Elena Stocco

Tailored PVA/ECM Scaffolds for Cartilage Regeneration

Articular cartilage 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. The objective of our study was to design in vitro a supporting structure for autologous chondrocyte growth. We realized a biohybrid composite scaffold combining a novel and nonspecific extracellular matrix (ECM), which is decellularized Whartons jelly ECM, with the biomechanical properties of the synthetic hydrogel polyvinyl alcohol (PVA). Whartons jelly ECM was tested for its ability in promoting scaffold colonization by chondrocytes and compared with polyvinyl alcohol itself and the more specific decellularized cartilage matrix. Our preliminary evidences highlighted the chance of using Whartons jelly ECM in combination with PVA hydrogels as an innovative and easily available scaffold for cartilage restoration.

Partially oxidized polyvinyl alcohol as a promising material for tissue engineering.

The desired clinical outcome after implantation of engineered tissue substitutes depends strictly on the development of biodegradable scaffolds. In this study we fabricated 1% and 2% oxidized polyvinyl alcohol (PVA) hydrogels, which were considered for the first time for tissue‐engineering applications. The final aim was to promote the protein release capacity and biodegradation rate of the resulting scaffolds in comparison with neat PVA. After physical crosslinking, characterization of specific properties of 1% and 2% oxidized PVA was performed. We demonstrated that mechanical properties, hydrodynamic radius of molecules, thermal characteristics and degree of crystallinity were inversely proportional to the PVA oxidation rate. On the other hand, swelling behaviour and protein release were enhanced, confirming the potential of oxidized PVA as a protein delivery system, besides being highly biodegradable. Twelve weeks after in vivo implantation in mice, the modified hydrogels did not elicit severe inflammatory reactions, showing them to be biocompatible and to degrade faster as the degree of oxidation increased. According to our results, oxidized PVA stands out as a novel biomaterial for tissue engineering that can be used to realize scaffolds with customizable mechanical behaviour, protein‐loading ability and biodegradability. Copyright

In vitro assessment of TAT - Ciliary Neurotrophic Factor therapeutic potential for peripheral nerve regeneration.

In regenerative neurobiology, Ciliary Neurotrophic Factor (CNTF) is raising high interest as a multifunctional neurocytokine, playing a key role in the regeneration of injured peripheral nerves. Despite its promising trophic and regulatory activity, its clinical application is limited by the onset of severe side effects, due to the lack of efficient intracellular trafficking after administration. In this study, recombinant CNTF linked to the transactivator transduction domain (TAT) was investigated in vitro and found to be an optimized fusion protein which preserves neurotrophic activity, besides enhancing cellular uptake for therapeutic advantage. Moreover, a compelling protein delivery method was defined, in the future perspective of improving nerve regeneration strategies. Following determination of TAT-CNTF molecular weight and concentration, its specific effect on neural SH-SY5Y and PC12 cultures was assessed. Cell proliferation assay demonstrated that the fusion protein triggers PC12 cell growth within 6h of stimulation. At the same time, the activation of signal transduction pathway and enhancement of cellular trafficking were found to be accomplished in both neural cell lines after specific treatment with TAT-CNTF. Finally, the recombinant growth factor was successfully loaded on oxidized polyvinyl alcohol (PVA) scaffolds, and more efficiently released when polymer oxidation rate increased. Taken together, our results highlight that the TAT domain addiction to the protein sequence preserves CNTF specific neurotrophic activity in vitro, besides improving cellular uptake. Moreover, oxidized PVA could represent an ideal biomaterial for the development of nerve conduits loaded with the fusion protein to be delivered to the site of nerve injury.

