CN106421918A - Chondrocyte composition - Google Patents
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- CN106421918A CN106421918A CN201610980008.8A CN201610980008A CN106421918A CN 106421918 A CN106421918 A CN 106421918A CN 201610980008 A CN201610980008 A CN 201610980008A CN 106421918 A CN106421918 A CN 106421918A
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Abstract
The invention discloses chondrocyte composition. The chondrocyte composition is prepared from a cell suspension and a temperature-sensitive hydrogel, wherein the cell suspension is formed by mixing mesenchymal stem cells and chondrocyte and re-suspending the mixture in a collagen solution. The chondrocyte composition is convenient to obtain, safe and reliable, is high in chondrocyte differentiation efficiency and can provide cartilage injury parts with cells required for tissue repair; the temperature-sensitive hydrogel has good exogenous cell carrier functions, can sufficiently fill cartilage injury parts with cells in the composition and prevent the cells from moving to other parts, so that the cartilage injury parts can be effectively promoted to be rapidly and effectively repaired and healed.
Description
Technical field
The present invention relates to chondrocyte composition.
Background technology
It is that osteoarthritis (osteoarthritis, OA) occurs that articular cartilage (articular cartilage, AC) is damaged
Major Risk Factors, OA is irreversible, to be palliated the agonizing sufferings by joint replacement when PD is to late period and
Recover function.Because the repair ability of articular cartilage is limited it is necessary to early intervention is to prevent from developing into OA.Current controls
Treatment mode is intended to stimulate by primary treatment, adjacent tissue and transplanting carrys out repairing articular cartilage.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 bioengineering
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, repairing articular cartilage is damaged significant.
However, current bioengineered tissue repair cartilage damage aspect research still have to be strengthened.
Content of the invention
It is contemplated that at least solving one of technical problem present in prior art.For this reason, one object of the present invention
Be to propose a kind of convenient obtain, safe and reliable, can effectively repair the means of cartilage damage.
It should be noted that the present invention is to be completed based on the following discovery of inventor：
One of wide cartilage repair techniques of cellular level of application are Autologous Chondrocyte transplanting at present
(Autologous chondrocyte implantation, ACI) technology.This technology comprises internal operative treatment and thin in vitro
Born of the same parents' culture technique：First, the non-bearing region in damaged joints carries out operation acquisition cartilage slices, and is transferred into storing
And in the sterile nutrient solution of transport；Then, using collagenase digesting cartilaginous tissue in cell culture chamber, obtain cartilage cell,
Using monolayer cultivation, cultured chondrocytes are expanded to the quantity that can be used for transplanting, filling out of cartilage defects is carried out by surgeon
Fill.In this surgical procedure, the cartilage cell of amplification in vitro is expelled to fault location.In order that cartilage cell rests on transplanting portion
Position, prevents from floating in a large number, further synovial membrane lobe can be sewn to damage location.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 individual layer amplification, these cell masses lose its phenotype to born of the same parents, and the production of collagen is (typically transparent by II type
Cartilage) to I type and type III (typical fibrocartilage) transformation.The result of the change of these phenotypes is that have poor biology
The generation of the extracellular matrix of mechanical performance.3rd, the substantial amounts of cartilage cell's of donor site offer is limited in one's ability.4th, donor set
The incidence of disease be also Autologous Chondrocyte major obstacle.
And autologous bone marrow mesenchymal stem cells transplanting is also the cartilage repair techniques of the wide cellular level of current application.
