Cell-free biomimetic polyurethane-based scaffold for breast reconstruction following non-malignant lesion resection. A first-in-human study.

BACKGROUND: Based on the volume of tissue removed, conservative surgery (BCS) cannot always guarantee satisfactory cosmetic results, unless resorting to more complex oncoplastic approaches. Investigating an alternative to optimize aesthetic outcomes minimizing surgical complexity, was the purpose of this study. We assessed an innovative surgical procedure based on the use of a biomimetic polyurethane-based scaffold intended for regenerating soft-tissue resembling fat, in patients undergoing BCS for non-malignant breast lesions. Safety and performance of the scaffold, and safety and feasibility of the entire implant procedure were evaluated. METHODS: A volunteer sample of 15 female patients underwent lumpectomy with immediate device positioning, performing seven study visits with six-month follow-up. We evaluated incidence of adverse events (AEs), changes in breast appearance (using photographs and anthropomorphic measurements), interference with ultrasound and MRI (assessed by two independent investigators), investigator's satisfaction (through a VAS scale), patient's pain (through a VAS scale) and quality of life (QoL) (using the BREAST-Q© questionnaire). Data reported are the results of the interim analysis on the first 5 patients. RESULTS: No AEs were device related nor serious. Breast appearance was unaltered and the device did not interference with imaging. High investigator's satisfaction, minimal post-operative pain and positive impact on QoL were also detected. CONCLUSIONS: Albeit on a limited number of patients, data showed positive outcomes both in terms of safety and performance, paving the way to an innovative breast reconstructive approach with a potential remarkable impact on clinical application of tissue engineering. TRIAL REGISTRATION: ClinicalTrials.gov (NCT04131972, October 18, 2019).

Conditioning the microenvironment for soft tissue regeneration in a cell free scaffold.

The use of cell-free scaffolds for the regeneration of clinically relevant volumes of soft tissue has been challenged, particularly in the case of synthetic biomaterials, by the difficulty of reconciling the manufacturing and biological performance requirements. Here, we investigated in vivo the importance of biomechanical and biochemical cues for conditioning the 3D regenerative microenvironment towards soft tissue formation. In particular, we evaluated the adipogenesis changes related to 3D mechanical properties by creating a gradient of 3D microenvironments with different stiffnesses using 3D Poly(Urethane-Ester-ether) PUEt scaffolds. Our results showed a significant increase in adipose tissue proportions while decreasing the stiffness of the 3D mechanical microenvironment. This mechanical conditioning effect was also compared with biochemical manipulation by loading extracellular matrices (ECMs) with a PPAR-γ activating molecule. Notably, results showed mechanical and biochemical conditioning equivalency in promoting adipose tissue formation in the conditions tested, suggesting that adequate mechanical signaling could be sufficient to boost adipogenesis by influencing tissue remodeling. Overall, this work could open a new avenue in the design of synthetic 3D scaffolds for microenvironment conditioning towards the regeneration of large volumes of soft and adipose tissue, with practical and direct implications in reconstructive and cosmetic surgery.

Validity and reliability of the GYKO inertial sensor system for the assessment of the elbow range of motion.

BACKGROUND: The evaluation of joints range of motion (ROM) represents a fundamental step in the diagnosis of joint disorders. Assessors usually measure the ROM angle through a universal goniometer (UG). GYKO inertial system (Microgate, Bolzano, Italy) represent a noninvasive, easy-to-use, Inertial Measurement Unit (IMU) method for the measurement of the elbow ROM. The aim of this study was to validate the GYKO digital device comparing it with the gold standard UG in the measurement of elbow flexion-extension ROM in healthy subjects. METHODS: Thirty healthy subjects (15 females, 15 males; mean age: 34 years, range 25-58 years) were enrolled. The elbow ROM of the dominant arm was measured with two methods, UG and GYKO. Active flexion-extension movement of the elbow was measured by two operators with UG (A1_UG; A2_UG) and with GYKO (A1_GYKO; A2_GYKO; B_GYKO). Intra-rater reliability, inter-rater reliability, and concurrent validity were analyzed by intraclass correlation coefficient (ICC) values. Bland-Altman plot was used to compare UG and GYKO. RESULTS: Both methods were very reliable (P<0.001). Intra-rater reliability showed strong correlation respectively for the UG (ICC=0.798) and for GYKO (ICC=0.859) while inter-rater reliability showed moderate correlation with UG (ICC=0.726) and strong correlation with GYKO (ICC=0.942). The concurrent validity, obtained by three comparisons (A1, A2 and B) showed moderate correlation (ICC: 0.576-0.776). CONCLUSIONS: The results of this study support the use of GYKO as useful as the UG for the assessment of the active flexion-extension ROM of the elbow.

