Platelet-Rich Products and Their Application to Osteoarthritis

Journal Pre-proof PLATELET-RICH PRODUCTS AND THEIR APPLICATION TO OSTEOARTHRITIS Livia Camargo Garbin, Christine S. Olver PII:

S0737-0806(19)30569-6

DOI:

https://doi.org/10.1016/j.jevs.2019.102820

Reference:

YJEVS 102820

To appear in:

Journal of Equine Veterinary Science

Received Date: 27 January 2019 Revised Date:

4 August 2019

Accepted Date: 22 October 2019

Please cite this article as: Garbin LC, Olver CS, PLATELET-RICH PRODUCTS AND THEIR APPLICATION TO OSTEOARTHRITIS, Journal of Equine Veterinary Science (2019), doi: https:// doi.org/10.1016/j.jevs.2019.102820. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 The Author(s). Published by Elsevier Inc.

PLATELET-RICH PRODUCTS AND THEIR APPLICATION TO OSTEOARTHRITIS Authorship: Livia Camargo Garbina, Christine S. Olverb, a

Department of Clinical Veterinary Sciences, School of Veterinary Medicine, Faculty of

Medical Sciences, University of West Indies, St. Augustine Campus, Trinidad &Tobago, West Indies. [email protected] b

Veterinary Diagnostic Laboratory, Department of Microbiology, Immunology and

Pathology, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, Colorado,80523, U.S.A. [email protected] Corresponding author: Dr. Livia Camargo Garbin a

Department of Clinical Veterinary Sciences, School of Veterinary Medicine, Faculty of

Medical Sciences, University of West Indies, St. Augustine Campus, Trinidad &Tobago, West Indies. [email protected] Ethical considerations: The authors have no ethical considerations to declare.

Declarations of interest: None

Source of funding: No funding was required for this paper. The Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) offered financial support for the graduate student, author of this paper during her PhD.

1

PLATELET-RICH PRODUCTS AND THEIR APPLICATION TO

2

OSTEOARTHRITIS

3

ABSTRACT

4

Autologous platelet-rich plasma (PRP) is a biological preparation made from the patient’s

5

own plasma that contains a platelet concentration above the whole blood baseline. Due to the

6

release of growth factors and other cytokines after degranulation, platelets have a central role

7

in inflammation and in different stages of the healing process. For this reason, PRP-derived

8

products have been used to enhance healing of musculoskeletal injuries and modulate

9

progression of inflammatory processes, including osteoarthritis (OA). Osteoarthritis is one of

10

the main causes of musculoskeletal disabilities in horses and currently there is no effective

11

treatment for this disease. Treatments that focus on the modulation of inflammation and

12

disease progression offer new hope for OA. PRP provides a more practical and accessible

13

option of therapy compared to other forms of biological treatment (i.e. stem cell therapies),

14

and is believed to induce the production of functional matrix. However, several factors

15

related to PRP production, including methods of preparation and application, and intra-

16

individual variability, lead to an inconsistent product, precluding reliable conclusions about

17

its efficacy for clinical use. The aim of this manuscript is to review the benefits related to the

18

clinical use of PRP in OA as well as factors that influence its use, the limitations of this

19

treatment, and future directions of PRP research and therapy.

20

Keywords: platelet-rich plasma, osteoarthritis, horse, growth factors

21

1. Introduction

22

Several studies have evaluated and demonstrated the beneficial biological effects of

23

platelet-rich products, reporting results in in vitro and in vivo research in musculoskeletal

24

disease [1, 2,3,4, 5, 6, 7, 8, 9, 10, 11]. Due to the growth factors released by the platelets and

25

their capacity to modulate tissue healing and inflammation, platelet-derived products have

1

26

been a focus of research as a potential treatment for osteoarthritis (OA). Yet, the use of these

27

products still results in contradictory outcomes. Although the proof of principle of platelet-

28

derived products has been demonstrated in vitro and anecdotally in vivo, the evidence-based

29

confirmation for its clinical efficacy remains to be elucidated.

30

The goal of this review is to clarify aspects of platelet physiology and activation that are

31

relevant to platelet-rich plasma (PRP) therapy, as well as the importance of growth factors in

32

OA and the use of PRP as a therapy for this disease. For this purpose, the authors did a

33

thorough analysis of the literature, investigating databases such as PubMed, ScienceDirect,

34

MEDLINE and Google Scholar. We also included references from manuscripts selected for

35

this review. All manuscripts selected were in English language and included key words such

36

as; platelet-rich plasma, platelet lysate, equine, osteoarthritis, autologous protein solution and

37

autologous platelet product.

38

2.0 Platelet physiology

39 40 41

2.1 The Platelet

42

megakaryocytes present in the bone marrow. Equine platelets normally circulate in the blood

43

in a concentration of 100 to 300 x103 /µL [12].The average concentration of platelets in blood

44

will vary according to age and breed [13]. For instance, platelet concentrations in

45

standardbred horses demonstrated to be 100 x103 platelets/µL lower compared to

46

thoroughbred horses (standardbred: 139 +/-44 x103 platelets/µL; thoroughbred: 284 +/- 22

47

x103 platelets/µL) [13]. Platelets are the first cells to respond to vascular damage. [2] The

48

damaged site attracts and activates platelets to release the contents of their α-granules, dense

49

granules and lysosomes [14]. Each one of those subcellular organelles contains different

50

types of proteins, essential for thrombus formation and hemostasis, chemotaxis of leukocytes,

51

as well as tissue healing [15]. In fact, platelets contain over 800 proteins, and considering

Platelets are anucleate cytoplasmic fragments derived from multinucleated

2

52

post translational modifications of these proteins there could be up to 1500 bioactive factors.

53

[16] Platelet’s bioactive factors are chemotactic for leukocytes and fibroblasts, contributing

54

to the debridement of the wound, proliferation of new matrix and neovascularization at the

55

wound site [17].

56

The α-granules are the most abundant granules in the platelet [18], containing the growth

57

factors that are chemotactic to cells and therefore stimulate cell migration, proliferation and

58

matrix synthesis [19]. These granules contain a large number of other bioactive molecules,

59

including hemostatic factors (i.e. factor V, fibrinogen), angiogenic factors (i.e. angiogenin,

60

vascular endothelial growth factor-VEGF), antiangiogenic factors (i.e. angiostatins, platelet

61

factor IV), proteases (i.e. metalloproteinases 9 and 2; MMP-9, MMP-2), and pro-

62

inflammatory factors (i.e. tumor necrosis factor alpha and interleukin-1 beta; TNF-α and IL-

63

1β). [20] In addition, α-granules present polypeptide growth factors such as basic fibroblast

64

growth factor – bFGF [21], transforming growth factor beta- TGF-β [22], platelet-derived

65

growth factor-PDGF [23], vascular endothelial growth factor -VEGF [24], epidermal growth

66

factors -EGF [25], and insulin-like growth factor -IGF [26]. Alpha-granules contribute to

67

primary and secondary hemostasis, secreting von Willebrand factor (vWf) and fibrinogen

68

[18][Table 1]. Each of those factors participate differently in the coagulation and

69

inflammatory process.

70

The dense granules or bodies (or δ-granules), contain adenine nucleotide (ADP and ATP),

71

serotonin, calcium and mediators that induce platelet aggregation, vasoconstriction and pro-

72

inflammatory cytokine production [27]. The platelet’s lysosomes contain enzymes such as

73

glucosidases, proteases and proteins with bactericidal activity (β-glucuronidase) [28]. The

74

factors released by platelet lysosomes can contribute to not only thrombus remodeling [14],

75

but pathogen clearance and breakdown of extracellular matrix as well [28].

3

76 77 78

2.2 Functions of platelets In the 90s, other functions of platelets besides coagulation started to be explored.

79

[29](Figure 1: Platelet Multifunction). Besides the numerous granules loaded with immune

80

mediators, the platelets contain a broad array of cell surface immune receptors and adhesion

81

molecules that could interfere with the platelet’s activation [30]. Platelets are capable of

82

secretion of pro-inflammatory cytokines such as IL-1β. Many α-granule mediators (e.g. P-

83

selectin) promote interaction between platelets and leukocytes, plasma proteins and

84

endothelium [28], which may allow platelets to initiate and propagate the inflammatory

85

process [18, 31]. Because of these characteristics, platelets may be able to initiate and

86

propagate the inflammatory process, influencing many inflammatory and immunomediated

87

disorders, such as rheumatoid arthritis [32]. In fact, platelets provide an amplifying role in the

88

pathophysiology of inflammatory arthritis. Collagen IV- induced platelet microparticles

89

expressed IL-1 leading to fibroblast-like synoviocytes to produce IL-8 [33]. These findings

90

demonstrate that platelets do play a role in the inflammatory process within the joint.

91

Although platelets participate in inflammation mainly as pro-inflammatory cells, they

92

also demonstrated potential anti-inflammatory effects by inhibiting NF-κB expression in

93

human chondrocytes [1, 2]. Such processes are still under investigation, and the balance

94

between pro-inflamatory and anti-inflammatory factors released by platelets will depend on

95

the circunstances [34].

