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.

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.

Biomaterial design in tissue engineering aims to identify appropriate cellular microenvironments in which cells can grow and guide new tissue formation. Despite the large diversity of synthetic polymers available for regenerative medicine, most of them fail to fully match the functional properties of their native counterparts. In contrast, the few biological alternatives employed as biomaterials lack the versatility that chemical synthesis can offer. Herein, we studied the HUVEC adhesion and proliferation properties of elastin-like recombinamers (ELRs) that were covalently functionalized with each three high-affinity and selectivity α _v β ₃- and α ₅ β ₁-binding bicyclic RGD peptides. Next to the bicycles, ELRs were also functionalized with various integrin-binding benchmark peptides, i.e. knottin-RGD, cyclo-[KRGDf] and GRGDS, allowing for better classification of the obtained results. Covalent functionalization with the RGD peptides, as validated by MALDI-TOF analysis, guarantees flexibility and minimal steric hindrance for interactions with cellular integrins. In addition to the covalently modified RGD-ELRs, we also synthesized another benchmark ELR comprising RGD as part of the backbone. HUVEC adhesion and proliferation analysis using the PicoGreen® assay revealed a higher short-term adhesion and proliferative capacity of cells on ELR surfaces functionalized with high affinity, integrin-binding bicyclic RGD-peptides compared with the ELRs containing RGD in the backbone.

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.

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.

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.

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.

Herein we present a system to obtain fibers from clickable elastin-like recombinamers (ELRs) that crosslink in situ during the electrospinning process itself, with no need for any further treatment to stabilize them. These ELR-click fibers are completely stable under in vitro conditions. A wrinkled fiber morphology is obtained. In addition to a random fiber orientation, oriented fibers with a high degree of alignment and coherence can also be obtained by using a rotational electrode. The production of multicomponent fibers means that different functionalities, such as cell-adhesion domains (RGD peptides), can be incorporated into them. In a subsequent study, two main cell lines present in the dermis and epidermis, namely keratinocytes and fibroblasts, were cultured on top of the ELR-click fibers. Adhesion, proliferation, fluorescence, immunostaining and histology studies showed the cytocompatibility of these scaffolds, thus suggesting their possible use for wound dressings in skin tissue engineering applications.

STATEMENT OF SIGNIFICANCE:

For the first time stable electrospun bioactive fibers are obtained by the in situ mixing of two "clickable" ELR components previously described by Gonzalez et al (Acta Biomaterialia 2014). This work describes an efficient system to prepare fibrous scaffolds based on peptidic polymers by electrospinning without the need of crosslinking agents that could be harmful for cells or living tissues. These bioactive fibers support cell growth due to the inclusion of RGD motifs (Staubli et al. Biomaterials 2017). Finally, the in vitro biocompatibility of the two main cell types found in the outer layers of skin, fibroblasts and keratinocytes, indicates that this system is of great interest to prepare elastic artificial skin substitutes for wound healing applications.

Biocompatibility studies, especially innate immunity induction, in vitro and in vivo cytotoxicity, and fibrosis, are often lacking for many novel biomaterials including recombinant protein-based ones, such as elastin-like recombinamers (ELRs), and has not been extensively explored in the scientific literature, in contrast to traditional biomaterials. Herein, we present the results from a set of experiments designed to elucidate the preliminary biocompatibility of 2 types of ELRs that are able to form extracellular matrix-like hydrogels through either physical or chemical cross-linking both of which are intended for different applications in tissue engineering and regenerative medicine. Initially, we present in vitro cytocompatibility results obtained upon culturing human umbilical vein endothelial cells on ELR substrates, showing optimal proliferation up to 9 days. Regarding in vivo cytocompatibility, luciferase-expressing hMSCs were viable for at least 4 weeks in terms of bioluminescence emission when embedded in ELR hydrogels and injected subcutaneously into immunosuppressed mice. Furthermore, both types of ELR-based hydrogels were injected subcutaneously in immunocompetent mice and serum TNFα, IL-1β, IL-4, IL-6, and IL-10 concentrations were measured by enzyme-linked immunosorbent assay, confirming the lack of inflammatory response, as also observed upon macroscopic and histological evaluation. All these findings suggest that both types of ELRs possess broad biocompatibility, thus making them very promising for tissue engineering and regenerative medicine-related applications.

