Controlled release of growth factors using synthetic glycosaminoglycans in a modular macroporous scaffold for tissue regeneration.

Healthy regeneration of tissue relies on a well-orchestrated release of growth factors. Herein, we show the use of synthetic glycosaminoglycans for controlled binding and release of growth factors to induce a desired cellular response. First, we screened glycosaminoglycans with growth factors of interest to determine kon (association rate constant), koff (dissociation rate constant), and Kd (equilibrium rate constant). As proof-of-concept, we functionalized an elastin-like recombinamer (ELR) hydrogel with a synthetic glycosaminoglycan and immobilized fibroblast growth factor 2 (FGF2), demonstrating that human umbilical vein endothelial cells cultured on top of ELR hydrogel differentiated into tube-like structures. Taking this concept further, we developed a tunable macroporous ELR cryogel material, containing a synthetic glycosaminoglycan and FGF2 that showed increased blood vessel formation and reduced immune response compared to control when implanted in a subcutaneous mouse model. These results demonstrated the possibility for specific release of desired growth factors in/from a modular 3D scaffold in vitro and in vivo.

Biohybrid elastin-like venous valve with potential for in situ tissue engineering.

Chronic venous insufficiency (CVI) is a leading vascular disease whose clinical manifestations include varicose veins, edemas, venous ulcers, and venous hypertension, among others. Therapies targeting this medical issue are scarce, and so far, no single venous valve prosthesis is clinically available. Herein, we have designed a bi-leaflet transcatheter venous valve that consists of (i) elastin-like recombinamers, (ii) a textile mesh reinforcement, and (iii) a bioabsorbable magnesium stent structure. Mechanical characterization of the resulting biohybrid elastin-like venous valves (EVV) showed an anisotropic behavior equivalent to the native bovine saphenous vein valves and mechanical strength suitable for vascular implantation. The EVV also featured minimal hemolysis and platelet adhesion, besides actively supporting endothelialization in vitro, thus setting the basis for its application as an in situ tissue engineering implant. In addition, the hydrodynamic testing in a pulsatile bioreactor demonstrated excellent hemodynamic valve performance, with minimal regurgitation (<10%) and pressure drop (<5 mmHg). No stagnation points were detected and an in vitro simulated transcatheter delivery showed the ability of the venous valve to withstand the implantation procedure. These results present a promising concept of a biohybrid transcatheter venous valve as an off-the-shelf implant, with great potential to provide clinical solutions for CVI treatment.

Impact of aromatic residues on the intrinsic disorder and transitional behaviour of model IDPs.

Understanding the interplay between order and disorder in intrinsically disorder proteins (IDPs), and its impact on the properties and features of materials manufactured from them, is a major challenge in the design of protein-based synthetic polymers intended for advanced functions. In this paper an elastin-like diblock co-recombinamer amphiphile (Phe-ELR) based on a hydrophobic block containing five phenylalanine (Phe) residues proximal to the carboxyl function of a glutamic acid (Glu) residue upon folding, and with Glu as the guest residue in the hydrophilic part, was engineered and its assembly behaviour compared with another amphiphilic ELR used as control. Phe-ELR was tailored in order to clarify the impact of the presence of aromatic residues in the amino acid sequence, which even in early studies by Urry's group already demonstrated a certain out-of-trend behaviour compared with other apolar amino acids, especially non-aromatic ones, on ELR behaviour. The combination of several experimental techniques indicates strong molecular interactions associated with the Phe residue, thus resulting in limited reversible character of the temperature-induced transitions during sequential thermal cycles, a lower than expected transition enthalpy, and clear differences in its supramolecular assembly with respect to the control ELR. A distinctive pre-aggregated state for the Phe-ELR under any condition of pH and temperature is found. Eventually, this state gives rise to Phe-core micelles or a solid jelly-like material, depending on the concentration, pH and presence of salts. In conclusion, it appears that the presence of aromatic residues and their ability to promote strong inter- and intramolecular interactions at any temperature and pH causes a complete modification of the order-disorder interplay present in other, non-aromatic ELRs. These molecular events have a profound impact on the physical properties of the resulting polymer when compared with other ELRs. This work helps to shed light on the limits that govern intrinsic disorder in ELRs beyond its inverse temperature transition.

Fast and reversible crosslinking of a silk elastin-like polymer.

