CN106581066A - Application of tissue-engineering bone - Google Patents
Application of tissue-engineering bone Download PDFInfo
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- CN106581066A CN106581066A CN201610980747.7A CN201610980747A CN106581066A CN 106581066 A CN106581066 A CN 106581066A CN 201610980747 A CN201610980747 A CN 201610980747A CN 106581066 A CN106581066 A CN 106581066A
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
Abstract
The invention discloses an application of tissue-engineering bone in preparation of medicines, wherein the medicine is used for treating bone injury diseases. The tissue-engineering bone includes mesenchymal stem cells and pre-induction cells. The tissue-engineering bone has a strong capability of being differentiated into bone cells, is high in yield of the bone cells, and can provides required cells for tissue repair in injured parts of bone and quickly repair and cure the injured parts of bone. The tissue-engineering bone is convenient to prepare and is safe and reliable.
Description
Technical field
The present invention relates to field of medicaments.In particular it relates to tissue engineered bone purposes.
Background technology
Bone injury tends to occur inactivate bone quilt when sclerite when wound passes limbs or betides open fracture debridement
Remove, mainly include Cranial defect and defect repair two large divisions.Bone injury is commonly encountered diseases clinically, is also the difficulty of orthopaedics therapy
One of topic.
However, still having to be developed suitable for the medicine of bone injury disease at present.
The content of the invention
It is contemplated that at least solving one of technical problem present in prior art to a certain extent.For this purpose, this
The bright purposes for proposing tissue engineered bone in medicine is prepared.
It should be noted that the present invention is completed based on the following discovery of inventor：
At present, clinically conventional Therapeutic Method mainly have autologous bone transplanting, homogeneous allogenic bone transplantation, bone xenograft and
Artificial bone graft etc., wherein autologous bone transplanting are considered as the goldstandard of repairing bone defect.But because autologous bone transplanting belongs to
Dig the eastern wall to repair the western one, the bone amount for not only providing is extremely limited, and may cause to supply wound, deformity, pain and the infection in area etc.
Problem, it is often more important that cause new bony defect；Allograph bone limited source, it is viral with generation rejection and infection
The risk of the infectious disease such as hepatitis, acquired immune deficiency syndrome (AIDS)；Simple artificial bone graft's cell necessary to due to lacking tissue repair, its skeletonization effect
Can be very low.And, for bone delay in healing, the patient of bone does not connect, said method often effect on driving birds is not good, Post operation is still suffered from
The situation of bone does not connect, bone delay in healing.
Inventor has found there is height self renewal based on stem cell in the research to the Therapeutic Method of bone injury disease
The characteristic such as ability, hyperproliferation and multi-lineage potential, implantable and reconstruction ability, can culturing stem cells amplification in vitro
Afterwards, it is input at bone injury site, bone injury disease can be effectively treated.
Inventor carries out further investigation discovery, by pre- to carrying out with the mescenchymal stem cell for being divided into osteocyte ability
Induction, can obtain having the stronger pre-induced cell for being divided into osteocyte ability.Further, inventor's discovery, by between
Mesenchymal stem cells and pre-induced cell collective effect in bone injury site, the reparation of bone injury site and healing efficiency apparently higher than
Pre-induced cell independent role.Thus, the tissue engineered bone comprising mescenchymal stem cell and pre-induced cell has stronger increasing
The ability of osteocyte is grown, is divided into, bone injury site can be made quickly and efficiently to repair and heal.
For this purpose, the present invention proposes purposes of the tissue engineered bone in medicine is prepared.Embodiments in accordance with the present invention, it is described
Medicine is used to treat bone injury disease, and the tissue engineered bone includes：Mescenchymal stem cell and pre-induced cell.
Inventor has found that mescenchymal stem cell has the ability for being divided into osteocyte, but differentiation direction is uncontrollable.Enter
And, by carrying out pre-induced to it, obtain that there is the pre-induced cell for being divided into osteocyte ability more by force, thus, pre-induced is thin
Born of the same parents can effectively be divided into osteocyte, and osteocyte yield is higher.Inventor is it was unexpectedly observed that by pre-induced cell and mesenchyme
During stem cell collective effect, osteocyte yield is higher than pre-induced cell independent role.Jing further investigation discoveries, mescenchymal stem cell
Osteocyte can not only be divided into, the quantity of osteocyte is improved, and some rush can be produced during Derived from Mesenchymal Stem Cells
Enter the factor such that it is able to promote propagation and the differentiation of pre-induced cell, osteocyte yield is improve on the whole.Thus, according to this
The tissue engineered bone of inventive embodiments has the stronger ability for being divided into osteocyte, and osteocyte yield is higher, can be bone
Damage location provides cell necessary to tissue repair, bone injury site is quickly and efficiently repaired and is healed, and conveniently obtains
, it is safe and reliable.
