**4. PRP formulations and regulatory requirements**

Innovative therapeutic tools appear in the horizon when basic knowledge and research surpasses a certain threshold and is ready for translation into the clinics. PRP technologies showed up in the late 80s, mainly based on increased knowledge about the functional role of platelets. Platelets are cytoplasmic fragments of the megakaryocyte in the bone marrow. A variety of molecules are stored in platelets' granules either synthesized by their parent cell the megakaryocyte or captured in the circulation. In PRP technologies platelets are used because of their capability to function as vehicles for growth factors and cytokine delivery.

Initially platelets were mainly studied because of their fundamental role in hemostasia. Allogeneic PRP, either derived from a single donor or pooled donors, has been used since the 60s as a transfusion product. In fact platelet transfusion is indicated in patients with platelet counts below 30,000 plt/ul or below 100,000 plt/ul if they are to follow a surgical procedure. Later in the 80s clinical researchers showed off that beyond their role in hemostasia, PRP derived product (PDWH) were effective in the management of chronic leg ulcers [35] and were an aid in cardiac surgery [36].

In the 90s, maxillofacial surgeons and oral implantologists introduced the clinical use of PRPs as autologous modifications of fibrin glue. They were confounded by the effects of PRP in bone regeneration in doing so accelerating the stability of dental implants. Of note, the antiinflammatory properties in soft tissues, presumably attributed to the presence of platelets in the preparation, was another hallmark in PRP findings.

In the new millennium, the use of PRP has been boosted not only by research in maxillofacial surgery and oral dentistry but also by new applications in orthopedics and sports medicine. In 2007, the term and definition of platelet rich plasma was introduce in Pubmed as a medical subject heading (MeSH) to be used for indexing scientific articles.

However, the definition of PRP in Pubmed is out of date by several reasons. First, PRP is not only used in surgical procedures but it is also used in the conservative management of nonhealing ulcers and as an injectable in the management of chronic pathologies such as tendi‐ nopathies or osteoarthritis. Secondly, the current definition claims that GFs in platelets enhance tissue regeneration this is true but only up to a point. In fact, not only GFs from platelets but also plasmatic GFs have a crucial role in repair. Besides, this definition overlooks the hundreds of proteins released from platelets that also participate in healing. Despite all these limitations, PRP inclusion as MeSH term has served to harbor PRP research under a unique term.

PRPs differ from conventionally synthesized drugs in that they are products derived from living sources. Indeed platelets are lively cells and they may experiment several temporary transformations from preparation to local tissue delivery. The process is known as platelet activation and involves changes in platelet morphology, aggregation, centralization of granules, and secretion of their content to the extracellular milieu. Another peculiarity is that PRP products are complex multi-molecular mixtures that cannot be readily characterized and reproducibility in the composition is influenced by biological inter-individual variability.

## **4.1. Types of PRP products**

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

Innovative therapeutic tools appear in the horizon when basic knowledge and research surpasses a certain threshold and is ready for translation into the clinics. PRP technologies showed up in the late 80s, mainly based on increased knowledge about the functional role of platelets. Platelets are cytoplasmic fragments of the megakaryocyte in the bone marrow. A variety of molecules are stored in platelets' granules either synthesized by their parent cell the megakaryocyte or captured in the circulation. In PRP technologies platelets are used because

Initially platelets were mainly studied because of their fundamental role in hemostasia. Allogeneic PRP, either derived from a single donor or pooled donors, has been used since the 60s as a transfusion product. In fact platelet transfusion is indicated in patients with platelet counts below 30,000 plt/ul or below 100,000 plt/ul if they are to follow a surgical procedure. Later in the 80s clinical researchers showed off that beyond their role in hemostasia, PRP derived product (PDWH) were effective in the management of chronic leg ulcers [35] and were

In the 90s, maxillofacial surgeons and oral implantologists introduced the clinical use of PRPs as autologous modifications of fibrin glue. They were confounded by the effects of PRP in bone regeneration in doing so accelerating the stability of dental implants. Of note, the antiinflammatory properties in soft tissues, presumably attributed to the presence of platelets in

of their capability to function as vehicles for growth factors and cytokine delivery.

