CN102485888A - Effect of pulse electromagnetic field on promotion of in vitro osteogenesis and differentiation of human umbilical cord mesenchymal stem cells - Google Patents
Effect of pulse electromagnetic field on promotion of in vitro osteogenesis and differentiation of human umbilical cord mesenchymal stem cells Download PDFInfo
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Abstract
The invention relates to a method of applying a pulse electromagnetic field in promoting in vitro differentiation of mesenchymal stem cells and especially to a method of applying a pulse electromagnetic field in promoting in vitro osteogenesis and differentiation of human umbilical cord mesenchymal stem cells and application of a compound of pulse electromagnetic field promoted human umbilical cord mesenchymal stem cells and hydroxyapatite in repairing bone defects.
Description
Invention field
The present invention relates to a kind of method of using pulse electromagnetic field to promote the mescenchymal stem cell vitro differentiation; Particularly relate to the method that pulse electromagnetic field promotes the external Osteoblast Differentiation of human umbilical cord mesenchymal stem cells, and pulse electromagnetic field promotes human umbilical cord mesenchymal stem cells and the application of hydroxylapatite compound in repairing bone defect.
Background of invention
(mesenchymal stem cells MSCs) is the multipotential stem cell with self and multidirectional differentiation potential in a group mesoderm source to mescenchymal stem cell.Research early and more deep be the mesenchymal stem cells MSCs that comes from the marrow of being grown up.From marrow other many tissues (like fat, muscle, placenta, umbilical cord etc.) in addition, separated at present and obtained MSCs.(human umbilical cord mesenchymal stem cells's human umbilical cord mesenchymal stem cells hUCMSCs) has drawn from always as the umbilical cord tissue of childbirth waste.Its convenience of drawing materials, wide material sources, have and adult bone bone marrow-drived mesenchymal stem similar biological, come from prenatal mescenchymal stem cell simultaneously and have more powerful multiplication capacity, the more differentiation potential, lower immunogenicity etc. of wide spectrum.Be expected to as organizational project or the good seed cell of cell replacement treatment.
(pulsed electyomagnetic fields PEMFs) is the pulsed current that is produced by surge generator and then generates an electromagnetic field pulse electromagnetic field.Surge generator can be regulated and control, and therefore can produce the pulsed current of CF, waveform, thereby produce specific pulse electromagnetic field.Because electricity with magnetic field be supplement and complement each other, interactive, the biological effect that enclosed pasture is different with Gauss's field energy generation, the change degree of each is carried out simultaneously, so the effect that can generate an electromagnetic field, and is referred to as the PEMFs energy at present.PEMFs firstly appears in the famous orthopedic specialist Bassett of U.S. apply pulse EM field therapy in 1977 in the application in the orthopaedic disease treatment as a kind of non-intruding therapy and successfully treats the patient of a routine congenital pseudarthrosis of tibia.So far, pulse electromagnetic field has been obtained significant curative effect at aspects such as the reparation of clinical promotion bone injury, osteoporosises.
Simultaneously, the generation of organizational engineering and regenerative medicine with develop into the damaged treatment of serious orthopaedics illness such as bone and brought new hope with bone does not connect etc.The propagation of seed cell and differentiation capability are Tissue Engineering Study and key in application place.Yet the Osteoblast Differentiation of bone tissue engineer seed cell adopts chemical reagent to induce or the growth factor-induced method more at present.These chemical reagent or growth factor can not guarantee that it plays a role with stable concentration in vivo, strengthen concentration and can produce CDCC again, and the seepage that surpasses physiological dose can cause the ectopic ossification effect of bone-inducing factor.If can be with PEMFs as the effective means that stimulates the mescenchymal stem cell Osteoblast Differentiation, with the use of avoiding the exogenous chemical inductor.Research shows that PEMFs can promote that scleroblast is cell proliferation, reaches the Osteoblast Differentiation in differential period, and MSCs is one of important progenitor cell that osteocyte forms in the bone reparation.Up to the present, PEMFs does not appear in the newspapers to the biological impact of hUCMSCs as yet; Can PEMFs promote the hUCMSCs Osteoblast Differentiation to wait the research confirmation.