Embodiments in accordance with the present invention, purposes of the above-mentioned tissue engineered bone in medicine is prepared can also have following additional
Technical characteristic：
Embodiments in accordance with the present invention, the administering mode of the medicine is injection.
Embodiments in accordance with the present invention, the content of the mescenchymal stem cell is 1 × 107～1 × 108Individual/mL, it is described pre-
The content of inducing cell is 1 × 107～1 × 108Individual/mL.Thus, tissue engineered bone has the stronger energy for being divided into osteocyte
Power, and osteocyte yield is higher, can provide cell necessary to tissue repair for bone injury site, makes bone injury site fast
Speed, effectively repair and heal, and it is convenient obtain, it is safe and reliable.
Embodiments in accordance with the present invention, the mescenchymal stem cell is 1 with the quantity ratio of pre-induced cell:1～4, preferably
1:2.Inventor obtains aforementioned proportion relation through many experiments, and thus, tissue engineered bone is divided into osteocyte with stronger
Ability, and osteocyte yield is higher, can provide cell necessary to tissue repair for bone injury site, makes bone injury site
Quickly and efficiently repair and heal, and it is convenient obtain, it is safe and reliable.If mescenchymal stem cell is excessive, the bone being divided into will be caused
Cell yield is low；If mescenchymal stem cell is very few, the osteocyte yield being divided into is equally low.
Embodiments in accordance with the present invention, the mescenchymal stem cell is mesenchymal stem cells MSCs, umbilical cord mesenchyma is dry thin
Born of the same parents, amniotic fluid stem cell or dental pulp stem cell, preferred umbilical cord mesenchymal stem cells.Umbilical cord mesenchymal stem cells are readily available.Thus,
Tissue engineered bone according to embodiments of the present invention has the stronger ability for being divided into osteocyte, and osteocyte yield is higher, energy
Enough cell necessary to tissue repair is provided for bone injury site, bone injury site is quickly and efficiently repaired and is healed.This
Outward, tissue engineered bone is in a liquid state, and is easy to operation, and it is convenient obtain, it is safe and reliable.
It will be appreciated to those of skill in the art that umbilical cord mesenchymal stem cells are the cell separation cultures originated by umbilical cord
And purification and come, can be by commercially available acquisition.In the same manner, to mesenchymal stem cells MSCs, amniotic fluid stem cell or dental pulp stem cell
Source repeat no more.
Embodiments in accordance with the present invention, the tissue engineered bone further includes at least one following：2×1010Individual/mL is rich
Thrombocyte plasma；1%w/v hydrogels；And 100 μM of three (2- dimethylaminoethyls) amine.There are various lifes in platelet rich plasma
The long factor, the ratio of each somatomedin is consistent with normal rates in human body, makes have optimal synergism between somatomedin, and
And contain a large amount of fibrins in platelet rich plasma, provide good support for repair cell.The addition of hydrogel can promote
Enter the adhesion of damaging cells, accelerate healing.Three (2- dimethylaminoethyls) amine can accelerate cell Osteoblast Differentiation.Thus, according to
The tissue engineered bone of the embodiment of the present invention has the stronger ability for being divided into osteocyte, and osteocyte yield is higher, Neng Gouwei
Bone injury site provides cell necessary to tissue repair, bone injury site is quickly and efficiently repaired and is healed.Additionally, group
Knit Engineering Bone to be in a liquid state, be easy to operation, and it is convenient obtain, it is safe and reliable.
Embodiments in accordance with the present invention, the mescenchymal stem cell expresses surface marker PROTEIN C D51.Inventor's discovery, energy
The Derived from Mesenchymal Stem Cells for enough expressing surface marker PROTEIN C D51 is stronger for the ability of osteocyte.
