Platelet-rich Plasma and Other Hemocomponents in Veterinary Regenerative Medicine

Platelet-rich plasma, platelet-rich fibrin, fibrin glue, and platelet lysate are the most widely used nontransfusional hemocomponents that, as biological and therapeutic aids, enhance the physiological reactions after an injury to facilitate the repair and regeneration processes. Recently, this type of therapy also has significantly expanded in veterinary medicine. Due to many similarities, the animal patient could be a good reference as a study model for humans, especially for chronic and difficult-to-heal injuries. This review discusses the current state of hemocomponent use for topical application in veterinary medicine, with a comparison with human medicine.

Introduction

In a ministerial decree from March 3, 2005, the Italian Board of Health decided to use the term hemocomponents to indicate “therapeutic constituents of blood that can be prepared using simple physical means intended to obtain their separation.”¹ Further, according to the Italian law of October 21, 2005, n.219, hemocomponents and hemoderivatives are considered blood products.² Hemocomponents are defined as “products obtained from the blood fractionation using simple physical means or apheresis,” whereas hemoderivatives are “plasma-derivatives medication or else as proprietary medicinal products extracted from the plasma by an industrial manufacturing process.”² In particular, platelet-rich plasma (PRP) or platelet gel (PG), platelet-rich fibrin (PRF), fibrin glue (FG), and platelet lysate (PL) are enclosed in the category of hemocomponents for nontransfusional (topical) use.

The aim of this review is to summarize the current state of hemocomponent use for topical application in veterinary medicine with parallelism and comparison to human medicine, defining differences and similarities, in order to shed light upon the potential mutual benefits arising from translational research to clinical practice.

Platelet-rich Plasma

Platelet-rich plasma, or PG, is a hemocomponent for topical use that has an autologous or allogeneic origin. It is obtained from the aggregation of a platelet concentrate mixed with calcium and biological (thrombin) or pharmacological aggregating factors.¹

Growth factors (GFs) in PRP
During the coagulation process, platelets release the factors contained in α-granules. This degranulation of platelet α-granules causes the release of several GFs, including platelet-derived growth factor (PDGF) αα, PDGFββ, and PDGFαβ; transforming growth factor (TGF) β1 and TGF-β2; vascular endothelial growth factor (VEGF); basic fibroblast growth factor (bFGF); platelet-derived epidermal growth factor (PDEGF); and insulin-like growth factor (IGF) I and IGF-II.^3-5

Platelet-derived GF is a thermostable GF composed by the α and β chains, which can give rise to 3 types of PDGF: αα, ββ, and αβ. Cells are sensitive to the action of the PDGF and may express 2 types of receptors, α and β.⁶ Although both receptors are involved in the transduction of mitogenic stimuli, only the β receptor is involved in the chemotactic stimulus.⁷ Platelet-derived growth factor is mitogenic for mesenchymal cells, such as fibroblasts, osteoblasts, and adipocytes, and stimulates the formation of collagen type I.^8-10 Furthermore, this factor indirectly promotes angiogenesis by activating the macrophages.^4,11

Transforming GF-β is involved in many physiological processes. It stimulates the proliferation and differentiation of mesenchymal stem cells and promotes the synthesis of collagen type I by the osteoblasts,¹² owns an angiogenic activity, and is able to inhibit osteoclast formation and epithelial cell proliferation in the presence of other GFs.⁴

Vascular endothelial GF stimulates the chemotaxis and proliferation of endothelial cells in order to promote the angiogenesis process.¹³ It also raises the hyperpermeability of the blood vessels. It is considered a mitogenic and proapoptotic factor that promotes the chemotaxis and differentiation of fibroblasts and epithelial, kidney, and glial cells.⁴

Basic fibroblast GF stimulates and coordinates the mitogenesis of mesenchymal stem cells during growth; it also maintains and repairs tissues. This effect has been reported for fibroblasts, osteoblasts, chondrocytes, and smooth and striated muscle cells.⁴ In addition, angiogenesis is enhanced by the stimulation of mitosis and migration of endothelial cells.^4,14

Platelet-derived epidermal GF has chemotactic and mitogenic effects on fibroblasts and epithelial cells. It also has been described as an inducer of cell migration and granulation tissue formation.⁴ Fibroblasts, as well as preosteoblasts and prechondrocytes, express a high number of PDEGF receptors.^4,15

