Three-component complex for cell therapy in ophthalmology

FIELD: medicine.

SUBSTANCE: invention relates to medicine, in particular to ophthalmology, and can be applied for cell therapy in case of different ophthalmopathologies, accompanied by dystrophic and atrophic processes as well. Three-component complex for cell therapy contains mesenchymal stem cells, labeled by magnetic microparticles. Cells are translocated into biological or synthetic fine-pore material, which in its turn is strongly fastened with polymer magnetic material with induction of constant magnetic field 1.5 mT, with multipolar reverse magnetisation.

EFFECT: invention ensures directed supply of stem cells to pathological nidus and holding stem cells for specified time with creation of possibility of giving complex any form, size and space configuration, suitable for extrascleral implantation to any area of eyeball or visual.

2 cl, 1 ex

 

The invention relates to medicine, namely to ophthalmology, and can be used for cell therapy in different eyes diseases, accompanied by dystrophic, and atrophic and other processes.

One of the promising methods of treatment of various eyes diseases, accompanied by dystrophic, and atrophic, etc. processes, for example, age-related macular degeneration, optic nerve atrophy, progressive myopia, etc. may be the use of mesenchymal stem cells (MSCS), however, such methods are not yet sufficiently developed.

Stem cells have a number of significant advantages: can ensure the regeneration of damaged areas through the production of various growth factors and key metabolites; able to expand the program of proliferation and differentiation, thereby restoring the lack of active cells; can be integrated into diseased areas of the retina and form the cells of the retinal phenotype. In ophthalmology therapeutic potential of stem cells has been studied in animals with hereditary retinal pathology, close to pigmented retinitis person (Lund et al. Subretinal transplantation of genetically modified human cell lines attenuates loss of visual function in dystrophic rats // PNAS. - 2001. - V.98. - N.17. P.9942-9947; Rander et al. Light-driven retinal ganglion cell responses in blind rd mice after neurona transplantation // Invest. Ophthalmol. Vis. Sci. - 2001. - V.42. - P.1057-1065; Woch et al., 2001; Sagdullaev et al. Retinal transplantation-induced recovery of retinotectal visual function in rodent model of retinitis pigmentosa // Invest. Ophthal. Vis. Sci - 2003. - V.44. - P.1686-1695).

Although mesenchymal stem cells of an adult organism have a more restricted differentiation potential than embryonic stem cells obtained by culturing cells of a blastocyst, their use safer. In addition, from the point of view of ethics, they are more acceptable for clinical use material.

A significant problem of cell therapy is targeted delivery of stem cells to pathological lesion and keeping them there for the time required to achieve a therapeutic effect.

The objective of the invention is the creation of a three-component system for cell therapy in different eyes diseases, accompanied by dystrophic, and atrophic and other processes.

The technical result of the invention is the possibility of modeling a three-component complex to give it any shape, size and spatial configuration suitable for extrascleral implantation to any area of the eyeball or the optic nerve and hold it there for the time required to achieve the desired therapeutic effect.

The technical result reached is highlighted by that:

1. Ternary complex contains MSCS labeled with magnetic microparticles, transposed in biological or synthetic porous material, which is firmly bonded with the polymer magnetic material with induction of a constant magnetic field of 1.5 MT, with multi-pole reversing the magnetization.

2. Translotsirovannoi MSC labeled with magnetic particles in biological or synthetic porous material in combination with a tight attachment to the polymer magnetic material with induction of a constant magnetic field of 1.5 MT, with multi-pole reversing magnetization provides deep penetration MSC in layers of biological or synthetic porous material, retention and even distribution of MSC with extrascleral implantation of a three-component complex to any area of the eyeball or the optic nerve within the time required to achieve therapeutic effect, due to the interaction of the magnetic fields of the particles and the magnetic material.

3. Implantation of ternary complex to the target area of the eyeball or the optic nerve is accompanied directed selective adhesion of MSC in the pathological focus, which contributes to the activation of reparative processes by healing transplanter the bathrooms stem cells in the immediate vicinity of the damaged area.

The ability of such processes to embryonic stem cells and adult stem cells of the body shown in experiments on animals (Lamba D.A., Karl M.O., C.B. Ware, Reh T.A. Efficient generation of retinal progenitor cells from human embryonic stem cells // PNAS, 2006, v.103, n.34, pp.12769-12774. Meyer J.S., Katz M.L., Maruniak J.A., M.D. Kirk Embrionic stem cell-derived neural progenitors incorporate into degenerating retina and enhance survival of host photoreceptora // Stem Cells, 2006, v.24, n.2, pp.274-283. Fiedlander M. Fibosis and diseases of the eye // J. Clin. Invest., 2007, v.117, n.3, pp.576-586), although many of the implementation mechanisms of therapeutic effects were not studied thoroughly.

