CN102232970A - Cell injection for treating bone injury and preparation method thereof - Google Patents
Cell injection for treating bone injury and preparation method thereof Download PDFInfo
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- CN102232970A CN102232970A CN2010101524612A CN201010152461A CN102232970A CN 102232970 A CN102232970 A CN 102232970A CN 2010101524612 A CN2010101524612 A CN 2010101524612A CN 201010152461 A CN201010152461 A CN 201010152461A CN 102232970 A CN102232970 A CN 102232970A
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Abstract
The invention relates to a cell injection for treating bone injury and a preparation method thereof, belonging to the field of tissue engineering. The injection is a compound formed by mesenchymal stem cells from autologous bone marrow, temperature sensitive gel and effective trophic factors separated and extracted from autoblood. The preparation method comprises the steps of: after extracting the bone marrow of a patient, separating and extracting the mesenchymal stem cells from the bone marrow, adding no animal serum in the amplification culture process, and mixing 1*(105-108) cells with 1-5ml of biological scaffold material to form the injectable compound. The cell injection for treating bone injury is mainly used for treating bone injuries including osteoarthritis, femoral head necrosis and delayed union and disunion of bone fracture, is convenient to use, has notable effect without immunological rejection, and can alleviate the great pain of patients caused by bone injury.
Description
Technical field
The present invention relates to a kind of treatment bone injury injection and preparation method thereof, be applicable to the treatment of orthopaedics, belong to field of tissue engineering technology osteoarthritis, femur head necrosis, fracture delayed union or disunion.
Background technology
Along with China's life-time dilatation and variation of dietary structure per capita, the raising day by day of living standard, bone injury patient is also more and more.The most common such as arthritis, femur head necrosis, fracture delayed union or disunion etc. at present, the sick rate of sending out all shows a rising trend every year, these diseases have brought very big influence for the people's life, and still from the means of present treatment, effect is not clearly yet.
First kind bone injury: arthritis, arthritis is a kind of common chronic disease, and modal is two kinds of osteoarthritis and rheumatoid arthritis, and the present arthritic of China estimates at more than 100,000,000, and number is also in continuous increase, and the people is to the old osteoarthritis of suffering from various degree simultaneously.Osteoarthritis is a kind of modal arthropathy, and the title of osteoarthritis is extremely many, as HOA, degenerative osteoarthritis, degeneration arthritis, hyperplastic osteoarthritis or osteoarthritis, all refers to a kind of disease, the domestic unified osteoarthritis of using.The sick reason of sending out of osteoarthritis mainly contains: 1, the aging articular cartilage degeneration of tissue; 2, the injured cartilage that causes in joint damages; 3, obesity increases the joint holding capacity, and cartilage takes place damaged thereupon; 4, work habit causes, causes the articular cartilage wearing and tearing as long-term sitting posture; 5, genes of individuals changes and causes cartilage lesion.Its prevalence increased along with the age, and the women is than male pilosity.The position of the regular incidence of osteoarthritis is that knee joint, finger, neck, lumbar vertebra etc. are located, and symptom is mainly arthralgia, stiff (can feel pain relief) behind light activity, and arthroncus, amyotrophy etc. can appear in weight person.Second class: femur head necrosis, femur head necrosis full name aseptic necrosis of femoral head, or ischemic necrosis of femoral head, be that the local blood fortune of femoral head that causes owing to multiple reason is bad, thereby cause a kind of pathological changes of the further ischemia of osteocyte, necrosis, bone trabecula fracture, collapse of the femoral head.Be familiar with this disease of femur head necrosis first so far from world medical circle in 1888, femur head necrosis changes frequently-occurring disease, commonly encountered diseases into by rare disease.Especially since the appearance and extensive use thereof of hormone, the sickness rate of femur head necrosis rises gradually.The vehicles are changed increasing of back vehicle accident in addition, and the change of people life style all makes this patient's quantity increase severely.According to incompletely statistics, present whole world femur head necrosis 3,000 ten thousand people, China has 4,000,000 people approximately.Femur head necrosis can betide any age but be maximum with 31-60 year, asexuality difference, any age all can be ill, and had hormone to use the probability showed increased of history, hip trauma history, excessive drinking history, relevant disease history person morbidity, beginning shows as hip joint or its dull pain, the dull pain in joint on every side more, the activity postemphasis, further develop the dysfunction that can cause hip joint, femur head necrosis has a strong impact on patient's quality of life and work capacity, if treatment is untimely, also can cause lifelong deformity.The 3rd class: fracture delayed union and nonunion.The fracture delayed union phalangeal fracture is not healing fully in the time of expection.Mainly granulation tissue or jejune osseous tissue abrim in the fracture gap.Appropriate as follow-up treatment, fracture still may heal.There are tenderness and indirect percussion pain in the patient part of delayed union, accompanies swelling in various degree, and local skin temperature can raise.Delayed union patient part can present abnormal movement, can be with becoming angle or shortened deformity.The nonunion phalangeal fracture is not healing in the time of expection, and the cellular activity and the healing process of fracture stop fully, is dense fibrous tissue in the fracture gap.Unless take intervening measure, otherwise fracture can't connect.
