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Journal of Veterinary Science logoLink to Journal of Veterinary Science
. 2024 Feb 22;25(2):e22. doi: 10.4142/jvs.23281

Effect of platelet-rich plasma in Achilles tendon allograft in rabbits

Seok-Hong Park 1,†, Dong-Yub Kim 1,†, Won-Jae Lee 1, Min Jang 1, Seong Mok Jeong 2, Sae-Kwang Ku 3, Young-Sam Kwon 1, Sungho Yun 1,✉
PMCID: PMC10990913  PMID: 38568824

Abstract

Background

Achilles tendon is composed of dense connective tissue and is one of the largest tendons in the body. In veterinary medicine, acute ruptures are associated with impact injury or sharp trauma. Healing of the ruptured tendon is challenging because of poor blood and nerve supply as well as the residual cell population. Platelet-rich plasma (PRP) contains numerous bioactive agents and growth factors and has been utilized to promote healing in bone, soft tissue, and tendons.

Objective

The purpose of this study was to evaluate the healing effect of PRP injected into the surrounding fascia of the Achilles tendon after allograft in rabbits.

Methods

Donor rabbits (n = 8) were anesthetized and 16 lateral gastrocnemius tendons were fully transected bilaterally. Transected tendons were decellularized and stored at −80°C prior to allograft. The allograft was placed on the partially transected medial gastrocnemius tendon in the left hindlimb of 16 rabbits. The allograft PRP group (n = 8) had 0.3 mL of PRP administered in the tendon and the allograft control group (n = 8) did not receive any treatment. After 8 weeks, rabbits were euthanatized and allograft tendons were transected for macroscopic, biomechanical, and histological assessment.

Results

The allograft PRP group exhibited superior macroscopic assessment scores, greater tensile strength, and a histologically enhanced healing process compared to those in the allograft control group.

Conclusions

Our results suggest administration of PRP on an allograft tendon has a positive effect on the healing process in a ruptured Achilles tendon.

Keywords: Achilles tendon, allograft, platelet-rich plasma, rabbit

INTRODUCTION

Tendons are dense connective tissues responsible for transmitting muscle force to the bone [1]. The Achilles tendon is the largest tendon in the body and begins at the middle of the calf and inserts at the calcaneus as three tendons fuse together [2,3]. The three tendons of the rabbit Achilles tendon are the medial gastrocnemius, lateral gastrocnemius, and superficial digital flexor [4].

In humans medicine, acute rupture of the Achilles tendon can occur due to sporting activities [5]. In veterinary medicine, acute ruptures are associated with impact injury or sharp trauma [6,7]. Chronic ruptures are associated with degeneration, which is characterized by thickening of the distal portion of the Achilles tendon in dogs [6]. The decrease of blood flow and stiffness in the Achilles tendon increases the risk to withstand stress in humans [8]. The clinical signs of ruptures present with non-weight bearing and plantigrade stance in the affected hindlimb in dogs [6].

The healing of the Achilles tendon occurs in three phases including inflammation, proliferation, and remodeling in humans and rabbits [9,10]. The remodeling phase can be divided into consolidation and maturation stages [11]. The consolidation stage commences at about 6 weeks and continues up to 10 weeks. After 10 weeks, maturation stage occurs, which can last to a year [11]. However, the healing of the ruptured tendon is challenging due to poor blood and nerve supply, as well as the low residual cell population within the tendon extracellular matrix (ECM) [10]. These factors limited self-healing capacity of the tendon, the healed tendon shows scar tissue formation and disorganized fiber architecture. This result contributes to reduced elasticity and mobility, increased risk of rupture recurrence [1]. Challenging factors for repairing Achilles tendon rupture also include the large gap formation and secondary contracture of the muscles [12,13]. For the treatment of the Achilles tendon, recent studies suggest that the implantation of a bioresorbable biomaterial into a tendon defect represent a potential strategy to facilitate the attainment of biomechanical stability in the injured tendon. These biomaterials play a crucial role in the assembly of the ECM and establishment of cellular interaction, both cell to cell and cell to ECM, through their intricate architecture [10]. Additionally, Achilles tendon repair can be using an autograft or allograft. The advantages of an autograft are decreased risk of disease transmission, better acceptance of the graft, increased biomechanical integrity, and lower cost, compared to those of an allograft [14,15]. However, an allograft decreases donor site morbidity, provides larger graft availability, and has less postoperative pain compared to an autograft [14,15]. Because of the large gap formation after rupture, obtaining sufficient graft material seems to be necessary, which is why allografts might be a better choice for repairing tendons.

