CN110269957A - A kind of umbilical cord MSCs modified bacteria cellulose compound support frame material and preparation method thereof - Google Patents
A kind of umbilical cord MSCs modified bacteria cellulose compound support frame material and preparation method thereof Download PDFInfo
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- 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
- A61L27/14—Macromolecular materials
- A61L27/16—Macromolecular materials obtained by reactions only involving carbon-to-carbon unsaturated bonds
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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
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- A—HUMAN NECESSITIES
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- 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
- A61L27/38—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 containing added animal cells
- A61L27/3804—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 containing added animal cells characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
- A61L27/3834—Cells able to produce different cell types, e.g. hematopoietic stem cells, mesenchymal stem cells, marrow stromal cells, embryonic stem cells
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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
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
- A61L27/54—Biologically active materials, e.g. therapeutic substances
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
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- C08B15/00—Preparation of other cellulose derivatives or modified cellulose, e.g. complexes
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- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
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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
- A61L2430/00—Materials or treatment for tissue regeneration
Abstract
The invention discloses modified bacteria cellulose compound support frame materials of a kind of umbilical cord MSCs and preparation method thereof, belong to stem cell transplantation field and bacteria cellulose compound support frame material field, now propose following scheme, it includes modified bacteria cellulose composite membrane and plantation in the umbilical cord MSCs on modified bacteria cellulose composite membrane, obtains P3 for umbilical cord MSCs through MSCs culture medium culture with neonatal umbilical cord China Tong Shi glue；The physical property of measurement composite membrane after freeze-dried.The bracket that the present invention can be adhered to using bacteria cellulose composite membrane as mescenchymal stem cell; the effect that mescenchymal stem cell promotes neurological functional recovery is given full play in cerebral injury local transplantation; solve the problems, such as how stem cell efficiently transplants approach; the endocranium of artificial meninx surgical repair defect can be substituted simultaneously; Hard meninges in reconstruction integrality; brain tissue is protected, the complication such as leakage of cerebrospinal, intracranial infection, Naoning tablet, epilepsy are prevented.
Description
Technical field
The present invention relates to stem cell transplantation fields and bacteria cellulose compound support frame material technical field, more particularly to one kind
Umbilical cord MSCs modified bacteria cellulose compound support frame material and preparation method thereof.
Background technique
Hypertensive cerebral hemorrhage is the global disease of a kind of high incidence, high disability rate and high lethality rate, is to endanger the mankind
Healthy not only common but also serious disease.China's cerebral hemorrhage annual morbidity is up to 50.6/10 ten thousand~80.7/10 ten thousand.In recent years, with
The acceleration of aging of population process and living-pattern preservation, disease incidence is in the trend that rises year by year, fall ill anxious, poor prognosis and medical treatment
Expense is high, seriously endangers health and life quality, is an arduous clinic and social public health problem.Traditional internal medicine is protected
Keep treatment or operation of opening cranium, therapeutic effect is undesirable, disable with the cause of death be mainly acute hematoma treated intracranial mass lesion and
Bleeding itself is to a series of pathological changes caused by brain and vascular lesion.Nervous system because by self-repairing capability limited without
The nerve cell for being same as other tissues, especially adult lacks power of regeneration, although there are neural stem cell in brain tissue,
But its ability for generating active serotonergic neuron is extremely limited after brain injury.Therefore, cell transplantation is for nervous system injury
Or the reparation after disease has great importance.In recent years, it is dry to report various tissue-derived mesenchymas for existing lot of documents
Cell is applied to the treatment of central lesion, disease, especially umbilical cord mesenchymal stem cells, because its differentiation capability is strong,
Materials are easy, and graft versus host disease is lighter, and infectious diseases recall rate is low, negative for tumor cells pollution, harmless to donor
Etc. advantages, it has also become a kind of economic, safety, for transplantation treatment source of human stem cell and be used for cell transplantation and increasingly at
It is ripe.But an efficient transplanting approach how is established, becomes the difficult point of stem cell transplantation always.