It is main at present by (1) non-drug therapy to treat above bone injury; (2) Drug therapy; (3) operative treatment; (4) from body cartilage/4 kinds of methods such as osteoblast transplanting.The method of non-drug therapy, Drug therapy and operative treatment all can not fundamentally be improved the cartilage injury, Drug therapy can only alleviate patient's misery in the short time, and the method for operative treatment not only costs an arm and a leg, most sufferers are difficult to bear, the wound that the while operative treatment brings to sufferer is big, the surgical technic content requirement is higher in addition, and general hospital is difficult to carry out because of the condition deficiency.Can play certain repair from body cartilage/osteoblast transplanting to the cartilage injury, but because sampling cartilage/skeletonization is from sufferer self, so just inevitably certain damage is caused at original healthy position, it is not only cartilage/one-tenth damaged bone that the disease of while bone injury is sent out reason, also relevant with bone injury position local microenvironment, blood confession deficiency etc.The local microenvironment of defect can't be changed from body cartilage/osteoblast implantation, certainly will the healing of essence can not be carried out damage location.
Summary of the invention
At the defective of above bone injury Therapeutic Method, the purpose of this invention is to provide a kind of safe, reliable and effective treatment bone injury product.
The selected seed cell of the present invention derives from patient's bone marrow, and it is fast simple that bone marrow extracts operation, and general clinician all can operate separately.It is very little to patient's self influence to extract a certain amount of bone marrow, the general postoperative recovery operate as normal of can leaving hospital in a couple of days.
The mescenchymal stem cell in autologous bone marrow of the present invention source is a pluripotent stem cell, can be in external evoked or body be divided into polytype cell under the effect of microenvironment, comprise chondrocyte, osteoblast, vascular endothelial cell etc., the present invention simultaneously is in the process of amplification bone marrow mescenchymal stem cell, do not add any animal serum, effectively avoid the infection of xenogenesis virus, reduced the risk of cell transplantation.The bone marrow mescenchymal stem cell can be secreted a large amount of cytokines such as angiogenesis factor etc. after being transplanted to diseased region, so just can impel the diseased region revascularization, and then improve the blood circulation of diseased region.
The present invention utilize tissue engineering technique with temperature sensitive type gel and autologous bone marrow source mescenchymal stem cell according to 1 * 10
5～10
8Individual cell combines with 1～5ml biomaterial to form and transplants complex, adds a certain amount of autoserum trophic factors simultaneously in complex, so just can effectively control the concentration of patient part transplanted cells, and improve the survival rate of transplanted cells.
Advantage of the present invention is: utilizes the clinical cases such as mescenchymal stem cell materials for binding biological tissue engineering technique treatment osteoarthritis, femur head necrosis, fracture delayed union or disunion in autologous bone marrow source, avoided immunologic rejection, and safe and effective.With the mode re-injection of injection treatment bone injury position, operating time is short, implement simple, workable, to patient cause painful little.
