Role of embedded pure xenogenous bovine platelet gel on experimental tendon healing, modelling and remodelling.

BioDrugs (2014) 28:537–556 DOI 10.1007/s40259-014-0107-0

ORIGINAL RESEARCH ARTICLE

Role of Embedded Pure Xenogenous Bovine Platelet Gel on Experimental Tendon Healing, Modelling and Remodelling Ahmad Oryan • Ali Moshiri • Abdolhamid Meimandi-Parizi

Published online: 17 September 2014 Ó Springer International Publishing Switzerland 2014

Abstract Background and Objectives Surgical reconstruction of large tendon defects is technically demanding. In addition, tendon healing has poor quality and is associated with development of peritendinous adhesions. Tissue engineering and regenerative medicine is an option. A combination of scaffolds and factors that promote healing, such as a bioactive graft, could be a valuable strategy for treatment of the injured tendons. Different forms of platelets have been used for tendon healing. Since the availability and cost effectiveness of biomaterials are important in tissue engineering, bovine platelets could be a valuable alternative option for the autograft platelets. We investigated whether bovine platelet gel embedded within an artificial tendon could be effective in tendon healing and regeneration, in vivo. Methods After in vitro evaluations, a large tendon defect model was produced in rabbits and the defect maintained align using Kessler suture. The animals were divided into four groups of control (no implant), treatment with collagen implant, collagen implant—polydioxanone sheath, and collagen implant—polydioxanone

Electronic supplementary material The online version of this article (doi:10.1007/s40259-014-0107-0) contains supplementary material, which is available to authorized users. A. Oryan Department of Pathology, School of Veterinary Medicine, Shiraz University, Shiraz, Iran A. Moshiri (&) A. Meimandi-Parizi Division of Surgery and Radiology, Department of Clinical Sciences, School of Veterinary Medicine, Shiraz University, Shiraz, Iran e-mail: [email protected]

sheet—bovine platelet gel. The healing and regeneration were assessed by gross- micro- and nano-morphologic analyses, biomechanical testing, biochemistry, bioelectricity, and clinical evaluations at 60 and 120 days after injury. Results Bovine platelet gel induced cellular proliferation and enhanced cell viability in vitro. In vivo, it significantly increased inflammation in the short term, enhanced cellular distribution, proliferation, migration, differentiation and matrix production at mid-term and finally it facilitated graft degradation, incorporation and acceptance in the newly regenerated tendon. Compared with the control groups, the platelet-treated neotendon had significantly higher mechanical strength which was due to the collagen fibril’s better density, diameter, number, differentiation and distribution, collagen fibril to fiber and fiber bundle differentiation and lower peritendinous adhesion, muscle fibrosis and atrophy. Conclusion Bovine platelet gel-embedded artificial tendon could be considered as a new option in reconstruction and healing of large tendon defects.

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Key Points Bovine platelet gel-embedded tissue-engineered artificial tendon was cytocompatible in vitro and biocompatible and biodegradable in vivo. Bovine platelet gel increased the biocompatibility of the collagen-polydioxanone sheath implants in vivo because the platelets modulated the inflammation and controlled the inflammatory cells distribution and behavior inside the implant. Bovine platelet gel was found to be a powerful tenoinductive agent so that it developed the large and aligned mature collagen fibrils at ultrastructure which were responsible for the increased biomechanical properties of the treated tendons. Bovine platelets had no side effects in rabbits and influenced tendon healing by increasing and modulating the inflammation which triggered the fibroplastic response and remodeling quality.

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the healing efficacy of the platelets could be due to their ability to induce inflammation, which is important for tendon healing [13, 16, 22–24]. Previous studies have used auto and allogenous forms of platelets with some satisfactory results [17, 18, 25, 26]. Recently, a series of studies used human platelets in a rabbit bone healing model with some promising results [27–29]. Availability of the platelet source is important for clinical application [13]. Bovine platelets are accessible, cost effective, and may be a reliable source for platelets which could be formed into gel; in a novel modification it could be used as a healing promotive factor in combination with scaffolds or artificial grafts. Given the above explanations, we produced a bovine platelet gel (BPG) and embedded it within a three-dimensional, tissue-engineered collagen implant-polydioxanone sheath (CI-PDS) artificial tendon. Our hypothesis was that the xenogenous bovine platelet could trigger inflammation in the short term and, due to its growth factors, it may be effective in tendon healing and regeneration. We tested our hypothesis and the healing efficacy of the bovine platelets by various in vitro and in vivo evaluations. 2 Materials and Methods

1 Introduction

2.1 Ethics

Large Achilles and other tendon injuries could happen due to tumors, traumas, gangrenous and infective ulcers, tendinopathies and several other conditions [1–3]. In such cases, resection of the diseased tissue leads to defect formation [4]. If small, tendon defects could be sutured directly or reconstructed with autogenous tissues [1, 2]. However, reconstruction of large tendon defects with autografts has considerable limitations including graft availability and donor site morbidity and this technique requires double surgery [5–7]. Using allografts increases the risk of disease transmission and rejection. The ethical concerns are another limitation of using allografts [4–8]. Tissue engineering is a newer approach than the classic options [9, 10]. Routinely, scaffolds are used to reconstruct such defects, while recent studies have shown that using scaffolds alone may not be able to produce a functional tendon comparable to normal uninjured tendons [11, 12]. Bioactive molecules and strategies are needed to combine with scaffolds to enhance the healing response [13]. Various forms of platelets such as platelet-rich plasma, platelet fibrin glue or platelet gel and direct injection of pure platelets have been used to accelerate tendon healing in vivo [13–18]. Currently, platelets are the focus of many studies due to their growth factors [19–21]. Recent studies suggest that

The investigators who undertook the measurements and analyses of the results were unaware of the experimental design and grouping details. All animals received human care in compliance with the Guide for Care and use of Laboratory Animals published by the National Institutes of Health (NIH publication No. 85-23, revised 1985). The study was approved by the local ethics committee of our veterinary school. 2.2 Preparation of the Collagen Implant and Polydioxanone Sheath Collagen type I was extracted from the bovine superficial digital flexor tendon. The purity of the type I collagen was confirmed by SDS/PAGE [11]. The acid solubilized collagen molecules were electrospinned onto a dual plate device to produce the large and aligned electrospun collagen fibers. After electrospinning, the acid-solubilized bovine tendon type I collagen molecules were mixed with the electrospun collagen fibers and polymerized in an incubator at 4 °C for 48 hours to produce a tridimensional collagen gel [6]. The collagens were aligned under 12 Tesla magnetic fields (CRETA, Grenoble) during polymerization [30]. The collagen composite was cut into several pieces of the same size and shape as the rabbit’s

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Achilles apparatus (L = 2 cm, H = 3.5 mm, W = 3 mm). The collagen composites were cross-linked after suspension in iso-osmolar 0.1 % riboflavin solution, using UV (wavelength of 365 nm) irradiation [8]. Polydioxanone sheet was purchased (J&J, CA, USA), sectioned, melted, and wrapped around each collagen piece to produce a CIPDS. The final product was repeatedly washed with distilled water, received 100 Gray g-radiation, and was suspended in ethanol 96 % to produce and maintain its sterility until surgery.

of CaCl2 10 % with a proportion of 10 platelet solution : 1 activator [15]. This produced a platelet gel embedded within the CI-PDS implant. The hybrid scaffolds were then air dried and placed in a sterile package for further use. The platelet gel embedded within the scaffolds were subjected to scanning electron microscopy (SEM), inverted microscopy and histology and the presence of the platelets in the internal parts of the scaffold was confirmed.