The essential measure for treating hypertensive cerebral hemorrhage is that hemotoncus is promoted to absorb as early as possible, control brain edema, mitigation or reverse
Perihematoma brain tissue impairment.Heavy hypertensive cerebral hemorrhage tends to the treatment of Surgery hemotoncus, especially for effectively slow
Secondary lesion caused by intracranial hypertension after cerebral injury, brain edema caused by solution bleeding is often taken bone flap to open wide endocranium and is subtracted
It presses and causes defect of meninges, generate the complication such as the excessive, intracranial infection of cerebrospinal fluid.Cranial surgery operation of opening cranium patient has 10%~
15% needs artificial dura mater to repair defect of meninges.Therefore find suitable endocranium repair materials, carry out duramater reparation for
Hard meninges in reconstruction integrality protects brain tissue, prevents the complication such as leakage of cerebrospinal, intracranial infection, Naoning tablet, epilepsy to have important
Effect also becomes an important topic in neurosurgery work.
Endocranium material, which is applied to clinical research, starts from early 20th century, but still fails to develop Utopian artificial hard
Meninx alternative materials.The problem of being primarily present be such as: tissue rejection reaction, endocranium and surrounding tissue adhesion, infection, bleeding, brain
Cerebrospinal fluid leakage, epilepsy, induced tumor etc..We are controllable when having utilized bacteria cellulose biosynthesis in the experiment of early period
Characteristic adds different water-soluble substances in the fermentation medium and prepares modified bacteria cellulose, and synthesizes and construct physics, changes
The bacteria cellulose composite membrane for learning superior performance, is tested by vitro and in vivo, has inquired into its possibility as artificial dura mater
Property.
It establishes on pertinent literature and experiment basis, we are done using modified bacteria cellulose composite membrane as umbilical cord mesenchyma
The bracket that cell depends on, the unique nanometer ultra microstructure of bacteria cellulose composite membrane itself, excellent mechanical mechanics property, high ratio
The features such as surface area and strong H-bonding capability all determine that it, with good biocompatibility, is cell in three-dimensional
Growth is attached under structure and proliferation provides feasibility.It is applied in surgery for hypertensive intracerebral hemorrhage, can not only be damaged in brain
Hurt local transplantation and play the effect of stem cell promotion neurological functional recovery, and artificial meninx surgical repair defect can be substituted
Endocranium has wide potential applicability in clinical practice.
For this purpose, the present invention proposes a kind of umbilical cord MSCs modified bacteria cellulose compound support frame material and preparation method thereof.
Summary of the invention
The purpose of the present invention is to solve disadvantages existing in the prior art, and a kind of umbilical cord MSCs proposed is modified thin
Fungin compound support frame material and preparation method thereof.
To achieve the goals above, present invention employs following technical solutions:
A kind of umbilical cord MSCs modified bacteria cellulose compound support frame material, including modified bacteria cellulose composite membrane and plantation
In the umbilical cord MSCs on modified bacteria cellulose composite membrane.
A kind of preparation method of umbilical cord MSCs modified bacteria cellulose compound support frame material, includes the following steps,
S1, umbilical cord MSCs's is separately cultured, and obtains P3 for navel through MSCs culture medium culture with neonatal umbilical cord China Tong Shi glue
Band MSCs；
S2, using Acetobacter xylinum M12 as starting strain, in the culture medium of bacteria cellulose biosynthesis
Carboxymethyl cellulose, sodium alginate and polyvinyl alcohol are added, bacteria cellulose composite membrane is extracted after being left to ferment, is then measured
Yield, the wet film water content of bacteria cellulose composite membrane；Then the physical property of freeze-dried rear measurement composite membrane；
S3 takes bacteria cellulose film, moist heat sterilization, and PBS impregnates for 24 hours, MesencultTMCulture solution impregnates for 24 hours, by P3 generation
Umbilical cord MSCs is with 1.0x106cells/cm2Density plantation on modified bacteria cellulose composite membrane, temperature is at 37 DEG C, volume point
The C0 that number is 0.052In incubator, using MesencultTMCulture solution culture, every 3d change liquid, and it is fine to obtain umbilical cord MSCs modified bacteria
Tie up plain compound support frame material.
