Enhanced Rotator-Cuff Repair Using Platelet-Rich Plasma Adsorbed

Subscriber access provided by Access provided by University of Liverpool Library

Article

Enhanced Rotator-cuff Repair Using Platelet-Rich Plasma Adsorbed on Branched Poly(ester urea)s Erin P. Childers, Nathan Z. Dreger, Alex B. Ellenberger, Mary Beth Wandel, Karen Domino, Yanyi Xu, Derek Luong, Jiayi Yu, David Orsini, Robert H. Bell, Christopher Premanandan, Stephen D Fening, and Matthew L. Becker Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.8b00725 • Publication Date (Web): 15 Jun 2018 Downloaded from http://pubs.acs.org on June 16, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 31 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

1

Enhanced Rotator-cuff Repair Using Platelet-Rich

2

Plasma Adsorbed on Branched Poly(ester urea)s

3

Erin P. Childersa‡, Nathan Z. Dregera‡, Alex B. Ellenbergera Mary Beth Wandela, Karen

4

Dominoa, Yanyi Xua, Derek Luonga, Jiayi Yua, David Orsinib, Robert H. Belld, Christopher

5

Premanandan e, Stephen D. Fening*c, and Matthew L. Becker* a f

6 7 8 9 10 11

a

Department of Polymer Science, The University of Akron, Akron, OH 44325, USA,b Summa Health System, Akron, OH 44304, USA,c Department of Biomedical Engineering, Case Western Reserve University, Cleveland, OH 44106, USA,d Department of Orthopaedics, Crystal Clinic Inc., Akron, OH 44333, USA, e Department of Veterinary Biosciences, College of Veterinary Medicine, The Ohio State University, Columbus, OH, 43210, USA, f Department of Biomedical Engineering, The University of Akron, Akron, OH 44325, USA,

12 13 14

KEYWORDS: poly(ester urea), platelet-rich plasma, rotator cuff, soft-tissue engineering

15

1

ACS Paragon Plus Environment

Biomacromolecules 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

16

ABSTRACT

17

Platelet-rich plasma (PRP) is a clinically relevant source of growth factors used commonly by

18

surgeons. The clinical efficacy of PRP use as reported in the literature is widely variable which is

19

likely attributed to poorly defined retention time of PRP at the repair site. To overcome this

20

limitation, branched poly(ester urea) (PEU) nanofibers were used to adsorb and retain PRP at the

21

implant site in an acute rotator-cuff tear model in rats. The adsorption of PRP to the branched-

22

PEU 8% material was characterized using quartz crystal microbalance (QCM) and immuno-

23

protein assay. After adsorption of PRP to the nanofiber sheet, the platelets actively released

24

proteins. The adhesion of platelets to the nanofiber material was confirmed by

25

immunofluorescence using a p-selectin antibody. In vivo testing using a rat rotator-cuff repair

26

model compared five groups; no repair (control), suture repair only, repair with disc implant

27

(Disc), repair with PRP-soaked disc (Disc PRP), and a PRP injection (PRP). Mechanical testing

28

at 84 d for the four surgical repair groups resulted in a higher stiffness (11.8 ± 3.8 N/mm, 13.5 ±

29

3.8 N/mm, 16.8 ± 5.8 N/mm, 12.2 ± 2.6 N/mm, respectively) for the Disc PRP group.

30

Histological staining using trichrome, hematoxylin and eosin Y (H&E), and safranin O

31

confirmed more collagen organization in the Disc PRP group at 21 d and 84 d. Limited

32

inflammation and recovery towards preoperative mechanical properties indicate PEU nanofiber

33

discs as translationally relevant.

34 35 36 37 38 39 40 41 42 43 44 45 46 47 48

2


Page 2 of 31

Page 3 of 31 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

49 50

Biomacromolecules

1. Introduction One of the most challenging repairs in orthopedic surgery is the reattachment of tendon to

51

bone. Rotator cuff repairs are particularly difficult for tears larger than 3 cm that require surgical

52

intervention or when chronic symptoms and pain last longer than 6 months. 1-3 The current repair

53

techniques (>250,000 per year) use sutures and anchors to secure the tendon to the bone. Despite

54

surgical intervention, healing is often slow and incomplete leading to poor remodeling and

55

frequent failures. Re-tear rates exceed 20% for partial tears and 90% for full rotator cuff tears.1-4

56

The high failure rates highlight the need for new approaches to enhance tendon-bone healing.

57

There has been increased interest in alternative or supplemental treatments in orthopedic

58

medicine utilizing platelet-rich plasma (PRP) to aid healing.5-10 PRP is isolated from a patient’s

59

whole blood using a serial centrifugation procedure that results in a concentrated solution of

60

platelets.11 Administration of PRP to the surgical site has been employed as a way to capitalize

61

on the hemostatic role that platelets play in clot formation, growth factor secretion, and healing.6,

62

12-14

63

involved in platelet activation and tendon-bone healing with great success.15,16-18 This makes the

64

lack of clinical efficacy all the more confounding and has limited widespread use of PRP

65

implementation in orthopedic procedures.5, 6, 9, 10, 19, 20 The lack of clarity in clinical procedures

66

may be attributed to the administration methods and lack of sustained delivery vehicle. With

67

current administration methods, there is little to keep PRP localized at the surgical site. It would

68

be ideal to have a PRP vehicle to provide sustained release throughout the healing process.

69

A great amount of in vitro work has been done to characterize platelets and the mechanisms

A number of synthetic polymers have been widely investigated for biomedical uses, motivated

70

by the ability to tailor the chemical, mechanical, and degradative properties to the desired

71

application. 22-28 The dominant degradable polymers utilized clinically are polyesters which

3



72

include poly(ε-caprolactone) (PCL), poly(lactic acid) (PLA), poly(glycolide) (PGA), and their

73

copolymers.29-33 While these materials have been used with great success in drug release

74

applications and as resorbable sutures, their degradation byproducts are prone to creating local

75

acidic environments and inflammation which precludes them from long term applications.29–31

76

Additionally, the rigid mechanical properties of these materials limit their use in soft-tissue

77

regeneration.32, 33 Efforts have been made to diversify the pool of degradable synthetic polymers

78

for more advanced medical applications, including the investigation of polycarbonates,34, 35

79

poly(α-amino acids),36, 37 and poly(ester urea)s (PEU)s.38-44 PEUs in particular are synthetically

80

flexible and the resulting properties afford opportunities to target multiple tissue applications

81

including bone repair,39, 41, 43, 44 vascular grafts,38 hernia repair,42 and other tissues while not

82

producing local acidic degradation products leading to limited inflammation.45 Previous work on

83

L-phenylalanine

84

on these substrates and the degradation products are non-toxic in vitro and in vivo.38, 41

85

Additionally, PEUs have the ability to be fabricated in to nanofiber films through electrospinning

86

with porosity ideal for cellular integration and small molecule adsorption.

and L-leucine-based PEUs has demonstrated that cells proliferate phenotypically

87

Considering the challenges involved in sustained PRP release, PEUs have the potential to be a

88

translatable delivery vehicle for sustained PRP release at the surgery site. In this initial study, a

89

branched-PEU (with 8 % branching composition) was synthesized into nanofiber discs43 and

90

adsorbed with PRP to enhance the healing response in an acute rotator-cuff tear in a rat model

91

with hopes of overcoming the pitfalls of current PRP administration methods.