2.3 Preparation of the Platelet Gel Embedded Within the Collagen Implant

2.4.1 Platelet Aggregation Test

Blood was harvested from healthy bovines and transferred into the sterile ethylene-diamine-tetraacetic-acid (EDTA) tubes (1.5 mg/mL of blood). The animals were free of any contagious and particularly zoonotic diseases such as bovine spongiform encephalitis, rabies, tuberculosis, and brucellosis and the safety of the blood samples were tested and approved by a certified laboratory. The samples were centrifuged at 1,500 rpm (2159g) for 15 minutes [15]. On centrifugation of the anti-coagulated blood, the following three layers were formed: red blood cells (bottom); white blood cells (WBCs)/platelets (buffy coat) (middle); and plasma (top) [22]. The plasma and buffy coat were suctioned into new tubes (platelets obtained after the first step centrifugation [PFSC]) and centrifuged again. Three layers including WBCs (bottom), platelet-rich plasma (PRP, middle) and platelet-poor plasma (PPP, top) were formed. The PPP and PRP were suctioned into the new tubes (platelets obtained after the second step centrifugation [PSSC]). The purity of the solution was tested by light microscopy, and for this purpose three samples were fixed over glass slides and stained with Wright–Giemsa staining; it was confirmed that the samples were free of bovine WBCs. The platelets were counted with a standard hemocytometer, and the total platelet count was calculated for each sample. After PRP and PPP preparation, the samples were lyophilized and pulverized. The powder was sterilized via UV irradiation and the sterile powder was dissolved in the sterile phosphate buffered saline (PBS) (0.9 % NaCl) (platelets obtained after the lyophilization and saline solving procedure [PLSSP]). The final platelet concentration per each lL was set to 2,000,000 platelets. This concentration was about 6–7 times the physiological platelet concentration (200,000–300,000 9 platelets/lL) in the blood. PLSSP 2 mL was transferred into a sterile custommade rectangular dish. The fully dehydrated CI-PDS implants were weighed and then placed in the dish. After 30 minutes, the scaffolds fully absorbed the solution. Two mL of the platelets were activated (platelet gel [PG]), using a combination of bovine thrombin (5,000 units) and 5 mL

2.4 In Vitro Evaluations

Platelet aggregation was tested by light transmission aggregometry (LTA). LTA measures the changes in transmission of a beam of light through a sample of PRP or platelet suspensions in buffer, which occur when platelets change shape and aggregate upon stimulation. In the LTA method (Chrono-log series 400, Harvertown, PA, USA), collagen (3.2 lg/mL) and TRAP-6 (32 lM) (Verum Diagnostica, Munich, Germany) were used as agonists. The results were expressed as maximal light transmission. The LTA of the PFSC (750,000 platelets/lL, n = 10 samples), PSSC (1,500,000 platelets/lL, n = 10 samples) and PLSSP (2,000,000 platelets/lL, n = 10 samples) were statistically compared. 2.4.2 Platelet Growth Factor Level The platelet-derived growth factor (PDGF) and insulin-like growth factor 1 (IGF-I) level of the PFSC (750,000 platelets/lL, n = 10 samples), PSSC (1,500,000 platelets/lL, n = 10 samples), PLSSP (2,000,000 platelets/lL, n = 10 samples) and the PG (2,000,000 platelets/lL, n = 10 samples) were measured, using commercially available Quantikine ELISA kits (DHD00, DG100 respectively, R&D Systems, Minneapolis, MN, USA). To measure the PDGF-AB level, the samples and standards were prepared according to the manufacturer’s protocol. The samples were incubated for 2 hours, washed and incubated with enzyme conjugated antibodies directed against PDGF-AB for an additional 2 hours at room temperature (RT). The wells were then washed and the substrate was added for 20 minutes at RT. The stop solution was added to each well and the absorbance was determined at 450 nm, using a microtiter plate reader. To measure IGF-I, a dilution series of IGF standards was prepared in 100 lL volumes in 96 well microtiter plates coated with a monoclonal antibody specific for IGF-I. The microtiter plate was incubated for 2 hours at 2–8 °C. The wells were washed three times and incubated with enzyme conjugated IGF-I for 1 hour at 2–8 °C. The wells were washed three times, the substrate solution was added and the plates were incubated for

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30 minutes at RT. The stop solution was added to each well and the absorbance for each was determined, at 450 nm, using a microtiter plate reader. 2.4.3 Sterility Status and Endotoxin Content of the Scaffolds The scaffolds were tested for sterility immediately after sterilization. They were immersed in a Nutrient Agar Broth (NEOGEN Co. Lansing, MI, USA) to cultivate fastidious micro-organisms and then maintained under agitation at 25 °C for 48 hours. Non-sterile scaffolds (n = 10) were used as negative control while the Graft jacket (Regenerative Tissue Matrix, Wright Medical Technology, Inc. San Antonio, TX, USA) was used as positive control (n = 10). Bioburden challenge test was also performed in the following manner: test organisms that included methicillinresistant Staphylococcus aureus (MRSA; DMST 20645 lot No. 3273, NIH) and Bacillus subtilis (ATCC 6633 DMST 15896 lot No. 3479, NIH) were suspended in tryptic soy broth (TSB) (Sigma-Aldrich Co. LLC) to provide a final concentration of 104 cfu/mL. Inoculation of the test carrier was performed by using a micropipette to place 20 lL of the test suspension on the surface of scaffolds and left to dry in the incubator at 37 °C for 18 hours. The scaffolds were then sterilized as described before. After sterilization, the scaffolds were transferred to test tubes containing TSB and were incubated at 25 °C for 7 days. The turbidity of the TSB was measured every day using a UV spectrophotometer at 625 nm wavelength and using McFarland standards as a reference to estimate the number of colonies. The limulus amebocyte lysate (LAL) (ToxinSensorTM Gel Clot Endotoxin Assay Kit, GenScript Inc. Piscataway, NJ, USA) test was used according to the manufacturer’s recommendation. The LAL reagent was mixed with the samples into the endotoxin-free vials, and then incubated at 37 °C for 60 minutes. Each vial was then inverted and checked to see whether a gel had formed. 2.4.4 Morphology of the Platelet Gel-CollagenPolydioxanone Sheath Implant For SEM, the samples were fixed in cold glutaraldehyde 2.5 %, coated by hexa-methyl-desalysin (TAAB, Co., London, UK) and finally gold-coated. The samples were then viewed under a scanning electron microscope (Cambridge, London, UK). The number of activated or inactivated platelets (based on the presence of pseudopodia) in the scaffolds (n = 10) were counted in the SEM photomicrographs (n = 10; Mag = 4009) and the proportion of the activated/total platelets were reported. To confirm that the platelets have been distributed in all parts of the scaffold, homogenously, ten oblique sections were provided

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from different areas of the scaffolds (n = 10) and the number of platelets were counted in ten photomicrographs from each section. Morphology of the platelets inside the scaffolds was also studied by histology. A total of ten histologic sections were provided from different parts of each scaffold (n = 10) and the number of platelets together with their distribution pattern were counted and studied in ten photomicrographs obtained from each section. 2.4.5 Cell Seeding Rat skin fibroblasts (cell line CRL-1213) were seeded on the CI, CI-PDS and CI-PDS-PG implants. The scaffolds were grown in vitro in a 5 % CO2 incubator at 37 °C for 24 hours, 5, 10 and 20 days, with the medium (Dulbecco’s Modified Eagle Medium supplemented with 10 % fetal bovine serum, 20 U/mL penicillin, and 20 lg/mL streptomycin [Invitrogen, Carlsbad, CA, USA]) being replaced every 3 days. 2.4.6 Cell Viability Cell viability was determined by live/dead cell assay, using fluorescein diacetate (FDA, Molecular Probes, Invitrogen Corporation) (live) and propidium iodide (Cayman Chemical Company, Michigan, USA) (dead). The scaffolds (n = 10 CI; n = 10 CI-PDS; n = 10 CI-PDS-PG) with the fluorescence stained cells were viewed under a Nikon fluorescent microscope. The number of live stained cells and dead stained cells were counted by the computer software (Image J, NIH, USA). The viability index was analyzed as: viability index = (number of viable cells/total number of cells) 9 100. 2.4.7 Immunofluorescence Microscopy Cell morphology and cell scaffold interaction were also studied by immunofluorescence microscopy. The scaffolds with cells on a 25 mm coverslip were washed twice with PBS. The cells were fixed in 4 % paraformaldehyde, at RT, for 60 minutes. The fixed cells were then washed twice with 0.02 % PBS/sodium azide and permeabilized with 0.2 % saponin for 10 minutes. The non-specific sites were blocked by incubation in 0.02 % PBS/1 % bovine serum albumin (BSA)/0.02 % sodium azide for 10 minutes at RT. The primary antibody (anti-Grp78, also known as BiP at a 1:100 dilution) was added to the coverslip to completely cover the surface and allowed to incubate at RT for 45 minutes. The coverslip was rinsed three times with 0.02 % PBS/sodium azide, and then blocked again with PBS/BSA/sodium azide for 10 minutes. The secondary antibody (Alexa 488 goat anti-rabbit at a 1:100 dilution) was added to the coverslip and incubated at RT for


45 minutes. The coverslip was rinsed three times with PBS/sodium azide, and then incubated in 2 mL of a DAPI (40 ,6-diamidino-2-phenylindole) solution of 0.5 ng mL-1 for 15 minutes. The coverslip was rinsed with PBS/sodium azide, then with deionized water and mounted on a glass slide with Fluoromount G. 2.5 In vivo Experiment 2.5.1 Animals and Grouping Details One hundred and sixty adult male white New Zealand rabbits were randomly divided into four groups: control (no implant; n = 40), treated with CI (n = 40), CI-PDS (n = 40), and bovine PG embedded within the CI-PDS (CI-PDS-PG) (n = 40), respectively. The animals of each group were then divided into two equal subgroups of 60-day (n = 20) and 120-day (n = 20) time points after injury to assess early and late stages of tendon remodeling. Another 80 rabbits were considered as pilot animals and divided in the same way as the experimental animals as mentioned above. Each group (n = 20) was then divided into four subgroups of 10-day (n = 5), 20-day (n = 5), 30-day (n = 5) and 40-day (n = 5) time points after injury to assess events of inflammation and fibroplasia stage of tendon healing.

