Platelet-rich plasma, an adjuvant biological therapy to assist peripheral nerve repair

Mikel Sánchez, Ane Garate, Diego Delgado,, Sabino Padilla

1 Arthroscopic Surgery Unit, Hospital Vithas San José, Vitoria‐Gasteiz, Spain

2 Department of Biomedical Engineering, University of Minnesota, Minneapolis, MN, USA

3 BTI Biotechnology Institute, Vitoria‐Gasteiz, Spain

Platelet-rich plasma, an adjuvant biological therapy to assist peripheral nerve repair

Mikel Sánchez1,2, Ane Garate2, Diego Delgado2,＊, Sabino Padilla3,＊

1 Arthroscopic Surgery Unit, Hospital Vithas San José, Vitoria‐Gasteiz, Spain

2 Department of Biomedical Engineering, University of Minnesota, Minneapolis, MN, USA

3 BTI Biotechnology Institute, Vitoria‐Gasteiz, Spain

How to cite this article:Sánchez M, Garate A, Delgado D, Padilla S (2017) Platelet-rich plasma, an adjuvant biological therapy to assist peripheral nerve repair. Neural Regen Res 12(1):47-52.

Open access statement:is is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 3.0 License, which allows others to remix, tweak, and build upon the work non-commercially, as long as the author is credited and the new creations are licensed under the identical terms.

erapies such as direct tension‐free microsurgical repair or transplantation of a nerve autogra, are now‐adays used to treat traumatic peripheral nerve injuries (PNI), focused on the enhancement of the intrinsic regenerative potential of injured axons. However, these therapies fail to recreate the suitable cellular and molecular microenvironment of peripheral nerve repair and in some cases, the functional recovery of nerve injuries is incomplete.us, new biomedical engineering strategies based on tissue engineering approach‐es through molecular intervention and scaffolding offer promising outcomes on the field. In this sense, evidence is accumulating in both, preclinical and clinical settings, indicating that platelet‐rich plasma products, and fi brin sca ff old obtained from this technology, hold an important therapeutic potential as a neuroprotective, neurogenic and neuroin fl ammatory therapeutic modulator system, as well as enhancing the sensory and motor functional nerve muscle unit recovery.

peripheral nerve injuries (PNI); Schwann cells ; axons, platelet-rich plasma; biomolecules; fi brin; sca ff old; intraneural; perineural; microenvironment

Accepted: 2016-12-16

Introduction

Every year, 350,000 patients are a ff ected by traumatic periph‐eral nerve injuries, which accounts for $150 billion in annual health care costs (Gri ffi n et al., 2013). Direct tension‐free mi‐crosurgical repair and/or the transplantation of a nerve auto‐grato bridge the gap are the gold standard treatments aimed at enhancing the intrinsic regenerative potential of injured axons (Fowler et al., 2015). However, such treatments do not recreate the suitable cellular and molecular microenvironment and in some cases, the functional recovery of nerve injuries is incomplete (Faroni et al., 2015). Platelet‐rich plasma (PRP) products hold an important therapeutic potential as a neuro‐protective, neurogenic, and neuroinflammatory therapeutic modulator system (Anitua et al., 2013; Kuffl er, 2014; Anitua et al., 2015a; Zheng et al., 2016) and as an enhancer of sensory and motor functional nerve‐muscle unit recovery (Anjayani et al., 2014; Kuffler, 2015; Sanchez et al., 2015), emerging as a biological adjuvant in peripheral nerve injuries (PNIs) and neuropathies.ese autologous products are applied, as a fi ll‐er of nerve conduits or vein‐muscle grafts across nerve gaps post trauma by in fi ltrating the nerve stumps perineurally and intraneurally which is guided with ultrasound probes, or as sca ff olds to bridge or wrap the injured nerve stumps (Farrag et al., 2007; Giannessi et al., 2014; Kim et al., 2014; Malahias et al., 2015). Moreover, there are non‐traumatic peripheral injuries such as compression, adhesion and fibrosis (as in the case of carpal tunnel syndrome and fi brotic post‐surgical side e ff ects) (Dodla et al., 2008), where this novel approach applied may ad‐ditionally diminish undesirable consequences such as fi brotic scars and denervated organ atrophy since this adjuvant therapy can speed up the functional recovery of the nerve‐muscle unit (Sariguney et al., 2008; Takeuchi et al., 2012; Wu et al., 2012; Ye et al., 2012; Sanchez et al., 2014).erefore, PRPs may be applied to assist and synergize with the gold standard therapies in nerve regeneration and neuropathies, and may be harnessed by surgeons in the operating room and in the clinical setting as an “o ff the shelf” alternative.