Due to the limitations of repairing ruptured tendons, promoting the healing process through the use of platelet-rich plasma (PRP) may be useful. PRP contains numerous bioactive agents and growth factors, such as vascular endothelial growth factor (VEGF), insulin-like growth factor (IGF), fibroblast growth factor (FGF), platelet-derived growth factor (PDGF), platelet-derived epidermal growth factor (EGF), transforming growth factor-beta (TGF-β), and EGF [16,17]. In these factors, VEGF exhibits angiogenic activity, promoting the recruitment, growth and proliferation of vascular cells while also increasing vessel wall permeability. IGF is antiapoptotic and anabolic activities in the early healing response and the expression of IGF, TGF-β and EGF precedes the promoting of collagen synthesis, a crucial role in tendon healing. PDGF, a mitotic and chemotactic factor for fibroblasts, induces the collagen synthesis. TFG-β is promote the maintenance and recruitment progenitor cells during tendon healing [18].

The purpose of this study was to evaluate the healing effect of PRP injected into the surrounding fascia of the Achilles tendon after allograft in rabbits, using macroscopic assessment, tensile strength testing, and histological evaluation. The results of present study anticipate that injecting PRP for Achilles tendon injury have a positive impact in healing process, such as reducing inflammation, decreasing scar formation and restoring tendon function and present an effective approach to the tendon treatment.

MATERIALS AND METHODS

Animal model

The subjects of this study were 24 male New Zealand white rabbits, aged 12 weeks and weighing 3.2 ± 0.3 kg. All rabbits were considered clinically healthy by physical examination and complete blood count (CELL-DYN 3700, Abbott, USA). Eight rabbits each were designated as intact control, allograft control, and allograft PRP group. Ethnical approval was confirmed by the Institutional Animal Care and Use Committee of Kyungpook National University (2020-0096).

Lateral gastrocnemius tendon harvest

Eight rabbits were selected as the donor group. They were anesthetized by intramuscular injection of ketamine (35 mg/kg, Yuhan Corporation, Korea) + xylazine (5 mg/kg, Bayer Korea, Korea) in the right quadriceps region. The left hindlimb was shaved and disinfected and an incision initiated from the calf, extending to the calcaneus. After dissection of skin, subcutaneous tissue, and tendon fascia, the Achilles tendon was revealed. The superficial digital flexor was dissected and retracted from the tendon group. The medial gastrocnemius tendon was then dissected, leaving the lateral gastrocnemius tendon available for harvest. The lateral gastrocnemius tendon was fully transected and inserted in a conical tube filled with 10 mL of DNase-RNase-free water [10,19].

Decellularization

Conical tubes containing DNase-RNase-free water and harvested tendons were placed onto a rotating shaker at 200 rpm, 37°C, for 12 h. After 12 h, water was discarded and 10 mL of 0.05% trypsin-EDTA was added to each tube. Tubes were placed onto a rotating shaker at 200 rpm, 37°C, for 1 h. After 1 h, the trypsin-EDTA was discarded and 10 mL of Dulbecco’s modified Eagle’s medium (DMEM) low-glucose containing 10% fetal bovine serum (FBS), 100 IU/mL penicillin, and 100 IU/mL streptomycin was added to each tube. Tubes were placed onto a rotating shaker at 200 rpm, 37°C, for 24 h. After 24 h, the DMEM-FBS solution was discarded and 10 mL of 2.0% Triton-X solution was added to each tube. Tubes were placed onto a rotating shaker at 200 rpm 37°C for 4 h. After 4 h, 2.0% Triton-X solution was discarded and 10 mL of deionized water (diH2O) was added to each tube. Tubes were placed onto a rotating shaker at 200 rpm, 37°C, for 12 h. After 12 h, diH2O was discarded. Samples were placed in cryotubes and preserved at −80°C. Methods of preparing allograft materials in this study were modified from those in a previous study [20].