Preferably, in the S1, neonatal umbilical cord tissue block will be cut into 1 × 1 × 1mm3Size is laid in culture bottle
Bottom is taken out in acquisition for 24 hours, and suitable physiology is added into culture dish in the fresh umbilical cord of viral diagnosis and Bacteria Detection qualification
Salt water washs umbilical cord to surface completely without bloodstain, umbilical cord is cut into the segment of 2~3cm, is cut off along vein blood vessel, careful to remove
Fahrenheit glue is removed after venous blood tube wall and arteries, the Fahrenheit glue of removing is shredded with tissue shear to 1mm size, by 100g/L
It is inoculated in the T75 culture bottle of the DMEM-F12 culture solution containing 10% fetal calf serum, sets CO2It is cultivated in incubator, every 2~3d
Liquid is changed, inverted microscope observes the cellular morphology climbed out of, after the fusion of cell 90%, passes on P1.
Preferably, when cultured cell line fusion 90%, the culture solution in bottle is removed in superclean bench, it is raw with 20ml
It manages salt water to clean cell aufwuchsplate 2 times, 37 DEG C of the digestive ferment digestion of 3ml 0.125% is added, observe quilt under inverted microscope
The cell of digestion is added 3ml complete medium after being not connected in flakes and neutralizes, and add 20ml normal saline dilution to cell rounding
Afterwards, cell suspension is made in piping and druming, and cell suspension suction is set in centrifuge tube, and 2000r/min is centrifuged 5min, is centrifuged radius 10cm,
With 1.0x10 after cell count4Cell/cm2Being inoculated into culture medium is MesencultTMT75 culture bottle in, 2~4h, that is, visible
Most cells are adherent, and 90% can be merged after 3~4d, need to be passed on again, and propagating method is same as above, until being passaged to
P3。
Preferably, carboxymethyl cellulose 0.5g/L, seaweed are added respectively in the culture medium of bacteria cellulose biosynthesis
Sour sodium 1.0g/L and polyvinyl alcohol 1.5g/L.
Compared with prior art, the beneficial effects of the present invention are: promoting neuron regeneration, again by people's umbilical cord MSCs
The characteristics of building, and played important function restored to nervous function, using modified bacteria cellulose composite membrane as
The bracket that MSCs is depended on, it is MSCs is disposable effectively, be largely transplanted to encephalic, and can for a long time in local action,
The efficiency of MSCs is given full play to, while utilizing the good Biocompatibility of bacteria cellulose composite membrane itself, is applied to brain
In bleeding operation, the effect that stem cell promotes neurological functional recovery can be played in cerebral injury local transplantation, solve stem cell
The problem of approach how is efficiently transplanted, while the endocranium of artificial meninx surgical repair defect can be substituted, Hard meninges in reconstruction is complete
Whole property protects brain tissue, prevents the complication such as leakage of cerebrospinal, intracranial infection, Naoning tablet, epilepsy.
Specific embodiment
The technical scheme in the embodiments of the invention will be clearly and completely described below, it is clear that described implementation
Example is only a part of the embodiment of the present invention, instead of all the embodiments.
Embodiment
A kind of umbilical cord MSCs modified bacteria cellulose compound support frame material proposed by the present invention, including modified bacteria cellulose
Composite membrane and plantation are in the umbilical cord MSCs on modified bacteria cellulose composite membrane.
A kind of preparation method of umbilical cord MSCs modified bacteria cellulose compound support frame material, includes the following steps,
S1, umbilical cord MSCs's is separately cultured, and obtains P3 for navel through MSCs culture medium culture with neonatal umbilical cord China Tong Shi glue
Band MSCs；
S2, using Acetobacter xylinum M12 as starting strain, in the culture medium of bacteria cellulose biosynthesis
Carboxymethyl cellulose, sodium alginate and polyvinyl alcohol are added, bacteria cellulose composite membrane is extracted after being left to ferment, is then measured
Yield, the wet film water content of bacteria cellulose composite membrane；Then the physical property of freeze-dried rear measurement composite membrane；
S3 takes bacteria cellulose film, moist heat sterilization, and PBS impregnates for 24 hours, MesencultTMCulture solution impregnates for 24 hours, by P3 generation
Umbilical cord MSCs is with 1.0x106cells/cm2Density plantation on modified bacteria cellulose composite membrane, temperature is at 37 DEG C, volume point
The C0 that number is 0.052In incubator, using MesencultTMCulture solution culture, every 3d change liquid, and it is fine to obtain umbilical cord MSCs modified bacteria
Tie up plain compound support frame material.
