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Effect of platelet-rich plasma and platelet-rich fibrin matrix on healing of vertical meniscal tears in a rabbit model

Objective: This study aimed to investigate the effect of platelet-rich plasma (PRP) and platelet-rich fibrin matrix (PRFM) on the healing of vertical medial meniscal tears in a rabbit model. Methods: The study was conducted on 72 New Zealand mature rabbits aged more than 6 months. Rabbits were randomly assigned to six groups: control (C) group, meniscal repair (MR) group, PRP group, PRFM group, MR+PRP group, and MR+PRFM group, with 12 rabbits in each group. A 5-mm full-thickness vertical tear was created in the avascular zone of the medial meniscus corpus in the right knee of all rabbits. The respective treatment for each group was given to the meniscal tear in each rabbit. Histological evaluation of healing was performed 6 and 12 weeks after surgery. Results: Defect filling and collagen formation remained low in the C group. However, in all other groups, there was no significant difference in the 6th week. In the 12th week, similar results as those obtained in the 6th week were obtained. In the C group, there was a difference in defect filling and cell type. This difference was that the defect filling and collagen formation remained low in the 12th week. No significant difference was observed between MR, MR + PRP and MR + PRFM groups. The MR group significantly differed from the other groups in the defect-filling rate and cell type; however, the use of PRP and PRFM did not provide an obvious benefit. Conclusion: The contradictory results obtained in previous studies emphasize the need for further research on the use of PRP in meniscal recovery and repair. We believe that if surgery is indicated, repair is absolutely necessary to improve the healing of the tissue in meniscal tears. Studies using human meniscal tissue for meniscal injury and those that evaluate clinical applications of PRP are warranted. Effect of platelet-rich plasma and platelet-rich fibrin matrix on healing of vertical meniscal tears in a rabbit model Recep Kurnaz1 , Orhan Balta2 1Department of Orthopaedics and Traumatology, Eskişehir Acıbadem Hospital, Eskişehir, Turkey 2Department of Orthopaedics and Traumatology, Gaziosmanpaşa University, School of Medicine, Tokat, Turkey The most important factor affecting the success of repair is the location of the tear. The success rate of repairs in the red-red region is >95% (1). However, as the location of the tear moves away from the vascular region, the success rate reduces to 70% (2, 3). Many repair techniques and suture configurations have been developed to increase the success rate according to the shape and location of the tear (4). In the white-white region, the chance of healing is low if there are no methods to improve the healing after the repair. In such cases, improvement methods should be used. Platelet-rich plasma (PRP) is a suitable and effective alternative when growth factors need to be applied in the clinical setting. Many autologous blood products are increasingly being used to improve tissue healing in clinical practice. Nowadays, our knowledge about the growth factors that play a critical role in tissue healing is increasing. Thus, high platelet concentrate products can provide more effective benefits for tissue healing (5). PRP is a natural product that includes growth factors such as platelet-derived growth factor (PDGF), transforming growth factor-beta (TGF-β), basic fibroblast growth factor (FGF), insulin-like growth factor (IGF), vascular endothelial growth factor (VEGF) and endothelial cell growth factor  (ECGF) (6, 7). These have demonstrated positive effects on cell proliferation, cell migration, angiogenesis, and extracellular matrix production in numerous cell types in both in vivo and in vitro models (6-10). There is no risk of blood-borne diseases because they are used as autologous products. PRP is a widely used and proven method in bone and soft tissue healing (5). For the wide clinical application of tissue repair and regeneration technologies, they should not only be scientifically effective but also be easily applicable and cost-effective. PRP has many advantages over animal-derived products and recombinant growth factors (11). There is no risk of carcinogenesis and A R T I C L E I N F O Article history: Submitted 23 December 2017 Received in revised form 23 May 2018 Last revision received 11 June 2019 Accepted 11 November 2019