Elastin-like polymers (ELPs) and their chimeric subfamily the silk elastin-like polymers (SELPs) exhibit a lower critical solvation temperature (LCST) behavior in water which has been extensively studied from theoretical, computational and experimental perspectives. The inclusion of silk domains in the backbone of the ELPs effects the molecular dynamics of the elastin-like domains in response to increased temperature above its transition temperature and confers gelation ability. This response has been studied in terms of initial and long-term changes in structures, however, intermediate transition states have been less investigated. Moreover, little is known about the effects of reversible hydration on the elastin versus silk domains in the physical crosslinks. We used spectroscopic techniques to analyze initial, intermediate and long-term states of the crosslinks in SELPs. A combination of thermoanalytical and rheological measurements demonstrated that the fast reversible rehydration of the elastin motifs adjacent to the relatively small silk domains was capable of breaking the silk physical crosslinks. This feature can be exploited to tailor the dynamics of these types of crosslinks in SELPs. STATEMENT OF SIGNIFICANCE: The combination of silk and elastin in a single molecule results in synergy via their interactions to impact the protein polymer properties. The ability of the silk domains to crosslink affects the thermoresponsive properties of the elastin domains. These interactions have been studied at early and late states of the physical crosslinking, while the intermediate states were the focus of the present study to understand the reversible phase-transitions of the elastin domains over the silk physical crosslinking. The thermoresponsive properties of the elastin domains at the initial, intermediate and late states of silk crosslinking were characterized to demonstrate that reversible hydration of the elastin domains influenced the reversibility of the silk crosslinks.

Topographically guided hierarchical mineralization.

Material platforms based on interaction between organic and inorganic phases offer enormous potential to develop materials that can recreate the structural and functional properties of biological systems. However, the capability of organic-mediated mineralizing strategies to guide mineralization with spatial control remains a major limitation. Here, we report on the integration of a protein-based mineralizing matrix with surface topographies to grow spatially guided mineralized structures. We reveal how well-defined geometrical spaces defined within the organic matrix by the surface topographies can trigger subtle changes in single nanocrystal co-alignment, which are then translated to drastic changes in mineralization at the microscale and macroscale. Furthermore, through systematic modifications of the surface topographies, we demonstrate the possibility of selectively guiding the growth of hierarchically mineralized structures. We foresee that the capacity to direct the anisotropic growth of such structures would have important implications in the design of biomineralizing synthetic materials to repair or regenerate hard tissues.

Antibiofilm coatings based on protein-engineered polymers and antimicrobial peptides for preventing implant-associated infections.

Implant-associated infections (IAIs) are one of the leading concerns in orthopedics and dentistry as they commonly lead to implant failure. The presence of biofilms and, increasingly frequently, drug-resistant bacteria further impairs the efficacy of conventional antibiotics. Immobilization of antimicrobial peptides (AMPs) on implant surfaces is a promising alternative to antibiotics for prevention of IAIs. In addition, the use of functional linkers for the AMP tethering enables to increase the antimicrobial potential and the bioactivities of the coating. In this study, an extracellular-matrix-mimicking system based on elastin-like recombinamers (ELRs) has been developed for the covalent anchoring of AMPs and investigated for use as a hybrid antibiofilm coating. A drip-flow biofilm reactor was used to simulate in vivo environmental dynamic conditions, thus showing that the presence of the AMPs in the hybrid coatings provided strong antibiofilm activity against monospecies and microcosm biofilm models of clinical relevance. These results, together with an excellent cytocompatibility towards primary gingival fibroblasts, encourage the use of ELRs as multivalent platforms for AMPs and open up a wide range of possibilities in the biofabrication of advanced coatings combining the antibiofilm potential of AMPs and the outstanding tunability and biomechanical properties of the ELRs.

Layer-by-layer modification effects on a nanopore's inner surface of polycarbonate track-etched membranes.

