RU2290939C2 - Agent for treatment of spinal cord and brain damages - Google Patents

Abstract

FIELD: medicine, neurosurgery, neurology, transplantation.

SUBSTANCE: invention proposes a biological plasmatic transplant representing a clot of human plasma blood containing cells of fetus nervous and hemopoietic tissue after 16-22 weeks of gestation and taken in the ratio = 1:(5-30). Invention provides preparing a matrix from plasma blood biologically compatible with cells added in it and representing a nutrient medium that stimulates proliferation of added cells, strong bioadhesive capacity of transplant providing its dense attachment to a patient tissue, high elasticity and moisture-content of transplant that provides the possibility for acquiring form by transplant corresponding to the nervous tissue defect, possibility for using standard surgery procedures for placing transplant in spinal cord and brain, capacity to tolerate prolonged implantation in a patient nervous tissue without lysis abut with retaining viability of cells of transplant, capacity for substituting defect in spinal cord and brain and recovering their function.

EFFECT: valuable medicinal properties of agent.

2 cl, 2 ex

Description

The invention relates to medicine and can be used in neurosurgery, neurology, transplantation.

Known means for the treatment of brain damage, which are either fragments of biological tissues or polymer prosthetic materials.

The first group includes donor tissue grafts, or autografts, or allografts, or xenografts used to connect damaged areas of the spinal cord (Houle and Reier, J. Comp. Neurol., 269, P.535, 1988). They are either suspension transplants consisting of cells of nerve tissue (Goldberg and Bernstein, J. Neuroscience Res., 19, P.34, 1988; Hoovler and Wrathall, Acta NeuropathoL, 81, P.303, 1991); or Schwan cells recombined with cultural sensory neurons (Kuhlengel et al., J. Comp. Neurol., 293, P. 74, 1990); or peripheral nerve segments (NN Burdenko, Russian doctor, No. 2, 1910, pp. 762-766; Wrathall et al., Acta NeuropathoL, 57, P.59, 1982). These and similar grafts fill the spinal cord defect. However, these grafts do not perform the function of restoring the spinal cord. Placed in a defect in brain tissue, they, deprived of nutrition, die, turning into a scar.

The second category of transplants includes various modifications of polymer neurogels. Authors Woerli S, Plant G.W, Harvey A.R. Cultured Rat Neuronal and Glial Cells Entrapped within Hydrogel Polymer Matrices: A Potential Tool for Neural Tissue Replacement (Neurosci. Lett., 1996, 205, P.197-201) describe technology for capturing neural tissue cells with homogeneous transparent polymer gels from poly N- (2-hydroxypropyl) methacrylic amide, which may contain collagen as an additional substrate. This procedure involves the additional introduction of the cell suspension to the polymer mixture and the polymerization process of the cell-polymer mixture. The resulting gel is optically transparent, and the cells are randomly scattered in the gel.

Known US patent No. 5863551 (1996, author - Woerli S.) "Implantable polymer hydrogel for therapeutic uses", which describes an implantable polymer hydrogel for therapeutic purposes. The invention relates to a polymer hydrogel, which can be used to replace parts of damaged soft organs, including nerve tissue. Live cells can be introduced into it.

All proposed varieties of the gel are based on various combinations of chemical substances that are not a nutrient medium for the cells placed in them. For this reason, immediately after transplantation, when the vascular network has not yet had time to develop, stem cells placed in a helium transplant die. In addition, in most cases, the gel, without biocompatibility, turns into a scar, which prevents the growth of axons.

The well-known "Method for the treatment of traumatic injuries of the spinal cord" (RF patent No. 2195941). This invention uses a fetus spinal cord transplant enriched with cells of the lining of the olfactory nerve bulbs. The graft is introduced into the area of the spinal cord defect after cyst removal. The cells of the lining of the bulbs of the olfactory nerves can also be introduced into the peripheral and proximal ends of the spinal cord. A suspension of fetal cells of the nervous and hematopoietic tissue is introduced into the subarachnoid space of the spinal cord. A cell suspension is administered without any matrix. The absence of a matrix does not allow the whole cell mass to be concentrated in the damage zone; it spreads throughout the entire subarachnoid space, which reduces the effectiveness of the treatment. For the same reason, the method does not create conditions for the development of the vasculature in the area of damage.

SUMMARY OF THE INVENTION

A tool is proposed (biological plasma transplant) for the treatment of injuries of the spinal cord and brain, representing a plasma clot (a clot of human blood plasma) containing fetal cells of the nervous and hematopoietic tissue.

The remaining cells of the nervous and hematopoietic tissue are in the graft in a ratio of 1: (5-30). They are obtained from fetus 16-22-week gestation.

The proposed tool (transplant) is obtained as follows (the calculation for obtaining a transplant with a volume of 2-2.5 ml is given).