96

After the discovery of the growth factors present within the alpha-granules, the idea of

97

using platelet concentrates for other than hemostatic therapy started in the field of

98

Regenerative Medicine [29]. Several studies in vitro and in vivo demonstrated that platelets

99

can modulate inflammation and promote healing [1, 2, 3, 4, 5, 6, 7, 9, 10, 11, 35, 36].

100 4

101 102

3.0 Growth Factors and their function in osteoarthritis Growth factors (GFs) are grouped in families of structurally related proteins and have

103

multiple functions in tissue healing and inflammation [37]. Growth factors released by

104

platelets have an important role in modulating the effect of catabolic cytokines such as IL-1

105

and IL-4, [38] and in inducing tissue remodeling and healing as well. [39] Specifically, GFs

106

stimulate a wide range of biologic effects in different types of cells inducing normal gene

107

expression to stimulate a wide range of actions within the cell (i.e. synthesis of matrix

108

components and mitosis). Therefore, either the presence or absence of specific receptors will

109

determine the cell’s capacity to respond to GF [37]. Growth factors are not mutagenic and

110

through modulation of gene expression, are responsible for regulating the normal reaction to

111

injury, healing and tissue regeneration [37].

112

Some GF are well known for promoting tissue healing, for example PDGF is an

113

important mitogenic factor inducing cell proliferation and proteoglycan production [40].

114

Insulin-like growth factor-1induces mesenchymal stem cell (MSC) proliferation and

115

modulates chondrogenesis [41], and this influences homeostasis by controlling proteoglycan

116

synthesis and breakdown [40]. Another example is Fibroblast Growth Factor-2 (FGF-2),

117

which stimulates proliferation of MSCs and chondrocytes. [42]

118

One of the main growth factors released by platelets, especially important for cartilage

119

matrix homeostasis and growth, is TGF-ß. This growth factor has anabolic effect in cartilage,

120

inducing cell proliferation, matrix production and osteochondrogenic differentiation [43].

121

TGF-ß was demonstrated to increase gene expression of collagen type II and aggrecan [43].

122

The constant release of TGF-ß is important for the entire process of chondrogenesis,

123

sustaining chondrocyte phenotype and regulating sulfation of glycosaminoglycan. In fact,

124

chondrocyte proliferation demonstrated to have a dose-dependent correlation to the

125

concentration of TGF-ß within the PRP [44]. However, increased TGF-ß1 activity has been 5

126

suggested to be associated with increase in bone mass and OA [45], in addition to synovial

127

fibrosis [46]. The concentration of TGF-β in PRP may vary though, depending on the method

128

used for preparation and activation [47]. The determination of this growth factor’s

129

concentration (?) within the PRP and its correlation with PRP outcomes in osteoarthritic joins

130

should be investigated.

131

For therapeutic purposes, GF can be delivered individually or in combination with other

132

factors directly into the site of the lesion. Thus, PRP show advantages due to the synergistic

133

effects of the many growth factors that are contained in it, having modulatory effects within

134

the OA joint (Figure 2 and 3). Consequently, PRP has been used as an additional treatment

135

option for musculoskeletal tissue healing.

136

Yet, while synergic effects of growth factors within the PRP are potentially beneficial, it

137

adds complexity to platelet -derived treatments. This happens because the growth factor

138

content varies according to the protocol used and PRP composition, as discussed below. In

139

addition, such growth factors have different effects in the different tissues within the joint

140

[24, 46].

141

4.0 Platelet-derived products

142

Platelet-rich plasma has been described in the literature as an autologous volume of

143

plasma with an increased concentration of platelets, in comparison with the whole blood from

144

which it was processed [39, 48]. Generally, this product is prepared by centrifuging

145

peripheral whole blood. [49] However, different methods for platelet separation and creation

146

of platelet-derived products have been described. Those include using gravity filtration

147

systems [50, 51], centrifugation protocols [51, 52] or apheresis. [53, 54]. Essentially,

148

apheresis and gravitational systems have been developed to create platelet concentrate

149

products for transfusion [55]. In apheresis, a cell separator submits the blood to

150

ultracentrifugation (3000 g) that separates the platelets, leucocytes and erythrocytes from the

6

151

plasma. The equipment has an optical reader that is capable of detecting the buffy coat cells

152

which are separated in a bag as the platelet concentrate. When the optical reader detects the

153

erythrocytes, the platelet collection is interrupted and erythrocytes, with leucocytes and

154

remaining platelets are directed to a third separated bag [56, 48]. Platelet-derived products

155

created by apheresis methods have demonstrated greater concentrations of TGF-β1 compared

156

to buffy coat concentrates, but both methods have been described as reliable for equine

157

platelet concentration [54]. In addition, apheresis method is less practical and is more costly

158

compared to gravitational and filtration methods, and thus may not offer the most practical

159

method for use in equine practice.

160

Manual centrifugation protocols for platelet concentration [52, 57, 58], could offer a

161

more economical alternative in comparison with centrifugation protocols that require the use

162

of kits [51]. However, such protocols are more laborious. Numerous gravitational kits have

163

been described [51], offering a more practical alternative compared to the manual method,

164

but still require basic laboratory settings (i.e. a centrifuge). In equine medicine, the following

165

systems have been used successfully in scientific reports; Harvest SmartPReP2 [10], Secquire

166

[59], GPSII Biomet [60] and Autologous Conditioned Plasma (ACP, Arthrex) [61] (Table 2).

167

The use of filtration kits are likely to offer the most user friendly alternative to the

168

field clinician, once it can be used by the stall site (Equine Platelet Enhancement Therapy -

169

“E-PET”, PallLife Sciences), not requiring centrifugation [50,51] (Table 2). In this method,

170

the platelet concentrate is suspended in a clear harvest medium before application into the

171

site of the lesion. It is important to observe that this product will not present (contain?)

172

plasma, and thus the concentration of certain growth factors that are produced by other

173

tissues (i.e. IGF-1 is produced by liver [62]), may be reduced in the final platelet-derived

174

product in the absence of plasma.

7

175

Platelet-rich plasma was initially implemented in 1970s, [49] and has been used to

176

enhance healing of bone from mandibular defect in human patients. [63] In the following

177

years, greater attention was given to forces and duration of centrifugation, which later were

178

noticed to be essential in determining the composition of the PRP. In the 1990’s, PRP was

179

implemented as a potential new biomaterial in the field of regenerative medicine. [29]

180

4.1 Forms of platelet-rich plasma use and delivery

181

Based on the different preparation methods, PRP was later categorized in a variety of

182

final products according to their cellular or fibrin content [48]. Platelet-derived products have

183

been classified as pure platelet-rich plasma (P-PRP), platelet-rich in growth factor (PRGF),

184

leucocyte- and platelet-rich plasma (L-PRP), pure platelet-rich fibrin (P-PRF) and leucocyte-

185

and platelet-rich fibrin (L-PRF) [48], and autologous conditioned plasma (ACP) [61] (Table

186

3).

187

Platelet and leucocyte-and platelet-rich fibrin preparations can be used as potential

188

biomaterial [48]. For platelet-rich fibrin (Choukroun’s PRF) preparation, blood is collected

189

without any anticoagulant and immediately centrifuged. During the centrifugation,

190

coagulation occurs leading to a formation of a leucocyte- and platelet-rich fibrin clot without

191

the need for biochemical modification of the blood. The fibrin clot is supposed to trap most

192

of platelets and leucocytes as well as growth factors and other circulating proteins [48] (Table

193

3).

194

Leucocyte-poor or pure-platelet-rich fibrin (P-PRF) is another preparation develop in

195

human medicine (Fibrinet platelet-rich fibrin matrix-PRFM by Cascade Medical, New Jersey,

196

USA) (Table 3). This essentially consist in blood collection and centrifugation at high speed

197

using tubes containing tri-sodium citrate as anticoagulant and a separator gel. Platelet-poor

198

plasma and buffy coat are transferred to another tube containing CaCl2 with help of a tube

199

connection system [48].

8

200

Leucocyte-poor or pure platelet-rich plasma were developed as another application of

201

transfusion platelets units and were reported initially to be used in maxillofacial surgery [64].

202

This product can be generated by plasmapheresis [56] as mentioned previously, or manually

203

as described by Anitua [65] (Table 3). This preparation is also denominated as plasma rich in

204

growth factors (PRGF). In this case, blood is collected with anticoagulants and centrifuged,

205

to separate the different components of the blood. A fraction of the platelet-poor plasma is

206

discarded and a small portion of the plasma, including the buffy coat layer (containing

207

platelets and leucocytes) [65], or the portion of the plasma immediately above the buffy coat

208

is collected [66]. The objective of the later approach is to avoid the collection of leucocytes.

209

The main issue with this technique is that it can be imprecise leading to irreproducible results

210

[48].

211

In addition to these forms, recently a product that has been developed and available in

212

equine practice is the autologous protein solution (APS), enriched in platelets and white

213

blood cells. After separation of the plasma enriched in platelets and white blood cells, the

214

plasma is placed in a concentrator that desiccates the product through polyacrylamide beads.

