ULOGA FAKTORA RASTA U ZARASTANJU EKSTRAKCIONE RANE THE ROLE OF GROWTH FACTORS IN EXTRACTION WOUND HEALING

Zarastanje rane je složeni proces koji uključuje hemostazu, inflamaciju, proliferaciju i remodelaciju tkiva. Faktori rasta su prirodni biološki posrednici koji regulišu najznačajnije ćelijske procese uključene u regeneraciju tkiva, kao što su DNA sinteza, angiogeneza, metabolička aktivnost, migracija, hemotaksa, proliferacija, diferencijacija i sinteza matriksa. Najznačajniji faktori rasta koji učestvuju u zarastanju ekstrakcione rane i regeneraciji koštanog tkiva su: trombocitni faktor rasta-PDGF, transformacioni faktor rasta-TGF β ,faktor rasta sličan insulinuIGF, koštani morfogenetski proteini-BMP-2, BMP – 7 , vaskularni endotelijalni faktor rasta, VEGF, ,fibroblastni faktor rasta-FGF. Faktori rasta pojavljuju se u različitim koncentracijama u različito vreme, pa se na osnovu njihovog prisustva može proceniti starost rane. Osim zarastanja rane faktori rasta mogu se primeniti za bolju oseointegraciju implanata,augmentaciju alveolarnog grebena, alveolita itd. Studije fizioloških procesa u kojima faktori rasta imaju regulatornu ulogu ukazuju da ovi molekuli retko kada svoje aktivnosti vrše u biološkoj izolaciji. Proučavanje interakcije između faktora rasta u alveolarnoj kosti može pružiti objašnjenje o sposobnosti tkiva da zaraste i pod nepovoljnim uslovima, kao što suinfekcija i zračenje


Introduction
Wound healing is a complex process that includes hemostasis, inflammation, proliferation and tissue remodeling.The process involves different types of cells, complex signaling events and a number of growth factors.
Growth factors are natural biological mediators that regulate crucial cellular processes involved in the tissue repair, such as DNA synthesis, angiogenesis, metabolic activity, migration, chemotaxis, proliferation, differentiation and matrix synthesis.Growth factors are cell-specific, with each factor regulating particular type of cells 1 .
Polypepitide growth factors work by binding to tyrosine kinase 2 -transmembrane receptor on the surface of the target cell membrane -via their active parts called "dimers" 2 .The activated T-K transmembrane receptor triggers the cell activity by raising the intracellular concentration of circular adenosine monophosphate and leads to the release of intracytoplasmic transducer signaling proteins.Transducer signaling proteins, once disengaged from the tyrosine kinase transmembrane receptors, proceed to the nucleus where they unlock specific gene sequences for controlling the cell function and inducing the expression of normal gene activity.
The various growth factors released during hemostasis have different roles, including stimulation and activation of monocytes, neutrophils and macrophages causing inflammation at the site of injury.Endothelial vasculogenesis and organogenesis takes place, and fibroblasts are recruited to the site of injury to form an extracellular matrix for the further healing process and tissue remodeling 5 .Most types of cells necessary for wound healing come from various depots, such as bone marrow, blood vessels, the epithelium basal layer, hair follicle and fatty tissue, that are rich in stem or progenitor cells which can differentiate into specialized cells influencing the selection of signaling molecules or growth factors during the healing process.
During the proliferative phase, fibroblasts infiltrate the wound, produce matrix metalloproteinase MMP 6 and interact with extracellular proteins thus promoting granulation.Fibroblasts further undergo the phenotypic change into miofibroblasts that even out the edge the wound, generating contractile force and facilitating wound closing.
Granulation tissue collagen fibers act as a provisional matrix that binds undifferentiated osteoprogenitor cells that, influenced by growth factors, primarily BMp ones, further differentiate into the cartilage and bone.Hematopoetic stem cells and endothelial cells precursors have their common origin in hemangioblast stem cells and are present in the bone marrow.
The finial phase of the wound healing process-remodeling, involves the regression of blood vessels and the replacement of the granulation tissue with extracellular matrix as well as its reconstruction.
It seems that PGDF is the first growth factor present in the wound and is a key mediator in wound healing.PDGF is a glycoprotein that is primarily released from platelets (their alpha granules) after injury and attracts neutrophils and macrophages.Neutrophils clean the wound from bacteria and debris and macrophages as mediators of wound healing continue with the release of growth factors attracting fibroblasts, and induce the next wound healing phase 8 .It is present in the highest concentration at the very beginning of the tissue healing, which peaked the third day [9][10][11] .The most important specific activities of PDGF include STEM cells mitogenesis, angigenesis and osteogenesisso as well as the modulation of other grow factors' activities 12 .During wound maturation, the platelet count reduces and the level of PDGF falls.Nevertheless, the PDGF actively participates in all stages of healing.Later, in the proliferative stage, it stimulates the differentiation of fibroblasts into myofibroblasts, leading to a contraction of collagen matrix and extraction wound 13 .
As far as PDGF concentration in extraction wound is concerned, Lalani et al. 14 who tested a rabbit model extraction wound, found that in the period from 2 to 4 days, there is a smaller amount of this growth factor in the alveolar bone, and from the 4 th day until the end of the 1 st week the level does not change significantly.Between the 1 st and 2 nd week the levels of PDGF increase, which in turn decreases between the 2 nd and 4 th week.Between the 4 th and 8 th week, the level of PDGF increases by six times.A small increase in the PDGF was observed between the 8 th and 12 th week, which then declined between the 12 th to 16 th week 14 .
The remodelation role reflects in stimulating production of collagen and fibroblasts.As wound debridement progress increases, PDGF amount also increases until bone formation start.PDGF is localized around the spicules of bone and around the osteocytes during the second week.From the 2 nd to 4 th week, a slow increase in the osseus mass coincides with a reduction in PDGF.However, as of the 4 th to 8 th week bone remodeling progresses, and the amount of PDGF increases as a result of the activities of osteoblasts and osteoclasts.The process of remodeling is slowed down from the 12 th to the 16 th week and the PDGF-level gradually declines 14 .This growth factor has been isolated, and nowadays it is recommended in the treatment of bone defects as highly purified recombinant human rh PDGF-BB 15 .