Insulin-like GF-I stimulates the formation of bone matrix by promoting the preosteoblast proliferation.^16,17 In addition, IGF-I stimulates osteoblasts to synthesize osteocalcin, alkaline phosphatase, and type I collagen.¹⁸ It also stimulates the proliferation and differentiation of mesenchymal stem cells in chondrogenesis, adipogenesis, and myogenesis.¹⁹ This GF promotes neuronal differentiation²⁰ and induces a chemotactic effect on vascular endothelial cells.⁴

Production, indications, and procedures for using PRP
Topical use of PRP promotes and accelerates the healing process in both soft tissues^21,22 and orthopedic conditions.^23,24 This ability is related to its plasticity and modeling at the level of the application site. The final product may be obtained by fractionation of whole blood, which can be collected as a predeposited or allogeneic donation (with or without red blood cell reinfusion) or by an autologous and allogeneic platelet apheresis. The entire process must be conducted to guarantee asepsis. The final product must be used as quickly as possible; otherwise, it should be frozen according to times and methods similar to those used for fresh frozen plasma (FFP).¹ However, despite the fact that current Italian legislation allows the conservation of PG and other hemocomponents, the present authors are unaware of any studies demonstrating evidence that hemocomponents maintain their properties after storage.

Substantially, PRP is a platelet concentrate that is positioned in situ on the lesion. Following the activation of PRP and (similarly to what occurs physiologically) platelet degranulation, GFs, chemokines, and other bioactive metabolites are released. The anabolic and chemotactic actions of all these factors contribute to a more rapid tissue repair. A variety of protocols and activating agents have been proposed in recent years, such as bovine thrombin,^25-28 the agonist peptide of the thrombin receptor,²⁸ the gelling agent ITA (NATREX Technologies, Inc, Greenville, NC),²⁵ the batroxobin (clotting enzyme isolated from the venom of the snake Bothrops atrox, belonging to the Viperidae family),²⁹ ascorbic acid,³⁰ pulse electric field,²⁸ and autologous thrombin.^27,31 Other protocols do not require activating agents.³²

Different methods of preparation can yield 2 types of PRP. The first, leukocyte-PRP, is marked by the presence of leukocytes³³; in the second type, pure-PRP, leukocytes are absent.³⁴ The presence or absence of leukocytes in platelet preparation is widely discussed.^35-41 Some authors have suggested avoiding tissue exposure to leukocytes as they may promote an inflammatory reaction that potentially can affect the healing process.^36-39 Other authors reported beneficial effects related to the increase of immunological and antibacterial resistances^38,40 as well as to increased growth factors that have been released.⁴¹ Once PRP is activated, it can be applied in combination with mesenchymal stromal stem cells (MSCs). Once combined with MSCs, PRP has a dual function: direct contribution to tissue repair and serving as an essential biological scaffold for cellular preservation at the injury site.^42-44

Recently, PRP has been used in many branches of human medicine, with the most interesting results published in oral-maxillofacial surgery,^4,45,46 cartilage and tendon repair,^5,47-49 orthopedic surgery and bone reconstruction (eg, delayed union,⁴⁸ nonunion,^46,48ischemic osteonecrosis,⁴⁸ bone defects,⁵ and tendon-muscular diseases^5,47,48), skin ulcers (pressure ulcers^{31,46,48,50,51}and diabetic ulcers^{31,48,50,52,53}), plastic-reconstructive and cosmetic surgery,⁴ and ophthalmology (corneal ulcers^4,46,48). Despite a growing interest in veterinary medicine, the scientific literature is still limited to case reports^54,55 or studies performed on experimentally induced lesions in several domestic species, such as dogs,^56-63 horses,^30,64-66 goats,⁶⁷ and pigs.^68,69 To the best of the authors’ knowledge, the only randomized controlled clinical trials are focused on the treatment of osteoarthritis^23,70 and chronic pressure ulcers.²² The findings of these randomized veterinary studies showed favorable results with significant improvement of clinical conditions after PRP treatment.^22,23,70