In the present invention, in the S1, neonatal umbilical cord tissue block will be cut into 1 × 1 × 1mm3Size is laid in culture bottle
Bottom is taken out in acquisition for 24 hours, and suitable physiology is added into culture dish in the fresh umbilical cord of viral diagnosis and Bacteria Detection qualification
Salt water washs umbilical cord to surface completely without bloodstain, umbilical cord is cut into the segment of 2~3cm, is cut off along vein blood vessel, careful to remove
Fahrenheit glue is removed after venous blood tube wall and arteries, the Fahrenheit glue of removing is shredded with tissue shear to 1mm size, by 100g/L
It is inoculated in the T75 culture bottle of the DMEM-F12 culture solution containing 10% fetal calf serum, sets CO2It is cultivated in incubator, every 2~3d
Liquid is changed, inverted microscope observes the cellular morphology climbed out of, after the fusion of cell 90%, passes on P1.
In the present invention, when cultured cell line merges 90%, the culture solution in bottle is removed in superclean bench, uses 20ml
Physiological saline cleans cell aufwuchsplate 2 times, and 37 DEG C of the digestive ferment digestion of 3ml 0.125% are added, observe under inverted microscope
The cell being digested is added 3ml complete medium after being not connected in flakes and neutralizes, and add 20ml physiological saline dilute to cell rounding
After releasing, cell suspension is made in piping and druming, and cell suspension suction is set in centrifuge tube, and 2000r/min is centrifuged 5min, is centrifuged radius
10cm, with 1.0x10 after cell count4Cell/cm2Being inoculated into culture medium is MesencultTMT75 culture bottle in, 2~4h is
It can be seen that most cells are adherent, 90% can be merged after 3~4d, needs to be passed on again, and propagating method is same as above, until passage
To P3.
In the present invention, carboxymethyl cellulose 0.5g/L, sea are added respectively in the culture medium of bacteria cellulose biosynthesis
Mosanom 1.0g/L and polyvinyl alcohol 1.5g/L.
Cytotoxicity experiment:
After the l cell strain L929 cell growth confluent cultures bottom of bottle for passing on 48h, with trypsin digestion, system
At cell suspension, it is inoculated in 96 well culture plates.37 DEG C, 5%CO2Preculture for 24 hours, keeps cell adherent in incubator.By material
Leaching liquor carries out continuing culture for 24 hours after equivalent exchanges with original fluid in culture plate.Positive control is the culture of 0.64% phenol
Liquid, negative control are the polytetrafluoroethyl-ne propylene material leaching liquor of known non-toxic.Group of cells shape is carried out with inverted microscope simultaneously
State observation.After continuing incubation in incubator, MTT liquid is added in every hole, continues to be incubated for 4h.Culture plate is taken out, liquid in hole is sucked out,
DMSO is added in every hole, surveys its absorbance value with microplate reader.OD value is higher, illustrates that the increment of L929 cell is stronger.The inspection of MTT colorimetric method
It surveys, cell Proliferation curve is drawn by absorbance, calculates cell opposite proliferation rate RGR.
RGR=experimental group OD/ feminine gender is according to group OD × 100%
Toxic level, cytotoxicity analysis standard are as follows: RGR >=100%0 grade: 75% are converted into according to 6 grades of toxicity standards of grading
~99%I grades: 50%~74%II grade: 25%~49%III grades: 1%~24%IV grade: V grades of O.
Hemolytic experiment:
Respectively take 10mL material soak, sterile purified water (positive control), 0.9% physiological saline (negative control) in three
Centrifuge tube is put into pre-temperature in water-bath.After taking new zealand white rabbit venous blood structure rafter acid sodium anticoagulant, the anticoagulant new fresh rabbit of dilution is added
Blood, water-bath.Aspirate supernatant spectrophotometer reads optical density OD value in 545nm wavelength.Hemolytic is calculated according to the following formula
Hemolysis rate:
Hemolysis rate (%)=(sample absorbance-feminine gender absorbance)/(positive absorbance-feminine gender absorbance) × 100%；
Acute toxicity testing:
20 healthy male white mouses are selected, is divided to two groups at random, every group 10, records original body mass.Experimental mice vein
The physiological saline leaching liquor of injection material sample, control group mice inject and prepare leaching liquor with the physiological saline criticized, after injection
At once, 4,24,48 and 72h observes mouse general state and toxicity performance and death toll, after injection for 24 hours, 48h and 72h observe body
Change again.And the performance of observation small white mouse toxicity and death toll in subsequent 3 weeks.Toxicity shows Assessment for classification: a is nontoxic: nothing is appointed
What symptom；B is slight: having mild, but without motion reduction, expiratory dyspnea or abdominal symptoms；C is obvious: abdomen irritation is exhaled
Inhale difficult, movement reduction, ptosis；D severe: failure is issued, is trembled, severe abdominal irritation, ptosis, breathing
It is difficult.
