Lyophilized L-PRF enhances the bioactivity and rheological properties of 3D-printed and bioprinted scaffolds containing Dental pulp stem cells.

Osteoarthritis (OA) is a degenerative and inflammatory joint disorder with cartilage loss. Dental pulp stem cells (DPSCs) can undergo chondrogenic differentiation and secrete growth factors associated with tissue repair and immunomodulation. Leukocyte- and platelet-rich fibrin (L-PRF) emerges in regenerative medicine because of its growth factor content and fibrin matrix. This study evaluates the therapeutic application of DPSCs and L-PRF in OA via immunomodulation and cartilage regeneration. Chondrogenic differentiation of DPSCs, with or without L-PRF exudate (ex) and conditioned medium (CM), and of bone marrow-mesenchymal stem cells was compared. These cells showed differential chondrogenesis. L-PRF was unable to increase cartilage-associated components. Immature murine articular chondrocytes (iMACs) were cultured with L-PRF ex, L-PRF CM, or DPSC CM. L-PRF CM had pro-survival and proliferative effects on unstimulated and cytokine-stimulated iMACs. L-PRF CM stimulated the release of IL-6 and PGE2, and increased MMP-13, TIMP-1 and IL-6 mRNA levels in cytokine-stimulated iMACs. DPSC CM increased the survival and proliferation of unstimulated iMACs. In cytokine-stimulated iMACs, DPSC CM increased TIMP-1 gene expression, whereas it inhibited nitrite release in 3D culture. We showed promising effects of DPSCs in an in vitro OA model, as they undergo chondrogenesis in vitro, stimulate the survival of chondrocytes and have immunomodulatory effects.

BackgroundPlatelet concentrates have been used in tissue regeneration. The purpose of this study was to examine effects of growth factors released from leukocyte- and platelet-rich fibrin (L-PRF) and concentrated growth factor (CGF) on the osteogenic differentiation of periodontal ligament fibroblasts (PDLFs).MethodsLeukocyte- and platelet-rich fibrins, CGFs and PDLFs were obtained from New Zealand rabbits. The release of basic fibroblast growth factor (bFGF), bone morphogenetic protein 2 (BMP-2) and transforming growth factor β1 (TGF-β1) from L-PRFs and CGFs was measured at 5 h and 1, 3, 5, 7 days, using the enzyme linked immunosorbent assay. The PDLFs were treated with exudates of L-PRF or CGF. After the treatment, cell counting kit-8 assay was performed at day 1, 3, 5 and 7. Alkaline phosphatase (ALP) assay and Western blotting were applied at day 7. Three blocking antibodies were used to neutralize the proteins of bFGF, BMP-2 and TGF-β1.ResultsLeukocyte- and platelet-rich fibrin and CGF showed different growth factor release pattern, but similar accumulated concentration of these growth factors. PDLFs proliferation was significantly promoted by both L-PRF and CGF at day 1, 3 and 7, and CGF group was superior to L-PRF group at day 1 and 3. Both L-PRF and CGF significantly enhanced PDLFs ALP activity and protein expression of osteogenic markers. The osteopontin level was higher in CGF group than in L-PRF group, but no significant differences were found between two groups for ALP activity. Three blocking antibodies significantly downregulated both L-PRF and CGF induced osteogenic markers expression.ConclusionBoth CGF and L-PRF can promote the proliferation and osteogenic differentiation of PDLFs. The bFGF, BMP-2 and TGF-β1 are involved in both L-PRF and CGF induced osteogenic differentiation of PDLFs.

L-PRF (leukocyte- and platelet-rich fibrin) is one of the four families of platelet concentrates for surgical use and is widely used in oral and maxillofacial regenerative therapies. The first objective of this article was to evaluate the mechanical vibrations appearing during centrifugation in four models of commercially available table-top centrifuges used to produce L-PRF and the impact of the centrifuge characteristics on the cell and fibrin architecture of a L-PRF clot and membrane. The second objective of this article was to evaluate how changing some parameters of the L-PRF protocol may influence its biological signature, independently from the characteristics of the centrifuge.In the first part, four different commercially available centrifuges were used to produce L-PRF, following the original L-PRF production method (glass-coated plastic tubes, 400 g force, 12 minutes). The tested systems were the original L-PRF centrifuge (Intra-Spin, Intra-Lock, the only CE and FDA cleared system for t...