Embodiments in accordance with the present invention, the pre-induced cell is obtained in the following manner：Inducing mesenchymal will be treated
Stem cell contacts with amplification culture medium, carries out amplification cultivation, to obtain amplification cultivation product；And produce the amplification cultivation
Thing is contacted with pre-induced culture medium, carries out pre-induced culture, to obtain the pre-induced cell.Thus, according to of the invention real
The tissue engineered bone for applying example has the stronger ability for being divided into osteocyte, and osteocyte yield is higher, can be bone injury portion
Position provides cell necessary to tissue repair, bone injury site is quickly and efficiently repaired and is healed.Additionally, tissue engineered bone
Be in a liquid state, be easy to operation, and it is convenient obtain, it is safe and reliable.
It should be noted that for mescenchymal stem cell to be induced and the mescenchymal stem cell of composition tissue engineered bone
Source do not do considered critical, can be identical source, will mescenchymal stem cell be divided into two parts, a part of Jing pre-induced is formed
Pre-induced cell, another part is grouped weaver's journey bone with pre-induced cell；Can also be separate sources, for example, belong to different batches
Secondary, different manufacturers etc..
Embodiments in accordance with the present invention, it is to train the amplification that the amplification cultivation product is contacted with pre-induced culture medium
Foster product is according to 3 × 103～8 × 103Individual/cm2, preferably 5 × 103Individual/cm2Inoculum concentration be inoculated in the pre-induced culture medium
In.Inventor obtains above-mentioned optimum inoculum concentration through many experiments, disclosure satisfy that cell breeds demand with this understanding.
Embodiments in accordance with the present invention, the pre-induced incubation time is 6～8 days, preferably 7 days.Inventor has found, induces
Culture 6～8 days, the cell needed for induction is adapted to transplanting, if overlong time, cell transition differentiation is even dead.
Embodiments in accordance with the present invention, the amplification culture medium includes：α-MEM containing 10 volume % hyclones cultures
Liquid.Inventor is had found, is cultivated using the culture medium for including the α-MEM culture fluid containing 10 volume % hyclones, Neng Gouyou
Breed mescenchymal stem cell to effect, be easy to follow-up pre-induced to process.
It should be noted that " α-MEM culture fluid " is known in those skilled in the art, acquisition pattern is not made strictly
Limit, for example voluntarily preparation, commercially available acquisition.Embodiments in accordance with the present invention, α-MEM culture fluid is purchased from Gibco, model
12561-056。
Embodiments in accordance with the present invention, the pre-induced culture medium includes：Basal medium, the basal medium bag
Include：DMEM culture fluid containing 10 volume % hyclones；50 μM of ascorbic acid；10mM sodium β-glycerophosphates；And 0.1mM ground plug
Meter Song.Inventor obtains above-mentioned optimum inducing culture through many experiments, in this culture medium, being capable of effectively induced synthesis
Pre-induced cell, and induced efficiency is high.
Understand for convenience, the method for preparing above-mentioned tissue engineered bone is described more fully below：
Embodiments in accordance with the present invention, methods described includes：(1) will treat that inducing mesenchymal stem cell is carried out at pre-induced
Reason, obtains pre-induced cell；It is described to obtain and (2) are mixed the pre-induced cell with mescenchymal stem cell
Tissue engineered bone.
Inventor has found that mescenchymal stem cell has the ability for being divided into osteocyte, but differentiation direction is uncontrollable.Enter
And, by carrying out pre-induced to it, obtain that there is the pre-induced cell for being divided into osteocyte ability more by force, thus, pre-induced is thin
Born of the same parents can effectively be divided into osteocyte, and osteocyte yield is higher.Inventor is it was unexpectedly observed that by pre-induced cell and mesenchyme
During stem cell collective effect, osteocyte yield is higher than pre-induced cell independent role.Jing further investigation discoveries, mescenchymal stem cell
Osteocyte can not only be divided into, the quantity of osteocyte is improved, and some rush can be produced during Derived from Mesenchymal Stem Cells
Enter the factor, to promote propagation and the differentiation of pre-induced cell, and then improve osteocyte yield.Thus, according to present invention enforcement
Tissue engineered bone obtained by the method for preparing tissue engineered bone of example has the stronger ability for being divided into osteocyte, and bone is thin
Born of the same parents' yield is higher, can provide cell necessary to tissue repair for bone injury site, bone injury site is quickly and efficiently repaiied
It is multiple and heal, and it is convenient obtain, it is safe and reliable.
