Assessment of the in vivo biofunctionality of a biomimetic hybrid scaffold for osteochondral tissue regeneration.

Chondral and osteochondral lesions represent one of the most challenging problems in the orthopedic field, as these types of injuries lead to disability and worsened quality of life for patients and have an economic impact on the healthcare system. The aim of this in vivo study was to develop a new tissue engineering approach through a hybrid scaffold for osteochondral tissue regeneration made of porous polyurethane foam (PU) coated under vacuum with calcium phosphates (PU/VAC). Scaffold characterization showed a highly porous and interconnected structure. Human amniotic mesenchymal stromal cells (hAMSCs) were loaded into scaffolds using pectin (PECT) as a carrier. Osteochondral defects in medial femoral condyles of rabbits were created and randomly allocated in one of the following groups: plain scaffold (PU/VAC), scaffold with hAMSCs injected in the implant site (PU/VAC/hAMSC), scaffold with hAMSCs loaded in pectin (PU/VAC/PECT/hAMSC), and no treated defects (untreated). The therapeutic efficacy was assessed by macroscopic, histological, histomorphometric, microtomographic, and ultrastructural analyses at 3, 6, 12, and 24 weeks. Histological results showed that the scaffold was permissive to tissue growth and penetration, an immature osteocartilaginous tissue was observed at early experimental times, with a more accentuated bone regeneration in comparison with the cartilage layer in the absence of any inflammatory reaction.

Electrophoretic bottom up design of chitosan patches for topical drug delivery.

Clobetasol propionate (CP) is a high-potency corticosteroid, representing the standard of care for the symptomatic treatment of different skin disorders as well as oral mucosal diseases. Several topical delivery systems are available for treating oral lesions, but the ideal one is still lacking. In this work, we propose a novel class of chitosan (CS) patches, loaded with CP, for the topical treatment of inflammatory chronic oral diseases. Chitosan patches have been fabricated via electrophoretic deposition (EPD), by using a one-pot approach in order to load controlled quantity of CP. Optimized structures showed a water uptake in the range of 200-360% and mechanical properties that allow the design of flexible patches in wet state (E = 0.6 MPa and σbr = 0.55 MPa). Ultraviolet-visible (UV-Vis) spectroscopy was used for the evaluation of both loading and release profile of CP in CS patches. The CP loading has been tuned by adjusting CP concentration in deposition bath-in the range 0.002-0.12 mg cm-2-while releasing curves show an in vitro CP burst of about 80% in the first two hours. Overall, the obtained properties paved the way for the application of this new class of patches for the local oral release of CP.

Fabrication of photothermally active poly(vinyl alcohol) films with gold nanostars for antibacterial applications.

The unique photothermal properties of non-spherical gold nanoparticles under near-infrared (NIR) irradiation find broad application in nanotechnology and nanomedicine. The combination of their plasmonic features with widely used biocompatible poly(vinyl alcohol) (PVA) films can lead to novel hybrid polymeric materials with tunable photothermal properties and a wide range of applications. In this study, thin PVA films containing highly photothermally efficient gold nanostars (GNSs) were fabricated and their properties were studied. The resulting films displayed good mechanical properties and a pronounced photothermal effect under NIR irradiation. The local photothermal effect triggered by NIR irradiation of the PVA-GNS films is highly efficient at killing bacteria, therefore providing an opportunity to develop new types of protective antibacterial films and coatings.

Polyurethane foam scaffold as in vitro model for breast cancer bone metastasis.

