Choukroun’s PRF Protocol for Oral Tissue Regeneration

Learning Objectives

• Understand the functions of various growth factors in regenerating oral hard and soft tissues.
• Learn about the wound healing process in extraction sites and how the PRF protocol supports this process.
• Recognize how different fibrin products have evolved to improve oral wound healing.
• Get familiar with the PRF protocol developed by Choukroun.
• Identify the benefits of using PRF in comparison to traditional bone grafting materials.
• Gain an understanding of why enhancing the ridge preservation protocol is necessary before implant placement.
• Be acquainted with the definition of L-PRF.
• Understand the unique physical and biochemical characteristics of PRF.

Advancements in Oral Tissue Regeneration

It has been a long-standing observation that soft and hard tissues within an extraction socket do not always heal predictably or ideally. The removal of a tooth often leads to a compromise in the alveolar ridge, resulting in decreased bone quality and quantity at prospective dental implant sites. This unpredictable healing can also have a negative effect on soft tissue contours, impacting both the functional and esthetic outcomes of implant restorations. Over the past few decades, immediate treatment protocols have emerged that facilitate more favorable healing at these extraction sites.

One commonly employed ridge preservation technique involves placing native bone or bone substitutes into a fresh extraction socket. This approach, which has become well-established, usually involves the use of a granular bone substitute. The material functions as a space maintainer or scaffold within the socket and gradually resorbs as new bone forms. However, these materials lack vascularization, leading to delayed resorption and bone remodeling. Moreover, these granular substitutes do not contain any intrinsic growth factors that could expedite the healing process. Additionally, controlling the movement of the particulates can be challenging, as they often migrate out of the socket if not properly retained. This necessitates an extra surgical procedure involving suturing a soft tissue flap in place, which subsequently reduces micro-vascularization at the wound margins.

Several surgical protocols have been developed in recent decades to optimize tissue regeneration and hemostasis at extraction sites in preparation for dental implants. Fibrin-based biological additives in oral surgery have been reported in dental literature as early as 1970. Specifically, Ross and his team, in 1974, acknowledged the regenerative capabilities of platelets because of the growth factors they contain. Initially, platelet-derived materials were primarily used in the form of fibrin glues to assist in clot retention and surgical closure. These autologous materials were derived from the patient's own tissues and had no risk of rejection. The hypothesis was that fibrin-based substances would improve healing since fibrin plays a major role in the early stages of extraction site closure.

Blood platelets, containing considerable fibrinogen levels, led researchers to explore platelet-based surgical additives further. In 1997, Whitman and colleagues introduced the concept of using platelet-rich gels during oral surgeries. These gels, later called Platelet-Rich Plasma (PRP) by Marx and colleagues, were created from the patient’s own blood and were intended to enhance wound healing. The increased levels of fibrin, fibrinogen, and growth factors introduced at the extraction site were theorized to form an improved blood clot and a biologically enriched environment for bone regeneration.

The PRP protocol involved drawing the patient's blood and adding an anticoagulant, followed by two separate centrifugation steps. To induce fibrin polymerization, bovine thrombin or calcium chloride was added. In a natural blood clot, the composition would be approximately 95% Red Blood Cells (RBCs), 5% platelets, and 1% leukocytes. However, a clot prepared with PRP typically contained 4% RBCs, 95% platelets, and 1% leukocytes.

Studies on the effects of PRP on alveolar ridge healing showed mixed results, with no clear consensus on their effectiveness. Consequently, the use of PRP outside of research remained limited. The process for creating PRP was not only time-consuming but also required specific blood additives such as anticoagulants, calcium chloride, and bovine thrombin, making it a relatively costly approach.

A significant advancement in autologous regenerative therapy occurred when the role of leukocytes in the initial healing of extraction sockets was recognized. In 2001, Choukroun and his colleagues in France introduced a simplified technique for preparing a leukocyte-, platelet-, and fibrin-rich biomaterial. This new biomaterial required no additional blood additives and was obtained through a simple blood draw on the day of surgery. Termed Leukocyte and Plasma-Rich Fibrin (L-PRF or PRF), this procedure has been well-documented, with over 300 peer-reviewed articles published on PubMed in the past five years from respected journals.

PRF: An Autologous Healing Material

PRF is an autologous biomaterial introduced into fresh extraction sites or other surgical sites to enhance healing. This effect is due to the suspension of platelets, leukocytes, and growth factors in a fibrin matrix. Its biologic and physical properties allow it to be used in four different ways within extraction sockets:

• Combined with granular bone substitutes to fill the socket.
• As a primary graft material to fill the socket.
• Sutured in place as a membrane for closure, protecting the graft from external factors.
• Sutured over the grafts for Guided Bone Regeneration (GBR).

