**3. Outlook for the control of tissue healing using PRP**

#### **3.1. Inflammation**

#### *3.1.1. Cell death and DAMPs in the extracellular space*

Injury in multicellular organisms is accompanied by cell damage and death, proportional to the magnitude of tissue injury that triggers a sophisticated sequence of reactions to cope with the insult. The degree of the inflammatory response depends on the severity of the injury that can induce different magnitudes of cell damage and death. Loss of cell integrity activates innate immune sensors by releasing to the extracellular space a myriad of intra-cytoplasmic mole‐ cules, known as DAMPs (Danger Activating Molecular Patterns). Among the DAMPs released by dying cells there is a growing list including cytosolic and nuclear proteins such as high mobility group box 1 (HMGB1), alarmins such as S100, and non-proteins including uric acid, DNA, RNA, and ATP. The inflammatory response triggered by the detection of DAMPs is an evolutionary conserved mechanism present in both vertebrates and invertebrates.

DAMPs transmit stress signals to the organism, and stimulate innate immune responses, starting by leukocyte infiltration, following by macrophage polarization and closing with the resolution of inflammation. This set of mechanisms is known as the inflammatory response, and serves to minimize the insult, and repair the damaged tissue in doing so contributes to the recovery of tissue homeostasis.

Cell death can result from injury but can also occur physiologically as a component of tissue homeostasis, since all tissues in accordance with their physiologic turnover rate replace old cells by new ones. In tissue turnover cell death is not accompanied by any inflammatory reaction, probably because DAMPs in the extracellular space do not reach a threshold con‐ centration. Importantly, errors in the control of immune homeostasis may be behind chronic diseases.

The administration of PRP during this phase can rescue damaged cells as PRP contains cytokines that can promote cell survival, as shown both in vivo and in vitro. For example, during cell auto-transplantation for the treatment of tissue defects in plastic surgery, the use of PRP increases the survival of pre-adipocytes and adipocytes. Pre-adipocytes treated with PRP showed anti-apoptotic activities and decreased the expression of molecular mediators of cell death including Bcl-2-interacting mediator of cell death [7]. Additionally PRP can protect human tenocytes against cell death induced by ciprofloxacin and dexamethasone [8]. Further‐ more, PRP could alleviate BMSC death under hostile conditions increasing the levels of paracrine interactions via stimulation of PDGFR/PI K/AKT/NF-kB signaling pathway [9]. PRP also promoted rejuvenation of aged and senescent MSC in vitro [10].

TLR receptors and DAMP-TLR activation is thought to be important in restoring homeostasis after cell death. Recent research has added layers of complexity to our understanding of PRP, and information about how molecular components of PRP interfere with DAMP signaling through NF-kb illustrates the anti-inflammatory effect of PRP in several tissues [11].

#### *3.1.2. Pattern of leukocyte infiltration*

All these regenerative events constitute different layers of biological control that can be

influenced by PRP administration.

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**Figure 1.** Potential layers for PRP influence in tissue regeneration

*3.1.1. Cell death and DAMPs in the extracellular space*

**3.1. Inflammation**

**3. Outlook for the control of tissue healing using PRP**

Injury in multicellular organisms is accompanied by cell damage and death, proportional to the magnitude of tissue injury that triggers a sophisticated sequence of reactions to cope with the insult. The degree of the inflammatory response depends on the severity of the injury that can induce different magnitudes of cell damage and death. Loss of cell integrity activates innate immune sensors by releasing to the extracellular space a myriad of intra-cytoplasmic mole‐ cules, known as DAMPs (Danger Activating Molecular Patterns). Among the DAMPs released by dying cells there is a growing list including cytosolic and nuclear proteins such as high mobility group box 1 (HMGB1), alarmins such as S100, and non-proteins including uric acid, DNA, RNA, and ATP. The inflammatory response triggered by the detection of DAMPs is an

evolutionary conserved mechanism present in both vertebrates and invertebrates.

The magnitude, pattern and timing of leukocyte infiltration are better described when tissue stress is induced by pathogens. However, in the case of sterile injuries, the extravasation of leukocytes in response to tissue damage is less understood. Actually, it is uncertain how PRP influences these three parameters: first, the magnitude of leukocyte infiltration, second, the pattern, and lastly, the timing.