PRP preparation

A 10 ml-syringe was prepared filled with 1.5 ml ACD-A solution. An 8–8.5 mL blood sample was drawn from the saphenous vein from the rabbits in the allograft PRP group (Fig. 1A). Blood samples were centrifuged twice (Gyrozen-1580MGR, Daejeon, Korea). Initial centrifugation was carried out at 1,800 rpm, 4°C for 10 min (Fig. 1B). After 10 min, plasma and buffy coat were collected from the blood sample and centrifuge d at 3,000 rpm, 4°C for 15 min (Fig. 1C). After 15 min, platelet-poor plasma was discarded (Fig. 1D) and 0.3 mL of PRP was collected into an Eppendorf tube (Fig. 1E). Platelet count was measured by a complete blood count (CELL-DYN 3700, USA). PRP was used only when platelet counts exceeded 1,000,000 cells/μL [21].

Fig. 1. PRP preparation. (A) Whole blood. (B) 1st centrifugation. (C) Plasma and buffy coat isolation. (D) 2nd centrifugation and discard platelet-poor plasma. (E) Isolated PRP.

Fig. 1

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PRP, platelet-rich plasma.

Tendon allograft and PRP injection

We anesthetized and disinfected identically as in the previous surgery 16 rabbits belonging to the allograft control group and allograft PRP group. The frozen gastrocnemius tendon was thawed in a water bath at 37°C for 10 min before placing the allograft. After revealing the Achilles tendon, the midpoint of the medial gastrocnemius was partially resected with a width of 2 mm and length of 10 mm. Thawed tendons were molded to the appropriate size for allografting. Five simple interrupted sutures were placed between the defect and the allograft tendon using monofilament absorbable suture material (Maxon, Covidien, Ireland). After suturing the fascia, 0.3 mL of PRP were injected in 0.15 mL portions, adjacent to the allograft tendon, proximally into the gastrocnemius muscle fascia and distally into the fascia around the calcaneus in the allograft PRP group. Subcutaneous tissues and skin were sutured in the same manner in both groups. All of the rabbits were bandaged in casts. Postoperative analgesia and antibiotics were administreted by subcutaneous injection of tramadol (4 mg/kg) and intramuscular injection of enrofloxacin (10 mg/kg) for 1 week [19,22].

Sampling

Rabbits were monitored for 8 weeks after surgery. At the day of sampling, rabbits were anesthetized by intramuscular injection of ketamine (35 mg/kg, Yuhan Corporation) and xylazine (5 mg/kg, Bayer Korea). Soon after anesthesia, rabbits were euthanized by intravenous injection of potassium chloride (100 mg/kg). Macroscopic assessment was carried out immediately. For histological analysis, three samples of allografted medial gastrocnemius tendon from each allograft group and intact medial gastrocnemius tendon from the intact control group were placed in 10% formalin. For tensile strength measurement, five samples of allografted medial gastrocnemius tendon from each allograft group and intact medial gastrocnemius tendon from the intact control group were sampled and stored at 10°C [10].

Macroscopic assessment

Macroscopic assessment was preceded by assessing rupture, adhesion, shape, inflammation, and abnormal changes in the neighboring tendon. Ruptured tendons were graded 0 and non-ruptured tendons were graded 1. Adhesion to the skin or subcutaneous tissue was graded 0 and no adhesion observed was graded 1. Abnormal shape was assessed by the increased thickness of the tendon. Normal thickness was graded 3, a slightly thickened tendon was graded 2, a moderately thickened tendon was graded 1, and an intensely thickened tendon was graded 0. Inflammation was assessed by the existence of edema, swelling, and redness. Existence of inflammation was graded 0 and lack of inflammation was graded 1. Abnormal changes in the neighboring tendons were graded 0, and those with no changes observed were graded 1. Both groups were assessed immediately after revealing the corresponding tendons. The scoring system was modified from a previous study [10].

