BIOLOGY AND BIOTECHNOLOGY

. In the modern era, tissue engineering is actively developing based on the utilization and enhancement of endogenous repair resources. Due to neurodegenerative processes that occur in traumatic brain injuries, vascular diseases of the central nervous system, and natural aging, the percentage of disability is steadily increasing, particularly in developed countries. The most pressing task today is to find optimal measures for prevention and therapy. Changes in neurodynamics, ischemia, inflammation, accumulation of toxic products, activation of catabolism, and a decrease in the activity of anabolic processes have both local and systemic implications. Neurons of nervous tissue are particularly sensitive. It is known that nervous tissue is capable of regeneration, but spontaneous regenerative processes do not fully restore the structure and function of the central nervous system. Contemporary research indicates that chemokines play a crucial role in regulating the viability, self-renewal, and attraction of stem cells. The dynamic interaction between neural stem cells is regulated by the chemokine CXCL12 (C-X-C motif chemokine 12) and its receptor CXCR4 (C-X-C motif chemokine receptor 4). Elevated levels of CXCL12 create conditions for the active recruitment of neural progenitor cells to sites of injury. Platelets serve as an endogenous reservoir for more than 1500 biofactors that influence various metabolic processes in the body's cells. Many of them exhibit neurotrophic activity.


Abstract.
In the modern era, tissue engineering is actively developing based on the utilization and enhancement of endogenous repair resources.Due to neurodegenerative processes that occur in traumatic brain injuries, vascular diseases of the central nervous system, and natural aging, the percentage of disability is steadily increasing, particularly in developed countries.The most pressing task today is to find optimal measures for prevention and therapy.Changes in neurodynamics, ischemia, inflammation, accumulation of toxic products, activation of catabolism, and a decrease in the activity of anabolic processes have both local and systemic implications.Neurons of nervous tissue are particularly sensitive.It is known that nervous tissue is capable of regeneration, but spontaneous regenerative processes do not fully restore the structure and function of the central nervous system.Contemporary research indicates that chemokines play a crucial role in regulating the viability, self-renewal, and attraction of stem cells.The dynamic interaction between neural stem cells is regulated by the chemokine CXCL12 (C-X-C motif chemokine 12) and its receptor CXCR4 (C-X-C motif chemokine receptor 4).Elevated levels of CXCL12 create conditions for the active recruitment of neural progenitor cells to sites of injury.Platelets serve as an endogenous reservoir for more than 1500 biofactors that influence various metabolic processes in the body's cells.Many of them exhibit neurotrophic activity.