Activated PRP can be used as a liquid state or under a coagulated form. Liquid PRP is likely to be used for infiltration or injection at the injury site (eg, skin, joints, tendons). Coagulated PRP is properly named platelet gel; it can be applied on skin surfaces, mucous membranes, or in internal tissues during open surgery.^34,61,71 Particularly, in horses, PRP can be advantageously applied by articular infiltration or at the level of tendon injury, especially in the frequent flexor tendon injuries of the phalanges in athletic horses, where such biological treatment enabled a shorter rehabilitation time and a higher number of horses that returned to racing.^72,73 Platelet-rich plasma was used recently with effectiveness in bone healing after percutaneous PRP injection in a donkey with delayed consolidation of a tibial fracture.⁷⁴

Randomized clinical trials, conducted both in human²¹ and in veterinary medicine,²² have shown that topical application of PRP has a real efficacy in the healing process of skin lesions compared with traditional therapeutic techniques. Both in humans⁷⁵ and in dogs,⁶¹ the use of PRP during reconstructive plastic surgery increases the perfusion of pedicle flaps and free grafts, facilitating engraftment and survival. In the complex field of wound healing, PRP can be associated with topical application of honey, exploiting a potential synergistic effect on the healing process.⁷⁶ The use of PRP to treat wounds localized in the distal parts of the limbs in horses brought about controversial results.^30,55,65,77 Some authors found an excessive formation of granulation tissue and delayed healing.^65,77

Despite many recent in vivo studies showing a significant therapeutic efficacy,^{54,55,59,64,66,70,78,79} these results apparently conflict with previous studies.^{56-58,65,69,80} These previous studies did not find any benefits from PG application on tendon injuries,⁵⁸ bone defects,⁵⁶ and skin⁵⁷ and palatal lesions⁸⁰ in dogs; pig enteric lesions⁶⁹; or equine wounds.⁶⁵ In vivo regenerative medicine studies using nontransfusional hemocomponents are summarized in the eTable (part 1 and part 2).^{22,23,30,31,42,44,45,50-75,77-85}

An important aspect to consider is that animal studies showing negative outcomes evaluated the effects of PRP on acute or experimentally induced injuries.^{56-58,65,69,77,80} It may be possible to hypothesize that PRP is more effective when it is applied on tissues that are biologically compromised or on spontaneous chronic lesions. In these cases, a resource of bioactive molecules might be necessary to induce and sustain a reparative response from adjacent tissue to reach complete healing.²² In fact, GF and cytokine imbalances were identified in chronic lesions, and this can explain how stimuli to healing can originate from these types of injuries.^{31,50,51,53,81} In accordance with this theory, chronic lesions have a higher deficit of GFs and cytokines, such that they will benefit from PRP application.^86,87Some studies, however, showed a significant effect, even in experimental acute injuries.^88,89A recent meta-analysis of animal studies suggested advantageous effects of PRP in the treatment of experimentally induced full-thickness skin wounds.⁹⁰

In a Cochrane systematic review, Martinez-Zapata et al⁹¹ stated that although PRP may improve chronic wound healing in people, the overall quality of evidence is low. Randomized controlled clinical trials evaluating PRP are limited and underpowered to detect treatment effect, and they are generally at a high or unclear risk of bias. Therefore, well-designed and adequately powered clinical trials are still needed to clarify this aspect.⁹¹

Some authors would empirically suggest that PRP could promote infections by acting as a substrate for microorganisms, according to the idea that compares PRP to culture media containing blood and derivatives.⁹² Today, in contrast with this theory, PRP is considered to inhibit the growth of microorganisms, even if there are no results that express this effect in a definitive manner.^40,92-95Although the exact mechanisms of interaction with bacteria need further investigation, a systematic review of current preclinical studies evidenced a bacteriostatic activity of platelet concentrates.⁹⁶

The complexity of establishing functional and nonfunctional parameters to define the qualitative properties of platelet concentrates is linked to aspects such as the variability and the bioavailability of biomolecules contained in the platelets and the variability in the product preparation. Although it is very difficult to understand what the ideal parameters to be evaluated in practice are, the platelet concentration (1 x 10⁹ mL^-1) would seem a reasonable compromise to determine the quality of the product.⁹⁷ In addition, PRP products containing concentrations of platelets ≥ 1 x 10⁹ mL^-1 seem to have a better effect on animals.⁹⁰ To remove an important variable related to the clinical effect of the product, the nontransfusional platelet concentrates should be standardized at least according to the number of platelets; this will allow for assessment and comparison of the results of clinical trials to be published in the near future.⁹⁷