Autologous chondrocytes as a novel source for neo-chondrogenesis in haemophiliacs

Haemophilic arthropathy is the major cause of disability in patients with haemophilia and, despite prophylaxis with coagulation factor concentrates, some patients still develop articular complications. We evaluate the feasibility of a tissue engineering approach to improve current clinical strategies for cartilage regeneration in haemophiliacs by using autologous chondrocytes (haemophilic chondrocytes; HaeCs). Little is known about articular chondrocytes from haemophilic patients and no characterisation has as yet been performed. An investigation into whether blood exposure alters HaeCs should be interesting from the perspective of autologous implants. The typical morphology and expression of specific target genes and surface markers were therefore assessed by optical microscopy, reverse transcription plus the polymerase chain reaction (PCR), real-time PCR and flow-cytometry. We then considered chondrocyte behaviour on a bio-hybrid scaffold (based on polyvinyl alcohol/Wharton’s jelly) as an in vitro model of articular cartilage prosthesis. Articular chondrocytes from non-haemophilic donors were used as controls. HaeC morphology and the resulting immunophenotype CD44+/CD49c+/CD49e+/CD151+/CD73+/CD49f−/CD26− resembled those of healthy donors. Moreover, HaeCs were active in the transcription of genes involved in the synthesis of the extracellular matrix proteins of the articular cartilage (ACAN, COL1A, COL2A, COL10A, COL9A, COMP, HAS1, SOX9), although the over-expression of COL1A1, COL10A1, COMP and HAS was observed. In parallel, the composite scaffold showed adequate mechanical and biological properties for cartilage tissue engineering, promoting chondrocyte proliferation. Our preliminary evidence contributes to the characterisation of HaeCs, highlighting the opportunity of using them for autologous cartilage implants in patients with haemophilia.

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.

Partially oxidized polyvinyl alcohol conduitfor peripheral nerve regeneration

Surgical reconstruction of peripheral nerves injuries with wide substance-loss is still a challenge. Many studies focused on the development of artificial nerve conduits made of synthetic or biological materials but the ideal device has not yet been identified. Here, we manufactured a conduit for peripheral nerve regeneration using a novel biodegradable hydrogel we patented that is oxidized polyvinyl alcohol (OxPVA). Thus, its characteristics were compared with neat polyvinyl alcohol (PVA) and silk-fibroin (SF) conduits, through in vitro and in vivo analysis. Unlike SF, OxPVA and neat PVA scaffolds did not support SH-SY5Y adhesion and proliferation in vitro. After implantation in rat model of sciatic nerve transection, the three conduits sustained the regeneration of the injured nerve filling a gap of 5 mm in 12 weeks. Implanted animals showed a good gait recovery. Morphometric data related to the central portion of the explanted conduit interestingly highlighted a significantly better outcome for OxPVA scaffolds compared to PVA conduits in terms of axon density, also with respect to the autograft group. This study suggests the potential of our novel biomaterial for the development of conduits for clinical use in case of peripheral nerve lesions with substance loss.

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.

The infrapatellar fat pad and the synovial membrane: an anatomo-functional unit

The infrapatellar pad, a fibro‐adipose tissue with peculiar microscopic and mechanical features, is gaining wide attention in the field of rheumatological research. The purpose of this descriptive review is to summarize the most recent published evidence on the anatomic, physiologic and biomechanical inter‐relationship between the infrapatellar fat pad and the knee synovial membrane. As an extrasynovial tissue, the infrapatellar fat pad does not directly interact with the articular cartilage; based on its location in close contact with the synovial membrane, and due to the metabolic properties of adipose tissue, it may influence the behavior of the synovial membrane. In fact, considering evidence of macroscopic and microscopic anatomy, the infrapatellar fat pad is the site of insertion of the infrapatellar and medial synovial plicae. Also biochemically, there is much evidence highlighting the interaction among these two structures; in the case of inflammation, the mutual interplay is ascribable to the release of pro‐inflammatory mediators stimulating the proliferation of inflammatory cells and promoting tissue modifications in both. All these assumptions could support the emerging idea that the infrapatellar fat pad and the synovial membrane may be considered a morpho‐functional unit.