The specific embodiment
The specific embodiment one: the mescenchymal stem cell extraction separation of derived from bone marrow, cultivation and evaluation
1. the mescenchymal stem cell extraction separation and the cultivation of derived from bone marrow
(1) patient's bone marrow extracts
The method that adopts anterior superior iliac spine to get the bone marrow art extracts patient's bone marrow.Routine disinfection, shop list after one side or the place's local anesthesia of bilateral anterior superior iliac spine, in the puncture of bilateral anterior superior iliac spine, are got bone marrow through different directions with the 10ml syringe with bone puncture needle.
(2) the blood samples of patients trophic factors extracts and preserves
When gathering bone marrow, collect a certain amount of blood of patient, and separate nutritional labeling (platelet and blood plasma) wherein in laboratory, the hemotrophic nutrition factor that packing is finished places-20 ℃ of freezing preservations, and preservation information is registered in real time.
(3) extraction separation of mesenchymal stem cells MSCs
Fresh patient's bone marrow with after the DMEM dilution, is added on suspension on the Percoll separating medium liquid level.20 ℃, 2500r/min, centrifugal 30min.Remove supernatant, draw the tunica albuginea layer, PBS washes 2 times, and 20 ℃, 1 800r/min, centrifugal 10min.With the DMEM suspension cell of the nutritional labeling that contains the extraction of 10% sufferer autoserum, counting transfers to 2 * 10 with cell density
5/ ml is inoculated in the 75mL culture bottle.Place the CO2 incubator, cultivate under 37 ℃, saturated humidity, 5%CO2 condition.Changed liquid once every 3 days.When treating that two weeks, the back mesenchymal stem cells MSCs merged, add and contain the digestion of 0.25%Trypsin-0.02%EDTA Digestive system, end to blow and beat cell repeatedly after the digestion, bottle graft kind, amplifying cells are divided with certain proportion in the washing back.
(4) amplification culture of mesenchymal stem cells MSCs
According to the demand of patient's symptom decision cell, constantly amplifying cells is until reaching the operation demand.
2. the evaluation of mesenchymal stem cells MSCs
With mesenchymal stem cells MSCs 0.25% trypsinization of the 3rd, 4,5 generations, PBS washing 2 times, preparation 10
6/ ml single cell suspension adds fluorescently-labeled CD29, CD90, CD106, CD105, CD44, CD71, CD34, CD45 monoclonal antibody respectively, puts 4 ℃ and hatches 30min, PBS washing 2 times, add 500 μ lPBS mixings, carry out flow cytometer and detect, set up homotype IgG control tube simultaneously.Experimental result is kinds of surface antigen positives such as three generations's mesenchymal stem cells MSCs CD29, CD44, CD106, CD71, CD90, CD105 as can be seen, and antigens c D34, CD45 feminine gender.
The specific embodiment two: mesenchymal stem cells MSCs does not have the cultural method of external source animal serum
For the viral pair cell of effectively avoiding animal serum to exist pollutes, the present invention is in cultivating the mesenchymal stem cells MSCs process, additive in the employed culture medium is to separate to obtain effective nutritional labeling platelet and blood plasma from patient's blood, and is mixed with into complete medium according to certain ratio with low sugar DMEM basal medium.
The specific embodiment three: mesenchymal stem cells MSCs and the safety of gel rubber material complex detect
Collect the third generation to the mesenchymal stem cells MSCs in five generations, according to 1 * 10
5～10
8Individual cell mixes formation injectable type complex with 1～5ml biogel, add the somatomedin that a certain amount of blood extracts therein.According to survival rate, mushroom, virus, the oncogenicity of relevant criterion detection cell, the standard of main reference has: " the 73rd page of 2005 editions the 3rd appendix XII A of Chinese pharmacopoeia and " organizational project medical product the 12nd part: the processing guide of cell, tissue, organ " relevant regulations; " 2005 editions the 3rd appendix regulations of Chinese pharmacopoeia; People's treated autologous cell is declared clinical trial guideline, management of laboratory animal regulations etc.The result shows: the mesenchymal stem cells MSCs of cultivating five generations of the third generation to the of amplification combines the pollution that the complex that forms is not subjected to any microorganism and virus with gel rubber material, animal oncogenicity is tested and do not observed animal body surface and intracorporeal organ generation canceration.