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(Matrifen, Roskilde, DK; 0.0015 mg/kg/h) patch was provided for 3 days [6, 32]. 2.5.4 Clinical Evaluations The physical and behavioral status of the animals was monitored weekly. Tarsal flexion degree, weight distribution per limb, pain on palpation, heel and toe position and swelling of the injured area were weekly monitored and scored. The sums of the weekly scores were statistically analyzed to define the differences between the groups [7, 32] (electronic supplementary material [ESM] 1: Table S1). 2.5.5 Bioelectrical Characteristics The importance of the method in assessing tendon healing has been previously discussed [8]. Direct transmission electrical current (DTEC; measured in micro-amp), and tissue resistance to DTEC (TRDTEC; measured in microohm) of the injured and contralateral normal tendons, were measured with a digital multi-pen-type meter (Mastech Seoul, South Korea). The negative probe was placed on the skin of the medial side and the positive probe on the skin of the lateral side of the Achilles tendon. DTEC and TRDTEC were recorded at weekly intervals over days 0–120 postinjury [8].

2.5.2 Premedication and Anesthesia

2.5.6 Platelet Derived Growth Factor (PDGF) Level

Premedication was provided by intra-muscular injection of 1 mg/kg acepromazine maleate. The animals were anesthetized by intra muscular injection of 15 mg/kg ketamine combined with 0.05 mg/kg xylazine hydrochloride (All from Alfasan Co, Woerden, Netherlands) [31].

Immediately before euthanasia, 10 mL of the peripheral blood was collected from each animal, placed in test tubes without EDTA (glass test tubes 16 9 150 mm, Karter Scientific, Labware manufacturing Co., CA, USA), and centrifuged for 10 minutes at 3,500 RPM to separate the serum. Serum PDGF concentration was measured with a commercial ELISA kit: PDGF-AA and AB immunoassay (Biotrend Chemicals, LLC136 South Holiday Road, Unit C, Destin, FL 32550) [5].

2.5.3 Injury Induction and Surgical Reconstruction Under aseptic conditions, a longitudinal skin incision was made over the Achilles tendon complex. Two centimeters of the Achilles tendon with the covering paratenon were completely excised by transverse incisions. Primary reconstruction of the tendon was undertaken, using doublestranded modified Kessler core pattern, by monofilament absorbable polydioxanone suture material (PDS 0-4, Ethicon, INC.1997, Johnson & Johnson, USA). This aligned the remaining tendon extremities in a normal anatomical position, and produced a 2-cm gap between the remaining tendon edges. The same method was applied for all the groups by the same surgeon. For insertion of the implants in the tendon gap, a double-stranded suture was passed through the longitudinal axis of the implants. The subcutaneous tissue and skin over the lesion were closed in a routine fashion. Postoperative analgesia with fentanyl

2.5.7 Euthanasia The animals were anesthetized by intra-muscular injection of a combination of 15 mg/kg ketamine, 2 mg/kg xylazine, and 1 mg/kg acepromazine maleate (all from Alfasan Co, Woerden, Netherlands). The animals were then euthanized by intra-cardiac injection of 1 mg/kg gallamine triethiodide (Specia Co., Paris, France) [31]. 2.5.8 Sample Collection After performing gross morphology, in each group, the harvested tendons were randomly divided equally into two groups (n for each group = 10). The first group was used

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for biomechanical testing. The second group was longitudinally sectioned into three parts. Part A (n = 10) was used for histopathology, part B (n = 10) for transmission electron microscopy (TEM) and part C for biochemistry (n = 10). 2.5.9 Gross Morphology Hyperemia, peritendinous adhesion, general appearance, muscle atrophy and fibrosis and tendon diameter were scored and measured [6] (ESM 1: Table S2). 2.5.10 Histopathology After fixation in 10 % neutral buffered formalin, the tendons were washed, dehydrated in a graded series of ethanol, cleared in xylene, embedded in paraffin wax, longitudinally and transversely sectioned at 4 lm thickness, mounted on glass slides and stained by hematoxylin and eosin. Under light microscopy, digital photomicrographs were captured and transferred into Adobe Photoshop cc (ADOBE Co, CA, USA). In the micrographs, the number of different types of cells and their total number, together with the number of blood vessels, were counted. The counting was performed in triplicate. Density of the collagen fibers and diameter of various structures were measured. Other histopathologic features were scored. In each group, a total of 250 photomicrographs were counted and scored [6, 12] (ESM 1: Table S3). 2.5.11 Transmission Electron Microscopy The method has been previously described [8]. Briefly, the samples were fixed in cold glutaraldehyde (4 %), dehydrated in a graded series of ethanol, and embedded in epoxy resin 811 (TAAB CO., London, UK). Transverse sections (n = 250 in each group) of 70–80 nm thickness were prepared, and standard methods were employed for production of the ultra-micrographs. The ultra-micrographs were transferred to Adobe Photoshop cc (ADOBE Co, CA, USA); number, diameter and density of the collagen fibrils were directly counted and other ultrastructural features were reported and or scored [8] (ESM 1: Table S4). 2.5.12 Biomechanical Testing The harvested tendon samples were mounted between the two cryoclamps and preconditioning was done before performing the mechanical testing (InstronÒ Tensile Testing Machine, London, UK). Maximum load, yield load, stiffness, strain, yield strain, maximum tensile stress and modulus of elasticity of the samples were then extracted

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from the force-displacement and stress-strain curves regenerated from the computer [12]. 2.5.13 Biochemistry Dry matter content was calculated as the following equation: percentage dry matter content = weight dry/weight wet 9 100. The hydroxyproline concentration was measured by spectrophotometry. The samples were hydrolyzed in 6 M HCl at 105 °C for 14 hours and the hydroxyproline was oxidized by chloramine T, and then by adding Ehrlich’s reagent and incubating at 60 °C, a chromophore was formed. To remove the interfering chromophores, the hydroxyproline product in alkaline media was extracted into toluene and then into acid phase. The absorbance of acid phase was read at 543 nm and the hydroxyproline content was calculated from the calibration curve based on the standard solutions run in the same way as the samples [32]. 2.6 Statistical Analyses All the quantitative values were expressed as mean ± standard deviation. The differences of the measured values between multiple groups at one time point (e.g., 60 or 120 days post-injury [DPI]) were tested, using one-way ANOVA and for two time points (60 vs 120 DPI), using two-way ANOVA with subsequent Tukey’s post hoc tests. All scored values were expressed as median (min– max). Kruskal–Wallis H test was performed to analyze the scored values. A p value of \0.05 was considered statistically significant [6].

3 Results 3.1 In vitro Evaluations 3.1.1 Morphology of the Platelets The non-activated PFSCs, PSSCs and also the PLSSPs were biconvex discoid structures shaped like a lens, 2–3 lm in greatest diameter. The activated platelets observed in the platelet gel had pseudopodia emission as confirmed by TEM and SEM. Collecting the whole blood samples in the EDTA did not have any deleterious effect on platelet morphology and their activation. 3.1.2 Light Transmission Aggregometry The LTA of the platelets of the whole plasma and buffy coat derived after the first centrifugation (PFSC; number of platelets = 750,000/lL), platelets derived after the second step centrifugation (PSSC; 1,500,000 platelets/lL), and the