The way PRP influences infiltrating immune cells is important because the latter play a major role in determining the outcome of tissue repair along with the secretory phenotype of local cells

#### *3.1.2.1. Polimorphonuclear cell (PMNs) infiltration*

The increase in vessel permeability and chemotactic signals from the injured tissues facilitates extravasation and movement of leukocytes within tissues by diapedesis. The use of PRP in this stage of healing modifies several aspects, first PRP increases vessel permeability by releasing

VEGF (also known as permeability factor, PF); in addition catecholamines such as dopamine and noradrenaline are delivered from dense granules in addition to histamine, all with synergistic effects in augmenting vessel permeability [12].

Polymorphonuclear cell (PMNs), including neutrophils (60-65% of the total leukocytes), eosinophils and basophils extravasate from the blood stream and perform a graded infiltration that reaches maximums in 12-24 h and is followed by decline, stop and apoptose. Excessive PMNs infiltration may be detrimental for the tissue because PMNs release a wide array of cytotoxic molecules. Granule components include several non-selective proteolytic enzymes, cytotoxins, antimicrobial peptides; in addition to the production of reactive oxygen species (ROS). The lifespan of neutrophils in the bloodstream is limited to hours but when they extravasate, the presence of DAMPs' agonists in the infiltrated tissues prolongs neutrophil survival.

PRP may influence both the amount of neutrophil infiltration and the survival of neutrophils in the injured tissues. In fact, PRP delivers both CCL and CXCL chemokines that attract different leukocyte subsets. In particular, CXCL7 (very abundant in platelets) in collaboration with NAP2 provides a strong chemotactic signal for neutrophil infiltration. In addition, PRP releases a known chemotactic cytokine for neutrophils, CXCL8/IL8. Moreover, we have recently shown that these chemotactic signals are reinforced and augmented by local cell synthesis in vitro [13]. PRP can also modify the lifespan of infiltrated leukocytes by modifying the molecular environment of the injury.

Thus, the administration of PRP would presumably modify the innate immune response, mainly by altering the molecular environment and the chemotactic driven pattern of neutro‐ phil infiltration, the intensity and the timing. However, these effects may be dependent on the tissue conditions and anatomical location.

#### *3.1.2.2. Monocyte/macrophage infiltration and polarization*

During the initial days subsequent to injury (from 2 h to 72 h) monocyte/macrophages gradually infiltrate the tissue, ready to clean up apoptotic neutrophils. Indeed, macrophages are specialized in clearance of death cells.

The expression "macrophage polarization" refers to the ability of macrophages to change their functional phenotype in response to molecular signals they sense in their microenvironment. Macrophages have been categorized conventionally into pro-inflammatory M1 and tissue repairing M2 phenotypes. In the presence of LPS or IFN-gamma macrophages are "classically" polarized and denominated M1 macrophages. They have an inflammatory phenotype as they express IL-1b, IL-6, IL-8 and TNF-a.

Instead, in the presence of high levels of IL-4, M2 macrophages are "alternatively" polarized and they produce anti-inflammatory cytokines, including IL-10, IL-1Ra, CD-36, scavenger receptor A or mannose receptor. However, growing knowledge about macrophage plasticity indicates that M1/M2 polarization is an over-simplified view. As a matter of fact, a continuum range of polarization states exist between the two extremes M1 and M2.

Inflammatory mechanisms are protective mechanisms that should be ideally self-limited and lead to complete resolution returning to tissue homeostasis. Recent data indicate that M1/M2 activation states are extremely plastic to external signals and macrophages can be repolarized from M2 to M1 states although the mechanism is unknown [14]. Resolution of inflammation is an active process involving the biosynthesis of specialized pro-resolving mediators by M2 polarized macrophages.

