It is currently accepted that platelets play a determining role in wound regeneration (22). Platelet-rich fibrin rational use stems from the fact that the clot acts as a natural tissue filler, being an important trigger for the local healing progress, promoting both neoangiogenesis and tissue remodeling, by releasing high concentrations of bioactive structural proteins (9). Nevertheless, platelets are not the single cellular elements with central role in the healing process. The concentration of leukocytes observed within the PRF also directly guarantees the tissue remodeling and regeneration (23).
The PRF use in canine patients has been rarely described. Studies have demonstrated the efficacy of PRF in dogs for tissue healing and regeneration of post-extraction sites in animals with spontaneous periodontal disease, reducing the expression of inflammatory cytokines such as TNF-α and IL-1β, and stimulating the expression of collagen production-associated genes such as COL1A1, COL3A1, and TIMP1, and of the GFs – PDGF-B, TGF−β1, and VEGF-A (24, 25). Additionally, PRF has gain interest in human context, being considered a hemoderivative with regenerative potential. There are several advantages accredited to this autologous biomaterial, being its preparation high reproducibility one of them, with low donor-to-donor variability in its composition (19, 26–28).
The GFs released are recognized as endogenous peptides that regulate both fibroblast and peripheral stem cell migration, proliferation, and differentiation, also promoting angiogenesis, which is crucial for wound healing processes (2, 29, 30). Also, progenitor stem cells were detected in platelet concentrates, the majority from the hematopoietic lineage (CD34+/CD45+), having the capacity to promote the maturation of endothelial cells and local neoangiogenesis; stem cells from nonhematopoietic lineages (CD34+/CD45-) were also found, having the ability to differentiate into mesenchymal cells (e.g. osteoblasts, chondrocytes) (31).
Data collected within this study demonstrated a significant difference in wound progress, confirmed by the reducing of all injury areas over the time, suggesting a sustained healing pattern provided by a local delivery of high concentration of both GFs and cytokines, released from the PRF matrix (19, 32).
Granulation tissue is an important component in the skin injury repair process, comprising new thin-walled capillaries, which fill the wound, during the healing by second intention (33). This tissue has been described as a contractile material, characterized by the proliferation of fibroblasts, endothelial cells and keratinocytes, colonized by local inflammatory cell population, in a complex cellular and molecular crosstalk within the wound region (34). In the present study, each PRF-grafting procedure was initially performed two times per week in order to increase the in situ concentration of bioactive peptides at the wound site. Overall, single PRF treatments were applied from the second week onward, until the acquisition of a notably proliferative and exophytic granulation tissue. Closed bandage with an imbued vaseline gauze was then applied, until epithelization and wound resolution was reached. The cases presenting extended or profound injuries (1, 6, 7 and 8), and one case with moistening crusted lesions due to its localization (case 8, lesion in the interdigital location) required additional regenerative biomaterial in the second week. In all the cases, PRF clots were perfectly integrated into the wound site, being locally degraded, with no rejection or necrosis.
A progressive wound contraction was documented in this study, more evident within the initial two weeks of PRF-therapy: the second PRF-grafting procedure was executed 4.5 days (median) after the initial wound presentation, associated with a wound contraction of 30.88%, at the first week. A median wound contraction of 62.76% was achieved between days 8 and 15 after two PRF-grafting treatments (n = 8), where case 1 accomplished the complete wound closure. A statistically positive correlation was found between the initial wound area, the wound healing duration, and the number of PRF treatments performed. Therefore, larger wounds require a higher number of PRFs, and longer time to heal. Nevertheless, the duration of the wound healing process is not associated with the number of PRF clots applied at the first treatment (day 1), or with the total number of PRF clots applied during the complete treatment. Contaminated wounds or wounds with higher scores required higher number of PRF clots applied.
Two of the seven PRF-assisted cases that achieved wound closure, experienced wound relapse. In these cases, histopathological examination exposed the presence of neoplastic lesions (an extraskeletal chondrosarcoma in case 2, and a dermic squamous cell carcinoma in case 3), that were deemed to be the relapsing cause. In case 2, previous imprint cytologic exam was negative for neoplastic cells. Recent works have studied platelet-derived formulations, such as platelet-rich plasma (PRP), as a co-adjuvant therapy in cancer treatment, helping the slower growth of the tumor (35, 36). In the past, the role of GFs on tissue angiogenesis and tumor progression has been suggested, but there is insufficient data regarding this matter. The authors strongly recommend biopsy examination on recurrent or chronic lesions before start PRF therapy.
