The brain tissue engineering. A.S.Brukhovetsky. Clinic of Restorative Interventional Neurology and Therapy 'NeuroVita', Russian Oncology Research Center of the Russian Academy of Medical Sciences, Moscow, Russia

In modern medicine anaplastic surgery means surgery operation of transplantation or implantation of organs and/or tissues. As a matter of fact it is well known vary means of plastic and transplantation aiming at change of organ anatomical structure or its substitution for the sake of providing its proper function. No doubt donors organ grafting is maximum radical treatment solution. However, the investigators practically don't look at brain as transplantation object due to comlexity and unsufficient study of its structure functional organization as well as lack of technical possibility of engraftment nowadays and also taking into consideration absolute unsolvment of engrafted brain problem ethics aspects. In one case the object of transplantation was met brain of a grown up donor but nerve tissue and/or embrio nerve cells suspension or human fetus. Let's take another case of different approach. It was engrafting of neuroepithetium stem cells of own patient debrided from osmetic linning of nose mucons membrane. The choice of embryonal nerve tissue is due to its capability to establish synaptic contacts with recipient's neurons meanrohile the opportunity of transplantation of grown up donor nerve tissue is strictly restricted. When due surgery technique is observed the implantation tissue engraftment comes to 80-95 percent. Nevertheless cells grafting surgery technique used nowadays only temporary improves brain functions and enable to provide full scale restoration of disrupted functions. The most probable reasons for unsufficient effectivity of employed methods of treatment are the following:

1. Donor cells transfer is effected to the locations of own dead cells (necrosis or sells atrophy or apoptasis (programmed cell death))
2. Unproper circulation of the blood in microcirculation basin of pathologic location cannot provide adequate metabolic support to transplant tissue and throw away gleam of hope of self revasculization.
3. Sometimes neurologia brain tissue has already substituted in different forms intramedullary cell defects causing cicatrix or parthy lysised. On top of all, lysis location is filled with liquor thereby disrupted cell sustaining structure.
4. Metabolism of remaining cells in pathologic locations is ruined resulting athrophy or demyelization of nervous fibre and disrupting of central nervous system.
5. Blocated vegetovascular support of vessels and somatic structure of pathologic nidus.

We have suggested one of the ways of solving these problems: bioengineering brain tissue plasty. By the way, we have invented some brand new treatment. For sake of realization of our concept we only tried to use legally approved in USA and Europe modern hightechnology surgery methods of treatment according to intratissue cerebral reconstruction ideology suggested by us. It enables by means of algorithmic employment of small scale invasion grafting information and telesurgery methods achieve rebuilding of tissue morphology structure of brain pathology locations, remodeling its vascular and vegetable supply and after all by application of selective electromagnetic radiation of High-frequency band or neurostimulation, try to get dynamic integration of its components. Hospital recovery of best cerebral functions is provided by specially directed rehabilitation.

Take into account that tissue engineering in the one hand is not pure technological way out for the problem of cerebral and bone marrow transplantation but intracorpus programmed substitution of some pathologically injured nerve tissue structures, methabolic defense for undamaged elements plus its reintegration in some new morphologic-functional structure. On the other hand,m incapability of solving in general ethic side of brain transplantation makes it enjoying full rights alternative for whole donor brain grafting to illing human being suffering from uncurable organic disease of central nervous system.

Technological aspect of surgery plasty is elaboration of special method of tissue engineering open neurosurgery operation or remotable intervention impact technique dealing with brain pathological location by means of multiple-channel modeling operators (intra-urterial, intravenous, subdurals, subarachnoid and so on and so forth) enabling to provide targetable vectors impact to tissue structure elements of pathological location, create necessary conditions for its transformation alter vasomatoric reactions and vasomotorical anomally deviations in tissue using some defined algorithms of applying physiologic surgery methods, endovascular X-ray surgery, cell neurografting as well as electromagnetic information influence. As a matter of fact all means stated here above enable to provide remodeling, reconstruction, self reinnervation of curable location and sustain integration of rebuilding morphological structure elements. This target was located and hit by deviding action into some succesive stages when intra-tissue brain plasty fulfilment.

The stages of brain tissue engineering.

