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PRP, PRF, and Advanced PRF: A Quick Comparative Analysis


In contemporary regenerative medicine, diverse methodologies have been employed to accelerate the regeneration of both hard and soft tissues. While recent endeavours have concentrated on biologics as pivotal mediators for tissue regeneration, challenges associated with the use of recombinant growth factors, including elevated supra-physiological doses and prohibitive costs, have been acknowledged. Notwithstanding these limitations, the application of growth factors such as recombinant platelet-derived growth factor (PDGF) has demonstrated positive outcomes in enhancing tissue formation across a spectrum of clinical procedures in both medicine and dentistry .


In addition to recombinant growth factors, autologous platelet concentrates, particularly platelet-rich plasma (PRP), have emerged as a supportive modality for tissue regeneration. PRP, derived from the patient's whole blood through centrifugation to achieve heightened concentrations, was initially introduced as "fibrin glue" in the 1970s and has gained popularity in medical and dental realms for regenerating both hard and soft tissues. Early investigations unveiled the capacity of key growth factors, including PDGF, in the blood to significantly modulate tissue repair and wound healing events. Studies have illustrated successful combinations of PRP with diverse biomaterials, such as collagen membranes and bone grafting materials. However, a notable drawback of PRP is the presence of anticoagulants, impeding the natural healing process despite containing growth factors implicated in tissue repair.


Subsequent research on platelet concentrates led to the development of platelet-rich fibrin (PRF), also known as leukocyte-PRF or L-PRF, which involves the use of a fibrin clot as a membrane containing autologous growth factors hypothesized to gradually release during wound healing. Recent advancements in PRF protocols, such as advanced platelet-rich fibrin (A-PRF), with modified centrifugation procedures have shown increased platelet cell numbers and enhanced behavior of monocytes/macrophages. Therefore, in this blog, we will compare the release of growth factors from PRP, PRF, and A-PRF.


Understanding Their Key Differences



Delineating the nuanced disparities between PRP, PRF, and A-PRF necessitates a comprehensive understanding of their respective composition, preparation methodologies, and therapeutic applications within the realm of regenerative medicine.


  • PRP

PRP is a therapeutic modality harnessed from the patient's own blood, employing a process of centrifugation to concentrate platelets and growth factors. The rationale behind this technique lies in the platelets' innate capacity to release bioactive substances that stimulate tissue repair and regeneration. The clinical utility of PRP has been substantiated by its demonstrable efficacy in promoting tissue regeneration across various medical and dental applications.


The dynamic process of growth factor release in PRP is intricately connected to the biological mechanisms governing platelet activation and degranulation. Through the process of centrifugation, platelets are concentrated in PRP, enriching it with bioactive molecules, including growth factors like Platelet-Derived Growth Factor (PDGF), Transforming Growth Factor-beta (TGF-β), Vascular Endothelial Growth Factor (VEGF), Epidermal Growth Factor (EGF), and Insulin-like Growth Factor (IGF). Activated platelets, responding to stimuli such as exposure to collagen at the site of tissue injury, initiate the crucial role they play in tissue repair and regeneration. This activation leads to degranulation, during which growth factors stored in alpha granules are released into the surrounding microenvironment.


The temporal dynamics of growth factor release in PRP are influenced by factors such as platelet concentration, the method of activation, and the presence of coagulants. Generally, an initial burst release of growth factors occurs immediately after platelet activation, contributing to the early phases of tissue repair. This is followed by a sustained and gradual release, extending over a more prolonged period, which is vital for the protracted processes of tissue healing and remodeling.


The unique composition of PRP, with its concentrated platelets and encapsulated growth factors within the plasma, establishes a bioactive reservoir that can be leveraged to modulate and enhance tissue regeneration. However, the utility of PRP is not without its caveats. The inclusion of anticoagulants in the preparation of PRP, while facilitating its fluidity, introduces an element that may potentially interfere with the natural healing cascade. Moreover, there exists the risk of achieving supra-physiological concentrations of growth factors, a phenomenon that necessitates careful consideration due to its potential implications for the biological response.


  • PRF

PRF signifies a significant advancement in platelet concentrate technology, representing an evolution beyond conventional methods. The hallmark of PRF lies in its utilization of a fibrin clot matrix, rendering it a biologically potent autologous substance. The preparation of PRF involves a meticulous centrifugation process, distinct from other techniques, leading to the formation of a fibrin matrix that is notably rich in platelets and leukocytes.


This unique structural composition is not merely incidental; rather, it is strategically designed to confer distinct advantages in the context of tissue regeneration. The fibrin matrix acts as a three-dimensional scaffold, fostering a conducive environment for cell migration, proliferation, and differentiation during the healing process. Furthermore, the richness in platelets and leukocytes within the matrix contributes to a bioactive milieu, enhancing the regenerative potential of the PRF.