Transformation growth factor TGF-β
Transforming growth factors are peptides that have an influence on the growth and phenotype of cells with the ability to initiate the transformation of non-neoplastic fibroblasts phenotype into malignant-like cells 16 .TGF-β is the representative member of this group of growth factors, also called the TGF-β superfamily.It is composed of several growth factors: TGF-β (β 1, β 2 and β 3), activin (A, B and AB), bone morphogenetic proteins (BMP-s) and inhibin.In mammals and birds three TGF-β isoforms were isolated (TGF-β1,-β2 and-β3), while in lower vertebrates there are three additional isoforms 17 .
The transformation growth factor beta plays an important role in a variety of cellular functions during all phases of wound healing, including: ECM production, protease expression and migration, chemotaxis, differentiation and proliferation of different cell types 18 .All three isoforms are synthesized and found in platelets and macrophages, and also produced by osteoclasts incorporated in the mineralized bone matrix.TGF-β1 plays a role in the phase of inflammation, angiogenesis, granulation tissue formation, and remodeling of the ECM and has an essential role in the re-epithelization.TGF-β1 is a haemostatic and mitogenic factor, which attracts undifferentiated cells at the site of injury and stimulates their proliferation and differentiation in fibroblasts, chondroblasts and osteoblasts.Bone cells synthesize this growth factor and store it in an inert form in the ECM creating reserves of TGF β in the body 19 .
TGFβ is expressed mainly in mature osteoblasts on the surface of the bone during bone development and growth and calluses of the healing fractures.The main functions of TGF-β1 and TGF-β2 are chemotaxis and mitogenesis of precursors of osteoblasts, osteoblast proliferation and differentiation, and the ability to delay osteoblasts on the collagen matrix in the healing wound in the bone.TGF β inhibits the formation of osteoclasts and bone resorption, allowing bone formation 20 .