Assuming that manipulation of macrophage polarization can be a tool for therapeutic exploitation, it is imperative to gain knowledge about how PRP influences macrophages. In fact, PRP modifies the environment and macrophages can gain distinct functions supporting their participation in inflammation or alternatively in the resolution of inflammation. Previous data showed that CXCL4/PF4 induces a polarization state distinct from M1 or M2 [15], and the term M4 polarization has been proposed. This is relevant because PF4 is one of the most abundant cytokines stored in platelets' alpha-granules (micromolar concentrations), and is released from platelets upon activation. However, M4 polarization has been studied in the context of atherosclerosis, but not in tissue repair.

Therefore, further research is indispensable to establish how PRP would influence the activation state of macrophages, and whether resolution of inflammation can be achieved by exposing macrophages to determined molecular environments.

#### *3.1.3. Regulation of fibrotic pathways*

VEGF (also known as permeability factor, PF); in addition catecholamines such as dopamine and noradrenaline are delivered from dense granules in addition to histamine, all with

Polymorphonuclear cell (PMNs), including neutrophils (60-65% of the total leukocytes), eosinophils and basophils extravasate from the blood stream and perform a graded infiltration that reaches maximums in 12-24 h and is followed by decline, stop and apoptose. Excessive PMNs infiltration may be detrimental for the tissue because PMNs release a wide array of cytotoxic molecules. Granule components include several non-selective proteolytic enzymes, cytotoxins, antimicrobial peptides; in addition to the production of reactive oxygen species (ROS). The lifespan of neutrophils in the bloodstream is limited to hours but when they extravasate, the presence of DAMPs' agonists in the infiltrated tissues prolongs neutrophil

PRP may influence both the amount of neutrophil infiltration and the survival of neutrophils in the injured tissues. In fact, PRP delivers both CCL and CXCL chemokines that attract different leukocyte subsets. In particular, CXCL7 (very abundant in platelets) in collaboration with NAP2 provides a strong chemotactic signal for neutrophil infiltration. In addition, PRP releases a known chemotactic cytokine for neutrophils, CXCL8/IL8. Moreover, we have recently shown that these chemotactic signals are reinforced and augmented by local cell synthesis in vitro [13]. PRP can also modify the lifespan of infiltrated leukocytes by modifying

Thus, the administration of PRP would presumably modify the innate immune response, mainly by altering the molecular environment and the chemotactic driven pattern of neutro‐ phil infiltration, the intensity and the timing. However, these effects may be dependent on the

During the initial days subsequent to injury (from 2 h to 72 h) monocyte/macrophages gradually infiltrate the tissue, ready to clean up apoptotic neutrophils. Indeed, macrophages

The expression "macrophage polarization" refers to the ability of macrophages to change their functional phenotype in response to molecular signals they sense in their microenvironment.

synergistic effects in augmenting vessel permeability [12].

the molecular environment of the injury.

tissue conditions and anatomical location.

are specialized in clearance of death cells.

*3.1.2.2. Monocyte/macrophage infiltration and polarization*

survival.

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Fibrotic tissue is characterized by excessive type 1 collagen accumulation that hinders tissue regeneration. The presence of myofibroblasts is central to fibrotic tissue production. They originate from a spectrum of cellular sources, and several molecular pathways can induce the transition of cells to myofibroblasts. In fact, myofibroblasts can describe a functional status rather than a fixed cell phenotype. Fibrosis is predominantly controlled by TGF-b1, which is secreted as an inactive protein associated to a latent protein. TGF-b1 enhances strongly the synthesis of type 1 collagen by creating an autocrine loop; additionally it is an antiapoptotic agent for myofibroblasts. TGF-b1 is abundant in PRP, stored in considerable amounts in agranules and secreted upon platelet activation. Additionally leukocytes secrete TGF-b1. TGFbeta-stimulated M2-like macrophages have profibrotic activity [16]. Instead, serum amyloid protein present in plasma has been shown to inhibit fibrosis in different models by regulating macrophage function. Thus, PRP actions are theoretically paradoxical regarding the develop‐ ment of fibrosis. However, clinical practitioners using PRP injections rarely report the presence of fibrosis.

Scarring is also a key problem for axon regeneration because fibrotic tissue may block axon growth and impair axon function.