Three-component complex is made as follows.

MSC grown in the culture of bone marrow cells taken from the patient during a diagnostic puncture of the sternum or Ilium (volume 0.5-1.0 ml). Cultivation of rice is carried out in a special box for cell cultures using the following equipment - centrifuge with disposable sterile centrifuge tubes 50 ml, thermostat air, laminar box, inverted and conventional microscopes, automatic pipettes, cylinders with carbon dioxide and air, the camera Goryaeva to calculate cell concentration. For culturing cell source bone marrow using a sterile disposable plastic culture flasks with square bottom 25 and 150 cm2. When the reproduction of the MSC uses the following environment and the solution is: medium RPMI-1640, medium 199, antibiotics: penicillin, amphotericin, a solution of L-glutamine, fetal calf serum. For 12-14 consecutive doublings (within 25-30 days) from the initial amount of undifferentiated MSCS contained in the received punctate the patient's bone marrow and approximately 103 cells produced approximately (1-2)×107 MSC necessary for a successful stem cell transplant.

Magnetic particles (d=2,8 µm) was injected into the cytoplasm of mesenchymal stem cells by the following method. Upon reaching 80-90% of confluently to culture MSCS add a suspension of magnetic particles. Magnetic particles pre-treated with surfactants to create the conditions for penetration into the cytoplasm of a cell. Cells incubated with particles for 24 h in a CO2 incubator. After incubation, the culture medium is changed and cells 5-fold washed free from magnetic particles with Hanks solution. The efficiency of labeling MSK magnetic particles for this technology is about 90%. The viability of stem cells is 95%.

Ready ternary complex can have any shape, size and spatial configuration that you choose and model depending on the shapes, sizes and configuration section of the eyeball or the optic nerve, to which plan of extras the scleral implantation of the complex. To do this, carry out the procurement of the required shape, size and configuration of the polymer magnetic material system samarium-cobalt or Neodym-iron-boron with the induction of a constant magnetic field of 1.5 MT with multi-pole reversing the magnetization. The thickness of the magnetic material is 0.2 mm, the magnetization of the polymer magnetic material can be produced, for example, as described in the patent of Russian Federation №2187162.

From a biological material, e.g., Dura mater or sclera, or collagen, or injection, or synthetic porous material, such as hydrogel or degele, or polyester, or silicone sponge, etc. carry out the procurement of such form and dimensions, so that in future could be attached to the billet of polymeric magnetic material.

MSC-labelled magnetic microparticles layer on the surface of the workpiece from biological synthetic porous material. Then under the Petri dish with layered material of MSC labeled with magnetic particles, the sum of the external magnetic field with a strength of 30 MT. The size and shape of the external magnet corresponds to the size and shape of the workpiece from biological or synthetic material. Thus, between the external magnet and MSC containing magnetic particles, biological engineering is the th or synthetic porous material. MSC containing magnetic particles, quickly and deeply translucida in layers of biological or synthetic porous material under the action of magnetic fields. Their fastening and raspletanie occurs within 1-1,5 hours after application. Fixed cells are held tightly in layers of porous material even in the current culture fluid that was simulated in the experiment using a peristaltic pump. Cells containing magnetic microparticles, proliferate, completely covering the surface of biological or synthetic porous material that proves the preservation of the functional properties of MSC.

In conclusion, the obtained structure of MSC in biological or synthetic porous material firmly fasten, for example, using suture fixation or biological glue for the preparation of magnetic polymer material.

The use of triple complex for cell therapy in the treatment of various eyes diseases, accompanied by dystrophic, and atrophic etc. processes is illustrated by the following data.

The use of triple complex for the surgical treatment of progressive and complicated myopia.

Use the ternary complex in the form of a flat blade length of 16 mm and a thickness of 0.8-1.0 mm, with a cloth, it is the missing structure MSC - biological or synthetic porous material, a length of 13 mm and an area of 50-65 mm2intended for implantation to the posterior pole of the eye, and foot devoid of structure "MSC - biological or synthetic porous material", width up to 2 mm and a length of 3 mm, intended for the filing of the implant to the sclera. The canvas of the complex may be oval, or rectangular, or trapezoidal, for example, isosceles. Rectangular and trapezoidal sheet can be made with rounded corners for ease of implantation. The leg may be, for example, semi-oval or rectangular shape. Rectangular leg can be made with rounded corners.