Embodiments in accordance with the present invention, the mescenchymal stem cell is 1 with the quantity ratio of pre-induced cell:1～4, preferably
1:2.Inventor obtains aforementioned proportion relation through many experiments, and thus, resulting tissue engineered bone has stronger differentiation
For the ability of osteocyte, and osteocyte yield is higher, can provide cell necessary to tissue repair for bone injury site, makes bone
Damage location is quickly and efficiently repaired and healed, and it is convenient obtain, it is safe and reliable.If mescenchymal stem cell is excessive, will cause point
The osteocyte yield of chemical conversion is low；If mescenchymal stem cell is very few, the osteocyte yield being divided into is equally low.
Embodiments in accordance with the present invention, the pre-induced process time is 6～8 days, preferably 7 days.Inventor has found, induces
Culture 6～8 days, the cell needed for induction is adapted to transplanting, if overlong time, cell transition differentiation is even dead.
Embodiments in accordance with the present invention, step (2) is further included：By platelet rich plasma, hydrogel and three (2- bis-
Methylaminoethyl) amine at least one, described mescenchymal stem cell and the pre-induced cell mixed, and will be resulting
Cell mixing is resuspended in normal saline, to obtain the tissue engineered bone.There are various somatomedin in platelet rich plasma,
The ratio of each somatomedin is consistent with normal rates in human body, makes have optimal synergism between somatomedin, and rich blood
Contain a large amount of fibrins in platelet-poor plasma, for repair cell good support is provided.The addition of hydrogel can promote to damage
The adhesion of cell, accelerates healing.Three (2- dimethylaminoethyls) amine can accelerate cell Osteoblast Differentiation.Thus, according to the present invention
Tissue engineered bone obtained by the method for embodiment has the stronger ability for being divided into osteocyte, and osteocyte yield is higher,
Cell necessary to tissue repair can be provided for bone injury site, bone injury site is quickly and efficiently repaired and is healed.This
Outward, tissue engineered bone is in a liquid state, and is easy to operation, and it is convenient obtain, it is safe and reliable.
Embodiments in accordance with the present invention, the pre-induced is processed to be included：Inducing mesenchymal stem cell and amplification cultivation will be treated
Base is contacted, and amplification cultivation is carried out, to obtain amplification cultivation product；And by the amplification cultivation product and pre-induced culture medium
Contact, carries out pre-induced culture, to obtain the pre-induced cell.Thus, obtained by method according to embodiments of the present invention
Tissue engineered bone there is the stronger ability for being divided into osteocyte, and osteocyte yield is higher, can carry for bone injury site
For cell necessary to tissue repair, bone injury site is set quickly and efficiently to repair and heal.Additionally, tissue engineered bone is in liquid
State, is easy to operation, and it is convenient obtain, it is safe and reliable.
Embodiments in accordance with the present invention, it is to train the amplification that the amplification cultivation product is contacted with pre-induced culture medium
Foster product is according to 3 × 103～8 × 103Individual/cm2, preferably 5 × 103Individual/cm2Inoculum concentration be inoculated in the pre-induced culture medium
In.Inventor obtains above-mentioned optimum inoculum concentration through many experiments, disclosure satisfy that cell breeds demand with this understanding.
Embodiments in accordance with the present invention, the amplification culture medium includes：α-MEM containing 10 volume % hyclones cultures
Liquid.Inventor is had found, is cultivated using the culture medium for including the α-MEM culture fluid containing 10 volume % hyclones, Neng Gouyou
Breed mescenchymal stem cell to effect, be easy to follow-up pre-induced to process.
It should be noted that " α-MEM culture fluid " is known in those skilled in the art, acquisition pattern is not made strictly
Limit, for example voluntarily preparation, commercially available acquisition.Embodiments in accordance with the present invention, α-MEM culture fluid is purchased from Gibco, model
12561-056。
Embodiments in accordance with the present invention, the pre-induced culture medium includes：Basal medium, the basal medium bag
Include：DMEM culture fluid containing 10 volume % hyclones；50 μM of ascorbic acid；10mM β-phosphoglycerol；And 0.1mM ground plug rice
Pine.Inventor obtains above-mentioned optimum inducing culture through many experiments, in this culture medium, can effectively induced synthesis it is pre-
Inducing cell, and induced efficiency is high.