Platelet-rich Fibrin

Platelet-rich fibrin, first developed by Choukroun et al,⁹⁸ is a second-generation platelet concentrate for surgical use. It is defined as an autologous leukocyte and PRF biomaterial. The protocol for PRF production is very simple and inexpensive; it does not require anticoagulants or gelling agents, but it is based on speed of blood collection, centrifugation, and handling to obtain a clinically usable clot. The PRF clot contains serum, many healing and immunity promoters, and innumerable platelets trapped in the fibrin mesh. This product can be used directly as a clot or as a strong fibrin membrane, driving out the fluids trapped in the fibrin matrix by compression. Potential clinical indications of PRF are numerous, including the improvement of soft tissue healing and bone graft protection and remodelling.^99-105 Studies considering PRF in animals are still limited in number.^58,62,106

Fibrin Glue

Fibrin glue is a hemocomponent for topical use that may have autologous or allogeneic origin. Its topical use has different functions: facilitating tissue adhesion, promoting hemostasis, and assisting the surgical suture during the wound healing process.¹ It is used with success in different surgical applications; FG can be a replacement or a supplement to surgical sutures (eg, sutures and anastomosis applied at different levels such as the esophagus, intestine, duodenum, ileum, trachea, and vessels).^107-110Moreover, FG also can find application in the healing process of parenchymal organs (eg, spleen, liver, pancreas, kidney, lung)^{107,108,111,112} and in all cases where its adhesive and hemostatic action can be exploited (eg, biliary bed, hepatic resection, bone defects).^107,108,113 For all these reasons, FG finds a greater use in cardiovascular, thoracic, and hepatic surgery but also neurosurgery, dentistry, and plastic-reconstructive surgery.^83,114

The formation of physiological covalent bonds between fibrin, fibronectin, and collagen tissue is the basis of FG adhesive properties. By mixing materials such as bioglass, bone substitute, and bio-autologous material (eg, autograft and bone fractions), the autologous FG can be very helpful in the case of significant large bone defects.¹¹³ The association of bone substitute with biological glue allows for easy placement of materials or biomaterials at the injury site so that bone substitute remains in situ more smoothly; the mixing of these 2 components creates a stable support that facilitates the use of biomembranes and allows for an easy fill of large cavities.^82,115,116Moreover, FG can act as an excellent scaffold for the topical application of mesenchymal stem cells used for the regeneration and healing of tissue injuries.⁸⁴ The most common production techniques can involve the use of a chemical method (ethanol extraction), different physical methods (cryoprecipitation), or centrifugation and physical separation by means of a special membrane that is suitable to concentrate the fibrogen. The fibrin is formed by the action of the thrombin on fibrinogen, a glycoprotein of 340 000 D.¹¹⁶ The product is obtained from the autologous or allogenic plasma through a procedure that ensures asepsis. After the preparation, FG has to be used as quickly as possible or should be frozen according to times and methods similar to those of fresh frozen plasma.¹

Platelet Lysate

Platelet lysate is a protein extract obtained from the fractionation of platelet concentrate by physical and chemical means. The PL induces the release of chemotactic and growth factors. This hemocomponent is mostly used as a supplement of cell growth in vitro. It also can be used as an alternative to fetal bovine serum to promote the expansion and differentiation of multipotent MSCs.^117-123 This possibility avoids the risk of potential contamination caused by pathogens coming from bovine species.^117-123 Regardless of the typical PRP properties, such as adhesion and hemostasis, the platelet soluble factors in the PL can influence the tissue formation and development through migration and cell replication.^124,125 More rarely, PL is studied and clinically used as a therapeutic agent for topical application.^85,126

Considerations for Clinical Use

The nontransfusional hemocomponents available in both human and veterinary medicine can act in synergy with conventional physical, pharmacological, and surgical therapies. Hemocomponents can complement standard of care, making treatment more modern and potentially more effective. Used topically for regenerative medicine purposes, hemocomponents improve the natural physiological response that should occur in wound healing. This innovative therapeutic approach is continuously evolving, as testified by the number of scientific studies published on this topic in recent years, proving the great interest of international scientific communities. Continual updating is therefore essential.

