Tissue-engineered grafts from human decellularized extracellular matrices: A systematic review and future perspectives

Tissue engineering and regenerative medicine involve many different artificial and biologic materials, frequently integrated in composite scaffolds, which can be repopulated with various cell types. One of the most promising scaffolds is decellularized allogeneic extracellular matrix (ECM) then recellularized by autologous or stem cells, in order to develop fully personalized clinical approaches. Decellularization protocols have to efficiently remove immunogenic cellular materials, maintaining the nonimmunogenic ECM, which is endowed with specific inductive/differentiating actions due to its architecture and bioactive factors. In the present paper, we review the available literature about the development of grafts from decellularized human tissues/organs. Human tissues may be obtained not only from surgery but also from cadavers, suggesting possible development of Human Tissue BioBanks from body donation programs. Many human tissues/organs have been decellularized for tissue engineering purposes, such as cartilage, bone, skeletal muscle, tendons, adipose tissue, heart, vessels, lung, dental pulp, intestine, liver, pancreas, kidney, gonads, uterus, childbirth products, cornea, and peripheral nerves. In vitro recellularizations have been reported with various cell types and procedures (seeding, injection, and perfusion). Conversely, studies about in vivo behaviour are poorly represented. Actually, the future challenge will be the development of human grafts to be implanted fully restored in all their structural/functional aspects

Nanopatterned acellular valve conduits drive the commitment of blood-derived multipotent cells

Considerable progress has been made in recent years toward elucidating the correlation among nanoscale topography, mechanical properties, and biological behavior of cardiac valve substitutes. Porcine TriCol scaffolds are promising valve tissue engineering matrices with demonstrated self-repopulation potentiality. In order to define an in vitro model for investigating the influence of extracellular matrix signaling on the growth pattern of colonizing blood-derived cells, we cultured circulating multipotent cells (CMC) on acellular aortic (AVL) and pulmonary (PVL) valve conduits prepared with TriCol method and under no-flow condition. Isolated by our group from Vietnamese pigs before heart valve prosthetic implantation, porcine CMC revealed high proliferative abilities, three-lineage differentiative potential, and distinct hematopoietic/endothelial and mesenchymal properties. Their interaction with valve extracellular matrix nanostructures boosted differential messenger RNA expression pattern and morphologic features on AVL compared to PVL, while promoting on both matrices the commitment to valvular and endothelial cell-like phenotypes. Based on their origin from peripheral blood, porcine CMC are hypothesized in vivo to exert a pivotal role to homeostatically replenish valve cells and contribute to hetero- or allograft colonization. Furthermore, due to their high responsivity to extracellular matrix nanostructure signaling, porcine CMC could be useful for a preliminary evaluation of heart valve prosthetic functionality

Work Function Tuning in Hydrothermally Synthesized Vanadium-Doped MoO3 and Co3O4 Mesostructures for Energy Conversion Devices

The wide interest in developing green energy technologies stimulates the scientific community to seek, for devices, new substitute material platforms with a low environmental impact, ease of production and processing and long-term stability. The synthesis of metal oxide (MO) semiconductors fulfils these requirements and efforts are addressed towards optimizing their functional properties through the improvement of charge mobility or energy level alignment. Two MOs have rising perspectives for application in light harvesting devices, mainly for the role of charge selective layers but also as light absorbers, namely MoO3 (an electron blocking layer) and Co3O4 (a small band gap semiconductor). The need to achieve better charge transport has prompted us to explore strategies for the doping of MoO3 and Co3O4 with vanadium (V) ions that, when combined with oxygen in V2O5, produce a high work function MO. We report on subcritical hydrothermal synthesis of V-doped mesostructures of MoO3 and of Co3O4, in which a tight control of the doping is exerted by tuning the relative amounts of reactants. We accomplished a full analytical characterization of these V-doped MOs that unambiguously demonstrates the incorporation of the vanadium ions in the host material, as well as the effects on the optical properties and work function. We foresee a promising future use of these materials as charge selective materials in energy devices based on multilayer structures