This technology carries out bone marrow aspiration in the ilium of patient, collects marrow；Sample is transferred to toilet and carries out cell separation；To 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 then seeded to culture in blake bottle, so that by gained cell amplification cultivation, passes to the second generation and prepares to move
Plant；Before transplanting using colony forming unit (Colony-forming unit, CFU) and Flow cytometry cell gram
Grand Forming ability and the expression of cell surface marker；Then, the MSCs pancreatin of the second generation is digested, and be resuspended in life
During regulating blood condition is clear, and it is loaded in asepsis injector, under perspective, at cell infusion to the knee injuries of patient.However, inventor
Find, autologous bone marrow mesenchymal stem cells transplanting has following defect：1st, bone marrow aspiration brings misery to patient.2nd, cell is direct
It is expelled to injury region, does not do other measures, cell may float in a large number.3rd, brachymedial phase effect is preferable, but long-time after
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 the very promising strategy of one kind,
Because MSCs has high proliferation ability and has the potential being divided into cartilage cell, and then form cartilage.MSCs is that fusiformis is thin
Born of the same parents, have fast breeding and self-renewal capacity, are present in a large amount of tissues, including 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 to repair mesenchyma group
Knit.MSCs can be to one-tenth cartilage, skeletonization, fatty development ways differentiation.Regulatory factor can promote the cartilage of MSC to occur, for example
TGF (TGF-β) and dexamethasone, this is confirmed with simple external model.
And then, inventor passes through a series of researchs and finds, using the mescenchymal stem cell (Umbilical in umbilical cord source
Cord Mesenchymal stem cells, UCMSC) it is transplanted at the injury of knee joint of patient, can damage effectively treatment cartilage
Wound.And, existing ripe UCMSC isolated culture method, and umbilical cord at present easily obtains it is not necessary to take invasive to patient
Operation, it is not required that extracting cartilage in other positions, can effectively reduce the misery of patient.And MSC can fast breeding and from
I updates, and has into the potential of cartilage differentiation, ensure that the vigor of neocartilage cell, and there is not the hidden of the incidence of disease
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 cartilage cell of UCMSC and induction is combined with collagen and temperature-sensitive hydrogel, and result can effectively 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, play a part exogenous cells carrier, can promote cell movement, stick, breed and
Differentiation, and provide structural support so that cell activity at defect, will not large area move.
Thus, in one aspect of the invention, the invention provides a kind of chondrocyte composition.Reality according to the present invention
Apply example, this chondrocyte composition includes：Cell suspension and temperature-sensitive hydrogel, wherein cell suspension are by doing mesenchyma
Cell and cartilage cell mix and are resuspended in collagen solution formation.
It is surprisingly found by the inventors that, the chondrocyte composition of the present invention is convenient to be obtained, safe and reliable, and, to cartilage
The efficiency high of cell differentiation, can provide cell necessary to tissue repair for cartilaginous lesion site, and temperature-sensitive hydrogel tool
There iss good exogenous cells carrier, the cell in composition can be made to be sufficient filling with cartilage damage and not to other
Local migration is quickly and efficiently repaired and is healed such that it is able to effectively facilitate cartilaginous lesion site.
According to embodiments of the invention, in described cell suspension, described mescenchymal stem cell and described cartilage cell's
Mixed proportion is 1:1～4:1.Thus, cartilage differentiation efficiency high, can provide necessary to tissue repair for cartilaginous lesion site
Cell.
According to embodiments of the invention, in described 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.
According to embodiments of the invention, described collagen is typeⅡ Collagen.Thus, cell mixture and collagen and temperature
The composite effect of quick type hydrogel is good, can effectively prevent cell extensive migration, such that it is able to reach the effect of long-term treatment.
According to embodiments of the invention, the concentration of described collagen solution is 1 mass %～8 mass %.Thus, mixing with cells
Thing is good with the composite effect of collagen and temperature-sensitive hydrogel.
According to embodiments of the invention, the volume ratio of described cell suspension and described 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 cartilage cell's mixture, Jin Erneng
The cell in composition is enough made to be sufficient filling with damaging such that it is able to effectively facilitate cartilage at cartilage damage and not to other local migrations
Traumatic part position is quickly and efficiently repaired and is healed.
According to embodiments of the invention, described temperature-sensitive hydrogel is temperature sensitive type poly lactic acid hydrogel.Thereby, it is possible to effective
Carry mescenchymal stem cell and cartilage cell's mixture, and so that it is sufficient filling with cartilage damage and not to other local migrations.