92 93 94

4


Page 4 of 31

Page 5 of 31 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

95

2. Materials and methods

96

2.1. General materials

97

All reagents and solvents were purchased from Sigma Aldrich (St. Louis, MO) or Acros

98

(Morris Plains, NJ) and used without further purification. The branched-PEU 8% (Scheme 1) has

99

an extent of branching of 8 %. The synthesis and characterization has been described

100

previously.43

101 102 103

2.2. Adsorption of PRP onto branched-PEU 8% using quartz-crystal microbalance The adsorption profiles of PRP were obtained using a quartz crystal microbalance with

104

dissipation (QCM-D) (Qsense E4, Biolin Scientific AB). The branched-PEU 8% was spin coated

105

onto SiO2 QCM chips (Qsense, 335 SiO2) using a 2 % (wt/wt) solution of polymer in N, N-

106

dimethylformamide (DMF) at 2000 rpm for 1 min. The film thickness on the QCM chips was

107

measured using an ellipsometer (J.A Woolham, M-2000) over a spectral range of 250 nm to

108

17000 nm, between angles 50°-70°, with incrememnts taken every 5°. The measurements were

109

fit using a model for the blank chip and for chips coated with branched-PEU 8%. The thickness

110

of the spun coated polymer films were ~39 ± 4 nm.43

111

The PRP was isolated from whole blood taken from Sprague Dawley female rats through

112

cardiac puncture (IACUC protocol (15-01-2-BRD). Acid-citrate-dextrose (ACD) anticoagulant

113

solution (85.0 mM trisodium citrate dihydrate, 66.6 mM citric acid monohydrate, and 111.0 mM

114

D-glucose) was prepared and used at a ratio of 1 : 6 (v:v) (anticoagulant : blood).45 The isolation

115

of PRP was prepared according to a modified version to previous methods.46, 47 Samples were

116

centrifuged at 600 × g for 30 mins to isolate plasma from the red blood cells. The plasma was

117

then centrifuged at 1400 × g for 15 mins to isolate the platelets. The platelet pellet was 5



118

resuspended with platelet-poor plasma at 10 % (v/v) of the original volume. The PRP adsorption

119

profiles were obtained using PRP solutions (× 250 dilution). The flow rate on the QCM was 150

120

µL/min and the shifts in frequency and dissipation were measured at overtones (n = 3, 5, 7, and

121

9). The 3rd overtone was plotted and reported. The chips were equilibrated in air and then in

122

phosphate buffered saline solution (PBS, 7.4 pH; Fisher Scientific, Pittsburgh, PA). PRP

123

solutions were added until the frequency stabilized, and equilibrium was achieved. Lastly, a PBS

124

wash was passed over the sensors. The QTools (Qsense) program normalizes the data by the

125

overtone, therefore the reported ߂f is actually (߂f/n). The frequency at the nth harmonic was

126

converted to real mass (∆m) (ng ● cm-2) by the Sauerbrey equation:

127 ∆m=-CQCM x ∆f 128 129

where CQCM is the mass sensitivity constant (17.7 ng/cm Hz).

130 131 132

2.3. Branched-PEU 8% implant fabrication Well-defined nanofibers of branched-PEU 8% were fabricated by dissolving the PEU in

133

1,1,1,3,3,3 hexafluoroisopropanol (HFIP) (6 wt %) and the solution was electrospun at a voltage

134

of 12 kV using a 25-gauge needle and collected at a distance of 15 cm on grounded aluminum

135

foil. The morphology of the nanofibers was characterized dry at room temperature (RT), and one

136

year later using field-emission scanning electron microscopy (SEM) (JSM-7401F, JEOL,

137

Peabody, MA). Acceleration voltage for the SEM imaging was 5.00 kV. All samples were

138

vacuum dried at room temperature and sputter coated with silver prior to scanning.43 The

139

surgical samples were cut into disc shapes (Figure 1) using a hole punch with an outer diameter

6


Page 6 of 31

Page 7 of 31 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

140

of 4 mm and inner diameter of 2 mm. Sterilization by ethylene oxide (EtO) gas treatment was

141

performed using a standard EtO 24 h cycle sterilization procedure (Andersen Sterilizers, Inc. AN

142

74i Anprolene gas sterilizer) followed by a 48 h purge.

143 144 145

2.4. Protein adsorption and release profiles To characterize the adsorption of plasma proteins from PRP (50 × dilution) onto a branched-

146

PEU 8% electrospun nanofiber sheet (60 mg), a Pierce 660 nm protein assay reagent

147

(ThermoFisher Scientific) was utilized. A plate reader was first used to determine the protein

148

concentration of the original PRP solution against a standard curve. The branched-PEU 8%

149

nanofiber sheet was submerged in the solution for 30 min and then the concentration of plasma

150

proteins adsorbed to the nanofiber sheet was measured. Additionally, the release of proteins from

151

activated platelets adsorbed onto the nanofiber sheet was determined. The nanofiber sheet was

152

submerged in a solution of PRP for 30 mins, then washed three times with Tyrode’s buffer (2.73

153

M sodium chloride, 53.6 mM potassium chloride, 238.0 mM sodium bicarbonate, and (1.16 g)

154

8.6 mM monosodium phosphate in distilled H2O (1 L) stored at 4 °C). After the washes, the

155

protein content was measured to determine if any adsorbed proteins were removed. The

156

activation solution (10 % wt/v of calcium chloride) was added, and after 20 min of incubation,

157

the protein content was measured (n = 3).

158 159

2.5. Characterizing platelet adhesion with immunofluorescence using p-selectin

160

Platelet adhesion to branched-PEU 8% nanofiber sheets was determined by an

161

immunofluorescence assay. The PRP solution was prepared as described above. Platelets without

162

serum were isolated as noted: whole blood from Sprague Dawley female rats was first

7



163

centrifuged at 600 × g for 30 mins to isolate the plasma from the blood cells, followed by plasma

164

centrifugation at 1400 × g for 15 mins separating out the platelets. The platelet pellet was

165

suspended in Tyrode’s buffer, and was then centrifuged one more time at 1400 × g for 10 mins.

166

The final pellet was suspended in Tyrode’s Buffer at 10 % of the original volume of the whole

167

blood. Three groups were prepared; PRP, platelets, and a buffer control (Tyrode’s buffer

168

solution). Each group’s solution was pipetted onto a separate nanofiber sheet (n = 3) for 30 min.