**4. PRP formulations and regulatory requirements**

peripheral neuropathy [34].

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an aid in cardiac surgery [36].

the preparation, was another hallmark in PRP findings.

PRP is prepared by taking a given volume of blood from a patient and processing it to separate blood components and concentrate the platelets and optionally the leukocytes. Importantly, the manipulation of blood in order to obtain PRP is minimal.

The nomenclature of PRP products reached a zenith of confusion at the beginning of the new millennium. In fact, more names than products appeared and the number of commercial terms was endless, including platelet concentrates (PC), autologous growth factors (AGF), plasma rich in growth factors (PRGF), platelet gel (PG), platelet rich fibrin matrix (PRFM) etc... It was evident that there were more names than PRPs to be named.

In 2009, Dohan [37] inspired the present nomenclature and classification of PRPs. Broadly speaking PRPs were categorized as pure PRP and leukocyte and platelet rich plasmas. Considering fibrin architecture and platelet counts we can differentiate further PRP subsets. Different PRP devices and harvest yield in terms of platelet and leukocyte count lead to the proposal of classification systems for PRP.

Alternatively to commercial automatic systems, PRP can be prepared in blood banks with highly standardized procedures. In this setting, PRP is prepared from a higher volume of blood, quality is assessed and aliquots of PRP are frozen for posterior applications. PRP obtained in blood banks are less expensive than PRPs obtained with automatic devices.

For blood withdrawal, many PRP protocols use anticoagulants to prevent blood from clotting. Most kits use ACDA or sodium citrate to chelate calcium ions in doing so preventing pro‐ thrombin conversion into thrombin. Other anticoagulants (i.e. heparin, EDTA) are avoided because they may compromise platelet stability and activation. Notwithstanding, ACDA and sodium citrate make the plasma acidic and some protocols recommend buffering the PRP back to a physiologic range prior to injection. Alternatively, PRP products such as leukocyte and platelet rich fibrin (L-PRF) do not use anticoagulants and fibrin is formed during the centri‐ fugation step. Evidently, these products have a physiological pH but cannot be used as injectable.

Importantly, PRP activation is needed to induce the secretion of granule contents i.e. platelet secretome. This occurs spontaneously in blood but is inhibited if the blood is withdrawn in tubes containing anticoagulants. Reversion of anticoagulants inhibition of coagulation and platelet activation can be achieved by several procedures. One possibility is the addition of calcium or thrombin/Ca2+ to cleave fibrinogen with subsequent polymerization of fibrin monomers. Alternatively, physiological activation can be achieved by injecting the unactivated PRP that once in contact with collagen and other tissue factors will get activated. The mode of delivery of PRP has also to be taken into account, since it is a more involved process than the delivery of a drug or a single recombinant protein. The application protocol and postapplication management involved will have a huge impact on determining whether the potential efficacy is seen.



**Table 2.** Main characteristics of the devices and/or kits used to prepare PRP. Modified from: http:// www.perfusion.com/perfusion/prpdevicesummary.asp

#### **4.2. Regulatory**

For blood withdrawal, many PRP protocols use anticoagulants to prevent blood from clotting. Most kits use ACDA or sodium citrate to chelate calcium ions in doing so preventing pro‐ thrombin conversion into thrombin. Other anticoagulants (i.e. heparin, EDTA) are avoided because they may compromise platelet stability and activation. Notwithstanding, ACDA and sodium citrate make the plasma acidic and some protocols recommend buffering the PRP back to a physiologic range prior to injection. Alternatively, PRP products such as leukocyte and platelet rich fibrin (L-PRF) do not use anticoagulants and fibrin is formed during the centri‐ fugation step. Evidently, these products have a physiological pH but cannot be used as

