Leucocyte and Platelet-rich Fibrin: a carrier of autologous multipotent cells for regenerative medicine.

The wound healing is a complex process wherein inflammation, proliferation and regeneration evolve according to a spatio-temporal pattern from the activation of coagulation cascade to the formation of a plug clot including fibrin matrix, blood-borne cells and cytokines/growth factors. Creating environments conducive to tissue repair, the haemoderivatives are commonly proposed for the treatment of hard-to-heal wounds. Here, we explored in vitro the intrinsic regenerative potentialities of a leucocyte- and platelet-rich fibrin product, known as CPL-MB, defining the stemness grade of cells sprouting from the haemoderivative. Using highly concentrated serum-based medium to simulate wound conditions, we isolated fibroblast-like cells (CPL-CMCs) adhering to plastic and showing stable in vitro propagation, heterogeneous stem cell expression pattern, endothelial adhesive properties and immunomodulatory profile. Due to their blood derivation and expression of CXCR4, CPL-CMCs have been suggested to be immature cells circulating in peripheral blood at quiescent state until activation by both coagulation event and inflammatory stimuli such as stromal-derived factor 1/SDF1. Expressing integrins (CD49f, CD103), vascular adhesion molecules (CD106, CD166), endoglin (CD105) and remodelling matrix enzymes (MMP2, MMP9, MMP13), they showed a transendothelial migratory potential besides multipotency. Taken together, our data suggested that a standardized, reliable and economically feasible blood product such as CPL-MB functions as an artificial stem cell niche that, under permissive conditions, originate ex vivo immature cells that could be useful for autologous stem cell-based therapies.

Combining a novel leucocyte-platelet-concentrated membrane and an injectable collagen scaffold in a single-step AMIC procedure to treat chondral lesions of the knee: a preliminary retrospective study.

BACKGROUND: Different surgical approaches are currently available to treat knee chondral defects. Microfracture is the most commonly applied, but it often leads to a mechanically inferior fibrocartilaginous tissue. To overcome this shortcoming, the Autologous, Matrix-Induced Chondrogenesis (AMIC) technique has been proposed. To further enhance the outcome of AMIC, the addition of haemoderivatives containing growth factors that stimulate cartilage healing has emerged as a new treatment method. Recently, a novel leucocyte-platelet-concentrated membrane (CLP-MB), highly enriched in platelets, monocytes/macrophages, fibrinogen, CD34+ and CD34+VEGFR-2+CD133+ cells, has been developed. Additionally, an injectable collagen scaffold (Cartifill) has been proposed as a replacement of the AMIC standard collagen membrane. AIMS: This preliminary study is aimed to evaluate the short-term safety and efficacy of the use of the CLP-MB membrane and injectable collagen scaffold when combined in single-step AMIC procedures for the treatment of knee chondral lesions. METHODS: Medical records of patients who underwent an AMIC procedure with the CLP-MB membrane combined with Cartifill were reviewed retrospectively. Follow-up assessments were conducted at 6 and 12 months after surgery. Clinical function was assessed on the basis of the International Knee Documentation Committee (IKDC) score. Pain was evaluated using the visual analogue scale (VAS). RESULTS: Twenty-five patients were identified as meeting the inclusion criteria. Mean IKDC and VAS scores significantly improved during the follow-up time. The postoperative course was uneventful. CONCLUSIONS: AMIC combined with the CLP-MB membrane, and Cartifill seems to be a promising approach to treat knee chondral defects.

Biofabrication of a novel leukocyte-fibrin-platelet membrane as a cells and growth factors delivery platform for tissue engineering applications.

Autologous platelet-rich hemocomponents have emerged as potential biologic tools for regenerative purpose, but their therapeutic efficacy still remains controversial. This work represents the characterization study of an innovative autologous leukocyte-fibrin-platelet membrane (LFPm), which we prepared according to a novel protocol involving multiple cycles of apheresis. The high content in fibrinogen gave to our hemocomponent the appearance of a manipulable and suturable membrane with high elasticity and deformation capacity. Moreover, being highly enriched with platelets, leukocytes, and monocytes/macrophages, the LFPm sustained the local release of bioactive molecules (platelet derived growth factor, vascular endothelial growth factor, interleukin-10, and tumour necrosis factor alpha). In parallel, the evaluation of stemness potential highlighted also that the LFPm contained cells expressing pluripotency and multipotency markers both at the messenger ribonucleic acid (NANOG, SOX2, THY1, NT5E, and ENG) and surface-protein level (CD44high /CD73+ /CD34+ /CD117+ /CD31+ ). Finally, biodegradation analysis interestingly showed a good stability of the membrane for at least 3 weeks in vitro and 1 week in vivo. In both cases, biodegradation was associated with progressive exposure of fibrin scaffold, loss/migration of cellular elements, and release of growth factors. Overall, collected evidence could shed some light on the regenerative effect that LFPms may exert after the autologous implant on a defect site.