169

The samples were then fixed with 4% paraformaldehyde in PBS. Samples were washed with a

170

0.5% triton × -100 in cytoskeletal stabilization (CS) buffer for 9 mins to extract and digest cell

171

walls. The samples were washed (3 ×) with CS buffer before adding 0.05% sodium borohydride

172

in CS buffer to reduce autofluorescence caused by Schiff bases. Next, the samples were washed

173

with 5% donkey serum in CS buffer for 20 min to block nonspecific binding. Samples were then

174

washed in CS buffer (3 ×) before adding anti-rat CD62P (p-selectin) (Biolegend, San Diego, CA)

175

at 1:200 for 1 h. CS buffer (3 ×) wash was followed by addition of a secondary antibody labeled

176

with tetramethylrhodamine (TRITC) for 1 h. Finally, the samples were washed in CS buffer (4 ×)

177

then mounted using mounting media (Vectashield). The films were pressed between a slide and a

178

cover slip. Photomicrographs were taken at 10 × magnification.

179 180

2.6. Surgical procedure

181

The following procedure was reviewed and approved by the University of Akron IACUC

182

committee (Protocol: 15-01-2-BRD). An incision (approximately 2.5 cm) was made over the

183

right shoulder on female Sprague-Dawley rats weighing 250 grams. The acromioclavicular

184

ligament was detached, and the deltoid was subsequently split to visualize the supraspinatus

185

tendon (Supplemental Figure 3A). A 4-0 nylon suture was used to secure the tendon using a

8


Page 8 of 31

Page 9 of 31 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

186

Modified-Mason-Allen stitch (Supplemental Figure 3B). Once secured, the supraspinatus

187

tendon was completely dissected from the humeral head (Supplemental Figure 3C). A 26-

188

gauge needle was used to puncture a hole through the humeral head and the suture was threaded

189

through the resulting hole (Supplemental Figure 3D, E). At the repair site, the rats either

190

received no additional surgical manipulation (Repair Only), an implanted branched-PEU 8%

191

nanofiber disc (Disc Only), a presoaked disc (20 mins) in a PRP solution (250 ×) (Disc PRP), or

192

an injection (20 µL) of a concentrated PRP solution (PRP Only). The tendon was then secured to

193

the bone, the deltoid stitched together, and the incision site closed (Supplemental Figure 3F).

194 195

2.7. Harvest

196

Rats were euthanized according to an approved IACUC protocol (15-01-2-BRD). Samples

197

were harvested at 7 d, 21 d, and 84 d time points. The supraspinatus tendon was identified and

198

marked with a suture. Samples for mechanical testing (n = 12) were put in PBS (1 ×) and

199

immediately prepared for analysis. Samples for histochemical analysis (n = 3) were placed in a

200

50:50 solution of 4% paraformaldehyde and 10% Immunocal solution (Stat Lab, McKinney, TX)

201

for 1 week to decalcify the bone.

202 203

2.8. Mechanical testing

204

Harvested samples were cleared of extra tissue until only the supraspinatus tendon attachment

205

to the humoral head remained. Sandpaper (80 grit) and super glue was used to sandwich and hold

206

the tendon. The samples were wrapped in PBS moistened towels until testing. Samples were

207

placed in a tensile test apparatus (TA. XTplus texture analyzer, Stable Micro Systems,

208

Godalming, UK) and the tendon/sandpaper was placed in the clamp and a custom jig was used to

9



209

hold the bone in place. The samples were subjected to a preload force of 0.2 N and then pulled to

210

failure at a rate of 0.1 mm/min. Samples were marked as interfacial tears or jaw break. Only

211

samples which tore at the interface were included in the calculation. Stiffness (N/mm), yield

212

point (N), maximum stress at break (N), and maximum strain (%) were calculated.49 Statistical

213

analysis was completed using IBM SPSS Statistics 24 software using a two-way ANOVA with

214

Tukey post hoc analysis. A value of p ≤ 0.05 was considered significant for statistical

215

comparisons. All quantitative data is presented as the average ± standard deviation. All sample

216

groups were done in replicate (n = 9-12).

217 218

2.9. Histological Characterization

219

Samples were washed in PBS (3 ×) for 10 min each, followed by a dehydrating series of

220

ethanol washes; 70 %, 80 %, 90 %, and 100 % for 10 min each to prepare the samples for

221

embedment in paraffin wax. Samples were sectioned and stained with hematoxylin and eosin

222

(H&E), trichrome, and safranin O. Stained slides were mounted with DPX. Photomicrographs

223

were taken on an Olympus SC-100.

224 225

3. Results and discussion

226

3.1. Synthesis and characterization of poly(ester urea)

227

Previous work has been done on linear and branched L-phenylalanine PEUs to assess fiber

228

morphology and stability under several conditions.43 It was observed that the branched-PEU 8%

229

had superior fiber longevity when compared to linear and other branched analogues. This was

230

attributed to the branching unit increasing the glass transition temperature resulting in retained

231

fiber morphology. Therefore, synthesis of the branched-PEU 8% was selected for this study. 10


Page 10 of 31

Page 11 of 31 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

232

Linear monomer (1-PHE-6) was synthesized through coupling hexanediol to the carboxylic acid

233

of L-phenylalanine using p-toluenesulfonic acid to prevent amidation. The branched monomer

234

(Triol-TYR) was synthesized through an esterification reaction between 1,1,1-

235

tri(hydroxylmethyl)ethane and boc-O-benzyl-L-tyrosine using N, N diisopropyl carbodiimide

236

(DIC) as a coupling reagent. The branched monomer was purified using silica gel

237

chromatography. Linear (1-PHE-6) and branched (Triol-TYR) monomers were used with

238

triphosgene to synthesize the branched-PEU 8% random copolymer using a 92:8 monomer molar

239

feed ratio (Scheme 1). Extent of monomer incorporation matched the molar feed ratio which

240

was confirmed using 1H-NMR spectroscopy as previously described.

11



241 242

Scheme 1. The branched-PEU 8% random copolymer is synthesized using defined

243

stoichiometric ratios of (1-PHE-6) and (Triol-TYR) monomers with a molar feed ratio of 92:8

244

respectively and triphosgene in an interfacial polymerization. The extent of branching can be

245

controlled precisely.

246 247 248

3.2. Fabrication and characterization of PEU disc

249

Discs of branched-PEU 8% were generated using electrospinning through a charged needle onto

250

aluminum foil (Figure 1a, 1b). The fabrication process was stable throughout and samples were 12


Page 12 of 31

Page 13 of 31 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

251

hole-punched into discs prior to EtO sterilization (Figure 1c). The disc size and shape were

252

designed to fit around the supraspinatus tendon (outer diameter = 4.00 mm, inner diameter =

253

2.00 mm, thickness = 0.25 mm). Retained fiber morphology is desired for any electrospun

254

material that hopes to become industrially relevant. To observe shelf-life stability, SEM fiber

255

morphology images were taken at room temperature initially and after one year under ambient

256

conditions. Promisingly, minimal fiber degradation or collapse had occurred (Figure 1d, 1e)

257

over this extended time frame which was desirable as fiber stability and shelf-life are a hurdle

258

that can preclude biomaterials from coming to market.