Principles of Cell Chemotaxis
Cell chemotaxis, the directed migration of cells toward a chemical signal, plays a crucial role in both pathological and physiological processes, including embryogenesis, wound healing, and cancer metastasis [1].During cell migration, cells are exposed to multiple signals in complex chemical and physical environments.The nature of these signals changes in space and time [2].Today, many questions remain regarding how cells sense and decipher numerous chemical signals.
Recently, there has been a hypothesis that the chemotactic productivity of cells under the influence of complex signals is regulated by the cell's ability to process signals through a cellular sensory mechanism.
Cell migration is a complex process involving several biological functions, including cell adhesion to the extracellular matrix (ECM), detachment from the substrate, and cytoskeletal remodeling.During migration, material within the cell is displaced, resulting in an expansion of cell content in the direction of increased chemotactic gradient, while the opposite part narrows [3].To perform chemotaxis, cells must have increased motility and the ability to establish polarity.Direction sensing is a property of cells to compare the occupancy of chemotactic receptor sites on their surface and determine where the concentration is highest [4].In the presence of a chemoattractant gradient, cells sense it, align their polarity with it, and ultimately migrate along the gradient.Chemotaxis is possible for individual cells and coordinated groups of cells.Collective migration allows a cluster of cells to migrate faster and follow a weak gradient of chemoattractant, to which individual cells may be insensitive.The clustering of migrating cells is determined by leader cells and follower cells, which differ in gene expression [5].
To implement the process of chemotaxis, a necessary condition is the presence of a chemoattractant or chemokine in the environment, and there must also be receptors on the cell's surface capable of interacting with this chemokine.Chemokines belong to a large family of small (7 kDa to 11 kDa) proteins.Currently, there are known 53 human chemokines and 23 chemokine receptors.Chemokines can be No 171 divided into four subgroups: CC, CXC, CX3C, and XC, based on the positioning of cysteine residues nearest to the N-terminus.For example, CC-chemokines have two consecutive cysteines near the N-terminus, while the first two cysteines in CXC-chemokines are separated by a single amino acid [6].
Chemokines exert their biological activity through surface chemokine receptors (CRs), which can be divided into two families: typical chemokine receptors, belonging to classical G protein-coupled receptors, and atypical chemokine receptors.Ligands and their receptors are known for their lack of selectivity, as different chemokines can bind to more than one receptor, and a receptor can bind to multiple chemokines [6].
Over the last decade, scientific research has emerged regarding the expression of chemokines and their receptors in the central nervous system (CNS).It is now understood that all types of brain cells synthesize various chemokines and can respond to chemokines through their receptors.In the CNS, both during development and in adulthood, the chemokine CXCL12 and the receptors CXCR4 and ACKR3 are constitutively expressed.These molecules are present in almost all types of CNS cells: neuronal, glial, endothelial.The interaction of chemokines with their receptors is essential for maintaining brain homeostasis [7] No 171 syndrome, or Waardenburg syndrome [9].Chemical substances that interfere with the migration of NCCs often result in craniofacial developmental defects in the fetus.This is welldocumented for ethanol or pesticides like triadimefon [10,11].Disruptions in retinoic acid levels are a significant cause of impaired migration and differentiation of NCCs.Under such conditions, craniofacial defects are observed in both animals and humans [12].
Various factors have been proposed as chemoattractants for NCCs, depending on the species and location.For example, for cranial NCCs in chicken embryos [13], vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF) have been suggested as chemoattractants.For cranial NCCs in zebrafish [14], fibroblast growth factor (FGF) has been identified as a chemoattractant.In the case of cranial NCCs in mice [15], different chemoattractants come into play.It's important to note that the mentioned chemoattractants may not be applicable to human NCCs.

Platelets -Biomaterial for Obtaining Preparations to Enhance the Chemotaxis of Human Neural Stem Cells (NSCs)
Considering the significant role of chemotaxis in the functioning and repair processes of the central nervous system (CNS), modern research is focused on finding effective chemoattractants to enhance chemotaxis for human NSCs.It has been found that lysate of human platelets (LHP) contains a potent chemoattractant for NSCs.
Platelets are formed from megakaryocytes found in the bone marrow and are continually released into the blood, maintaining normal physiological levels of 150 -400 × 106/ml.Platelets are characterized by the absence of a nucleus, relatively small size (2 µm -4 µm) in a resting state, the presence of many surface membrane markers, especially GPIb-IX-V, GPVI, and GPIIb/IIIa complexes, and an open canalicular system that allows bidirectional exchange of molecules, including proteins It is believed that in vivo platelet activation leads to the release of factors that stimulate growth, enhances neurogenesis under physiological conditions, probably partly through PF4 (CXCL4) [18].Another example is the chemokine CCL5, which is abundant in platelets and has recently been shown to contribute to aerobic glucose metabolism and have a positive impact on synaptic plasticity in the hippocampus [19].
In experiments with stem cells, a strong bioactivity of platelet lysate is observed.Initial control showed that in the absence of LHP, suspended NSCs migrate in all directions.When cells were exposed to the same concentration of chemoattractant from different sides, they migrated equally toward both stimuli.In cases of unilateral localization of LHP, NSCs migrated towards the chemoattractant.The direction of chemotaxis occurs parallel to the gradient.