According to embodiments of the invention, described temperature sensitive type poly lactic acid hydrogel is MPEG-PLGA.Thus, it is as external source
Property the making good use of of cell carrier, the cell in composition can be made to be sufficient filling with cartilage damage and to other local migrations,
Quickly and efficiently repair and heal so as to effectively facilitate cartilaginous lesion site.
According to embodiments of the invention, described mescenchymal stem cell is umbilical cord mesenchymal stem cells.Thus, mesenchyma is done carefully
Born of the same parents' wide material sources, easily obtain, safe and harmless, and the efficiency high to cartilage differentiation.
According to embodiments of the invention, described cartilage cell is by the preferred umbilical cord mesenchymal stem cells of mescenchymal stem cell
Induction obtains.And, the abductive approach of cartilage cell is not particularly limited, as long as can be from the preferred umbilical cord of mescenchymal stem cell
Mescenchymal stem cell effectively induces acquisition cartilage cell.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 illustrate, the present invention has at least one of following advantages：
1st, the present invention obtains mescenchymal stem cell, wide material sources by discarded object umbilical cord, can obtain in a large number, and will not be to patient
Damage.
2nd, the present invention carries out the induction differentiation in cartilage cell direction to part UCMSC, promotes UCMSC to cartilage differentiation
Efficiency.
3rd, temperature-sensitive hydrogel can play the effect of good exogenous cells carrier so that cell can be sufficient filling with
At cartilage damage, and not to other local migrations.
The additional aspect of the present invention and advantage 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
Below in conjunction with embodiment, the solution of the present invention is explained.It will be understood to those of skill in the art that it is 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 described by document in the art or condition or according to product description.Agents useful for same or instrument
The unreceipted production firm person of device, be can by city available from conventional products.
Embodiment 1
Prepare chondrocyte composition according to following steps, and skin grafing and mending is carried out to cartilage damage patient：
1st, being separately cultured of UCMSC
The fresh umbilical cord got is sent into GMP workshop, separates and obtain UCMSC.UCMSC is cultivated amplification；And take out
Part cell carries out chondroblast induction, expands, prepare transplanting after obtaining a number of cartilage cell.
2nd, prepare before transplanting
The cartilage cell that UCMSC obtained above is obtained with induction differentiation, is digested with 0.25% pancreatin, is used in combination
Brine, then by UCMSC and cartilage cell 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 %.This 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 it fully mix for a period of time, to obtain the chondrocyte composition of the present invention.
Then, the cell sampling in the chondrocyte composition of the present invention is carried out microorganism, virus five, mycoplasma,
Endotoxin and cell function detection, to guarantee security.
3rd, transplant
Using joint cavity injection method.Wherein, patient carries out arthroscopy before accepting injection, 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 adopt the improvement situation of nuclear magnetic resonance image check knee joint function.
It was found that the cell in the chondrocyte composition of the present invention can be sufficient filling with cartilage damage and not to it
Its local migration, cartilaginous lesion site healing is quick, effectively.
Comparative example 1
Method according to embodiment 1 prepares chondrocyte composition, and cartilage damage patient is carried out with skin grafing and mending, difference
It is only that：
Chondrocyte induction is not carried out to UCMSC, that is, the cell in cell suspension is all using UCMSC.
Observe after transplanting, patient's cartilaginous lesion site chondroid tissue, Principle of Pain is alleviated, damage to have and to a certain degree heal
Close.
Comparative example 2
Method according to embodiment 1 prepares chondrocyte composition, and cartilage damage patient is carried out with skin grafing and mending, difference
It is only that：
The ratio of the UCMSC in cell suspension and cartilage cell 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 cartilage damage patient is carried out with skin grafing and mending, 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 cartilage cell's formation efficiency is relatively low, thus healing speed is slow.
Comparative example 4
Method according to embodiment 1 prepares chondrocyte composition, and cartilage damage patient is carried out with skin grafing and mending, 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 cartilage cell's formation efficiency is relatively low, and healing speed is slow.
Comparative example 5
Method according to embodiment 1 prepares chondrocyte composition, and cartilage damage patient is carried out with skin grafing and mending, difference
It is only that：
Do not adopt temperature sensitive type poly lactic acid hydrogel MPEG-PLGA (carrying out injection transplantation only with cell suspension).