259 260

Figure 1. Fabrication of the branched-PEU 8%. Branched-PEU 8% nanofibers for implant discs

261

were (a) electrospun (12 kV) onto (b) aluminum foil and (c) cut into discs using a hole punch

262

(inner diameter 2 mm, outer 4 mm). SEM nanofibers prepared (d) dry at RT and the same

13



263

nanofibers (e) one year later were characterized with scale bars equal to 10 µm. The (f) flexible

264

nature of the nanofiber sheet once removed from the aluminum foil can be observed.

265 266 267

3.3. Adsorption of PRP on branched-PEU 8% using QCM. Using QCM, an adsorption profile for PRP on QCM sensors coated in branched-PEU 8% was

268

obtained. A frequency drop of 70 Hz was recorded during PRP addition; using the Sauerbrey

269

equation, this correlated to mass adsorption of 1166 ± 86 ng/cm2 (Figure 2). After the adsorption

270

of PRP equilibrated, a second PBS wash was used. Adsorbed PRP remained on the surface after

271

the second PBS wash, indicating that PRP had irreversibly adsorbed to the surface. With respect

272

to PEUs from previous studies, the mass of adsorbed PRP to branched-PEU 8% is greater than

273

that of the linear derived L-valine-based PEU (926 ± 59 ng/cm2) which was desirable as PRP

274

deposition will encourage PRP retention in a scaffold compared to previously synthesized

275

PEUs.49

276 277

Figure 2. Adsorption profiles of PRP onto branched-PEU 8% using QCM. Branched-PEU 8%

278

was spin coated onto QCM chips at a thickness ~39 ± 4 nm. Samples (n = 3) were first

279

equilibrated in a PBS solution. The solution was then switched to PRP (250 ×) and after the

14


Page 14 of 31

Page 15 of 31 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

280

initial drop of 70 Hz (area mass adsorption of 1166 ±86 ng/cm2) the samples were equilibrated.

281

Finally, a PBS solution was flowed over the samples to wash off unbound proteins or platelets.

282 283

3.4. Protein adsorption and release profiles.

284

The amount of adsorbed plasma proteins from PRP onto branched-PEU 8% nanofiber sheets

285

was carried out in several steps and determined by a Pierce 660 nm dye assay (Figure 3a, 3b, 3c,

286

3d). The nanofiber sheet adsorbed 5050 ± 54 µg of plasma proteins from the PRP (Figure 3e).

287

The nanofiber sheets were 60 mg prior to adsorption, therefore it was calculated that 86 µg of

288

PRP was adsorbed per 1 mg of nanofiber sheet. The adsorption could be attributed to sufficient

289

porosity in the nanofiber sheets to allow for PRP integration and hydrogen bonding between the

290

urea moiety in the polymer backbone with plasma and plasma proteins.

291

Platelet adsorption to the branched-PEU 8% film and nanofiber sheets was promising, however

292

platelet fidelity after adsorption is critical for any PRP delivery vehicle to provide clinical

293

efficacy. To ensure that platelets were still active after being adsorbed on to the branched-PEU

294

8% nanofiber sheet, a 10% calcium chloride solution, which plays a role in platelet aggregation

295

and vesicle secretion pathways, was used to measure protein secretion.50 Upon activation, the

296

activated platelets released 3390 ± 287 µg of proteins from the nanofiber sheet demonstrating

297

that platelets are present on the nanofiber sheet and are still able to activate and release proteins

298

(Figure 3f). This maintained functionality is ideal as platelet activation in vivo could be expected

299

to occur endogenously, affording localized growth factor release at the repair site.

15



300 301

Figure 3. Protein adsorption and release after activation. A PRP solution was prepared and (a)

302

the original protein content was measured. Then the branched-PEU 8% nanofiber sheet was

303

immersed in the PRP solution for 30 mins and the (b) remaining proteins in solution were

304

measured. To determine the amount of released protein, the nanofiber sheet was then washed in

305

buffer (3 ×) and placed in an activation solution (c) for 30 min, (d) the amount of released

306

protein was measured. The results (e) show a 5050 ± 54 µg adsorption of PRP onto the nanofiber

307

scaffold. Of the adsorbed protein, the amount released (f) shows that 3390 ± 287 µg was

308

released after activation. All measurements are reported as an average ± standard deviation (n =

309

3).

310 311

3.5. Immunofluorescence using p-selectin.

16


Page 16 of 31

Page 17 of 31 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

312

Biomacromolecules

Platelets were visualized on branched-PEU 8% nanofiber sheets using an antibody for p-

313

selectin, (HI62P CD62P), a transmembrane glycoprotein that is expressed on activated platelets.

314

Both PRP (containing plasma proteins and platelets) and isolated platelets were used to visualize

315

platelet adsorption to the nanofibers. A Tyrode’s buffer solution was used as a control. The

316

nanofibers are clearly visible under fluorescence excitation (green) and the primary antibody was

317

labeled with a TRITC secondary antibody (red). Both the PRP and platelet samples show

318

activated platelets lining the nanofibers (Figure 4a, 4b) while no such staining is noted on the

319

blank nanofibers (Figure 4c). This gives further evidence to the presence of platelets on the

320

nanofibers.

321 322

Figure 4. Characterization of platelet adsorption onto branched-PEU 8% using

323

immunofluorescence. A solution of PRP (a), Platelets (b), and PBS (c) were adsorbed onto

324

branched PEU-8% nanofibers. A multichannel image was taken where platelets were stained

325

with the antibody HI62P labeled with TRITC while a FITC filter was used to observe the fibers.

326

Magnification 10 × with a scale bar of 50 µm.

327 328

3.6. Mechanical Testing

17



329

Page 18 of 31

Rat rotator cuff samples were mechanically tested to determine the strength of the surgically

330

repaired tendon-bone interfaces. Five groups were tested; Repair Only, Disc, Disc PRP, PRP,

331

and a non-surgical control. During testing, samples were observed for jaw breaks or interfacial

332

breaks. Interfacial breaks are a more significant measurement for determining the strength of the

333

tendon-bone repair. Jaw breaks were excluded from the data set.