Breast cancer (BC) represents the most incident cancer case in women (29%), with high mortality rate. Bone metastasis occurs in 20-50% cases and, despite advances in BC research, the interactions between tumor cells and the metastatic microenvironment are still poorly understood. In vitro 3D models gained great interest in cancer research, thanks to the reproducibility, the 3D spatial cues and associated low costs, compared to in vivo and 2D in vitro models. In this study, we investigated the suitability of a poly-ether-urethane (PU) foam as 3D in vitro model to study the interactions between BC tumor-initiating cells and the bone microenvironment. PU foam open porosity (>70%) appeared suitable to mimic trabecular bone structure. The PU foam showed good mechanical properties under cyclic compression (E=69-109kPa), even if lower than human trabecular bone. The scaffold supported osteoblast SAOS-2 cell line proliferation, with no cytotoxic effects. Human adipose derived stem cells (ADSC) were cultured and differentiated into osteoblast lineage on the PU foam, as shown by alizarin red staining and RT-PCR, thus offering a bone biomimetic microenvironment to the further co-culture with BC derived tumor-initiating cells (MCFS). Tumor aggregates were observed after three weeks of co-culture by e-cadherin staining and SEM; modification in CaP distribution was identified by SEM-EDX and associated to the presence of tumor cells. In conclusion, we demonstrated the suitability of the PU foam to reproduce a bone biomimetic microenvironment, useful for the co-culture of human osteoblasts/BC tumor-initiating cells and to investigate their interaction. STATEMENT OF SIGNIFICANCE: 3D in vitro models represent an outstanding alternative in the study of tumor metastases development, compared to traditional 2D in vitro cultures, which oversimplify the 3D tissue microenvironment, and in vivo studies, affected by low reproducibility and ethical issues. Several scaffold-based 3D in vitro models have been proposed to recapitulate the development of metastases in different body sites but, still, the crucial challenge is to correctly mimic the tissue to be modelled in terms of physical, mechanical and biological properties. Here, we prove the suitability of a porous polyurethane foam, synthesized using an appropriate formulaton, in mimicking the bone tissue microenvironment and in reproducing the metastatic colonization derived from human breast cancer, particularly evidencing the devastating effects on the bone extracellular matrix caused by metastatic spreading.

Alternating air-medium exposure in rotating bioreactors optimizes cell metabolism in 3D novel tubular scaffold polyurethane foams.

BACKGROUND: In vitro dynamic culture conditions play a pivotal role in developing engineered tissue grafts, where the supply of oxygen and nutrients, and waste removal must be permitted within construct thickness. For tubular scaffolds, mass transfer is enhanced by introducing a convective flow through rotating bioreactors with positive effects on cell proliferation, scaffold colonization and extracellular matrix deposition. We characterized a novel polyurethane-based tubular scaffold and investigated the impact of 3 different culture configurations over cell behavior: dynamic (i) single-phase (medium) rotation and (ii) double-phase exposure (medium-air) rotation; static (iii) single-phase static culture as control. METHODS: A new mixture of polyol was tested to create polyurethane foams (PUFs) as 3D scaffold for tissue engineering. The structure obtained was morphologically and mechanically analyzed tested. Murine fibroblasts were externally seeded on the novel porous PUF scaffold, and cultured under different dynamic conditions. Viability assay, DNA quantification, SEM and histological analyses were performed at different time points. RESULTS: The PUF scaffold presented interesting mechanical properties and morphology adequate to promote cell adhesion, highlighting its potential for tissue engineering purposes. Results showed that constructs under dynamic conditions contain enhanced viability and cell number, exponentially increased for double-phase rotation; under this last configuration, cells uniformly covered both the external surface and the lumen. CONCLUSIONS: The developed 3D structure combined with the alternated exposure to air and medium provided the optimal in vitro biochemical conditioning with adequate nutrient supply for cells. The results highlight a valuable combination of material and dynamic culture for tissue engineering applications.

The effect of polyurethane scaffold surface treatments on the adhesion of chondrocytes subjected to interstitial perfusion culture.