Work Function Tuning in Hydrothermally Synthesized Vanadium-Doped MoO3 and Co3O4 Mesostructures for Energy Conversion Devices

The wide interest in developing green energy technologies stimulates the scientific community to seek, for devices, new substitute material platforms with a low environmental impact, ease of production and processing and long-term stability. The synthesis of metal oxide (MO) semiconductors fulfils these requirements and efforts are addressed towards optimizing their functional properties through the improvement of charge mobility or energy level alignment. Two MOs have rising perspectives for application in light harvesting devices, mainly for the role of charge selective layers but also as light absorbers, namely MoO3 (an electron blocking layer) and Co3O4 (a small band gap semiconductor). The need to achieve better charge transport has prompted us to explore strategies for the doping of MoO3 and Co3O4 with vanadium (V) ions that, when combined with oxygen in V2O5, produce a high work function MO. We report on subcritical hydrothermal synthesis of V-doped mesostructures of MoO3 and of Co3O4, in which a tight control of the doping is exerted by tuning the relative amounts of reactants. We accomplished a full analytical characterization of these V-doped MOs that unambiguously demonstrates the incorporation of the vanadium ions in the host material, as well as the effects on the optical properties and work function. We foresee a promising future use of these materials as charge selective materials in energy devices based on multilayer structures

In vitro assessment of a novel composite scaffold for articular cartilage restoration

Articular cartilage (AC) lesions are a particular challenge for regenerative medicine due to cartilage low self-ability repair in case of damage. Hence, a significant goal of musculoskeletal tissue engineering is the development of suitable structures in virtue of their matrix composition and biomechanical properties [1]. The objective of our study was to design in vitro a supporting structure for cartilage chondrocytes to treat focal articular joint defects. We realized a bio-hybrid composite scaffold combining decellularized Wharton’s jelly (W’s J) with the biomechanical properties of the synthetic hydrogel polyvinyl alcohol (PVA). The hydrogel itself and the more specific decellularized cartilage matrix were used as controls. Immunohistochemical analysis highlighted a similar histomorphology for W’s J and AC matrices. Human chondrocytes were isolated from articular cartilage by collagenase II digestion and then characterized by flow-cytometry and RT-PCR to assess the expression of specific markers. CD44+/CD73+/CD151+ chondrocytes were seeded on PVA, PVA/AC and PVA/W’s J scaffolds to test their ability to support cell colonization. According to SEM micrographs and MTT proliferation assay, PVA/W’s J revealed a singular attitude to sustain cell proliferation despite its aspecific origin. Our preliminary evidences highlighted the chance of using Wharton’s jelly in combination with PVA hydrogels as an innovative and easily available scaffold for cartilage restoration

Radicals and Ions Formed in Plasma-Treated Organic Solvents: A Mechanistic Investigation to Rationalize the Enhancement of Electrospinnability of Polycaprolactone

This paper reports and discusses the beneficial effects on the quality of electrospun polycaprolactone nanofibers brought about by pretreatment of the solvent with non-thermal plasma. Chloroform/dimethylformamide 9:1 (CHCl3:DMF 9:1) and pure chloroform were pretreated by a few minute exposure to the plasma generated by an atmospheric pressure plasma jet (APPJ). Interestingly, when pure chloroform was used, the advantages of plasma pretreatment of the solvent were way less pronounced than found with the CHCl3:DMF 9:1 mixture. The chemical modifications induced by the plasma in the solvents were investigated by means of complementary analytical techniques. GC-MS revealed the formation of solvent-derived volatile products, notably tetrachloroethylene (C2Cl4), 1,1,2,2-tetrachloroethane (C2H2Cl4), pentachloroethane (C2HCl5), hexachloroethane (C2Cl6) and, in the case of the mixed solvent, also N-methylformamide (C2H5NO). The chlorinated volatile products are attributed to reactions of ·Cl and Cl-containing methyl radicals and carbenes formed in the plasma-treated solvents. ·Cl and ·CCl3 radicals were detected and identified by EPR spectroscopy analyses. Ion chromatography revealed the presence of Cl−, NO3-, and HCOO− (the latter only in the presence of DMF) in the plasma-treated solvents, thus accounting for the observed increased conductivity and acidification of the solvent after plasma treatment. Mechanisms for the formation of these solvent derived products induced by plasma are proposed and discussed. The major role of radicals and ions in the plasma chemistry of chloroform and of the chloroform/dimethylformamide mixture is highlighted. The results provide insight into the interaction of plasma with organic solvents, a field so far little explored but holding promise for interesting applications