Degeneration and Regeneration after PNI: Molecular and Cellular Events

Following a PNI, an orchestrated multicellular and pleiotropic molecular response will ensue.is response consists in the interplay among Schwann cells (SCs), resident macrophages, endothelial cells (ECs), and fi broblasts, mainly modulated by injured axons, myelin breakdown products, soluble factors, and hypoxia as main signals. It will end up regrowing and guiding axons, and reconnecting them with the target organs at a rate of about 1 mm per day in humans (Parrinello et al., 2010; Zochodne, 2012; Cattin et al., 2015) (Figure 1).

Disruption of the regeneration unit by the noxious agent results in loss of axonal contact with SCs whose phenotype is drastically modi fi ed, thereby contributing to SC activation or transdifferentiation. Macrophages will collaborate with the activated‐dedi ff erentiated SCs in clearing the myelin and other tissue debris. Moreover, these SCs come into direct contact with resident fi broblasts that accumulate in large numbers at the site of injury in fl uencing SC migration and dedi ff erentia‐tion (Parrinello et al., 2010; Arthur‐Farraj et al., 2012; Jessen etal., 2015). SCs show a striking plastic response to the biological battlefield they are exposed to inside a damaged nerve and are the early detectors of damage (Figure 1). In a context‐ and time‐dependent manner, transdifferentiated SCs perform a variety of cell repair tasks from phagocytosing myelin debris to secreting neurotrophic and neurotropic factors (laminin), pro‐liferation and migration, which results in the formation of SC cords and Bungner Bands in the proximal and distal nerve seg‐ment, respectively (Gaudet et al., 2011; Zochodne, 2012; Jessen et al., 2015). Although SCs have the reputation of being the engine of peripheral nerve repair, in the nerve repair complex process, they are fuelled by axon growth cones and supportive stromal cells such as macrophages and fibroblasts, the very elements of Wallerian degeneretion as a neuroinflammatory process (Figure 1) (Parrinello et al., 2010; Gaudet et al., 2011; Cattin et al., 2015; Chen et al., 2015; Jessen et al., 2015). Emerg‐ing evidence suggests that macrophage plasticity contributes to peripheral nerve regenerationviadistinct mechanisms: by phagocyitosing myelin debris, synthesizing trophic factors such as vascular endothelial growth factor (VEGF) and promoting angiogenesis, producing collagen type VI, modulating the proliferation and migration of SCs, and in fl uencing the resolu‐tion of in fl ammation through the polarization from M1 to M2 phenotype (Mokarram et al., 2012; Cattin et al., 2015; Chen et al., 2015). Cattin et al. (2015) con fi rmed an idea suggested by Chen et al. (2005) that blood vessels might provide substrate or signalling for axon growth guidance and SC migration, by showing that macrophages selectively sense hypoxia in the area of nerve bridge and drive angiogenesisviathe VEGF‐secretion pathway at the nerve bridge (Figure 1). Despite the robust repair capacity to regrow peripheral nervous axons shown in the adult mammal (Gaudet et al., 2011; Cattin et al., 2015) and meticulous microsurgical nerve repair techniques there are some limiting factors, including the poor vascularization, the patients age, the chronic denervation of SCs, the endoneurial and perineurial fi brosis, the misguided axonal growth, the vast distance that axon growth cones must cover to reinnervate target organs/tissues, as well as their atrophy, and the rate of regeneration (Hall, 2005; Zochodne, 2012; Scheib and Hoke, 2013; Painter et al., 2014).