Experimental animal:
Adult Wistar rats 40, random sampling is grouped after number.
All animals are divided into 4 groups:
1st group: false damage group (Sham-injured), n=8 are not damaged and are injected；
2nd group: bleeding group (CH), n=8 not intervene after cerebral hemorrhage mold preparation；
3rd group: treatment control group (CH+M), n=8, bacteria cellulose composite membrane covers after cerebral hemorrhage mold preparation
(Membrane)；
4th group: treatment group (CH+MSCs), n=16, cerebral hemorrhage mold prepare posterior umbilicus band MSCs bacteria cellulose composite membrane
Repairing；
Reagent, kit: yellow Jackets；
Instrument, consumptive material: knife blade, eye scissors, drill, syringe, the test tube containing anticoagulant, suture policy and
Line etc..
Experimental procedure:
1) the fixed rat of:
A. the rat prostrate (back up) anaesthetized is fixed on mouse plate with rope, rope slip-knot, is not easy to beat in this way
It is sliding.Two front foots that rat is kept when fixed and two rear feet difference are point-blank.
B. with cotton that the head of rat is pico- padded so that it is convenient to cut skin.
2) drills:
A. the skull of mouse is exposed into along lambdoidal suture incision of skin about 3-5cm in the head of rat.
B. the lambdoidal suture of rat carries out drilling on the right of it label at 1-2mm is found.
C. drill vertical drilling at mark is used, depth should not be too big.(prevent from piercing brain tissue, have fall through feel when
Stop)
D. stop blooding.
3) is injected:
A. the tail portion of rat is cut one section, is squeezed out blood with finger, and with being equipped with the test tube of anticoagulant for blood
It collects, blood volume substantially 0.5ml.
B. needle tubing is directed at the aperture on rat skull, needle tubing is inserted into rat cranial cavity, insertion depth is about
1.5cm。
C. the piston for pushing needle tubing, rat blood is slowly injected into cranial cavity, promotes about 0.5ml.
D. it after the completion of injecting, waits about after five minutes, needle tubing is extracted out.
4) attaching of .MSC modified bacteria cellulose composite membrane:
Experimental group randomly selects 24 Rat Cerebral hemorrhage Models, is modified at dura defect with the umbilical cord MSCs prepared thin
The repairing of fungin composite membrane, control group are covered with modified bacteria cellulose composite membrane (Membrane) and are repaired.
5) is sutured, and puts back to recovery, observes animal.
Observer's umbilical cord MSC modified bacteria cellulose composite membrane promotes a possibility that neurological functional recovery；
Neurologic score:
According to the neurotrosises severity scale such as Chen, NSS by: movement, feel, reflection and four part group of blance test
At Neuroscore 0-18 is normal: 0, the afunction of most serious: 18；Score value is higher, and it is more serious to represent functional lesion.
13-18, it is serious to damage；7-12, moderate lesion；1-6, minor injury.
The processing and observation of brain tissue:
4 weeks after Hemorrhage Model preparation, groups of animals gives 10% chloraldurate intraperitoneal injection of volume fraction anesthesia, 40g/
L paraformaldehyde is perfused through left ventricle and fixes.Brain tissue is completely removed, fixed after same fixer room temperature to stay overnight, rear row paraffin packet
It buries, is sliced in MSC modified bacteria cellulose composite membrane transplanted sites continuous coronal, piece is 5 μm thick.1 is extracted every 10 slices
Row BrdU immunohistochemical staining, after determining BrdU positive cell under mirror, take its contiguous slices respectively row HE, cresyl viollet,
NSE, GFAP, vascular endothelial growth factor (vascularendothelial growth factor, VEGF) immunohistochemistry
Dyeing.