#### **3.2. Angiogenesis**

The supply of oxygen is essential for cell metabolism and wound healing. Indeed, poor tissue perfusion creates a hypoxic environment that impairs the healing process. Angiogenesis involves multiple biological mechanisms including cell migration, proliferation, and differ‐ entiation.

Vascularization occurs through outgrowth of preexisting blood vessels (angiogenesis) and involves cell migration and proliferation. Upon injury, vessels consisting of naked endothelial cell channels have potential to sprout and branch providing nutrients and oxygen to regener‐ ating tissues. Vessel sprouting and enlargement are driven by migratory endothelial cells (EC) called tip cells. Additional types of cells, i.e. smooth muscle cells and pericytes, are involved in vessel stabilization. Both mechanisms, angiogenesis and arteriogenesis, involve a wide array of cytokines and growth factors that can be supplied in physiological concentrations by PRP administration (Table 1). Essentially, PRP cause endothelial cell proliferation and capillary tube formation in vitro.

Alternatively to angiogenesis, new vessels can be formed through mobilization and domicil‐ iation of progenitors of endothelial cells to sites of tissue injury (vasculogenesis) or ischemic tissues, a process mediated by VEGF and SDF-1a binding to CXCR4 receptors on EPCs.

PRP augments ischemic neovascularization presumably due to the stimulation of the three above described mechanisms: angiogenesis, arteriogenesis and vasculogenesis. Arteriogenesis is the main driver of restoration of blood perfusion in ischemia [17].

Indeed, the importance of coagulation factors and of platelet secretome (VEGF, TGF-b1, PDGF, bFGF, angiopoietin) is evident for angiogenesis, not only because of their individual actions but because of beneficial synergies between these GFs. For example, Ang-1, an EC survival factor, stimulates capillary tube formation synergistically with VEGF. Also synergy between both PDGF and VEGF results in the formation of a more mature vascular network than when each factor is given alone [18]. Also the angiopoietin system contributes to vessel maintenance growth and stabilization.

Paradoxically, platelets also provide several antiangiogenic factors necessary for vessel downregulation. Angiogenesis inhibitors (CXCR3 agonists) such as PF4 and TSP-1 are very abun‐ dantly stored in platelets. Additionally, angiostatin, a product of plasminogen proteolysis, inhibits angiogenesis. Both pro- and anti-angiogenic properties have been attributed to TGFb1. At low doses it contributes to the angiogenic switch in part by upregulation of VEGF and uPA, whilst at high doses contributes to the resolution of angiogenesis by inhibiting EC proliferation and migration, promoting the reformation of the basement membrane.


ment of fibrosis. However, clinical practitioners using PRP injections rarely report the presence

Scarring is also a key problem for axon regeneration because fibrotic tissue may block axon

The supply of oxygen is essential for cell metabolism and wound healing. Indeed, poor tissue perfusion creates a hypoxic environment that impairs the healing process. Angiogenesis involves multiple biological mechanisms including cell migration, proliferation, and differ‐

Vascularization occurs through outgrowth of preexisting blood vessels (angiogenesis) and involves cell migration and proliferation. Upon injury, vessels consisting of naked endothelial cell channels have potential to sprout and branch providing nutrients and oxygen to regener‐ ating tissues. Vessel sprouting and enlargement are driven by migratory endothelial cells (EC) called tip cells. Additional types of cells, i.e. smooth muscle cells and pericytes, are involved in vessel stabilization. Both mechanisms, angiogenesis and arteriogenesis, involve a wide array of cytokines and growth factors that can be supplied in physiological concentrations by PRP administration (Table 1). Essentially, PRP cause endothelial cell proliferation and capillary

Alternatively to angiogenesis, new vessels can be formed through mobilization and domicil‐ iation of progenitors of endothelial cells to sites of tissue injury (vasculogenesis) or ischemic tissues, a process mediated by VEGF and SDF-1a binding to CXCR4 receptors on EPCs.