The most important factor affecting the success of repair is the location of the tear. The success rate of repairs in the red-red region is >95% (1). However, as the location of the tear moves away from the vascular region, the success rate reduces to 70% (2,3). Many repair techniques and suture configurations have been developed to increase the success rate according to the shape and location of the tear (4). In the white-white region, the chance of healing is low if there are no methods to improve the healing after the repair. In such cases, improvement methods should be used. Platelet-rich plasma (PRP) is a suitable and effective alternative when growth factors need to be applied in the clinical setting. Many autologous blood products are increasingly being used to improve tissue healing in clinical practice. Nowadays, our knowledge about the growth factors that play a critical role in tissue healing is increasing. Thus, high platelet concentrate products can provide more effective benefits for tissue healing (5). PRP is a natural product that includes growth factors such as platelet-derived growth factor (PDGF), transforming growth factor-beta (TGF-β), basic fibroblast growth factor (FGF), insulin-like growth factor (IGF), vascular endothelial growth factor (VEGF) and endothelial cell growth factor (ECGF) (6,7). These have demonstrated positive effects on cell proliferation, cell migration, angiogenesis, and extracellular matrix production in numerous cell types in both in vivo and in vitro models (6)(7)(8)(9)(10). There is no risk of blood-borne diseases because they are used as autologous products. PRP is a widely used and proven method in bone and soft tissue healing (5). For the wide clinical application of tissue repair and regeneration technologies, they should not only be scientifically effective but also be easily applicable and cost-effective. PRP has many advantages over animal-derived products and recombinant growth factors (11). There is no risk of carcinogenesis and immunological reaction. Finally, it is much cheaper than other recombinant factors.
Few studies have investigated the efficacy of PRP on meniscal healing. Various PRP-containing growth factors may increase bone and soft tissue healing (12)(13)(14). Although there is insufficient evidence for this, it is commonly used today (15).
There are four families of PRP products based on the fibrin structure and leukocyte content: pure PRP, leukocyte and PRP (L-PRP), pure platelet-rich fibrin (PRF), and leukocyte and PRF (L-PRF) (16)(17)(18). Among these four families, L-PRF slowly releases growth factors for 7 days (17,19). This suggests that the combination of leukocytes and fibrin matrix significantly influences the capture and release of growth factors. In addition, they help growth factors act as a combination of platelets and leukocytes in a complex fibrin matrix (20)(21)(22)(23).
Recently, L-PRF has shown promising experimental and clinical results in trauma cases in fields such as sports medicine and orthopedics (21,24,25). Nevertheless, L-PRF cannot be used as an injectable product because of its viscosity. PRP has a more fluid viscosity than L-PRF; therefore, PRP can penetrate into the meniscus better. Based on the positive effects of PRP on cell proliferation, collagen synthesis, and vascularization, PRP has been used as a bioaccumulant for the regeneration of meniscal tissue (17,26). Injection of PRP into the area with the intrasubstance tear can help the torn meniscus to heal and prevent the tear from progressing into uneven tears that require meniscus resection. However, there are limited reports on the effect of PRP on the healing of the torn meniscus.
Therefore, this study aimed to investigate the effect of PRP and platelet-rich fibrin matrix (PRFM) on the healing of vertical meniscal tears in a rabbit model. The hypothesis of this research was that PRP and PRFM would accelerate meniscal healing.

Experimental animals
This study was performed on 72 New Zealand mature rabbits aged >6 months. The rabbits were fed the same feed 1 week before the experiments and kept under the same conditions. The study was approved by the Tokat Gaziosmanpaşa University Animal Experiments Local Ethics Committee. Rabbits were randomly assigned to the following six groups: control (C) group, meniscal repair (MR) group, PRP group, PRFM group, MR+PRP group, and MR+PRFM group, with 12 rabbits in each group. The right knee joint of each rabbit was operated, and the contralateral knee was normal. The rabbit model has been used in previous studies (27,28).
In the control group, the right lower extremity of each rabbit was shaved following sterile staining and covering, and the skin was incised in a longitudinal anterior midline fashion to the knee joint. After medial parapatellar arthrotomy, the patella was dislocated laterally, and the knee was exposed. A 5-mm full-thickness vertical tear was created with a number 11 scalpel in the avascular zone of the medial meniscus corpus in the right knee in all rabbits. The patella was then reduced, and the incision was closed with 4/0 vicryl sutures; the skin was closed with continuous sutures using 3/0 silk. The rabbits in the control group were left without any additional intervention, and the tear was left as it was.
In the MR group, the knee joint was incised using the same surgical method. In all the rabbits, a 5-mm full-thickness vertical tear was created in the avascular zone of the medial meniscus corpus in the right knee, and the tear was repaired using vertical single suture using 4/0 PDS. The patella was then reduced, and the incision was closed with 4/0 vicryl; the skin was closed with continuous sutures using 3/0 silk ( Figure 1).
In the PRP group, after the tear was formed, the patella was reduced, and the incision was closed with 4/0 Vicryl; the skin was closed with continuous sutures using 3/0 silk. At the 1 st , 2 nd , and 3 rd postoperative weeks, PRP (1 cc) obtained from each rabbit was injected into the joints. Along with the PRP injection, 0.2 cc of calcium gluconate was added for platelet activation.
In the MR+PRP group, the tear was repaired with a vertical single suture using 4/0 PDS. The patella was then reduced, and the incision was closed with 4/0 vicryl; the skin was closed with continuous sutures using 3/0 silk. At the 1 st , 2 nd , and 3 rd postoperative weeks, PRP (1 cc) obtained from each rabbit was injected into the joints.
In the PRFM group, after the tear was formed in the meniscus, PRFM (2 cc) obtained from each rabbit was in injected to their defect area in the same session.
In the MR+PRFM group, the defect was repaired with vertical single suture using 4/0 PDS, and PRFM (2 cc) obtained from each rabbit was injected into their tear area in the same session.
• PRP and PRFM applications do not accelerate the healing in meniscal tears.
• It is not right to expect benefits from PRP and PRFM applications by not repairing the meniscus tear. In this study, rabbits in which meniscus tears are repaired are among the groups with the highest recovery results.
• However, no significant difference was observed in terms of healing with the application of PRP and PRFM among the meniscus repair groups.