Plasma Rich in Growth Factors: an Injectable Sca ff old to Assist in Nerve Repair

Plasma rich in growth factors (PRGFs) consist of a pool of growth factors (GFs), microparticles, and other bioactive mediators many of them trapped, through fibrin heparan sulfate‐binding domains, in a three‐dimensional transient fi brin matrix (Figure 2) (Anitua et al., 2015b). Once PRP is in fi ltrated intraneurally as a liquid‐to‐gel injectable sca ff old, or wrapped around the injured nerve gap as a matrix‐like viscous and malleable structure, or both, (Sanchez et al., 2015) (Figure 3) tissue fi brinolysis breaks the fi brin down, thereby releasing cell signalling molecules such as neuro‐trophic (nerve growth factor (NGF), brain‐derived neuro‐trophic factor (BDNF), insulin‐like growth factor 1 (IGF‐1), platelet‐derived growth factor (PDGF), VEGF, hepatocyte growth factor (HGF)) and neurotropic factors (fibrin, fi‐bronectin, and vitronectin) (Anitua et al., 2015c).

Growingin vitroandin vivoevidence suggests that the biomolecules conveyed by PRPs are instrumental agents that modulate early inflammation, stem cell‐like myelinating SC activation, macrophage polarization, as well as the active reso‐lution of in fl ammation, angiogenesis, and fi brogenesis, thereby acting as key drivers of full nerve functional recovery (Sondell et al., 1999; Jiang et al., 2013; Zheng et al., 2014; Cattin et al., 2015; Jessen et al., 2015).ere are so far six lines of evidence pointing the therapeutic potential of PRPs as follows (Figure 3).

Neuroprotection and prevention of cell apoptosis

Several growth factors present in PRP including NGF, BDNF, PDGF, VEGF, IGF‐1, transforming growth factor beta (TGFB) alone or in combination have been shown to exert an antia‐poptotic and neuroprotective e ff ect on mesenchymal stem cells (MSCs), neurons, SCs, and human neural stem cells (Sondell et al., 1999; Lee et al., 2003; Borselli et al., 2010; Emel et al., 2011; Luo et al., 2012; Rao and Pearse, 2016). PRP fibrin scaffolds enriched with NGF, BDGF, and retinoic acid and loaded with bone marrow stromal cells (BMSCs), enhance their survival and differentiation into the neural phenotype (Zurita et al., 2010). In addition, when this PRP scaffold was transplanted into the brain, the viability and biologic activity of allogenic BMSC increased (Vaquero et al., 2013). Moreover, neuropro‐tective and anti fi brotic bene fi cial e ff ects (Cho et al., 2010; Wu et al., 2012) were reported with the injection of PRP into the corpus cavernosum in a bilateral cavernous nerve injury rat model and applying PRP in a facial nerve suture in a guinea pig model. A recentin vitrostudy on neuronal cultures from mouse model of Alzheimer’s disease (Anitua et al., 2015a) showed that the neurotoxicity induced by aggregated β‐amyloid added in primary neuronal cultures was signi fi cantly reduced, and the living cell number aer the co‐treatment with PRP in‐creased. In addition, chronic intranasal administration of PRP in Alzheimer’s disease mouse model elicits neuroprotection which is likely mediated by the activation of the antiapoptotic PIEK/Akt signalling pathway (Anitua et al., 2014).

Stimulation of angiogenesis

Borselli et al. (2010) showed in an ischemic limb rodent model with loss of neuromuscular junction (NMJ) innerva‐tion that an injectable sca ff old loaded with VEGF and IGF‐1 accelerated regeneration of damaged NMJs together with an enhancement of angiogenesis. In a rat model it has been reported that sciatic nerve gaps of 10 mm repaired with vein grafi lled with PRP exhibited a more prominent early neoangiogenesis than sciatic nerve gaps treated with nerve autograft alone (Kim et al., 2004). In this regard, it should be taken into account that fi brin is a pivotal element within PRP that provides ECM tissue with a robust and permissive 3D matrix for angiogenesis (Hall et al., 2007).