We are conducting analysis of pathologic nidus diagnostic parametors, elaborating strategy of bioengineering plasty, estimating volume of surgery manipulations, need of biologic substances and tissue elements, finding out indications and contraindications for its execution, fixing time for every stage subject to data of patient examination, carrying out modeling of surgery intrusion, working out plans of each plasty stage.

Creating and preparation of operators of plasty location modeling and construction elements:

1. Carrying out sampling, culturing and standartization of cerebral vesicle tissue of human or animal embrio (fetus), conducting tissue splitting to separate cells or accomplishing culturing of neuroepithelial stem cells of patient nose mucous membrane linning.
2. Dissociation of cells en vitro (in culture substance) in accordance with its type and later on separate culturing as well as estimation of cells vatability.
3. Preparation of specialized cell transplantant (neuro, neuro-neruoglial, glial of vary prescription and quantaty), culturing neuro or neuroepithelial stem cells.
4. Conducting so to say "focusing" of plasty location, preparation for surgery access and mounting of necessary manipulators:
   
   • In X-ray surgery premises we investigate the condition of vascular red in bram pathologic location planned for bioengineering plasty by vasographic method ending in mounting of permanent intra-arterial and intravenous multiple-channel fixed in maximum short range to brain plasty location.
   • Mounting subarachroid multiple-channel modeling operators or preparation of all necessary tools and instruments as well as medicines for intrusion inward introduction of biomaterial and bioactive substances in brain substance that is puncture of subarachoid locations, cisterns and ventricles of brain, stereotaxis or transvascular introduction and so on
   • The patient is provided with standard preliminary procedures before operation to start, preanesthetic medication and illing man is moved to resuscitation department, where subclavium vein catheterization is made to him and then man see himself connected to cardio- and encephalopathy monitoring units.
   • Simpathetic deinervation of plasty location is made and also provided protection against local angiocramp by means novocain injection or artificial hybernation.

Artificial hybernation is effected by prolonging manipulations which slow down brain methabolic process:

1. Central control unesthesia.
2. Surface iranial rubber ice-water bottle linning.
3. Applying hypothermic devices..

Vessel remodeling.

1. To reveal reserve capabilities of vessels we use prolonged intra-arterial perfusion in brain plasty location of antiaggrents, vasonctive substances and medicine, improving local mycrocirculation and blood heology as well as creating specific bioconditions (acidity, gases mixture, partial blood pressure) in plasty location.
2. To bring to active life reserve vessels and colaterals we make long antiaggrents and medicine transfusion, improving mycrocirculation and mechanically creating perfusion pressure increase.
3. We alter vessel bloodstream geometry by increasing location bloodstream volume which enables create or restore regional collateral microcirculation network of general brain plasty.
4. Forced sanation of plasty location is provided by means of intra-arterial transfusion in plasty location of low-molecular compounds adsorlents and active extratrunk remove of metabolic destructions decay products by venous and subarachnoid manipulater using methods of extrabody desintoxication (aortal venous hemodialysis liqour shoresis, lymphophoresis, lymphosorption and so on) together with artificially forced kidney blood stream (artificial diuresis).

Cell transplantation.

At this stage of operation the following actions are conducted:

1. Methabolic sustain and defense of remaining tissue structures of pathologic brain region by means of prolonged intra-arterial infusion into cerebral main arteries of solutions of noctropics nucleic acids and neurospecific proteins that results in activation of genetic system of pathology location cells, enchancing sanation genesis of brain reserves.
2. Substitution of damaged cell structures is made by open neurosurgery operation-brain grafting of some embrio cells quantuty of nerve tissue (neuroblasts, neuroglioblasts and its combinations) or applying methods of subarachnoid nerve cells transfusion, microendoscopy grafting endovascular cell transplantation, stereotoxic grafting or during open neurosurgery operations.
3. Bioassembly is conducted by transplantation of human beings neurostem cells. For this purpose, transfusion of neuro-stem cells suspention through intro-arterial manipulator is made, which under strict X-ray surgery appliances control is moved the maximum short range to puthology location and depends on type of brain cells injury (neuroglial enlargement or domination of atrophic process) or it may be stereotactic neurografting that is neurotransplantation of stem cells into the plasty location.

Vegetative reinnervation.