In vitro and in vivo study of a novel biodegradable synthetic conduit for injured peripheral nerves

In case of peripheral nerve injury (PNI) with wide substance-loss, surgical reconstruction is still a challenge. Bridging the gap by autologous sensory nerves as grafts is the current standard; nevertheless, the related issues have prompted the research towards the development of effective artificial synthetic/biological nerve conduits (NCs). Here, we manufactured a novel NC using oxidized polyvinyl alcohol (OxPVA) that is a biodegradable cryogel recently patented by our group [1]. Thus, its characteristics were compared with neat polyvinyl alcohol (PVA) and silk-fibroin (SF) NCs through in vitro/in vivo analysis. Considering in vitro studies, a morphological characterization was performed by Scanning Electron Microscopy (SEM). Thereafter, cell adhesion and proliferation of a Schwann-cell line (SH-SY5Y) were evaluated by SEM and MTT assay. Regarding in vivo tests, the NCs were implanted into the surgical injured sciatic nerve (gap: 5 mm) of Sprague-Dawley rats, and the functional recovery was assessed after 12-weeks. The NCs were then processed for histological, immunohistochemical (anti-CD3; -β-tubulin; -S100) and Transmission Electron Microscopy (TEM) analyses. In particular, morphometric analyses (section area, total number and density of nerve fibers) were performed at the level of proximal, central and distal portions with respect to NC. In vitro results by SEM showed that PVA and SF supports have a smoother surface than OxPVA scaffolds. Moreover, unlike SF scaffolds, PVA-based ones do not support SH-SY5Y adhesion and proliferation. Regarding the in vivo study, all animals showed a functional recovery with normal walk, even though only animals implanted with PVA and SF NCs sometimes showed spasms while walking. On the contrary, animals implanted with OxPVA NCs exhibited a normal movement. Anti-CD3 immunohistochemistry assessed the absence of severe inflammatory reactions in all the grafts. A strong positive immunoreaction for β-tubulin and S100 demonstrated the good regeneration of nervous fibers. TEM highlighted regeneration of myelinated/un-myelinated axons and Schwann cells in all the grafts. However, morphometric analysis demonstrated that OxPVA assure a better outcome in nerve regeneration in terms of total number of nerve fibers. Our results sustain the potential of OxPVA for the development of NCs useful for PNI with substance loss with the advantage of biodegradation

Short Bowel Syndrome and Tissue Engineering: a preliminary study towards the development of a new regenerative approach in paediatric patients

Pediatric Short Bowel Syndrome (SBS) is a malabsorption state following massive surgical resections of the small intestine. The current therapeutic options issues (i.e. parental nutrition, surgical lengthening, transplantation) have prompt the research in Tissue Engineering. Thus, our aim was to preliminary investigate in vitro/in vivo two composite scaffolds for engineering the small intestine without resorting to autologous intestinal epithelial organoid units which, to date, are the cell source mainly considered for this purpose. In particular, we developed composite supports consisting of a novel biocompatible/resorbable cryogel that is oxidized polyvinyl alcohol (OxPVA) [1] crosslinked with intestinal mucosa whole (wIM/OxPVA) or homogenized (hIM/OxPVA). After evaluating the scaffolds by histology and Scanning Electron Microscopy (SEM), we assessed their interaction with adipose mesenchymal stem cells. Thereafter, the in vivo behavior of scaffolds was studied implanting them in the omentum of Sprague Dawley rats. At 4 weeks, explants were processed by histology and immunohistochemistry (CD3; F4/80; Ki-67; desmin; α-SMA; MNF116). Considering the in vitro evidence, both histological and SEM results proved the effectiveness of the decellularization, and allowed to appreciate the preservation of intestinal villi of the wIM as well as the characteristic features of the hIM. At 7 days from cell seeding, MTT assay showed that hIM/OxPVA scaffolds could support cell adhesion/proliferation even if the wIM/OxPVA ones seem to significantly increase cell growth (

