At 3 weeks, host vessels infiltration toward the center of the construct was reported only for the HA-FIB group, giving rise to a process eventually leading to the breakdown of the tissue-engineered cartilage at later time points (Fig. quality. Here, we propose a clinically compatible fibrin/hyaluronan scaffold seeded with nasal chondrocytes (NC) and functionalized with an FDA-approved anti-angiogenic drug (bevacizumab; Avastin?), which sequestrates vascular endothelial growth factor from the surrounding environment. Our results show that this sustained bevacizumab release from NC-loaded scaffolds after subcutaneous implantation in nude mice efficiently blocked host vessels ingrowth (five occasions lower CD31+ cells infiltration vs. control group, at 3 weeks after implant), and enhanced constructs survival rate (75% vs. 18% for the control, at 6 weeks after implant). assays, developed to elucidate the role of specific construct components in the remodeling, allowed to determine that Mupirocin fibrin degradation products enhanced the endothelial cell proliferation, as well as the macrophage migration; whereas the presence of bevacizumab was capable of counteracting these effects. The proposed pharmacological control of angiogenesis by a therapeutic drug released from a scaffold might enhance cartilage regeneration by MACI approaches, possibly allowing it to bypass the complex and costly phase of graft preculture to gain increased functionality. Introduction Damaged articular cartilage has a limited capacity of self-repair due to its avascular nature and low cellular mitotic activity.1C4 Cell-based repair techniques, such as matrix-induced autologous chondrocyte implantation, showed positive clinical outcomes, despite the formation of a fibrocartilaginous/fibrous repair tissue that is characterized by inferior mechanical properties and limited durability.5 Most likely, the lack of essential extracellular matrix (ECM) components, including high-molecular-weight hyaluronic acid, and other anti-angiogenic factorssuch as chondromodulin, endostatin, and angiostatin6exposes the not fully mature designed tissues to an early blood vessel invasion. Such a host reaction might lead to final poor cartilaginous quality7 and, eventually, to premature implant degradation. Thus, control of angiogenesis might be crucial for both the development and the maintenance of physiological articular cartilage.8 In particular, it has been demonstrated that vascular endothelial growth factor (VEGF), one of the most potent angiogenic factors, plays an essential role in the ossification process at the level of the growth plate, modulating cartilage vascularization and hypertrophy.9 Recent data also uncover that chondrocyte-derived VEGF promotes catabolic pathways in the osteoarthritic cartilage.10 Neo-angiogenesis is also accompanied by the massive infiltration of mononuclear cells, such as monocytes. VEGF acts as a powerful chemoattractant for monocytes,11 which could potentially lead to a fast macrophage-driven resorption of the implanted designed cartilage. Taken together, these aspects strongly underline the importance to control angiogenesis, and, in particular, the signaling of VEGF in cartilage tissue engineering (CTE). To this extent, cell-based anti-angiogenic gene therapies for cartilage regeneration have been already successfully investigated by inducing overexpression of either endostatin12,13 or chondromodulin.14 In addition, overexpression of soluble VEGF receptor-1 combined with the release of growth factors belonging to the transforming growth factor beta (TGF-) superfamily enhanced cartilage regeneration in both rat osteoarthritic10 and osteochondral defect models.15 We hypothesized that VEGF blockade by using a biomaterial-based anti-angiogenic drug release system could provideright upon implantationan appropriate environment for the formation of stable cartilage by freshly seeded engineered constructs. In particular, we developed a hyaluronan/fibrin-based porous scaffold which Mupirocin was functionalized by the incorporation of a humanized monoclonal anti-VEGF antibody (bevacizumab)16 that binds to human VEGF17 and is currently used as an anti-angiogenic therapeutic drug in the treatment of metastatic colorectal cancer, metastatic kidney cancer, and glioblastoma. The use of a drug-eluting scaffold would overcome the limitations of gene therapy in terms Rabbit Polyclonal to DHRS2 of a direct clinical translation.18 High-molecular-weight hyaluronan and fibrin were chosen in virtue of their biocompatibility, chondro-supportive nature,1,4 and extensive clinical use.2,19C21 Among the promising Mupirocin cell sources for CTE, we opted for nasal chondrocytes (NC), as they represent one of the most interesting candidates for clinical application in virtue of (1) the relative ease and low morbidity of the harvest procedure22; (2) a better retained capacity on cell growth to re-differentiate and generate hyaline-like tissue23 as compared with chondrocytes of other origin; and (3) their capacity to properly respond to mechanical forces which are typically associated with joint loading.24 Despite the orthotopic model being a more clinically relevant approach, in this study, we decided to use a subcutaneous implantation Mupirocin in nude mice, since it represents probably the most efficacious model that’s useful for tests the intrinsic capability of constructs to create stable cartilage cells,7 becoming seen as Mupirocin a a far more hostile and vascularized microenvironment and, therefore, representing a far more arduous tests floor for our reasons. Strategies and Components All reagents had been bought from Sigma Aldrich, unless stated otherwise, and were utilised without additional purification. Tradition health supplements and press were from.
Our case was analyzed in this series, and em /em -light chains were found in the macrophage intracellular crystals by LC-MS/MS (Rodriguez et al
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