SECEC Research Grant 2008 II: Use of platelet- and leucocyte-rich fibrin (L-PRF) does not affect late rotator cuff tendon healing: a prospective randomized controlled study

Surgical repair of the rotator cuff repair is one of the most common procedures in orthopedic surgery. Despite it being the focus of much research, the physiological tendon-bone insertion is not recreated following repair and there is an anatomic non-healing rate of up to 94%. During the healing phase, several growth factors are upregulated that induce cellular proliferation and matrix deposition. Subsequently, this provisional matrix is replaced by the definitive matrix. Leukocyte- and platelet-rich fibrin (L-PRF) contain growth factors and has a stable dense fibrin matrix. Therefore, use of LPRF in rotator cuff repair is theoretically attractive. The aim of the present study was to determine 1) the optimal protocol to achieve the highest leukocyte content; 2) whether L-PRF releases growth factors in a sustained manner over 28 days; 3) whether standard/gelatinous or dry/compressed matrix preparation methods result in higher growth factor concentrations. 1) The standard L-PRF centrifugation protocol with 400 x g showed the highest concentration of platelets and leukocytes. 2) The L-PRF clots cultured in medium showed a continuous slow release with an increase in the absolute release of growth factors TGF-β1, VEGF and MPO in the first 7 days, and for IGF1, PDGF-AB and platelet activity (PF4=CXCL4) in the first 8 hours, followed by a decrease to close to zero at 28 days. Significantly higher levels of growth factor were expressed relative to the control values of normal blood at each culture time point. 3) Except for MPO and the TGFβ-1, there was always a tendency towards higher release of growth factors (i.e., CXCL4, IGF-1, PDGF-AB, and VEGF) in the standard/gelatinous- compared to the dry/compressed group. L-PRF in its optimal standard/gelatinous-type matrix can store and deliver locally specific healing growth factors for up to 28 days and may be a useful adjunct in rotator cuff repair.