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544 b Fig. 1 a Schematic view of the xenogenous-based platelet-rich plasma (PRP) preparation and its embedding within the collagenpolydioxanone sheath (PDS) implant. The peripheral blood was obtained through intravenous catheterization of healthy bovines, and was transferred into the ethylene-diamine-tetraacetic acid (EDTA) tubes to prevent blood coagulation. The samples were centrifuged twice. After the first centrifugation, three layers were formed, including (i) platelet-poor plasma (PPP), (ii) PRP ? white blood cells (WBCs) = buffy coat, and (iii) red blood cells (RBCs). The PPP and buffy coat layers were suctioned and transferred into a new tube. The RBCs were discarded. The PPP and buffy coat were centrifuged again. This resulted in the formation of three layers, including PPP, PRP, and WBC. WBCs were discarded, and the PPP and PRP were suctioned into the sterile tubes. The solution was mixed and lyophilized to concentrate the platelets and produce a lyophilized platelet powder. The powder was then sterilized through ultraviolet (UV) irradiation, and then dissolved in sterile phosphate buffer saline (PBS) to produce the desired concentration (6–7 times greater than the physiological blood concentration). The sterile, fully dehydrated three-dimensional collagen-PDS implant was immersed in the PRP solution, and the implant was left to absorb the platelets in its architecture. The PRP solution was then activated, using bovine thrombin and CaCl2. Consequently, the absorbed PRP in the collagenPDS implant was activated and the gel was formed in the implant. The final product was a platelet gel embedded in the collagen-PDS implant. Each arrow shows a subsequent step. b–d Scanning electron microscopic (SEM) images of the collagen-PDS-platelet gel implant. The platelets are indicated by arrows. e and f are micrographs of the bovine platelets after second-step centrifugation captured by light and inverted microscopies, respectively. g–i are live dead cell assays of the cultured fibroblasts over the collagen implant (CI), collagen implant-PDS (CI-PDS), and collagen implant-PDS-platelet gel (CIPDS-PG), respectively, after 20 days of cell culture. The live cells have been stained by fluorescein dye acetate and dead cells have been stained with red propidium iodide. Note that the platelets increased the cell viability of the implants. j–l are SEM images of the cultured fibroblasts over the CI, CI-PDS, and CI-PDS-PG, respectively, after 20 days of cell culture. Note that the bovine platelets improved cellular proliferation and distribution over the implant. m–p are fluorescence microscopic images of the fixed rat fibroblasts on CI, CIPDS, and CI-PDS-PG, respectively, with rabbit anti-GRP 78 conjugated with Alexa 488 goat anti-rabbit (green)/40 ,6-diamidino-2phenylindole (DAPI; blue) stained cells after 20 days of cell seeding and culture. Note that the bovine platelets improved cellular migration and proliferation of the cultured fibroblasts. q and r are surgical figures captured during implantation of the artificial tendons. Note that 2 cm of Achilles tendon has been discarded (q) and the artificial graft has been inserted in the defect area

platelet solution prepared after lyophilization and saline solving procedures (PLSSP; 20,000,000 platelets/lL) were 78.34 ± 3.55, 74.36 ± 6.91, and 68.32 ± 5.92 %, respectively. There were no significant differences between the LTA of PFSC and the PSSC (p [ 0.05). There were also no significant differences between the LTA of the PSSC and the PLSSP (p [ 0.05). 3.1.3 Platelet Growth Factor Level The PDGF-AB levels of the PFSC (number of platelets = 750,000/lL), PSSC (1,500,000 platelets/lL),

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PLSSP (20,000,000 platelets/lL) and the platelet gel (activated platelets; 20,000,000 platelets/lL) were 74.18 ± 7.91, 243.27 ± 34.12, 169.88 ± 27.02 and 692.55 ± 102.38 ng/mL, respectively. The IGF-I levels of the PFSC (number of platelets = 750,000/lL), PSSC (15,000,000 platelets/lL), PLSSP (20,000,000 platelets/ lL) and the platelet gel (activated platelets; 20,000,000 platelets/lL) were 32.78 ± 12.32, 78.43 ± 8.19, 68.54 ± 10.92 and 132.47 ± 22.93 ng/mL, respectively. Activation of the platelets significantly increased the growth factors levels of the platelet gel compared with the controls (p = 0.001). 3.1.4 Morphology of the Collagen-Polydioxanone Sheath-Platelet Implant Number of platelets/mm3 of the CI-PDS was 2 9 106. At SEM, the platelets and fibrin were well distributed and infiltrated throughout the CI-PDS-PG so that the implant absorbed the platelets, homogenously. The structure of the platelets was confirmed by SEM, TEM and light microscopy. No significant differences were seen between the number of the platelets in ten different areas of the implants (p [ 0.05). In the CI-PDS-PG scaffolds, the proportion of the activated platelets/total platelets was 89.61 ± 6.54 %. Such a proportion suggests that the activation method was effective and most of the platelets were activated inside the scaffolds. 3.1.5 In vitro Cell Viability Tests In all the CI (89.52 %), CI-PDS (88.15 %), and CI-PDSPG (92.19 %), almost all the fibroblasts were green, indicating the cells were alive. Lack of propidium iodide (red) stained dead cells supports the idea that normal rat fibroblasts were attached to the scaffold and that the majority of the cells were viable. Xenogenous-based BPG-embedded CI-PDS significantly increased the cellular proliferation and cell viability (p \ 0.05). Number of FDA-stained viable cells in the CI, CI-PDS and CI-PDS-PG (2009) were 127.82 ± 12.01CI-PDS-PG versus 83.92 ± 6.37CI-PDS versus 84.71 ± 9.41CI (day 5, p = 0.001), 274.82 ± 25.91CI-PDS-PG versus 180.47 ± 16.93CI-PDS versus 186.33 ± 27.91CI (day 10, p = 0.001), and 572.42 ± 57.77CI-PDS-PG versus 297.48 ± 58.11CI-PDS versus 321.34 ± 49.23CI (day 20, p = 0.001) and was 97.23CI-PDS-PG versus 92.85CI-PDS versus 94.83CI % (day 5), 95.32CI-PDS-PG vs. 91.64CI-PDS versus 92.16CI % (day 10) and 92.19CI-PDS-PG versus 87.44CI-PDS versus 89.52CI % (day 20) of the total cellularity (vitality index). After 5, 10, and 20 days of fibroblast seeding and culture into the CI, CI-PDS, and CI-PDS-PG, the qualitative


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Fig. 2 Bioelectrical characteristics including direct transmission electrical current and tissue resistance to direct transmission electrical current of the injured healing tendon from days 0–120 post-injury and serum platelet-derived growth factor (PDGF) level of the animals at 60 and 120 days post-injury (DPI). Note that micro-amp of a tissue increases with inflammation during the first 2 weeks post-injury while it decreases as the tissue becomes remodeled and the healing is in a

more advanced stage. Micro-ohm of a tissue decreases during inflammation (i.e., the first 2 weeks post-injury) but it gradually increases during the fibroplasia and remodeling stages of tendon healing. The serum PDGF level is an indicator of platelet growth factors and healing quality. Note that the platelet-treated group has a significantly higher serum PDGF level at 60 and 120 DPI compared with the controls

results of the SEM and immunofluorescence microscopic studies showed that bovine platelets resulted in a better distribution of the cultured fibroblasts inside the scaffold and the cells were more mature and produced more collagenous matrix in the scaffold. In the CI-PDS-PG, the cultured fibroblasts proliferated all over the scaffold both on the surface and also in the internal architecture of the scaffolds so that the fibroblasts oriented in an aligned manner along the direction of the scaffold collagen fibers. There were no significant differences between numbers of the cultured fibroblasts in different parts of the CI-PDS-PG scaffolds as confirmed by immunofluorescence microscopy, SEM and light microscopy (p [ 0.05) (Fig. 1). In contrast to the embedded platelet gel scaffolds, although the fibroblasts proliferated in an aligned manner outside and inside the CI and CI-PDS scaffolds, the number of cultured fibroblasts was significantly higher in the peripheral parts than the inner parts of the scaffolds (p \ 0.05). These results confirmed that the BPG triggered and accelerated the fibroblasts proliferation more homogenously and continuously all over the scaffolds.

3.2 In vivo Findings 3.2.1 Clinical Evaluations None of the animals died during the course of the experiment and treatment with BPG did not alter the weight gain of the animals. All the animals had good appetite during the course of the study. Those animals treated with artificial tendon-platelet gel (AT-PG) significantly had superior scored values for the tarsal flexion degree of the injured limb (15 [10–18]CI-PDS-PG vs 24 [22–29]CI-PDS vs 29.5 [27–35]CI vs 34.2 [31–42]Control (defect only), p \ 0.05), heel and toe position of the injured limb (14.5 [12–17]CI-PDS-PG vs 19 [16–22]CI-PDS vs 28 [24–36]CI vs 38 [34–44]Control, p \ 0.05), weight distribution on the legs (19 [15–24]CI-PDS-PG vs 34 [29–37]CI-PDS vs 39 [36–44]CI vs 45 [41–49]Control, p \ 0.05) and pain on palpation (12.5 [9–14]CI-PDS-PG vs 18 [14–21]CI-PDS vs 18.5 [15–22]CI vs 28 [24–32]Control, p \ 0.05) compared with those treated with artificial tendon (CI-PDS), collagen implant or left untreated (control, no implant).

