CN106540326A - Purposes of the chondrocyte composition in medicine is prepared - Google Patents
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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
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Abstract
The invention discloses purposes of the chondrocyte composition in medicine is prepared, for repairing cartilage injury, the chondrocyte composition includes the medicine：Cell suspension and temperature-sensitive hydrogel, wherein cell suspension are by mixing mescenchymal stem cell and chondrocyte and being resuspended in collagen solution formation.The chondrocyte composition of the present invention is convenient to be obtained, safe and reliable, and, to the efficiency high of Chondrocyte Differentiation, cell necessary to tissue repair can be provided for cartilaginous lesion site, and temperature-sensitive hydrogel has the effect of good exogenous cells carrier, the cell in chondrocyte composition can be made to be sufficient filling with cartilage injury and not to other place migrations such that it is able to effectively facilitate cartilaginous lesion site and quickly and efficiently repair and heal.
Description
Technical field
The present invention relates to purposes of the chondrocyte composition in medicine is prepared.
Background technology
It is that osteoarthritis (osteoarthritis, OA) occur that articular cartilage (articular cartilage, AC) is damaged
A Major Risk Factors, OA is irreversible, to be palliated the agonizing sufferings by joint replacement when PD is to late period and
Recover function.As the repair ability of articular cartilage is limited, it is necessary to which early intervention is preventing from developing into OA.Current controls
Treatment mode is intended to be stimulated and transplanted come repairing articular cartilage by primary treatment, adjacent tissue.Can by rational repairing and treating
To obtain comparatively ideal result in short-term to mid-term, but after longer period of time, easily recur.In recent years, based on biological engineering
The tissue engineering technique of the cellular level of tissue is widely studied, and this bioengineered tissue can reappear hyaline cartilage
Characteristic, and combine together with original tissue, for repairing articular cartilage damages significant.
However, the research in terms of current bioengineered tissue repairs cartilage injury still has to be strengthened.
The content of the invention
It is contemplated that at least solving one of technical problem present in prior art.For this purpose, one object of the present invention
Be propose it is a kind of convenient obtain, it is safe and reliable, can effectively repair the means of cartilage injury.
It should be noted that the present invention is completed based on the following discovery of inventor：
At present using one of wide cartilage repair techniques of cellular level be Autologous Chondrocyte transplanting
(Autologous chondrocyte implantation, ACI) technology.The technology is thin comprising internal operative treatment and in vitro
Born of the same parents' culture technique：First, operation is carried out in the non-bearing region of damaged joints and obtain cartilage slices, and be transferred into storing
And in the sterile nutrient solution of transport；Then, the collagenase digesting cartilaginous tissue used in cell culture chamber, obtains chondrocyte,
Cultured chondrocytes are expanded to using monolayer culture the quantity that can be used to transplanting, filling out for cartilage defects is carried out by surgeon
Fill.In the operation process, the chondrocyte of amplification in vitro is expelled to into fault location.In order that chondrocyte rests on transplanting portion
Position, prevents a large amount of drifts from walking, synovial membrane lobe can be sewn to damage location further.However, it is found by the inventors that, Autologous Chondrocyte
There is following defect in transplanting：1st, Autologous Chondrocyte transplanting needs Two intrusions to perform the operation, larger to damage to patient.2nd, cartilage is thin
In vitro during monolayer amplification, these cell masses lose its phenotype to born of the same parents, and the production of collagen protein is (typical transparent by II types
Cartilage) to the transformation of I types and type III (typical fibrous cartilage).The result of the change of these phenotypes is with poor biology
The generation of the extracellular matrix of mechanical performance.3rd, donor site provides the limited in one's ability of substantial amounts of chondrocyte.4th, donor set
Sickness rate be also Autologous Chondrocyte major obstacle.
And autologous bone marrow mesenchymal stem cells transplanting is also cartilage repair techniques at present using wide cellular level.
The technology carries out bone marrow aspiration in the ilium of patient, collects bone marrow；Sample is transferred to toilet carries out cell separation；To bone marrow
Add phosphate buffer in puncture fluid, be then transferred in lymphocyte separation medium, be centrifuged and collect mononuclearcell and count
Number, is washed with PBS, is cultivated in being then seeded to culture bottle, prepares to move by gained cell amplification culture, to pass to the second filial generation
Plant；Adopt before transplanting colony forming unit (Colony-forming unit, CFU) and Flow cytometry cell gram
The expression of grand Forming ability and cell surface marker；Then, the MSCs pancreatin of the second filial generation is digested, and is resuspended in life
During treating blood disorderss are clear, and it is loaded in asepsis injector, under perspective, at the knee injuries of cell infusion to patient.However, inventor
It was found that, there is following defect in autologous bone marrow mesenchymal stem cells transplanting：1st, bone marrow aspiration brings pain to patient.2nd, cell is direct
Injury region is expelled to, other measures are not done, cell may float in a large number.3rd, brachymedial phase effect is preferable, but after long-time
The injury region pain of patient has risen, and ability to act has also declined.