One of the defining characteristics of PRF is its hypothesized ability to facilitate a gradual and sustained release of growth factors during the entire course of wound healing. This temporal aspect is critical, as it aligns with the protracted timeline of tissue repair and regeneration, ensuring a continuous supply of bioactive molecules at various stages of the process. In clinical practice, PRF has garnered substantial attention and adoption due to its observed favorable outcomes in both hard and soft tissue regeneration. Its versatile applicability across diverse medical and dental procedures has positioned PRF as a valuable adjunct in regenerative therapies.


The intricacies of growth factor release in PRF underscore its unique biological properties and strategic preparation methodology. PRF, distinguished by its fibrin matrix enriched with platelets and leukocytes, exhibits a distinctive temporal and spatial profile in the release of growth factors, contributing to its regenerative efficacy.


The preparation of PRF involves a specific centrifugation process, generating a fibrin clot matrix that encapsulates platelets and leukocytes. This matrix acts as a reservoir for growth factors, including Platelet-Derived Growth Factor (PDGF), Transforming Growth Factor-beta (TGF-β), Vascular Endothelial Growth Factor (VEGF), and others. The intrinsic architecture of the fibrin matrix is hypothesized to play a crucial role in facilitating the gradual and sustained release of these growth factors during the entire course of wound healing. The temporal dynamics of growth factor release in PRF are integral to its regenerative potential. Unlike PRP, where an initial burst release is followed by a decline, PRF exhibits a more prolonged and controlled release profile. This sustained release aligns with the protracted timeline of tissue repair and regeneration, ensuring a continuous supply of growth factors at various stages of the healing process.


The fibrin matrix of PRF serves as a three-dimensional scaffold that supports cellular activities, including migration, proliferation, and differentiation. This structural aspect, coupled with the gradual release of growth factors, creates an environment conducive to optimal tissue regeneration. The interplay between platelets, leukocytes, and the fibrin matrix contributes to the bioactive milieu within PRF, enhancing its regenerative capabilities.


  • A-PRF

A-PRF stands as a refined iteration of PRF, characterized by modified centrifugation parameters. Specifically, A-PRF is centrifuged at lower speeds and for extended durations, yielding an augmented concentration of platelets and influencing the behavior of monocytes/macrophages. This modification is anticipated to enhance the regenerative potential of A-PRF, representing a notable advancement in platelet concentrate technology.


The growth factor release dynamics in A-PRF represent a nuanced and sophisticated aspect of platelet concentrate technology, emphasizing refinement in centrifugation protocols to optimize regenerative potential. A-PRF, an evolved iteration of PRF, introduces modifications in centrifugation parameters, specifically employing lower speeds and extended durations.


The distinctive centrifugation protocol for A-PRF, at 1500 rpm for 14 minutes, deviates from the standard PRF protocol (2700 rpm for 12 minutes). This alteration is not arbitrary but strategically designed to enhance the concentration of platelets and influence the behavior of monocytes/macrophages. The rationale is rooted in the premise that this modified centrifugation process results in increased platelet cell numbers and elicits more pronounced responses from key immune cells, thereby augmenting the regenerative milieu.


While the growth factor release in A-PRF has not been extensively explored in the existing body of knowledge, the rationale behind the modifications in centrifugation parameters offers insights into its potential impact. The heightened concentration of platelets, known for their role in growth factor secretion, and the altered behavior of immune cells suggest a dynamic interplay that may influence the release kinetics of growth factors.


The temporal aspect of growth factor release in A-PRF, similar to PRF, remains a critical consideration. A-PRF is anticipated to exhibit a sustained and gradual release of growth factors, aligning with the protracted timeline of tissue repair and regeneration. However, the specific kinetics and magnitude of growth factor release in A-PRF, especially over extended periods, necessitate further empirical investigation.


The growth factor release in A-PRF is intricately linked to the modified centrifugation parameters. The unique combination of lower speeds and prolonged duration holds the potential to influence platelet concentration and immune cell behavior, thereby shaping the regenerative milieu. While the principles align with the broader understanding of platelet concentrate technology, the specificities of growth factor release kinetics in A-PRF warrant dedicated research endeavors for a comprehensive comprehension and optimization of its therapeutic applications.