334

Table 1. The mechanical results for the surgically repaired rotator-cuff rat model.a

Groups

Stiffness Yield Point Max Force Strain at failure (N/mm) (N) (N) (%)

21 days

Repair only

7.9 ± 3.1

7.2 ± 4.0

7.9 ± 4.0

11.2 ± 7.3

Disc

6.9 ± 3.4

5.7 ± 3.0

7.2 ± 2.9

14.6 ± 6.7

Disc-PRP

8.6 ± 1.9

4.6 ± 2.0

5.7 ± 2.9

13.5 ± 6.0

PRP

7.3 ± 3.3

3.6 ± 1.9

4.5 ± 2.6

10.0 ± 8.3

Repair only

11.8 ± 3.8

15.8 ± 5.5

17.3 ± 5.1

12.6 ± 5.1

Disc

13.5 ± 3.8

13.3 ± 4.7

15.6 ± 4.8

16.8 ± 6.4

Disc-PRP

16.8 ± 5.8

13.6 ± 6.1

17.2 ± 5.6

17.2 ± 8.0

PRP

12.2 ± 2.6

13.0 ± 4.3

16.2 ± 4.2

16.1 ± 5.9

Control

22.6 ± 5.1

21.1 ± 5.1

22.6 ± 3.7

8.4 ± 2.3

84 days

335 336 337

a

samples were tested at a rate of 0.1 mm/min, the reported results include stiffness (N/mm), yield point (N), Max force (N), and strain at failure (%). The results are reported as the average ± standard deviation.

338 339

The mechanical results showed no major difference between the four experimental groups at

340

21 d. The only significant difference observed was greater stiffness in the non-surgical control

18


Page 19 of 31 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

341

compared to the experimental groups (Figure 5c). The stiffness measurements at 21 d for the

342

Repair Only, Disc, Disc PRP, and PRP are 7.9 ± 3.1 N/mm, 6.9 ± 3.4 N/mm, 8.6 ± 1.9 N/mm,

343

and 7.3 ± 3.3 N/mm respectively (Table 1). The Disc PRP group showed a higher average,

344

however the differences were not statistically significant. The stiffness results at 84 d (11.8 ± 3.8

345

N/mm, 13.5 ± 3.8 N/mm, 16.8 ± 5.8 N/mm, 12.2 ± 2.6 N/mm, respectively) show a significant

346

difference between the Disc PRP group and all of the 21 d results, and 84 d results for the Disc,

347

and PRP (Figure 5c). This was promising as the Disc PRP group had a significant enhancement

348

compared to other groups as time progressed. Moreover, there was no statistically significant

349

difference between the Disc PRP and the non-surgical control at 84 d (22.6 ± 5.1 N/mm) or

350

Repair only group at 84 d. A significant difference was found between the Repair Only and the

351

control group which was expected as stiffness of a healthy tendon was expected to outperform

352

the Repair Only group. The lack of statistical significance between the Disc PRP group and the

353

control at 84 d was ideal as it indicates the Disc PRP group was approaching the strength of the

354

healthy unaltered tendon-bone interface of the control faster than other groups. The mechanical

355

data provide evidence for the regenerative properties of the Disc PRP implants, which resulted in

356

enhanced stiffness at 84 d.

19



357 358

Figure 5. Mechanical characterization for interface tendon-bone repair in rats. The mechanical

359

plots force vs. distance for a) 21 d and b) 84 d were completed using a texture analyzer, and c)

360

stiffness measurements were calculated from the slope of the force vs. distance plots at the yield

361

point for each sample and compared across the five experimental groups. Statistical analysis was

362

performed with a p ≤ 0.05. All data is presented as the average ± standard deviation. All sample

363

groups were done in replicate: Repair Only 21 d (n=10), Disc Only 21 d (n=10), Disc PRP 21 d

364

(n=10), PRP Only 21 d (n=12), Control (n=13), Repair Only 84 d (n=10), Disc Only 84 d (n=11),

365

Disc PRP 84 d (n=11), and PRP Only 84 d (n=10). * indicates p value < 0.05 between the disc-

366

PRP repair and other experimental groups. ¥ indicates p value < 0.05 between the control other

367

experimental groups.

368

20


Page 20 of 31

Page 21 of 31 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

369

Biomacromolecules

Additional analysis using a Boxplot (Figure 6) analysis reveals that at 84 d, the Disc-PRP has

370

an outlier outside of the maximum. The upper quarter, and average are greater than all other

371

experimental groups. The outlier is increasing the average for the Disc-PRP at 84 d, however this

372

outlier was not a jaw break and is a true data point. The rest of the experimental groups show no

373

significance between the groups at 21 d and 84 d.

374 375

Figure 6. Mechanical rotator-cuff results. Texture analyzer results were extrapolated from force

376

vs. distance plots for Repair Only, Disc, Disc-PRP, PRP Only, and control at 21 d and 84 d time

377

points.

378 379

3.7. Histological Characterization

380

Rat rotator cuffs were surgically repaired four different ways; Repair Only, Disc Only, Disc

381

PRP, and PRP Only. A non-surgical control was used as an unaltered tissue comparison. Each

382

group was stained with H&E, trichrome, and safranin O at 7 d, 21 d, and 84 d. With standard

21



383

H&E staining, the experimental groups at 7 d display high cell densities, and disorganization of

384

proliferating fibrous connective tissue within repaired tendon is expected as it is in the

385

inflammation and remodeling phase (Figure 7). The high cell densities are partially

386

characterized by increased numbers of reactive fibroblasts with associated extracellular matrix

387

(fibrosis). A smaller proportion of the cellular population is comprised of inflammatory cells,

388

primary cells typical of chronic inflammatory responses, such as lymphocytes, plasma cells,

389

macrophages and multinucleated giant cells. Similar cell density is also observed in the rotator

390

cuff repairs dosed with PRP at 7 d by Beck et al., where they describe the high cell density.5

391

However, in this group, the overall reactive fibrous response is less than the other experimental

392

groups with the chronic inflammatory response present. By 21 d, the inflammatory component in

393

all experimental groups is reduced but still present. The inflammatory cells present are similar in

394

composition. The reactive fibroblasts remain similar in size; however, the cell population and the

395

extracellular matrix start to exhibit some degree of organization. The repair and remodeling

396

phases of tendon-bone repair are slower than most tissues. The tendon at the enthesis is avascular

397

which limits the oxygen and nutrients available for healing and may explain why little to no

398

organization has occurred at this point. Fibroblast cell density and size are much lower in the

399

experimental groups at 84 d compared to 21 d and more collagen and matrix is present and

400

partially organized/remodeled. Some residual chronic inflammation remains but is far less than

401

the degree of inflammation observed at 7 and 21 days. The remodeling does not appear complete

402

when compared to the healthy unaltered control. The Disc PRP group is in a more advanced

403

stage of remodeling as compared to the other experimental groups as seen in the observable

404

reformation of the tendon-bone interface and a higher degree of organization of the collagen in

405

the tendon.

22


Page 22 of 31

Page 23 of 31 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

406 407

Figure 7. The H&E staining of surgically repaired rat rotator cuffs. Four surgical groups (Repair

408

Only, Disc Only, Disc PRP, and PRP Only) were compared to a non-surgical control at 7 d, 21 d,

409

and 84 d. Cell nuclei appear basophilic (dark purple). Extracellular matrix (collagen and bone)

410

appear eosinophilic (pink). Large aggregates of inflammatory cells can be observed in all

411

experimental groups at day 7. This inflammation progressively reduces in severity by day 84 in

412

all experimental groups. The photomicrographs are oriented so the bone is towards the bottom,

413

tendon is on the top, and an arrow indicates the tendon-bone interface. Photomicrographs are at

414

40 × magnification and the scale bar is 200 µm.