Tensile strength measurement

Tensile strength was measured in each group (intact control, allograft control, allograft PRP) using a computerized testing machine (SV-H1000, Japan Instrumentation System Co., Ltd., Japan). Tensile loads were denoted as Newtons (N) and measured at maximum strength in constant speed (300 mm/min) with a bilateral stretch of 5 mm [1,23].

Histological process

Three samples from each group were trimmed and re-fixed in 10% neutral buffered formalin for 24 h. Subsequently, three samples from each group were placed into one paraffin block using an automated tissue processor (Shandon Citadel 2000, Thermo Scientific, USA) and an embedding center (Shandon Histostar, Thermo Scientific). Then, 10 paraffin sections with a thickness of 3–4 μm from each block were prepared using an automated microtome (RM2255, Leica Biosystems, Germany) equipped with a tungsten bladder (TC-65, Leica Biosystems). In each Achilles tendon, total three Achilles tendon samples in each group, and proximally and distally histological field in each sectioned Achilles tendon part and total six Achilles tendon histological fields in each group were statistically analyzed, in the histopathological analysis. For general histopathology, sections were stained by hematoxylin and eosin (H&E) [1,24], Masson’s trichrome (MT) for collagen fibers [1,25], Alcian blue (AB) for proteoglycans [24], and an avidin-biotin peroxidase complex (ABC)–based immunohistochemical stain for collagen types I (COL I) and collagen type III (COL III) [1,26,27]. Samples were observed using a light microscope (Model Eclipse 80i, Nikon, Japan) equipped with histological camera system (ProgResTM C5, Jenoptik Optical Systems GmbH, Germany) and a computer-assisted automated image analyzer (i-Solution FL ver. 9.1, IMT i-Solution Inc., Canada).

Immunohistochemistry

In order to block endogenous peroxidase activity, samples were incubated in methanol and 0.3% H2O2 for 30 min. Normal horse serum blocking solution was used to block non-specific binding of immunoglobulin for 1 hour in an humidity chamber after heating-based (95–100°C) epitope retrievals in 10 mM citrate buffers (pH 6.0) [28]. Primary antisera (Abcam, UK) were used at 4°C in an humidity chamber for one night and subsequently incubated with biotinylated universal secondary antibody and ABC reagents for 1 hour at room temperature. A peroxidase substrate kit (Vector Lab. Inc., USA) was used for 3 min at room temperature to induce reaction in the samples. Samples were all rinsed in 0.01M phosphate buffered saline for three times [26,27].

Histomorphometrical analysis

Modified semiquantitative grading scores ranging from 0 to 3 were applied to assess histopathological changes in the Achilles tendon samples. Normal samples, flattened and spindle-shaped nucleus and straight parallel fibers with slight waviness, were graded 0, mildly to moderately abnormal samples, separation of fibers and increasing waviness, were graded 1 or 2, and severely abnormal samples, increasing rounded nucleus and total loss of fine fiber structure, were graded 3 [1,24,29]. Infiltration of mean inflammation cell numbers (cells/mm2) [1,24], mean AB-positive proteoglycan-occupied regions (%) [24], MT positive collagen fiber-occupied regions (%) [1,25], and COL I and COL III immunopositive regions (%) [1,27,30] were measured by an automated image analyzer and histological camera system.

Statistical analysis

All numerical values were expressed as mean ± standard deviation for five samples of tensile strength and six samples of histomorphometry. Variance homogeneity was determined using the Levene test. If there were no significant deviations from the Levene test, data were analyzed by one-way ANOVA test followed by Tukey’s honest significant difference test. If there was a significant deviation from the Levene test, the Kruskal-Wallis H test was used. If the Kruskal-Wallis H test showed a significant difference, a Mann-Whitney (MW) U test was conducted. The level of significance was set at p < 0.05.