The purpose of this study was to measure chondrocytes detachment from cellularized constructs cultured in a perfusion bioreactor, and to evaluate the effect of different scaffold coatings on cell adhesion under a fixed flow rate. The scaffolds were polyurethane foams, treated to promote cell attachment and seeded with human chondrocytes. In a preliminary static culture experiment, the scaffolds were imbibed with fetal bovine serum (FBS) and then cultured for 4 weeks. To quantify cell detachment, the number of detached cells from the scaffold treated with FBS was estimated under different interstitial perfusion flow rates and shear stress levels (0.005 mL/min equivalent to 0.05 mPa, 0.023 mL/min equivalent to 0.23 mPa, and 0.045 mL/min equivalent to 0.45 mPa). Finally, groups of scaffolds differently treated (FBS, plasma plus FBS, plasma plus collagen type I) were cultured under a fixed perfusion rate of 0.009 mL/min, equivalent to a shear stress of 0.09 mPa, and the detached cells were counted. Static cultivation showed that cell proliferation increased with time and matrix biosynthesis decreased after the first week of culture. Perfused culture showed that the number of detached cells increased with the perfusion rate on FBS-treated constructs. The plasma-treated/collagen-coated scaffolds showed the highest resistance to cell detachment. To minimize cell detachment, the perfusion rate must be maintained in the order of 0.02 mL/min, giving a shear stress of 0.2 mPa. Our set-up allowed estimating the resistance to cell detachment under interstitial perfusion in a repeatable manner, to test other scaffold coatings and cell types.

Exploiting novel sterilization techniques for porous polyurethane scaffolds.

Porous polyurethane (PU) structures raise increasing interest as scaffolds in tissue engineering applications. Understanding the effects of sterilization on their properties is mandatory to assess their potential use in the clinical practice. The aim of this work is the evaluation of the effects of two innovative sterilization techniques (i.e. plasma, Sterrad(®) system, and ozone) on the morphological, chemico-physical and mechanical properties of a PU foam synthesized by gas foaming, using water as expanding agent. In addition, possible toxic effects of the sterilization were evaluated by in vitro cytotoxicity tests. Plasma sterilization did not affect the morphological and mechanical properties of the PU foam, but caused at some extent degradative phenomena, as detected by infrared spectroscopy. Ozone sterilization had a major effect on foam morphology, causing the formation of new small pores, and stronger degradation and oxidation on the structure of the material. These modifications affected the mechanical properties of the sterilized PU foam too. Even though, no cytotoxic effects were observed after both plasma and ozone sterilization, as confirmed by the good values of cell viability assessed by Alamar Blue assay. The results here obtained can help in understanding the effects of sterilization procedures on porous polymeric scaffolds, and how the scaffold morphology, in particular porosity, can influence the effects of sterilization, and viceversa.

Nano/micro hybrid scaffold of PCL or P3HB nanofibers combined with silk fibroin for tendon and ligament tissue engineering.

A novel biodegradable nano/micro hybrid structure was obtained by electrospinning P3HB or PCL nanofibers onto a twisted silk fibroin (SF) structure, with the aim of fabricating a suitable scaffold for tendon and ligament tissue engineering. The electrospinning (ES) processing parameters for P3HB and PCL were optimized on 2D samples, and applied to produce two different nano/micro hybrid constructs (SF/ES-PCL and SF/ES-P3HB).Morphological, chemico-physical and mechanical properties of the novel hybrid scaffolds were evaluated by SEM, ATR FT-IR, DSC, tensile and thermodynamic mechanical tests. The results demonstrated that the nanofibers were tightly wrapped around the silk filaments, and the crystallinity of the SF twisted yarns was not influenced by the presence of the electrospun polymers. The slightly higher mechanical properties of the hybrid constructs confirmed an increase of internal forces due to the interaction between nano and micro components. Cell culture tests with L929 fibroblasts, in the presence of the sample eluates or in direct contact with the hybrid structures, showed no cytotoxic effects and a good level of cytocompatibility of the nano/micro hybrid structures in term of cell viability, particularly at day 1. Cell viability onto the nano/micro hybrid structures decreased from the first to the third day of culture when compared with the control culture plastic, but appeared to be higher when compared with the uncoated SF yarns. Although additional in vitro and in vivo tests are needed, the original fabrication method here described appears promising for scaffolds suitable for tendon and ligament tissue engineering.