Statistical procedures:
Respectively after the transfer 1d, 1w, 2w, 3w, 4w, 5w carry out NSS scoring, whole data using SPSS statistical package into
Row statistical analysis.Each group of data withIt indicates.It is tested using Independent-Samples t Test.p<
0.05, which is considered difference, significant.
As a result:
1, the clinical manifestation of cerebral hemorrhage mold:
After Hemorrhage Model preparation, rat may occur in which 10-15 seconds apneas, generally can be self-healing, and be expert at compared with elder
It can restore autonomous respiration after artificial respiration, it is then dead to be unable to recuperator.Regaining consciousness after about 1-3 hours after cerebral injury, rat has hemiplegia,
It is slow in action, rolls to the right when walking about.
2, the pathological study of rat cerebral trauma:
2.1. brain tissue gross examination of skeletal muscle:
The visible appearance for having focal zone brain edema of 3d after cerebral hemorrhage damage；14d shows as the increasing of graft area endocranium after wound
Thickness, brain surface have the cortex defect of diameter 8mm or so to be recessed, and invaginate about 2-3mm, in light yellow, when with observing after transplanting
Between extension, thickness wounds stove color gradually deepens, and is finally in glassy yellow.
2.2. histopathology dyeing observation:
The perihematoma of rat cerebral tissue visible tissue structure under HE dyeing is destroyed, falls off, neuronal degeneration, cell space wrinkle
Contracting, there is spongiocyte and inflammatory cell infiltration.Cerebral hemorrhage group rat is in the dyeing of 14d brain tissue hippocampus cresyl viollet and normal sea
Horse structure compares, and dentate fascia, upper and lower granular cell layer are shown in that pycnotic cell and neuronal degeneration, necrosis, Nissl body obviously subtract
It is few.
3. the Neuroscore after Intracerebral Hemorrhage in Rats:
The NSS appraisal result of tri- groups of the rat 1d after bleeding, CH, CH+PBS, CH+MSCs is through homogeneity test of variance, F=
0.620, p > 0.05, illustrate to make the neurological dysfunction degree between brain injury animal model each group than more consistent (table 1).
Neuroscore result after 1. Intracerebral Hemorrhage in Rats of table
Group1:Sham-injured,Group2:CH,Group3:CH+M,Group4:CH+MSCs.
1.p<0.05,compared with Sham-injured group at the same time point.
2.p>0.05,compared with CH at the same time point.
3.p<0.05,compared with CH+PBS at the same time.
Rat nerve function is in bleeding group (CH) compared with false damage group (Sham), and each time point NSS scoring increases after wound
Height, statistical analysis have significant difference (p < 0.05)；Treatment group (CH+MSCs) and bleeding group (CH), treatment control group (CH+
M it) compares, there was no significant difference (p > 0.05) for 1d NSS scoring after wound；1 week later each time point compared after wound, received
MSCs transplanting treatment group's Neuroscore compared with bleeding group (CH) and treat control group (CH+M) improve significantly (p <
0.01).Illustrate that bleeding of the umbilicus MSCs transplanting is effective to the post-traumatic neurological functional recovery of rat brain.
Histological observation after bleeding of the umbilicus MSCs transplanting:
The 2w after rat cerebral trauma dyes the Specific marker NSE of neuron, is damaged around bleeding of the umbilicus MSCs transplanting
The survival of the visible brown color NSE positive cell of the cortex and hippocampus of wound, cell aggregation, and a part of cell are in nerve
Spongiocyte marker GFAP stained positive.In the cell of the visible Hochest stained positive of fluorescence microscope, it was demonstrated that be outer
It is damaged the regulation of brain tissue local environment after the bleeding of the umbilicus MSCs transplanting of source property and breaks up；Tissue after cerebral injury 4w is cut
Piece compares when by with 2w in fluorescence microscopy microscopic observation, it is seen that stronger migration occurs in the MSCs of transplanting, farthest up to 2mm
Left and right.