The control of the morphology, as well as the physical and chemical properties, of nanopores is a key issue for many applications. Reducing pore size is important in nanopore-based sensing applications as it helps to increase sensitivity. Changes of other physical properties such as surface net charge can also modify transport selectivity of the pores. We have studied how polyelectrolyte layer-by-layer (LBL) surface modification can be used to change the characteristics of nanoporous membranes. Studies were performed with a custom made three-dimensional multilayer microfluidic device able to fit membrane samples. The device allowed us to efficiently control LBL film deposition over blank low-cost commercially available polycarbonate track-etched (PCTE) membranes. We have demonstrated pore diameter reduction and deposition of the layers inside the pores through confocal and SEM images. Posterior impedance measurement studies served to evaluate experimentally the effect of the LBL deposition on the net inner nanopore surface charge and diameter. Measurements using direct current (DC) and alternative current (AC) voltages have demonstrated contrasted behaviors depending on the number and parity of deposited opposite charge layers. PCTE membranes are originally negatively charged and results evidenced higher impedance increases for paired charge LBL depositions. Impedance decreased when an unpaired positive layer was added. These results showed a different influence on the overall ion motility due to the effect of different surface charges. Results have been fit into a model that suggested a strong dependence of nanopores' impedance module to surface charge on conductive buffers, such as Phosphate Buffer Saline (PBS), even on relatively large nanopores. In AC significant differences between paired and unpaired charged LBL depositions tended to disappear as the total number of layers increased.

A double safety lock tumor-specific device for suicide gene therapy in breast cancer.

The complexity and continuous evolution of cancer make the design of novel strategies of treatment a constant challenge in biomedicine. Moreover, most of cancer treatments are still not tumor-specific and provoke high systemic toxicity. Herein we have developed a novel selective nanodevice to eliminate tumor cells while leaving healthy ones intact. To achieve this objective, a polyplex carrier, comprising an elastin like-recombinamer covalently conjugated to an aptamer and complexed with therapeutic DNA, was tested. This carrier forms a double-lock multifunctional device due to specific binding to a tumor cell marker and the selective expression of therapeutic DNA inside human breast-cancer cells. Due to the stability provided by ELRs, the homogeneous population of polyplexes obtained showed selective toxicity against cancer cells in in vitro and in vivo assay. Inhibition of tumor progression was detected early being very significant at the end point, with a dose-dependent reduction in tumor mass. Histological studies revealed a specific reduction in tumor parenchyma and in specific tumor cell markers. These results represent an important step toward the rational development of an efficient, safe and more specialized gene-delivery device for tumor therapy.

Self-Assembling ELR-Based Nanoparticles as Smart Drug-Delivery Systems Modulating Cellular Growth via Akt.

This work investigates the physicochemical properties and in vitro accuracy of a genetically engineered drug-delivery system based on elastin-like block recombinamers. The DNA recombinant techniques allowed us to create this smart complex polymer containing bioactive sequences for internalization, lysosome activation under acidic pH, and blockage of cellular growth by a small peptide inhibitor. The recombinant polymer reversibly self-assembled when the temperature was increased above 15 °C into nanoparticles with a diameter of 72 nm and negative surface charge. Furthermore, smart nanoparticles were shown to enter in the cells via clathrin-dependent endocytosis and properly blocked phosphorylation and consequent activation of Akt kinase. This system provoked apoptosis-mediated cell death in breast and colorectal cancer cells, which possess higher expression levels of Akt, whereas noncancerous cells, such as endothelial cells, fibroblasts, and mesenchymal stem cells, were not affected. Hence, we conclude that the conformational complexity of this smart elastin-like recombinamer leads to achieving successful drug delivery in targeted cells and could be a promising approach as nanocarriers with bioactive peptides to modulate multiple cellular processes involved in different diseases.

Use of proteolytic sequences with different cleavage kinetics as a way to generate hydrogels with preprogrammed cell-infiltration patterns imparted over their given 3D spatial structure.

Control over biodegradation processes is crucial to generate advanced functional structures with a more interactive and efficient role for biomedical applications. Herein, a simple, high-throughput approach is developed based on a three-dimensional (3D)-structured system that allows a preprogramed spatial-temporal control over cell infiltration and biodegradation. The 3D-structured system is based on elastin-like recombinamers (ELRs) characterized by differences in the kinetics of their peptide cleavage and consists of a three-layer hydrogel disk comprising an internal layer containing a rapidly degrading component, with the external layers containing a slow-degrading ELR. This structure is intended to invert the conventional pattern of cell infiltration, which goes from the outside to the inside of the implant, to allow an anti-natural process in which infiltration takes place first in the internal layer and later progresses to the outer layers. Time-course in vivo studies proved this hypothesis, i.e. that it is possible to drive the infiltration of cells over time in a given 3D-structured implant in a controlled and predesigned way that is able to overcome the natural tendency of conventional cell infiltration. The results obtained herein open up the possibility of applying this concept to more complex systems with multiple biological functions.