RESULTS

Macroscopic assessment

On the grade scale ranging from 0 to 7, the intact control group (Fig. 2A) averaged 7 in macroscopic assessment. The allograft control group (Fig. 2B) scored 4.37 ± 1.31, and the allograft PRP group (Fig. 2C) scored 5.75 ± 0.66. The allograft PRP group scored significantly higher on average (p < 0.05) in macroscopic assessment compared to allograft control group (Fig. 3A).

Fig. 2. Macroscopic features of intact control, allograft control, and allograft PRP groups. (A) Intact tendons showed no signs of adhesion, thickness, or abnormal changes in the neighboring tendon. (B, C) Allograft control and PRP tendons were grafted successfully without any signs of rupture. Both allograft groups had adhesion to surrounding tissues but no changes to the neighboring tendons (yellow arrow). The allograft control group tendons showed slight inflammation (edema and swelling) and thickness (yellow arrowhead) compared to tendons in the allograft PRP group. (D) Tensile strength measurement procedure (yellow circles).

Fig. 2

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PRP, platelet-rich plasma.

Fig. 3. (A) Macroscopic scoring of the intact control, allograft control, and allograft PRP groups. (B) Changes in tensile strengths in the intact control, allograft control, and allograft PRP groups.

Fig. 3

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PRP, platelet-rich plasma.

*p < 0.05 and **p < 0.01 as compared with intact controls; #p < 0.05 and ##p < 0.01 as compared with allograft controls.

Tensile strength testing

In tensile strength measurement (Fig. 3B), the intact control group scored the highest mean tensile strength (Newtons, N) of 61.74 ± 8.71. The allograft control group had significantly the lowest tensile strength, 30.90 ± 5.28 (p < 0.01). The allograft PRP group scored significantly higher tensile strength compared to allograft control group. 48.96 ± 1.41 (p < 0.05).

Immunohistochemistry

General histopathology using H&E, AB, and MT showed severe abnormal cell density, nuclear roundness fiber arrangement and structure, inflammatory cell infiltration, accumulation of AB positive proteoglycans and decrease of MT positive collagen fibers were observed in the allograft control group as compared to those of intact control group. However, significant increases of MT positive collagen fiber occupied regions, and decreased abnormalities against cell density, nuclear roundness, fiber arrangement and structure, mean AB positive proteoglycan occupied regions and inflammatory cell numbers infiltrated were demonstrated by the allograft PRP group as compared to those of allograft control group (Fig. 4). ABC-based immunohistochemical staining also revealed marked decreases of COL I and related decreased of COL III were observed in the allograft control group as compared to those of intact control group. However, significantly increases of COL I immunostained regions, and decreases of COL III immunostained regions were demonstrated by allograft PRP group as compared to those allograft control group (Fig. 5).

Fig. 4. Representative general histopathological profiles of intact control, allograft control, and allograft PRP groups. The allograft control group showed nonparallel fiber arrangement (yellow asterisk) and a loss of fiber structure (yellow arrowhead) compared to the allograft PRP group. The allograft control group had higher cell density (yellow arrow) and (e) proteoglycans were significantly stained by Alcian blue and (f) decreased of MT positive collagen fibers compared to results in the allograft PRP group. a, b, c = hematoxylin and eosin; d, e, f = Alcian blue; g, h, i = MT. Scale bars = 100 μm.

Fig. 4

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PRP, platelet-rich plasma; MT, Masson’s trichrome.

Fig. 5. ABC-based immunohistochemical staining of intact control, allograft control, and allograft PRP groups. The allograft control group showed nonparallel fiber arrangements compared to the allograft PRP group. The allograft PRP group had more stained COL I and less stained COL III compared to the allograft control group. a, b, c = COL I; d, e, f = COL III. Scale bars = 100 μm.

Fig. 5

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PRP, platelet-rich plasma; COL I, collagen type I; COL III, collagen type III.