Choukroun’s PRF Protocol: A Simplified Approach

Choukroun’s PRF protocol is recognized for its simplicity and affordability compared to other PRP protocols documented in the literature. This method requires only a standard blood draw and basic laboratory equipment, although specific centrifuges and preparation instruments are recommended. Instrumentation can be sourced from Process, Inc. in Nice, France, or Intra-Lock, Inc. in the United States. This setup transforms the patient’s blood into usable PRF “membranes” for surgical applications. The protocol includes the following steps:

• A blood draw of 10 mL from the patient on the day of surgery, no longer than 2 hours before surgery and PRF membrane application.
• Quick transfer of the blood sample to the centrifuge after collection to avoid premature coagulation.
• Use of a calibrated centrifuge (Process, Inc., Nice, France) for a single centrifugation run, following Choukroun’s specifications for time, angulation, and RPMs.
• Post-centrifugation, three distinct layers become visible in the blood vial:
  • Red blood cells (RBCs) at the bottom,
  • A dense opaque white PRF “clot” in the middle (comprising platelets suspended in fibrin),
  • Straw-colored serum of platelet-poor plasma at the top.
• The platelet-poor plasma, rich in essential growth factors, is poured into a sterilized PRF Box (a specially designed surgical steel instrument with a perforated floor and a weighted press) to hydrate and bathe the PRF during membrane production.
• The PRF clot is extracted from the vial, with the RBC layer cut off and discarded.
• The remaining PRF clots (which are flexible and resilient) are placed in the “PRF box” floor, and the PRF press plate is applied to flatten the clots into PRF membranes for application at the extraction site.
• After compression, standardized PRF membranes of consistent size and thickness are produced.
• The extraction site is curetted, and the PRF membranes are utilized to fill the socket (with or without granular bone substitute, as determined by the surgeon).
• A small volume of 0.5% metronidazole solution can be added for protection against anaerobic bacteria.
• If the socket's diameter is too wide for primary closure, the final PRF membrane is sutured in place at the extraction socket's crest to retain the PRF and/or bone graft material and shield the site from oral trauma. The dense fibrin structure of the PRF membranes provides strength, elasticity, and protection, making them ideal for suturing.
• Healing is monitored for 7-14 days, with sutures removed at the appropriate time.

Enhancing Wound Healing with PRF

PRF membranes significantly promote wound healing and oral tissue regeneration due to their prolonged bioactivity and resilient physical properties. Initially, a natural blood clot forms a fibrin matrix, followed by cell migration and differentiation, leading to tissue remodeling. The PRF membrane acts as an enhanced autologous clot, containing substantial amounts of growth factors and glycoproteins that aid tissue healing and regeneration:

• Transforming Growth Factor-Beta (TGFB-1)
• Platelet-Derived Growth Factor (PDGF-AB)
• Insulin-like Growth Factor 1 (IGF-1)
• Vascular Endothelial Growth Factor (VEGF)
• Glycoproteins (Fibronectin, Vitronectin, Thrombospondin-1)

The PRF membranes serve as optimized blood clots capable of initiating healing and releasing vital growth factors into the socket core for over 7 days post-operatively. This promotes the proliferation of various cell types, including:

• Osteoblasts
• Fibroblasts
• Keratinocytes
• Adipocytes

Additionally, the growth factors in PRF stimulate cellular differentiation and angiogenesis, leading to enhanced vascularization within the healing socket. PRF membranes are thus excellent enhancers of osteogenesis and help prevent early bone resorption at potential implant sites.

Besides protecting the healing socket from oral trauma with their dense fibrin matrix, PRF membranes facilitate internal cell migration into the healing socket. Externally, they replicate ideal tissue architecture, encouraging early epithelialization and assisting in the joining of gingival margins. This is because PRF provides an excellent matrix for gingival fibroblast migration across the healing area.

Improved healing outcomes are typically observed in extraction sites where all four surrounding alveolar walls remain intact. In cases where multiple walls are compromised, a granular bone substitute is often added to the PRF to enhance bone volume regeneration for implant placement.

Benefits of Using PRF

In summary, Choukroun’s PRF protocol offers numerous advantages for regenerating oral tissues at extraction sites, particularly in implant site development:

• Autologous (natural and patient-specific)
• No additives required (no bovine thrombin, calcium chloride, or anticoagulants)
• Bioactive with multiple growth factors released
• Gradual, sustained release of growth factors over 7 days
• Excellent matrix for cell migration
• Facilitates regeneration of both hard and soft tissues
• Promotes post-operative hemostasis
• Efficient migration of stem cells through the matrix
• Accelerates cell differentiation
• Regulates inflammatory responses in the socket
• Supports immune system function
• Can be used alone or alongside traditional bone substitutes
• Applicable to a wide range of surgical and periodontal procedures
• Exceptional mechanical properties and easy suturing
• Strong protective capabilities for wound closure
• Relatively simple and rapid membrane preparation
• Minimal complexity in blood handling
• Cost-effective per membrane
• Reduced healing time before implant placement
• Less post-operative discomfort for patients
• No reported adverse effects in literature
• Increasing applicability in various surgical and periodontal procedures (e.g., walled defect repair, peri-implant defect repair, gingival grafting, sinus lift procedures, etc.)

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