Embodiments in accordance with the present invention, the mescenchymal stem cell and treat that inducing mesenchymal stem cell is separately advance
Through screening, to obtain the mescenchymal stem cell of expression surface marker PROTEIN C D51.Inventor's discovery, expresses surface marker albumen
The Derived from Mesenchymal Stem Cells of CD51 is stronger for the ability of osteocyte, thus, by screening (such as flow cytometer screening), with
Obtain and there is the stronger mescenchymal stem cell for being divided into osteocyte ability, then carried out pre-induced, and pre- lure what is obtained
Guided cell and the mescenchymal stem cell collective effect for expressing surface marker PROTEIN C D51, to obtain the osteocyte of higher yields.
The administration frequency and dosage of the medicine of the present invention can be determined by multiple correlative factors, and the factor includes will quilt
The order of severity of the disease type for the treatment of, route of administration, patient age, sex, body weight and disease and as active component
Drug type.Some embodiments of the invention, daily dose can be divided into 1 dose, 2 doses of suitable form or multi-agent, with whole
With 1 time, 2 times or multiple dosing in time period, as long as reaching therapeutically effective amount.
Term " treatment " is used to refer to the desired pharmacology of acquisition and/or physiologic effect.The effect is just wholly or in part
Can be preventative for prevention disease or its symptom, and/or just partially or completely cure diseases and/or disease are caused not
Can be curative for good action." treatment " used herein covers the disease of mammal, particularly people, including：(a)
Prevention disease (for example preventing bone injury) or disease occur in the individuality fallen ill still not yet is made a definite diagnosis in easily illness；B () suppresses
Disease, for example, block disease development；Or (c) alleviates disease, for example, mitigate the symptom related to disease." treatment " used herein
Cover any use that medicine or compound are given the individual disease to treat, cure, alleviate, improve, mitigate or suppress individuality
Medicine, will including but not limited to contain tissue engineered bone described herein and give individuality in need.
Embodiments in accordance with the present invention, the medicine of the present invention can be used in combination with conventional treatments and/or therapy, or
Person can be used separately with conventional treatments and/or therapy.When the medicine of the present invention is using the conjoint therapy with other medicines
During middle administration, they can sequentially or simultaneously give individuality.Or, the medicine of the present invention can include the organizational project of the present invention
Bone, pharmaceutically acceptable carrier or pharmaceutically acceptable excipient and other curatives known in the art or preventive drug
Combination.
The additional aspect and advantage of the present invention will be set forth in part in the description, and partly will become from the following description
Obtain substantially, or recognized by the practice of the present invention.
Description of the drawings
The above-mentioned and/or additional aspect and advantage of the present invention will become from the description with reference to accompanying drawings below to embodiment
Substantially and by force understand, wherein：
Fig. 1 shows mescenchymal stem cell aspect graph according to an embodiment of the invention；
Fig. 2 shows mescenchymal stem cell surface marker protein expression analysis figure according to an embodiment of the invention；With
And
Fig. 3 shows Osteoblast Differentiation efficiency analysiss figure according to an embodiment of the invention.
Specific embodiment
Embodiments of the invention are described below in detail.The embodiments described below is exemplary, is only used for explaining this
It is bright, and be not considered as limiting the invention.
The solution of the present invention is explained below in conjunction with embodiment.It will be understood to those of skill in the art that following
Embodiment is merely to illustrate the present invention, and should not be taken as limiting the scope of the invention.Unreceipted particular technique or bar in embodiment
Part, carry out according to the technology or condition described by document in the art or according to product description.Agents useful for same or instrument
The unreceipted production firm person of device, be can pass through city available from conventional products.
The flow cytometry of embodiment 1 identifies the surface marker of mescenchymal stem cell
(1) mescenchymal stem cell (cellular morphology is as shown in Figure 1) is incubated at (favourable public purchased from three containing serum-free medium
Department, China) 10cm Tissue Culture Dishs in.Whne cell growth and when reaching more than 90% degree of converging, suction is abandoned after culture medium and uses PBS
Buffer rinse cell once, adds TrypLe Express enzymic digestion cells, when coming off whne cell rounding and from culture plate
Terminate digestion with the α MEM culture medium containing 10% hyclone in time, collect cell suspension into 15ml centrifuge tubes, 1200rpm from
The heart 5 minutes.Suction is abandoned after supernatant and uses fresh serum-free media re-suspended cell, the generation of Secondary Culture 3, takes the cell of the culture third generation,
Use trypsinization.