215

It is claimed that this product is enriched not only in growth factors but anti-inflammatory

216

factors as well [67] (Table 2).

217

PRP can be delivered in multiple forms, as through local injections or infiltrations, with

218

biomaterials and scaffolds [3, 4, 44], contained in hydrogel microspheres [4] or through the

219

use of PRP gel seeded with cells [68]. Several papers exploring platelet-derived products

220

have been published in the past 10 years. Specifically, hundreds of papers were published

221

investigating the therapeutic potential of PRP in musculoskeletal disease.

222

4.2 Use of PRP as a therapy

223 224

PRP has been used as a therapy for musculoskeletal injuries as it contains several growth factors that can potentially optimize tissue healing [37]. Most GF are released within 1 hour 9

225

of platelet activation and their half-life usually ranges from minutes to hours [37]. The

226

growth factors’ release is influenced by agonist used in PRP and the differences between

227

agonist and its effects are discussed below. PRP has been used in musculoskeletal tissue

228

lesions and shown anti-inflammatory [69] and anabolic effects by stimulating

229

chondrogenesis. [44] In addition, platelet-rich plasma decreased apoptosis and increased

230

autophagy in osteoarthritic chondrocytes [69], although recent evidence suggest autophagy

231

happens independently of PRP use [70]. Still, positive modulatory effects of platelet-derived

232

products and its matrix restoration properties have been well demonstrated previously in

233

different models [44, 69, 70].

234 235 236

4.2.1 Platelet-derived products in osteoarthritis

237

of OA in the horses, similar to the definition in humans, and is described as a set of disorders

238

with a common end stage of progressive degeneration of the articular cartilage with changes

239

in the bone and soft tissues [71].

240

Spontaneous OA is a common cause of lameness observed in horses [71]. The definition

The articular cartilage is a highly-specialized tissue that has a high matrix/cell ratio. In

241

homeostatic conditions, anabolic and catabolic agents are balanced. In osteoarthritis though,

242

there is an imbalance between anabolic and catabolic agents, triggered by pro-inflammatory

243

cytokines such as IL-1β and TNF-α, which are among the most important catabolic regulators

244

in OA [ 71, 72]. These cytokines induce their own production, as well as further cytokines

245

(such as IL-6, IL-8, nitric oxide-NO, prostaglandin) and enzymes (cyclooxygenase 2-COX-2)

246

in chondrocytes and synoviocytes, disseminating the inflammation within the joint. In

247

cartilage, the most important enzymes are the metalloproteinases (such as MMP-1, MMP-3,

248

MMP-8 and MMP-13) and aggrecanases (such as A disintegrin and metalloproteinase with

249

thrombospondin motifs 1-ADAMTS) [73]. The increase in these degradative enzymes results

10

250

in destruction of important matrix components, such as collagen type II and proteoglycans

251

[73].

252

Most of the current treatments for OA focus on treating the symptoms, leading to an

253

increase in interest for disease modifying treatments, such as platelet-rich plasma. Due to its

254

potentially anabolic and anti-catabolic effects and the ease of preparation, PRP became a

255

promising treatment option for OA in several species [ 4, 69, 74, 75]. Autologous protein

256

solution (APS), a platelet-derived product, reduced pain and lameness compared to saline in

257

client-owned dogs [5] and human patients with OA after 1 year of treatment [76]. In a

258

meniscal-tear rat model, the APS did not demonstrate clinical improvement, but it did reduce

259

cartilage degeneration. [77]

260

Specifically, in the horse, PRP has demonstrated promising results in vitro. In one study,

261

equine cartilage explants were stimulated with lipopolysaccharide (LPS) and then treated

262

with leukocyte-and platelet-rich gel or pure platelet-rich gel supernatants in different

263

concentrations. The authors observed that lower concentrations of leukocyte-and platelet-rich

264

gel supernatant presented the best therapeutic potential; reducing gene expression of pro-

265

inflammatory enzymes (MMP-13 and ADAMT-4), promoting an anabolic effect (increase in

266

cartilage oligomeric matrix protein -COMP) and reducing cell apoptosis. At higher

267

concentrations (of platelets and leukocytes) though, the same product showed downregulation

268

of anabolic extra-cellular matrix genes, such as collagen type 2 and COMP [75]. In a similar

269

study done in synovium explants stimulated with LPS, lower concentrations of leukocyte-

270

and platelet-rich gel induced higher gene expression levels of interleukin –1 receptor

271

antagonist protein (IL-1Ra).[78] In normal equine synovium, leukocyte-and platelet-rich gel

272

supernatant produced similar positive results when used in either higher or lower

273

concentrations. [79] These results may indicate that platelet-derived products with higher

11

274

concentrations of leukocytes and platelets may induce anabolic effects [79] however, type of

275

tissue and condition (inflamed versus normal) should be taken into consideration.

276

These in vitro results indicate that the anti-inflammatory and anabolic effects of the

277

platelet products depend upon the cellular and molecular profile of the PRP-derived product

278

used as well as the concentration. The concentration of leukocyte and the condition of the

279

tissue (inflamed versus normal) seemed to interfere directly in the response to PRP in both

280

synovium [78, 79] and in cartilage [75] experiments. Leucocyte–rich PRP-derived products

281

demonstrated potential positive effects however, only when used in lower concentrations or

282

in non-stimulated tissues. In fact, high concentrations of white blood cells (WBC) in platelet-

283

derived products were believed to be detrimental to joints due to the enzymes and cytokines

284

released by the WBC, [80] especially considering that in OA, both synovium and cartilage

285

are under pro-inflammatory condition.

286

Curiously when used in a human co-culture model of OA, the anti-inflammatory

287

competence of PRP was observed with the use of both leukocyte-rich and leukocyte poor

288

PRP. Platelet-rich plasma in different formulations reduced the expression of pro-

289

inflammatory markers and induced the expression of anti-inflammatory markers at 24, 48 and

290

72 hours of study [7]. Indeed, increased levels of mononuclear cells in platelet-derived

291

products such as APS demonstrated short- and long-term pain relief in human [81] and

292

equine [67].

293

In client-owned horses with naturally occurring OA, leucocyte-rich platelet-derived

294

product (APS) demonstrated significant improvement in lameness grade, range of motion and

295

asymmetry indices of vertical peak force in kinetic gait analysis by 14 and by 52 weeks after

296

treatment with no adverse effects [67]. Curiously when used in an in vitro study, APS did not

297

present significant anti-inflammatory effects in chondrocyte stimulated with IL-1β and TNF-

298

α. Although samples treated with APS or ACS presented increase secretion of IL-10 and IL-

12

299

1ra compared to controls, neither APS nor ACS reduced the expression of pro-inflammatory

300

genes in stimulated chondrocytes [82].

301

Additionally, in horses with moderate to severe forelimb OA, PRP treatment did not

302

present statistically significant improvement in gait, although authors did suggest a possible

303

beneficial effect despite the lack of statistical difference [9]. In another study, platelet lysate

304

(PL) was used to treat OA of the distal interphalangeal joint and treated animals were

305

compared to controls. From the ten horses that received treatment, nine returned to normal

306

athletic activities. However, no significant radiographic improvements were observed in

307

osteoarthritic lesions and horses gradually returned to their initial degree of lameness after the

308

seventh month of treatment. [83] Thus, the effects of platelet-products vary greatly according

309

to the model of research implemented and cellular content of the platelet-derived product,

310

and such facts should be taken into consideration when interpreting in vitro data to support

311

the clinical application of PRP. A summary of the in vivo studies using PRP in equine joints

312

can be observed on Table 4.

313

4.3 Factors that influence PRP effects

314

As PRP is usually used as an autologous treatment, standardization of this product is

315

impossible because of the individual characteristics. In horses, intrinsic factors such as age,

316

horse’s breed and sex [84], affected whole blood cellular composition, which could

317

potentially influence in PRP’s effects. For instance, Colombian creole horses (CCH)

318

presented significant increase in PDGF in platelet-derived products compared to Argentinean

319

creole horses (ACH) [84]. Similar findings were observed for young horses (less than 5 years

320

of age) compared to older horses and in females compared to males [84]. The authors

321

suggested that clinicians should consider intrinsic characteristics of the horse when using

322

platelet-derived products, since cellular and molecular composition could be influenced by

323

such factors [84].

13

324

Currently there is no standardization for PRP preparation and application, and several

325

methods have been described [52, 53, 57], which is one of the main reasons that cause

326

difficulty for comparison between manuscripts. There are many kits available for the

327

preparation of autologous platelet-derived products, and each preparation method will lead to

328

different products in terms of cellular composition and growth factor content [51] (Table 2).