And mescenchymal stem cell (mesenchymal stem cells, MSCs) transplanting is a kind of very promising strategy,
Because MSCs has high proliferation ability and with the potential for being divided into chondrocyte, and then forms cartilage.MSCs is that fusiformis is thin
Born of the same parents, with fast breeding and self-renewal capacity, are present in a large amount of tissues, including bone marrow, synovial tissue, blood, fatty group
Knit and periosteal tissue.They have polyphyly differentiation potential, can be divided into various kinds of cell type to create and repair mesenchyme group
Knit.MSCs can be to into cartilage, skeletonization, fatty development ways differentiation.Regulatory factor can promote the cartilage of MSC to occur, for example
Transforming growth factor (TGF-β) and dexamethasone, this is confirmed with simple external model.
Further, inventor is by a series of research discoveries, the mescenchymal stem cell (Umbilical originated using umbilical cord
Cord Mesenchymal stem cells, UCMSC) it is transplanted at the knee joint injury of patient, being capable of effectively treatment cartilage damage
Wound.Also, existing ripe UCMSC isolated culture methods, and umbilical cord at present are easily obtained, it is not necessary to take invasive to patient
Operation, it is not required that extract cartilage in other positions, can effectively reduce the pain of patient.And MSC can fast breeding and from
I updates, and with the potential into cartilage differentiation, ensure that the vigor of neocartilage cell, and there is no the hidden of sickness rate
Suffer from.
Further, inventor before transplantation, carries out into chondrocyte induction differentiation, to promote UCMSC to cartilage to part UCMSC
Direction breaks up, and the chondrocyte of UCMSC and induction is combined with collagen and temperature-sensitive hydrogel, as a result effectively can prevent
Cell extensive migration, reaches the effect of long-term treatment.Wherein, temperature sensitive type poly lactic acid hydrogel is liquid below 37 DEG C,
Gel will be formed after reaching 37 DEG C, will play a part of exogenous cells carrier, can promote cell movement, stick, breed and
Differentiation, and structural support is provided so that cell is movable at defect, will not large area movement.
Thus, in one aspect of the invention, the invention provides purposes of the chondrocyte composition in medicine is prepared,
The medicine is used to repair cartilage injury.Embodiments in accordance with the present invention, the chondrocyte composition include：Cell suspension and temperature
Quick type hydrogel, wherein the cell suspension is by mixing mescenchymal stem cell and chondrocyte and being resuspended in collagen solution
Formed.
It is surprisingly found by the inventors that, the chondrocyte composition of the present invention is convenient to be obtained, safe and reliable, also, to cartilage
The efficiency high of cell differentiation, can provide cell necessary to tissue repair for cartilaginous lesion site, can be effective for cartilage
Injury repairing；And temperature-sensitive hydrogel has the effect of good exogenous cells carrier, in chondrocyte composition being made
Cell be sufficient filling with cartilage injury and not to other place migrations such that it is able to effectively facilitate cartilaginous lesion site it is quick,
Effectively repair and heal.
Embodiments in accordance with the present invention, in the cell suspension, the mescenchymal stem cell and the chondrocyte
Mixed proportion is 1:1～4:1.Thus, cartilage differentiation efficiency high, can provide necessary to tissue repair for cartilaginous lesion site
Cell.
Embodiments in accordance with the present invention, in the cell suspension, the content of collagen is 1 mass %～8 mass %.By
This, cell mixture is good with the composite effect of collagen and temperature-sensitive hydrogel, can effectively prevent cell extensive migration, from
And the effect of long-term treatment can be reached.
Embodiments in accordance with the present invention, the collagen are II collagen protein.Thus, cell mixture and collagen and temperature sensitive
The composite effect of type hydrogel is good, and chondrocyte composition can effectively prevent cell extensive migration, such that it is able to reach length
The effect of phase treatment.
Embodiments in accordance with the present invention, the concentration of the collagen solution is 1 mass %～8 mass %.Thus, mixing with cells
Thing is good with the composite effect of collagen and temperature-sensitive hydrogel.
The volume ratio of embodiments in accordance with the present invention, the cell suspension and the temperature-sensitive hydrogel is 1:1～2.By
This, temperature-sensitive hydrogel is good to the supporting effect of the mescenchymal stem cell in cell suspension and chondrocyte mixture, Jin Erneng
The cell in chondrocyte composition is enough made to be sufficient filling with cartilage injury and not to other place migrations such that it is able to effectively to promote
Enter cartilaginous lesion site quickly and efficiently to repair and heal.