The foregoing is only a preferred embodiment of the present invention, but scope of protection of the present invention is not limited thereto,
Anyone skilled in the art in the technical scope disclosed by the present invention, according to the technique and scheme of the present invention and its
Inventive concept is subject to equivalent substitution or change, should be covered by the protection scope of the present invention.
Claims (5)
1. a kind of umbilical cord MSCs modified bacteria cellulose compound support frame material, which is characterized in that multiple including modified bacteria cellulose
Film and plantation are closed in the umbilical cord MSCs on modified bacteria cellulose composite membrane.
2. a kind of preparation method of umbilical cord MSCs modified bacteria cellulose compound support frame material described in claim 1, feature
It is, includes the following steps,
S1, umbilical cord MSCs's is separately cultured, and obtains P3 for umbilical cord through MSCs culture medium culture with neonatal umbilical cord China Tong Shi glue
MSCs；
S2 is added in the culture medium of bacteria cellulose biosynthesis using Acetobacter xylinum M12 as starting strain
Carboxymethyl cellulose, sodium alginate and polyvinyl alcohol extract bacteria cellulose composite membrane after being left to ferment, then measure bacterium
Yield, the wet film water content of cellulose composite membrane；Then the physical property of freeze-dried rear measurement composite membrane；
S3 takes bacteria cellulose film, moist heat sterilization, and PBS impregnates for 24 hours, MesencultTMCulture solution impregnates for 24 hours, by P3 for umbilical cord
MSCs is with 1.0x106cells/cm2Density plantation on modified bacteria cellulose composite membrane, temperature is in 37 DEG C, volume fraction
0.05 C02In incubator, using MesencultTMCulture solution culture, every 3d change liquid, obtain umbilical cord MSCs modified bacteria cellulose
Compound support frame material.
3. a kind of preparation method of umbilical cord MSCs modified bacteria cellulose compound support frame material according to claim 2,
It is characterized in that, in the S1, neonatal umbilical cord tissue block will be cut into 1 × 1 × 1mm3Size is laid in culture bottle bottom, takes out
In acquisition for 24 hours, the fresh umbilical cord of viral diagnosis and Bacteria Detection qualification is added suitable physiological saline, washes into culture dish
Umbilical cord is washed to surface completely without bloodstain, umbilical cord is cut into the segment of 2~3cm, is cut off along vein blood vessel, vein blood vessel is carefully removed
Fahrenheit glue is removed after wall and arteries, the Fahrenheit glue of removing is shredded with tissue shear to 1mm size, is inoculated in and is contained by 100g/L
In the T75 culture bottle of the DMEM-F12 culture solution of 10% fetal calf serum, CO is set2It is cultivated in incubator, changes liquid every 2~3d,
The cellular morphology that micro- sem observation climbs out of is set, after the fusion of cell 90%, passes on P1.
4. a kind of preparation method of umbilical cord MSCs modified bacteria cellulose compound support frame material according to claim 3,
It is characterized in that, when cultured cell line merges 90%, the culture solution in bottle is removed in superclean bench, with 20ml physiological saline
37 DEG C of digestive ferment of 3ml0.125% digestion are added in cleaning cell aufwuchsplate 2 times, observed under inverted microscope be digested it is thin
Born of the same parents, to cell rounding, be not connected in flakes after be added 3ml complete medium neutralize, and add 20ml normal saline dilution after, piping and druming system
At cell suspension, cell suspension suction is set in centrifuge tube, 2000r/min is centrifuged 5min, radius 10cm is centrifuged, after cell count
With 1.0x104Cell/cm2Being inoculated into culture medium is MesencultTMT75 culture bottle in, 2~4h, that is, visible most cells
It is adherent, 90% can be merged after 3~4d, needs to be passed on again, and propagating method is same as above, until being passaged to P3.
5. a kind of preparation method of umbilical cord MSCs modified bacteria cellulose compound support frame material according to claim 2,
It is characterized in that, adds carboxymethyl cellulose 0.5g/L, sodium alginate respectively in the culture medium of bacteria cellulose biosynthesis
1.0g/L and polyvinyl alcohol 1.5g/L.