Implant-supported restoration of the maxillary anterior segment that is biologically, functionally, and esthetically acceptable following traumatic injuries in the maxillary anterior segment is always complex.1 Careful extraction2 of the fractured root, residual labial bone preservation, proper flap design, ideal positioning of the implant, appropriate soft-tissue contour, and the crown emergence are all important steps necessary to achieve a predictable, stable, functional, and esthetic success. However, healing of the tissues is always difficult to control and the development of new techniques and materials to improve these treatments is still necessary. The use of platelet concentrates is an interesting approach.Platelet concentrates for surgical use are widely used and continuously investigated in oral and maxillofacial surgery.3 The objective is to gather platelet growth factors and to inject them on a surgical site to stimulate the healing process. A significant percentage of the literature is focused on the platelet-rich plasma (PRP) families. PRP4–7 is a liquid platelet suspension often activated into a platelet-rich gel (like fibrin glues).Another technology called leukocyte- and platelet-rich fibrin (L-PRF) allows for the preparation of strong fibrin membranes enriched with cells (activated platelets, leukocytes, circulating cells)8 and platelet growth factors.9 This autologous healing biomaterial is free of additives (no anticoagulant during blood harvest, no chemicals for activation), simple, inexpensive, and quick to prepare (15 minutes for all steps). This technique is specifically adapted to the practical needs in daily implant dentistry. Several articles have reported the use of these L-PRF membranes for the stimulation of bone and gingival healing during subantral sinus augmentations10 and global rehabilitations using dental implants.11,12 The effect of these membranes on soft tissue healing and maturation is particularly significant.13In this case letter, the replacement of a fractured central incisor with immediate postextraction implant and crown placement is presented step by step with the use of L-PRF.A 45-year-old woman presented with an endodontically treated maxillary right central incisor that was fractured during a recent sport-related accident (Figure 1). The tooth presented with a Class 2 crown mobility suggesting a longitudinal fracture of the crown corresponding to a deep infrabony pocket on the labial surface. Probing pocket depths and mobility of adjacent teeth were within normal limits. Radiographic examination of the tooth demonstrated an incomplete endodontic treatment but without periapical radiolucency (Figure 2).Following removal of the crown (Figure 3a and 3b), a vertical bony defect was observed on the labial surface of the tooth, what may lead to a lack of support for the soft tissue volume and an unesthetic implant restoration profile emergence. The fractured root was carefully removed and a thorough curettage of the remaining alveolus was performed to eliminate any residual infective tissue in the avulsion socket that could compromise the osseointegration of an immediately placed implant.A tapered 4.3 mm collar diameter and 11.5 mm length implant (Ossean, Intra-Lock, Boca Raton, Fla) was placed (Figure 4), reaching 60 Ncm primary stability. The final decision on immediate loading was made after implant placement using insertion torque and resonance frequency analysis as acceptance criteria. A straight titanium abutment was screwed into the implant at 35 Ncm and adapted to the incisal emergence profile (Figures 5 and 6).L-PRF was produced following the standard procedure (Process protocol, Nice, France).9 To fill the space between the labial surface of the implant and the residual labial bone wall, a mix of L-PRF and cortico-cancellous porcine bone (Gen-Os, Tecnoss, Turin, Italy) was placed. The bio-implant graft was covered and protected with a L-PRF membrane (Figure 7), and no suture was used.A provisional crown was then prepared and cemented with the provisional bonding cement, TempBond (Kerr, Orange, Calif) (Figures 8a and b). To reduce lateral forces, the provisional crown had no occlusal contact with the opposing arch. Some excess of L-PRF membrane was cut away on the labial and palatal sides. The patient was instructed to eat a soft diet and avoid placing food in the area of the provisional crown during the first 6 weeks.Two days following the surgery, the tissues presented a positive healing characteristic (Figure 9), and at 7 days postop the gingival esthetic profile was well defined (Figure 10). At this time, an esthetic adaptation of the collar was done by relining the crown with resin relining material. Regular clinical and radiographic controls were subsequently performed and no technical complications such as screw loosening, resin fracture or pain upon chewing were noted during the 3 month postsurgical osseointegration time period.After 3 months (Figure 11), a zirconia straight abutment was prepared and a full-ceramic crown was constructed with CEREC (Sirona, Bensheim, Germany) CAD/CAM technology (Figure 12a and b). The matured gingival tissue guided the emerging profile of the tooth. After 6 months, the final result appears to be satisfactory (Figure 13). Two years later (Figure 14), the restoration is stable and esthetic. The gingival tissue has continued to mature, as observed that the gingival collar has an improved contour and thicker biotype than in the initial months following surgery.Immediate postextraction implants in the maxillary esthetic area are currently used frequently and are subtle, exacting treatments. The use of healing materials such as L-PRF are well suited to these applications because this material has a robust stimulating effect on the healing of soft and osseous tissues.14 Moreover, as a strong solid fibrin membrane, it is particularly easy to use in implant dentistry and periodontology. It additionally offers a protective effect (both mechanical and biological) to the grafted area.11,12The immediate implant and bone graft allows for the maintenance and regeneration of the damaged labial bone wall. The dental implant serves as a support for three dimensional reconstruction (vertical, horizontal, and labial/lingual thickness).15 Without the immediate placement of the implant and graft material, the alveolar ridge after extraction would resorb significantly, resulting in the absence of adequate bone volume for ideal implant positioning. This is particularly true for patients with a thin alveolar ridge and gingival tissue. However immediate implant placement and bone grafts are always sensitive to the gingival quality, as the gingival tissue has to cover and protect the site. If the gingival tissue is weak or damaged, dehiscence can appear in the covering tissue leading to the contamination of the grafted site. For this reason, some authors recommend the use of connective tissue grafts to reinforce the peri-implant tissues.16 The L-PRF is therefore especially indicated in this application. The fibrin membrane of L-PRF acts as a bio-barrier, protecting the implant and the graft from the oral environment. Moreover, by providing growth factors, leukocytes, and a permeable fibrin matrix for the growth of endothelial and epithelial cells, this healing material stimulates neoangiogenesis and accelerates gingival healing and maturation.Many authors have shown the positive impact of immediate loading on the protection of peri-implant bone levels and osseointegration.17,18 This technique offers advantages for patient comfort and the healing process. It also facilitates a natural healing and maturation of the peri-implant bone and soft tissues around the crown, therefore to achieve a more esthetic and predictable emergence profile for the prosthetic restoration.19 Combined with L-PRF, this immediate crown offers the possibility to secure the L-PRF membrane in a stable position without suture, and also to use it as a transitory supporting material for the regeneration of the gingival tissue. Additionally, the temporary crown shapes the ideal profile emergence.In this type of treatment, the quality and design of the implant are similarly important. The macrodesign of the implant20 and its surface21 are aspects of the technologies that permit the clinician to have greater control and improve the treatment outcome.22 In this case, the use of a tapered implant was adapted to the shape of the alveolus, preserving the osseous structure surrounding the socket during immediate implant placement. The implant also presents a recent specific microrough nanorough chemically-enhanced surface (Ossean)23,24 which may also be an element of the success.Finally from a practical standpoint, the L-PRF is easy to use on the surgical site. The elastic consistency of the L-PRF membrane allows the clinician to punch it around a prosthetic pillar (abutment). The antihemorrhagic properties of L-PRF are also advantageous and convenient for this type of surgery.25 In this protocol, a flapless approach is used to avoid flaps and incisions that could significantly reduce micro-vascularization in critical areas like the interincisal papillae that would interfere with the cicatrization and the final esthetic result. Because of its texture and healing properties, the L-PRF membranes allowed for the use of a micro-surgical approach without incisions and sutures, leading to an optimal tissue healing.The authors have no conflict of interest to report.This work for the development of regenerative implantable materials is supported by a grant from the National Research Foundation of Korea (NRF) funded by the Korean government-MEST (No. 2011-0030121).