Modified Olsver solution (0.8 g of sodium citrate, 0.42 g of sodium chloride, 2.05 g of glucose, 0.8 ml of 10% citric acid is adjusted to 100 ml with double-distilled water), sterilized by filtration through a 0.22 μm filter and poured into a test tube in an amount of 3.5-4 ml. Then 5-6 ml of fresh donated blood is added to the same tube. The contents of the tube are thoroughly mixed and centrifuged to separate the plasma from the cells (for example, for 10 minutes at a speed of 1500 rpm). The plasma is aspirated, transferred to a new tube and centrifuged again under the same conditions. The plasma obtained after repeated centrifugation is again aspirated and sterilized by filtration. A suspension is prepared in a physiological solution of stem cells of the nervous (brain) and hematopoietic liver tissue of a human fetus of 16-22-week gestation (abortion material). The cells of the nervous and hematopoietic liver tissue are in suspension in a ratio of 1: (5-30). To 1 ml of this suspension containing (1.5-2) × 10 ⁸ cells, add 1-1.5 ml of twice centrifuged sterile plasma.

A mixture of plasma cells is well mixed on a stirrer. The clot is initiated by adding to the mixture a solution of calcium chloride at the rate of 50 μl of a solution of calcium chloride per 1 ml of a mixture of cells in plasma (a solution of calcium chloride is prepared by adding 100 mg of CaCl ₂ to 5 ml of deionized water). After adding CaCl _{2, the} tube was removed from the mixer. After initiation, a clot forms on average in 10 seconds. The test tube is left for 5-15 minutes for better "ripening". Then, holding the tube “near the neck”, and slightly tapping the finger of the other hand along the lower end of the tube, retract the clot and separate it from the walls of the tube. Then, with a simple “transfusion” of the bunch, it is transferred to a bottle, the cork of which is rolled up. A 2–2.5 ml transplant is obtained containing (1.5–2) × 10 ⁸ cells. The clot can be densified by retraction and separation of part of the liquid (20-30% of the clot volume).

Before surgery, the patient undergo MRI and determine the location and size of the damage. Based on the data obtained, a transplant of a predetermined size is prepared. If after the introduction of it during surgery into the area of damage, the graft is larger than necessary to fill the defect, the excess part is cut off.

A typical laminectomy is performed in the projection of the damaged area of the spinal cord. Dissect the dura mater. Excision of scar tissue and intraspinal cysts is performed. The lining of cysts is excised before exposing the spinal cord tissue in the proximal and distal stumps of it. The transplant described above is introduced into the resulting spinal cord defect - a plasma clot with stem cells. A clot completely replaces a defect in the spinal cord, adhering to its exposed surfaces. In the proximal stump of the spinal cord, a suspension of cells of the lining of the bulbs of the olfactory nerves of the fetus (RU 2195941) in physiological saline (1-20 thousand cells in 0.5-1 ml of suspension) in a volume of 1.5 ml and 3-5 ml of a suspension of the above nerve cells is injected and hematopoietic liver tissue containing (0.4-0.8) × 10 ⁸ cells in 1 ml. Suture on the dura mater and suture the wound.

The advantages of the proposed biological plasma transplant:

- a matrix was obtained that is biologically compatible with the cells introduced into it, which is a nutrient medium for them;

- a matrix obtained from blood plasma stimulates the proliferation of cells introduced into it;

- strong bioadhesive ability of the transplant, ensuring its tight attachment to the patient’s tissue;

- high elasticity and moisture content, which makes it possible for the transplant to acquire a form corresponding to the shape of a defect in nervous tissue;

- the ability to use standard surgical procedures to place the transplant in the spinal cord and brain

- the ability to withstand prolonged implantation in the patient’s nervous tissue without lysis of the clot and while maintaining the viability of the cells contained in the graft;

- the ability to replace a defect in the spinal cord and brain and restore their function.

Information confirming the possibility of carrying out the invention with the implementation of the appointment

Test 1. The ability to preserve the transplant qualities of a biological plasma graft was studied in vitro. It is known that cerebrospinal fluid has pronounced lytic properties. It was possible to assume that she would dissolve the clot and / or cells introduced into it.

Plasma clot was created according to the method described above. Fetal stem cells of the nervous tissue of the human fetus were introduced into it. Then, by spinal puncture, the patient received 8-10 ml of cerebrospinal fluid. The finished transplant was placed in a test tube with cerebrospinal fluid so that it freely floated in it. The tube was incubated in an incubator at 37 ° C for 7 days. After 7 days, a morphological study was carried out (stained with hematoxylin-eosin and according to Van Gieson). It was found that among the fibers of fibrin there is a large number of immature nerve cells located in groups and singly. Some cells are in apoptosis, but mostly the cell contours are preserved. Thus, cerebrospinal fluid not only did not destroy the plasma clot, but also contributed to cell survival.