329

It is also important to consider that there are differences in concentrations of such

330

components depending on the species in which the experiment is done [51]. Clear

331

divergences between results reported in human and equine studies were observed [51]. For

332

instance, equine platelet-derived products often present lower concentrations of platelets

333

compared to the same systems used in human [2, 51]. Different commercial kits have been

334

validaded for horses [51]. Autologous conditioned plasma (ACP) is one example, as well

335

as the equine platelet enhancement therapy (E-PET)[51]. E-PET use gravity for its

336

preparation and can be used in field conditions [51]. Manual techniques offer a low-cost

337

option for clinicians however, it is not only more laborious but also may increase the risk of

338

contamination during the preparation and is more prone to errors [51], Certain methods such

339

as apheresis for instance might provide more standardized and repeatable protocols [53], but

340

usually are more costly as previously discussed.

341

The response to PRP as a treatment is affected by several parameters related to the PRP

342

composition as well as the condition and type of tissue treated. In a wide range of variables in

343

PRP, the cellular composition of this product is one of the most discussed. In many studies

344

with different tissues, lower concentrations of platelets had an insufficient/sub-optimal

345

clinical effect, whereas higher concentrations were inhibitory for promotion of angiogenesis

346

in vitro [85]. An intermediate concentration of platelets in the PRP (2-6 fold the

347

concentration of the whole blood) exhibited optimal results in comparison with higher

348

concentrations on peri-implant bone regeneration [87]. The decrease of desired effects with 14

349

the use of highly concentrated platelet products might be due to a concentration-dependent

350

negative feedback. The higher concentration of platelets in the PRP do not necessarily

351

improve the healing capacity of this product [52]. The dose-response curve of the tissue to

352

PRP effects is not linear and inhibitory effects take place once a certain concentration of

353

platelets has been reached [88]. The excessive number of platelets can lead to apoptosis,

354

downregulation and desensitization of growth factor receptor resulting in a paradoxical

355

inhibitory effect [89, 90]. Such characteristics can justify an unsatisfactory effect of PRP-

356

derived products used in higher concentrations observed in some studies [52, 86, 87].

357

Therefore, intermediate concentrations of platelets might be more adequate for clinical use.

358

An additional factor that could contribute to observed deleterious effects of highly

359

concentrated PRP could be the concurrent higher level of leukocytes present within the

360

product. Leukocytes are believed by many authors to be deleterious for tissue healing

361

because of the release of reactive oxygen species (ROS), pro-inflammatory cytokines and

362

degradative enzymes [37, 80, 91]. Such cytokines may incite or intensify undesirable

363

inflammatory effects and delay tissue healing [ 80, 91], because of stimulation of the high

364

number of activated neutrophils, which can release catabolic factors [92]. However, these

365

conclusions are based on data from in vitro cell culture models [52, 91, 92].

366

Additionally, the type of leucocyte present within the product (neutrophils versus

367

monocytes), seems to play a role as well. While neutrophil- leucocyte enriched autologous

368

products can potentially have a deleterious effect [9, 92], increased number of monocytes

369

could have potential anti-inflammatory effects [67, 82, 93], especially after stimulation with

370

borosilicate beads (Autologous conditioned serum - ACS) [93] or polyacrylamide beads

371

(APS) [67].

15

372

The ratio between IL-1ra:IL-1β was significantly correlated with improved pain scores

373

six months after treatment in human patients diagnosed with early joint disease and treated

374

with a single intra-articular injections of APS [77]. In fact, IL-1Ra:IL-1β ratios of 10-1000

375

have been shown to inhibit activity of IL-1β in cell culture [94]. A possible explanation for

376

the increase levels of anti-inflammatory cytokine could be through microparticles from

377

platelets polarizing monocytes contained in APS inducing those to become M2, a pro-healing

378

macrophage during or after delivery of the product into the joint [95]. It is important to note

379

that APS and PRP are essentially different platelet-derived products (Table 2). In addition, in

380

order to have an enriched IL-Ra final product, cells need to be stimulated in special

381

conditions as previously mentioned [96]. The protocols used and consequently the final

382

cytokine concentration of the product influences on the observed results. Thus, not only the

383

concentration of WBC, but rather the type of blood cell (and stimulation of such cells) and

384

how those factors can influence on cytokine content and effects of platelet-derived products

385

need to be further clarified.

386

The activation methods used in PRP are another factor of variability in this therapy.

387

Platelets can be activated through different stimuli such as physical or chemical. In vivo,

388

platelets are mainly activated by the subendothelial collagen in combination with shear

389

forces, von Willebrand’s factor, thrombin, ADP or all those combined [29]. The exogenous

390

activation of platelet products was described initially for preparation of PRP in humans, to

391

induce maximum growth factor release and to use the fibrin clot formed as scaffold [63]. In

392

experimental conditions, platelets within the PRP products can be activated through

393

endogenous and exogenous methods using collagen, thrombin, ADP and calcium ionophores

394

[29]. The different methods of activation are an essential point in growth factor concentration

395

and dynamics of release [97]. Close to 70% of the growth factors are released within the first

396

10 minutes of activation and almost 100% of the growth factor content within the platelets

16

397

are believed to the secreted in the first few hours [99] . Further growth factors release may

398

happen depending on where the platelets are in their life cycle. Once activated, the platelets

399

release an initial “burst” of growth factors that is followed by a more sustained release [98,

400

100]. The physiological activation of platelets is more a continuous and stable process instead

401

of an acute “all-or-nothing” response. [101]

402

The kinetics of growth factor release can potentially influence PRP effects within the

403

tissue and it can vary according to the preparation method implemented [100]. Resting PRP

404

increased TGF-β1 and PDGF-BB over time, while platelet lysate (rehydrated lyophilized

405

platelets) reduced growth factor release over time [102]. In comparison to platelet-rich fibrin

406

(PRF), PRP demonstrated to release more growth factors after activation, but PRF

407

demonstrated greater TGF-β release over time. Both materials demonstrated a cumulative

408

TGF-β yield at the end of the study [103]. Platelet-derived growth factor did not show a

409

cumulative yield over time and presented a different kinetics of release depending on the

410

preparation used [103]. The results of the study indicated that PRF may trap PDGF-BB,

411

which would lead to a slower release [103]. These studies suggest that growth factor content

412

and kinetic of release depends not only on the agonist agent used but also the preparation

413

method which may influence on tissue healing.

414

In the horse, PRP activation is not as common as in humans and different methods have

415

been described, usually with using thrombin, CaCl2 [47] or an association of both [2].

416

Because of the lack of a consensus for PRP activation, some may suggest to use PRP in

417

resting form. The main issue with this practice is the fact that only a fraction of the growth

418

factors is released. A standardized approach could lead to a more effective and consistent

419

product for clinical use. For instance, equine platelets activated by calcium chloride had

420

significantly greater PDGF release compared to freeze-thaw and thrombin (equine and

421

bovine). It’s important to consider that a significant amount of growth factors were actually 17

422

retained in the clots formed after fibrin polymerization induced by thrombin. Transforming

423

growth factor β though, was comparable between the methods [47]. Interestingly, the same

424

experiment reviewed that PDGF levels were even greater in platelets treated with supra-

425

physiologic concentration of bovine thrombin compared to positive controls [47].

426

The main downside in using thrombin are the potential risks associated with its use,

427

especially when bovine thrombin is used for application of PRP in other species such as

428

equine [47]. Reactions such as an immune response to a foreign protein or nonspecific

429

proteolytic tissue degradation are potential side effects of use of bovine thrombin [104 ,105].

430

Autologous equine thrombin on the other hand, was not significantly effective in activating

431

platelets compared to bovine thrombin and CaCl2 [47]. The authors of this study

432

recommended CaCl2 for PRP activation as an effective and inexpensive method that is able to

433

provide up to 80% of total growth factor release [47]. However, in another study calcium

434

gluconate promoted greater gelation time, significant growth factor release without evident

435

calcium deposition in PRP clots compared to calcium chloride [106]. Since this is an

436

additional factor of variability and can influence on the growth factor composition of the final

437

product, a standardized method of activation is indicated for optimal results.

438

On top of the PRP treatment itself, the condition of the tissue prior to the use of platelet

439

products (acute versus chronic inflammation) and age of the subject submitted to treatment

440

should be considered. Better outcomes were reported in younger in comparison with older

441

patients and in more mild OA cases in comparison with severe ones [39, 107] . This might be

442

since platelets from aged human patients release less GF and seem to be less effective in

443

stimulating MSCs [107, 108]. Also, the use of non-steroidal anti-inflammatories (NSAID)

444

such as ketoprofen affected the platelet concentration in equine PRP, possibly due to the

445

decreased platelet aggregation caused by NSAIDs [109].

18

446

Finally, the protocol for PRP application is another factor that influences clinical results.

447

Additional PRP injections are not necessarily related to better outcome. In a double blinded

448

randomized placebo-controlled study, no statistical difference was observed between OA

449

patients treated with either one or two PRP injections [36].

450

In sum, the protocols applied for PRP might vary according to the tissue and condition in

451

which the product is used. Because of variation in processing techniques and application

452

protocols, as well as the patient’s own characteristics and operator’s variability, the efficacy

453

of a specific PRP product for a certain condition cannot be broadly applied to all PRP

454

products and conditions.