Biomimetic hybrid scaffolds for osteo-chondral tissue repair: Design and osteogenic differentiation of human placenta-derived cells (hPDC).

A novel functionally-graded hybrid (FGHY) scaffold was designed and developed with a load-bearing structure represented by a PU foam loaded with a graded composition of CaPs (biomimetic component) and pectin gel as cell carrier. hPDC populations encapsulated in pectin gels and injected into the FGHY scaffolds demonstrated the ability to differentiate toward the osteogenic lineage. The ability of these biomimetic hybrid scaffolds to stimulate cell adhesion and proliferation and to support differentiation of hPDCs make these scaffolds excellent candidates for an use in bone regeneration.

Design of 2D chitosan scaffolds via electrochemical structuring.

Chitosan (CS) is a versatile biopolymer whose morphological and chemico-physical properties can be designed for a variety of biomedical applications. Taking advantage of its electrolytic nature, cathodic polarization allows CS deposition on electrically conductive substrates, resulting in thin porous structures with tunable morphology. Here we propose an easy method to obtain CS membranes with highly oriented micro-channels for tissue engineering applications, relying on simple control of process parameters and cathodic substrate geometry. Cathodic deposition was performed on two different aluminum grids in galvanostatic conditions at 6.25 mA cm(-2) from CS solution [1g L(-1)] in acetic acid (pH 3.5). Self-standing thin scaffolds were cross linked either with genipin or epichlorohydrin, weighted, and observed by optical and electron microscopy. Swelling properties at pH 5 and pH 7.4 have been also investigated and tensile tests performed on swollen samples at room temperature. Finally, direct and indirect assays have been performed to evaluate the cytotoxicity at 24 and 72 h. Thin scaffolds with two different oriented porosities (1000 µm and 500 µm) have been successfully fabricated by electrochemical techniques. Both cross-linking agents did not affected the mechanical properties and cytocompatibility of the resulting structures. Depending on the pH, these structures show interesting swelling properties that can be exploited for drug delivery systems. Moreover, thanks to the possibility of controlling the porosity and the micro-channel orientation, they should be used for the regeneration of tissues requiring a preferential cells orientation, e.g., cardiac patches or ligament regeneration.

In vitro study on silk fibroin textile structure for anterior cruciate ligament regeneration.

A novel hierarchical textile structure made of silk fibroin from Bombyx mori capable of matching the mechanical performance requirements of anterior cruciate ligament (ACL) and in vitro cell ingrowth is described. This sericin-free, Silk Fibroin Knitted Sheath with Braided Core (SF-KSBC) structure was fabricated using available textile technologies. Micro-CT analysis confirmed that the core was highly porous and had a higher degree of interconnectivity than that observed for the sheath. The in vivo cell colonization of the scaffolds is thus expected to penetrate even the internal parts of the structure. Tensile mechanical tests demonstrated a maximum load of 1212.4±56.4 N (under hydrated conditions), confirming the scaffold's suitability for ACL reconstruction. The absence of cytotoxic substances in the extracts of the SF-KSBC structure in culture medium was verified by in vitro tests with L929 fibroblasts. In terms of extracellular matrix production, Human Periodontal Ligament Fibroblasts (HPdLFs) cultured in direct contact with SF-KSBC, compared to control samples, demonstrated an increased secretion of aggrecan (PG) and fibronectin (FBN) at 3 and 7 days of culture, and no change in IL-6 and TNF-α secretion. Altogether, the outcomes of this investigation confirm the significant utility of this novel scaffold for ACL tissue regeneration.

Preparation and characterization of shape memory polymer scaffolds via solvent casting/particulate leaching.

