The effect of autologous platelet rich fibrin (PRF), a second generation platelet product, on the healing of experimental articular cartilage lesions was evaluated in an animal model. Full thickness cartilage lesions with a diameter of 6 mm and depth of 5 mm were created in the weight bearing area of femoral condyles of both hind limbs in 12 adult mixed breed dogs. Defects in the left hind limb of each dog were repaired by PRF implantation whereas those in the right hind limb were left empty. The animals were euthanized at 4, 16, and 24 weeks following surgery and the resultant repair tissue was investigated macroscopically and microscopically. The results of macroscopic and histological evaluations indicated that there were significant differences between the PRF treated and untreated defects. In conclusion, the present study indicated that the use of platelet rich fibrin as a source of autologous growth factors leads to improvement in articular cartilage repair.
Although articular cartilage injuries are among the most frequently encountered orthopaedic problems of the knee joint, their treatment has always been challenging for clinicians. The avascular nature of hyaline articular cartilage along with the low number of chondrocytes present and their minimum mitotic activity means that this particular connective tissue has a limited capacity for self-repair particularly in partial thickness injuries. The resultant repair tissue with its inferior biochemical and biomechanical characteristics ultimately fails, leading to the development of osteoarthritis or degenerative joint disease [
Tissue engineering and the use of bioactive agents, that is, growth factors and cytokines, are the latest methods which are being employed for the treatment of articular cartilage injuries. Studies have indicated that a number of growth factors, namely, transforming growth factor
Platelet rich fibrin (PRF) is a second generation platelet concentrate which has many advantages over the first generation platelet rich plasma (PRP). PRF is produced by collecting autologous blood in glass tubes without any anticoagulant and immediate centrifugation. The resultant product is a true biomaterial containing fibrin clot, platelets, and leukocytes with a high concentration of growth factors. PRF does not require any activation prior to use and the growth factors are released slowly over a sustained period of time [
The aim of the present study was to investigate the effect of PRF as an autologous source of growth factors on healing of full thickness articular cartilage defects of the knee in an experimental animal model and to determine whether PRF use would have a positive effect on cartilage regeneration. For this purpose, macroscopic and microscopic characteristics of the repair tissue obtained following the use of PRF in articular cartilage defects were investigated.
Twelve skeletally mature mixed breed male dogs with the body weight of 20–30 kg were used in this study. The study was approved by the Experimentation Ethics Committee and Research Council of the Faculty of Veterinary Medicine, Islamic Azad University, Tabriz branch, and the animals were kept under the institutional laws for animal experiments.
The dogs were judged to be healthy based on the findings from physical examination and laboratory tests (complete blood cell count, blood biochemistry profiles, and urinalysis). The stifle joint (equivalent of the human knee) of each animal was carefully examined to rule out any joint instability. Skeletal maturity was determined by radiography prior to start of the experiment.
A total of 48 articular cartilage defects were created on the femoral condyles of the stifle joint (4 defects per dog). Defects in the left hind limb of each dog were repaired by PRF implantation whereas those in the right hind limb were left empty and considered as the controls.
Autologous PRF was prepared according to the method described by Dohan et al. [
Venous blood sample following centrifugation indicating the PRF clot in the middle layer of the test tube (a) and the removed clots placed inside sterile petri dish (b).
Food was withheld from the animals for 12 hours before the surgery. Each dog was premedicated by intramuscular injection of xylazine (1 mg/kg) and atropine (0.04 mg/kg). Anesthesia was induced by intravenous injection of 2.5% solution of thiopental and maintained with halothane in oxygen following endotracheal intubation. Cefazolin (20 mg/kg) was given as preoperative antibiotic immediately following induction and lactated ringer’s solution (10 mL/kg/hr) was infused during the surgery. The animal was placed in dorsal recumbency and under aseptic conditions; the medial approach to the stifle joint with lateral patellar luxation was used to gain access inside the joint. The joint was fully flexed to gain access to the weight bearing areas of the femoral condyles. Full thickness articular cartilage defects with a diameter of 6 mm and depth of 5 mm were created in the weight bearing area of each femoral condyle using a drill equipped with 6 mm drill bit. Bleeding was observed in all the defects confirming the involvement of subchondral bone and full thickness nature of the injury. Defects in the left stifle of dogs (
Full thickness articular cartilage defects left empty in the control group (a) and press-fitted with PRF clot in the treatment group (b).