Macroencapsulation devices provide the dual possibility of immunoprotecting transplanted cells while also being retrievable, the latter bearing importance for safety in future trials with stem cell–derived cells. However, macroencapsulation entails a problem with oxygen supply to the encapsulated cells. The βAir device solves this with an incorporated refillable oxygen tank. This phase 1 study evaluated the safety and efficacy of implanting the βAir device containing allogeneic human pancreatic islets into patients with type 1 diabetes. Four patients were transplanted with 1‐2 βAir devices, each containing 155 000‐180 000 islet equivalents (ie, 1800‐4600 islet equivalents per kg body weight), and monitored for 3‐6 months, followed by the recovery of devices. Implantation of the βAir device was safe and successfully prevented immunization and rejection of the transplanted tissue. However, although beta cells survived in the device, only minute levels of circulating C‐peptide were observed with no impact on metabolic control. Fibrotic tissue with immune cells was formed in capsule surroundings. Recovered devices displayed a blunted glucose‐stimulated insulin response, and amyloid formation in the endocrine tissue. We conclude that the βAir device is safe and can support survival of allogeneic islets for several months, although the function of the transplanted cells was limited (Clinicaltrials.gov: NCT02064309).

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.

Tissue engineering for cartilage repair requires biomaterials that show rapid gelation and adequate mechanical properties. Although the use of hydrogel is the most promising biomaterial, it often lacks in rigidity and anchorage of cells when they are surrounded by synovial fluid while they are subjected to heavy loads. We developed and produced the Silk Elastin-Like co-Recombinamer (SELR), which contains both the physical interaction from elastin motifs and from silk motifs. In the first part of this work, we set up and optimized a preannealing treatment based on the evolution of silk motifs into β-sheet structures in order to fulfill the required mechanical properties of hydrogels for cartilage repair. The new preannealed SELRs (pA(EIS)2-(I5R)6) were characterized with the combination of several experimental techniques (CD, TEM, SEM, and rheology) to provide a deep insight into the material features. Finally, the regeneration properties of the pA(EIS)2-(I5R)6hydrogel embedded with chondrocytes were evaluated. After 4 weeks of culturing in a standardized and representative ex vivo model, the biochemical and histological analysis revealed the production of glycosaminglycans and collagen. Moreover, the immunohistochemistry showed the absence of fibro-cartilage and the presence of hyaline cartilage. Hence, we conclude that the pA(EIS)2-(I5R)6 hydrogel presents improved mechanical properties while conserving the injectability, which leads to successful regeneration of hyaline cartilage in an ex vivo model.

A simple system for cell selectivity and spatially controlled cell adhesion has been developed using model gold surfaces grafted with a combination of two ELRs containing into their backbone cell-adhesion domains such as RGD and REDV. Grafting onto gold was achieved via redox reaction through thiol groups present in amino terminal cysteine tails of the ELRs. The correlation among contact angle, SEM micrographs, AFM, XPS and QCM-D have been carried out.
After in-depth adhesion studies, a mixture of 75% ELR-REDV and 25% ELR-RGD was found to exhibit high selectivity for endothelial cells, promoting strong adhesion thereof.
Consequently, certain areas of gold surfaces (strips) were cleaned by laser ablation and functionalized with the mixture 75% ELR-REDV – 25% ELR-RGD leading to a spatial segregation of the co-culture made of HUVEC and HFF1 cells. This platform therefore exhibits selective spatial control over cell adhesion associated with the bioactive epitopes (RGD and REDV) contained in the ELR sequence, since each functionalized surface (including strips) have similar topographic and hydrophobic characteristics and mechanical properties are in the same order of magnitude.

A bone tissue replacement with relevant anatomical size requires the production of 3D scaffolds, which in turn limits the mass transport of nutrients and oxygen to sustain cell survival. A viable vascular network is required to overcome this problem. However, this cannot be established immediately after the implantation of a scaffold. The aim of this study was to develop a 3D wet-spun bone tissue engineering scaffold, coated with an elastin-like recombinamer (ELR) peptide with an endothelial cell-attracting REDV sequence to promote early vascularization. Scaffolds were produced using biodegradable poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), and an ELR was immobilized onto it after oxygen plasma treatment (PHBV-O2-ELR-REDV). O2 plasma treatment and ELR modification of the PHBV changed the wettability, topography, and composition of the surface. A moderately hydrophilic surface was obtained after oxygen plasma treatment and ELR-REDV coating with a contact angle of 66.63 ± 0.77°. The surface roughness decreased after plasma treatment from 343.4 to 160.0 nm and increased to 280.3 nm after ELR-REDV coating. FTIR-ATR showed amide I and amide II bonds after ELR-REDV coating showing that the coating was successful. Scaffolds were tested in vitro with rabbit bone marrow mesenchymal cells. ELR modification did not cause a significant difference in adhesion or proliferation compared to unmodified controls. On the other hand, ELR-modified scaffolds attracted a higher number of human umbilical vein endothelial cells (HUVECs) due to the REDV sequence. The Alamar Blue test and confocal laser scanning microscopy micrographs showed that HUVEC migration and attachment on PHBV-O2-ELR-REDV scaffolds was around 2.5-fold higher than untreated PHBV scaffolds after 14 d. Plasma-treated scaffolds (PHBV-O2) showed an increase in the number of adhered HUVECs due to increased surface wettability. It can, therefore, be suggested that PHBV-O2-ELR-REDV scaffolds have significant potential to induce early vascularization due to increased attractiveness for endothelial cells. This could alleviate the vascularization problem of 3D implants for bone tissue engineering.