Goal of the invention
An object of the present invention is to provide a kind of method of using pulse electromagnetic field to promote the external Osteoblast Differentiation of human umbilical cord mesenchymal stem cells; Be characterised in that pulse electromagnetic field as the effective means that stimulates the external Osteoblast Differentiation of mescenchymal stem cell; (use chemical reagent or growth factor can not guarantee that it plays a role with stable concentration in vivo the use of avoiding the exogenous chemical inductor; Strengthen concentration and can produce CDCC again; The seepage that surpasses physiological dose can cause the ectopic ossification effect of bone-inducing factor), avoid the use of the untoward reaction that exogenous chemical inductor or growth factor cause.
According to a preferred embodiment of the invention, wherein said mescenchymal stem cell is a human umbilical cord mesenchymal stem cells.
According to a preferred embodiment of the invention, wherein said pulse electromagnetic field is produced by the pulse electrical signal generating unit.
According to a preferred embodiment of the invention, the pulse electromagnetic field stimulation parameter is: frequency 50Hz; Voltage 500mv/cm; Waveform rectangle positive pulse; Dutycycle 50%; Thorn flyback cycle 3h/d.
Another object of the present invention provides pulse electromagnetic field and promotes human umbilical cord mesenchymal stem cells and the application of hydroxylapatite compound in repairing bone defect.
According to a preferred embodiment of the invention, wherein said bone is damaged is that the segmental bone that utilizes operation method to cause is damaged.
Description of drawings
Fig. 1 shows that pulse electromagnetic field promotes human umbilical cord mesenchymal stem cells Osteoblast Differentiation alkaline phosphatase activities detected result.
Fig. 2 shows that pulse electromagnetic field promotes human umbilical cord mesenchymal stem cells Osteoblast Differentiation 21 days, Von Kossa ' s coloration result.Wherein 2A is that the induced liquid+pulse of traditional chemical reagent becomes induced by magnetic field group (PE+CR+); 2B is that basic culture solution adds pulse electromagnetic field and induces group (PE+CR-); 2C is that chemical reagent is induced group (PE-CR+); 2D is the conventional culture group (PE-CR-) of basic culture solution.
Fig. 3 shows that pulse electromagnetic field promotes human umbilical cord mesenchymal stem cells Osteoblast Differentiation 14 days, sodium alizarinsulfonate dyeing.Wherein 3A is that the induced liquid+pulse of traditional chemical reagent becomes induced by magnetic field group (PE+CR+); 3B is that basic culture solution adds pulse electromagnetic field and induces group (PE+CR-); 3C is that chemical reagent is induced group (PE-CR+); 3D is the conventional culture group (PE-CR-) of basic culture solution.
Fig. 4 shows that pulse electromagnetic field promotes human umbilical cord mesenchymal stem cells Osteoblast Differentiation Real-time PCR detected result.Wherein 4A is RUNX2 genetic expression; 4B is Collagen I genetic expression; 4C is that OPN expresses.
Fig. 5 shows that pulse electromagnetic field promotes human umbilical cord mesenchymal stem cells Osteoblast Differentiation western-blot detected result.Wherein 5A is that western-blot detects Collagen I; 5B is that western-blot detects RUNX2; 5C is that western-blot detects OPN.
Fig. 6 shows that pulse electromagnetic field promotes the radiological examination result of human umbilical cord mesenchymal stem cells and hydroxylapatite compound repairing bone defect.Wherein 6A is human umbilical cord mesenchymal stem cells+Win 40350+pulse electromagnetic field group; 6B is human umbilical cord mesenchymal stem cells+Win 40350 group; 6C is the blank group.
Summary of the invention
The present invention relates to a kind of method of using pulse electromagnetic field to promote the mescenchymal stem cell vitro differentiation; Particularly relate to pulse electromagnetic field and promote the external Osteoblast Differentiation of human umbilical cord mesenchymal stem cells, and pulse electromagnetic field promotes human umbilical cord mesenchymal stem cells and the application of hydroxylapatite compound in repairing bone defect.