M A I N P O I N T S
Previous studies have reported that long-term inactivity adversely affects meniscal healing (29)(30)(31). Thus, no restriction was applied to the rabbits after surgery. For analgesia, acetaminophen was added to the drinking water of all rabbits at 1-2 mg/kg per 100 ml, and standard rabbit feed was given. In addition, cefazolin sodium 20 mg/kg was administered intramuscularly for 5 days.

Preparation of PRP
Blood samples were obtained from each rabbit. Using a readymade PRP kit (Double Syringe Autologous Carrier Plasma [ACP] System, Arthrex, Inc.), 10 mL of blood was taken from the ears of rabbits under anesthesia. Each tube was centrifuged at 1500 rpm for 5 min using the single centrifuge method. After centrifugation, the remaining plasma portion was separated and a total of 4 cc of separated plasma was obtained (PRP). Platelet counting was performed in 1 cc PZP material. The remaining 3 cc of PRP was stored at −80 °C in 1-cc amounts ( Figure 2).

Preparation of PRFM
Using a ready-made PRP kit (Double Syringe ACP System, Arthrex, Inc.), 10 mL of blood was taken from the rabbits under anesthesia. Each tube was centrifuged at 3000 rpm for 10 min with their own centrifuge instrument using the single centrifuge method. The PRFM gel was applied to the defect region using 3

Sacrification of rabbits
Half of the subjects in each group were sacrificed at the 6 th week after the surgery and the remaining subjects were sacrificed at the 12 th week with high-dose anesthetic (pentothal sodium, Abbott). Histopathological examination was performed with disarticulation such that the tibial connections of the meniscus were not disturbed.

Histopathological examination
For histopathological examination, the right knee meniscus of each rabbit was separated and first kept in 10% neutral formalin at least for 2 days. After the tissues were washed, they were embedded in paraffin; thereafter, 4-μm sections were collected using a microtome. Finally, the sections were stained with hematoxylin and eosin. Stained sections were evaluated using a light microscope (original magnification X4, X10). In the histopathological evaluation, a semi-quantitative assessment scheme summarized in Table 1 was used by single pathologist (17,26,32).

Statistical analysis
Descriptive analyses were performed to obtain information about the general characteristics of the experimental groups.
When p-values were <0.05, they were considered statistically significant. One-way analysis of variance was used to compare the defect-filling rate, cell type, cellularity, collagen organization, and vascularity mean between the groups. Readyto-use the Statistical Package for Social Sciences version 19.0 software was used in calculations (IBM Corp.; Armonk, NY, USA).