A major goal in materials science is to develop bioinspired functional materials based on the precise control of molecular building blocks across length scales. Here we report a protein-mediated mineralization process that takes advantage of disorder-order interplay using elastin-like recombinamers to program organic-inorganic interactions into hierarchically ordered mineralized structures. The materials comprise elongated apatite nanocrystals that are aligned and organized into microscopic prisms, which grow together into spherulite-like structures hundreds of micrometers in diameter that come together to fill macroscopic areas. The structures can be grown over large uneven surfaces and native tissues as acid-resistant membranes or coatings with tuneable hierarchy, stiffness, and hardness. Our study represents a potential strategy for complex materials design that may open opportunities for hard tissue repair and provide insights into the role of molecular disorder in human physiology and pathology.

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.

In the last decades, recombinant structural proteins have become very promising in addressing different issues such as the lack of traceability of biomedical devices or the design of more sensitive biosensors. Among them, we find elastin-like recombinamers (ELRs), which can be designed to self-assemble into diverse structures, such as hydrogels. Furthermore, they might be combined with other protein polymers, such as silk, to give silk-elastin-like recombinamers (SELRs), holding the properties of both proteins. In this work, due to their recombinant nature, we have fused two different fluorescent proteins (FPs), i.e., the green Aequorea coerulescensenhanced green fluorescent protein and the near-infrared eqFP650, to a SELR able to form irreversible hydrogels through physical cross-linking. These recombinamers showed an emission of fluorescence similar to the single FPs, and they were capable of forming hydrogels with different stiffness (G′ = 60–4000 Pa) by varying the concentration of the SELR-FPs. Moreover, the absorption spectrum of SELR-eqFP650 showed a peak greatly overlapping the emission spectrum of the SELR-Aequorea coerulescens enhanced green fluorescent protein. Hence, this enables Förster resonance energy transfer (FRET) upon the interaction between two SELR molecules, each one containing a different FP, due to the stacking of silk domains at any temperature and to the aggregation of elastin-like blocks above the transition temperature. This effect was studied by different methods, and a FRET efficiency of 0.06–0.2 was observed, depending on the technique used for its calculation. Therefore, innovative biological applications arise from the combination of SELRs with FPs, such as enhancing the traceability of hydrogels based on SELRs intended for tissue engineering, the development of biosensors, and the prediction of FRET efficiencies of novel FRET pairs.

The control of the in vivo vascularization of engineered tissue substitutes is essential in order to obtain either a rapid induction or a complete inhibition of the process (e.g. in muscles and hyaline-cartilage, respectively). Among the several polymers available, Elastin-Like Recombinamers (ELR)-based hydrogel stands out as a promising material for tissue engineering thanks to its viscoelastic properties, non-toxicity, and non-immunogenicity. In this study, we hypothesized that varying the cell adhesion properties of ELR-hydrogels could modulate the high angiogenic potential of adipose tissue-derived stromal vascular fraction (SVF) cells, predominantly composed of endothelial/mural and mesenchymal cells. Human SVF cells, embedded in RGD-REDV-bioactivated or unmodified ELR-hydrogels, were implanted in rat subcutaneous pockets either immediately or upon 5-day-culture in perfusion-bioreactors. Perfusion-based culture enhanced the endothelial cell cord-like-organization and the release of pro-angiogenic factors in functionalized constructs. While in vivo vascularization and host cell infiltration within the bioactivated gels were highly enhanced, the two processes were strongly inhibited in non-functionalized SVF-based hydrogels up to 28 days. ELR-based hydrogels showed a great potential to determine the successful integration of engineered substitutes thanks to their capacity to finely control the angiogenic/inflammation process at the recipient site, even in presence of SVF cells.

Stimuli-responsive polymers are capable of changing their physico-chemical properties in a dynamic way, to respond to variations on the surrounding environment. These materials have gained increasingly importance for different areas, such as drug delivery, biosensors, microelectronic systems and also for the design and modification of biomaterials to apply on tissue engineering field. In the last years, different strategies have been envisaged for the development of stimuli-responsive biomaterials. Layer-by-layer (LbL) is a promising and versatile technique to modify biomaterials' surfaces, and has allowed tailoring interactions with cells. In this study, LbL is used to construct biomimetic stimuli-responsive coatings using elastin-like recombinamers (ELRs). The recombinant nature of ELRs provides the ability to introduce specific bioactive sequences and to tune their physicochemical properties, making them attractive for biomedical and biological applications. By using complementary clickable ELRs, we were able to construct multilayer coatings stabilized by covalent bonds, resulting from the Huisgen 1,3-dipolar cycloaddition of azides and alkynes. Herein, we exploited the switchable properties of the ELRs-based coatings which are dependent on lower critical solution temperature (LCST) transition. Above LCST, the polymers collapsed and nanostructured precipitates were observed on the surface's morphology, increasing the water contact angle. Also, the influence of pH on prompting reversible responses on coatings was evaluated. Finally, in vitro cell studies using a C2C12 myoblastic cell line were performed to perceive the importance of having bioactive domains within these coatings. The effect of RGD incorporation is clearly noted not only in terms of adhesion and proliferation but also in terms of myoblast differentiation.