455

4.4 Limiting factors for PRP clinical use

456

Regardless of the positive results obtained in several studies, it is important to take into

457

consideration that many of the referred studies were not randomized and/or blinded

458

controlled trials [11,83, 107], and therefore should be regarded with caution. In fact, in a

459

recent meta-analysis, from 10 manuscripts selected for the study, 8 presented high risk of bias

460

[110]. The high heterogeneity among studies is another critical limitation of PRP [111].

461

Additionally, many of these studies suggest that autologous PRP is a safe treatment, however,

462

the potentially negative effects of PRP were not fully investigated at this point. Common

463

intra-articular adverse effects such as bleeding, bruising, peripheral nerve injury, stiffness and

464

soreness may occur as possible adverse effects in PRP injections [112].

465

As mentioned in this review, the variability of PRP composition which depends on the

466

commercial PRP kit used (processing technique), patient characteristics, as well as the PRP

467

application protocols, which make the comparison between studies challenging. [37] Based

468

on these issues, the Platelet-Activation-White blood cells (PAW) classification system was

469

proposed as a standardization method for researchers to more accurately compare the PRP

470

used between studies. The PAW system takes the following parameters into consideration;

19

471

the absolute number of platelets, the activation method and the presence or absence of white

472

blood cells [113]. Some papers have used this classification method for their PRP, however is

473

still not widely adopted.

474

The limited use of classification systems for PRP and lack of quality of clinical trials (as

475

double–blinded, placebo-controlled studies) creates difficulty for sustaining the clinical use

476

of PRP. Hence, because of the lack of convincing pre-clinical and clinical data, researchers

477

defy adversities to support the clinical use of PRP.

478 479 480

5.0 Future directions

481

[110, 111] lead to an increase interest in its application in sports medicine and orthopedic

482

surgery fields. The market in human medicine for PRP in 2009 was valued at 45 million

483

dollars and currently this value is estimated to have gone up to 126 million [114].

484

Although challenges still exist for the use of PRP clinically, positive anecdotal effects

Considering the vast demand for PRP use in orthopedic clinical practice, there are several

485

aspects of PRP application that could be optimized and standardized to allow a more

486

consistent and reliable use [37]. For instance, the determination of optimal characteristics in

487

PRP content to augment healing in different tissues and consequently, the development of

488

customized methods for preparation and application are potential targets for future research

489

[37, 115]. In addition, the clarification of appropriate timing, dosing and frequency of

490

treatment and how this should be applied in each tissue type, injury or surgical procedures are

491

also crucial [37]. Protocols that can allow customization of PRP specifically to the tissue and

492

condition as well as the removal of deleterious components might be one of the key factors

493

for an optimal use of PRP [37].

494

In conclusion, although several studies involving PRP use have been published in the past

495

10 years, the clinical efficiency of this product in the treatment of osteoarthritis remains to be

496

determined. The anecdotal positive results and the possibility of a lower cost regenerative

20

497

therapy lead companies to invest in the development of kits for the easy preparation of PRP,

498

which is appealing to the field clinician. As with any emerging treatment, improvement of

499

PRP use as a therapy is required and clinicians should be mindful of the realistic outcomes

500

and applications of PRP as a therapy for OA.

501

Acknowledgments: We would like to thank the Conselho Nacional de Desenvolvimento

502

Científico e Tecnológico (CNPq) for the concession of the scholarship for the graduate

503

student.

504

Funding: This review manuscript did not receive any specific grant from funding agencies in

505

the public, commercial, or not-for-profit sectors.

506

LIST OF FIGURE LEGENDS:

507

Figure 1: Biological functions of platelets. Indirect and direct roles of platelets. These

508

functions include the hemostasis process, anti-microbial defense, inflammation, tumor

509

biology, tissue healing and fib

510

is, and maintenance of vascular tone and integrity. Diagram based on the references;

511

Golebiewska and Poole[19], 2014; Jenne, Urrutia and Kubes, 2013[27], and Ghoshal and

512

Bhattacharva, 2014[116].

513

Figure 2: Platelet-rich plasma effects in osteoarthritic joints. Platelet-rich plasma

514

demonstrated to decrease the negative effects caused by IL-1β stimulation in chondrocytes

515

such as decreasing gene expression of collagen type 2 alpha 1 chain (COL-2A1) and

516

aggrecan (ACAN) genes and increasing expression of A disintegrin and metalloproteinase

517

with thrombospondin motifs (ADAMTS-4), prostaglandin-endoperoxide synthase-2 (PTGS-

518

2) and nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB) expression

519

[1].This product also demonstrated to reduce expression of C-X-C chemokine receptor type 4

520

(CXCR4), cyclooxygenase 2 (COX-2) and metalloproteinases (MMPs) in chondrocytes,

521

demonstrated to decrease apoptosis[69], and to reduce the expression of MMPs in synovium.

21

522

Platelet-rich plasma demonstrated to decrease expression of ADAMTS-5, TIMP-1 and to

523

increase expression of aggrecan in osteoarthritic cartilage [7]. In addition, this product

524

promoted resident-macrophage differentiation to M2 [95], and to decrease gene expression of

525

ADAMTS-5, vascular endothelial growth factor (VEGF) and tissue metalloproteinase

526

inhibitor (TIMP-1) in synovium from osteoarthritic joints, co-cultured with cartilage [7].

527

Finally, PRP demonstrated to induce increase expression of Col II, TIMP, transforming

528

growth factor beta (TGF-β), interleukins (IL-4, IL-10, IL-13) and glycosaminoglycan (GAG)

529

in chondrocytes [69], and to stimulated migration and proliferation of stem cells and

530

chondrogenesis [39]. Green arrows represent stimulation or upregulation, full red arrows

531

represent inhibition and dotted red arrows represent downregulation.

532

Figure 3: Anti-inflammatory effect of platelet-rich plasma. It has been demonstrated that

533

the anti-inflammatory effects of PRP are explained at least partially by the growth factors

534

present within it. Hepatocyte growth factor (HGF), present within PRP, demonstrated to

535

disrupt the NF-κB transactivating activity. This mainly happens due to the effect of HGF in

536

increasing nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor alpha

537

(IΚΒα) expression, leading to NF-κB-p65 subunit retention within the cytosol, and impeding

538

it to translocate to the nucleus to induce the transcription of pro-inflammatory factors such as

539

MMPs, ADAMTs and IL-1β [2]. Green arrow represents upregulation, purple arrow

540

represents stimulation and dotted red arrow represents inhibition.

541 542 543 544 545 546 547 548 549 550

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a modified Curasan kit, with preparations received from a local blood bank. Clin Oral Implants Res. 2003; 14(3):357-62. [57]. Textor J.A., Norris J.W., Tablin F. Effects of preparation method, shear force and exposure to collagen on release of growth factors from equine platelet-rich plasma. AJVR 2011; 72 (2):271-8. [58]. Cavallo C, Filardo G, Mariani E, Kon E , Marcacci M, Ruiz MTP, et al. Comparison of Platelet-rich plasma formulations for cartilage healing. An in vitro study. J Bone Joint Surg Am, 2014; 96:423-9. [59]. Waselau M, Sutter WW, Genovese RL, Bertone AL. Intralesional of platelet-rich plasma followed by controlled exercise for treatment of midbody suspensory ligament desmitis in Standardbred racehorses. J Am Vet Med Assoc 2008; 232(10):1515-20. [60]. Bosch g, Van Schie HT, de Groot MW, Cadby JA, van de Lest CH, Barneveld A, van Weeren PR. Effects of platelet-rich plasma on the quality of repair of mechanically induced core lesions in equine superficial digital flexor tendons: a placebo-controlled experimental study. J Orthop Res 2010; 28 (2):211-7. [61]. Rindermann Georg, Cislakova Maria, Arndt Gisela, Carstanjen Bianca. Autologous conditioned plasma as Therapy of tendon and ligament. J. Vet. Sci. 2010; 11 (2): 173-175. [62]. Yakar S, Liu JL, Stannard B, Butler A, Accili D, Sauer B., et al. Normal growth and development in the absence of hepatic insulin-like growth factor I. Proc Natl Acad Sci USA 1999;96:7324-9. [63]. Marx RE, Carlson ER, Eichstaedt RM, Schimmele SR, Strauss JE, Georgeff KR. Platelet-rich plasma: Growth factor enhancement for bone grafts. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 1998;85:638-646. [64]. Whitman DW, Berry RL, Green DM. Platelet gel: an autologous alternative to fibrin glue with applications in oral and maxillofacial surgery. J. Oral Maxillofac Surg 1997; 55:1294-9. [65]. Anitua E, Sanchez M, Orive G, Andia I. The potential impact of the preparation rich in growth factors (PRGF) in different medical fields. Biomaterials 2007; 28: 4551-4560. [66]. Sanchez M, Anitua E, Andia I, Padilla S, Mujika I. Comparison of surgically repaired Achilles tendon tears using platelet-rich fibrin matrices. Am J sports Med 2007; 35: 245-51. [67]. Bertone AL, Ishihara A, Zekas LJ, Wellman ML, Lewis KB, Schwarze RA, et al. Evaluation of a single intra-articular injection of autologous protein solution for treatment of osteoarthritis in horses. Am J Vet Res 2014;75:141-51. [68]. Tobita M, Tajima S, Mizuno H. Adipose tissue-derived mesenchymal stem cells and platelet-rich plasma: stem cell transplantation methods that enhance stemness. Stem Cell Res Ther 2015;6:215. [69]. Moussa M, Lajeunesse D, Hilal G, El Atat O, Haykal G, Sehal R, et al. Platelet rich plasma (PRP) induces chondroprotection via increasing autophagy, anti-inflammatory markers, and decreasing apoptosis in human osteoarthritic cartilage. Exp Cell Res 2017;352:146-56. [70]. Yang F., Hu H., Yin W., Li G., Yuan T., Xie X., et al Autophagy is independent of the chondroprotection induced by platelet-rich plasma releasate. BioMed Research International [Internet]. 2018 [cited 2019 May 31]; 2018: 9726703 [about 11p.]. Available from: https://www.ncbi.nlm.nih.gov/pubmed/?term=Autophagy+is+independent+of+the+chondrop rotection+induced+by+platelet-rich+plasma+releasate. [71]. McIlwraith CW. Current concepts in equine degenerative joint disease. J Am Vet Med Assoc 1982;180:239-50.