PRP augments ischemic neovascularization presumably due to the stimulation of the three above described mechanisms: angiogenesis, arteriogenesis and vasculogenesis. Arteriogenesis

Indeed, the importance of coagulation factors and of platelet secretome (VEGF, TGF-b1, PDGF, bFGF, angiopoietin) is evident for angiogenesis, not only because of their individual actions but because of beneficial synergies between these GFs. For example, Ang-1, an EC survival factor, stimulates capillary tube formation synergistically with VEGF. Also synergy between both PDGF and VEGF results in the formation of a more mature vascular network than when each factor is given alone [18]. Also the angiopoietin system contributes to vessel maintenance

Paradoxically, platelets also provide several antiangiogenic factors necessary for vessel downregulation. Angiogenesis inhibitors (CXCR3 agonists) such as PF4 and TSP-1 are very abun‐ dantly stored in platelets. Additionally, angiostatin, a product of plasminogen proteolysis, inhibits angiogenesis. Both pro- and anti-angiogenic properties have been attributed to TGFb1. At low doses it contributes to the angiogenic switch in part by upregulation of VEGF and uPA, whilst at high doses contributes to the resolution of angiogenesis by inhibiting EC

proliferation and migration, promoting the reformation of the basement membrane.

is the main driver of restoration of blood perfusion in ischemia [17].

of fibrosis.

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entiation.

**3.2. Angiogenesis**

tube formation in vitro.

growth and stabilization.

growth and impair axon function.

**Table 1.** PRP-associated positive and negative regulators of angiogenesis. Some of these molecules may have both proand anti-angiogenic potential depending on the situation at the time of their release and/or the expression of cryptic sites. Reproduced from International Journal of Clinical Rheumatology, August 2012, Vol.7, No4, Pages 397-412 with permission of Future Medicine Ltd.[21]

As shown above, PRP provides the opportunity to therapeutically manipulate angiogenesis by targeting multiple cell phenotypes. Crosstalk between cell types along with multiple signals constitutes the complex system that regulates angiogenesis. Not only the activities of endo‐ thelial cells, but also of endothelial progenitors, smooth muscle cells and pericytes are influenced by PRP signals. Pericytes, crucial for vessel stabilization, can arise from different cell sources since they can transdifferentiate from the endothelium, a common vascular progenitor or a mesenchymal progenitor. The association of pericytes with newly formed vessels regulates EC proliferation, survival, migration, differentiation and vascular branching, blood flow and vascular permeability. Pericytes have important roles in tissue repair. For example, in skeletal muscle perycites arise from blood vessels and express NG2 proteoglycan and alkaline phosphatase and they efficiently regenerate the muscle expressing myogenic markers only when fully differentiated [19-20]. However, no information is yet available about the interaction of the molecular pool released from PRP and pericytes.

Other cooperative mechanisms such as partial degradation of the ECM and basement mem‐ brane are necessary to facilitate cell migration during angiogenesis. Actually, several protease families released from PRP including plasminogen activators (uPA and PAI-1), and MMPs have been characterized as having a role in the proteolytic degradation and remodeling of the subendothelial basement membrane and the surrounding ECM. By digesting ECM proteins, these enzymes create a path for tip cell migration. Platelets contain fibrinolytic factors and enzymes that may regulate precisely the pericellular proteolytic environment required for the control of cell migration and matrix remodeling. For example urokinase plasminogen (uPA) and plasminogen activator inhibitor type I (PAI-1) proceed as modifiers of the pathway that impact migratory events. Almost all cell types need to migrate under physiological or pathological conditions. Clearly the binding of PAI-1 with its several targets has the potential to influence the motile program at multiple levels. Further complexity is provided by the presence of endogenous proteases inhibitors (TIMPs) that control the activity of proteases.

#### **3.3. Stem/precursor cell activation and differentiation**

It has been demonstrated in last years that most organs have a resident pool of somatic, tissue specific cells. These stem cells are located in niches characterized by a typical spatial localiza‐ tion, the anchorage of stem cells to supporting cells, and the presence of typical extracellular matrix. These cells are docked in specific microenvironments that control their survival and self-renewal capabilities preventing them from exhaustion. In the niche, the integration of stimulatory and inhibitory signals determines cell quiescence.

In general, these cells are mitotically quiescent. PRP contains agents able to restore mitosis in quiescent precursor cells, consequently mitotically arrested cells are able to divide again. By definition, these precursor stem cells are capable of self-renewal and have various potentialities for differentiation. In general they are committed to differentiate in the local cell phenotype and are designed to substitute dying cells during turnover, trauma or pathology. PRP participates in mobilization of progenitor cells and proliferation but its effects in differentiation are controversial [22].