Understanding Their Growth Factors



  • PRP


PRP's mechanism of action involves an acceleration of neovascularization, a process critical for establishing new blood vessels. This, in turn, amplifies the supply of blood and nutrients to the damaged tissue, expediting the healing process. Additionally, the augmented blood supply induced by PRP plays a multifaceted role by stimulating the requirements, proliferation, and differentiation of cells intricately involved in the overall healing process. PRP acts as a therapeutic catalyst by promoting neovascularization, ameliorating blood supply, and providing an enriched milieu of growth factors and proteins. These concerted effects ultimately contribute to the accelerated regeneration and repair of damaged tissues.


The functional properties of PRP predominantly hinge on the synthesis and secretion of various growth factors, which are released upon platelet activation. These growth factors, stored in thrombocyte α-granules, play a crucial role in regulating cellular processes, including chemotaxis, mitogenesis, and differentiation. When activated, the secreted growth factors exert direct stimulation on local mesenchymal and epithelial cells, promoting their migration, division, and increased synthesis of collagen and matrix. This cascade of events leads to the formation of fibrous connective tissue and scar formation.


In the context of damaged tissue, the synergy of growth factors released by PRP becomes evident. The released growth factors not only independently stimulate various cellular activities but also interact with each other, activating different intracellular signaling pathways that collectively enhance tissue repair.


PRP Growth Factors and Their Specific Roles:


  1. PDGF, discovered in platelets, stimulates chemotaxis and mitosis of fibroblasts, collagen synthesis, and extracellular matrix remodeling. It also plays a role in macrophage and neutrophil chemotaxis and enhances the secretion of TGF-β from macrophages.

  2. TGF-β, part of the TGF-β superfamily, includes three isoforms: TGF-β1, TGF-β2, and TGF-β3. TGF-β1, actively secreted by thrombocytes following injury, promotes collagen production, inhibits collagen breakdown, stimulates angiogenesis, connective tissue regeneration, and chemotaxis of immune cells.

  3. VEGF, secreted by activated thrombocytes and macrophages in damaged tissue, is crucial for stimulating new blood vessel formation.

  4. EGF stimulates chemotaxis and angiogenesis of endothelial cells, mitosis of mesenchymal cells, and promotes epithelialization, thereby expediting the healing process.

  5. IGF-1, a polypeptide hormone, is a normal component of plasma and can be transported into platelets by IGF binding proteins. Released during platelet activation, IGF-1 stimulates differentiation and mitogenesis of mesenchymal cells and promotes bone formation by influencing osteoblast proliferation and differentiation.

  6. FGF, a potent mitogen, exerts multiple actions on various cell types. It is a crucial mitogen for mesenchymal cells, chondrocytes, and osteoblasts.


  • PRF


PRF stands out as an immune concentrate with a specific composition and a three-dimensional architecture. This biomaterial contains a plethora of growth factors, including PDGF, TGF β1, and IGF, each exhibiting diverse potent local properties such as cell migration, attachment, proliferation, and differentiation. PRF is not only a healing biomaterial but also functions as an interpositional material, accelerating wound closure and mucosal healing through its fibrin bandage and growth factor release. Importantly, it acts as a barrier preventing the early invagination of undesired cells, creating a separation between desired and undesired cellular elements.


Biochemical analysis reveals that PRF consists of a well-integrated assembly of cytokines, glycan chains, and structural glycoproteins embedded within a slowly polymerized fibrin network. These components, known for their synergistic effects on healing processes, play a crucial role in supporting angiogenesis and immunity. The proposed mechanism of action for PRF involves the in vitro release of growth factors and in vivo studies support the notion that PRF is a concentrated suspension of platelet-derived growth factors, known promoters of tissue regeneration and wound healing.


PRF Growth Factors and Their Specific Roles:


  1. PDGF: Enhances cell migration, attachment, and proliferation.

  2. TGF β1: Involved in wound healing and tissue regeneration.

  3. IGF: Stimulates cellular proliferation and differentiation.

In the context of tissue engineering, the success of cell growth and differentiation is closely linked to an appropriate scaffold. PRF emerges as a promising scaffold for regenerative endodontics due to its ability to selectively bind and localize cells, as well as undergo biodegradation over time. The literature review on regenerative endodontic applications of PRF underscores its versatility and efficacy in various clinical scenarios. Studies demonstrate the combined use of PRF with graft materials, such as hydroxyapatite (HA), in treating large periapical lesions. The combination accelerates graft crystal resorption and induces rapid bone formation. PRF has also shown potential in bone tissue engineering, with membranes suitable for cultivating periosteal cells. Additionally, PRF in combination with beta tricalcium phosphate (β-TCP) or HA has been successful in bone augmentation, offering faster healing than biomaterials alone.