415 416

Masson’s trichrome is helpful when distinguishing collagen from skeletal muscle surrounding

417

the joint. With this stain, the nuclei appear black/purple, the cytoplasm of cells (including

418

skeletal myofibers) red, and the collagen, blue. The presence and organization of collagen can be

419

easily visualized using this type of histological stain (Figure 8). Disorganized collagen and

420

higher cell densities as described above can be observed at 7 d. At 21 d, more collagen was

421

present near the interface in the repair only, Disc PRP, and PRP groups. The Disc PRP group is

422

the only group that exhibit higher degrees of collagen bundle organization at the interface 23



423

resembling the control tissue. This appearance provides support for the increase in mechanical

424

strength for Disc PRP at 21 d. The groups at 84 d are in the remodeling phase with low cell

425

densities and more collagen at the tendon-bone interface. The Repair Only and Disc PRP groups

426

at 84 d have an observable reintegration of the tendon into the bone that is more advanced than

427

the other groups at 84 d. The reformation of the interface lends support for the increase in

428

stiffness for the Repair Only and Disc PRP groups shown in the mechanical results.

429 430

Figure 8. The trichrome staining of surgically repaired rat rotator cuffs. Four surgical groups

431

(Repair Only, Disc, Disc PRP, and PRP) were compared to a non-surgical control at 7 d, 21 d,

432

and 84 d. The nuclei are stained black, the cellular cytoplasm and skeletal muscle is stained red.

433

Collagen is stained blue. The photomicrographs are oriented so the bone is towards the bottom,

434

tendon is on the top, and an arrow indicates the tendon-bone interface. Photomicrographs are at

435

40 × magnification and the scale bar is 200 µm.

436

The final stain, safranin O, is helpful in highlighting cartilaginous tissue (hyaline cartilage,

437

fibrocartilage or hyaline cartilage) from other extracellular matrix. Cell densities and some

438

organization along with fibrocartilage in the enthesis can be visualized in each of the groups

24


Page 24 of 31

Page 25 of 31 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

439

(Figure 9). The control group possessed normal fibrocartilage at the tendon-bone interface.

440

Similar to H&E and trichrome, the 7 d time point showed higher cell densities as described

441

above and disorganization of the tendon tissue. It is important to note that fibrocartilage was not

442

present near the tendon-bone interface at 7 d.

443 444

Figure 9. The Safranin O staining of surgically repaired rat rotator cuffs. Four surgical groups

445

(Repair Only, Disc, Disc PRP, and PRP) were compared to a non-surgical control at 7 d, 21 d,

446

and 84 d. The nuclei are stained black, the cytoplasm green, and the cartilage (fibrocartilage) red.

447

The photomicrographs are oriented so the bone is towards the bottom, tendon is on the top, and

448

an arrow indicates the tendon-bone interface. Photomicrographs are at 40 × magnification and

449

the scale bar is 200 µm.

450 451

No red staining can be seen at the 21 d either, and the organization seen in the H&E and

452

trichrome sections is less visible in the safranin O staining. An increase in organization of the

453

cytoplasm and lower cell densities can be seen at 84 d. Reformation of the fibrocartilage layer is

454

noticeable in the Disc PRP group. A faint amount of fibrocartilage can be seen at the tendon-

25



455

bone interface which suggests why the Disc PRP group at 84 d had a slightly higher stiffness

456

than the Repair Only group even though they both appeared to have organized integration in the

457

trichrome stain. The reformation of the fibrocartilage layer could have some impact on

458

reinforcing the tendon-bone interface during mechanical testing.

459 460 461

4. Conclusion A branched-PEU 8% was used as a PRP delivery vehicle and was characterized for PRP and

462

plasma protein adsorption, platelet adhesion, and platelet activation using a number of

463

techniques. PRP was found to adsorb at 1166 ng/cm2 with PRP retention after a PBS wash

464

indicating irreversibly adsorbed PRP to the PEU substrates. A protein assay revealed that the

465

plasma proteins in PRP adsorbed at 86 µg/mg and that after activation 3390 µg of proteins were

466

released from adsorbed platelets, strengthening the evidence that functional platelets were

467

adsorbed. An acute rotator cuff injury model in rats was used to assess the utility of PEUs in a

468

clinically relevant repair model. The model evaluated five different experimental groups; Repair

469

Only, Disc, Disc PRP, PRP, and a non-surgical control was used for a healthy enthesis

470

comparison. Tensile measurements at 21 d and 84 d showed that the Disc PRP group had

471

increased mechanical properties, which was supported by histological staining. This work

472

indicates that PRP could find more clinical efficacy and improve healing rates if coupled with a

473

PEU, acting as a local release vehicle, to provide sustained PRP release. While a chronic or

474

delayed model are likely to show more distinct differences between the control and PRP loaded

475

discs, the lack of inflammation and recovery of preoperative mechanical properties show that the

476

use of PEU nanofiber discs have the potential to be translationally relevant.

477

26


Page 26 of 31

Page 27 of 31 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

478

ASSOCIATED CONTENT

479

Supporting Information

480

Spectral, other characterization data, and surgical scheme not included in the main text can be

481

found in the supporting information.

482

AUTHOR INFORMATION

483

Corresponding Author

484 485 486 487 488 489 490 491 492

*To whom correspondence should be addressed Matthew L. Becker Department of Polymer Science The University of Akron 170 University Ave Akron, OH 44325-3909 Email: [email protected] Phone: (330) 972-2834

493

Author Contributions

494

The manuscript was written through contributions of all authors. All authors have given approval

495

to the final version of the manuscript. ‡These authors contributed equally. (match statement to

496

author names with a symbol)

497 498

Funding sources

499

This project is based upon work supported by the NSF Graduate Research Fellowship Program

500

to E.P.C. The authors acknowledge The Biomaterials Division of the Nation Science Foundation

501

(DMR 1507420) and the Robert F. Kepley Grant in orthopedic research from the Summa

27



502

Foundation for supporting this research. MLB is grateful for support from the W. Gerald Austen

503

Endowed Chair in Polymer Science and Polymer Engineering from the Knight Foundation.

504

Notes

505

The authors declare no competing financial interest.

506

Acknowledgements

507

The authors would like to thank Andre Domino and David Navratil from B&B Microscope

508

Limited, for their help and expertise with automated microscopy of tissue histology. The authors

509

would like to thank Walter Horne for training and advice for the surgical procedures.

510

Additionally, the authors would like to sincerely thank Dr. Christopher Premanandan for

511

histological characterization and insight.