It may be carrying into effect by impact on regional sympathetic nodes. In some cases it is fully enough to mount perfusion cutheter on sympathetic node projection and provide its activation alternating injection of sympathetic node blokers (novocain, lydecain and others) or some inhibitors which irritate the nodes, for example. But in some heaviest curable cases of brain parenchyma damage using X-ray surgery appliances or microendoscopy tools, punction of sympathetic node (star sympathetic nodes, paravertebral sympathetic nodes and other regional vegetative nodes) is conducted and then comes the grafting into it of embrio Schwann nerve cells.

Somatic and vegetovascular integration of tissue constituents.

The best way to achieve proper results is physical regional impact on plasty location aiming at synchronization of all tissue donor material elements as well as brain functions of recipient. Solving this problem we applied intramuscular electrostimulation of limbs muscles and nerves, peridural implantation of electrodes and programmed neurostimulators. Light conductor of helium-neon lase may also be of help in part. Nevertheless, we began to prefer lately solving this problem the method of transcranial magnetostimulation as noninvasive medical technology enables to achieve the same results as invasive treatment methods do.

Rehabilitation and creation of new brain functions.

This stage is characterized by mass regional symptomatic intra-arterial perfusion of antihypoxanthines, ozone, blood speciment plazma and so on; control angiography to exclude local trombosis; removal of intra-arterial and subarachnoidal modeling operators; active rehabilitation and training of former brain functions; recovery of dissupted cerebral functions which due to previous treatment or its active stimulation employing traditional methods together with special medical physical exercises with biofeddback bond methods. Algorithm of this method application is net strict and order of every stage fulfillment is individual in each particular case. We may add that its main features are persistent, cascade, possibility to miss plasty stages or do it simultaneously but in condition of critical cure therapy and resuscitation departments only.

Plasty may be finished on every stage subject to achievment of fine clinical results of disrupted brain functions restoration satisfactory both to patient and doctor.

The theoretical ground of suggested biomedical technology is contemporary fundamental investigations on physiological surgery (R. Yerish, 1961) wave genetic code of eucariots (p.p. garyev, 1997, A.A. Beresin, 1998), echievments of endovascular intervention X-ray surgery dealing with regional perfusion of pharmaceutical substances (E.A. Selyvanov, M.D. Hanevitch, 1997, 1998; V.S. Savelyev, 1986, V.F. Zubritsky, A.S. Breyhovetsky, 1998) and cell transplantology (g.T. Sukhyh, E. Molnar, 1995; B.V. Gaydar with co-authors, 1996; A.S. Breyhovetsky, 1998) resulting in proof that tissue somatic cells are of highest plasty (B.L. Cotlyar, 1986) and capable of altering morphologic structure of its cells DNA nucleus under influence of acoustic electromagnetic radiation as well as under extremly high-frequency band radiations (Devyatkov, golant, Beletsky, 1996). On top of all somatic tissue cells are capable to get cell methabolic cooperation, activation and transformation of genetic cell reserves for sake of organ functions recovery. Bioengineering method is also based on ten years long fundamental experimental and practical investigations conducted by immunochemical lab at Sebsky Mdical Center. It was working on the problem of human neurospecific proteins influence on separaqte structure elements of nerve, cerebrovascular tissue and hematoencelophalic barrier (V.P. Chekhonin with co-authors, 1996, 1997, 1998).

It is theoretically proofed and experimentally confirmed the possibility of substitution tissue organ pathological cells elements for donor embrio cells subject to straight transvascular citotransfusion (V.Repin, 1995) as well as organ functions condition, modulation when transplantation of cells into its submembrane room (A.S. Bryohovetsky, 1997, 1998, 2003). To cap it all each of methods in question unable to achieve persistant clinical effect. Programmed employment of all these methods within framework of hihly specialized technological algorithm of bioengineering tissue plasty can do it.

Here is clinical example of brain plasty tissue engineering of vascular pathology patient.

Example.