The Effects of Transcutaneous Spinal Direct Current Stimulation on Neuropathic Pain in Multiple Sclerosis: Clinical and Neurophysiological Assessment.

Background: Central neuropathic pain represents one of the most common symptoms in multiple sclerosis (MS) and it seriously affects quality of life. Spinal mechanisms may contribute to the pathogenesis of neuropathic pain in MS. Converging evidence from animal models and neurophysiological and clinical studies in humans suggests a potential effect of transcranial direct current stimulation (tc-DCS) on neuropathic pain. Spinal application of DCS, i.e., transcutaneous spinal DCS (ts-DCS), may modulate nociception through inhibition of spinal reflexes. Therefore, ts-DCS could represents an effective, safe and well-tolerated treatment for neuropathic pain in MS, a largely unexplored topic. This study is a pilot randomized double-blind sham-controlled trial to evaluate the efficacy of ts-DCS on central neuropathic pain in MS patients. Methods: Thirty-three MS patients with central neuropathic pain were enrolled and randomly assigned to two groups in a double-blind sham-controlled design: anodal ts-DCS group (n = 19, 10 daily 20-min sessions, 2 mA) or sham ts-DCS group (n = 14, 10 daily 20-min sessions, 0 mA). The following clinical outcomes were evaluated before ts-DCS treatment (T0), after 10 days of treatment (T1) and 1 month after the end of treatment (T2): neuropathic pain symptoms inventory (NPSI), Ashworth Scale (AS) for spasticity and Fatigue Severity Scale (FSS). A subgroup of patients treated with anodal ts-DCS (n = 12) and sham ts-DCS (n = 11) also underwent a parallel neurophysiological study of the nociceptive withdrawal reflex (NWR) and the NWR temporal summation threshold (TST), two objective markers of pain processing at spinal level. Results: Anodal ts-DCS group showed a significant improvement in NPSI at T1, which persisted at T2, while we did not detect any significant change in AS and FSS. Sham ts-DCS group did not show any significant change in clinical scales. We observed a non-significant trend towards an inhibition of NWR responses in the anodal ts-DCS group at T1 and T2 when compared to baseline. Conclusions: Anodal ts-DCS seems to have an early and persisting (i.e., 1 month after treatment) clinical efficacy on central neuropathic pain in MS patients, probably through modulation of spinal nociception. Clinical Trial Registration: www.ClinicalTrials.gov, identifier #NCT02331654.

Exploring the potential of polyurethane-based soft foam as cell-free scaffold for soft tissue regeneration.

Reconstructive treatment after trauma and tumor resection would greatly benefit from an effective soft tissue regeneration. The use of cell-free scaffolds for adipose tissue regeneration in vivo is emerging as an attractive alternative to tissue-engineered constructs, since this approach avoids complications due to cell manipulation and lack of synchronous vascularization. In this study, we developed a biodegradable polyurethane-based scaffold for soft tissue regeneration, characterized by an exceptional combination between softness and resilience. Exploring the potential as a cell-free scaffold required profound understanding of the impact of its intrinsic physico-chemical properties on the biological performance in vivo. We investigated the effect of the scaffold's hydrophilic character, degradation kinetics, and internal morphology on (i) the local inflammatory response and activation of MGCs (foreign body response); (ii) its ability to promote rapid vascularisation, cell infiltration and migration through the scaffold over time; and (iii) the grade of maturation of the newly formed tissue into vascularized soft tissue in a murine model. The study revealed that soft tissue regeneration in vivo proceeded by gradual infiltration of undifferentiated mesenchymal cells though the periphery toward the center of the scaffold, where the rapid formation of a functional and well-formed vascular network supported cell viability overtime. STATEMENT OF SIGNIFICANCE: Exploring the potential of polyurethane-based soft foam as cell-free scaffold for soft tissue regeneration. In this work, we address the unmet need for synthetic functional soft tissue substitutes that provide adequate biological and mechanical support to soft tissue. We developed a series of flexible cross-linked polyurethane copolymer scaffolds with remarkable fatigue-resistance and tunable physico-chemical properties for soft tissue regeneration in vivo. Accordingly, we could extend the potential of this class of biomaterials, which was so far confined for bone and osteochondral tissue regeneration, to other types of connective tissue.