P.244 Infraorbital modiflcation of the Weber-Ferguson flap

Effects of leukocyte- and platelet-rich fibrin alone and combined with hyaluronic acid on early soft tissue healing after surgical extraction of impacted mandibular third molars: A prospective clinical study

Leukocyte and platelet-rich fibrin (L-PRF) is a 3-D autogenous biomaterial derived via simple and rapid centrifugation of whole blood patient samples, in the absence of anti-coagulants, bovine thrombin, additives or any gelifying agents. A relatively new “revolutionary” step in second generation platelet concentrate-based therapeutics, clinical effectiveness of L-PRF remains highly-debatable, whether due to preparation protocol variability, limited evidence-based clinical literature and/or inadequate understanding of its biocomponents. This critical review provides an update on the application of L-PRF during oral surgery procedures, in human Randomized and Controlled Clinical Trials only (up to February 2016). Accordingly, autologous L-PRF is often associated with early bone formation and maturation; accelerated soft-tissue healing; and reduced post-surgical pain and discomfort. L-PRF is a simple, malleable and safe biomaterial suitable for use in oral surgery. An innovative tool in Regenerative Dentistry, L-PRF is a strong alternative and possibly cost-effective biomaterial for oral-tissue regeneration. Preparation protocols require revision and standardization. Furthermore, a good analysis of its rheological properties, biocomponents and their bioactive function would enhance the validity, comprehension and therapeutic potential of the reported findings or observations; a step closer towards a new era of “super” dental biomaterials and bioscaffolds.