(2) postdigestive mescenchymal stem cell cell is put in 1.5mL EP pipes, is separately added into 1 μ l streaming antibody
(CD73, CD90, CD34, CD19, CD14, HLA-DR), 4 DEG C of lucifuges are incubated 30 minutes.
(3) with PBS washed cell twice, then with flow cytomery mescenchymal stem cell surface marker
Expression, as a result as shown in table 1 and Fig. 2, it can be seen that cell phenotype is homogeneous, cell expression mesenchymal cell markers CD73, CD90,
Hematopoietic cell markers CD34, CD14, CD19 are not expressed, and does not express human leucocyte antigen (HLA) HLA-DR, comply fully with " international cell
The standard that treatment association " specifies with regard to mescenchymal stem cell surface marker.
The flow cytometry of table 1 is identified
| Cell phenotype | Detected value | Normal reference value |
| CD73 | 95.92% | >95% |
| CD90 | 99.99% | >95% |
| CD34 | 0.11% | <2% |
| CD19 | 0.00% | <2% |
| CD14 | 0.00% | <2% |
| HLA-DR | 0.00% | <2% |
The mescenchymal stem cell of the screening expression CD51 of embodiment 2
(1) mescenchymal stem cell is incubated in the 10cm Tissue Culture Dishs containing serum-free medium.Treat cell growth simultaneously
When reaching more than 90% degree of converging, suction is abandoned after culture medium and uses PBS rinse cell once, adds TrypLe Express enzymes
Peptic cell, uses in time the α MEM culture medium containing 10% hyclone to terminate disappearing when coming off whne cell rounding and from culture plate
Change, collect cell suspension into 15ml centrifuge tubes, 1200rpm is centrifuged 5 minutes.Suction is abandoned after supernatant and uses fresh serum-free media weight
Outstanding cell, in the generation of Secondary Culture 3, takes the cell of the culture third generation, uses trypsinization.
(2) postdigestive mescenchymal stem cell cell is put in 1.5mL EP pipes, 10 μ l CD51 streaming antibody of addition, 4
DEG C lucifuge is incubated 30 minutes.
(3) with PBS washed cell twice, the mesenchyme for then going out to express CD51 with selected by flow cytometry apoptosis does thin
Born of the same parents, after testing, the expression of CD51 is 4% or so in mescenchymal stem cell.
(4) cell is collected, in being inoculated in 10cm Tissue Culture Dishs, is then placed in 37 DEG C, 5%CO2Cultivate in incubator.
The comparison of 3 mescenchymal stem cells of embodiment+pre-induced cell and mescenchymal stem cell Osteoblast Differentiation
(1) the mescenchymal stem cell culture that obtains of screening of example to be performed 2 to during 80～90% fusion by cell Trypsin
Count after enzymic digestion, then according to 8000 cell/cm2(α-MEM containing 10 volume % hyclones are cultivated to be seeded to 6 orifice plates
Liquid) in, then cell is put into into 37 DEG C, 5%CO2Cultivate in incubator.
(2) second days, Osteogenic Induction Medium is changed, medium component is DMEM, 10% hyclone, 0.1mM ground plug
Meter Song, 10mM sodium β-glycerophosphate and 50 μM of ascorbic acid.
(3) changed liquid once every two days, induce 7 days, the cell for being obtained is pre-induced cell.
After (4) 7 days, culture medium is abandoned in suction, adds TrypLe Express enzymic digestions with PBS rinse cell once
Cell, is received in time when coming off whne cell rounding and from culture plate with the termination digestion of the α MEM culture medium containing 10% hyclone
Into 15ml centrifuge tubes, 1200rpm is centrifuged 5 minutes collection cell suspension.The pre-induced cell 6 × 10 that will be collected7Individual/mL, enforcement
The mescenchymal stem cell 3 × 10 that the screening of example 2 is obtained7Individual/mL, 2 × 1010Individual/mL platelet rich plasmas；1%w/v hydrogels；With
And 100 μM of three (2- dimethylaminoethyls) amine mixing, in being inoculated in 6 orifice plates.The only embodiment 2 of setting simultaneously is screened between obtaining
Mesenchymal stem cells are used as control experiment group.