Mescenchymal stem cell (MSCs) is the multipotential stem cell with self and multidirectional differentiation potential in a group mesoderm source.Mescenchymal stem cell has and is divided into multiple histiocytic potential, but has lost the ability that develops into complete individuality, and multipotential stem cell therefore is otherwise known as.MSCs distributes extensively, is dispersed throughout the whole mescenchymal tissue of body.From marrow, fat, skin, bone, bleeding of the umbilicus, placenta and peripheral blood, be separated to MSCs at present.In addition, in the tissue on each bar blood vessel side of health mesenchymal cell is arranged all, the center is divided into mescenchymal stem cell.Umbilical cord mesenchymal stem cells (hUCMSCs) separates with the circumvascular jelly of Wharton of Umbilical artery and obtains from being arranged in umbilical vein just.
HUCMSCs has the similar many differentiation potentials with MSCs; Confirm at present; Under external evoked condition; HUCMSCs not only can like scleroblast, chondrocyte, adipocyte etc., can also break up to the neuron cell and the liver cell like cell in ectoderm and entoderm source to the histocyte differentiation in multiple mesoderm source.
An object of the present invention is to provide a kind of method of using pulse electromagnetic field to promote the external Osteoblast Differentiation of human umbilical cord mesenchymal stem cells; Be characterised in that pulse electromagnetic field as the effective means that stimulates the external Osteoblast Differentiation of mescenchymal stem cell; (chemical reagent or growth factor can not guarantee that it plays a role with stable concentration in vivo with the use of avoiding the exogenous chemical inductor; Strengthen concentration and can produce CDCC again, the seepage that surpasses physiological dose can cause the ectopic ossification effect of bone-inducing factor).
According to a preferred embodiment of the invention, wherein said mescenchymal stem cell is a human umbilical cord mesenchymal stem cells.The present invention is a research object with isolating human umbilical cord mesenchymal stem cells (hUCMSCs) from aborted fetus umbilical cord jelly of Wharton, and basic biological characteristics such as its morphology, immunophenotype and many differentiation potentials is studied.Confirm that institute's isolated cells is a mescenchymal stem cell.For carrying out pulse electromagnetic field, next step induce hUCMSCs cell source to be provided, for the clinical application of hUCMSCs provides theoretical foundation and technical support to osteoblast differentiation.
According to a preferred embodiment of the invention, wherein said pulse electromagnetic field is produced by the pulse electrical signal generating unit.Surge generator is adjustable, therefore can produce the pulsed current of CF, waveform, thereby produce specific pulse electromagnetic field.
According to a preferred embodiment of the invention, the pulse electromagnetic field stimulation parameter is: frequency 50Hz; Voltage 500mv/cm; Waveform rectangle positive pulse; Dutycycle 50%; Thorn flyback cycle 3h/d.The differentiation behavior of stem cell is influenced by its microenvironment of living in.Discover that pulse electromagnetic field has " window effect " to bone forming, promptly cell only produces reaction to the magnetic field of CF, intensity, and magnetic field and intercellular effect are not linear.The magnetic field of different frequency, intensity can produce different biological effects.
Another object of the present invention provides pulse electromagnetic field and promotes human umbilical cord mesenchymal stem cells and the application of hydroxylapatite compound in repairing bone defect.
Fracture and bone injury are common clinicals, because bone structure is special, even can heal, also need reach several thoughtful several months fixed constraints activities, often cause multiple complications such as limbs disuse atrophy, joint accretion, bedsore, breathing and urinary system infection.The clinical especially insoluble disease of fracture compound comminuted or large segmental bone defect, its quantity, form and had relatively high expectations by the matching degree of plot structure for graft.Simple creeping substitution and induced osteogenesis effect of leaning on implantable bone becomes knitting slower, has a strong impact on patient's work capacity and life quality.Utilize pulse electromagnetic field to stimulate the support carrier to combine stem cell to make it to Osteoblast Differentiation, and the implantable bone defect, thereby promote bone defect healing, be expected to become the good approach of bone tissue engineer repairing bone defect.The present invention has confirmed that the hUCMSCs multiplication capacity is powerful, immunogenicity is weak or do not have, have the osteoblast differentiation ability, is the seed cell source of ideal bone tissue engineer; PEMFs can effectively promote the hUCMSCs Osteoblast Differentiation.We form mixture with hUCMSCs and Win 40350 (HA), implant the large segmental bone defect place, and carrying out PEMFs stimulates, and can accelerate knitting, recovers the integrity and the bio-mechanical performance (seeing embodiment 2 for details) of bone rapidly.