Leukocyte and platelet rich fibrin (L-PRF) is one of the platelet concentrates used to support regeneration and healing process. Many studies showed possible immunological and antibacterial properties of L-PRF. We perform an in vitro study to analyze the effect of L-PRF on platelet activation, platelet-leukocytes interactions and antimicrobial activity, important components in the healing process. Molecular biomarkers related with platelet activation and platelet-leukocyte interactions were analyzed by means of flow cytometry when L-PRF exudate was added to whole blood platelets. L-PRF membrane was used to evaluate antimicrobial activity using Enterococcus faecalis (ATCC 29212), Pseudomonas aeruginosa (ATCC 27853) and Candida albicans (ATCC 90028). Our experimental design allows to evaluate platelet activation and analyze molecular biomarkers of other immune cells and platelet-leukocyte interactions. From the results obtained we can conclude that L-PRF can be a valuable tool in healing process, efficient in activating platelets of whole blood and inhibiting microbial growth. In our opinion, the use of L-PRF exudate, in addition to L-PRF membrane, presents some advantages that have to be considered in clinical trials. Additional research on the characterization and quantification of cells and its products present in the L-PRF exudate, as well as on the temporal factor released. Also, further studies using strains isolated from clinical cases are needed.

Platelet-rich fibrin (PRF) has been widely used owing to its ability to stimulate tissue regeneration. To date, few studies have described the antibacterial properties of PRF. Previously, PRF prepared by horizontal centrifugation (H-PRF) was shown to contain more immune cells than leukocyte- and platelet-rich fibrin (L-PRF). This study aimed to compare the antimicrobial effects of PRFs against Staphylococcus aureus and Escherichia coli in vitro and to determine whether the antibacterial effects correlated with the number of immune cells. Blood samples were obtained from eight healthy donors to prepare L-PRF and H-PRF. The sizes and weights of L-PRF and H-PRF were first evaluated, and their antibacterial effects against S. aureus and E. coli were then tested in vitro using the inhibition ring and plate-counting test methods. Flow-cytometric analysis of the cell components of L-PRF and H-PRF was also performed. No significant differences in size or weight were observed between the L-PRF and H-PRF groups. The H-PRF group contained more leukocytes than the L-PRF group. While both PRFs had notable antimicrobial activity against S. aureus and E. coli, H-PRF demonstrated a significantly better antibacterial effect than L-PRF. Furthermore, the antimicrobial ability of the PRF solid was less efficient than that of wet PRF. In conclusion, H-PRF exhibited better antibacterial activity than L-PRF, which might have been attributed to having more immune cells.

Background Medication-related osteonecrosis of the jaws (MRONJ) is a serious condition associated with bone modifying agents (BMAs) intake, leading to impaired bone healing and increased morbidity. Despite various therapeutic approaches, an optimal treatment strategy remains elusive. Leukocyte- and Platelet- Rich fibrin (L-PRF) has emerged as a promising autologous biomaterial due to its regenerative properties. This study aimed to evaluate the efficacy of L-PRF in the treatment and prevention of MRONJ.Material and Methods A prospective cohort study was conducted, including a total of 30 patients diagnosed with MRONJ (stage I or II) or at risk of developing it (non-MRONJ). Patient underwent standardized treatment involving surgical debridement followed by L-PRF application. Clinical and demographic data were collected, and healing outcomes were assessed at multiple follow-up intervals (7 days, 14 days, 1 month, 3 months and 6 months). Statistical analyses, including Kaplan-Meier survival estimates, were performed to evaluate treatment effectiveness.Results The study demonstrated an overall healing of 90%, with a complete recovery in 82.4% of confirmed MRONJ cases and 100% of at-risk patients. L-PRF exhibited good clinical outcomes, including reduced inflammation and pain, accelerated epithelialization, and improved tissue regeneration. The median healing time was estimated at 33.41 days for MRONJ patients and 11.00 for non-MRONJ. No significant differences in healing rates were observed based on age, sex, or systemic conditions.Conclusions L-PRF represents a promising adjunct in MRONJ management, improving healing outcomes and postoperative recovery. Its autologous nature and growth factor release enhance bone regeneration, suggesting its potential as both a therapeutic and preventive strategy. Further larger-scale clinical trials are needed to standardize protocols and validate long-term efficacy. Key words:Medication-related osteonecrosis of the jaws, leukocyte- and platelet-rich fibrin, prevention, adjuvant therapies, bone healing.