At 4, 16, and 24 weeks following surgery, the dogs were euthanized by an overdose of thiopental sodium injection and the distal femurs were harvested for macroscopic and histological evaluation of the repair tissue. Four dogs were randomly assigned to each of the sampling periods; therefore the number of PRF treated and control defects was 8 at each time period. Immediately after euthanasia, digital photographs of the defect area were taken and the International Cartilage Repair Society (ICRS) evaluation score [
ICRS macroscopic evaluation of cartilage repair.
Categories | Score |
---|---|
Degree of defect repair | |
In level with surrounding cartilage | 4 |
75% repair of defect depth | 3 |
50% repair of defect depth | 2 |
25% repair of defect depth | 1 |
0% repair of defect depth | 0 |
Integration with border zone | |
Complete integration with surrounding cartilage | 4 |
Demarcating border <1 mm | 3 |
3/4 of graft integrated, 1/4 with a notable border >1 mm width | 2 |
1/2 of graft integrated with surrounding cartilage, 1/2 with a notable border >1 mm | 1 |
From no contact to 1/4 of graft integrated with surrounding cartilage | 0 |
Macroscopic appearance | |
Intact smooth surface | 4 |
Fibrillated surface | 3 |
Small, scattered fissures or cracks | 2 |
Several, small or few but large fissures | 1 |
Total degeneration of grafted area | 0 |
Overall repair assessment | |
Grade I: normal | 12 |
Grade II: nearly normal | 11–8 |
Grade III: abnormal | 7–4 |
Grade IV: severely abnormal | 3–1 |
Following macroscopic assessment, each femoral condyle was fixed in 10% buffered neutral formalin, decalcified, and embedded in paraffin for routine histological sectioning. Three sagittal sections (5
O’Driscoll histological cartilage repair score.
Characteristics | Score |
---|---|
Nature of predominant tissue | |
Cellular morphology | |
Hyaline articular cartilage | 4 |
Incompletely differentiated mesenchyme | 2 |
Fibrous tissue or bone | 0 |
Safranin-O staining of the matrix | |
Normal or nearly normal | 3 |
Moderate | 2 |
Slight | 1 |
None | 0 |
Structural characteristics | |
Surface regularity | |
Smooth and intact | 3 |
Superficial horizontal lamination | 2 |
Fissures 25–100% of the thickness | 1 |
Severe disruption including fibrillation | 0 |
Structural integrity | |
Normal | 2 |
Slight disruption including cysts | 1 |
Severe disintegration | 0 |
Thickness | |
100% of normal adjacent cartilage | 2 |
50–100% of normal cartilage | 1 |
0–50% of normal cartilage | 0 |
Bonding to the adjacent cartilage | |
Bonded at both ends of graft | 2 |
Bonded at one end or partially at both ends | 1 |
Not bonded | 0 |
Freedom from cellular changes of degeneration | |
Hypocellularity | |
Normal cellularity | 3 |
Slight hypocellularity | 2 |
Moderate hypocellularity | 1 |
Severe hypocellularity | 0 |
Chondrocyte clustering | |
No clusters | 2 |
<25% of the cells | 1 |
25–100% of the cells | 0 |
Freedom from degenerative changes in adjacent cartilage | |
Normal cellularity, no clusters, and normal staining | 3 |
Normal cellularity, mild clusters, and slight staining | 2 |
Mild or moderate hypocellularity, slight staining | 1 |
Severe hypocellularity, poor or no staining | 0 |
Total |
|
Comparison between the values obtained from the macroscopic and histological scores of PRF treated and control groups at each time point was made using the Mann-Whitney
The distribution of mean ICRS scores between experimental groups at 3 different sampling times is shown in Figure
Mean ICRS scores for macroscopic evaluation of the repair tissue in the control and PRF treated groups at different time intervals. Error bars indicate standard deviation (SD) and the
Four weeks after PRF implantation, the defects were filled with a bright red coloured fibrous repair tissue with discernible margins and white coloured areas resembling normal articular cartilage (Figure
Macroscopic appearance of the representative defects from the two treatment groups at different time intervals: (a) control (4 weeks), (b) PRF treated (4 weeks), (c) control (16 weeks), (d) PRF treated (16 weeks), (e) control (24 weeks), and (f) PRF treated (24 weeks).