Modern Experience in the Use and Types of Platelet Preparations for Neuroregeneration
Scientific research has provided a basis for the clinical use of platelets and bioproducts obtained from platelets.Besides the wide range of biologically active molecules present in alpha granules, the non-toxicity of platelets and the bioproducts derived from them are crucial factors.This is evident from the use of platelet preparations in human cell culture, particularly as a substitute for fetal bovine serum [20].Platelet-derived preparations are also used for the culture of primary mesenchymal stem cells (MSCs) isolated from various tissues to enhance in vitro expansion.The addition of platelet lysate (PL) is especially effective in accelerating cell expansion.Importantly, this does not alter the cell phenotype, and the expanded cells retain their ability to differentiate according to criteria established by the International Society for Cellular and Gene Therapy [21].There are no signs of teratogenicity either.Recent scientific studies have confirmed the advantages of PL for the expansion of differentiated human cells, demonstrating the non-toxicity and functional activity of PL preparations for a wide range of cell types [22].
Today, international protocols exist for the preparation of platelet products for introduction into the cerebrospinal system during neurodegenerative processes.Platelets are obtained from blood, which is held at (22 ± 2) °C for several hours before the initial centrifugation.The procedure involves centrifugation at approximately 1000 g for 10 minutes at 22 °C, mainly used in the USA, to obtain platelet-enriched plasma (PEP).PEP is then obtained by centrifugation at 3000 g for 5 minutes at 22 °C and resuspension of the sediment in a small volume of plasma (50 ml -70 ml).Platelet concentrates (PCs) are obtained from PEP [23].
In Europe, a centrifugation procedure involving blood at 3000 × g for 5 minutes at 22 °C is mainly used, resulting in a product with a high platelet content -PCs located between erythrocytes (lower part) and platelet-poor plasma (upper part) [23].
Preclinical assessments using cellular and animal models of neurological disorders, while preliminary, suggest that both types of procedures can be used to prepare PCs.PCs are used as standalone therapeutic preparations and also to obtain other therapeutic derivatives, such as platelet lysates, extracellular vesicles.It is recognized that for clinical use, platelet preparations must meet standards common to bioproducts, such as bacterial sterility, low endotoxin content, physicochemical characteristics, protein content, and levels of proteolytic and thrombogenic activity [24].

Human Platelet Lysates (HPL)
Experimental evidence has demonstrated the effectiveness of HPL in stimulating chemotaxis of neural stem cells (NSCs) and neuroregeneration.HPL contains growth factors and neurotrophins, including BDNF, TGF-β, PDGF-AA and -BB, IGF, EGF, VEGF, bFGF, MSP, M-CSF, angiopoietin-1, angiogenin, and IGF-binding protein 3 (IGF-BP3) [25].HPL also contains the crucial platelet factor 4 (PF4 or CXCL4) for chemotaxis of neural stem cells, as well as a wide range of other chemokines: CC-chemokine ligand 3 (CCL3), CCL4 (MIP-1β), CCL5, CXCL1, MCP1/CCL2, CXCL2 [26)].The physiological secretion of these chemokines is observed during platelet activation, explaining their presence in HPL preparations obtained by platelet lysis.It has been shown that platelet granules contain both pro-inflammatory and anti-inflammatory molecules.Among the molecules found in HPL preparations are interleukins (IL)-1α, IL-1 receptor antagonist (IL-1Ra), IL-4, cell surface receptors (cluster of differentiation 14 (CD14) and CD40L), tissue inhibitor of metalloproteinase (TIMP)-1.Interleukins are typically associated with inflammation modulation but can also act as trophic factors, stimulating cell migration, differentiation, and playing an anti-inflammatory role [27].Additionally, HPL is enriched with antioxidants such as glutathione peroxidase, glutathione S-transferase, catalase, ceruloplasmin, superoxide dismutase, and thioredoxin [28].Glutathione and thioredoxin are considered thiol antioxidants.Glutathione actively participates in antioxidant and detoxification reactions in conjunction with glutathione-dependent enzymes, directly scavenges OH radicals, and plays a role in homocysteine reduction [29].Thioredoxin has redox activity, and its levels increase in diseases associated with oxidative/nitrosative stress and inflammation [29].Superoxide dismutase and catalase are enzymatic antioxidants that protect against oxidative stress.Biochemical studies have characterized the content of biochemical components in HPL: albumin, total cholesterol, triglycerides, vitamin B12, calcium, iron, and sodium [25].Importantly, HPL contains the neurotransmitter serotonin, which plays various roles, including vascular tone regulation, hemostasis, immune response, tissue remodeling, and energy metabolism.Serotonin is stored in platelet granules [30].
The trophic factors present in HPL are key signaling molecules that have specific functions in fundamental CNS biological processes.For example, using an in vitro neuronal cell culture model, the ability of BDNF and PDGF to protect neuronal cells from apoptotic death has been demonstrated [31].Other factors, including EGF, mesencephalic astrocytederived neurotrophic factor (MANF), and IGF-1, are crucial for enhancing neuronal survival, providing neuroprotection, proliferation, and differentiation of neuronal stem cells [32,33].In vitro and in vivo experiments have demonstrated the functions of growth factors and neurotrophins in the formation of neurospheres and the differentiation of neurons.Neurotrophic factors present in platelet lysates, such as VEGF, EGF, FGF-2, PDGF, BDNF, PF-4, TGF-β, IGF-1, BMP-2, -4, and -6, participate in neurogenesis and neuroepithelial cell proliferation, differentiation, migration, and survival [34].