Koštani morfognetski proteni
Jedinstvenu podfamiliju u okviru superfamilije TGF-a formiraju BMP-i.Do sada je otkriveno 15 koštanih morfognetskih proteina koji se dele u podfamilije po sličnosti u njihovoj aminokiselinskoj sekvenci.Njihova It also stimulates the production of collagen and fibronectin and plasminogen activating factor in bone cells.It seems that TGF-β1 and TGF-β2 usually alternately operate in most systems 19 .Their most pronounced effect is in the process of morphogenesis and epithelization.
In a study that examined the temporal and spatial presentation of growth factors during extraction wound healing in rabbits, TGF-β1 increasing was observed from the 2 nd to the 4 th day after the extraction, mostly present in the ECM, which was expected as platelets and inflammatory cells release proteins during the acute inflammatory phase; the concentrations of TGF-β1 were reduced by the end of the first week 14 .The proteins can generally be found on the outskirts of the newly formed osteoid, and minimum quantities are present in the ECM.
Between the 1 st and the 2 nd week, there was an increase in the concentration of TGF-β1.From the second week, the concentration continued to increase but in lower amounts, so that in the 4 th week the concentration was twice as high compared to the initial level.From the 12 th week, TGF-β1 and TGF-β2 declines were noticed again.It seems as the TGF-β1 is a trigger for the beginning of bone formation and that increase in its concentration means that bone mass is increased too.In all periods, the concentration of TGF-β1 was greater in the maxilla than in mandible 14 .
Active bone formation takes place in the period between the 4 th and the 12 th week, and then the osteoclasts begin to resorb the bone.TGF-β1 is released in its latent form from the bone matrix, and converts into the active form in the acid environment.TGF-β1 is mostly present in the ECM and along the periphery of the remodeling bone.As the number of osteoclasts in the Haversian channels decreases, the concentration of TGF-β1 stabilizes and grows no more 14 .

Insulin-like growth factor IGF-I and IGF-II
Insulin-like growth factors are a class of polypeptides with the amino-acid sequence that is similar to insulin.IGF I and IGF II are the most abundant growth factors present in the bone matrix.biološka aktivnost je pokazala sposobnost za ektopično formiranje enhondralne kosti 22 .Korišćenjem tehnike molekularnog kloniranja, 6 članova iz porodice BMP-a svrstano je u posebnu grupu od BMP-2 do BMP-7 (koji se još naziva i osteopontin OP-1).
Produkcija BMP-7, koji se u velikoj meri detektuje kod reparacije posle ekstrakcije zuba, najizraženija je u periodu između druge i 8. nedelje posle ekstrakcije, sa progresivnim padom u periodu od 12. do 24.nedelje 26,27 .To bi značilo da je ovaj faktor važan za ranu i intermedijsku fazu zarastanja kosti, tj.za mineralizaciju koštanog matriksa i privremenu sintezu trabekularne kosti.Nasuprot tome, količine The main source of these growth factors is the liver but they are also synthesized in other tissues, fetus and placenta, being particularly rich sources, from which they reach the extracellular fluid.Insulin-like growth factor IGF-II is nutritionally regulated.The high amount of IGF-I and IGF-II indicates the presence of sufficient amount of energy and proteins during the growth.These growth factors have significant effects on bone mineral homeostasis, with proanabolic and anticatabolic activity.They are responsible for developing and maintaining musculoskeletal system 21 .IGF-I and IGF-II stimulate bone cells in an autocrine or paracrine manner by increasing DNA synthesis, osteocalcin synthesis and alkaline phosphatase activity 13 .Depending on the dose they also stimulate directed preosteoblast migration.IGF-I is a chemotactic factor for fibroblasts, osteoblasts and osteoblast-progenitor cells, and it promotes production of bone matrix (i.e. the synthesis of collagen and non-collagen proteins).IGF-II influences the bone metabolism mith marked anabolic eftects,aftechug affecting oteoblast mitogenesis.