Tethering QK peptide to enhance angiogenesis in elastin-like recombinamer (ELR) hydrogels.

The development of new capillary networks in engineered constructs is essential for their survival and their integration with the host tissue. It has recently been demonstrated that ELR-based hydrogels encoding different bioactivities are able to modulate their interaction with the host after injection or implantation, as indicated by an increase in cell adhesion and the ability to trigger vascularization processes. Accordingly, the aim of this study was to increase their angiogenic ability both in vitro and in vivo using a small VEGF mimetic peptide named QK, which was tethered chemically to ELR-based hydrogels containing cell-adhesion sequences in their backbone, such as REDV and RGD, as well as a proteolytic site (VGVAPG). In vitro studies were performed using a co-culture of endothelial and fibroblast cells encapsulated into the ELR-based hydrogels in order to determine cell proliferation after 21 days of culture, as well as the number of cell-cell interactions. It was found that although the presence of this peptide does not influence the morphological and rheological properties of these hydrogels, it has an effect on cell behaviour, inducing an increase in cell proliferation and the formation of endothelial cell clusters. In vivo studies demonstrate that the QK peptide enhances the formation of prominent functional capillaries at three weeks post-injection, as confirmed by H&E staining and CD31 immunohistochemistry. The newly formed functional microvasculature ensures perfusion and connection with surrounding tissues. These results show that ELR-QK hydrogels increase capillary network formation and are therefore attractive candidates for application in tissue regeneration, for example for the treatment of cardiovascular diseases such as myocardial infarction or ischemia.

A transferrin receptor-binding mucoadhesive elastin-like recombinamer: In vitro and in vivo characterization.

The development of mucoadhesive materials is of great interest and is also a major challenge. Being adsorption sites, mucosae are suitable targets for drug delivery, but as defensive barriers they are complex biological surfaces to interact with, mainly due to their protective mucus layer. As such, first- and second-generation mucoadhesives focused on material-mucus interactions, whereas the third generation of mucoadhesives introduced structural motifs that are able to interact with the cells beneath the mucus layer. The combination of different prerequisites (water solubility, soft gel formation at body temperature and able to interact with the mucus) in a single molecule is easily achieved using elastin-like recombinamers (ELRs) given their multiple block design. Moreover, we have been able to introduce a short amino-acid sequence known as T7 that is able to bind to transferrin receptors in the epithelial cell layer. The T7 sequence enhances the cell-binding properties of the mucoadhesive ELR (MELR), as demonstrated using a Caco-2 epithelial cell model. In vivo experiments confirmed the mucoadhesive properties found in vitro. STATEMENT OF SIGNIFICANCE: The development of a mucoadhesive material is a major challenge. Mucosae are suitable targets for drug delivery, but as defense barriers, they are complex surfaces to interact with. In this work we report the first ELR that combines different functional blocks, in a single molecule, which provide it with the properties of soft-gel forming at body temperature and being able of efficiently adhering to the mucus layer of mucosas, as well as to the underlying epithelial cell layer, as demonstrated in vitro and in vivo. The rationally designed materials presented in this work sets the basis for developing ELR-based, mucosa-directed drug delivery systems, which could improve patient's compliance, enhancing drug retention at the mucosal site.

Silk-ELR co-recombinamer covered stents obtained by electrospinning.

In the field of tissue engineering the choice of materials is of great importance given the possibility to use biocompatible polymers produced by means of biotechnology. A large number of synthetic and natural materials have been used to this purpose and processed into scaffolds using Electrospinning technique. Among materials that could be used for the fabrication of scaffold and degradable membranes, natural polymers such as collagen, elastin or fibroin offer the possibility to design structures strictly similar to the extracellular matrix (ECM). Biotechnology and genetic engineering made possible the advent of a new class of biopolymers called protein-based polymers. One example is represented by the silk-elastin-proteins that combine the elasticity and resilience of elastin with the high tensile strength of silk-fibroin and display engineered bioactive sequences. In this work, we use electrospinning technique to produce a fibrous scaffold made of the co-recombinamer Silk-ELR. Obtained fibres have been characterized from the morphological point of view. Homogeneity and morphology have been explored using Scanning Electron Microscopy. A thorough study regarding the influence of Voltage, flow rate and distance have been carried out to determine the appropriate parameters to obtain the fibrous mats without defects and with a good distribution of diameters. Cytocompatibility has also been in vitro tested. For the first time we use the co-recombinamer Silk-ELR for the fabrication of a 2.5 angioplasty balloon coating. This structure could be useful as a coated scaffold for the regeneration of intima layer of vessels.