Patient Mr. Z., born in 1949, was examined and cured in neurological hospital from 03.09.1997 till 13.11.1997 because of hemorragic stroke in area of left middle cerebral artery, middle hemorrage in backside area of internal capsule and adjucent parts of left hemisphere thalamus together with rightside hemiplegia and sensory motor aphasia. Discirculational encephalopathy of second stage due to cerebral atherosclerosis, decompensation. Hypertension of third stage. Atherosclerosis of aorta and coronary atherosclerosis, atherosclerotic cardiosclerosis. H-O Urinary bladder stone disease in remission stage. Small concrement of sinister kidney. Secondary pyelonephritic at exarcerbation stage. Cronic rena linsufficiency, second stage. Insulin independent diabetis of slight clinical course. Chronic hyperacid gastritis in remission stage. The patient was placed in charge of intensive case department by emergency indications because of acute brain blood supply disturbance of soporouse stage. When he was examined, intracerebral medical hemotoma in sitister hemisphere was revealed. After reduction of brain syndroms of the patient. Dextral hemiplegia and rough sensory and motor aphasic disorders were detected. In spite of intensive therapy treatment during mounth there was no effect of any kind. The treatment was following; Diet # 10, polarizing compound, clofeline, capoten, cavinton, nootropil, nimotop, stageron, reopoliglukine, fenobarbital, sedative therapy, hyperbaric oxigenation, physical therapy, injection therapy. When injection therapy the patient health condition stabilized. After all treatment course patients condition remained stable and even still neurological deficit remained as it was before. We fixed unstable arterial blood pressure; till 180-200/110-100 Taking into account unefficiency of undertaken conservative therapy during long period, spinal fluid sonation, persisted noncorrectional hemiplegia and rough afatic disorder, hospital doctors consultation diagnostic decision of 13.10.1997 was to offer the patient and relatives tissue engineering brain plasty.

The operation began on day of 16.11.1997 with patients relatives informed consent and within ten days all stages of operation were carried out. Postoperation period was complicated by acute cronic pyelonephritis which was replied by mass antibiotic therapy (gentamicin, metrogyl). The bioengineering plasty was resulted in fully reduction of afatic disorder, movements in right limbs were recovered; the power of them reached four points, almost in full scale restored movement in hand fingers, weakness of leg diminished, the patient started to serve himself and became available for rehabilitation. The patient became to stand and walk with his wife's assistant. In satisfactory condition he was moved from the intensive care department ot rehabilitation Center for rehabilitation-recovery treatment.

Analysis of blood and urine due to treatment in question were of positive dynamics. Here is the analysis of blood of 28.10.1997; hemoglobin-112 units, erythrocytes (red blood cells) - 3,5 in mm, leukocytes (white blood cells) - 9,5 in mm, hematocrit (packed cell volume (31 single units of 66 mm for an hour. Basophiles (basifilic leucocytes) - 1, eosinophiles - 1, rodnuclens cells - 1, segmentnucleus cells - 77, lymphocytes 18, monocytes - 2. And now lets look at the analysis of urine of 7.10.1997; protein - 0,913 gr.per lt., single flat epithely in sight, leukocytes up to 5 in sight, erythrocytes - 0-5 in sight. Coagulogram of 19.10.1997 moved to normal level. Biochemical blood indicators of 23.10.1997 without pathology: Electoencephalogram - diffusion changes of brain bioelectric potencial with disfunction of stem - mesocerebral structures. Electrocariogram; rythm is in order, moderate changes of rheoencephalogram - slow increase of vascular tone, venus drainage is strain. Brain computer tomography of 03.09.1997 in projection of pesterior round of inner capsule of sinister hemysphere and adjusting parts of hypotalamus detected medial hematoma without blood burst into ventricles. Photoroentgenography of chest organs; thoracic aorta is indurated, heart enlarged in transverse, all the rest is in standard.

It is recommended to patient to observe schedule of work and rest to avoid neural and athletic overexertion, regular cdontrol of cheoencephalogram. The control ultrasound imaging is to be carried in six months. Repeated courses of noatropics; pyrocetam (noatropil) one capsule three times a day. Trental must be used three times a day of two pills. After two months has to be taken a course of B-group vitamins ( that is thiamine, riboflavin, choline, pyridoxine, inosital, cyanocolalamin and orotic acid). In three months should be repeated electrocardiogram under control and observation of dispancery neurologist.

Catamnesis: After eight months the patient arrived for control examination. Capability to speak is recovered fully, there is some little spasmodic gait disorder of wernicke-mann type. He is able to serve himself and doesn't need help any more. Fourteen months later the patient started to work again.