Specific embodiment mode four: the zoopery of mesenchymal stem cells MSCs and gel rubber material complex
Choose some of Thirty male rats, utilize meniscectomy to bring out the arthritic generation of rat bone.The osteoarthritis rat is divided into experimental group and matched group (medicine group and placebo group), experimental group utilizes rat allogeneic mesenchymal stem cells MSCs injection (the same the present invention of method) treatment, and medicine matched group and placebo group are used treatment osteoarthritis medicine and normal saline respectively.Observe the local activity situation of joint of osteoarthritis rat in the 1st week, the 2nd week, the 4th week, the 6th week, the 8th week of treatment, and in the 4th week, the dissection of the 8th week, pathology section examination, the result shows: the activity situation of osteoarthritis experimental group after accepting cell transplantation is significantly better than the medicine group, and placebo group is the poorest.The osteoarthrosis place of experimental group damage has the variant cell of transplanting to exist, and cartilage recovers significantly better than medicine group and placebo group.
Concrete burn example five: the clinical experiment of autologous bone marrow mesenchymal stem cells treatment osteoarthritis
1. case is selected:
According to orthopaedics and tranmstology branch of Chinese Medical Association: osteoarthritis diagnosis and treatment guide (version in 2007) is selected case.The preferential sufferer of selecting arthroncus and ankylosis, selected the case all chamber of experimentizing are checked (routine blood test, protein electrophoresis, immune complex and SC etc.) and X ray examination (observe asymmetry joint space situation of change and have or not edge, joint hypertrophy and hydrarthrosis).
2. Therapeutic Method:
After extracting patient's bone marrow, the extraction separation mesenchymal stem cells MSCs, amplification in vitro is cultivated mesenchymal stem cells MSCs, according to the method for the specific embodiment three mesenchymal stem cells MSCs and gel composite is carried out the safety detection simultaneously.Symptom according to sufferer prepares a certain amount of injection, and before operation was implemented, the complex of getting 20～50 μ l carried out skin test to the clinical trial patient and detects, and observes to have or not immunoreation to occur.Behind skin test, the patient does not go out a typical immunological rejection.After the anesthesia of osteoarthritis position partly sterilised, inject injection of the present invention, the injection site is the osteoarthrosis chamber.After operation finishes, ice compress injection site 1 hour, patient remained in a hospital under observation 2～3 hours, noticed that the patient injection position has or not symptoms such as redness, pain to take place.
3. effect analysis
Osteoarthritis position symptom variation situation is observed in the 1st week after the patient undergos surgery, the 4th week, the 6th week, the 8th week, the institute's further consultation of the 24th week.And in the 8th the week and the 24th week the injection site was carried out x-ray observation.The result shows: after injection treatment of the present invention, patient's daily life is restored gradually, and the pain during motion has remarkable alleviation.By the treatment of x-ray observation discovery by autologous bone marrow mesenchymal stem cells and gel composite, not only effectively stoped the development trend of cartilage defect, the position of original cartilage defect has simultaneously also obtained reparation to a certain degree.
Claims (5)
1. treat bone injury injection and preparation method thereof for one kind, it is characterized in that: get patient's autologous bone marrow, through obtaining from body pluripotency mescenchymal stem cell after separation and Extraction, the cultivation amplification, temperature sensitive type gel rubber material in conjunction with degradable, good biocompatibility forms the injectable type complex simultaneously, the injection site is mainly the bone injury position, as osteoarthrosis chamber, femur head necrosis place, the lacuna place of not healing etc. of fracturing.
2. as treatment bone injury injection as described in claims, it is characterized in that: mesenchymal stem cells MSCs derives from from body, immunologic rejection and ethical issues have been avoided, in its incubation, utilize the effective nutritional labeling of autoblood as the cytotrophy additive, no external source animal serum has improved the cell transplantation safety; Can be divided into cartilage or osteoblast behind the autogenous cell implantable bone damage location, the repairing bone defect position;
3. as the method for treatment bone injury injection preparation as described in claims 1, it is characterized in that: 1 * 10
5～10
8Individual cell mixes the formation complex with 1～5ml biologic bracket material.