P(NIPAAM-co-HEMA) thermoresponsive hydrogels: an alternative approach for muscle cell sheet engineering.

Loss of skeletal muscle tissue caused by traumatic injury or damage due to myopathies produces a deficit of muscle function for which there is still no clinical treatment. Transplantation of myogenic cells, themselves or combined with materials, has been proposed to increase the regenerative capacity of skeletal muscle but it is hampered by many limitations, such as low cell survival and engraftment or immunological reaction and low biocompatibility of the exogenous materials. Recently, myoblast sheet engineering, obtained with thermoresponsive culture dishes, has attracted attention as a new technique for muscle damage treatment. For this purpose, a series of thermoresponsive hydrogels, constituted by poly(N-isopropylacrylamide-co-2-hydroxyethylmethacrylate) [p(NIPAAM-co-HEMA)] were synthesized by a simple and inexpensive free-radical polymerization of the two co-monomers with a redox initiator. Different ratios of N-isopropylacrylamide (NIPAAm) and 2-hydroxyethylmethacrylate (HEMA) have been examined to evaluate the effects on physicochemical, mechanical and optical hydrogel properties. The murine muscle cell line C2 C12 has been exploited to test the cytotoxicity of the thermoresponsive hydrogels, depending on different synthesis conditions. In this study, we have identified a thermoresponsive hydrogel that allows cell adhesion and viability, together with the detachment of viable sheet of muscle cells, giving the chance to develop further applications for muscle damage and disease. Copyright © 2014 John Wiley & Sons, Ltd.

Photocrosslinked poly(amidoamine) nanoparticles for central nervous system targeting.

This study presents an innovative method for the synthesis of polymeric nanoparticles (NPs) for central nervous system (CNS) targeting. The method is based on Ultraviolet light (UV)-induced crosslinking of diacrylamide-terminated oligomers of poly(amidoamine)s (PAAs), a widely used class of synthetic polymers in biomedical field research, especially in drug delivery thanks to their excellent biocompatibility and controlled biodegradability. Previous attempts aiming at preparing PAA-based NPs by self-assembly were challenged by lack of structural stability and consequently their early degradation and premature drug release. Here, the UV-induced crosslinked PAA NPs demonstrated to overcome main disadvantages of the self-assembled ones, as they showed improved stability and controlled release properties. Besides the remarkable efficiency to produce monodisperse and stable PAA NPs, the UV-induced crosslinking method is featured by great versatility and low environmental impact, since it does not require use of organic solvents and multiple purification steps. The capability of PAA NPs to encapsulate a fluorescently labelled model protein was experimentally demonstrated in this study. Cell culture experiments showed that PAA NPs were biocompatible and highly permeable across an in vitro blood-brain barrier model, thus highlighting their great potential as drug delivery vectors for CNS delivery.

Biologically and mechanically driven design of an RGD-mimetic macroporous foam for adipose tissue engineering applications.

Despite clinical treatments for adipose tissue defects, in particular breast tissue reconstruction, have certain grades of efficacy, many drawbacks are still affecting the long-term survival of new formed fat tissue. To overcome this problem, in the last decades, several scaffolding materials have been investigated in the field of adipose tissue engineering. However, a strategy able to recapitulate a suitable environment for adipose tissue reconstruction and maintenance is still missing. To address this need, we adopted a biologically and mechanically driven design to fabricate an RGD-mimetic poly(amidoamine) oligomer macroporous foam (OPAAF) for adipose tissue reconstruction. The scaffold was designed to fulfil three fundamental criteria: capability to induce cell adhesion and proliferation, support of in vivo vascularization and match of native tissue mechanical properties. Poly(amidoamine) oligomers were formed into soft scaffolds with hierarchical porosity through a combined free radical polymerization and foaming reaction. OPAAF is characterized by a high water uptake capacity, progressive degradation kinetics and ideal mechanical properties for adipose tissue reconstruction. OPAAF's ability to support cell adhesion, proliferation and adipogenesis was assessed in vitro using epithelial, fibroblast and endothelial cells (MDCK, 3T3L1 and HUVEC respectively). In addition, in vivo subcutaneous implantation in murine model highlighted OPAAF potential to support both adipogenesis and vessels infiltration. Overall, the reported results support the use of OPAAF as a scaffold for engineered adipose tissue construct.