Scleroplastic operation using a three-component complex is performed as follows. In the lower - and upper outer quadrant, some distance from the limbus 6 mm, produce incisions of the conjunctiva and tenon's membrane length 8-9 mm, with a spatula to form two tunnel towards the posterior pole of the eye so that each of them freely accommodate the three-component complex. Each three-component complex is immersed under tenon's membrane distal part to the posterior pole of the eye and is fixed to the sclera at 10 mm from the limbus to the leg. The surgery is completed by suturing on the conjunctiva and the introduction of n is e antibiotic.

Clinical example. Patient Z., 14 years. Diagnosis: progressive high myopia OU. He complained of progressive vision loss, since 8 years. Significant visual impairment noted during the last year.

Visual acuity OD 0.04 sph-7.0=0.8

OS 0.05 sph-6.5=0.8

PZO OD 26.2 mm

OS 26.0 mm

Acoustic density of the sclera at the posterior pole OU=40,25 DB.

Indicators electroretinogram in white light, showing a decrease of the amplitude "a" and "b" waves: wave ' a ' =41 µv wave "b"=69 mV.

When viewed in the peripheral parts of the ocular fundus revealed areas of lattice degeneration of the retina OD in the upper sector, OS - outer sector.

Conducted scleroplastic operation. During the operation, to the rear pole of the eye implanted two trekhkomponentnykh complex containing MSC-labelled magnetic microparticles, translotsirovannoi in the hydrogel, which was firmly bonded using suture fixation of the polymer magnetic material system samarium-cobalt with the induction of a constant magnetic field of 1.5 MT, with multi-pole reversing the magnetization. The complexes were in the form of a flat blade length of 16 mm and a thickness of 0.8 mm, with oval blade length of 13 mm and an area of 65 mm2and rectangular leg width of 2 mm and a length of 3 mm.

Within 1.5 years after the operation:

Visual acuity OD 0.05 sph-7.0=0.9

PZO OD 26.2 mm

OS 26.0 mm

Acoustic density of the sclera at the posterior pole OU=45,4 DB.

Indicators electroretinogram in white light show increasing values of the amplitude "a" and "b" waves: wave "a"=43 µv wave "b"=74 mV. When viewed in the peripheral parts of the ocular fundus retina condition is stable, without increasing degenerative changes.

Scleroplastic surgery with implants to the posterior pole of the eye of ternary complexes was performed in 7 patients (10 eyes) with progressive and complicated myopia.

Used ternary complexes containing MSC-labelled magnetic microparticles, translotsirovannoi in biological material: Dura or sclera, or collagen, or injection, or synthetic porous material: hydrogel or digel, or polyester fabric or silicone sponge, which was firmly bonded using suture fixation or biological adhesive polymer magnetic material system samarium-cobalt or Neodym-iron-boron with the induction of a constant magnetic field of 1.5 MT, with multi-pole reversing the magnetization. The complexes were in the form of a flat blade length of 16 mm and a thickness of 0.8-1.0 mm, oval, or rectangular, or trapezoidal blade length of 13 mm and an area of from 50 to 65 mm2and semi-oval or rectangular is through leg width up to 2 mm and a length of 3 mm In each case, implanted two three-component complex.

The follow - up period of up to 2 years. In all cases, the observed stabilization of visual functions and parameters of the PHC, the increase in acoustic density of the sclera at the posterior pole from 41.1±0.3 to operations to 45.3±0,5 2 years after surgery, no signs of degenerative changes in the peripheral retina or their progression.

The use of triple complex for the treatment of optic nerve atrophy of various etiologies.

Use the ternary complex in the form of two semi-cylinders. The height of each half is 4 mm, the inner radius of the base is 4 mm.

Three-component complex is implanted as follows. In the lower inner and upper outer quadrants, some distance 5 mm from the limbus, perform two incision of the conjunctiva and tepanovoi shell for 7-8 mm, then dumb by forming two tunnel toward the posterior pole of the eyeball towards each other, and then injected into the tunnels and place the three-component complex consisting of two semi-cylinders, which cover the optic nerve, posterior short ciliary arteries and part of the retro-bulbar tissue without closing them. The surgery is completed by overlapping seam on the conjunctiva.

The study included 8 patients (8 eyes) aged from 40 to 65 years with atrophy SP is positive nerve of various etiologies. Visual acuity before treatment patients ranged between 0.05 and 0.125.