PRP influences the number of stem cells by virtue of its mitotic effect and maintains stemness in most settings. Actually, the effects of PRP on the differentiation of synovium derived MSCs are negative in all three lineages and PRP alone maintains MSCs stemness [23]. Besides PRP inhibits differentiation of adult rat tendon stem cells towards nontenocyte lineage [24]. Recent research in skeletal muscle repair has shown that PRP maintains stemness of muscle progenitor cells [25]. Though, PRP did not interfere with the osteogenic, chondrogenic and myogenic differentiation in the appropriate differentiation conditions.

In many cases of traumatic injury or disease the quantity and potency of this endogenous pool of precursor cells is insufficient to regenerate compromised tissues and migration and homing of mesenchymal stem cells circulating in the blood stream is required.

In fact, bone marrow contains several types of stem cells including hematopoietic cells that differentiate into mature blood cells, endothelial progenitor cells and MSCs which are proposed to give rise to the majority of marrow stromal cell lineages including chondrocytes, osteoblasts, fibroblasts, adipocytes, endothelial cells. Circulating MSCs in the blood stream can home injured tissue in response to chemoattractants released from platelets such as SDF-1a/CXCL12.

PRP was shown to be effective in promoting the migration of MSCs. In addition PRP can increase the number of MSC by stimulating proliferation. Actually the number of CMOs manufacturing cells for in-human trials are taking advantage of the mitogen properties and fetal calf serum [FCS) the typical cell culture supplement is being substituted by PRP. Several studies showed that population cell doublings is enhanced by PRP, that is to say PRP reduces the time needed to get a predefined cell number necessary for efficacious cell therapy [26].

## **3.4. Modulation of nerve repair**

families released from PRP including plasminogen activators (uPA and PAI-1), and MMPs have been characterized as having a role in the proteolytic degradation and remodeling of the subendothelial basement membrane and the surrounding ECM. By digesting ECM proteins, these enzymes create a path for tip cell migration. Platelets contain fibrinolytic factors and enzymes that may regulate precisely the pericellular proteolytic environment required for the control of cell migration and matrix remodeling. For example urokinase plasminogen (uPA) and plasminogen activator inhibitor type I (PAI-1) proceed as modifiers of the pathway that impact migratory events. Almost all cell types need to migrate under physiological or pathological conditions. Clearly the binding of PAI-1 with its several targets has the potential to influence the motile program at multiple levels. Further complexity is provided by the presence of endogenous proteases inhibitors (TIMPs) that control the activity of proteases.

It has been demonstrated in last years that most organs have a resident pool of somatic, tissue specific cells. These stem cells are located in niches characterized by a typical spatial localiza‐ tion, the anchorage of stem cells to supporting cells, and the presence of typical extracellular matrix. These cells are docked in specific microenvironments that control their survival and self-renewal capabilities preventing them from exhaustion. In the niche, the integration of

In general, these cells are mitotically quiescent. PRP contains agents able to restore mitosis in quiescent precursor cells, consequently mitotically arrested cells are able to divide again. By definition, these precursor stem cells are capable of self-renewal and have various potentialities for differentiation. In general they are committed to differentiate in the local cell phenotype and are designed to substitute dying cells during turnover, trauma or pathology. PRP participates in mobilization of progenitor cells and proliferation but its effects in differentiation

PRP influences the number of stem cells by virtue of its mitotic effect and maintains stemness in most settings. Actually, the effects of PRP on the differentiation of synovium derived MSCs are negative in all three lineages and PRP alone maintains MSCs stemness [23]. Besides PRP inhibits differentiation of adult rat tendon stem cells towards nontenocyte lineage [24]. Recent research in skeletal muscle repair has shown that PRP maintains stemness of muscle progenitor cells [25]. Though, PRP did not interfere with the osteogenic, chondrogenic and myogenic

In many cases of traumatic injury or disease the quantity and potency of this endogenous pool of precursor cells is insufficient to regenerate compromised tissues and migration and homing