A Tailor-Made Synthetic Polymer for Cell Encapsulation: Design Rationale, Synthesis, Chemical-Physics and Biological Characterizations.

This study presents a custom-made in situ gelling polymeric precursor for cell encapsulation. Composed of poly((2-hydroxyethyl)methacrylate-co-(3-aminopropyl)methacrylamide) (P(HEMA-co-APM) mother backbone and RGD-mimicking poly(amidoamine) (PAA) moiteis, the comb-like structured polymeric precursor is tailored to gather the advantages of the two families of synthetic polymers, i.e., the good mechanical integrity of PHEMA-based polymers and the biocompatibility and biodegradability of PAAs. The role of P(HEMA-co-APM) in the regulation of the chemico-physical properties of P(HEMA-co-APM)/PAA hydrogels is thoroughly investigated. On the basis of obtained results, namely the capability of maintaining vital NIH3T3 cell line in vitro for 2 d in a 3D cell culture, the in vivo biocompatibility in murine model for 16 d, and the ability of finely tuning mechanical properties and degradation kinetics, it can be assessed that P(HEMA-co-APM)/PAAs offer a cost-effective valid alternative to the so far studied natural polymer-based systems for cell encapsulation.

In vivo imaging study of angiogenesis in a channelized porous scaffold.

The main scientific issue hindering the development of tissue engineering technologies is the lack of proper vascularization. Among the various approaches developed for boosting vascularization, scaffold design has attracted increasing interest over the last few years. The aim of this article is to illustrate a scaffold design strategy for enhancing vascularization based on sacrificial microfabrication of embedded microchannels. This approach was combined with an innovative poly(ether urethane urea) (PEUtU) porous scaffold to provide an alternative graft substitute material for the treatment of tissue defects. Fluorescent and chemiluminescent imaging combined with computed tomography were used to study the behavior of the scaffold composition within living subjects by analyzing angiogenesis and inflammation processes and observing the variation in x-ray absorption, respectively. For this purpose, an IntegriSense 680 probe was used in vivo for the localization and quantification of integrin αvß3, due to its critical involvement in angiogenesis, and a XenoLight RediJect Inflammation Probe for the study of the decline in inflammation progression during healing. Overall, the collected data suggest the advantages of embedding a synthetic vascular network into a PEUtU porous matrix to enhance in vivo tissue integration, maturation, and regeneration. Moreover, our imaging approach proved to be an efficient and versatile tool for scaffold in vivo testing.

RGD-mimetic poly(amidoamine) hydrogel for the fabrication of complex cell-laden micro constructs.

The potential of the 3D cell culture approach for creating in vitro models for drug screening and cellular studies, has led to the development of hydrogels that are able to mimic the in vivo 3D cellular milieu. To this aim, synthetic polymer-based hydrogels, with which it is possible to fine-tune the chemical and biophysical properties of the cell microenvironment, are becoming more and more acclaimed. Of all synthetic materials, poly(amidoamine)s (PAAs) hydrogels are known to have promising properties. In particular, PAAs hydrogels containing the 2,2-bisacrylamidoacetic acid-agmatine monomeric unit are capable of enhancing cellular adhesion by interacting with the RGD-binding αVß3 integrin. The synthesis of a new photocrosslinkable, biomimetic PAA-Jeffamine®-PAA triblock copolymer (PJP) hydrogel is reported in this paper with the aim of improving the optical, biocompatibility and cell-adhesion properties of previously studied PAA hydrogels and providing an inexpensive alternative to the RGD peptide based hydrogels. The physicochemical properties of PJP hydrogels are extensively discussed and the behavior of 2D and 3D cell cultures was analyzed in depth with different cell types. Moreover, cell-laden PJP hydrogels were patterned with perfusable microchannels and seeded with endothelial cells, in order to investigate the possibility of using PJP hydrogels for fabricating cell laden tissue-like micro constructs and microfluidic devices. Overall the data obtained suggest that PJP could ultimately become a useful tool for fabricating improved in vitro models in order to potentially enhance the effectiveness of drug screening and clinical treatments.