17.15 ± 2.64 51.16 ± 4.3 2.67 ± 1.3 43.23 ± 3.89 33.44 ± 3.93

87.31 ± 7.4 4.19 ± 1.54 59.32 ± 8.19 12.35 ± 2.96

Lymphocyte (n)

Plasma cell (n)

Macrophage (n)

1.32 ± 0.04

7.93 ± 1.23 3.7 ± 0.15 1.69 ± 0.04

Immature fibroblast (diameter; lm)

Mature fibroblast (diameter; lm)

49.63 ± 2.98

15.81 ± 0.98

35.19 ± 3.3

82.47 ± 3.68

15.64 ± 1.03 42.9 ± 2.65

6.72 ± 0.87

3.09 ± 0.98

35.4 ± 2.16

21.49 ± 2.32

45.08 ± 1.98

92.6 ± 3.61

15.36 ± 0.82 45.29 ± 2.24

1.3 ± 0.03

3.57 ± 0.14

6.29 ± 0.43

5.05 ± 1.39

5.17 ± 1.01

19.88 ± 1.29

25.19 ± 3.65

53.67 ± 3.19

12.54 ± 2.24

66.44 ± 5.31

23.47 ± 3.66

24.94 ± 3.28

131.82 ± 6.11

85.70 ± 5.07

401.2 ± 9.59 241.17 ± 9.1

15.98 ± 1.24

21.32 ± 2.42

60.52 ± 3.32

119.01 ± 5.25

24.43 ± 1.98 54.18 ± 2.13

1.2 ± 0.02

2.76 ± 0.37

5.06 ± 0.61

12.52 ± 1.93

16.22 ± 1.45

15.48 ± 1.45

51.85 ± 4.36

56.3 ± 2.54

10.19 ± 1.37

59.58 ± 6.59

29.31 ± 4.75

44.56 ± 3.05

195.60 ± 12.33

49.10 ± 5.96

428.64 ± 7.44 280.21 ± 5.33

48.46 ± 2.9

19.86 ± 1.65

23.52 ± 4.03

81.62 ± 4.31

15.24 ± 0.97 42.57 ± 1.85

1.52 ± 0.08

3.31 ± 0.39

7.82 ± 1.13

4.74 ± 1.65

6.83 ± 0.75

17.29 ± 2.24

32.87 ± 6.26

33.22 ± 5.67

2.56 ± 1.12

63.51 ± 5.6

1.09 ± 0.69

22.01 ± 2.53

121.44 ± 11.34

167.48 ± 12.11

443.11 ± 16.37 322.14 ± 15.19

32.9 ± 2.84

10.62 ± 1.36

62.38 ± 3.02

101.04 ± 3.57

21.6 ± 1.93 50.48 ± 1.65

1.24 ± 0.05

2.79 ± 0.36

5.49 ± 0.33

3.1 ± 0.93

4.72 ± 0.65

10.16 ± 0.98

16.9 ± 3.28

22.44 ± 2.64

1.15 ± 0.87

39.63 ± 5.2

0.97 ± 0.63

33.31 ± 2.69

122.91 ± 3.84

68.73 ± 6.37

289.17 ± 8.36 222.48 ± 9.68

CI

21.65 ± 3.02

15.19 ± 1.47

70.65 ± 3.76

122.31 ± 5.51

25.13 ± 1.09 51.03 ± 2.08

1.18 ± 0.02

2.55 ± 0.2

5.55 ± 0.36

1.94 ± 0.66

3.38 ± 0.38

6.32 ± 1

11.06 ± 1.27

24.21 ± 2.95

1.84 ± 0.92

46.34 ± 3.44

4.08 ± 2.58

46.31 ± 4.65

109.22 ± 6.58

41.05 ± 5.36

273.71 ± 7.59 191.54 ± 7.91

CI-PDS

11.6 ± 1.27

9.23 ± 0.84

86.99 ± 4.69

192.9 ± 8.05

28.32 ± 1.19 56.86 ± 2.18

0.97 ± 0.03

2.49 ± 0.19

4.88 ± 0.51

4.3 ± 1.72

2.33 ± 0.35

1.34 ± 0.6

7.24 ± 0.81

7.55 ± 0.95

1.34 ± 0.62

15.81 ± 1.93

3.33 ± 1

84.8 ± 5.32

96.46 ± 6.07

28.39 ± 5.24

236.62 ± 11.3 217.41 ± 8.85

CI-PDS-PG

CI collagen implant, DPI days post-injury, PDS polydioxanone, PG platelet gel

One- and two-way ANOVA with their subsequent post hoc tests were used to analyze the differences between the groups. Number of tissue samples in each group = 10, number of tissue sections for each sample = 5, number of histologic fields used to count and measure different variables in each tissue section = 5. In total, 250 histopathologic fields were used to analyze the histopathologic features of different groups

24.77 ± 3.4 63.16 ± 2.27

Cell density (%)

Background density (%)

8.21 ± 1.86

69.19 ± 4.08

Large vessels (diameter; lm)

Collagen density (%)

11.9 ± 0.86 34.41 ± 1.58

Small vessels (diameter; lm) Medium vessels (diameter; lm)

Fibrocyte (diameter; lm)

3.35 ± 0.18

0.62 ± 0.56

Large vessels (n)

3.67 ± 0.7

3.07 ± 0.88

Medium vessels (n)

26.7 ± 2.02

7.84 ± 0.63

Small vessels (n)

14.18 ± 2.04

3.72 ± 1.64 7.29 ± 1.73

Fibrocyte (n)

Neutrophil (n)

96.41 ± 5.63 144.68 ± 8.99

221.82 ± 10.83 111.44 ± 6.43

Immature fibroblast (n)

Mature fibroblast (n)

373.44 ± 8.01 257.06 ± 5.31

523.41 ± 19.14 334.24 ± 10.9

Vessels (n)

CI-PDS-PG

Control (no implant)

CI-PDS


CI

120 DPI time point

60 DPI time point

Total cell (n) Total fibroblast (n)

Table 1 Histopathologic analysis

546 A. Oryan et al.


547

Fig. 3 Host–graft interaction mechanism after implantation of various implants in an experimentally induced tendon defect. Note that combination of platelet gel with the artificial tendon considerably improved cellular proliferation, migration, differentiation and matrix production. Platelets also improved and enhanced implant degradation,

incorporation, and acceptance compared with the controls. Long arrows show the direction of the newly regenerated collagen fibers. All the figures are longitudinal histologic sections. Stained with H&E. A.C.R accepted collagen remnant (as a part of new tendon), B.V. blood vessel, C.R collagen remnant, I Inflammation, R.T regenerated tissue

3.2.2 Bioelectricity of the Injured Tendons

than the control groups. The inflammation persisted for up to 2 weeks. Then, the inflammation gradually decreased and the fibroplasia stage developed in all the groups. At gross morphology, the CI-PDS-PG was degraded and replaced by the new tendon (day 20) faster than those of the collagen (day 30) and CI-PDS (day 30) implants. In the control group (no implant), no tendon was formed. At 60 and 120 DPI, the platelettreated tendons had less hyperemia and peritendinous adhesion but also had higher short and long transverse diameters compared with the controls. At the histopathologic level, compared with those tendons treated with either the collagen or CI-PDS implants, treatment with BPG increased the migration and proliferation of the inflammatory cells in the artificial tendon more homogenously. In the collagen and CI-PDS groups, the implants were mostly degraded and replaced by the new tissue but the remnants of the implants were still present at 60 DPI. In contrast, in the platelet gel group, the implant was degraded faster than the controls. In addition, considerable parts of the implant were infiltrated by the fibroblasts and were accepted as parts of the new tendon so that after 60 DPI, no evidence of implant remnant was seen. At 60 DPI, the BPG increased inflammation and total cellularity but, interestingly, it also increased collagen production so that the collagen fibers were oriented in an aligned manner and the cells were laid along the direction of the collagen fibers. The BPG also increased angiogenesis and angiomaturation at that stage. At 120 DPI, those tendons treated with BPG had superior tissue maturity,

Bovine platelets increased DTEC of the injured area at 7 and 14 DPI more than that observed for those treated with implants alone but, at 21 DPI to the end of the experiment, the DTEC of the platelet-treated group decreased gradually but faster than the control groups so that after 120 DPI the DTEC of the platelet groups was closer to the normal value measured at day 0 and was significantly lower than that of the control groups (p \ 0.05). From 21 DPI to the end of the experiment, bovine platelets increased the TRDTEC of the injured area gradually but faster than in the control groups so that, at 120 DPI, the TRDTEC of the platelet group was significantly higher than those of the control groups and was closer to the normal value (p \ 0.05) (Fig. 2). 3.2.3 Serum PDGF Level At 60 DPI, BPG significantly increased the serum PDGF AA and AB level of the injured animals compared with the controls (p = 0.001 for all). Compared with 60 DPI, the serum PDGF level of all the groups significantly decreased at 120 DPI. At this stage (120 DPI), the serum PDGF level of the PG group was still significantly higher than the control groups (p = 0.001 for all) (Fig. 2). 3.2.4 Gross Morphologic and Histopathologic Findings Treatment with bovine platelets increased transverse diameter, hyperemia, and inflammatory response faster

