Method of regenerating articular cartilage

 

(57) Abstract:

The invention relates to medicine, namely to traumatology and can be used for regeneration of articular cartilage. For this intra-articular injected fetal cartilage 16-20 weeks of gestation. 1 ml of suspension contains (4-6)106chondrocytes. The method allows to stimulate the regeneration of cartilage. It helps to restore the function of damaged joint.

The invention relates to medicine, namely to traumatology and can be used to treat patients with injuries of the articular cartilage.

Damage to articular cartilage is typical for intra-articular fractures and injuries of the joints, microclimatically disease - repetitive repetitive microtraumas of this section, deforming osteoarthritis, inflammatory joint disease.

Known methods of treatment of damaged articular cartilage, including the elimination of the causes of damage - reduction of intra-articular fractures, the removal of the damaged meniscus, strengthening ligaments, removal of debonded cartilage [1] and the creation of favorable conditions for the functioning of the joint - articular oxygenotherapy is they do not provide a complete regeneration of damaged articular cartilage, which consists in providing an enabling environment for sliding in the joint and chondrogenic diffentiate granulation tissue, and only contribute to the prevention of progression of degenerative changes in the joint.

Closest to the present invention is a method for the diagnosis and treatment of diseases of cartilage by applying immortalizing (immortal) human chondrocytes [3]. The method is a variant of cultural technology using genetic engineering, which would produce immortalized chondrocytes, saved with differentiated characteristics, allowing their use in gene therapy of diseases of the cartilage.

The disadvantages of this method include the difficulty of obtaining immortalized chondrocytes in comparison with the proposed method and possible complications of the body (the reaction is immediate or delayed-type) associated with the introduction into the body of the cultures of immortalized chondrocytes obtained by genetic engineering. Emerging immunological conflict in damaged cartilage, exploring the process of chondrogenesis.

The objective of the invention is a method for the regeneration of articular cartilage to prevent the graft-versus-host and simplify it by using more affordable hondroprotektor when his injuries.

The problem is solved by the method lies in the fact that as hondroprotektor use fetal cartilage fetal 16-20 weeks of gestation in the form of a suspension containing (4-6)106chondrocytes in ml.

The difference of the proposed method consists in the use of fetal chondrocytes - fetal cartilage containing biologically active substances vector organ-specific orientation.

Know the use of fetal tissues and cells for the treatment of various diseases. Is diabetes mellitus [4], neurological degenerative diseases: multiple sclerosis, Alzheimer's disease, amyotrophic lateral sclerosis [5, 6] , cardiovascular disease [7], violations of the integrity of the skin (burns, frostbite [8, 9].

The main aspect of the use of fetal tissue and cells for treatment is high cosmetic and functional potential of biologically active organosi graft-versus-host. The last in the transplantation of fetal tissue and cells are practically not observed.

Known methods of introducing drugs into the joint cavity [2] to stimulate repair of articular cartilage.

Distinctive technique proposed method is disposable intra-articular introduction fetal cartilage 16-20 weeks of gestation (the content of chondrocytes (4-6)106one ml of suspension) as the main chondroprotective biomaterial.

Clinical research of the authors of the proposed method is established that in comparison with traditional methods of treatment of intra-articular transplantation of fetal chondrocytes promotes more short-term rehabilitation of patients with injuries of the joints, on average by 15%. The main positive aspect of the proposed method is the regeneration and restoration of the surface of the damaged cartilage of the joint, which is confirmed by the data of intra-articular arthroscopy.

Comparative analysis of the proposed solutions and prototype shows that the proposed method differs from the known fact that as hondroprotektor for intra-articular injection using a suspension of fetal cartilage, stawley way meets the patentability criteria of "novelty."

The method constituting the invention, intended for use in health care. The possibility of its fulfillment is confirmed as described in the application techniques and equipment. The claimed method provides achievement perceived by the applicant of the technical result, namely the provision of high reparative ability of regeneration of articular cartilage and the shortening of terms of rehabilitation of patients with diseases of the joints.

From the above it follows that the claimed invention meets the condition of patentability "industrial applicability".

In the analysis of known methods revealed in them, the lack of information on the impact of the distinctive features of the method on the achievement of the technical result, therefore, the invention meets the requirement of "inventive step".

The inventive method is as follows.

Methods of obtaining a suspension of fetal chondrocytes.

Fetal material was obtained when carrying out legal abortions for medical or social reasons in terms 18-20 weeks of gestation. In terms of operating with sterility dissect the femoral head of the fetus abeauty material mechanically desynchronous until a homogeneous suspension mass, used during the centrifugation filtralite through a nylon filter (1000 rpm for 1.5 min). The resulting sedimentation of a suspension of chondrocytes are placed in a nutrient medium 199 with antibiotics (in the ratio 1:3) and add one part of cryoconserved in relation to the total volume. As a last use 10% solution of dimethylsulphoxide (DMSO). Next, a suspension of chondrocytes placed in an ultralow freezer for 24 hours at a temperature of -80oC, then placed in liquid nitrogen (-196oC) for long-term storage.

The essence of the proposed method is as follows.

A suspension of fetal chondrocytes transfer to operating in liquid nitrogen. Pre-prepare a water bath with a temperature-40oWith. Cryoprobes with cell suspension of chondrocytes slowly immersed in water. The process of thawing continue for 5 minutes until a temperature of the body. After that, the contents of the test tube under conditions of sterility is transferred into a syringe and injected directly into the affected joint in number (4-6)of 106fetal chondrocytes in 1 ml Total solution volume of 5 ml containing 20106fetal chondrocytes. Maintenance of cell suspension, remove the needle and perform several movements in the joint for uniform spread of the introduced cell suspension on the articular surface. No restrictions on the behavior of the patient after the procedure is not administered.