Result of study of the present invention shows that pulse electromagnetic field can promote the external Osteoblast Differentiation of human umbilical cord mesenchymal stem cells, and pulse electromagnetic field can promote the application in repairing bone defect of human umbilical cord mesenchymal stem cells and hydroxylapatite compound.
Below by embodiment preferred forms of the present invention is described.These embodiment are intended to further illustrate for example the present invention, rather than limit the await the reply scope of claim of the present invention by any way.
Embodiment
Embodiment 1: pulse electromagnetic field promotes the external Osteoblast Differentiation of human umbilical cord mesenchymal stem cells
One, the separation and Culture of hUCMSCs
1.hUCMSCs separation
Take from and be willing to contribute artificial abortion fetus corpse (gestational age was 12～18 weeks), umbilical cord is cut in alcohol-pickled sterilization, and umbilical vein and Umbilical artery are peeled off in the PBS flushing.Jelly of Wharton is cut into the tissue block of the about 5mm of length, adopts enzyme digestion to separate hUCMSCs and add cellular segregation liquid II (DMEM/F12,10%FBS, 0.01%II Collagen Type VI enzyme, 0.05% Unidasa), at 37 ℃, 5%CO
2With digest 12h under 95% conditions of air.The centrifugal 10min of 1500rpm abandons supernatant, with the DMEM/F12 suspension cell that contains 10%FBS, with 5 * 10
4The density inoculation of/ml.
2.hUCMSCs former be commissioned to train foster
Institute's isolated cells is at 37 ℃, 5% CO
2, change liquid after cultivating 48h under 95% the air gas phase, wash gently 3 times with PBS earlier, to remove most not attached cell, change basic culture solution (DMEM/F12 that contains 10%FBS), 37 ℃, 5% CO
2, 95% air conditions continues down to cultivate.
The cultivation 3.hUCMSCs go down to posterity
Treat that cell grows to 80% o'clock of accounting for the petridish bottom surface and goes down to posterity; Inhale earlier and go old nutrient solution, PBS to wash 2 times, bounce back with cellular segregation liquid digestion to most of cell cytoplasm; Will be when the petridish diapire comes off; Add basic culture solution and stop digestion, blow and beat gently with suction pipe and process single cell suspension, cell inoculation is continued to cultivate in another petridish with 1: 3 ratio.
4.hUCMSCs frozen with the recovery
Frozen: cell grows at 80% o'clock that accounts for the petridish bottom surface, digestion and collecting cell suspension, and the centrifugal 5min of 1000rpm abandons supernatant, with 5 * 10
6The ratio of cells/ml adds the precooling liquid storage, fully in the frozen pipe of the rearmounted precooling of mixing.By 4 ℃ of 30min ,-20 ℃ of 30min ,-80 ℃ of programs of spending the night are carried out frozen.Place-150 ℃ of Ultralow Temperature Freezers to preserve at last.Long-term frozen after certain hour recovery cell at interval, the observation of cell growing state.
Recovery: from-150 ℃ of Ultralow Temperature Freezers, take out frozen pipe, drop into rapidly in 37 ℃ of water-baths and rock fast, dissolve fully, in 1～2min, accomplish rewarming until frozen storing liquid.The sucking-off cell suspension inoculation adds basic culture solution, in 37 ℃, 5%CO in petridish then
2, cultivate under 95% the air gas phase.Recovery back cell inoculation density can be big slightly, and the small portion cell may lose vigor can not be adherent, should when changing liquid in second day, remove.