Receptor–Receptor Interactions of G Protein-Coupled Receptors in the Carotid Body: A Working Hypothesis

In the carotid body (CB), a wide series of neurotransmitters and neuromodulators have been identified. They are mainly produced and released by type I cells and act on many different ionotropic and metabotropic receptors located in afferent nerve fibers, type I and II cells. Most metabotropic receptors are G protein-coupled receptors (GPCRs). In other transfected or native cells, GPCRs have been demonstrated to establish physical receptor–receptor interactions (RRIs) with formation of homo/hetero-complexes (dimers or receptor mosaics) in a dynamic monomer/oligomer equilibrium. RRIs modulate ligand binding, signaling, and internalization of GPCR protomers and they are considered of relevance for physiology, pharmacology, and pathology of the nervous system. We hypothesize that RRI may also occur in the different structural elements of the CB (type I cells, type II cells, and afferent fibers), with potential implications in chemoreception, neuromodulation, and tissue plasticity. This ‘working hypothesis’ is supported by literature data reporting the contemporary expression, in type I cells, type II cells, or afferent terminals, of GPCRs which are able to physically interact with each other to form homo/hetero-complexes. Functional data about cross-talks in the CB between different neurotransmitters/neuromodulators also support the hypothesis. On the basis of the above findings, the most significant homo/hetero-complexes which could be postulated in the CB include receptors for dopamine, adenosine, ATP, opioids, histamine, serotonin, endothelin, galanin, GABA, cannabinoids, angiotensin, neurotensin, and melatonin. From a methodological point of view, future studies should demonstrate the colocalization in close proximity (less than 10 nm) of the above receptors, through biophysical (i.e., bioluminescence/fluorescence resonance energy transfer, protein-fragment complementation assay, total internal reflection fluorescence microscopy, fluorescence correlation spectroscopy and photoactivated localization microscopy, X-ray crystallography) or biochemical (co-immunoprecipitation, in situ proximity ligation assay) methods. Moreover, functional approaches will be able to show if ligand binding to one receptor produces changes in the biochemical characteristics (ligand recognition, decoding, and trafficking processes) of the other(s). Plasticity aspects would be also of interest, as development and environmental stimuli (chronic continuous or intermittent hypoxia) produce changes in the expression of certain receptors which could potentially invest the dynamic monomer/oligomer equilibrium of homo/hetero-complexes and the correlated functional implications.

Composite Scaffolds Based on Intestinal Extracellular Matrices and Oxidized Polyvinyl Alcohol: A Preliminary Study for a New Regenerative Approach in Short Bowel Syndrome

Pediatric Short Bowel Syndrome is a rare malabsorption disease occurring because of massive surgical resections of the small intestine. To date, the issues related to current strategies including intestinal transplantation prompted the attention towards tissue engineering (TE). This work aimed to develop and compare two composite scaffolds for intestinal TE consisting of a novel hydrogel, that is, oxidized polyvinyl alcohol (OxPVA), cross-linked with decellularized intestinal wall as a whole (wW/OxPVA) or homogenized (hW/OxPVA). A characterization of the supports was performed by histology and Scanning Electron Microscopy and their interaction with adipose mesenchymal stem cells occurred by MTT assay. Finally, the scaffolds were implanted in the omentum of Sprague Dawley rats for 4 weeks prior to being processed by histology and immunohistochemistry (CD3; F4/80; Ki-67; desmin; α-SMA; MNF116). In vitro studies proved the effectiveness of the decellularization, highlighting the features of the matrices; moreover, both supports promoted cell adhesion/proliferation even if the wW/OxPVA ones were more effective (p < 0.01). Analysis of explants showed a continuous and relatively organized tissue wall around the supports with a connective appearance, such as myofibroblastic features, smooth muscle, and epithelial cells. Both scaffolds, albeit with some difference, were promising; nevertheless, further analysis will be necessary.