Enhancing axonal outgrowth capacity

Overcoming the in fl ammatory microenvironment

Dampening the denervated target muscle atrophy

Several animal studies have demostrated that the application of PRP as a fi ller, a fi brin membrane, or both, induce an earli‐er axonal regeneration and functional recovery (Farrag et al., 2007; Sariguney et al., 2008; Emel et al., 2011; Wu et al., 2012; Gianessi et al., 2014; Kim et al., 2014; Sanchez et al., 2015). This is the case reported by Sanchez et al. (2015) on sheep, where nerves repaired with PRP were associated with an ear‐lier electrophysiological recovery and lower muscle atrophy, suggesting that PRP application may dampen the target mus‐cle atrophy. In addition, another recovery burden in nerve re‐pair is scarring, which has been reported to be minimized by the repair of sciatic injured nerve assisted with PRP (Gianessi et al., 2014). Anitua et al. (2015d) showed that intramuscular injection of PRP 24 hours aer the induction of limb ischemia in mice, mitigates fi brosis and muscle atrophy.ese results are in agreement with the reduction of atrophy in denervat‐ed muscle reported when muscle was infiltrated with cells (Schaakxs et al., 2013), e ff ects suggested to be mediated by the IGF‐1 (Shavlakadze et al., 2005). Moreover, TGFB, an import‐ant GF within PRP, attenuates the adverse effects of chron‐ically denervated Schwann cells, and reactivated SCs support axon regenerationin vivo(Sulaiman and Gordon, 2012).

In the wake of promising results in animal experimentation, PRP has been applied either as fi ller of nerve conduits across post traumatic nerve gaps (Kuffler, 2011, 2014), as a liquid dynamic sca ff old in fi ltrated perineurally (Hibner et al., 2012; Anjayani et al., 2014; Malahias et al., 2015), intraneurally, or both (as in the case of a peroneal nerve palsy (Sanchez et al., 2014) and other damaged nerves. Furthermore, it also has been applied as sca ff old or fi brin membranes (Kuffler, 2011, 2014; Scala et al., 2014) with bene fi cial outcomes and better functional recovery. Kuffler applied autologous platelet rich fi brin as a fi ller of a collagen tube, proceeding to bridge the 12 cm nerve gap 3.25 years aer an ulnar nerve trauma, and to recovery of both muscle and sensory function (Kuffler, 2011). In a recent series of cases of surgical nerve repair, Kuffler (2014) reported functional recovery in patients un‐der 58 years whose nerve gaps of 2–16 cm were treated with collagen tube fi lled with PRP, from 0.5–3 years post trauma.

In a double‐blind, randomized, clinical trial, the application of perineural PRP injections in tibial and ulnar nerves has shown sensory improvement in leprosy peripheral neurop‐athy (Anjayani et al., 2014). In a retrospective analysis of 10 patients with persistent pudendal neuralgia, who underwent a second trans‐gluteal decompression of the pudendal nerve, they injected activated PRP around the coated nerve, report‐ing a significant reduction in pain (Hibner et al., 2012). In a case series of 14 patients with carpal tunnel syndrome, a single ultrasound‐guided injection of PRP around the median nerve led to the disappearance of pain in eight patients, and pain alleviation in three patients at three months of follow‐up(Malahias et al., 2015). Another case report, in this case ap‐plying sequential proximal and distal ultrasound‐guided PRP injections intraneurally and perineurally (Figure 3) in a com‐mon peroneal nerve palsy, Sanchez et al. (2014) reported a signi fi cant functional recovery assessed by electromyographic signs of reinnervation for both peroneus longus and tibialis anterior muscles as well as almost full recovery of sensiviity. It has been reported that the intravenous injection of 25 cc of concentrated PRP in a 6‐year‐old‐boy with perinatal cerebral palsy is safe, and can signi fi cantly improve the cognitive and language functions (Alcaraz et al., 2015).

Figure 1 Spontaneous peripheral nerve regeneration is a multicellular and pleiotropic process.

Figure 2 Illustration of some biological mediators of platelet-rich plasma (PRP) that govern tissue repair by still poorly undestood mechanisms.

Figure 3 Six lines of evidence suggest the therapeutic potential use of PRPs on neural tissue repair and regeneration.

Conclusion

Author contributions:MS, DD, AG and SP contributed to the conception and design of the review. MS, DD, AG and SP contributed to dra-ing, writing, critical revision and fi nal approval of the article.

Con fl icts of interest:SP is a researcher at BTI (Biotechnology Institute) a dental implant company that investigates in the fi elds of oral implantology and PRGF-Endoret technology.

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Diego Delgado, Ph.D. or Sabino Padilla, M.D., Ph.D., diego.delgado@ucatrauma.com or sabinopadilla@hotmail.com.

10.4103/1673-5374.198973

＊< class="emphasis_italic">Correspondence to: Diego Delgado, Ph.D. or Sabino Padilla, M.D., Ph.D., diego.delgado@ucatrauma.com or sabinopadilla@hotmail.com.

orcid: 0000-0001-9992-6927 (Mikel Sánchez) 0000-0002-0494-0804 (Diego Delgado)