Two, PEMFs acts on hUCMSCs
Get the hUCMSCs of P3 logarithmic phase, adherent growth is inhaled and is abandoned basic culture solution when accounting for petridish floorage 60%.D-Hanks liquid is given a baby a bath on the third day after its birth inferior, and single cell suspension is processed in 0.25% trypsinase+0.02%EDTA digestion.Inoculate 6 orifice plates, inoculum density is 5 * 10
3Cells/cm
2Inoculating cell is divided into four groups, is respectively: the 1. conventional culture group (PE-CR-) of basic culture solution; 2. basic culture solution adds pulse electromagnetic field and induces group (PE+CR-); 3. chemical reagent is induced group (PE-CR+); 4. induced liquid+the pulse of traditional chemical reagent becomes induced by magnetic field group (PE+CR+).All cells all places 37 ℃, 5%CO
2, 95% air conditions is cultivated down, and every 3d changes corresponding basic culture solution and inducing culture liquid respectively.Pulse electromagnetic field induces group (2., 4. organizing) pulse electromagnetic field stimulation parameter to be: frequency 50Hz; Voltage 500mv/cm; Waveform rectangle positive pulse; Dutycycle 50%; Thorn flyback cycle 3h/d.
Three, cellular form changes observation
After giving the PEMFs stimulation, growth of inverted phase contrast microscope observation of cell every day and metamorphosis situation.About PE+CR-group, PE-CR+ group and PE+CR+ group cell induction 7d, cellular form does not have obvious change, still is spindle shape, and cell is arranged and still is radiation or whirlpool shape.Prolong with incubation time, the cell cell space increases gradually, becomes ellipse or polygon by spindle shape.The cell that cell appearance meets around the scleroblast form calcification tubercle is spindle shape, radial distribution.
Four, SEAP (ALP) is active detects
The ALP detection kit is used in the SEAP analysis, presses the operation of test kit specification sheets.SEAP decomposes disodium phenyl phosphate, produces free phenol and phosphoric acid, and phenol generates red quinone derivative with the amino antipyrine effect of 4-through Tripotassium iron hexacyanide oxidation in basic soln, can measure the height of enzyme activity according to the red depth.Get P3hUCMSCs with 5 * 10
4/ ml inoculates 24 orifice plates, is divided into PE-CR-group, PE+CR-group PE-CR+ group, PE+CR+ group.Respectively at 1d, 3d, 5d, 7d, 14d, 21d, 28d collecting cell.Cell adds protein lysate 100ul with PBS washing 2 times ,-80 ℃ of multigelations 3 times, and abundant lysing cell, the centrifugal 10min of 12000g, supernatant transfer in the new centrifuge tube.In enzyme plate, add 50ul damping fluid, 50ul matrix liquid, 3ul cellular proteins lysate respectively, 37 ℃ hatch 15min after, add 150ul colour developing liquid, 520nm detects the photoabsorption of each sample.Use Pierce BCA Assay kit protein quantification test kit to carry out determination of protein concentration: get the 25ul protein lysate and add the 200ulBCA working fluid, hatch 30min for 37 ℃, 562nm detects photoabsorption, calculates the protein concentration of cell pyrolysis liquid.ALP relative content=OD520nm/ protein concentration, each sample are got 3 parallel appearance and are averaged, and calculate the SD deviation simultaneously.With time is transverse axis, and relative quantification is that the longitudinal axis is drawn the ALP activity curve.
The ALP activity of PE+CR-group, PE-CR+ group and PE+CR+ group all obviously increases, and ALP obviously increased when wherein the PE+CR-group stimulated 3d, and 14d can reach the peak, after slightly descend.The action intensity of PE+CR+ is the strongest, possibly be that pulse electromagnetic field and chemical reagent have produced co-induction effect (seeing accompanying drawing 1).