Test 2. The possibility of using the proposed biological plasma transplant for the indicated purpose was studied in the experiment by the example of implantation in parts of the damaged brain of rats. We used a plasma clot obtained by the above method and containing fetal stem cells of human nervous and hematopoietic liver tissue. Rats underwent trepanation of the skull, a piece of the brain was removed from the cortex with a volume of about 0.5 cm ³ . A plasma clot with stem cells according to the invention was introduced into this defect. Rats were sacrificed after 14 and 30 days, the brain was removed, and preparations were prepared using sections of the brain in the area of transplantation using the standard method. The preparations were stained with hematoxylin-eosin.

After 14 days, a cavity limited by glia proliferates with a large number of capillary vessels is visible in the brain tissue. Inside the damage zone, loose connective tissue, the “reticular stroma” (thin reticular fibrils), is visible. Among the fibrous structures, groups of neuroblasts are observed, in places of 3-4, sometimes single. These are large cells with a centrally located nucleus and nucleolus. Nissl substance (Nissl stain) is detected in the cytoplasm of these cells, which confirms the preservation of the viability of transplanted fetal neuroblasts.

30 days after the transplantation of neuroblasts into the damage zone, a cavity is revealed made by proliferates of glia and loose connective tissue with a large number of capillary vessels. Among the array of glia cells, cells of loose connective tissue, neurons are visible in groups of 3-4 and single. These are large cells, the cytoplasm of which gives a positive Nissl reaction. A rather homogeneous matrix, a neuropil, is formed around these cells. These cells are located in all fields of view in the "transplant" area.

Thus, the following data were obtained:

1. Smooth conjugation of a plasma clot with recipient tissues over the entire surface.

2. Neoplasm of capillaries and the ingrowth of capillaries into a plasma clot.

3. Preservation of viability and ability to proliferate cells of the nervous tissue introduced into the plasma clot

4. The ingrowth of glial cells into a plasma clot, creating a trophic microenvironment for the introduced stem neuronal cells.

Clinical observations:

1) Patient A.-c. A., on 08.06.2002, when falling from a height, received a compression fracture of the Th11 vertebra with contusion of the spinal cord. Immediately developed a syndrome of a complete transverse break of the spinal cord (lower flaccid paraplegia; violation of all types of sensitivity; acute urinary retention, followed by incontinence after 3 weeks). In an emergency, rear stabilization by metal construction was performed. The complex of rehabilitation measures did not give a recovery effect. Magnetic resonance imaging (MRI) determines myelomalacia with cyst formation at the level of damage. November 13, 2003 a laminectomy was performed. A 2 ml cyst was emptied and an appropriate volume containing a fetus nerve and hematopoietic cells (17 weeks of gestation) in a ratio of 1:10 was introduced into the plasma biological transplant cavity. 1.5 ml of a suspension of olfactory nerve lining cells (10 thousand cells in 1 ml) and 4 ml of a suspension of fetal cells of nerve tissue and hematopoietic liver cells (0.5 × 10 ⁸ cells in 1 ml) were injected into the proximal stump of the spinal cord.

After 6 months, the patient had a reduction in the adductors and abductors of the thigh muscles, and the level of sensitivity disorders decreased by 4 segments. Fully controls the act of urination and defecation.

2) Patient A.V. T. (b. 1981). When diving into the pool on 10.01.2003, she received a C5 vertebral fracture with the syndrome of a complete transverse break of the spinal cord. In order of emergency, a front propping corporedesis was made with the replacement of the C5 body with an autobone and a titanium nickelide implant. Despite the ongoing intensive rehabilitation, restoration of spinal cord function did not occur. On November 11, 2004, an operation was performed: C5 laminectomy, transplantation of a plasma biological transplant impregnated with fetal (21-week gestation) stem cells of nervous and hematopoietic tissue in a ratio of 1:25. 1.5 ml of a suspension of olfactory nerve lining cells (20 thousand cells in 1 ml) and 3 ml of a suspension of fetal cells of nerve tissue and hematopoietic liver cells (0.8 × 10 ⁸ cells in 1 ml) were injected into the proximal stump of the spinal cord.

After 5 months, a significant restoration of spinal cord function was noted: 1) the contraction of the pectoral and intercostal muscles was restored and the chest type of breathing was restored (speech was difficult before the operation, and after the operation not only speaks loudly, but also sings loudly); 2) the tone of the back muscles was restored, which allowed the patient to sit in a stroller (before that, she could only lie); 3) the strength in the fingers recovered to 2 points (it became possible to hold a glass, spoon, handle in hands); 4) severe spasticity in the legs almost disappeared while maintaining muscle tone; 5) the level of pain sensitivity decreased to Th 7 segment, 6) the restoration of urination function began, can hold urine for 4-5 hours, feels urge (there was true incontinence before transplantation). Neuromyography data: after transplantation, conductivity is restored to the right by 30%, to the left by 42% (before surgery, there is a complete absence of electrical signal conductivity.

Claims (2)

1. A tool for the treatment of injuries of the spinal cord and brain, characterized in that it is a clot of human blood plasma containing fetal cells of the nervous and hematopoietic tissue in a ratio of 1: (5-30). 2. The tool according to claim 1, containing fetus cells 16-22-week gestation.