Bone morphogenetic proteins BMP -2,BMP-7
BMPs form a unique subfamily within the TGF superfamily.Fifteen bone morphognetic proteins that have been discovered so far are divided into subfamilies according to their similarities in terms of amino-acid sequence.Their biological activity has been shown to enable endochondral bone formation 22 .Using molecular cloning technique, six members of BMP family, from BPM-2 to BPM-7, have been classified into a special group (BPM-7 that also known as osteopontin OP-1).
BMPs are growth factors with strong osteoinductive potential, with bone tissue healing regulation as their basic role.They have the strongest effect on differentiation of mesenchymal cells into osteoblasts and hondroblasts 23,24 .Some studies 24 suggest that BMPs do not stimulate mature osteoblasts and that mature fibroblasts are unable to induce osteogenic activity after BMPtreatment.This would mean that BMPs have an osteogenic effect exclusively on immature and multi-potent cells.Many local and systemic factors affect the BMPs mechanism of action during bone formation.
Local factors that show synergism with BMP are basic fibroblast factors and prostaglandins, while BMP-2 and TGF-â1 have the opposite effect on osteoblasts differentiation 25 .
A study that examined the concentration of growth factors at the site of tooth extraction in rabbits showed that concentration of BMP-2 is low immediately after tooth extraction but increase progressively between the second and the fourth day.The BMP-2 values then increase minimally until the week four.In the period between the 4 th and the 8 th week the values are tripled, however, there were no changes in the period between the 8 th and 12 th week.In the very beginning, most of BMP-2 is present in the ECM where it stimulates the differentiation of mesenchymal cells, while towards the end of the first week its presence is more prominent in the osteoid part.An interesting fact is that as the concentration of BMP-2 increases, whereas the concentration of TGF-â1 decreases.The inverse relationship between TGF-â1 and BMP-2 is an example of the growth factors interaction in the wound healing process.BMP-2 increases between the 4 th and the 12 th week during the period of active bone formation and remodelation 14 .As the bone remodeling process weakens, TGF-â1 and BMP-2 concentrations become more stable.
The production of BMP-7 which is largely detected during the reparation process after tooth extraction is most pronounced in the period between the 2 th and the 8 th week after extraction, with a progressive decline in the period from week 12 to 24 26,27 .This would mean that this particular factor is important in early to intermedial stage of bone healing.i.e. during the mineralization of the bone matrix and provisional synthesis of trabecular bone.In contrast, the amount of BMP-7 decreases as osteocalcin accumulation increases in maturing bone matrix.

5.Vascular endothelial growth factor
Angiogenesis and vasculogenesis are the primary requirements for tissue regeneration 28 .VEGF expression is regulated by tissue hypoxia and ischemia.Increased levels of VEGF that are induced by hypoxia make VEGF-driven angiogenesis central response to low oxygen pressure and a major factor in the therapeutic angiogenesis 29,30 .
VEGF binds to receptors of endothelial cells leading to their growth, proliferation and migration.Activated endothelial cells aggregate into tubular structures (morphogenesis) and further connect with pericytes (maturation).Pericytes provide a variety of regulatory signals, including TGF-β1 and others, which leads to the endothelium standstill and blood vessel survival irrespective of further angiogenesis stimulation (stabilization).The spatial localization of angiogenetic signals in the extracellular matrix (ECM) plays a fundamental role in ensuring an appropriate completion of all steps in vasculogenesis 31 .
Vascular endothelial growth factor is an important mediator in lymphangiogenesis and vascular permeability.It also stimulates epithelization and collagen deposition 32 .Certain cytokines and growth factors such as TGF-β1, TGF-β2, KGF, FGF-2, PDGF-BB and EGF act in a paracrine manner and stimulate VEGF expression, suggesting their high biological interaction [33][34][35] .
A study on growth factors presence at the site of the extraction wound in rabbits showed no change in the values of VGDF in the maxilla in the period from 48 hours to the 4 th day after extraction.From the 4 th day to the end of the 1 st week, VEGF levels increase and this value did not change between the 1 st and the 2 nd week.However, this level almost tripled in the period between week 4 and 8.In the period between weeks the 12 th to the 16 th week there was no change in the amount of VEGF 36 .Another study assessing the level of VEGF in the blood of rats after tooth extraction has confirmed that the level of VEGF in the venous blood increases in the first 24 hours after extraction, especially in young rats, although VEGF levels at the site of extraction are not significantly different in young and adult rats 37 .