Histomorphometrical analysis

Semiquantitative grading score

The intact control group scored 0.17 ± 0. 41 with respect to cell density (Fig. 6A). The allograft control group scored 2.33 ± 0.52 (p < 0.01) and the allograft PRP group scored 1.50 ± 0.55 (p < 0.01) were significantly higher as compared to intact control group. However, allograft PRP group score were significantly lower as compared to allograft control group (p < 0.05). The intact control group scored 0.33 ± 0.52 with respect to nuclear roundness (Fig. 6B). The allograft control group scored 2.33 ± 0.52 (p < 0.01) and the allograft PRP group scored 1.33 ± 0.52 (p < 0.05) were significantly higher as compared to intact control group. With respect to fiber arrangement (Fig. 6C), the intact control group scored 0.17 ± 0.41. The allograft control group scored 2.17 ± 0.41 (p < 0.01) and the allograft PRP group scored 1.17 ± 0.41 (p < 0.01) were significantly higher as compared to intact control group. With respect to fiber structure (Fig. 6D), the intact control group scored 0.17 ± 0.41. The allograft control group scored 2.17 ± 0.41 (p < 0.01), and the allograft PRP group scored 1.33 ± 0.52 (p < 0.01) were significantly higher as compared to intact control group. However, the scores of all 4 criteria in allograft PRP group were significantly lower compared to those in allograft control group.

Fig. 6. Evaluation of semiquantitative scores comparing intact control, allograft control, and allograft PRP groups. (A) Cell density. (B) Nuclear roundness. (C) Fiber arrangement. (D) Fiber structure.

Fig. 6

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PRP, platelet-rich plasma.

*p < 0.05 and **p < 0.01 as compared with intact controls; #p < 0.05 and ##p < 0.01 as compared with allograft controls.

Quantitative histomorphometry

In the assessment of mean inflammatory cell numbers (cells/ mm2) (Fig. 7), the intact control group had 6.00 ± 1.79 cells. The allograft control group had 96.33 ± 23.78 cells (p < 0.01) and the allograft PRP group had 32.00 ± 13.08 cells (p < 0.01) were significantly higher as compared to intact control group. In assessing mean AB-positive proteoglycan-occupied regions (%) (Fig. 8A), the intact control group was 10.81% ± 5.69% occupied. The allograft control group had an occupancy of 46.66% ± 12.38% (p < 0.01) and the allograft PRP group had occupancy of 26.70% ± 8.10% (p < 0.05), both significantly higher compared to the intact control group. In assessment of mean MT positive collagen fiber-occupied regions (%) (Fig. 8B), the intact control group were 86.69% ± 10.50% occupied. The allograft control group had an occupancy of 53.17% ± 12.70% (p < 0.01) and the allograft PRP group had an occupancy of 77.44% ± 10.06% (p < 0.01), the allograft control group were significantly lower compared to the intact control group. In assessing the mean COL I immunoreactive regions (%) (Fig. 8C), the intact control group had an occupancy of 82.32% ± 10.53%. The allograft control group had occupancy of 20.25% ± 6.62% (p < 0.01) and the allograft PRP group had occupancy of 44.93% ± 10.18% (p < 0.01), both significantly lower compared to the intact control group. In assessing the mean COL III immunopositive regions (%) (Fig. 8D), the intact control group had an occupancy of 7.39% ± 2.73%. The allograft control group had an occupancy of 44.48% ± 10.03% (p < 0.01) and the allograft PRP group had an occupancy of 19.16% ± 4.60% (p < 0.05), both significantly higher compared to the intact control group. Furthermore, the values for all 5 criteria in allograft PRP group showed significantly lower mean inflammatory cell numbers, proteoglycan and COL III, along with significantly higher collagen fiber and COL I, compared to those in allograft control group.

Fig. 7. Mean inflammatory cell numbers (cells/mm2) comparing intact control, allograft control, and allograft PRP groups.

Fig. 7

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PRP, platelet-rich plasma.

**p < 0.01 as compared with intact controls; #p < 0.05 as compared with allograft controls.

Fig. 8. Mean percentage of occupied regions comparing intact control, allograft control, and allograft PRP groups. (A) Proteoglycan. (B) collagen. (C) collagen I. (D) collagen III.