Example 1.

Patient M. , 38 years. When a car accident has been closed fracture of the left inner ankle with displacement of fragments. Upon admission to the hospital the patient was fractured bone block S. Novokaini 1% to 20 ml, manual reposition and fixation of a joint in the rear and a V-shaped plaster tire to the middle third of the thigh. After 3 days in the control x-ray examination showed a secondary displacement of fragments. When performing arthroscopy at the seat of fracture of the inner ankle defect of articular cartilage size 2,02,5 see Made osteosynthesis ankle spokes. Made a puncture of the ankle joint between mm. Extensor digitorum longus and extensor hallulis longus. Under arthroscopic control the needle is in the joint cavity, after which the arthroscope is removed imposed stitches on the wound. Through the needle into the joint cavity is entered in cell suspension of chondrocytes in the number of 20106fetal chondrocytes. After injection the needle is removed. Produced more passive movements in the joint. Superimposed posterior plaster splint to the upper third of the leg.

Postoperative period was without immunocomplexes 3-5 times a day. Active movement is allowed after 3 weeks, the fixation of the joint plaster splint is discontinued. Full load on the operated limb is allowed after 10 weeks.

When viewed through 6 months of complaints, the patient does not show. Runs with a full load on the operated limb, not lame. Motion in the ankle joint in full, soft tissue swelling and effusion into the joint there.

After 6 months of repeated ankle arthroscopy and removal of the spokes of the ankle. On arthroscopy: in the damage zone of the line of fracture is not defined, on the former site of the defect of the articular cartilage visible white gloss articular surface, not dissimilar in appearance from healthy cartilage.

Thus, it was established that the introduction of a suspension of fetal chondrocytes in the joint cavity has a reparative effect on cartilage and promotes full restoration of the functionality of the damaged joint, does not cause immune conflict and is more accessible in part used chondrocytes.

Just under our supervision by the 5 patients who produced the transplantation of fetal chondrocytes when performing arthroscopy. In nastolatka. Surgery of the joints of the limbs. - Kiev, 1975, S. 120.

2. A. P. Sacks. Joint disease, diagnosis and treatment. N. Novgorod, 1994, S. 210.

3. Biotechnol, Newswateh, - 1994, 4 July. - S. 7. - Eng. ISSN 0275-3685 European patent W 094/09118 prototype. Internet site, 621.33.31. Nimap and animal cells and tissue cultivation.

4. Transplantology. The leadership. Ed. by Acad. C. I. Shumakov. - M.: Medicine, 1995. - S. 317.

5. Transplantation of fetal tissue and cells. Sat. scient. Art. under the editor. C. I. Kulakov, G. T. Dry // Bull. the experts. biology and medicine. - 1998. - So 126. Mgr. 1. C. 72.

6. Transplantation of fetal tissue and cells. Sat. scient. Art. under the editor. C. I. Kulakov, G. T. Dry // Bull. the experts. biology and medicine. - 1998. - So 126 - Mgr. 1. - S. 81.

7. Treatment of coronary heart disease by transplanting fetal tissue. Methodical recommendations, Irkutsk, 1999 - 8 C.

8. The use of cultured fibroblasts to restore the skin in severely burned. D. S. Sarkisov, V. D. Fedorov, E. C. Glushchenko, etc. // Bulletin of the experts. biologist. and medicine 6, 1995. S. 567 to 570.

9. Prospects for the use of fetal fibroblasts in the treatment of wounds of various etiologies. So Kolokoltsov, D. Yurchenko, N. G. Kolosov, etc. //Bulletin of the ECA, M.: Mir, 1976. - S. 23.

Method of regenerating articular cartilage, including intra-articular injection of chondrocytes person, characterized in that as hondroprotektor use fetal cartilage 16-20 weeks of gestation in the form of a suspension containing (4-6)106chondrocytes in 1 ml.

 

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FIELD: surgical facilities.

SUBSTANCE: invention provides material suitable as hemostatic and wound-healing remedy in low-invasive internal surgery involving endoscopic techniques. Allogenic connective-tissue formations (tendons, fascias, dermis) are subjected to mechanical cleaning to remove the rests of adjoining tissues and foreign bodies, washed with running water during 5-10 min, degreased by cold (+4оС) acetone, placed for 5-10 min in 3% hydrogen peroxide solution to remove blood, and thrice rinsed with 0.9% sodium chloride solution. Treated tissues are frozen in cryogenic chamber at -45оС and dried in vacuo to constant weight using liophilic drying technique. Dried material is then wetted at 1:5 weight ratio in solution containing calculated quantities of hemostatic (fibrinogen), fibrinolysis inhibitor (aminocapronic acid), and antibiotic (cephalexin) for 10-20 min until biomaterial structure is completely and uniformly impregnated. Solution id prepared at +4оС by consecutively dissolving 0.2 g cephalexin and 1 g fibrinogen in 10 ml of 5% aminocapronic acid solution. Thereafter, biomaterial is once again frozen and subjected to liophilic drying, then ground on blade-type mill (e.g. "Cyclotec", Foss Tecator) to particle size 0.1-0.25 mm, which are packaged by 0.5 g doses into glass 20-ml bottles, tightly sealed, and sterilized by gamma radiation in dose 2.5 MRad (25 kGr).

EFFECT: simplified technology, improved quality and structure of material.

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