Fibroblast growth factors
Fibroblasts and fibrocytes play an important role in tissue reconstruction by replacing the old collagen type III with collagen type I and forming a network of collagen molecules.Tension forces on the edges of the wound increase owing to generated elastin, thus completing the process of wound edges approximation and the scar formation 38 .
Fibroblast growth factors make a large polipeptide family of 22 members which have a key role in neurological function, development and metabolism 39,40 .FGF are produced by keratinocytes, fibroblasts, endothelium, smooth muscle cells, chondrocytes and fat cells.Acidic fibroblast growth factor (aFGF-1) and basic fibroblast growth factor (bFGF-2) have been identified in the wound fluid, especially in the early stages of the healing process 41 .The role of FGF in wound healing has been demonstrated in vitro and in vivo through its action in the early activation of macrophages and increase of the components of the extracellular matrix, as well as in the proliferation and differentiation of neuroectodermal and mesodermal derivatives, endothelial cell proliferation, adipogenesis, angiogenesis and epithelia-lization 42,43 .
The acidic fibroblast growth factor (aFGF-1) which influences the proliferation of fibroblasts and endothelial cells, promoting angiogenesis and wound healing has the most important role in the wound healing process.FGF-2, on the other hand, increases the level of osteocalcin in the medium, which suggests that it modulates the function of osteoblastic cells 44 .Keratinocyte growth factor KGF-1 or FGF-7, a member of the FGF family induces faster re-epithelization by accelerating the proliferation and differentiation of epithelial cells and inhibiting apoptosis 45,46 .In addition, it is a potent mitogen for vascular endothelial cells and up-regulation of VEGF.Keratinocyte growth factor-2 (KGF-2) or FGF-10 has a similar mechanism of action, but also it stimulates the granulation tissue and collagen formation.
A study that examined the spatial and temporal concentrations of FGF-2 in rabbit extraction wound showed no changes in the concentration of FGF-2 in the period from 48 hours to four days after tooth extraction in the maxilla.From the end of the 1 st to the end of the 2 nd week FGF-2 concentration is doubled, and then decreased from the 2 nd to 4 th week.Between the 4 th and the 8 th week there was a significant increase in the concentration of FGF-2 which then continued to grow moderately from week 8 to 12.A decrease in the concentration of FGF-2 was observed from the 12 th to the 16 th week 36 .
Similar changes in the concentration of FGF2 have been noticed in the mandible after tooth extraction.From the 1 st to the 2 nd week, an increase in the concentration was approximately six-fold.The values of FGF2 in the period from the 2 nd to the 8 th week were significantly higher in the mandible than in the maxilla.The concentrations of FGF-2 did not change over a period from the 4 th to the 8 th week but, they doubled from the 8 th to the 12 th week 36 .
Purified recombinant FGF-2 in optimal concentrations induces the proliferation of pulp cell predecessors, periodontal ligament fibroblasts and mesenchymal cells, while at the same time affecting their osteogenic potential 47 .

The use of growth factors in extraction wound healing
Autologous platelet concentratesplatelet-rich plasma (PRP) and platelet-rich fibrin (PRF) contain high levels of growth factors including three PDGF isomers, transforming growth factor beta, insulin-like growth factor, epidermal growth factor and vascular endothelial growth factor, which is why they have been long used in oral and maxillofacial surgery.
Bone defect regeneration was experimentally induced by the isolation and long-term cultivation of autologous stem cells.BMP-2 is the product of stem cells which are abundant in fat tissue.The fat tissue stem cells application can be carried out as promptly as within 24 hours owing to a special method of tissue engineering, and has positive results in healing bone defects 48 .In addition to wound healing, some authors propose the use of growth factors in the treatment of alveolitis 49 , implant osseointegration 50 and alveolar ridge augmentation 51 .There is evidence that the postoperative healing was better and postoperative pain lower in patients treated with growth-factors-rich plazma 52 .Arany et al. 53 report the increase in TGF-β1 growth factor concentration after low-level laser treatment of extraction wound, which explains the accelerated wound healing after low-level laser radiation 54 .In a study that used dogs as test subjects, Munkhdulami et al. 55 found that both bone quality and quantity were better after rhBMP-2 was injected into dentoalveolar defects.
The effects of BMP depend on its concentration, which is specific for each animal species, as well as on the carrier the concentrations are released from.These carriers are in most cases demineralized bone matrix, bioactive glass, non-absorbable hydroxyapatite or resorbable beta calcium tree-phosphate.

Conclusion
Isolated characterization of the respective growth factors' effects is practically impossible due to their actions being pleiotropic and mutually overlapping.Studies of physiological processes in which growth factors have a regulatory role indicate that these molecules rarely act in biological isolation.
The study of the interaction between the growth factors in the alveolar bone can explain tissue ability to heal even under adverse conditions, such as infection and radiation.The application of growth factors should certainly be considered in cases where difficult and slow wound healing can be expected.