Extracellular Vesicles (EVs) -Derived from Platelets
In recent times, attention has been given to the therapeutic properties of vesicles generated from platelets [35].EVs constitute a heterogeneous population of nanosized vesicles of cellular origin, enclosed by a lipid bilayer [36].EVs play a crucial role in intercellular transport of biomolecules.The functions of EVs depend on the receptors present on their membrane and the content of biomolecules they carry.EVs contain trophic factors/proteins, lipids, messenger (m)RNA, microRNA, which can be delivered to distantly located cells.EVs are considered potential standalone therapeutic products and hold promise as drug delivery vehicles in various areas of regenerative medicine and disease therapy [37], such as Parkinson's disease, gliomas [38], Batten's disease [39], and Alzheimer's disease [40].The scientific rationale for using EVs in treating CNS disorders includes lower risk of immunogenicity and tumorigenicity compared to mesenchymal stem cells (MSCs) [41]; biocompatibility compared to synthetic nanocarriers [37]; and the ability to cross the blood-brain barrier (BBB) [42].

Extracellular Vesicles in Platelet Lysates (PLs)
It is believed that the pool of EVs accounts for approximately 50% of the available pool of platelets and TBI is a pathology that requires a multifaceted therapeutic strategy to provide rapid anti-inflammatory effects and neuroregeneration by enhancing angiogenesis and neurogenesis, remodeling the neurovascular system, and reconstructing neural connections [46].Experimental studies have shown that in focal TBI modeling, there is an accumulation of activated platelets surrounding the site of injury, correlating with a significant reduction in neuronal cell death [47].The protective capacity of Platelet Lysates (PLs) has also been demonstrated using other in vitro and in vivo TBI models.Stimulation of SH-SY5Y neuroblastoma cells with PLs in vitro promotes the faster maturation and growth of neurites in the injury zone (a model of traumatic injury) [48].Intranasal administration of PLs after modeling penetrating TBI (cortex injury) for 6 days improves behavioral, motor, and memory functions.At the molecular level, the use of PLs reduces cortical neuroinflammation, oxidative stress, and synaptic loss.These data are consistent with the aforementioned physiological role of platelets and indicate the clinical significance of platelet-derived products in TBI.These studies also suggest that in the case of severe trauma, local administration of PLs followed by continuous intranasal administration may be beneficial.
Proceedings of the 6th International Scientific and Practical Conference «Scientific Trends and Trends in the Context of Globalization» (September 19-20, 2023).Umeå, Kingdom of Sweden No 171

Chemotaxis in the Development and Functioning of the CNS
Additionally, platelets contain from three to eight dense granules approximately 150 nm in size, which serve as a reservoir of neurotransmitters, including serotonin, dopamine, histamine, adrenaline, gamma-aminobutyric acid, and glutamate, as well as polyphosphate and ATP [17], many of which have neurotrophic activity.Alpha granules are also rich in potent intercellular signaling molecules such as CCL5 and chemokine ligand CXCL4 (PF4).
Proceedings of the 6th International Scientific and Practical Conference «Scientific Trends and Trends in the Context of Globalization» (September 19-20, 2023).

of Bioproducts for Traumatic Brain Injury (TBI) Therapy
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