Self-assembly in elastin-like recombinamers: a mechanism to mimic natural complexity.

The topic of self-assembled structures based on elastin-like recombinamers (ELRs, i.e., elastin-like polymers recombinantly bio-produced) has released a noticeable amount of references in the last few years. Most of them are intended for biomedical applications. In this review, a complete revision of the bibliography is carried out. Initially, the self-assembly (SA) concept is considered from a general point of view, and then ELRs are described and characterized based on their intrinsic disorder. A classification of the different self-assembled ELR-based structures is proposed based on their morphologies, paying special attention to their tentative modeling. The impact of the mechanism of SA on these biomaterials is analyzed. Finally, the implications of ELR SA in biological systems are considered.

A novel lipase-catalyzed method for preparing ELR-based bioconjugates.

Herein we present a novel one-pot method for the chemical modification of elastin-like recombinamers (ELRs) in a mild and efficient manner involving enzymatic catalysis with Candida antarctica lipase B. The introduction of different functionalities into such ELRs could open up new possibilities for the development of advanced biomaterials for regenerative medicine and, specifically, for controlled drug delivery given their additional ability to respond to stimuli other than pH or temperature, such as glucose concentration or electromagnetic radiation. Candida antarctica lipase B immobilized on a macroporous acrylic resin (Novozym 435) was used to enzymatically couple different aminated substrates to a recombinamer containing carboxylic groups along its amino acid chain by way of an amidation reaction. A preliminary study of the kinetics of this amidation in response to different reaction conditions, such as solvent, temperature or reagent ratio, was carried out using a phenylazobenzene derivative (azo-NH2) as a model. The optimal amidation conditions were used to couple other amine reagents, such as phenylboronic acid (FB-NH2) or polyethylene glycol (PEG-NH2), thus allowing us to obtain photoresponsive, glucose-responsive or PEGylated ELRs that could potentially be useful as sensors in devices for controlled drug delivery.

Tuning the Stiffness of Surfaces by Assembling Genetically Engineered Polypeptides with Tailored Amino Acid Sequence.

We introduce elastin-like recombinamers (ELRs) as polypeptides with precise amino acid positioning to generate polypeptide coatings with tunable rigidity. Two ELRs are used: V84-ELR, a hydrophobic monoblock, and EI-ELR, an amphiphilic diblock. Both were modified with the amine-reactive tetrakis (hydroxymethyl) phosphonium chloride compound. We evaluated the affinity, conformation, and dissipative behavior of ELRs assembled on alkanethiol self-assembled coatings by quartz crystal microbalance with dissipation monitoring, multiparametric surface plasmon resonance, and atomic force microscopy. The thickness of the polypeptide coatings showcases the preferential affinity of ELRs to NH2- and CH3-terminated surfaces. We demonstrate that V84-ELR strongly bonded to the substrate and reorganizes into an extended and more hydrated layer as the adsorbed amount increases, whereas EI-ELR has a less dissipative behavior. The results suggest that ELR adsorption depends on the amino acid sequence and the substrate chemistry, ultimately influencing the stiffness of the polypeptide coatings.

Production of bioactive hepcidin by recombinant DNA tagging with an elastin-like recombinamer.

With the lack of new chemical antibiotics and increasing pathogen resistance to those available, new alternatives are being explored. Antimicrobial peptides (AMPs) with a broad range of effects, including antibacterial, antifungal, and antiviral actions, have emerged as one of the options. They can be produced by recombinant DNA technology, but the chromatographic methods used for peptide purification are expensive and time consuming. Here, we describe the design, production, purification and assessment of the antibacterial activity of the human peptide hepcidin, using an elastin-like recombinamer as fusion partner. The recombinant protein Hep-A200 was produced in Escherichia coli and purified by a non-chromatographic procedure, exploiting the thermal properties of the A200 elastin-like recombinamer. Recombinant Hep-A200 was found to retain antibacterial activity against Gram-positive and Gram-negative species.