Results
In the 6 th week, the defect-filling rate in the control group was <25%, no cell. There was no collagen organization, and there was fibrocartilaginous dissociation. In the MR group, the defect-filling rate was 25%-75%, and the cell type had 25%-75% chondrocyte dominance. There was hyaline or mucinous degeneration in the collagen fibers. In the PRP group, the defect-filling rate was <25%, no cell. There was no collagen organization, and there was fibrocartilaginous dissociation. In the PRFM group, the cell/extracellular matrix ratio was >0.5. The defect-filling rate was 25%-75%, and the cell type had 25%-75% chondrocyte dominance. In the MR+PRP group, the collagen fibers were organized, and hyaline or mucinous degeneration was present. The defect-filling rate was 25%-75%, with more than 75% chondrocyte dominance as  the cell type. In the MR+PRFM group, there was hyaline or mucinous degeneration with the collagen fibers organized. The defect-filling rate was 25%-75%, with >75% chondrocyte dominance as the cell type.
In the 12 th week, the defect-filling rate was >75% in the C group, with 25%-75% chondrocyte dominance. There was hyaline or mucinous degeneration in the collagen fibers. In the MR group, the defect-filling rate was 75%-100%, and the The defect-filling rate in the PRP group was 0%-75%. There was hyaline or mucinous degeneration in the collagen fibers.
The cell/extracellular matrix ratio in the PRFM group was >0.5. The defect-filling rate was 25%-100%, and the cell type had 25%-25% chondrocyte dominance. In the MR+PP group, the defect-filling rate was 75%-100%. The collagen fibers had an organized, homogenous eosinophilic matrix structure. In the MR+PRFM group, the defect-filling rate was between 75%-100%, and the cell type had a chondrocyte dominance of 75%-100%. The collagen fibers had an organized, homogenous eosinophilic matrix structure.
Histologically, mixed-type cellular proliferation was observed in most cases. Generally, in the 6 th -week biopsies, besides the occasional appearance of inflammatory cells, the dominant cell type was fibroblastic, whereas in the 12 th week, higher amounts of chondrocyte cells were observed. It was also observed that the process of inflammation and vascularization continued in the 12 th week in the meniscal repair groups (Figure 4). This condition may be associated with increased inflammation against the suture material used. In the 12 th week, similar results as those obtained in the 6 th week were observed; in the C group, there was a difference in defect filling and cell type ( Table 4, 5). Defect filling and collagen formation remained low (Table 6). Meanwhile, no statistically significant difference was observed between MR, MR + PRP and MR + PRFM groups.
Microscopic analysis showed that the improvement in the C group was significantly lower than that in the other groups. Al-  though the defect-filling rate was <25% in this group, the collagen bundles were not formed in the subjects yet. In the other groups, similar morphological features were observed in the meniscal repair groups in terms of defect filling, collagen organization, and cellularity; however, they had higher scores than the non-repaired groups. There was no significant difference among the MR, MR+PRP, and MR+PRFM groups. In this study, the MR group was significantly different from the other groups in terms of defect-filling rate and cell type. However, the use of PRP and PRFM did not provide an obvious benefit.