Versatile fabrication of vascularizable scaffolds for large tissue engineering in bioreactor.

Despite significant progresses were achieved in tissue engineering over the last 20 years, a number of unsolved problems still remain. One of the most relevant issues is the lack of a proper vascularization that is limiting the size of the engineered tissues to smaller than clinically relevant dimensions. Sacrificial molding holds great promise to engineered construct with perfusable vascular architectures, but there is still the need to develop more versatile approaches able to be independent of the nature and dimensions of the construct. In this work we developed a versatile sacrificial molding technique for fabricating bulk, cell-laden and porous scaffolds with embedded vascular fluidic networks. These branched fluidic architectures are created by highly resistant thermoplastic sacrificial templates, made of poly(vinyl alcohol), representing a remarkable progress in manufacturability and scalability. The obtained architecture, when perfused in bioreactor, has shown to prevent the formation of a necrotic core in thick cell-laden constructs and enabled the rapid fabrication of hierarchically branched endothelium. In conclusion we demonstrate a novel strategy towards the engineering of vascularized thick tissues through the integration of the PVA-based microfabrication sacrificial approach and perfusion bioreactors. This approach may be able to scale current engineered tissues to clinically relevant dimensions, opening the way to their widespread clinical applications.

Poly(amido-amine)-based hydrogels with tailored mechanical properties and degradation rates for tissue engineering.

Poly(amido-amine) (PAA) hydrogels containing the 2,2-bisacrylamidoacetic acid-4-amminobutyl guanidine monomeric unit have a known ability to enhance cellular adhesion by interacting with the arginin-glycin-aspartic acid (RGD)-binding αVß3 integrin, expressed by a wide number of cell types. Scientific interest in this class of materials has traditionally been hampered by their poor mechanical properties and restricted range of degradation rate. Here we present the design of novel biocompatible, RGD-mimic PAA-based hydrogels with wide and tunable degradation rates as well as improved mechanical and biological properties for biomedical applications. This is achieved by radical polymerization of acrylamide-terminated PAA oligomers in both the presence and absence of 2-hydroxyethylmethacrylate. The degradation rate is found to be precisely tunable by adjusting the PAA oligomer molecular weight and acrylic co-monomer concentration in the starting reaction mixture. Cell adhesion and proliferation tests on Madin-Darby canine kidney epithelial cells show that PAA-based hydrogels have the capacity to promote cell adhesion up to 200% compared to the control. Mechanical tests show higher compressive strength of acrylic chain containing hydrogels compared to traditional PAA hydrogels.

Effects of branched or linear architecture of bioreducible poly(amido amine)s on their in vitro gene delivery properties.

In this study, the gene delivery properties of new hyperbranched poly(amido amine)s (PAAs) with disulfide linkages in the main chain were investigated in comparison with their linear analogs. Eight different bioreducible PAAs were prepared by Michael addition of N,N'-bisacryloylpiperazine (BP) with cystamine (CYST) or N,N'-dimethylcystamine (DMC) and of N,N'-cystaminebisacrylamide (CBA) with N,N'-ethylenediamine (EDA) or N,N'-dimethylethylenediamine (DMEDA). In order to study the effect of terminal groups on the transfection efficiency, each polymer was terminated with 4-aminobutanol (ABOL) or with 2-aminoethanol (ETA). The hyperbranched and the linear PAAs generally formed polyplexes with plasmid DNA with sizes around 200nm and positive zeta potentials ranging from +10 to +22mV at polymer/DNA weight ratios equal or higher than 3/1. Remarkably low or no cytotoxicity was observed for both hyperbranched and linear PAAs. Hyperbranched CBA-containing PAAs showed higher gene expression in DNA transfection tests with COS-7 cells than their linear analogs and up to two times higher than linear PEI that was used as the reference polymer. Transfection efficiencies of the branched PAAs were generally enhanced by the presence of serum, which is a promising property for future in vivo studies with these hyperbranched PAAs. In this study the ease of synthetic modification of both linear and hyperbranched poly(amido amide)s and the versatility of hyperbranched PAAs in regulating DNA transfection and cytotoxicity are demonstrated. The results show the large possibilities for this class of polymers to provide polymeric vectors with controllable properties for gene therapy applications.