548

A. Oryan et al.

Fig. 4 Morphologic figures of injured and normal tendons at 120 days post-injury (DPI). Note that treatment with bovine platelets significantly improved tendon regeneration, reduced tendon adhesion and muscle fibrosis and atrophy. The treated tendons had better cellular differentiation and distribution, collagen fiber alignment and density. At the ultrastructural level (transmission electron microscopy; TEM), the platelets significantly improved collagen fibrillogenesis and differentiation so that the collagen fibrils of the platelet

group have a higher diameter and have been distributed multimodally, which is more similar to normal uninjured tendons when compared with the controls (defect, treated with collagen implant, and treated with collagen implant-polydioxanone sheath). In the TEM images, the collagen fibrils are indicated by a star. In the histologic figures of the gastrocnemius-soleus muscle, the stars show a muscle fiber

their collagen fibers were mature and align, were comparable to normal tendon, and their cellularity and number of blood vessels had decreased compared with 60 DPI. At this stage (120 DPI), the biggest proportions of the platelettreated tendons were collagenous matrix. At 60 DPI, treatment with BPG significantly increased the number of total cellularity, mature fibroblasts, fibrocytes, neutrophils, macrophages, total vascularity, mediumand large-sized blood vessels, transverse diameter of the small-, medium-, and large-sized blood vessels, and collagen density compared with the control groups (p \ 0.05). At 120 DPI, those tendons treated with BPG had significantly lower cellularity, immature tenoblasts, lymphocytes, and macrophages, total blood vessels, small- and mediumsized blood vessels, and cell density but also had significantly higher tenocytes, diameter of small-, medium-, and large-sized blood vessels and collagen density compared with the controls (p \ 0.05) (Table 1).

Those tendons treated with BPG showed superior collagen fiber alignment (0 [0–1]CI-PDS-PG vs 1 [1–2]CI-PDS vs 1.5 [1–3]CI vs 3 [2–3]control, p \ 0.05), perivascular edema (0 [0–1]CI-PDS-PG vs 1 [0–2]CI-PDS vs 1 [0–2]CI vs 3 [2–3]control, p \ 0.05), tissue maturity (0.5 [0–1]CI-PDS-PG vs 1 [1–2]CI-PDS vs 2 [1–3]CI vs 3.5 [3–4]control, p \ 0.05), crimp pattern (0 [0–1]CI-PDS-PG vs 1 [0–2]CI-PDS vs 1.5 [1–2]CI vs 4 [4–4]control, p = 0.001 for both) and vascularity (0.5 [0–2]CI-PDS-PG vs 1.5 [1–2]CI-PDS vs 1.5 [1–3]CI vs 4.5 [4–5]control, p \ 0.05) compared with those tendons treated with either the collagen or CI-PDS implants and also with those that were left untreated (Figs 3, 4). 3.2.5 Ultrastructural Findings At 60 DPI, treatment with BPG significantly increased number, transverse diameter and density of the collagen fibrils and elastic fibers compared with those that were

CI-PDS-PG

3,787.39 ± 146.91

1,848.42 ± 74.01

Mature fibroblast (diameter)

Fibrocyte (diameter)

0

Fibrils (256–307 nm) (diameter)

8,429.48 ± 557.24

0


Immature fibroblast (diameter)

0


2.33 ± 0.66

0


Elastic fiber (number)

47.96 ± 1.6

28.72 ± 2.46


28.72 ± 2.46

350.26 ± 21.71

Total (n)

47.15 ± 4.17

0

Fibrils (256–307 nm) (n)

Total (diameter)

0


Fibril density (%)

0

0


1,673.26 ± 98.62

3,582.81 ± 148.18

6,764.45 ± 345.7

8.93 ± 1.46

66.23 ± 5.28

0

0

72.96 ± 4.68

45.28 ± 5.19

590.08 ± 28.81

0

0

0

48.95 ± 8

0


549.8 ± 26

350.26 ± 21.71


1,521.9 ± 61.62

3,352.38 ± 158.55

6,456.23 ± 300.79

6.8 ± 0.93

72.24 ± 5.01

58.73 ± 2.75

0

0

0

86.66 ± 4.23

54.45 ± 2.03

652.77 ± 24.28

0

0

0

75.27 ± 7.65

597.18 ± 18.78

1,296.44 ± 64.78

2,904.54 ± 122.21

5,834.2 ± 698.12

16.39 ± 1.29

81.62 ± 4.64

66.95 ± 2.31

0

166.78 ± 6.94

118.18 ± 4.17

94.42 ± 4.58

58.39 ± 3.08

914.44 ± 47.08

0

8.68 ± 2.5

43.57 ± 5.61

112.49 ± 12.23

723.59 ± 32.00

1,560.14 ± 83.83

3,329.08 ± 282.39

7,580.23 ± 260.02

1.11 ± 0.388

55.99 ± 4.97

42.45 ± 2.37

0

0

0

70.68 ± 3.5

38.17 ± 3.89

290.47 ± 41.91

0

0

0

50.85 ± 16.71

228.35 ± 17.78


CI-PDS


CI

120 DPI time point

60 DPI time point

Table 2 Transmission electron microscopy

1,313.35 ± 61.09

2,912.29 ± 169.06

5,737.96 ± 330.73

5.29 ± 0.79

71.73 ± 4.68

61.61 ± 2.77

0

0

111.86 ± 3.81

92.39 ± 4.04

51.43 ± 1.33

498.62 ± 17.52

0

0

23.74 ± 12.12

91.36 ± 9.53

371.36 ± 12.49

CI

1,161.44 ± 68.15

2,788.05 ± 200.06

4,844.45 ± 330.93

3.27 ± 0.72

79.59 ± 4.07

68.78 ± 2.41

0

157.91 ± 5.71

122.01 ± 2.74

100.42 ± 1.65

53.51 ± 2.71

465.28 ± 35.08

0

2.17 ± 1

16.44 ± 6.91

124.93 ± 14.19

326.57 ± 12.00

CI-PDS

1,071.33 ± 58.09

2,320.14 ± 89.73

4,391.23 ± 247.51

7.31 ± 0.81

86.86 ± 6.11

82.25 ± 2.74

262.71 ± 4.69

194.08 ± 7.36

139.79 ± 7.6

100.40 ± 1.88

59.67 ± 2.55

732.77 ± 24.42

10.61 ± 2.5

25.24 ± 4.23

69.70 ± 11.42

237.42 ± 18.74

426.39 ± 17.97

CI-PDS-PG

Platelet Gel and Tendon Healing 549

treated with either the collagen or CI-PDS implants (p = 0.001 for all). At 120 DPI, due to the fibril aggregation and increase in the collagen fibril size, the number of collagen fibrils significantly decreased in all groups compared with that observed at 60 DPI. At this stage (120 DPI), those tendons treated with BPG had a significantly higher number, transverse diameter, and density of collagen fibrils and elastic fibers compared with those tendons treated with either the collagen or CI-PDS implants and the control group (p = 0.001 for all). Collagen fibrils in the BPG-treated group were differentiated and tetra-modally distributed all over the field. In longitudinal sections, they were continuous and highly aligned. Collagen fibrils of those tendons treated with either the collagen or CI-PDS implants were bimodal and trimodal at 60 and 120 DPI, respectively, and were continuously distributed in an aligned manner. Collagen fibrils of the control group were unimodal, amorphous, interrupted, and were haphazardly oriented throughout the field. Those tendons treated with BPG had higher numbers of tenocytes. In addition, their tenocytes were more mature than the controls (Fig. 4; Table 2). 3.2.6 Biochemistry of the Injured Tendons Treatment with BPG significantly increased the dry matter and hydroxyproline contents both at 60 and 120 DPI compared with the controls (p = 0.001 for all). At 120 DPI, those tendons treated with BPG reached 82.51 and 80.33 % of the dry matter and hydroxyproline contents of the normal uninjured tendons, respectively (Fig. 5). CI collagen implant, DPI days post-injury, PDS polydioxanone, PG platelet gel

One- and two-way ANOVA with their subsequent post hoc tests were used to analyze the differences between the groups. Number of tissue samples in each group = 10, number of tissue sections for each sample = 5, number of histologic fields used to count and measure different variables in each tissue section = 5. In total, 250 ultramicrographs were used to analyze the ultrastructural features of different groups

249.01 ± 14.56 207.99 ± 12.48 190.03 ± 9.68 113.03 ± 16.94 Elastic fiber (diameter)

56.26 ± 7.58

139.23 ± 9.63

95.98 ± 10.47

CI-PDS CI Control (no implant) CI-PDS-PG CI-PDS CI Control (no implant)

120 DPI time point 60 DPI time point Table 2 continued

374.9 ± 30.98

A. Oryan et al.

CI-PDS-PG

550

3.2.7 Biomechanical Findings Treatment with BPG significantly increased the ultimate load, yield load, stiffness and also significantly decreased maximum and yield strain and therefore it significantly increased maximum tensile stress and elastic modulus of the injured treated tendons (p \ 0.05). The BPG-treated tendons had significantly higher mechanical properties compared with the control groups both at 60 and 120 DPI (p \ 0.05). Compared with 60 DPI, the biomechanical properties of all the groups significantly increased at 120 DPI (p = 0.001 for all). At this stage, those tendons treated with BPG gained 62.04, 61.33, 44.56, 71.8, and 68.81 % of the ultimate load, yield load, stiffness, maximum stress and elastic modulus of their normal contralateral tendons, respectively (Fig. 5).