The potential of PRF as an ideal scaffold in revascularization of immature permanent teeth with necrotic pulps is supported by its richness in growth factors that enhance cellular proliferation and differentiation. The stabilizing sheath and mechanical sustenance provided by PRF fragments accelerate cellular migration critical for neo-angiogenesis and vascularization. Notably, platelet cytokines (PDGF, TGF-alpha, IGF-1) are gradually released during fibrin matrix resorption, creating a perpetual healing process.


  • A-PRF


A-PRF Growth Factors and Their Specific Roles:


1. VEGF Release: Vascular Endothelial Growth Factor (VEGF) is a key regulator of angiogenesis, crucial for tissue vascularization.

2. PDGF-AB Release: A study establishes that A-PRF stands out by releasing the highest levels of PDGF-AB.

3. TGF-β1 Release: A-PRF, when considered independently, exhibits the most significant release of TGF-β1.


A-PRF release abundant VEGF, PDGF-AB, and TGF-β1, positioning it as a potent biomaterial for angiogenesis, cell proliferation, and tissue regeneration. The lack of correlation between A-PRF clot size and growth factor release introduces intriguing complexities in the understanding of A-PRF biology. The negligible impact on SCAP viability, coupled with potential trends in migration, raises questions about the nuanced interplay between growth factor release and cellular responses in the context of dental regenerative applications. This study contributes pivotal insights into the intricate dynamics of A-PRF and calcium silicate-based materials, paving the way for refined approaches in dental biomaterial development.


Past Research Comparing PRP, PRF and A-PRF


Comparative release of growth factors from PRP, PRF, and advanced-PRF (2016)


This study used blood samples from six volunteer donors (total of 18 samples) collected with informed consent. The donors, aged 30 to 60, provided blood for the preparation of PRP, PRF, or A-PRF through centrifugation. PRP was prepared by centrifuging 10 mL of whole blood twice. PRF and A-PRF were isolated without anticoagulants, with different centrifugation parameters. The resulting clots were transferred to culture dishes for further investigation.


To assess growth factor release over time, samples were placed in a shaking incubator at 37 °C for 15 min, 60 min, 8 h, 1 day, 3 days, and 10 days. Culture media was collected at each time point, frozen, and replaced. ELISA assays were performed to quantify the released growth factors (PDGF-AA, PDGF-AB, PDGF-BB, TGF-β1, VEGF, IGF, and EGF). The quantification was carried out by measuring absorbance at 450 and 570 nm. Protein levels were measured at each time point, and statistical analysis was performed using two-way ANOVA with Bonferroni test.


The Results of this study include:


  • Total Protein Released after 10 Days: A-PRF released the highest total amount of growth factors compared to PRF and PRP. Specifically, A-PRF released 11048.19 ng/mL, significantly more than PRF (9261.89 ng/mL) and PRP (6176.15 ng/mL).

  • PDGF-AA, PDGF-AB, and PDGF-BB Release Over Time: PDGF-AA release from PRP was significantly higher at 15 min but dropped significantly at 60 min. A-PRF showed significantly higher release at 3 days. PDGF-AB and PDGF-BB showed similar trends, with A-PRF demonstrating significantly higher release at later time points (1 to 10 days). Total protein content for PDGF-BB was higher in PRP samples.

  • TGF-β1 and VEGF Release Over Time: PRP demonstrated higher levels of TGF-β1 and VEGF at early time points (15 min and 8 h) but dropped significantly by 10 days. A-PRF showed significantly higher levels at 1, 3, and 5 days for both TGF-β1 and VEGF.

  • EGF and IGF Release Over Time: PRP showed significantly higher EGF levels only at 15 days. A-PRF demonstrated significantly higher release of EGF at earlier time points (60 min, 8 h, 1 day, and 3 days). No significant differences were observed for IGF between PRF and A-PRF.


In summary, A-PRF exhibited the highest total growth factor release over 10 days compared to PRF and PRP. Growth factors showed varying release patterns over time, with A-PRF generally demonstrating higher levels at later time points. Few studies also provide insights into the temporal dynamics of growth factor release from different platelet concentrates, emphasizing the potential advantages of A-PRF in terms of sustained growth factor release for regenerative applications.


Reference:

Growth Factors Released from Advanced Platelet-Rich Fibrin in the Presence of Calcium-Based Silicate Materials and Their Impact on the Viability and Migration of Stem Cells of Apical Papilla (2023)

Platelet Rich Plasma: A short overview of certain bioactive components (2016)

A novel antimicrobial-containing nanocellulose scaffold for regenerative endodontics (2021)

Platelet rich fibrin - a novel acumen into regenerative endodontic therapy (2014)

Comparative release of growth factors from PRP, PRF, and advanced-PRF (2016)

 

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