The scientific references herein confirm the therapeutic potential of hemocomponents in regenerative and reconstructive medicine. In veterinary medicine, the variety of possible applications is wide, ranging from skin to orthopedic lesions, from ophthalmological to odontostomatological injuries, and so on. Finally, an important consideration is the versatility of use in any animal species, whether small or large.

The indications for use of hemocomponents deal with chronic, difficult-to-treat diseases that often lack a cure; in fact, under such conditions, traditional therapeutic approaches are often used just as symptomatic treatments with a final aim of stabilizing the clinical situation and preventing its worsening.

The physician must carefully evaluate, case by case, the real indications for treatment with hemocomponents by assessing the current clinical condition, the trend of the disease, the treatments already performed, and the prior results achieved. All this information should help in choosing the most appropriate treatment (type of blood product, method of production, type and frequency of application), relying on scientific knowledge and evidence, and avoiding the risk of creating false illusions about the final result.

Considerations for Future Research

Research, both in humans and in animals, still has many elements to investigate in the use of hemocomponents as topical therapy or for nontransfusional purposes. The analysis of the current literature is complicated by a lack of standardized study protocols, production techniques, and outcome measures. Therefore, production technique has to be standardized and adapted to real clinical needs. Moreover, the quality of the final product must be validated so that it can be applied in reproducible studies.

In this context, it is crucial to increase the number of preclinical studies, both in vitro and in vivo, to understand the exact mechanism of action of nontransfusional hemocomponents, their storage possibilities, properties, and potential for homologous and heterologous use as well as to identify new therapeutic indications, risks, and limitations. Besides, the scientific community cannot be exempt from execution of phase 1 clinical trials in order to evaluate the safety and tolerability of hemocomponents for nontransfusional use, and of phase 2 and 3 trials, which are randomized clinical trials used to assess the efficacy of these interesting therapeutic aids.

With so many similarities in etiopathogenetic, clinical, and therapeutic aspects, the animal patient with chronic injuries could be a good model for the development of this class of biological products for human therapy. The concept to simultaneously improve human and animal health finds its cultural background inspiration in the concept of “One World, One Health, One Medicine,” which features a profound interconnection between human and animal health. The animal model should be an effective study for alternative or adjuvant therapies for human difficult-to-treat, chronic injuries, such as pressure, diabetic, and corneal ulcers; primary and secondary degenerative joint disease; tendon and ligament injuries; pseudarthrosis; and central and peripheral neurological disorders. These conditions are debilitating for both the human and animal patient and frustrating for the physician. Moreover, these injuries often represent a substantial cost for the pet owner or, in the case of humans, for the national health system and caregivers. Besides, with these conditions, nontransfusional hemocomponents could reveal their peak of therapeutic indication and efficacy.

Conclusions

The use of autologous nontransfusional hemocomponents for tissue regeneration purposes, both in humans and in animals, represents a major advance in the field of personalized regenerative medicine. The rationale for the use of hemocomponents is derived from the assumption that such products will increase local concentration of growth factors, will provide a fibrin scaffold for cell maintaining and migration, and thereby will activate the endogenous tissue regeneration process.

Hemocomponents have been shown to be effective in many different therapeutic applications. Nevertheless, more intense and high-quality research activity is needed to clarify many aspects of molecular mechanisms supporting biological effects and evaluate clinical indications and applications. Human and veterinary regenerative medicine could benefit each other by carrying out a joint research activity.

Acknowledgments

The authors thank Dr. Paul Christopher Gatenby for the revision of the English language.

Authors: Adolfo Maria Tambella, DVM, MSc; Stefano Martin, DVM, PhD; Andrea Cantalamessa, DVM, PhD, PGDip; Evelina Serri, BSc; and Anna Rita Attili, DVM, PGDip

Affiliation: School of Biosciences and Veterinary Medicine, University of Camerino, Matelica, MC, Italy

Correspondence: Adolfo Maria Tambella, DVM, MSc, School of Biosciences and Veterinary Medicine, University of Camerino, Via Circonvallazione, 93/95, 62024 Matelica, MC, Italy; adolfomaria.tambella@unicam.it

Disclosure: The authors report no conflicts of interest. This work was supported by a research grant from the University of Camerino (Fondo di Ateneo per la Ricerca 2014-2015). The funding source had no involvement in study design; in the collection, analysis, and interpretation of data; in the writing of the report; and in the decision to submit the article for publication.

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