Characterization of novel autologous leukocyte fibrin platelet membranes for tissue engineering applications

Autologous hemocomponents have recently emerged as potential biologic tools for regenerative purpose, consisting mainly of platelet concentrates which locally release growth factors (GFs) to enhance the tissue healing process. Despite two decades of clinical studies, the therapeutic efficacy of platelet concentrates is still controversial. This work represents a first characterization of a novel autologous leukocyte fibrin platelet membrane (LFPm), which is prepared by the Department of Immunohematology of Belluno Hospital according to a well standardized protocol. The quantification of their specific content showed that LFPms are enriched not only with platelets, but also with monocytes/macrophages, fibrinogen and CD34+ cells. Mechanical properties of LFPms were investigated by tensile tests, revealing that the specific elasticity of membranes was maintained over time. Furthermore, the release kinetics of Platelet Derived Growth Factor, Vascular Endothelial Growth Factor, Tumor Necrosis Factor alpha and Interleukin-10 was assessed by ELISA, demonstrating that LFPms act as GF delivery systems which sustain the local release of bioactive molecules. For in vitro biodegradation analysis, LFPm samples were incubated into PBS solution for 4, 7, 14, 21 days. SEM micrographs showed a progressive loss in cellular elements associated to a simultaneous exposure of the fibrin scaffold, also confirmed by histological and immunohistochemical investigations. In parallel, LFPm disks were implanted into a subcutaneous dorsal pouch of healthy nude rats and explanted after 4, 7, 14, 21 days for in vivo biodegradation study. SEM, histological and immunohistochemical analysis revealed that the typical LFPm fibrin structure was maintained until day 7, with a contemporary loss of cellular elements. From day 14, the morphology and texture of samples became less and less recognizable, confirming that a progressive biodegradation occurred. Overall, collected evidences could support the rationale for the clinical use of LFPms, shading some light on the regenerative effect they may exert after the autologous implant on a defect site

Exploring a tissue engineering strategy as a novel approach for haemophilic arthropathy treatment

Among the most disabling complications of Haemophilia, repeated and sponta- neous intra-articular haemorrhages may cause irreversible damage to the joint. This leads to haemophilic arthropathy, a polyarticular disease characterized by joint stiff- ness, chronic pain and a severely limited range of motion. Occurrence of haemophilic arthropathy can be avoided by the prophylactic administration of clotting factors to prevent articular haemorrhages, but it can also be addressed using anti-inflammatory drugs and surgery to alleviate the effects of articular damage, up to arthroplasty as resolute option [1]. However, innovative strategies for the prevention and treatment of this common and serious complication are still required, due to some important limits of current therapies, first of all inhibitor development. In this work, we inves- tigated a tissue engineering approach to regenerate articular focal lesions in Haemo- philic patients by in vitro development of an autologous bio-hybrid prosthesis. For this purpose, we isolated articular chondrocytes from Haemophilic patients (HaeCs) and characterized them for the first time in literature, to verify whether they were altered by blood exposure. Using healthy chondrocytes as control, optical microscope morphological analysis, flow cytometry immunophenotype evaluation and gene expression study by qRT-PCR were performed. After that, an innovative compos- ite scaffold was obtained by combining decellularized Wharton’s Jelly (W’s J) from human umbilical cord with a novel biodegradable polyvinyl alcohol (PVA) hydrogel [2]. Finally, we assessed HaeCs capacity to re-populate biosynthetic scaffolds by Scan- ning Electron Microscopy and MTT assay on cells seeded on supports. Taken togeth- er, our results contributed to define HaeCs phenotype, highlighting the possibility to use these cells for autologous implant. What is more, HaeCs capacity to growth and proliferate on composite scaffolds set the stage for planning the development of autologous tissue substitutes for haemophilic cartilage regeneration