Fig. 8

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PRP, platelet-rich plasma.

*p < 0.05 and **p < 0.01 as compared with intact controls; ##p < 0.01 as compared with allograft controls.

DISCUSSION

In human medicine, there are several reports comparing the outcomes between conservative and surgical treatment of Achilles tendon rupture. Conservative treatment, such as cast immobilization and functional bracing, has higher rates of re-rupture compared to surgical treatment [31]. Suture of the ruptured tendon, autograft, allograft, and xenograft are the options for surgical treatment. Large gap formation and secondary contracture of the ruptured tendons make the surgery more challenging [12]. Therefore, grafting options such as autograft and allograft should be considered in augmenting surgical repair of the Achilles tendon. As mentioned before, allografts have significant advantages compared to autografts, which makes allografts a primary choice for tendon grafts.

PRP is an autologous blood product containing numerous growth factors that are widely used in regenerative medicine [32]. In human medicine, using PRP in tendon injuries, such as chronic degeneration and rupture, accelerates healing [16,18]. PRP contains alpha and delta granules that are located inside the platelets [32]. Alpha granules are the most abundant among them, containing growth factors such as PDGF, TGF-β, VEGF, EGF, IGF, and FGF [32,33]. Delta granules are composed of adenosine, histamine, and calcium, which also have a crucial role in the process of healing [33]. Platelets can be activated by using calcium or thrombin. However, in this study, PRP was not activated for following reasons. According to a recent study, non-activated PRP enhanced wound closure, fibroblast differentiation, and angiogenesis compared to thrombin-activated PRP [34]. Additionally, non-activated PRP excreted growth factors specifically to the receptors. In contrast, thrombin-activated PRP excreted all proangiogenic and antiangiogenic growth factors [34]. In order to maximize the effect of single administration of PRP, non-activated platelets were used in this study.

The process of tendon healing includes an inflammatory phase, regenerative phase, and remodeling phase [11]. The inflammatory phase is initiated by the intervention of erythrocytes and inflammatory cells. Angiogenesis and tenocyte proliferation also occur during this phase [11,35]. The regenerative phase starts few days after injury, maximizing type III collagen, water content, and glycosaminoglycan concentration [11,36]. At 6 weeks after injury, healing proceeds to a remodeling phase subdivided into consolidation and maturation stages [11,37]. Cellularity, collagen, and glycosaminoglycan synthesis are decreased and increased type I collagen synthesis is observed in the consolidation stage. Healing is completed in the maturation stage, which begins 10 weeks after injury and may last for a year [11]. The remodeling of the synthesized ECM typically commence around 6 weeks after the injury, with this process potentially lasting up to 8 weeks. This healing phage corresponds to the period when the consolidation phase typically occurs. Therefore, the observation period for this study was set at 8 weeks [4,10].

At 8 weeks after allograft, macroscopic assessment was carried out before tissue sampling. Both allograft control and allograft PRP group were successfully in place without rupture. Both groups had adhesion to near tissues and no abnormal changes in neighboring tendons were observed. However, allograft control group was presented with slight inflammation (edema and swelling) and thickness in tendon compared to allograft PRP group. Edema and swelling of tendon are associated with high content of proteoglycan which is responsible for water-binding in rabbits [10]. High content of proteoglycans occurs in tendinopathy which thickened tendons are characteristic in dogs [6]. These macroscopic results suggest the mitigated inflammation of allograft PRP group tendons compared to allograft control group, and higher vulnerability of allograft control group tendons compared to allograft PRP group tendons.

Measurement of tensile strengths is an important assessment in order evaluating recovery of a tendon’s biomechanical properties [1,23]. The intact control group had higher tensile strengths compared to both the allograft control group and allograft PRP group. However, the allograft PRP group had a significantly higher tensile strength compared to that in the allograft control group. A previous study suggests there is a correlation between histological properties and restoration of tensile strength in rat tendon repair [38]. Parallel arrangements of collagen fibers are responsible for the tensile strength of the tendon in rat [38]. In present study, the allograft PRP group had a lower score on fiber arrangements and higher tensile strengths compared to the control group. The results of this study, consistent with previous studies, support the effectiveness of aligning fiber in the direction parallel to the stress in enhancing tensile strength. These findings suggest that PRP had positive effects on tendon healing.