In all cases used the ternary complex in the form of two semi-cylinders, each with a height of 4 mm, an inner radius of the base is 4 mm, containing MSC-labelled magnetic microparticles, translotsirovannoi in biological or synthetic porous material, for example, Dura or sclera, or collagen, or injection, or synthetic porous material, for example, a hydrogel or digel, or polyester fabric or silicone sponge, which is bonded with a polymeric magnetic material system samarium-cobalt or Neodym-iron-boron with the induction of a constant magnetic field of 1.5 MT, with multi-pole reversing the magnetization.

In the lower inner and upper outer quadrants, some distance from the limbus 5 mm, was performed two incision of the conjunctiva and tenon's membrane during 7-8 mm, then dumb formed by two tunnel to the rear pole of the eyeball towards each other, then in the tunnels was introduced and placed a three-component complex consisting of two semi-cylinders, which covered the optic nerve, posterior short ciliary arteries and part of the retro-bulbar tissue without closing them. The operation ended with the imposition of blanket continuous methodologi seam on the conjunctiva.

After 12 months is in after treatment, marked improvement of visual acuity in all cases from 0.05 to 0.1. All patients identified the expansion of the boundaries of the field of view of 10 to 30 degrees, as well as the improvement according to EL and PAC. Linear blood flow velocity in the Central retinal artery in all cases increased and amounted to an average of 6.1±0,04 cm/sec compared to the baseline of 4.6±0,02 cm/sec.

The use of triple complex for the treatment of "dry" form of age-related macular degeneration.

Use the ternary complex in the form of a circle 6 mm in diameter, containing MSC-labelled magnetic microparticles, translotsirovannoi in biological or synthetic porous material, which is firmly bonded with the polymer magnetic material with induction of a constant magnetic field of 1.5 MT, with multi-pole reversing the magnetization.

Three-component complex is implanted extrascleral in the projection of the macular zone. In the upper outer quadrant 4 mm from the limbus perform incision of the conjunctiva length of 6 mm concentric limbu and with a spatula to form a tunnel in Sultanova space towards the rear pole of the eye. In the tunnel, enter the three-component complex, with a spatula produce sclerocornea to localize the position of the complex under constant control by using binocular Ophthalmoscope. Upon completion of the implantation of the conjunctiva is sutured with a continuous suture.

On the observation were 7 patients (7 eyes) with "dry" form of AMD in age from 60 to 68 years. Visual acuity before treatment in patients ranged from 0.08 to 0.12.

In all cases used a three-component complexes in the form of a circle 6 mm in diameter, containing MSC-labelled magnetic microparticles, translotsirovannoi in biological material: Dura or sclera, or collagen, or injection, or synthetic porous material: hydrogel or digel, or polyester fabric or silicone sponge, which was firmly bonded using suture fixation or biological adhesive polymer magnetic material system samarium-cobalt or Neodym-iron-boron with the induction of a constant magnetic field of 1.5 MT, with multi-pole reversing the magnetization.

Complexes implanted extrascleral in the projection of the macular zone. In the upper outer quadrant 4 mm from the limbus performed incision of the conjunctiva length of 6 mm concentric limbu and with a spatula shaped tunnel in Sultanova space towards the rear pole of the eye. The tunnel has introduced a three-component complex, with a spatula made sclerocornea to localize its position under constant control by using binocular Ophthalmoscope. Upon completion of the implantation of the conjunctiva sutured with a continuous suture.

12 months after treatment in all cases of sharpness of the vision 0.1 and 0.35, the observed increase in foveal sensitivity of the retina according to a computer perimetric studies, as well as the increase of the amplitude of a-wave and b-wave according to the macular electroretinogram, which testified to the increase of the functional activity of the retina. In the study of hemodynamics in all patients there was an increase in the average flow velocity in the ophthalmic artery, Central retinal artery and posterior short ciliary arteries. The results remained stable during the whole observation period. The follow-up period ranged from 12 to 36 months.

Thus, the proposed invention provides the possibility of modeling a three-component complex to give it any shape, size and spatial configuration suitable for extrascleral implantation to any area of the eyeball or the optic nerve and hold it there for the time required to achieve the desired therapeutic effect.

1. A three-component system for cell therapy in ophthalmology, containing mesenchymal stem cells labeled with magnetic microparticles, translotsirovannoi in biological or synthetic porous material, which is firmly bonded with the polymer magnetic material with induction of a constant magnetic field of 1.5 MT, the multi-pole reversing the magnetization.

2. The complex according to claim 1, characterized in that it has any shape, size and spatial configuration suitable for extrascleral implantation to any area of the eyeball or the optic nerve.



 

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