At 16 weeks, the reparative tissue in both PRF treated and control groups were opaque white resembling normal surrounding cartilage (Figures
Mean histological scores of the two treatment groups at different postoperative times are shown in Figure
Mean O’Driscoll scores for histological evaluation of the repair tissue in the control and PRF treated groups at different time intervals. Error bars indicate standard deviation (SD) and the
Control and PRF treated defects were filled with fibrous tissue with abundant fibroblasts 4 weeks after surgery. Numerous blood vessels along with small cystic cavities were also observed in the repair tissue. In the control group, the cysts and blood vessels were much closer to the defect surface and more abundant than the PRF treated group. The repair tissue had bonded well with the surrounding normal cartilage in both groups. The surface of the defects was covered by fibroblasts in both groups although the surface was much smoother in the PRF treated group. No obvious evidence of cartilage-like tissue formation was observed in the repair tissue of both treatment groups (Figures
Histologic sections of the representative defects from the control group at different time intervals: 4 weeks ((a)–(c)), 16 weeks ((d)–(f)), and 24 weeks ((g) and (h)). Haematoxylin-eosin staining; original magnification: 40x ((a), (d), and (g)), 100x ((b), (e), and (h)), and 400x ((c) and (f)).
Histologic sections of the representative defects from the PRF treated group at different time intervals: 4 weeks ((a)–(c)), 16 weeks ((d)–(f)), and 24 weeks ((g)–(i)). Haematoxylin-eosin staining; original magnification: 40x ((a), (d), and (g)), 100x ((b), (e), and (h)), and 400x ((c), (f), and (i)).
At 16 weeks, the number of fibroblastic cells in the repair tissue of the control defects had decreased and blood vessels were only observed in the deeper parts of the defect towards the subchondral bone area (Figure
At 24 weeks, the repair tissue in the control group had disintegrated to very large cysts containing tissue debris. The chondrocyte-like cells were only observed at the peripheral regions of the cysts and above the subchondral bone area (Figures
The main rationale behind the use of platelet rich products in wound healing is the presence of high concentrations of different growth factors in these biological products. Mammalian wound healing consists of distinct overlapping stages of haemostasis, inflammation, proliferation, and tissue remodelling [
Growth factors are the biological products which regulate the development and homeostasis of articular cartilage throughout life [
Choukroun’s platelet rich fibrin is a second generation platelet product developed by Choukroun et al. in 2001 [
The hypothesis of this study was that PRF could positively influence articular cartilage regeneration through the same mechanisms described for PRP. The results of macroscopic and histologic evaluations indicate that PRF use had a positive influence on cartilage repair. To the authors’ knowledge, this is the first study to describe the use of Choukroun’s PRF in articular cartilage repair. The only other study describing the use of Choukroun’s PRF in cartilage repair is the one conducted by Kuo et al. [
In this study, full thickness articular cartilage defects were created on the femoral condyles. This specific region was chosen because of its weight bearing and due to the fact that most clinical lesions, both in humans and animals, are seen in this region. All the necessary mechanisms of cartilage repair particularly cellular migration are thought to be activated in full thickness lesions intensifying the role of growth factors in repair [
The time course for the healing of canine full thickness articular cartilage defects consists of granulation tissue formation at 1 month, cellular tissue formation with high synthetic activity and low glycosaminoglycan content at 6 weeks, metaplasia to fibrocartilage by 4 months, and imperfect hyaline cartilage formation by 6 months [
Although higher macroscopic and histological grading scores were observed at 24 weeks compared with the 16-week sampling time in both control and PRF treated groups but no statistically significant within-group difference was observed between the two sampling periods. The major difference between the repair tissue formed at 16 and 24 weeks was the presence of central cysts in the latter sampling time in both treatment groups. These cysts were seen in 50 and 25 percent of the control and PRF treated defects, respectively, and their presence reflected central disintegration of the repair tissue. The defects containing cysts had lower macroscopic and histological grading scores; therefore it seems that the quality of repair is inferior in the defects that contain cystic lesions at 24 weeks. The findings of Jackson et al. [
A limitation of our study was that immunohistochemical staining to specify the type of collagen produced in the repair tissue was not conducted. The other limitation was that the study period could not be extended longer, that is, up to one year to evaluate the repair tissue alterations further. Both limitations were due to financial constraints.
It can be concluded that the use of autologous PRF leads to macroscopic and histological improvements in articular cartilage repair and regeneration. Further studies are required to examine the effect of PRF on partial thickness and chronic articular lesions.
The authors declare that there is no conflict of interests regarding the publication of this paper.
The authors would like to thank Tabriz Branch, Islamic Azad University, for the financial support of this research which is based on a Research Project Contract no. 2-17-5-102536.