Macroporous click-elastin-like hydrogels for tissue engineering applications.

Elastin is a key extracellular matrix (ECM) protein that imparts functional elasticity to tissues and therefore an attractive candidate for bioengineering materials. Genetically engineered elastin-like recombinamers (ELRs) maintain inherent properties of the natural elastin (e.g. elastic behavior, bioactivity, low thrombogenicity, inverse temperature transition) while featuring precisely controlled composition, the possibility for biofunctionalization and non-animal origin. Recently the chemical modification of ELRs to enable their crosslinking via a catalyst-free click chemistry reaction, has further widened their applicability for tissue engineering. Despite these outstanding properties, the generation of macroporous click-ELR scaffolds with controlled, interconnected porosity has remained elusive so far. This significantly limits the potential of these materials as the porosity has a crucial role on cell infiltration, proliferation and ECM formation. In this study we propose a strategy to overcome this issue by adapting the salt leaching/gas foaming technique to click-ELRs. As result, macroporous hydrogels with tuned pore size and mechanical properties in the range of many native tissues were reproducibly obtained as demonstrated by rheological measurements and quantitative analysis of fluorescence, scanning electron and two-photon microscopy images. Additionally, the appropriate size and interconnectivity of the pores enabled smooth muscle cells to migrate into the click-ELR scaffolds and deposit extracellular matrix. The macroporous structure together with the elastic performance and bioactive character of ELRs, the specificity and non-toxic character of the catalyst-free click-chemistry reaction, make these scaffolds promising candidates for applications in tissue regeneration. This work expands the potential use of ELRs and click chemistry systems in general in different biomedical fields.

Bioactive scaffolds based on elastin-like materials for wound healing.

Wound healing is a complex process that, in healthy tissues, starts immediately after the injury. Even though it is a natural well-orchestrated process, large trauma wounds, or injuries caused by acids or other chemicals, usually produce a non-elastic deformed tissue that not only have biological reduced properties but a clear aesthetic effect. One of the main drawbacks of the scaffolds used for wound dressing is the lack of elasticity, driving to non-elastic and contracted tissues. In the last decades, elastin based materials have gained in importance as biomaterials for tissue engineering applications due to their good cyto- and bio-compatibility, their ease handling and design, production and modification. Synthetic elastin or elastin like-peptides (ELPs) are the two main families of biomaterials that try to mimic the outstanding properties of natural elastin, elasticity amongst others; although there are no in vivo studies that clearly support that these two families of elastin based materials improve the elasticity of the artificial scaffolds and of the regenerated skin. Within the next pages a review of the different forms (coacervates, fibres, hydrogels and biofunctionalized surfaces) in which these two families of biomaterials can be processed to be applied in the wound healing field have been done. Here, we explore the mechanical and biological properties of these scaffolds as well as the different in vivo approaches in which these scaffolds have been used.

Biocompatibility and immunogenicity of elastin-like recombinamer biomaterials in mouse models.

Novel thermo-sensitive elastin-like recombinamers (ELRs) containing bioactive molecules were created for use as a biomimetic biomaterial for tissue regeneration. For effective use for in vivo applications, it is essential to ensure that they do not induce adverse inflammatory, immune, or allergic responses that inhibit tissue repair. Therefore, we sought to establish a pre-clinical approach to evaluate biocompatibility in experimental mice using ELRs as a prototype biomaterial. First, we measured in vitro proliferation and cytokine production from BALB/c and C57BL/6 mouse splenocytes incubated with ELRs. Second, we used a rapid, high throughput in vivo approach in which inflammatory cells and cytokines were measured following an intraperitoneal implantation. Lastly, a subchronic in vivo approach was used in which ELRs or positive controls were subcutaneously implanted and the implantation sites were assessed for inflammation and gene expression. We found that ELRs induced mild inflammation and minimal fibrosis compared to the intense response to Vitoss. Additionally, implantation increased antigen-specific antibody titers for both groups and gene expression profiling of the implantation sites revealed the upregulation of inflammation, fibrosis, and wound healing-related genes in ELR and positive control-implanted mice compared to sham controls. These data demonstrate that ELRs appear safe for use in tissue engineering. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 924-934, 2018.