Five, Von Kossa ' s dyeing
The hUCMSCs that gets P3 is inoculated in 6 well culture plates of inserting deckglass in advance, wait to be induced to the calcification tubercle appears and after, PBS washes 3 times; With the fixing 30min of 4% Paraformaldehyde 96 room temperature, distilled water flushing adds 1% silver nitrate solution lucifuge 30min; Daylight or uviolizing 10min, distilled water flushing adds hypo solution incubated at room 1h; Distilled water flushing, drying, neutral gum mounting.
Begin to occur the calcification tubercle about PE+CR-group 14d, the calcification tubercle occurs about the about 21d of PE-CR+.Along with stimulating fate to increase, tubercle quantity and area increase gradually, increase.Be brownish black through Von Kossa ' s dyeing.The PE-CR-group does not see that the calcification tubercle occurs and dyeing is negative.It is that PE+CR+ group＞PE+CR-group＞PE-CR+ group＞PE-CR-organizes that each group can sort by the difference of calcification tubercle formation time, quantity and area.(seeing accompanying drawing 2)
Six, sodium alizarinsulfonate dyeing
The hUCMSCs that gets P3 is inoculated in 6 well culture plates of inserting deckglass in advance, be induced to the calcification tubercle appears and after, PBS washes 3 times, 95% ethanol room temperature is 10min fixedly, distilled water flushing, the sodium alizarinsulfonate working fluid is hatched 30min for 37 ℃, the distilled water flushing.Observe, take the photograph phase under the inverted phase contrast microscope.
Begin to occur the calcification tubercle about PE+CR-group 14d, the calcification tubercle occurs about the about 21d of PE-CR+.Along with stimulating fate to increase, tubercle quantity and area increase gradually, increase.Sodium alizarinsulfonate is dyed orange with it, and the PE-CR-group does not see that the calcification tubercle occurs and dyeing is negative.It is many than other groups that PE+CR+ group calcification tubercle forms quantity, and the time relatively early.(seeing accompanying drawing 3)
Seven, Real-time PCR detects
The Trizol method is extracted blank control group and is induced the group cell total rna, after rt becomes cDNA, carries out the experiment of Real-time PCR relative quantification.Rt cDNA reaction system such as Table 1 are listed:
Table 1 reverse transcription reaction is formed
Reaction conditions: 42 ℃ of 60min------70 ℃ of 5min
Design of primers: listed like Talble2 with Beacon designer 7 software design Runx2, Co-I, OPN primer sequence, it is synthetic that primer is delivered the living worker in Shanghai:
Table 2Realtime PCR primer sequence
Realtime PCR reacts composition:
Table 3Realtime PCR reacts composition
Reaction conditions: after 40 circulating reactions of 95 ℃ 30S------58 ℃ 60S-------72 ℃ of 60S finished, the relative quantification result generated through MxPro software automatically.
Extract the people total RNA of osteogenic tissue, rt be cDNA as template DNA, cDNA is that relative content is 1,5,25,125 standard substance with 5 times of concentration dilute with waters respectively.Use standard substance to be template, carry out Realtime PCR reaction, drawing standard curve, the amplification rate of acquisition primer Runx2, collagen I, OPN according to the reaction system of Table 3.
The Real-time PCR detected result of PE+CR+, PE+CR-, each group of PE-CR+ shows that the genetic expression of Runx2, collagen I, OPN all changes with the induction time variation.General trend is a PE+CR+ group＞PE+CR-group＞PE-CR+ group.The genetic expression of PE-CR-group is not seen and is prolonged in time and obvious change takes place.Each gene expression amount of PE+CR+ is the highest, confirms that synergy has taken place when hUCMSCs is carried out osteogenic induction for pulse electromagnetic field and chemical inducer.The genetic expression of Runx2 peaks when inducing 7d, and after this expression amount is on a declining curve.(seeing accompanying drawing 4)
Eight, Western blot analyzes
When each group cultivation or stimulation 14d, lysis, the centrifugal 10min of cell lysate 12000g; 100 ℃, 5min makes protein denaturation, and 30mg albumen carries out electrophoresis with 12% SDS-Page glue; Transfer on the NC film 4 ℃ of sealing 1h in containing the PBST of 5% skim-milk then.With an anti-incubated overnight of dilution in 1: 500, add two of HRP mark and resist, take a picture.