Embodiments in accordance with the present invention, the temperature-sensitive hydrogel are temperature sensitive type poly lactic acid hydrogel.Thereby, it is possible to effective
Mescenchymal stem cell and chondrocyte mixture are carried, and which is sufficient filling with cartilage injury and not to other place migrations.
Embodiments in accordance with the present invention, the temperature sensitive type poly lactic acid hydrogel are MPEG-PLGA.Thus, which is used as external source
Sexual cell carrier is made good use of, and the cell in chondrocyte composition can be made to be sufficient filling with cartilage injury and not to other ground
Side migrates such that it is able to effectively facilitates cartilaginous lesion site and quickly and efficiently repairs and heal.
Embodiments in accordance with the present invention, the mescenchymal stem cell are umbilical cord mesenchymal stem cells.Thus, mesenchyme is dry thin
Born of the same parents' wide material sources, easily obtain, safe and harmless, and to the efficiency high of cartilage differentiation.
Embodiments in accordance with the present invention, the chondrocyte are by the preferred umbilical cord mesenchymal stem cells of mescenchymal stem cell
What induction was obtained.Also, the abductive approach of chondrocyte is not particularly limited, as long as can be from the preferred umbilical cord of mescenchymal stem cell
Mescenchymal stem cell effectively induces acquisition chondrocyte.Thus, chondrocyte composition, to the efficiency of Chondrocyte Differentiation
Height, can provide cell necessary to tissue repair for cartilaginous lesion site.
In addition it is also necessary to explanation, the present invention is with least one of following advantages：
1st, the present invention obtains mescenchymal stem cell by garbage umbilical cord, and wide material sources can be obtained in a large number, and will not be to patient
Damage.
2nd, the present invention carries out the induction differentiation in chondrocyte direction to part UCMSC, promotes UCMSC to cartilage differentiation
Efficiency.
3rd, temperature-sensitive hydrogel can play a part of good exogenous cells carrier so that cell can be sufficient filling with
At cartilage injury, and not to other place migrations.
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.
Specific embodiment
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.
Embodiment 1
Chondrocyte composition is prepared according to following steps, and skin grafing and mending is carried out to patient cartilage injury：
1st, the separation and Culture of UCMSC
The fresh umbilical cord got is sent into into GMP workshops, isolated UCMSC.UCMSC cultures are expanded；And take out
Part cell carries out chondroblast induction, expands, prepare transplanting after obtaining a number of chondrocyte.
2nd, prepare before transplanting
The chondrocyte that UCMSC obtained above is obtained with induction differentiation, is digested with 0.25% pancreatin, is used in combination
Brine, then by UCMSC and chondrocyte according to 1:1～4:1 ratio mixes and is resuspended in typeⅡ Collagen
In solution (concentration of collagen solution is 1-8 mass %), to obtain cell suspension, wherein in cell suspension, collagen contains
Measure as 1-8 mass %.The cell suspension is mixed with 1: 1～2 volume ratio with temperature sensitive type poly lactic acid hydrogel MPEG-PLGA,
Being placed in shaking table makes which fully mix for a period of time, to obtain the chondrocyte composition of the present invention.
Then, to the present invention chondrocyte composition in cell sampling carry out microorganism, virus five, mycoplasma,
Endotoxin and cell function detection, to guarantee safety.
3rd, transplant
Using joint cavity injection method.Wherein, patient carries out arthroscopy before injection is received, and determines at joint injury
Size, thickness etc., with every square centimeter 5 × 106The density of cell is injected.
4th, effect observation after transplanting
After treatment, periodically using the improvement situation of nuclear magnetic resonance image check knee joint function.
As a result find, the cell in the chondrocyte composition of the present invention can be sufficient filling with cartilage injury and not to which
Its place migration, cartilaginous lesion site healing are quick, effective.
Comparative example 1
Method according to embodiment 1 prepares chondrocyte composition, and skin grafing and mending is carried out to patient cartilage injury, difference
It is only that：
The cell not carried out to UCMSC in chondrocyte induction, i.e. cell suspension all adopts UCMSC.
Observe after transplanting, patient's cartilaginous lesion site chondroid tissue, Principle of Pain is alleviated, damage has to a certain degree heals
Close.
Comparative example 2
Method according to embodiment 1 prepares chondrocyte composition, and skin grafing and mending is carried out to patient cartilage injury, difference
It is only that：
The ratio of UCMSC and chondrocyte in cell suspension is 1：3.
Observe after transplanting, patient's cartilaginous lesion site healing speed is slow.
Comparative example 3
Method according to embodiment 1 prepares chondrocyte composition, and skin grafing and mending is carried out to patient cartilage injury, difference
It is only that：
The ratio of cell suspension and temperature sensitive type poly lactic acid hydrogel MPEG-PLGA is 1: 3.
Observe after transplanting, patient's cartilaginous lesion site chondrocyte formation efficiency is relatively low, so as to healing speed is slow.