PURPOSE: Porous Shape Memory Polymers (SMPs) are ideal candidates for the fabrication of defect fillers, able to support tissue regeneration via minimally invasive approaches. In this regard, control of pore size, shape and interconnection is required to achieve adequate nutrient transport and cell ingrowth. Here, we assessed the feasibility of the preparation of SMP porous structures and characterized their chemico-physical properties and in vitro cell response. METHODS: SMP scaffolds were obtained via solvent casting/particulate leaching of gelatin microspheres, prepared via oil/water emulsion. A solution of commercial polyether-urethane (MM-4520, Mitsubishi Heavy Industries) was cast on compacted microspheres and leached-off after polymer solvent evaporation. The obtained structures were characterized in terms of morphology (SEM and micro-CT), thermo-mechanical properties (DMTA), shape recovery behavior in compression mode, and in vitro cytocompatibility (MG63 Osteoblast-like cell line). RESULTS: The fabrication process enabled easy control of scaffold morphology, pore size, and pore shape by varying the gelatin microsphere morphology. Homogeneous spherical and interconnected pores have been achieved together with the preservation of shape memory ability, with recovery rate up to 90%. Regardless of pore dimensions, MG63 cells were observed adhering and spreading onto the inner surface of the scaffolds obtained for up to seven days of static in vitro tests. CONCLUSIONS: A new class of SMP porous structures has been obtained and tested in vitro: according to these preliminary results reported, SMP scaffolds can be further exploited in the design of a new class of implantable devices.

Effects of the magnetic resonance field on breast tissue expanders.

BACKGROUND: Tissue expansion for breast reconstruction after mastectomy is a safe and effective procedure. A magnetic resonance imaging (MRI) scan can be requested for patients with a breast expander to evaluate concurrent diseases. The electromagnetic field of the MR can interfere with biomedical devices, resulting in potential hazards, compromising the diagnosis, or creation of artifacts. METHODS: Four tissue expanders with an integrated magnetic valve were tested. The temperature increase was measured using an infrared camera in the MR scanner. The expanders were tested (half-full and full of saline solution) both free in air and immersed in a phantom. The ferromagnetic properties of the devices were assessed using the deflection angle method. To evidence artifacts due to the presence of the expander, MR images were acquired for expanders tested in air and in the phantom. A valve localization test was performed after MRI analysis. RESULTS: A slight increase in temperature was demonstrated, without any clinical significance. The deflection angle due to the magnetic field depends on the distance from the bore of the magnet. The angle is higher when the device is closer to the bore. The presence of the magnetic valve influences the MRI signal, creating artifacts on the acquired images, even far from the valve itself. The valve localization test allowed verification of correct valve functioning for all the expanders after the MRI analysis. CONCLUSIONS: Under selected conditions, MRI scans can be feasible. Heating is not expected to be a major concern, whereas valve displacement could happen in certain clinical conditions. The presence of artifacts is almost unavoidable. LEVEL OF EVIDENCE III: This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266.

Assessment of scaffold porosity: the new route of micro-CT.

A complete morphologic characterization of porous scaffolds for tissue engineering application is fundamental, as the architectural parameters, in particular porosity, strongly affect the mechanical and biological performance of the structures. Therefore, appropriate techniques for this purpose need to be selected. Several techniques for the assessment of scaffold porosity have been proposed, including Scanning Electron Microscopy observation, mercury and liquid extrusion porosimetry, gas pycnometry, and capillary flow porometry. Each of these techniques has several drawbacks and, a combination of different techniques is often required so as to achieve an in depth study of the morphologic properties of the scaffold. A single technique is often limited and suitable only for the assessment of a specific parameter. To overcome this limit, the most attractive option would be a single nondestructive technique, yet capable of providing a comprehensive set of data. It appears that micro-computed tomography (micro-CT) can potentially fulfill this role. Initially developed to characterize the 3D trabecular microarchitecture of bone, its use has been recently exploited by researchers for the morphologic characterization of porous biomaterials, as it enables obtaining a full assessment of the porous structures both in terms of pore size and interconnected porosity. This review aims to explore the use of micro-CT in scaffold characterization, comparing it with other previously developed techniques; we also focus on the contribution of this innovative tool to the development of scaffold-based tissue engineering application.