Nonetheless, the influence of PRF treatments on the development of the neoplasia has never been clearly stated by human clinical researchers using PRF therapy, and it is important to note that being a recent methodology, there are still many unexplored features (23, 37, 38).
PRF-therapy has claimed to have effective topical antimicrobial activity (39–41).The inexistence of wound infection along this research may uncover an important intrinsic antimicrobial property of PRF clots, especially considering that no local antiseptic was applied and that four wounds included in this study were unequivocally infected. This study found a statistically significant correlation between the score of cleanliness/ contamination of wounds and the total number of PRFs applied. Case 8 was treated only with PRF therapy (no systemic antibiotic or anti-inflammatory), and case 3 had antibiotic administration only in the first 3 days of PRF-therapy. Considering case 7, a topical ear treatment was administered due to an external otitis in the last third of the treatment (> day 24). The otitis may have delayed the wound healing process in this animal, as the owner reported that the dog was trying to scratch the affected ear.
Once that all the cases were referred from a general veterinary clinician already receiving systemic antimicrobials, the suspension of these therapy was not recommended with the integration of the animals in PRF treatments, due to the associated risk of a possible antimicrobial resistance drawback. The bacterial culture of the wounds was not performed once that antimicrobials were already being administrated, and the first PRF treatments contributed to a significant clinical improvement of the lesions. Nevertheless, the authors would like to state that a bacterial culture test with a respective sensitivity analysis is recommended when treating skin wounds with significative tissue loss, complicated, or chronic, in the alignment of current veterinary medical practices. Nevertheless, in all cases the antimicrobials administrated were aligned by current veterinary clinical guidelines, considering that: four wounds were clinically infected and with purulent discharge or exudate (cases 1, 2, 4 and 5); two cases have passed for surgical interventions immediately before PRF therapy (cases 5 and 6); two cases had open wounds with tissue loss (cases 3 and 7), being these animals living outdoor, contacting with soil frequently, and therefore, these wounds were assumed to be contaminated. Nonetheless, the clinical outcomes attained by PRF therapy support that this technique may reduce the use of additional antimicrobial/chemical agents generally required for wound management, adding to the ecological and biodegradability properties of this biomaterial.
Regarding the technical features of PRFs’ manufacturing, using fresh blood without anticoagulants, when the treated wound required more than one PRF and 5 mL syringes for the collection of each corresponding PRF, was crucial to obtain consistent PRF clots. The blood harvest should be constant without excessive pressure (that occurs when small volume syringes are used), inhibiting vacuum over the vein and its collapse.
The authors recognize the absence of a control group as a limitation of this study. However, the authors considered that having a group with no implemented treatment would be unethical. Nevertheless, a control group consisting in wounds treated with physiologic saline solution for comparison with wounds treated with PRF-therapy would not be representative of the healing process evolution, since the wounds documented in this study occurred spontaneously, and therefore, with different etiologies. Furthermore, the clinical experience of the research group allows to infer those treatments with PRFs are faster than conventional treatments with commercial ointments, and easier to handle during wound healing.
Beyond its efficiency performance, PRF-methodology revealed to be an easy, cost-effective therapy, with a simple obtention protocol, and with low variability in clot formation between individuals. Furthermore, PRF-therapy technique can be pointed as an environmental protective medical technique: polypropylene tubes used to produce the standardized PRFs were reused, envisioning the waste reduction, and supporting ecological practices in clinical research. The tubes were used after both appropriate decontamination and washing, followed by autoclave sterilization (14, 42). Polypropylene is a plastic polymer that is cost-efficiently produced, thermoresistant, robust, with antifouling properties, with low cell attachment, free from absorption of hydrophobic drug molecules and water evaporation (43). The reduced use of antibiotics is also a major feature of this technique, addressing one of our biggest issues in clinical approaches under the One Health context, the antibiotic resistance.
The results from this study sustain PRF therapy as an innovative biomedical methodology for the regeneration of skin lesions, from different etiologies, where second intention healing is required, leading to the stimulation of progressive wound regeneration.