CSF leakage is a major complication after cranial surgery, and although fibrin sealants are widely used for reinforcing dural closure, concerns exist regarding their safety, efficacy, and cost. Leukocyte- and platelet-rich fibrin (L-PRF), an autologous platelet concentrate, is readily available and inexpensive, making it a cost-effective alternative for commercially available fibrin sealants. This study aimed to demonstrate the noninferiority of L-PRF compared with commercially available fibrin sealants in preventing postoperative CSF leakage in supra- and infratentorial cranial surgery, with secondary outcomes focused on CSF leakage risk factors and adverse events. In a single-blinded, prospective, randomized controlled interventional trial conducted at a neurosurgery department of a tertiary care center (UZ Leuven, Belgium), patients undergoing elective cranial neurosurgery were randomly assigned to receive either L-PRF (active treatment) or commercially available fibrin sealants (control) for dural closure in a 1:1 ratio. Among 350 included patients, 328 were analyzed for the primary endpoint (44.5% male, mean age 52.3 ± 15.1 years). Six patients (5 in the control group, 1 in the L-PRF group) presented with CSF leakage requiring any intervention (relative risk [RR] 0.20, one-sided 95% CI -∞ to 1.02, p = 0.11), confirming noninferiority. Of these 6 patients, 1 (in the control group) presented with CSF leakage requiring revision surgery. No risk factors for reconstruction failure in combination with L-PRF were identified. RRs for adverse events such as infection (0.72, 95% CI -∞ to 1.96) and meningitis (0.36, 95% CI -∞ to 1.25) favored L-PRF treatment, although L-PRF treatment showed slightly more bleeding events (1.44, 95% CI -∞ to 4.66). Dural reinforcement with L-PRF proved noninferior to commercially available fibrin sealants, with no safety issues. Introducing L-PRF to standard clinical practice could result in important cost savings due to accessibility and lower cost. Clinical trial registration no.: NCT03812120 (ClinicalTrials.gov).

Leucocyte- and platelet-rich fibrin (L-PRF) has been tested for enhancing alveolar ridge preservation (ARP), but little is known about the local release profile of growth factors (GF), and the clinical equipoise related to its efficacy remains. This study compared the patterns of GF release, early soft tissue healing, and alveolar ridge resorption following unassisted healing and L-PRF application in non-molar extraction sockets. Atraumatic tooth extraction of two hopeless teeth per patient was followed by unassisted healing or L-PRF placement to fill the socket in 18 systemically healthy, non-smoking subjects. This intra-individual trial was powered to assess changes in horizontal alveolar ridge dimensions 1 mm below the crest of alveolar bone. GF concentrations in wound fluid were assessed with a multiplex assay at 6, 24, 72, and 168 h. Early healing was evaluated with the wound healing index and changes in soft tissue volumes on serial digital scans. Hard tissue changes were measured on superimposed CBCT images after 5months of healing. L-PRF resulted in higher GF concentrations in wound fluid (WF) than in the control, but no differences in release patterns or time of peak were observed. No inter-group differences in early healing parameters were observed. Alveolar bone resorption was observed in both groups. No significant inter-group differences were observed in hard tissue healing 1, 3, or 5 mm apical to the original bone crest or in the ability to digitally plan a prosthetically guided implant with or without bone augmentation. L-PRF increased the GF concentrations in WF of extraction sockets without shifting the pattern observed in unassisted healing, while the increased delivery did not translate into clinical benefits in early wound healing or ARP. The current findings question the assumption that increased local concentrations of GF by L-PRF translate into improved clinical outcomes. Additional definitive studies are needed to establish the benefits of L-PRF in ARP (ClinicalTrials.gov NCT03985033).

Selecting a relevant animal model for testing the in vivo effects of Choukroun's platelet-rich fibrin (PRF): Rabbit tricks and traps