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[72]. Murakami S, Lefebvre V, de Crombrugghe B. Potent inhibition of the master chondrogenic factor Sox9 gene by interleukin-1 and tumor necrosis factor-alpha. J Biol Chem 2000;275:3687-92. [73]. Lieberthal J, Sambamurthy N, Scanzello CR. Inflammation in joint injury and posttraumatic osteoarthritis. Osteoarthritis Cartilage 2015;23:1825-34. [74]. Mifune Y, Matsumoto T, Takayama K, Ota S, Meszaros LB,Usas A, et al. The effect of platelet-rich plasma on the regenerative therapy of muscle derived stem cells for articular cartilage repair. Osteoarthritis Cartilage 2013;21:175-85. [75]. Carmona JU, Ríos DL, López C, Alvares ME, Perez JE, Bohorquez ME. In vitro effects of platelet-rich gel supernatants on histology and chondrocyte apoptosis scores, hyaluronan release and gene expression of equine cartilage explants challenged with lipopolysaccharide. BMC Vet Res 2016;12:135. [76]. Kon E, Engebretsen L, Verdonk P, Nehrer S, Filardo G. Clinical Outcomes of Knee Osteoarthritis Treated With an Autologous Protein Solution Injection: A 1-Year Pilot Double-Blinded Randomized Controlled Trial. Am J Sports Med 2018; 46:171-80. [77]. King W, Bendele A, Marohl T, Woodell-May J. Human blood-based anti-inflammatory solution inhibits osteoarthritis progression in a meniscal-tear rat study. J Orthop Res 2017;35: 2260-8. [78]. Ríos DL, López C, Álvarez ME, Samudio IJ, Carmona JU. Effects over time of two platelet gel supernatants on growth factor, cytokine and hyaluronan concentrations in normal synovial membrane explants challenged with lipopolysaccharide. BMC Musculoskelet Disord 2015;16:153. [79]. Ríos DL, López C, Carmona JU. Platelet-Rich Gel Supernatants Stimulate the Release of Anti-Inflammatory Proteins on Culture Media of Normal Equine Synovial Membrane Explants. Vet Med Int 2015;2015:547052. [80]. Sundman EA, Cole BJ, Fortier LA. Growth factor and catabolic cytokine concentrations are influenced by the cellular composition of platelet-rich plasma. Am J Sports Med 2011;39:2135-40. [81]. van Drumpt RA, van der Weegen W, King W, Toler K,Macenski MM. Safety and Treatment Effectiveness of a Single Autologous Protein Solution Injection in Patients with Knee Osteoarthritis. Biores Open Access 2016; 5: 261-8. [82].Linardi R.L., Dodson M.E., Moss K.L., King W.J., Ortved K.F. The Effect of Autologous Protein Solution on the Inflammatory Cascade in Stimulated Equine Chondrocytes. Frontiers in Veterinary Science. March 2019; 6:64. [83]. Tyrnenopoulou P, Diakakis N, Karayannopoulou M, Savvas I, Koliakos G. Evaluation of intra-articular injection of autologous platelet lysate (PL) in horses with osteoarthritis of the distal interphalangeal joint. Vet Q 2016;36:56-62. [84]. Giraldo CE, López C, Álvarez ME, Samudio IJ, Carmona JU. Effects of the breed, sex and age on cellular content and growth factor release from equine pure-platelet rich plasma and pure-platelet rich gel. BMC Vet Res 2013;9:29. [85].Giusti I, Rughetti A, D'Ascenzo S, Millimaggi D, Pavan A, Dell-Orso L, Dolo V. Identification of an optimal concentration of platelet gel for promoting angiogenesis in human endothelial cells. Transfusion 2009;49:771-8. [86]. Yoshida R, Cheng M, Murray MM. Increasing platelet concentration in platelet-rich plasma inhibits anterior cruciate ligament cell function in three-dimensional culture. J Orthop Res 2014;32:291-5. [87]. Weibrich G, Hansen T, Kleis W, Buch R, Hitzler WE. Effect of platelet concentration in platelet-rich plasma on peri-implant bone regeneration. Bone 2004; 34:665-71.

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791 792 793 794 795 796 797 798 799 800 801 802 803 804 805 806 807 808 809 810 811 812 813 814 815 816 817 818 819 820 821 822 823 824 825 826 827 828 829 830 831 832 833 834 835 836 837 838 839

[88]. Alberts B BD, Lewis J, Raff M, Roberts K, Watson JD. Molecular Biology of the Cell. 3rd ed. New York: Garland Science; 1994. [89]. Haynesworth SE, Kadiyala KS, Liang LN, Bruder SP. Mitogenic stimulation of human mesenchymal stem cells by platelet release suggest a mechanism for enhancement of bone repair by platelet concentrates. Proceedings of the 48th Meeting of orthopedic Research Societ; 2002; Boston, USA. [90]. Gruber R, Varga F, Fischer MB, Watzek G. Platelets stimulate proliferation of bone cells: involvement of platelet-derived growth factor, microparticles and membranes. Clin Oral Implants Res 2002;13:529-535. [91]. McCarrel TM, Minas T, Fortier LA. Optimization of leukocyte concentration in platelet-rich plasma for the treatment of tendinopathy. J Bone Joint Surg Am 2012;94:e143(141-148). [92]. Boswell SG, Schnabel LV, Mohammed HO, Sundman EA, Minas T, Fortier LA. Increasing platelet concentrations in leukocyte-reduced platelet-rich plasma decrease collagen gene synthesis in tendons. Am J Sports Med 2014;42:42-9. [93]. Frisbie D.D., Kawcak CE, Werpy NM, Park RD, McIlwraith CW. Clinical, biochemical, and histological effects of intra-articular administration of autologous conditioned serum in horses with experimentally induced osteoarthritis. Am J Vet Res. 2007 Mar; 68(3):290-6. [94]. Argüelles D, Carmona JU, Pastor J, Iborra A, Vinals L, Martinez P, et al. Evaluation of single and double centrifugation tube methods for concentrating equine platelets. Res Vet Sci 2006;81:237-45. [95]. Vasina EM, Cauwenberghs S, Feijge MA, Heemskerk JW, Weber C, Koenen RR. Microparticles from apoptotic platelets promote resident macrophage differentiation. Cell Death Dis 2011;2:e211. [96]. Wehling P, Moser C, Frisbie D, McIlwraith CW, Kawcak CE, Krauspe R, et al. Autologous conditioned serum in the treatment of orthopedic diseases: the orthokine therapy. BioDrugs 2007;21:323-332. [97].Mazzocca AD, McCarthy MB, Chowaniec DM, Cote MP, Romeo AA, Bradley JP, et al. Platelet-rich plasma differs according to preparation method and human variability. J Bone Joint Surg Am 2012; 94:308-16. [98]. Anitua E, Zalduendo MM, Alkhraisat MH, Orive G. Release kinetics of platelet-derived and plasma-derived growth factors from autologous plasma rich in growth factors. Ann Anat 2013;195:461-6. [99]. Knezevic NN, Candico KD, Desai R., Kaye AD. Is Platelet-rich Plasma a Future Therapy in Pain Management? Med. Clin North Am 2016; 100:199-2017 [100]. Oh JH, Kim W, Park KU, Roh YH. Comparison of the cellular composition and cytokine-release kinetics of various platelet-rich plasma preparations. Am J Sports Med 2015; 43(12): 3062-70. [101]. Jurk K, Kehrel BE. Platelets: physiology and biochemistry. Semin Thromb Hemost 2005;31:381-92. [102]. McCarrel T., Fortier L. Temporal growth factor release from platelet-rich plasma, trehalose lyophilized platelets, and bone marrow aspirate and their effect on tendon and ligament gene expression. J Orthop Res 2009; 27(8):1033-42. [103]. McLellan J., Plevin S. Temporal Release of growth factors from platelet-rich fibrin (PRF) and Platelet-rich plasma (PRP) in the Horse: A comparative in vitro analysis. Intern J Appl Res Vet Med. 2014; 12 (1): 44- 53. [104]. Clark J, Crean S, Reynolds MW. Topical bovine thrombin and adverse events: a review of the literature. Curr Med Res Opin 2008;24:2071-87.

