Platelet-Rich Fibrin Scaffolds for Cartilage and Tendon Regenerative Medicine: From Bench to Bedside.

Nowadays, research in Tissue Engineering and Regenerative Medicine is focusing on the identification of instructive scaffolds to address the requirements of both clinicians and patients to achieve prompt and adequate healing in case of injury. Among biomaterials, hemocomponents, and in particular Platelet-rich Fibrin matrices, have aroused widespread interest, acting as delivery platforms for growth factors, cytokines and immune/stem-like cells for immunomodulation; their autologous origin and ready availability are also noteworthy aspects, as safety- and cost-related factors and practical aspects make it possible to shorten surgical interventions. In fact, several authors have focused on the use of Platelet-rich Fibrin in cartilage and tendon tissue engineering, reporting an increasing number of in vitro, pre-clinical and clinical studies. This narrative review attempts to compare the relevant advances in the field, with particular reference being made to the regenerative role of platelet-derived growth factors, as well as the main pre-clinical and clinical research on Platelet-rich Fibrin in chondrogenesis and tenogenesis, thereby providing a basis for critical revision of the topic.

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.

Leucocyte-platelet haemocomponents for topical use: regenerative potentiality.

Wider and wider is the interest in different clinical ambits in the application of haemocomponents produced from peripheral blood for regenerative purposes. These are mainly made up of concentrated platelets capable of releasing locally growth factor (GF) that stimulates tissue regeneration. Our group has devised a method to produce a new leucocyte-platelet haemocomponent enriched in fibrinogen that integrates the GF stimulus with the presence of cells involved in the regenerative processes: monocytes and stem cells. The use of the cell separator to collect these haemocomponents from peripheral blood has allowed us to realize a safe standardized product, with good regenerative potentiality and reasonable costs. This is obtained by modifying some parameters of separation, and without cell manipulation.

New method to produce hemocomponents for regenerative use from peripheral blood: integration among platelet growth factors monocytes and stem cells.

Recent studies have shown the importance of monocytes/macrophageses and of CD34+ progenitors in tissue regeneration processes. These cells, obtained generally from bone marrow, are seen in damaged tissue. We have studied a method to collect from the peripheral blood, using a cell separator and without stimulation of the patient/donor, a leukocyte platelet concentrated hemocomponent (CLP) for regenerative use which contains platelets, monocytes/macrophages, fibrinogen and CD34+ cells. We appraised the composition and cell functionality of the final hemocomponent during production and cryoconservation. The results show a positive increase in concentration values, in comparison with the pre-collection, of the cells that were involved in regeneration; i.e. the platelets, monocytes and CD34+ cells. These concentrations were also maintained at an effective level during cryoconservation of the hemocomponent. The CLP also demonstrated positive clonogenic potential in culture, showing that the CD34+ progenitors involved in CFU formation are functional in the fresh and thawed product. In brief we have shown that it is possible to produce, in a simple way, a hemocomponent for regenerative use that is standardized, reliable, and is economically feasible.

IgG Antibody Responses to the Aedes albopictus 34k2 Salivary Protein as Novel Candidate Marker of Human Exposure to the Tiger Mosquito.

Mosquitoes of the Aedes genus transmit arboviruses of great importance to human health as dengue, chikungunya, Zika and yellow fever. The tiger mosquito Aedes albopictus can play an important role as arboviral vector, especially when Aedes aegypti is absent or present at low levels. Remarkably, the rapid worldwide spreading of the tiger mosquito is expanding the risk of arboviral transmission also to temperate areas, and the autochthonous cases of chikungunya, dengue and Zika in Europe emphasize the need for improved monitoring and control. Proteomic and transcriptomic studies on blood feeding arthropod salivary proteins paved the way toward the exploitation of genus-specific mosquito salivary proteins for the development of novel tools to evaluate human exposure to mosquito bites. We previously found that the culicine-specific 34k2 salivary protein from Ae. albopictus (al34k2) evokes specific IgG responses in experimentally exposed mice, and provided preliminary evidence of its immunogenicity to humans. In this study we measured IgG responses to al34k2 and to Ae. albopictus salivary gland protein extracts (SGE) in individuals naturally exposed to the tiger mosquito. Sera were collected in two areas of Northeast Italy (Padova and Belluno) during two different time periods: at the end of the low- and shortly after the high-density mosquito seasons. Anti-SGE and anti-al34k2 IgG levels increased after the summer period of exposure to mosquito bites and were higher in Padova as compared to Belluno. An age-dependent decrease of anti-saliva IgG responses was found especially in Padova, an area with at least 25 years history of Ae. albopictus colonization. Moreover, a weak correlation between anti-saliva IgG levels and individual perception of mosquito bites by study participants was found. Finally, determination of anti-al34k2 IgG1 and IgG4 levels indicated a large predominance of IgG1 antibodies. Overall, this study provides a convincing indication that antibody responses to al34k2 may be regarded as a reliable candidate marker to detect temporal and/or spatial variation of human exposure to Ae. albopictus; a serological tool of this kind may prove useful both for epidemiological studies and to estimate the effectiveness of anti-vectorial measures.

New bioresorbable wraps based on oxidized polyvinyl alcohol and leukocyte-fibrin-platelet membrane to support peripheral nerve neurorrhaphy: preclinical comparison versus NeuraWrap.

Nerve wrapping improves neurorrhaphy outcomes in case of peripheral nerve injuries (PNIs). The aim of this preclinical study was to assess the efficacy of two novel biodegradable wraps made of a synthetic 1% oxidized polyvinyl alcohol (OxPVA) and a natural leukocyte-fibrin-platelet membrane (LFPm) versus the commercial product NeuraWrap. After rats sciatic nerve transection and neurorrhaphy, the wraps were implanted and compared for functional outcome, by sciatic function index assessment; structural characteristics, by histological/immunohistochemical analysis; ultrastructural features, by transmission electron microscopy. Moreover, a morphometric study was also performed and collagen distribution was observed by Second Harmonic Generation microscopy. After 12 weeks from implantation, all wraps assured nerve function recovery; no scar tissue/neuromas were visible at dissection. LFPm wraps were completely resorbed, while residues of OxPVA and NeuraWrap were observed. In all groups, biocompatibility was confirmed by the absence of significant inflammatory infiltrate. According to histological/immunohistochemical analysis and morphometric findings, OxPVA and LFPm wraps were both effective in preserving nerve integrity. These results assess that bioengineered OxPVA and LFPm wraps successfully guarantee favorable lesion recovery after PNI/neurorrhaphy and, in future, may be considered an interesting alternative to the commercial NeuraWrap.

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.