4 Discussion This is the first study to report the role of BPG on tendon healing. The results clearly showed that BPG, when


551

Fig. 5 Dry matter and hydroxyproline contents and biomechanical features of the injured and normal tendons at two time points of 60 and 120 days post-injury (DPI). CI collagen implant, N Newton, PDS polydioxanone, PG platelet gel

embedded within the artificial tendon graft, could improve the biocompatibility, biodegradability and healing incorporative properties of the artificial tendon. The most effective role of BPG was shown to be correlated with its effectiveness on the inflammatory mechanism. In fact, the BPG enhanced the inflammation in the short term, triggered a faster fibroplasia which led to improved remodeling characteristics. The BPG had no harmful effect in vivo and was well tolerated by the rabbits. Healing of large tendon defects is difficult [6]. The healing response is composed of three overlapping phases including inflammation, fibroplasia and remodeling [4]. The role of inflammation should be highlighted because without inflammation, no effective healing could be expected [8]. In the inflammatory phase, the neutrophils, macrophages, lymphocytes, and several macro and micro molecules are engaged to produce inflammatory events [4]. Macrophages have a highlighted role because they trigger cellular behavior and accelerate the transition to fibroplasia [6, 8]. In addition, they phagocytize the necrotic tissues and dead cells [4]. Woodall et al. [33], in an in vitro investigation, showed that human PRP is able to suppress the human macrophages. This could explain why the autogenous PRP may have invaluable outcomes in healing and regeneration. Unlike Woodall et al., we showed that bovine PRP is effective in increasing rabbit macrophages in vivo

and the outcome of tendon healing was found to be correlated with this novel finding. We followed inflammation and other stages of tendon healing using various methods including bioelectricity, clinical evaluations, and histopathology at multiple time points. Platelet gel increased inflammation in the short term; however, its duration was not prolonged. Galliera et al. [23] showed that platelets play an active role in the immunological and inflammatory aspects of tissue healing. Indeed, they can be directly involved in the inflammatory response through the production and release of several inflammatory mediators, including a variety of cytokines such as transforming growth factor (TGF)-b, interleukin (IL)-1b, CD40L, and chemokines such as CXCL7, CXCL4, CXCL4L1, CCl5, CXCL1, CXCL8, CXCL5, CXCL12, CCL2, and CCL3. Platelet are not only a source of several chemokines involved in the inflammatory response and tissue healing, but they also express chemokine receptors, in particular CCR1, CCR3, CCR4, and CXCR4, thus being able to regulate the inflammatory response associated with the healing process. However, this local inflammation must be taken under control, and platelets can prevent the excess of leukocyte recruitment by anti-inflammatory cytokines such as TGF-b. These findings are in complete accordance with our results.

552

In histopathology, the platelet gel increased cellularity in the short- to mid-term but also enhanced distribution throughout the healing tissue. Good distribution of the inflammatory cells and fibroblasts enhanced graft resorption, incorporation, and acceptance. The BPG also increased vascularity and angiogenesis throughout the healing tissue in the short- to mid-term, and thus established improved tissue circulation which was necessary for tendon regeneration. Bosch et al. [26] studied the role of PRP on neovascularization of experimentally induced tendon injuries in horses and showed that a single injection of PRP is able to significantly increase neovascularization of the healing tendon. Also, Vogrin et al. [34], in a prospective, randomized, double-blind clinical trial, showed that local application of platelet gel enhances early revascularization of the graft in the osteoligamentous interface zone after anterior cruciate ligament (ACL) reconstruction. Compared with the controls, the higher inflammatory response, together with a better tissue microcirculation observed in the BPG-treated group, improved cellular behavior in the healing tendon [6]. In fact, the migrated mesenchymal cells were highly differentiated into tenoblasts and tenocytes. These cells are responsible for matrix production during tendon healing. As a result, at ultrastructural level, superior collagen fibrillogenesis, fibril diameter, density and continuity together with an improved collagen fibril to fiber differentiation were seen in the BPGtreated tendons compared with the controls. Under light microscopy, the collagen fibers were aligned, densely packed in close contact with each other, and elongated between muscle to bone. In a systematic review on effectiveness of autologous platelets (e.g., PRP) on healing and repair of tendon ruptures and tendinopathies in human and animal models, Sadoghi et al. [35] concluded that platelets are effective in tendon ruptures because they have a role in tissue maturation, which is in line with our findings. These beneficial results may also be due to the role of PDGFs [21, 36]. Zhang and Wang [37] showed that PRPclot releasate, due to its growth factors, promotes differentiation of tendon stem cells into active tenocytes, exhibiting high proliferation rates and collagen production capability. Also, Hoppe et al. [36] showed that the tenocytes of chronic rotator cuff tendon tears can be stimulated by platelet-released growth factors so that the growth factors increased cellular proliferation, migration, differentiation, and matrix production of such cells in vitro, which is in accordance with our in vitro and in vivo findings. In addition, Anitua et al. [19] showed that releasates from both platelet-rich and platelet-poor clots stimulate tendon cell proliferation, in contrast to un-clotted PPP. The cultured tendon cells synthesized vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) in the presence of PPP clots and PRP-clot releasates; thus, the

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synthesized amount was significantly higher with supernatants from platelet-rich clots than supernatants from the platelet-poor clots. Our in vitro results suggest that the activated platelets in the BPG had significantly higher PDGF and IGF-I growth factor levels than the controls, which enhanced migration, proliferation, differentiation, and matrix production of the cultured fibroblasts in the scaffolds. We also measured the PDGF level of the animals and showed that those animals treated with BPG had significantly higher serum PDGF levels than the controls, suggesting that the platelets increased the level of growth factors during tendon healing. The importance of PDGF has been previously shown and it has been suggested that serum PDGF level is an important indicator of tendon healing [6, 32]. A higher serum PDGF level suggests a better healing response had occurred. The BPG also reduced tendon adhesion to the surrounding structures and prevented development of muscle fibrosis. All these evidences suggest that bovine platelets not only improved the tendoconductivity of the scaffold but also enhanced the tendoinductivity, tenointegration, and tenoincorporative properties of the graft. Collectively, treatment with BPG, by the above mechanisms, significantly increased the mechanical properties and functionality of the newly regenerated tendon. Less muscle atrophy, together with the better physical activity observed in the animals treated with BPG, confirmed that the structural and mechanical properties of the healing treated tendons led the animals to have better functionality during the course of the experiment. We showed that it is possible to increase the bioactivity of the engineered tissue-based constructs in vivo. A combination of BPG with CI-PDS is one such example, and has strong importance in the field of tissue engineering and regenerative medicine. This statement was confirmed by in vitro and in vivo evaluations as described in the present research. Numerous studies suggest and recommend PRP as an effective therapeutic approach for musculoskeletal injuries [20]. Recent trends suggest controversy regarding the effectiveness of autogenous platelets on tendon or other musculoskeletal tissue healing and regeneration. For example, Visser et al. [38] implanted autologous plateletrich fibrin (PRF) membrane in a patellar tendon defect model in eight dogs and showed that PRF membrane does not enhance the rate or quality of tendon healing in patellar tendon defects. However, it did increase the amount of reparative tissue within and around the defect. Schepull et al. [39], in a randomized controlled trial (level of evidence 2), showed that PRP is not useful for treatment of Achilles tendon ruptures. The mechanical variables showed a large degree of variation between patients that could not be explained by measuring error. No significant group