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[105]. Diesen DL, Lawson JH. Bovine thrombin: history, use, and risk in the surgical patient. Vascular 2008;16 Suppl 1:S29-36. [106]. Giraldo C.E.; alvarez M.E.; Carmona J.U. Influence of calcium salts and bovine thrombin on growth factor release from equine platelet-rich gel supernatants. Vet Comp Orthop Traumatol 2017; 30 (1):1-7. [107]. Salini V, Vanni D, Pantalone A, Abate M. Platelet Rich Plasma Therapy in Noninsertional Achilles Tendinopathy: The Efficacy is Reduced in 60-years Old People Compared to Young and Middle-Age Individuals. Front Aging Neurosci 2015;7:228. [108]. Lohmann M, Walenda G, Hemeda H, Joussen S, Drescher W, Jockenhoevel S, et al. Donor age of human platelet lysate affects proliferation and differentiation of mesenchymal stem cells. PLoS One 2012;7:e37839. [109]. Rinnovati R, Romagnoli N, Gentilini F, Lambertini C., Spadari A. The influence of environmental variables on platelet concentration in horse platelet-rich plasma. Acta Vet Scand 2016;58:45. [110]. Dai WL, Zhou AG, Zhang H, Zhang J. Efficacy of Platelet-Rich Plasma in the Treatment of Knee Osteoarthritis: A Meta-analysis of Randomized Controlled Trials. Arthroscopy 2017;33: 659-670.e651. [111]. Shen L, Yuan T, Chen S, Xie X., Zhang C. The temporal effect of platelet-rich plasma on pain and physical function in the treatment of knee osteoarthritis: systematic review and meta-analysis of randomized controlled trials. J Orthop Surg Res 2017;12:16. [112]. Dhillon RS, Schwarz EM, Maloney MD. Platelet-rich plasma therapy - future or trend? Arthritis Res Ther 2012;14:219. [113]. DeLong JM, Russell RP, Mazzocca AD. Platelet-rich plasma: the PAW classification system. Arthroscopy 2012; 28: 998-1009. [114]. GlobalData [Internet]. Platelet rich plasma:a market snapshot. c2013 -[cited 2019 May 31]. Available from: http://www.docstoc.com/docs/47503668/Platelet-Rich-Plasma-AMarket-Snapshot. [115]. Salamanna F, Veronesi F, Maglio M, Della Bella E., Sartori M., Fini M. New and emerging strategies in platelet-rich plasma application in musculoskeletal regenerative procedures: general overview on still open questions and outlook. Biomed Res Int 2015;2015:846045. [116]. Ghoshal K., Bhattacharva M. Overview of platelet physiology: its hemostatic and nonhemostatis role in disease pathogenesis. ScientificWorldJournal, 2014;1-16. [117]. Pichereau F., Decory M., Ramos G.C. Autologous Platelet Concentrate as a Treatment for Horses with Refractory Fetlock Osteoarthritis. Journal of Equine Veterinary Science 2014; 43: 489-93. [118]. Moraes A.P.L., Moreira J.J., Brossi P.M., Machado T.S.L., Michelacci Y.M., Baccarin R.Y.A. Short-and long-term effects of platelet-rich plasma upon healthy equine joints: Clinical and laboratory aspects. Can Vet J 2015;56:831-38. [119]. Bembo F., Eraud J., Philandrianos C., Bertrand B., Silvestre A., Veran J., et al. Combined use of platelet rich plasma & micro-fat in sport and race horses with degenerative joint disease: preliminary clinical study in eight horses. Muscles, Ligaments and Tendons Journal 2016;6 (2):198-204.

29

889

TABLES:

890

Table 1 Platelet granule α - granule

Dense bodies or granules (or δ-granules) Lysosomes

Content Growth factors (VEGF, PDGF, TGF-β, EGF etc), coagulation factors (factor V, XI, XIII), fibrinolytic inhibitors (α2-plasmin inhibitor, PAI-1), immunoglobulins, chemokines (CXCL7,4,5 etc), CD63, CD3, adhesive glycoproteins (fibrinogen, fibronectin, vWF, thrombospondin, Pselectin) Serotonin, histamine, Ca2+, Mg2+, nucleotides (ATP, ADP, GTP, GDP) Carboxypeptidases, cathepsins, acid phosphatase, collagenase, heparinase, βglucuronidase, β-galactosidase etc

891 892

Table 1: Platelet granules and their content. Platelets contain three main types of granules

893

that release their content once platelets are activated. Each granule contains different

894

molecules that are involved in platelet activation, modulation of the inflammatory process

895

and immunity (Jenne, Urrutia and Kubes, 2013 [27]).

896 897 898 899

30

900 901 Commercial Product

Type of Plateletrich product

Amount of blood

Anticoagulant

Equipment necessary

Protocol

Amount of platelet product

Amount of platelet (10 3 /µL)

Dilution of platelets

in:

Amount of leucocyte (10 3 /µL)

recovered

Angel

Autologous platelet concentrate/Platelet -rich plasma

60 mL

ACD-A

Angel spin system

ACP

Autologous conditioned plasma/Platelet-rich plasma Autologous platelet concentrate

10mL

ACD-A (Oh et al, 2015)

60 mL

ACD-A solution

GPSIII

Autologous platelet concentrate

60 mL

ACD-A solution

ACP double syringe and centrifuge E-PET system, no centrifuge required GPSIII and centrifuge

SmartPReP 2 system

Platelet-rich plasma

60 mL

ACD anticoagula nt

Secquire

Platelet concentrate/ Platelet-rich plasma

50 mL

Pro-Stride

Autologous plasma

55 mL

E-PET

One centrifugation 1200 xg for 16 minutes

1.5-2 mL collected and diluted to a 6mL final volume 6 mL

IL-1Ra and/or antiinflammator y cytokines

Reference

Not available

Hessel et al. 201551*

Oh et al, 2015100. Hessel et al. 201551* Hessel et al. 201551*

320 ±198.1

Plasma

9.1 ± 6

183.2± 39.7

Plasma

0.9 ± 0.3

0.85 and 0.28 ng/ml

Not available

11± 2.5

5.27 and 1.7 ng/ml

Not available

40.6 ± 3.9

5.16 and 0.68 ng/ml TGFβ1: 8-10 ng/mL; PDGFBB: 810 ng/mL and IGFI: < 2g/mL* TGFbeta 1: 15.3 ng/µL TGFbeta 2:1 and IGF: 107.4 ng/µL** Enriched

Not available

Hessel et al. 201551*

Not available

Schnabel et al, 200710*

Not available

Sutter et al, 200454

IL-

Bertone et al,

Centrifugation at 352xg for 5 minutes No centrifugation necessary

6 mL

533.3± 198.2

Centrifugation 15 minutes at 1100xg

6.5 mL

761±240

2% sodium chloride solution Plasma

SmartPReP 2 centrifuge system

*Protocol provided by manufacturer

10 mL

395± 44

Plasma

4.48

ACD anticoagula nt

Secquire Kit and Supplier centrifuge

First centrifugation 2100 xg for 9 minutes (to separate components of the blood) followed by another centrifugation 200 xg for 3 minutes

5 mL

1,472.5

Plasma

32.5

ACD

Pro-Stride -

Two centrifugation

5-6 mL

243± 53

Plasma

75± 4.8

31

Growth -factors (PDGFBB and TGF-b) 2.44 and 0.66 ng/ml

solution (APS)

902 903

• •

anticoagula nt

APS kit (separator and concentrator) and centrifuge

steps of 15 and 2 minutes (1 for separator and another for concentrator respectively)

with PDGFAB/BB, IGF-1, EGF and TGFb1*

1Ra:1,757±10 0 pg/ml sTNF receptor 1: 16.9±2.9 pg/ml IL-10: 3,271±807 ng/ml

201467

* Data based on mean calculated from N=6 horses (Hessel et al [51] and Schnabel et al, 2007 [10]). ** Product tested in 15 horses (Sutter et al, 2004 [54]).

904 905

Table 2: Commercial kits available for preparation of platelet-derived products in horses. Several kits are available to the clinician, each

906

one has a different protocol for preparation and may require additional equipment or material.