512 513

REFERENCES

514 515 516 517 518 519 520 521 522 523 524 525 526 527 528 529 530 531

1. Aleem, A. W.; Brophy, R. H.; Outcomes of Rotator Cuff Surgery What Does the Evidence Tell Us? Clinics in Sports Medicine 2012, 31, 665-675. 2. Gulotta, L. V.; Rodeo, S. A.; Growth Factors for Rotator Cuff Repair. Clinics in Sports Medicine 2009, 28, 13-23. 3. Hing, K. A.; Best, S. M.; Tanner, K. E.; Bonfield, W.; Revell, P. A.; Biomechanical assessment of bone ingrowth in porous hydroxyapatite. Journal of Materials Science: Materials in Medicine 1997, 8, 731-736. 4. Thomopoulos, S.; Genin, G. M.; Galatz, L. M.; The development and morphogenesis of the tendon-to-bone insertion - What development can teach us about healing. J. Musculoskelet. Neuronal Interact. 2010, 10, 35-45. 5. Beck, J.; Evans, D.; Tonino, P. M.; Yong, S.; Callaci, J. J.; The biomechanical and histologic effects of platelet-rich plasma on rat rotator cuff repairs. The American Journal of Sports medicine 2012, 40, 2037-44. 6. Everts, P. A. M.; Knape, J. T. A.; Weibrich, G.; Schönberger, J.; Hoffmann, J.; Overdevest, E. P.; Box, H. A. M.; van Zundert, A.; Platelet-Rich Plasma and Platelet Gel: A Review. The Journal of Extra-Corporeal Technology 2006, 38, 174-87. 7. Gandhi, A.; Bibbo, C.; Pinzur, M.; Lin, S. S.; The role of platelet-rich plasma in foot and ankle surgery. Foot and Ankle Clinics 2005, 10, 621-37, viii.

28


Page 28 of 31

Page 29 of 31 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

532 533 534 535 536 537 538 539 540 541 542 543 544 545 546 547 548 549 550 551 552 553 554 555 556 557 558 559 560 561 562 563 564 565 566 567 568 569 570 571 572 573 574 575 576

Biomacromolecules

8. Hapa, O.; Cakici, H.; Kukner, A.; Aygun, H.; Sarkalan, N.; Baysal, G.; Effect of platelet-rich plasma on tendon-to-bone healing after rotator cuff repair in rats: an in vivo experimental study. Acta Orthopaedica et Traumatologica Turcica 2012, 46, 301-7. 9. Mlynarek, R.; Kuhn, A.; Bedi, A.; Platelet-Rich Plasma (PRP) in Orthopedic Sports Medicine. The American Journal of Orthopedics 2016, 45, 290-294. 10. Textor, J., Platelet-Rich Plasma (PRP) as a Therapeutic Agent: Platelet Biology, Growth Factors and a Review of the Literature. In Platelet-Rich Plasma: Regenerative Medicine: Sports Medicine, Orthopedic, and Recovery of Musculoskeletal Injuries, Lana, D. J. F. S.; Andrade Santana, H. M.; Dias Belangero, W.; Malheiros Luzo, C. A., Eds. Springer Berlin Heidelberg: Berlin, Heidelberg, 2014; pp 61-94. 11. Campbell, N. A.; Reece, J. B.; Urry, L. A.; Cain, M. L.; Wasserman, S. A.; Minorsky, P. V., Biology. Pearson: San Francisco, 2008. 12. Blair, P.; Flaumenhaft, R.; Platelet α–granules: Basic biology and clinical correlates. Blood Reviews 2009, 23, 177-189. 13. Eppley, B. L.; Pietrzak, W. S.; Blanton, M.; Platelet-rich plasma: a review of biology and applications in plastic surgery. Plastic and Reconstructive Surgery 2006, 118, 147e-159e. 14. Whitman, D. H.; Berry, R. L.; Green, D. M.; Platelet gel: an autologous alternative to fibrin glue with applications in oral and maxillofacial surgery. Journal of Oral and Maxillofacial Surgery, 1997, 55, 1294-9. 15. Getgood, A.; Henson, F.; Brooks, R.; Fortier, L. A.; Rushton, N.; Platelet-rich plasma activation in combination with biphasic osteochondral scaffolds-conditions for maximal growth factor production. Knee Surg. Sports Traumatol. Arthrosc. 2011, 19, 1942-1947. 16. Angeline, M. E.; Rodeo, S. A.; Biologics in the Management of Rotator Cuff Surgery. Clinics in Sports Medicine 2012, 31, 645-663. 17. Apostolakos, J.; Durant, T. J. S.; Dwyer, C. R.; Russell, R. P.; Weinreb, J. H.; Alaee, F.; Beitzel, K.; McCarthy, M. B.; Cote, M. P.; Mazzocca, A. D.; The enthesis: a review of the tendon-to-bone insertion. Muscles, Ligaments and Tendons Journal 2014, 4, 333-342. 18. Docheva, D.; Müller, S. A.; Majewski, M.; Evans, C. H.; Biologics for tendon repair. Advanced Drug Delivery Reviews 2015, 84, 222-239. 19. Chung, S. W.; Song, B. W.; Kim, Y. H.; Park, K. U.; Oh, J. H.; Effect of platelet-rich plasma and porcine dermal collagen graft augmentation for rotator cuff healing in a rabbit model. The American Journal of Sports Medicine 2013, 41, 2909-18. 20. Marx, R. E.; Platelet-rich plasma (PRP): what is PRP and what is not PRP? Implant Dentistry 2001, 10, 225-8. 21. Kohane, D. S.; Langer, R.; Polymeric Biomaterials in Tissue Engineering. Pediatr Res 2008, 63, 487-491. 22. Seal, B. L.; Otero, T. C.; Panitch, A.; Polymeric biomaterials for tissue and organ regeneration. Materials Science and Engineering: R: Reports 2001, 34, 147-230. 23. Nair, L. S.; Laurencin, C. T.; Biodegradable polymers as biomaterials. Progress in Polymer Science 2007, 32, 762-798. 24. Laurencin, L. S. N. C. T.; Polymers as Biomaterials for Tissue Engineering and Controlled Drug Delivery. Adv Biochem Engin/Biotechnol 2006, 102, 47-90. 25. Latere, J.-P.; Lecomte, P.; Dubois, P.; Jérôme, R.; 2-Oxepane-1,5-dione: A Precursor of a Novel Class of Versatile Semicrystalline Biodegradable (Co)polyesters. Macromolecules 2002, 35, 7857-7859.