The western blot detected result of PE+CR+, PE+CR-, each group of PE-CR+ shows; Induce hUCMSCs when osteoblast differentiation 14d; The protein expression level of the Runx2 of PE+CR+ group, collagen I, OPN is the highest, and the PE+CR-group is little with PE-CR+ histone expression level difference.(seeing accompanying drawing 5)
Embodiment 2:PEMFs promotes the experimental study of hUCMSCs and hydroxylapatite compound repairing bone defect
1.hUCMSCs preparation with the HA mixture
Get third generation hUCMSCs, I processes single cell suspension with cellular segregation liquid, uses the saline water suspension cell, and the adjustment cell density is 2 * 10
7/ ml and 0.2g granule type porous hydroxyapatite are processed mixture, and TV is 1ml.
2. the making of the damaged model of bone
Select 40 of purebred adult new zealand white rabbits, body weight is about: 2.0～2.5kg, and male and female are not limit, with new (0.2ml/kg) intramuscular anesthesia of speed dormancy; Get dorsal position, field of operation is shaved a mao preserved skin, the routine disinfection drape; Under the aseptic condition, forearm oar side is about the operative incision into 3.0cm along radius in the left side, appears radius stage casing (away from the epiphysis end); Excise the 1.5cm radius together with periosteum, cause the segmental bone damaged, sew up.Handle the opposite side radius with quadrat method.Postoperative is with the small splints fixation forearm about 2 weeks.Preceding 3 days with tincture of iodine wiping wound disinfection.
3. animal divides into groups
A group: hUCMSCs+HA+PEMFs
B group: hUCMSCs+HA
C group: blank group
Each group is 10 models, respectively hUCMSCs+HA or HA is injected the bone defect through skin.Begin the A group is carried out electric pulse stimulation 2h every day in second day after operation.
4. postoperative routine inspection
The body temperature of clinical follow recording laboratory animal, activity and wound healing situation.All animal postoperatives do not have infection, and body temperature is normal, and wound healing is good.
5. radiological examination
Carry out forearm X ray examination (seeing accompanying drawing 6) in 2,4,8 weeks of postoperative.
Claims (3)
1. method of using pulse electromagnetic field to promote the external Osteoblast Differentiation of human umbilical cord mesenchymal stem cells.
2. according to the method for claim 1, pulse electromagnetic field is produced by the pulse electrical signal generating unit.The pulse electromagnetic field stimulation parameter is: frequency 50Hz; Voltage 500mv/cm; Waveform rectangle positive pulse; Dutycycle 50%; Thorn flyback cycle 3 hours/day.
3. provide pulse electromagnetic field to promote human umbilical cord mesenchymal stem cells and the application of hydroxylapatite compound in repairing bone defect.
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| CN109161523A (en) * | 2018-09-27 | 2019-01-08 | 海口市人民医院（中南大学湘雅医学院附属海口医院） | Culture method of human umbilical cord mesenchymal stem cells and culture method of chondrogenic differentiation of human umbilical cord mesenchymal stem cells |
| CN110819619A (en) * | 2018-08-10 | 2020-02-21 | 北京大学 | Application of nanosecond pulsed electric field in improving dryness of cells |
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| US20050084962A1 (en) * | 2003-08-20 | 2005-04-21 | Bruce Simon | Methods of treatment using electromagnetic field stimulated stem cells |
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| CN104017769A (en) * | 2014-05-23 | 2014-09-03 | 暨南大学 | Application of ruthenium complex modified nano selenium in promotion of mesenchymal stem cell in-vitro osteoblast differentiation |
| CN110819619A (en) * | 2018-08-10 | 2020-02-21 | 北京大学 | Application of nanosecond pulsed electric field in improving dryness of cells |
| CN109161523A (en) * | 2018-09-27 | 2019-01-08 | 海口市人民医院（中南大学湘雅医学院附属海口医院） | Culture method of human umbilical cord mesenchymal stem cells and culture method of chondrogenic differentiation of human umbilical cord mesenchymal stem cells |
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