Comparative example 4
Method according to embodiment 1 prepares chondrocyte composition, and skin grafing and mending is carried out to patient cartilage injury, difference
It is only that：
The ratio of cell suspension and temperature sensitive type poly lactic acid hydrogel MPEG-PLGA is 1：3.
Observe after transplanting, patient's cartilaginous lesion site chondrocyte formation efficiency is relatively low, and healing speed is slow.
Comparative example 5
Method according to embodiment 1 prepares chondrocyte composition, and skin grafing and mending is carried out to patient cartilage injury, difference
It is only that：
Temperature sensitive type poly lactic acid hydrogel MPEG-PLGA (carrying out injection transplantation only with cell suspension) is not adopted.
Observe after transplanting, walk in a large number by drift for the cell in chondrocyte composition, patient's cartilaginous lesion site chondrocyte
Formation efficiency is relatively low, and healing speed is slow.
Comparative example 6
Method according to embodiment 1 prepares chondrocyte composition, and skin grafing and mending is carried out to patient cartilage injury, difference
It is only that：
Collagen solution is substituted using normal saline.
Observe after transplanting, walk in a large number by drift for the cell in chondrocyte composition, patient's cartilaginous lesion site chondrocyte
Formation efficiency is relatively low, and healing speed is slow.
In the description of this specification, reference term " one embodiment ", " some embodiments ", " example ", " specifically show
Example ", or the description of " some examples " etc. mean specific features with reference to the embodiment or example description, structure, material or spy
Point is contained at least one embodiment or example of the present invention.In this manual, to the schematic representation of above-mentioned term not
Identical embodiment or example are referred to necessarily.And, the specific features of description, structure, material or feature can be any
One or more embodiments or example in combine in an appropriate manner.
Although an embodiment of the present invention has been shown and described, it will be understood by those skilled in the art that：Not
These embodiments can be carried out with various changes, modification, replacement and modification in the case of the principle and objective that depart from the present invention, this
The scope of invention is limited by claim and its equivalent.
Claims (10)
1. purposes of the chondrocyte composition in medicine is prepared, the medicine are used to repair cartilage injury, it is characterised in that institute
Stating chondrocyte composition includes：Cell suspension and temperature-sensitive hydrogel, wherein the cell suspension is by mesenchyme is done
Cell and chondrocyte mix and are resuspended in collagen solution formation.
2. purposes according to claim 1, it is characterised in that in the cell suspension, the mescenchymal stem cell and
The mixed proportion of the chondrocyte is 1:1～4:1.
3. purposes according to claim 1, it is characterised in that in the cell suspension, the content of collagen is 1 mass %
～8 mass %.
4. purposes according to claim 1, it is characterised in that the volume of the cell suspension and the temperature-sensitive hydrogel
Than for 1:1～2.
5. purposes according to claim 1, it is characterised in that the temperature-sensitive hydrogel is temperature sensitive type poly lactic acid water-setting
Glue.
6. purposes according to claim 4, it is characterised in that the temperature sensitive type poly lactic acid hydrogel is MPEG-PLGA.
7. purposes according to claim 1, it is characterised in that the mescenchymal stem cell is umbilical cord mesenchymal stem cells.
8. purposes according to claim 1, it is characterised in that the chondrocyte is by the preferred umbilicuss of mescenchymal stem cell
Induce what is obtained with mescenchymal stem cell.
9. purposes according to claim 1, it is characterised in that the collagen is typeⅡ Collagen.
10. purposes according to claim 1, it is characterised in that the concentration of the collagen solution is 1 mass %～8 matter
Amount %.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN112336751A (en) * | 2020-11-27 | 2021-02-09 | 福建华民生物科技有限公司 | Medicine for treating cartilage injury and arthritis and matched injection device thereof |
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| CN1507927A (en) * | 2002-12-18 | 2004-06-30 | 上海第二医科大学附属第九人民医院 | Method for inducing bone marrow substrate stem cell into cartilage |
| CN1626251A (en) * | 2003-12-09 | 2005-06-15 | 上海第二医科大学附属第九人民医院 | Tissue engineering transplantation of cartilage cell, and preparation method |
| CN101291586A (en) * | 2005-09-02 | 2008-10-22 | 英特菲思生物技术公司 | A method for cell implantation |
| CN101036805A (en) * | 2006-03-15 | 2007-09-19 | 尹飞 | Cell mixing method for constructing tissue engineering cartilage |
| CN103223194A (en) * | 2013-01-15 | 2013-07-31 | 广州莱德尔生物科技有限公司 | Cartilage graft for cartilage injury repair and preparation method thereof |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112336751A (en) * | 2020-11-27 | 2021-02-09 | 福建华民生物科技有限公司 | Medicine for treating cartilage injury and arthritis and matched injection device thereof |
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