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|---|---|---|---|---|
| WO2022119545A3 (en) * | 2020-12-04 | 2022-09-22 | Yildiz Teknik Üniversitesi | Bacterial cellulose based umbilical cord ring |
Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101041844A (en) * | 2007-04-29 | 2007-09-26 | 山东轻工业学院 | Method for improving bacteria cellulose output by adding sodium alginate |
| CN101509026A (en) * | 2009-03-27 | 2009-08-19 | 上海应用技术学院 | Bacteria cellulose compound film, preparation and uses thereof |
| CN101509025A (en) * | 2009-03-20 | 2009-08-19 | 武汉科技学院 | Method of preparing bacteria cellulose composite material |
| CN102961777A (en) * | 2012-12-11 | 2013-03-13 | 北京科技大学 | Method for preparing porous compound type high permeability absorption hemostasis coating with modified nano-crystalline cellulose |
| CN105176920A (en) * | 2015-10-12 | 2015-12-23 | 王泰华 | Method for differentiating human umbilical cord mesenchymal stem cells into myocardial-like cells |
| CN106267310A (en) * | 2016-08-16 | 2017-01-04 | 仇颖莹 | A kind of preparation method of composite bacterial cellulose medical dressing |
| CN108660110A (en) * | 2018-05-21 | 2018-10-16 | 山东大学 | A method of broken up using the aquagel evoked stem cell of bacteria cellulose piezoelectricity |
| CN108676194A (en) * | 2018-04-08 | 2018-10-19 | 北京林业大学 | A kind of complex polysaccharide hydrogel and its biology in situ synthetic method |
| WO2018200753A1 (en) * | 2017-04-25 | 2018-11-01 | Paul Gatenholm | Biocompatible conductive inks based on cellulse nanofibrils for 3d printing of conductive biomedical devices |
-
2019
- 2019-07-15 CN CN201910634780.8A patent/CN110269957A/en active Pending
Patent Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101041844A (en) * | 2007-04-29 | 2007-09-26 | 山东轻工业学院 | Method for improving bacteria cellulose output by adding sodium alginate |
| CN101509025A (en) * | 2009-03-20 | 2009-08-19 | 武汉科技学院 | Method of preparing bacteria cellulose composite material |
| CN101509026A (en) * | 2009-03-27 | 2009-08-19 | 上海应用技术学院 | Bacteria cellulose compound film, preparation and uses thereof |
| CN102961777A (en) * | 2012-12-11 | 2013-03-13 | 北京科技大学 | Method for preparing porous compound type high permeability absorption hemostasis coating with modified nano-crystalline cellulose |
| CN105176920A (en) * | 2015-10-12 | 2015-12-23 | 王泰华 | Method for differentiating human umbilical cord mesenchymal stem cells into myocardial-like cells |
| CN106267310A (en) * | 2016-08-16 | 2017-01-04 | 仇颖莹 | A kind of preparation method of composite bacterial cellulose medical dressing |
| WO2018200753A1 (en) * | 2017-04-25 | 2018-11-01 | Paul Gatenholm | Biocompatible conductive inks based on cellulse nanofibrils for 3d printing of conductive biomedical devices |
| CN108676194A (en) * | 2018-04-08 | 2018-10-19 | 北京林业大学 | A kind of complex polysaccharide hydrogel and its biology in situ synthetic method |
| CN108660110A (en) * | 2018-05-21 | 2018-10-16 | 山东大学 | A method of broken up using the aquagel evoked stem cell of bacteria cellulose piezoelectricity |
Non-Patent Citations (4)
| Title |
|---|
| C.M.C.O. SOUZA ET AL: "Regeneration of Skin Tissue Promoted by Mesenchymal Stem Cells Seeded in Nanostructured Membrane", 《TRANSPLANTATION PROCEEDINGS》 * |
| YI-MENG CAO ET AL: "Surface-structured bacterial cellulose loaded with hUSCs accelerate skin wound healing by promoting angiogenesis in rats", 《BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS》 * |
| 王海波: "细菌纤维素耐低温生产菌株的选育及其改性的研究", 《中国优秀硕士学位论文全文数据库 基础科学辑》 * |
| 郑敬彤等: "大鼠脂肪干细胞与细菌纤维素膜的复合培养", 《中国组织工程研究与临床康复》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2022119545A3 (en) * | 2020-12-04 | 2022-09-22 | Yildiz Teknik Üniversitesi | Bacterial cellulose based umbilical cord ring |
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