29



577 578 579 580 581 582 583 584 585 586 587 588 589 590 591 592 593 594 595 596 597 598 599 600 601 602 603 604 605 606 607 608 609 610 611 612 613 614 615 616 617 618 619 620 621

26. Gurusamy-Thangavelu, S. A.; Emond, S. J.; Kulshrestha, A.; Hillmyer, M. A.; Macosko, C. W.; Tolman, W. B.; Hoye, T. R.; Polyurethanes based on renewable polyols from bioderived lactones. Polymer Chemistry 2012, 3, 2941-2948. 27. Zhang, S.; Li, A.; Zou, J.; Lin, L. Y.; Wooley, K. L.; Facile Synthesis of Clickable, WaterSoluble, and Degradable Polyphosphoesters. ACS Macro Letters 2012, 1, 328-333. 28. Dwan'Isa, J.-P. L.; Lecomte, P.; Dubois, P.; Jérôme, R.; Hydrolytic and Thermal Degradation of Random Copolyesters of ε-Caprolactone and 2-Oxepane-1,5-dione. Macromolecular Chemistry and Physics 2003, 204, 1191-1201. 29. Huang, C. L.; Steele, T. W.; Widjaja, E.; Boey, F. Y.; Venkatraman, S. S.; Loo, J. S. The influence of additives in modulating drug delivery and degradation of PLGA thin films. NPG Asia Mater. 2013, 5 (7), e54 DOI: 10.1038/am.2013.26. 30. Gentile, P.; Chiono, V.; Carmagnola, I.; Hatton, P. V. An overview of poly(lactic-coglycolic) Acid (PLGA)-based biomaterials for bone tissue engineering. Int. J. Mol. Sci. 2014, 15 (3), 3640–3659 DOI: 10.3390/ijms15033640. 31. Suchenski, M.; McCarthy, M. B. eth; Chowaniec, D.; Hansen, D.; McKinnon, W.; Apostolakos, J.; Arciero, R.; Mazzocca, A. D. Material properties and composition of soft-tissue fixation. Arthroscopy 2010, 26 (6), 821–831 DOI: 10.1016/j.arthro.2009.12.026. 32. Angela, L. S.; Chia-Chih, C.; Bryan, P.; Todd, E., Strategies in Aliphatic Polyester Synthesis for Biomaterial and Drug Delivery Applications. In Degradable Polymers and Materials: Principles and Practice (2nd Edition), American Chemical Society: 2012; Vol. 1114, pp 237-254. 33. Madhavan Nampoothiri, K.; Nair, N. R.; John, R. P.; An overview of the recent developments in polylactide (PLA) research. Bioresource Technology 2010, 101, 8493-8501. 34. Tangpasuthadol, V.; Pendharkar, S. M.; Peterson, R. C.; Kohn, J.; Hydrolytic degradation of tyrosine-derived polycarbonates, a class of new biomaterials. Part II: 3-yr study of polymeric devices. Biomaterials 2000, 21, 2379-2387. 35. Ertel, S. I.; Kohn, J.; Evaluation of a series of tyrosine-derived polycarbonates as degradable biomaterials. Journal of Biomedical Materials Research 1994, 28, 919-930. 36. Anderson, J. M.; Gibbons, D. F.; Martin, R. L.; Hiltner, A.; Woods, R.; The potential for poly-α-amino acids as biomaterials. Journal of Biomedical Materials Research 1974, 8, 197207. 37. Chow, D.; Nunalee, M. L.; Lim, D. W.; Simnick, A. J.; Chilkoti, A.; Peptide-based biopolymers in biomedicine and biotechnology. Materials Science and Engineering: R: Reports 2008, 62, 125-155. 38. Gao, Y.; Childers, E. P.; Becker, M. L.; l-Leucine-Based Poly(ester urea)s for Vascular Tissue Engineering. ACS Biomaterials Science & Engineering 2015, 1, 795-804. 39. Li, S.; Yu, J.; Wade, M. B.; Policastro, G. M.; Becker, M. L.; Radiopaque, Iodine Functionalized, Phenylalanine-Based Poly(ester urea)s. Biomacromolecules 2015, 16, 615624. 40. Lin, F.; Yu, J.; Tang, W.; Zheng, J.; Xie, S.; Becker, M. L.; Postelectrospinning “Click” Modification of Degradable Amino Acid-Based Poly(ester urea) Nanofibers. Macromolecules 2013, 46, 9515-9525. 41. Stakleff, K. S.; Lin, F.; Smith Callahan, L. A.; Wade, M. B.; Esterle, A.; Miller, J.; Graham, M.; Becker, M. L.; Resorbable, amino acid-based poly(ester urea)s crosslinked with

30


Page 30 of 31

Page 31 of 31 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

622 623 624 625 626 627 628 629 630 631 632 633 634 635 636 637 638 639 640 641 642 643 644 645 646 647 648

Biomacromolecules

osteogenic growth peptide with enhanced mechanical properties and bioactivity. Acta Biomaterialia 2013, 9, 5132-5142. 42. Wade, M. B.; Rodenberg, E.; Patel, U.; Shah, B.; In vitro and in vivo evaluation of electrospun poly(ester urea)s as hernia repair mesh materials. Biomacromolecules 2016. 43. Yu, J.; Lin, F.; Becker, M. L.; Branched Amino Acid Based Poly(ester urea)s with Tunable Thermal and Water Uptake Properties. Macromolecules 2015, 48, 2916-2924. 44. Yu, J.; Lin, F.; Lin, P.; Gao, Y.; Becker, M. L.; Phenylalanine-Based Poly(ester urea): Synthesis, Characterization, and in vitro Degradation. Macromolecules 2014, 47, 121-129. 45. Zimmermann, J.; Loontjens, T.; Scholtens, B. J. R.; Mülhaupt, R.; The formation of poly(ester–urea) networks in the absence of isocyanate monomers. Biomaterials 2004, 25, 2713-2719. 46. Cazenave, J.-P.; Ohlmann, P.; Cassel, D.; Eckly, A.; Hechler, B.; Gachet, C., Preparation of Washed Platelet Suspensions From Human and Rodent Blood. In Platelets and Megakaryocytes: Volume 1: Functional Assays, Gibbins, J. M.; Mahaut-Smith, M. P., Eds. Humana Press: Totowa, NJ, 2004; pp 13-28. 47. Pryor, M. E.; Polimeni, G.; Koo, K. T.; Hartman, M. J.; Gross, H.; April, M.; Safadi, F. F.; Wikesjo, U. M.; Analysis of rat calvaria defects implanted with a platelet-rich plasma preparation: histologic and histometric observations. Journal of Clinical Periodontology 2005, 32, 966-72. 48. Galatz, L. M.; Sandell, L. J.; Rothermich, S. Y.; Das, R.; Mastny, A.; Havlioglu, N.; Silva, M. J.; Thomopoulos, S.; Characteristics of the rat supraspinatus tendon during tendon-tobone healing after acute injury. Journal of Orthopaedic Research, 2006, 24, 541-50. 49. Childers, E. P.; Peterson, G. I.; Ellenberger, A. B.; Domino, K.; Seifert, G. V.; Becker, M. L.; Adhesion of Blood Plasma Proteins and Platelet-rich Plasma on l-Valine-Based Poly(ester urea). Biomacromolecules 2016, 17, 3396-3403. 50. Roberts, D. E.; McNicol, A.; Bose, R.; Mechanism of collagen activation in human platelets. The Journal of Biological Chemistry 2004, 279, 19421-30.

649 650

Cc

651 652 653

31


Enhanced Rotator-Cuff Repair Using Platelet-Rich Plasma Adsorbed

Recommend Documents