Discussion
As stated above, this study aimed to investigate the effect of PRP and PRFM on the healing of vertical meniscal tears in a rabbit model. The hypothesis of the research was that PRP and PRFM would increase meniscal tear healing. However, no statistically significant difference was observed among the groups.
Meniscal repair is considered for traumatic tears that occur close to the peripheral and vascular area of the meniscus in young patients. The vascular supply is provided by the capillaries from the perimeniscal capillary plexus and exudes up to 30% of the width of the meniscus in the "red-red" region (14). Two-thirds of the internal meniscus, "red-white, " and "white-white" regions contain little or no nerves and blood vessels (33)(34)(35).
The meniscus has the ability to heal at the external 1/3 site because of a fibrin clot rich in inflammatory cells at the site of injury. Fibrous repair tissue grows on the damaged area because of undifferentiated mesenchymal cells (18). Owing to this mechanism, biological therapies aim to improve the ability of tissue healing and repair by increasing the vascularity of the meniscus. Various anabolic growth factors have been identified in meniscal tissue repair and regeneration.
Interleukin-1 and epidermal growth factors increase meniscal cell migration, and bone morphogenetic protein-2 and insulin-like growth factor-I stimulate the migration of fibrochondrocytes from the periphery to the avascular region (10).
PRP is the promising autologous source of these and other growth factors. However, few studies have assessed the use of PRP in the treatment of meniscal injury or in the strengthening of meniscal repair.
Iscida et al. investigated the use of PRP prepared using the double-spin technique for the regeneration of meniscal tissue in both in vitro and in vivo studies (11). In vitro cells were isolated from rabbit tissue collected from the internal two-thirds of the meniscus and treated with PRP or platelet-poor plasma (PPP) at concentrations of 3%, 10%, and 30% for 48 h. Treatment with PRP compared with that with PPP and control showed that PRP not only increased the proliferation of the meniscal cells but also increased in vitro glycosaminoglycan synthesis. Gene expression analysis showed that although there was no difference in type-I collagen, there was upregulation of biglycan and decorin with large aggregate expression compared with that in the cultures treated with PPP. In an in vivo study, 30 mL of PRP, PPP, or phosphate buffered saline was added to freeze-dried gelatin hydrogels and implanted in a 1.5-mm defect in the meniscus avascular region in adult female rabbits.
In order to increase the effect of growth factors in meniscal healing, it is important that growth factors are exposed to the target area for a long period (18). However, the half-life of growth factors is too short to maintain in vivo biological activity. Therefore, it is important to deal with the short half-life of growth factors when they are locally applied to the in vivo meniscal tear site.
Tabata et al. proposed a controlled release system, a method for limiting the short half-life disadvantage by using biodegradable acidic gelatin hydrogel (36). In this system, growth factors are immobilized in gelatin hydrogels via physicochemical interaction with gelatin molecules, and immobilized growth factors are released from the hydrogel by hydrogel disintegration. Growth factors included in the biodegradable acidic gelatin hydrogel are slowly released and protected against desorption; therefore, the target tissue can be exposed to growth factors continuously for 2 weeks (20,22).
After 4 weeks of study, Ishida et al. concluded that the hydrogel kept releasing growth factors in PRP for at least 4 weeks (11). Animals were euthanized at the 4 th , 8 th , and 12 th postoperative weeks, and tissue sections were stained with hematoxylin-eosin and safranin-O to assess tissue repair using a semi-quantitative scoring system. At the 12 th week, significantly higher scores were observed with regard to the number of fibrochondrocytes and extracellular matrix production in the PRP-treated tissue defects.
The sum of these in vitro and results led the authors to conclude that PRP increases the healing properties of internal avascular meniscus (11). In the present study, in addition to the inflammatory cells at the 6 th week biopsies, the dominant cell type was fibroblastic, while in the week-12, chondrocyte cells were observed.
Some studies have reported that long-term inactivity adversely affects meniscal healing. Considering these studies, no restriction was applied to the rabbits after surgery (25,26,29).
Narita et al. also used biodegradable gelatin hydrogel for PRP presentation on horizontal meniscal tears (26). They found that FGF-2 with GH significantly stimulated the proliferation of meniscal cells and inhibited meniscal cells by up to 4 weeks before they died, thereby increasing meniscal cell density and enhancing meniscal repair in the rabbit model. However, neither study (11,26) directly measured the amount of growth factors released from platelets in vivo; therefore, it is unclear how long the biological activity of the growth factors lasted. The present results showed no difference between the control group and the PRP group. This may be due to the premature burst release in meniscal tears and desorption of growth factors. In this study, the amount of growth factors released from the platelets was not directly measured; therefore, it is unclear whether the growth factors are appropriately released and activated. In addition, we are unaware as to how long the growth factors remain biologically active. There is limited information regarding the in vivo release properties of growth factors because previous studies also did not directly measure the amount of growth factors in the in vivo meniscal tear model (11,26). However, recent studies have shown that the composition of L-PRP adversely affects the mechanical properties of the fibrin skeletons and that the increased cell reduces a more preventative inflammatory environment and ultimately a cell proliferation response directly related to an inflammatory condition. This is harmful to tissue regeneration (37)(38)(39).
When Arnoczky and Warren created vascular access channels connecting the peripheral vascular region to the internal avascular region, the injury site in the avascular region could be exposed to vascular origin growth factors; they stated that it could promote meniscal regeneration (40). In horizontal tear patterns in both the C and PRP groups, there is a possibility that the vascular access channels links the peripheral vascular area with the inner avascular area of the meniscus. Hence, not only the PRP group but also the C group may be exposed to a certain growth factor level. Fibrochondrocytes are considered to migrate toward chemotactic growth factors released from both peripheral blood vessels and PRP.
Another study by Zellner et al. evaluated the use of mesenchymal stem cells (MSC) for meniscal tissue engineering (41). MSCs were implanted on an L-PRC-loaded hyaluronan-collagen composite matrix and implanted in a 2-mm defect region of the meniscus avascular region in adult male rabbits. The animals were euthanized at 6 and 12 weeks; macroscopic, histological, and immunohistochemical evaluation of the tissue was conducted. MSCs and PRP-treated defects did not show any improvement at 6 or 12 weeks compared with a cell-free implant framework (41).
In the microscopic analysis in the current study, the improvement in the C group was significantly lower than that in the other groups. Although the defect-filling rate was <25% in this group, the collagen bundles were not yet formed in the subjects. In particular, in groups with meniscal repair, defect filling, collagen organization, and cellularity have similar morphological features but higher scores than non-repaired groups. The authors presumed that the difference in our findings and those reported by Ishida et al. may be related to the larger defect size.
Shin et al. evaluated the effect of PRP on horizontal meniscal tears in an experimental rabbit model and concluded that, unlike their hypotheses, PRP did not contribute positively to the healing process (42). However, they applied PRP as a single injection in their study. In the study, three PRP injections were administered at 1-week intervals.
In a study by Griffin et al., 15 of the 35 patients who underwent meniscal repair were treated with PRP, and the remaining 20 were treated with the repair without PRP. There was no significant difference in the clinical and functional scores between the two groups (13).