(5) second days, Osteogenic Induction Medium is changed, medium component is DMEM, 10% hyclone, 0.1mM ground plug
Meter Song, 10mM sodium β-glycerophosphate and 50 μM of ascorbic acid, liquid was changed once every two days, and after inducing 20 days, calcium tuberosity is formed, and is carried out
Alizarin red staining, and examine under a microscope, as a result as shown in figure 3, wherein A is control experiment group, only containing mescenchymal stem cell；
B is experimental group, containing mescenchymal stem cell and pre-induced cell.As can be seen that mescenchymal stem cell and pre-induced groups of cells institute
The calcium tuberosity number of formation significantly more than only group containing mescenchymal stem cell, illustrates that mescenchymal stem cell is made jointly with pre-induced cell
With resulting osteoblast yield is higher.
In the description of this specification, reference term " one embodiment ", " some embodiments ", " example ", " specifically show
The description of example " or " some examples " etc. means to combine specific features, structure, material or spy that the embodiment or example are described
Point is contained at least one embodiment of the present invention or example.In this manual, to the schematic representation of above-mentioned term not
Identical embodiment or example must be directed to.And, the specific features of description, structure, material or feature can be with office
Combine in an appropriate manner in one or more embodiments or example.Additionally, in the case of not conflicting, the skill of this area
Art personnel can be tied the feature of the different embodiments or example described in this specification and different embodiments or example
Close and combine.
Although embodiments of the invention have been shown and described above, it is to be understood that above-described embodiment is example
Property, it is impossible to limitation of the present invention is interpreted as, one of ordinary skill in the art within the scope of the invention can be to above-mentioned
Embodiment is changed, changes, replacing and modification.
Claims (10)
1. purposes of the tissue engineered bone in medicine is prepared, it is characterised in that the medicine is used to treat bone injury disease,
The tissue engineered bone includes：Mescenchymal stem cell and pre-induced cell.
2. purposes according to claim 1, it is characterised in that the administering mode of the medicine is injection.
3. purposes according to claim 1, it is characterised in that the tissue engineered bone further include it is following at least it
One：2×1010Individual/mL platelet rich plasmas；1%w/v hydrogels；And 100 μM of three (2- dimethylaminoethyls) amine.
4. purposes according to claim 3, it is characterised in that
The content of the mescenchymal stem cell is 1 × 107～1 × 108Individual/mL,
The content of the pre-induced cell is 1 × 107～1 × 108Individual/mL,
Preferably, the mescenchymal stem cell and the quantity ratio of pre-induced cell are 1:1～4, more preferably 1:2.
5. purposes according to claim 3, it is characterised in that the mescenchymal stem cell be mesenchymal stem cells MSCs,
Umbilical cord mesenchymal stem cells, amniotic fluid stem cell or dental pulp stem cell, preferred umbilical cord mesenchymal stem cells.
6. purposes according to claim 3, it is characterised in that the mescenchymal stem cell expresses surface marker albumen
CD51。
7. purposes according to claim 3, it is characterised in that the pre-induced cell is obtained in the following manner：
To treat that inducing mesenchymal stem cell is contacted with amplification culture medium, carry out amplification cultivation, to obtain amplification cultivation product；With
And
The amplification cultivation product is contacted with pre-induced culture medium, pre-induced culture is carried out, it is thin to obtain the pre-induced
Born of the same parents.
8. purposes according to claim 7, it is characterised in that contact the amplification cultivation product with pre-induced culture medium
It is according to 3 × 10 by the amplification cultivation product3～8 × 103Individual/cm2, preferably 5 × 103Individual/cm2Inoculum concentration be inoculated in it is described
In pre-induced culture medium.
9. purposes according to claim 7, it is characterised in that the pre-induced incubation time is 6～8 days, preferably 7 days.
10. purposes according to claim 7, it is characterised in that
The amplification culture medium includes：α-MEM culture fluid containing 10 volume % hyclones,
The pre-induced culture medium includes：50 μM of ascorbic acid；10mM sodium β-glycerophosphates；0.1mM dexamethasone；And basis
Culture medium, the basal medium includes：DMEM culture fluid containing 10 volume % hyclones.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201610980747.7A CN106581066B (en) | 2016-11-08 | 2016-11-08 | Use of tissue engineering bone |
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| CN201610980747.7A CN106581066B (en) | 2016-11-08 | 2016-11-08 | Use of tissue engineering bone |
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| CN106581066A true CN106581066A (en) | 2017-04-26 |
| CN106581066B CN106581066B (en) | 2020-07-03 |
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