907 908 909 910

Table 3 Platelet Product P-PRP

Composition

L-PRP

Some PPP+ buffy coat+ residual RBC Fraction of PPP+ small fraction of buffy coat PPP+ buffy coat + CaCl2

Anitua’s PRGF P-PRF (Fibrinet PRFM) L-PRF (Choukroun’s method)

Small fraction of PPP + buffy coat fraction

Buffy coat (Platelets +leucocytes) +circulating molecules (growth factors) +fibrin

Platelet concentration High or low depending on the method Moderate

Leucocyte concentration Low

Fibrin density Low

Fibrin polymerization Weak

*Liquid/gel

Moderate

Low

Weak

Liquid

Low

Low

Low

Weak

Liquid

Moderate

Low

High

Strong

Clot

High

Moderate

High

Strong

Clot

911

32

Form

912 913 914 915 916 917 918 919

Table 3: Platelet-derived products. These products can vary according to the cellular and fibrin content, as well as the fibrin density and polymerization. Such factors will influence on how PRP can be delivered as well as its effects. Descriptions based on Dohan Ehrenfest et al (2009) [48]. Leucocyte and platelet concentration: Low (less than 40% of cells), Moderate (between 40-80%) and High (more than 80%). Percentage (%) values are refereed as percentage of cells recovered after the spin; Fibrin polymerization type: Strong – mainly trimolecular or equilateral junctions and Weak- mainly tetramolecular or bilateral junctions. Table information based mainly on kits and methods used in human therefore, the classification of the platelet concentrate is based on studies performed in humans. P-PRP: Pure platelet-rich plasma, L-PRP: leucocyte and platelet-rich plasma, PRGF: platelet-rich in growth factors, P-PRF: platelet-rich fibrin, L-PRF: leucocyte and platelet-rich fibrin.

920 921

Table 4 Study

Study design

Placebo/control used ( if any)

Length of the study

Number of horses

Sex

Type of lesion

Plateletderived product used

Bertone et al, 201467

Prospective randomized masked placebocontrolled clinical trial

Saline (control group) *comparison of treated horses with itself before treatment

52 weeks Outcome parameters evaluated for 15 days and owner’s lameness assessment up to 52 weeks.

40

M/F

Naturally occurring osteoarthritis

Autologous Protein Solution (NStride Arthritis Treatment) No platelet activation

16 weeks

12

Mirza et al, 20169

Case-control/ case series

Results of the treated horses were compared to itself in two

M/F

Naturally occurring osteoarthritis (moderate to severe)

33

Autologous plateletderived product (E-

Classification of plateletrich plasma (if PRP was used) PAW** classification system Platelet concentration (P2) leukocyte concentration (A) No exogenous activation Product diluted in plasma

Outcome parameters used

Results

Comments and Potential Pitfalls

-Subjective lameness -Kinetic gait analysis -Joint signs of pain, swelling -Synovial fluid and blood analysis -Radiography

-Significant improvement in subjective and objective lameness grade and range of motion by 14 days.

Platelet concentration (P3), leukocyte

-Kinetic analysis

-Kinetic gait changes were variable among

-Results were effective only in moderate to mild lameness and joints without significant degenerative changes -Long term evaluation (52 weeks) assessed based on client opinion only (higher subjectivity) -Symptom improvement, but no evidence of disease modifying effect -Definition of a positive response (improvement percentage),

-Radiographs

-Comfort levels improved at 12 and 52 weeks.

situations: before treatment and after intraarticular anaesthesia

Textor and Tablin, 201350

Experiment in vivo study/ safety study

Saline

5 days

7

M/F

Healthy/normal joints

PET system). No activation

level (A). Product diluted in hypertonic solution

Autologous

Platelet concentration (P2), leukocyte(A). Diluted in hypertonic solution

Plateletderived product (EPETTM) activated with (CaCl2 , bovine thrombin and resting platelet product)

horses treated - No significant improvement was observed

-Vital signs -Synovial effusion Periarticular

effects Flexion

-Synovial fluid cytology

-Significant increase in WBC in synovial fluid at 24 hours. -Thrombin activated PRP lead to greater effusion, periarticular signs, lameness and WBC concentration

after treatment.

Tyrnenopoulou

et al, 201683

Case-control study

Saline

1 year

15

M /F

Osteoarthritis of the distal interphalangeal joint

34

Plateletlysate (plateletlysate through freeze-

Platelet concentration (P2) leukocyte level (B)

Subjective lameness exam and radiographs

-Significant lower lameness grades 10 days after second

increased probability of bias. -Study did not use proper controls (compared treated horse with itself after intraarticular anaesthesia) -Not clear if horses were subjected to other treatments concurrently or previously to study -Great variability in the platelet product used -Treatments were applied simultaneously in different limbs in the same horse. -Mild systemic inflammatory response may have influenced in result. -Once all limbs were used, lameness could not be accurately measured. -No radiographs performed in horses before study (for confirmation that joints were healthy) -Lower number of controls compared to treated horses. -No objective analysis (besides radiograph).

thaw method diluted in plasma)

Pichereau, Decory and Ramos, 2014117

Case series

None * comparison of treated horses with itself before treatment

Outcome parameters evaluated for 15 days and owner’s lameness assessment up to1 year

20

M/F

Chronic OA of fetlock (metatarsusphalangeal joint)

Plateletplateletconcentrate (diluted in PBS) and activated with CaCl2

Platelet number(P2) and leukocyte level (B)

Moraes et al, 2015118

Experimental control in vivo study

Saline *Horses were treated with PRP and 15 days later, treated with Saline in the contralateral joint.

28 days

8

M/F

Healthy metacarpophalangeal joints

Plateletrich plasma. No platelet activation

Platelet concentration (P2). No concomitant increase in leucocyte.

Bembo et al,

Case series

None

10 months

8

M/F

Degenerative joint

Platelet-

High platelet

35

Subjective lameness analysis performed by clinicians and owners -Growthfactor analysis of plateletproduct (Plateletderived growth factor PDGF), and IL-1beta analysis of synovial fluid (during 15 initial days of study) -Synovial fluid cytology, verification of chondroitin sulphate and hyaluronic acid, IL1ra, TNFalfa, PGE2 and clinical exam Subjective

injection. -No significant radiographs changes were observed. -Horses relapsed after one year. -Significant clinical improvement of 80% of the horses in the study, up to 1 year after treatment. -Decrease of IL-1β levels in joints (after 15 days) -Decrease in lameness correlated with decrease in IL-1β levels.

-Significant results relied only on subjective measurements.

-PRP promoted a mild selflimiting inflammatory response in joints, compared to saline with no further compromise to articular cartilage

-Low number of horses -Not-blinded

-Improvement

-Low number of

-No controls -Positive results mainly based in subjective analysis -Long term positive results based on subjective analysis performed by owner (not clear if clinicians participated)

2016119

922 923 924 925 926 927 928 929 930

* comparison of treated horses with itself before treatment

disease (fetlock and carpus)

rich plasma in addition to microfat. No platelet activation.

concentration and leucocyte below baseline count

lameness scale and return to competition and training

in lameness score within three months of the study. -Most of horses returned to competition or intensive training

horses and variability of lameness degree included in study -Short follow-up -Lack of controls -Positive results only based on subjective analysis. -No justification of why micro-fat was used in addition to PRP and the potential synergic effects (if any) -Lack of proper groups in general (PRP only, microadipose only, PRP+ micro adipose only and control) -No characterization/or count of stem cells that authors claim to be present within the micro-fat treatment.

PAW Classification system: Platelet concentration (platelets/µ µL); P1- bellow the baseline, P2 – greater then 1x and below 4x baseline values, P3- 4x to 6x the baseline values and P4- greater than 6 x baseline values. Leucocyte concentration: A – above baseline levels, Bbelow baseline levels (DeLong et al, 2012)113. ** Based on PAW classification system. Manuscripts analyzed did not communicate fold-increase in platelet or leucocyte number in PRP. Classification was done by the authors of the current manuscript, based on the data provided on each clinical study cited and the guidelines according to the PAW classification system (DeLong et al, 2012)113.

36

931

Table 4: Summary of the publications about the use of platelet-rich plasma in equine joints.

37

PRP ACAN Col2A1 TIMP-1 PTGS2 ADAMTS

ADAMTS-5 VEGF TIMP

NFκ-B

IL-1β

COX-2 CXCR-4 MMPs

Articular cartilage

COL II GAG TGF- β IL- (4,10,13) TIMP

MMPs

Synovial membrane

LPS

TNF-α

IL-1β

PRP HGF

TNF-R

IL-1R

TLRs

IΚΚγ / NEMO IΚΚα

IΚΚβ

p50 IΚΒα p65

MMPs ADAMTs IL-1β

IΚΒα

HIGHLIGHTS •

Platelet-rich plasma (PRP) is a biological preparation containing platelet concentration above the whole blood baseline.

•

Due to the high concentrations of growth factors, this product has been extensively used in musculoskeletal disorders to modulate progression of the inflammatory process and promote healing.

•

Osteoarthritis is a main cause of musculoskeletal disabilities in horses

•

PRP could offer a more practical and accessible option of biological therapy for osteoarthritis compared to other biological therapies.

•

Several factors related to PRP preparation, application and intra-individual variability lead to inconsistent clinical results, which precludes the formation of reliable conclusions about the efficacy of this product for clinical use in osteoarthritis.

ETHICAL STATEMENT This manuscript is a review article and therefore the authors Dr. Livia Garbin and Dr. Christine Olver do not have any ethical considerations to declare.

CONFLICT OF INTEREST STATEMENT

The authors of this manuscript, Dr. Livia Garbin and Dr. Christine Olver, declare that there is no conflict of interest in the publication of this manuscript.