Enhanced Rotator-cuff Repair Using Platelet-Rich Plasma Adsorbed

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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

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Biomacromolecules

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Enhanced Rotator-cuff Repair Using Platelet-Rich

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Plasma Adsorbed on Branched Poly(ester urea)s

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

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

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

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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,

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

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ACS Paragon Plus Environment

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ABSTRACT

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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inflammation and recovery towards preoperative mechanical properties indicate PEU nanofiber

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discs as translationally relevant.

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Biomacromolecules

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

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

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

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

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

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

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

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

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

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

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

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

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12-14

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

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

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

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

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

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

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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

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

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

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

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

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

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

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

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

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

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

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

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

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

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L-phenylalanine

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

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

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with porosity ideal for cellular integration and small molecule adsorption.

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

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

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

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

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

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

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2. Materials and methods

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2.1. General materials

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

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

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

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previously.43

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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

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

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

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

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

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

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

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

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

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

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

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

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

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

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



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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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the protein content was measured (n = 3).

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2.5. Characterizing platelet adhesion with immunofluorescence using p-selectin

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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cover slip. Photomicrographs were taken at 10 × magnification.

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2.6. Surgical procedure

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

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

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

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

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

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

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

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

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

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

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

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

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

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2.7. Harvest

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

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

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

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

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

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for 1 week to decalcify the bone.

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2.8. Mechanical testing

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

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

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

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

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

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

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

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

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

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

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

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

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

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2.9. Histological Characterization

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

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

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

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

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were taken on an Olympus SC-100.

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3. Results and discussion

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3.1. Synthesis and characterization of poly(ester urea)

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

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

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

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

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


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

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

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

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

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

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

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

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

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

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

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

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

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controlled precisely.

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3.2. Fabrication and characterization of PEU disc

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

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


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

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

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

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

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

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

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

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that can preclude biomaterials from coming to market.

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

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

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

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

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

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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

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

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

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

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

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

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

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

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PEUs.49

276 277

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

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

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

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

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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.

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

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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).

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

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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

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

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

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

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

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

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

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

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

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

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

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300 301

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

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

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

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

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

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

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

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

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3).

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3.5. Immunofluorescence using p-selectin.

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

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

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

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

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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

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

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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

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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.

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Magnification 10 × with a scale bar of 50 µm.

327 328

3.6. Mechanical Testing

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

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

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

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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.

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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

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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

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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

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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-

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

367

experimental groups.

368

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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

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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.

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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

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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

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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

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Enhanced Rotator-cuff Repair Using Platelet-Rich Plasma Adsorbed

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