol, serial dehydration；
8) dimethylbenzene 5min × 2 time are transparent；
9) resinene sealing, microscopically observation after sunning.
(2) Toluidine blue staining step:
1) it organizes after fixing, conventional decalcification, embedding, slicing treatment；
2) 5 μ m-thick paraffin piece 30min are toasted at 60 DEG C, at room temperature rewarming 10min；
3) dimethylbenzene dewaxing 10min × 2 time；
4) each 3min of 95%, 80%, 70%, 30% alcohol, distilled water 2min, gradient enter water, aquation tissue；
5) toluidine blue disseminates 30-40min, and flowing water rinses 3-5min；
6) acetone breaks up to cartilage cell high-visible in hyacinthine；
6) 80%, 90%, 95%, 100% each 5sec of alcohol, serial dehydration；
7) dimethylbenzene 5min × 2 time are transparent；
8) resinene sealing, microscopically observation after sunning.Experimental result is as shown in Figure 4 and Figure 5, indicates postoperative 8 weeks
With 16 weeks, histological stain show fibrocartilage regeneration the case where.
From experimental result as can be seen that freeze-drying PRP load ICA bracket group fibrocartilage regeneration situation is good.
Claims (10)
1. a kind of composite biological material, which is characterized in that the ingredient of the composite biological material is by PRP, icariin and chlorination
Calcium composition.
2. composite biological material as described in claim 1, which is characterized in that the mass percent of the calcium chloride is
0.63%-1.49%.
3. composite biological material as described in claim 1, which is characterized in that the mass percent of the PRP is 63.6%--
88.9%.
4. composite biological material as described in claim 1, which is characterized in that the mass percent of the icariin is
21.6%-25.93%；
Preferably, the weight ratio of the PRP and icariin is 2.85-3.53:1.
5. the composite biological material as described in claim 1-4 is any, which is characterized in that the composite biological material is aggregate
Material, bone implant, bone graft, bone substitute or bone bracket；
Preferably, the composite biological material is bone tendon interface graft；
More preferably kneecap kneecap tendon graft.
6. composite biological material a method as claimed in any one of claims 1 to 5, which is characterized in that the composite biological material is by freeze-drying
Processing.
7. any composite biological material of claim 1-6 is applied in preparing tissue engineering bracket；
Preferably, the tissue engineering bracket is bone tendon Interface Microstructure engineering rack.
8. any composite biological material of claim 1-6 is in the bone material or bone implant of preparation synthesis or bone graft
Or the application in bone substitute or bone bracket；
Preferably, the bone bracket is whole or layering bone bracket.
9. a kind of preparation method of the arbitrarily described composite biological material of claim 1-6, which comprises the following steps:
(1) preparation includes the gel of PRP and icariin；
(2) gel in step (1) is lyophilized；
Preferably, the step further includes,
(3) it sterilizes；
It is further preferable that being carried out disinfection using ethylene oxide, disinfection by ultraviolet light or hydrogen peroxide fumigating system gas.
10. preparation method as claimed in claim 9, which is characterized in that the preparation of the gel includes:
PRP is concentrated, obtains PRP concentrate；
Icariin is dissolved into obtain icariin solution；
It is mixed again with the PRP concentrate after calcium chloride solution is added to the icariin solution, obtains mixed liquor；
Preferably, concentration PRP uses gradient centrifugation；
It is further preferred that the gradient centrifugation is differential centrifugation；
It is further preferable that the step of preparation of the PRP concentrate, includes:
A. by 20-30mL blood sample, 250-350g is centrifuged 10-15min, removes red blood cell；
B. then 800-1000g is centrifuged 10-15min, takes the blood sample of lower layer 2.4-3.0mL, remaining liquid removes to obtain PRP concentrate
PRP；
It is further preferred that the step of preparation of the PRP concentrate further include:
C. 30-60s is shaken uniformly to get concentration PRP concentrate；
Or preferably, the icariin is dissolved using DMSO；
It is further preferable that the concentration of the icariin solution is 45-55mM；
Or preferably, the concentration of the calcium chloride solution is 5-2010% (w/v) calcium chloride solution；
Or preferably, in the mixed liquor, the volume ratio of icariin solution, calcium chloride solution and PRP concentrate is 1-2:10-
15:100-110.
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