Importantly, PRP activation is needed to induce the secretion of granule contents i.e. platelet secretome. This occurs spontaneously in blood but is inhibited if the blood is withdrawn in tubes containing anticoagulants. Reversion of anticoagulants inhibition of coagulation and platelet activation can be achieved by several procedures. One possibility is the addition of calcium or thrombin/Ca2+ to cleave fibrinogen with subsequent polymerization of fibrin monomers. Alternatively, physiological activation can be achieved by injecting the unactivated PRP that once in contact with collagen and other tissue factors will get activated. The mode of delivery of PRP has also to be taken into account, since it is a more involved process than the delivery of a drug or a single recombinant protein. The application protocol and postapplication management involved will have a huge impact on determining whether the

injectable.

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**Device Commercial name**

**GPS III™ (Biomet)**

**Angel (Arthrex)**

**ACP (Arthrex)**

**AutoloGel System (Cytomedix)**

**GenesisCS (Emcyte)**

**Pure PRP 2 (Emcyte)**

**Harvest® SmartPrep2**

potential efficacy is seen.

**Technology FDA**

Computer Aided

system

Standard Centrifugation Thixotropic gel

Standard Centrifugation

Standard Centrifugation **approved**

**Total process time**

Direct Siphoning 510(k) 16 min \$1550 10±3x

**Disposable list Price**

510(k) 25 min \$495 4.3x 76% L-PRP

510(k) 5 min \$295 2.1±2x 60% Pure PRP

510(k) 1-2 min \$325 1x 78% L-PRP

Pending 5.5 min N/A 8-16x 76% Pure PRP

(4ml)

Floating Buoy 510(k) 15 min \$700 3.2x 90% L-PRP

Floating Shelf 510(k) 16 min \$395 4x 72.0±10% L-PRP

**Increase above baseline** **Platelet recovery** **PRP formulations**

68±17.1% L-PRP

Regulatory requirements for PRPs are not uniform across the world. For example, in the US, PRP is not a product instead administration of PRP is a procedure and is, therefore, not subject to regulation by the FDA. However, the devices used to prepare PRP are regulated by the FDA premarket approval process and have to get a 510(k) clearance.

Devices and procedures destined to prepare PRP are classified for their intended use as class III medical devices and reach the market via premarket approval application. The product is evaluated to ensure that the product is safe and effective and displays consistent performance characteristics. 510(K) premarket notification exists for products that are similar to those already marketed usually called predicate device. In these cases 510(k) clearance is evaluated only for substantial equivalency.

Table 1 shows devices and/or kits for PRP preparation

At the European level there is no harmonized regulatory framework for PRP therapies, and each country has its own approach to PRP regulation within the jurisdiction of national authorities. Devices must comply with Class II-a medical device directive 93/42/EEC. Device approval in EU is overseen in each EU country by a governmental body called a Competent Authority. Instead, the surgical use of PRP can be considered as an autologous graft within the surgical procedure as regulated by Directive 2004/23/EC.

Of note, if regulatory requirements for PRP therapies were over-interpreted, unnecessary work derived therein will increase costs and hamper the clinical use of PRP therapies. This hypo‐ thetical situation would be prejudicial for many patients since advancements in PRP science can provide effective treatments for pathologies with substantial social and economic burden.

#### *4.2.1. Reimbursement*

Currently most insurance plans do not reimburse for PRP treatment due to the lack of data about their efficacy. Interestingly, in the US Category III, code 0232T is used for emerging technologies and applies for nonsurgical uses of PRP. This code allows data collection to be used to document widespread use for FDA approval and potential reimbursement. Besides, this code will allow the AMA (American Medical Association) to track the use of PRP, since codes T are considered experimental they will require pre-authorization for payment. If a physician feels that the patient would benefit from PRP injections, typically as a step to avoid a more costly and invasive procedure preauthorization for PRP reimbursement should be requested. Presentation of cost savings rationale can be the key to successful preauthorization. In general managers are concerned about physician's plans to get injured patients back to productivity or work.