Stained histopathologic features support the superior semiquantitative and quantitative histomorphometry results in the allograft PRP group compared to the allograft control group. In 6 weeks after tendon injury, the characteristic of the consolidation stage, the repair tissue undergoes a transition from a cellular to a fibrous. Tenocyte metabolism remains high during this stage, and both tenocyte and collagen fibers align in the direction of stress in human [11]. The normal tendon, tenocyte nucleus, fiber structure and arrangement are characterized as flattened and spindle-shaped, straight parallel fibers with slight waviness. However, in the ruptured and tendinopathy injury, tenocytes initially decreased in number and then the pathological changes progressed, the nucleus became progressively rounded, and separation of the fibers with increased waviness in human [29]. In this study, a semiquantitative grading score system was applied based on histopathological evidence, and the criteria were cell density, nuclear roundness, fiber arrangement, and structure. According to this grading system, a normal tendon scored 0, mild and moderate prevalence scored 1 and 2, and severely abnormal tendon scored 3 [1,27]. As a result of histological analysis, the allograft PRP group had a lower semiquantitative score on all four criteria, suggesting a superior healing process compared to allograft control group. These results imply that PRP has the capability to promote tendon healing, thereby suggesting its suitability for tendon injury treatment.

Inflammation in acute tendon injury declines as fibroplasia and fibrillogenesis are initiated [39]. The higher number of mean inflammatory cell numbers in the allograft control group compared to the allograft PRP group suggests a slower healing process since fibroplasia and fibrillogenesis are delayed. Proteoglycans have been proved to bind with collagen I and II, thus impeding fibrillogenesis in rabbit [29]. Also, a high proportion of proteoglycans compared to collagen may reduce the interfiber cohesion of the collagen bundles in rabbit [29]. A lower mean AB-positive proteoglycan-occupied regions in the allograft PRP group suggests a stronger tendon structure compared to the allograft control group. Collagen comprises 60%–85% of the dry tendon weight, and high proportion of collagen contributes to a high tensile strength in tendons [30]. Type I consists approximately 95% of the healthy tendon [40]. Ruptured tendons have reduced quantities of type 1 collagen and increased type III. Type III collagen has a lower resistance to tensile forces, and large quantities of this collagen makes the tendon vulnerable and exposes to a higher risk of rupture in human [40]. PRP injected allograft group had a higher collagen-occupied region, higher mean COL I immunolabeled regions (%), and lower mean COL III immunostained regions (%), indicating a better healing process and tendon stability. In these immunohistochemistry results, it is evident that PRP stimulates fibroblast migration and proliferation, leading to increased number of migrating fibroblasts and enhanced production of collagen fibers. Consequently, the initiation of the remodeling phase in allograft PRP group precedes that of the allograft control group, thereby promoting tendon healing.

In this study, allograft PRP group demonstrated a superior healing process compared to the allograft control group with respect to macroscopic features, tensile strength, and histologic analysis. Therefore, our results suggest that administration of PRP on an allograft tendon has a positive effect on the healing process in a ruptured Achilles tendon.

Footnotes

Conflict of Interest: The authors declare no conflicts of interest.

Author Contributions:
  • Conceptualization: Park SH, Kim DY, Yun SH.
  • Data curation: Park SH, Kim DY.
  • Formal analysis: Park SH, Jeong SM, Ku SK.
  • Investigation: Park SH, Kim DY, Lee WJ, Jang M.
  • Methodology: Jang M, Lee WJ.
  • Project administration: Yun SH.
  • Software: Ku SK, Park SH.
  • Supervision: Ku SK, Yun SH.
  • Validation: Park SH, Kim DY, Yun SH.
  • Visualization: Ku SK, Jeong SM.
  • Writing - original draft: Park SH, Kim DY.
  • Writing - review & editing:, Kwon YS, Yun S.

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