differences in elasticity modulus could be shown. There was no significant difference in heel raise index. The Achilles tendon total rupture score was lower in the PRP group, suggesting a detrimental effect. Regarding the effectiveness of concentrated platelets on tendon healing, although the results of the recent human clinical studies are disappointing, animal studies showed more encouraging results. This controversy arises because clinical studies used autogenous platelets while animal studies used allogenous platelets. Aspenberg and Virchenko [25], in a study of a rat Achilles tendon defect model, showed that after intrahematoma injection of allogenous concentrated platelets, the tendon callus strength and stiffness increased by about 30 % after 1 week, which persisted for as long as 3 weeks after the injection. Histology showed treatment improved tendon callus maturation. Beck et al. [17] also showed that allogenous PRP is effective in the healing and regeneration of a tendon-from-bone supraspinatus tear model in rats because it increased fibroplastic response and vascular proliferation during the first 21 days of tendon injury but failed to improve mechanical strength of the healing tendons more than that observed for the controls. Matsunaga et al. [18], using the same methodology as ours but using allogenous compact platelet-rich fibrin scaffold (CPFS), implanted the CPFS in the injured area of the partial patellar tendon defect and ligament reconstruction model in rabbits and showed, after 12 weeks, that the ultimate failure load and stiffness are higher for the right patellar tendon than for the left patellar tendon (control, no implant) in the former model. The CPFS promoted ligament repair in contrast with that on the untreated side in the latter model. Finally, Lyras et al. [16], in an experimental study in a rabbit patellar tendon defect model, showed that application of allogenous platelet gel in the defect area significantly increases the mechanical properties of the healing tissue at 28 DPI and it appears that PRP has a strong effect in the early phase of tendon healing. This effect is probably due to the growth factors that are released from the platelets during activation. To date, only a few studies have used xenogenous-based platelets for the purpose of tissue healing. In a series of relevant studies, it has been shown that xenogenous-based human platelets have promising curative effects on healing and regeneration of a rabbit radial bone defect model [27– 29]. We showed that the pure xenogenous-based bovine platelets are effective in tendon healing, modeling, and remodeling. Based on the above explanations, it seems the platelet source is determinant on the healing outcome. Based on the recent literature and based on the results of the present investigation, it could be suggested that the autogenous form of platelets have less inflammatory effects on tissue healing, the allogenous platelets have moderate

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stimulation of the inflammation, and the xenogenous platelets have higher inflammatory effects during tendon healing. Based on the beneficial role of inflammation on tendon healing, it seems the allogenous and xenogenous forms of platelets play a superior role in triggering the inflammation which is correlated with the superior healing outcome observed in animal studies compared with clinical studies [16–18, 25, 27–29, 38, 39]. Since the availability and cost effectiveness of biomaterials are important in tissue engineering and regenerative medicine, bovine platelets could be considered as a suitable reagent for increasing the bioactivity and healing efficacy of tissue-engineered constructs. When xenogenous-based biomaterials such as BPG are considered for use in tissue engineering, some points should be taken into consideration. First, the material should be harvested from animals that are free from any contagious and zoonotic diseases. In the present study, the platelets were harvested from the whole blood of animals that had no contagious and zoonotic disease. Second, the material should be sterile and its endotoxin contents should be in the standard ranges. We sterilized both the scaffolds and bovine platelets and the microbiological results confirmed that the sterility method was effective. In addition, the endotoxin content of the scaffolds was in the standard range. Third, the biomaterial should be well tolerated by the host. Although BPG increased inflammation locally after implantation of the CIPDS-PG in the rabbit tendon defect, the animals didn’t show any hypersensitivity or allergic reactions in response to the BPG. In addition, inflammation was increased by the BPG for 2 weeks only and then subsided. This type of inflammatory pattern normally happens after many surgeries. Our clinical results suggest that BPG, when embedded within the CI-PDS, has no hazardous systemic effects because none of the animals showed signs of fever or died and their appetite and physical activity were not altered after surgery. This could be correlated with the preparation methods we used. We used pure platelets. Dragoo et al. [22] evaluated the inflammatory effect of two different commercially available PRP systems, Biomet GPS III leukocyte-rich PRP (LR-PRP) versus MTF Cascade leukocyte-poor PRP (LP-PRP), after intra-tendinous injection in an animal model. They indicated that compared with the leukocyte-poor Cascade PRP, the leukocyte-rich GPS III PRP initiated a significantly greater acute inflammatory response at 5 days after injection. Recently, Kaux et al. [40] reported a case of exuberant inflammatory reaction after a single injection of PRP to treat jumper’s knee in a 35-year-old male type 1 diabetic patient. Perhaps if we used bovine WBCs in combination with the pure platelets, the inflammation might be exaggerated, which is not beneficial for tendon healing. Another mechanism could be the lyophilization of the platelets. This process

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may have a role in decreasing the immunologic reactions related to the xenogenous bovine platelets. One of the major differences between the results of the platelet-based studies is the method of platelet preparation. In fact, many methodologies have been used in producing different types of PRPs, platelet gels, platelet fibrin matrix, etc. One of the most important factors during platelet preparation is the anticoagulant agent. It has been suggested that anticoagulant agents could alter the structure and function of the platelets during PRP preparation. EDTA and acid citrate dextrose (ACD) are used during PRP preparation as anticoagulant agents. Although it has been shown that EDTA may damage human platelet structure and function, other studies have shown different results [41]. For example, McShine et al. [42] compared the efficacy of EDTA, ACD-A and the combination of ACDA ? EDTA on platelet count of the whole blood samples. They showed that platelet counts in citrated blood samples were lower than those in EDTA and highlighted the need to present citrated samples mixed with dried EDTA when characterization or quality control of blood and blood components is required. In another study by Stokol and Erb [43] it was shown that the median platelet count was significantly lower in citrate-anticoagulated blood compared with EDTA-anticoagulated blood. Citrate also yielded inaccurate results for mean platelet volume and mean platelet count, likely because of inadequate sphering of platelets. Thus, they recommend that citrate should not be used as an anticoagulant when accurate platelet counts are desired in dogs. In another study, Araki et al. [44] used whole blood samples to optimize the preparation protocols for PRP, WBC-containing platelet-concentrated plasma, and noncoagulating PDGF concentrate. They showed that as an anticoagulant, EDTA inhibits platelet aggregation more efficiently than acid citrate dextrose solution, resulting in higher non-aggregated platelet yield and final PDGFBB content. In our preparation protocol, we used EDTA as an anticoagulant factor. Our in vitro results suggest that the bovine platelets had normal morphology both before and after platelet activation, had normal function and released their growth factors more effectively when concentrated. Based on the literature, there is no evidence suggesting EDTA is harmful for bovine platelets. Perhaps this issue should be studied in future investigations to show which anticoagulant agent is more effective in preserving the bovine platelet morphology and function. In addition, this issue should be considered when bovine platelets are going to be used in the clinical setting. This study had some limitations. First, it should be highlighted that although animal models have strong relevancy to humans, animals have different anatomy, physiology, evolution, and diet to humans. We did not evaluate other sources of platelet gel such as auto and allogenous

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forms. However, extensive research exists showing auto and allogenous forms are effective in tendon healing. Our purpose was to test the effectiveness of xenogenous-based bovine platelets on tendon healing. Comparison with other sources was not in the scope of this research and would have considerably increased the number of animal models. These limitations should be considered when the results of the present study are translated in the clinical setting.

5 Conclusion Embedding the BPG within the artificial tissue-engineered graft improved cytocompatibility, biocompatibility, and regenerative capability of the graft. In addition, BPG improved biodegradability of the graft in vivo and increased the acceptance rate and incorporation of the graft with the newly regenerated tendon. This treatment strategy increased inflammation in the short term, triggered cellular migration, differentiation, proliferation, maturation, and matrix deposition at mid-term and increased collagen content, collagen fibrillogenesis, differentiation and alignment in the long term. Also, platelet gel increased angiogenesis and vascular maturity and finally, due to the above mechanisms and by decreasing in peritendinous adhesion, muscle fibrosis and atrophy, it produced a new tendon that had significantly higher mechanical and functional properties than controls. Such a cost-effective, accessible, and reliable treatment strategy could be considered as an effective modality in the clinical setting. Acknowledgments We thank the Veterinary School, Shiraz University, for financial support and cooperation and much appreciate additional funding through Grant ISNF 87020247, from the Iranian National Science Foundation. Funding This work was supported by the Veterinary School, Shiraz University and the Iranian National Science Foundation (Grant ISNF 87020247). The funders had no role in study design, data collection, analysis, decision to publish, or preparation of the manuscript. Competing interests Ahmad Oryan, Ali Moshiri, and Abdolhamid Meimandi-Parizi have no conflicts of interest that are directly relevant to the content of this study. Author contribution of the study.

The authors had equal contribution in all parts

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