4. as the method for bone injury injection preparation as described in claims 1, it is characterized in that: biomaterial is good, the degradable temperature sensitive type gel stent of the compatibility, be mainly poloxamer (poloxamer) gel, add a certain amount of deriving from gel-cell complexes from the effective trophic factors of the serum of body.
5. as the method for claims 1 described bone injury injection preparation, its range of application is: the treatment of osteoarthritis, femur head necrosis, fracture delayed union or nonunion.
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Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103340904A (en) * | 2013-07-10 | 2013-10-09 | 西比曼生物科技（上海）有限公司 | Composition for treating osteoarthritis |
| CN104487569A (en) * | 2012-05-10 | 2015-04-01 | 生物材料细胞公司 | Osteogenic differentiation of mesenchymal stem cells |
| CN105062967A (en) * | 2015-09-01 | 2015-11-18 | 沈慧勇 | Preparation method and application of human mesenchymal stem cells |
| CN105106238A (en) * | 2015-08-10 | 2015-12-02 | 山东省药学科学院 | Cell therapy composition for treating osteoarthritis and cartilage defects |
| CN106474156A (en) * | 2016-11-08 | 2017-03-08 | 华南生物医药研究院 | Purposes in preparing medicine for the compositionss |
| 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 |
| CN108531451A (en) * | 2018-04-20 | 2018-09-14 | 山东智康医疗科技有限公司 | Obtain the cultural method of the cell subsets with treatment of ulcerative colitis effect |
| CN109266602A (en) * | 2018-08-01 | 2019-01-25 | 申意伟 | The bone marrow mesenchymal stem cells suspension for treating osteonecrosis |
| CN111067920A (en) * | 2019-12-25 | 2020-04-28 | 中国人民解放军联勤保障部队第九二〇医院 | Preparation for treating degenerative disease of lumbar intervertebral disc and preparation method thereof |
| CN111407785A (en) * | 2020-04-15 | 2020-07-14 | 山东隽秀生物科技股份有限公司 | Composite acellular matrix injection for treating femoral head necrosis and using method |
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2010
- 2010-04-22 CN CN2010101524612A patent/CN102232970A/en active Pending
Cited By (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104487569A (en) * | 2012-05-10 | 2015-04-01 | 生物材料细胞公司 | Osteogenic differentiation of mesenchymal stem cells |
| CN103340904A (en) * | 2013-07-10 | 2013-10-09 | 西比曼生物科技（上海）有限公司 | Composition for treating osteoarthritis |
| WO2015003623A1 (en) * | 2013-07-10 | 2015-01-15 | 西比曼生物科技（上海）有限公司 | Composition for treating osteoarthritis |
| CN103340904B (en) * | 2013-07-10 | 2016-03-23 | 西比曼生物科技（香港）有限公司 | The compositions for the treatment of osteoarthritis |
| CN105106238A (en) * | 2015-08-10 | 2015-12-02 | 山东省药学科学院 | Cell therapy composition for treating osteoarthritis and cartilage defects |
| CN105062967A (en) * | 2015-09-01 | 2015-11-18 | 沈慧勇 | Preparation method and application of human mesenchymal stem cells |
| CN106474156A (en) * | 2016-11-08 | 2017-03-08 | 华南生物医药研究院 | Purposes in preparing medicine for the compositionss |
| 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 |
| CN108531451A (en) * | 2018-04-20 | 2018-09-14 | 山东智康医疗科技有限公司 | Obtain the cultural method of the cell subsets with treatment of ulcerative colitis effect |
| CN109266602A (en) * | 2018-08-01 | 2019-01-25 | 申意伟 | The bone marrow mesenchymal stem cells suspension for treating osteonecrosis |
| CN111067920A (en) * | 2019-12-25 | 2020-04-28 | 中国人民解放军联勤保障部队第九二〇医院 | Preparation for treating degenerative disease of lumbar intervertebral disc and preparation method thereof |
| CN111407785A (en) * | 2020-04-15 | 2020-07-14 | 山东隽秀生物科技股份有限公司 | Composite acellular matrix injection for treating femoral head necrosis and using method |
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Application publication date: 20111109 |