In fact, bone marrow contains several types of stem cells including hematopoietic cells that differentiate into mature blood cells, endothelial progenitor cells and MSCs which are proposed to give rise to the majority of marrow stromal cell lineages including chondrocytes, osteoblasts, fibroblasts, adipocytes, endothelial cells. Circulating MSCs in the blood stream

**3.3. Stem/precursor cell activation and differentiation**

stimulatory and inhibitory signals determines cell quiescence.

differentiation in the appropriate differentiation conditions.

of mesenchymal stem cells circulating in the blood stream is required.

are controversial [22].

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#### **Peripheral nerve injury and regeneration**

Peripheral nerves have the capacity to regenerate after an axonal injury. There are several kinds of peripheral nerve damages depending on the damage of the axon and surrounding tissue. After an injury or a breakdown, axon is able to regrow expressing repair-related molecules and aided by Schwann cell activation, proliferation, phagocytic activity and production of neurotrophic factors. These factors activate signaling cascades promoting synthesis of molecules related to axonal regeneration events. In addition to producing bioactive molecules, Schwann cells form structures called Bünger bands, which have as a function the physical guidance of growing axons [27]. They also recruit macrophages to the injury site to remove debris from the injury and help them supporting axon repair by secreting chemokines. As a result, axon healing is the result of the interaction between molecular signals and cellular events which allow proper growth of the axonal stump and consequent recovery of its functional activity at the end of healing process.

Peripheral nerve fibers are stimulated immediately after injury and release several neuropep‐ tides into the microenvironment of the wound. Substance P, neuropeptide Y and calcitonin gene-related peptide (CGRP) influence endothelial cells, fibroblasts and are involved in vasoregulation and angiogenesis.

#### **Growth factor and cytokine involvement in nerve healing**

All these results have been attributed to growth factors released from platelets when activation occurs, but an accurate function and optimal concentration have not been identified [28]. Platelets release a high number of growth factors which may have precise effects on their own, or work synergistically depending on their concentration. Wound healing is influenced by diverse growth factors secreted by platelet such as PDGF, TGF-β, PF4, VEFG, EGF, PDEGF, IGF-I and others. Although these are not classically classified as neurotrophic, they have been demonstrated to have a role in Schwann cell migration, proliferation, neuron metabolism, synthesis of neurotrophic factors, matrix formation and myelinization and thus in axon regeneration [29-33]. Also, platelets release other molecules which are not growth factors such as catecholamines, histamine, serotonin, ADP, ATP and others which take part in blood vessel formation, immune reactions both innate and adaptive, and thus in tissue regeneration [30].

Nerve healing depends on equilibrium between Schwann cell proliferation and activation and neurotrophic molecules which create a regenerative milieu which helps axon repair and myelinization. Several growth factors present in PRP, such as PDGF, TGF-β1 and FGF-II, have shown to promote Schwann cell proliferation, activation and differentiation which may explain beneficial PRP effects shown in the previously commented studies [30, 33]. These growth factors, for which Schwann cells and neurons have membrane receptors, trigger the expression and subsequent synthesis of classic neurotrophic factors such as nerve growth factor [NGF), Glial derived growth factor (GDNF), brain-derived neurotrophic factor (BDNF) and ciliary neurotrophic factor (CTNF)[30, 32]. Also, PDGF expression has been shown to be enhanced in neurons after a nerve injury, which may support the theory that this growth factor has an important role in axon healing. Another growth factor present in PRP, which has been signaled as neurotrophic is VEGF. It has been shown to be neuroprotective, to also augment Schwann cell proliferation and axon growth [30]. IGF-I has also been pointed out as a central promoter of nerve healing*. In vitro,* it has been observed that IGF enhanced neuron axonal growth and that myelinization does not occur when IGF is removed. Also, IGF has been shown to promote Schwann cell proliferation and migration. In vivo, IGF injections in the site of nerve injury have been proved to ameliorate nerve healing and myelinization [28, 29,30, 31].

In the clinical arena, perineural injections of PRP induced sensorial recovery in leprosy peripheral neuropathy [34].