Observe after transplanting, a large amount of drift of the cell in composition is walked, patient's cartilaginous lesion site cartilage cell's formation efficiency
Relatively low, healing speed is slow.
Comparative example 6
Method according to embodiment 1 prepares chondrocyte composition, and cartilage damage patient is carried out with skin grafing and mending, difference
It is only that：
Collagen solution is substituted using physiological saline.
Observe after transplanting, a large amount of drift of the cell in composition is walked, patient's cartilaginous lesion site cartilage cell's formation efficiency
Relatively low, healing speed is slow.
In the description of this specification, reference term " embodiment ", " some embodiments ", " example ", " specifically show
The description of example " or " some examples " etc. means specific features, structure, material or the spy describing with reference to this embodiment or example
Point is contained at least one embodiment or the example of the present invention.In this manual, to the schematic representation of above-mentioned term not
Necessarily refer to identical embodiment or example.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
Multiple changes, modification, replacement and modification can be carried out to these embodiments in the case of the principle of the disengaging present invention and objective, this
The scope of invention is limited by claim and its equivalent.
Claims (10)
1. a kind of chondrocyte composition is it is characterised in that include：Cell suspension and temperature-sensitive hydrogel, wherein cell suspension
It is by mixing mescenchymal stem cell and cartilage cell and being resuspended in collagen solution formation.
2. chondrocyte composition according to claim 1 is it is characterised in that in described cell suspension, described between fill
The mixed proportion of matter stem cell and described cartilage cell is 1:1～4:1.
3. chondrocyte composition according to claim 1 is it is characterised in that in described cell suspension, the containing of collagen
Measure as 1 mass %～8 mass %.
4. chondrocyte composition according to claim 1 is it is characterised in that described cell suspension and described temperature sensitive type water
The volume ratio of gel is 1:1～2.
5. chondrocyte composition according to claim 1 is it is characterised in that described temperature-sensitive hydrogel is temperature sensitive type poly
Lactic acid hydrogel.
6. chondrocyte composition according to claim 5 is it is characterised in that described temperature sensitive type poly lactic acid hydrogel is
MPEG-PLGA.
7. chondrocyte composition according to claim 1 is it is characterised in that described mescenchymal stem cell is to fill between umbilical cord
Matter stem cell.
8. chondrocyte composition according to claim 1 is it is characterised in that described cartilage cell is to be done by mesenchyma
The induction of cell preferred umbilical cord mesenchymal stem cells obtains.
9. chondrocyte composition according to claim 1 is it is characterised in that described collagen is typeⅡ Collagen.
10. chondrocyte composition according to claim 1 is it is characterised in that the concentration of described collagen solution is 1 matter
Amount %～8 mass %.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107550935A (en) * | 2017-09-11 | 2018-01-09 | 上海亚睿生物科技有限公司 | A kind of biological gel for treating joint disease and its application |
| 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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| CN102065929A (en) * | 2008-06-26 | 2011-05-18 | 凯希特许有限公司 | Stimulation of cartilage formation using reduced pressure treatment |
| CN102123745A (en) * | 2008-02-29 | 2011-07-13 | 科洛普拉斯特公司 | Compositions and methods for augmentation and regeneration of living tissue in a subject |
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| CN101534747A (en) * | 2006-07-10 | 2009-09-16 | 哥伦比亚大学 | De novo formation and regeneration of vascularized tissue from tissue progenitor cells and vascular progenitor cells |
| CN102123745A (en) * | 2008-02-29 | 2011-07-13 | 科洛普拉斯特公司 | Compositions and methods for augmentation and regeneration of living tissue in a subject |
| CN102065929A (en) * | 2008-06-26 | 2011-05-18 | 凯希特许有限公司 | Stimulation of cartilage formation using reduced pressure treatment |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107550935A (en) * | 2017-09-11 | 2018-01-09 | 上海亚睿生物科技有限公司 | A kind of biological gel for treating joint disease and its application |
| 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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Application publication date: 20170222 |