A logical cure for type 1 diabetes (T1D) involves replacing the lost insulin-producing cells with new ones, preferably cells from a well-characterized and unlimited source of human insulin-producing cells. This straightforward and simple solution to provide a cure for T1D is immensely attractive but entails at least two inherent and thus far unresolved hurdles: 1) provision of an unlimited source of functional human insulin-producing cells and 2) prevention of rejection without the side effects of systemic immunosuppression. Generation of transplantable insulin-producing cells from human embryonic stem cells or induced pluripotent stem cells is at present close to reality, and we are currently awaiting the first clinical studies. Focus is now directed to foster development of novel means to control the immune system to enable large-scale clinical application. Encapsulation introduces a physical barrier that prevents access of immune cells to the transplanted cells but also hinders blood vessel ingrowth. Therefore, oxygen, nutrient, and hormonal passage over the encapsulation membrane is solely dependent on diffusion over the immune barrier, contributing to delays in glucose sensing and insulin secretion kinetics. This Perspective focuses on the physiological possibilities and limitations of an encapsulation strategy to establish near-normoglycemia in subjects with T1D, assuming that glucose-responsive insulin-producing cells are available for transplantation.

Bone regeneration is a complex process requiring highly orchestrated interactions between different cells and signals to form new mineralized tissue. Blood vessels serve as a structural template, around which bone development takes place, and also bring together the key elements for bone homeostasis into the osteogenic microenvironment, including minerals, growth factors and osteogenic progenitor cells. Vascular endothelial growth factor (VEGF) is the master regulator of vascular growth and it is required for effective coupling of angiogenesis and osteogenesis during both skeletal development and postnatal bone repair. Here, we will review the current state of knowledge on the molecular cross-talk between angiogenesis and osteogenesis. In particular, we will focus on the role of VEGF in coupling these two processes and how VEGF dose can control the outcome, addressing in particular: (1) the direct influence of VEGF on osteogenic differentiation of mesenchymal progenitors; (2) the angiocrine functions of endothelium to regulate osteoprogenitors; (3) the role of immune cells, e.g., myeloid cells and osteoclast precursors, recruited by VEGF to the osteogenic microenvironment. Finally, we will discuss emerging strategies, based on the current biological understanding, to ensure rapid vascularization and efficient bone formation in regenerative medicine.

Biomineralization of bone, a controlled process where hydroxyapatite nanocrystals preferentially deposit in collagen fibrils, is achieved by the interplay of the collagen matrix and non-collagenous proteins.Mimicking intrafibrillar mineralization in synthetic systems is highly attractive for the development of advanced hybrid materials with elaborated morphologies and outstanding mechanical properties, as well as understanding the mechanisms of biomineralization. Inspired by nature, intrafibrillar mineralization of collagen fibrils has been successfully replicated in vitro via biomimetic systems where acidic polymeric additives are used as analog of non-collagenous proteins in mediating mineralization. The developmentof synthetic templates that mimic the structure and functions of collagenous matrix in mineralization has yet to be explored.   In this study, we demonstrated that self-assembled fibrils of elastin-like recombinamers (ELRs) can induce intrafibrillar mineralization. The ELRs displayed a disordered structure at low temperature but self-assembled into nanofibrils above its inverse transition temperature. In the presence of the self-assembled ELR fibrils, polyaspartate-stabilized amorphous calcium phosphates ]preferentially infiltrated into the fibrils and then crystalized into hydroxyapatite crystals with their [001] axes aligned parallel to the long axis of the ELR fibril. As the recombinant technology enables designing and producing well-defined ELRs, their molecular and structural properties can be fine-tuned. By examining the ultrastructure of the self-assembled ELRs fibrils as well as their mineralization, weconcluded that the spatial confinement formed by a continuum β-spiral structure in an unperturbed fibrillar structure rather than electrostatic interactions or bioactive sequences in the recombinamer composition played the crucial role in inducing intrafibrillar mineralization.

The main clinical problems for dental implants are (1) formation of biofilm around the implant—a condition known as peri-implantitis and (2) inadequate bone formation around the implant—lack of osseointegration. Therefore, developing an implant to overcome these problems is of significant interest to the dental community. Chitosan has been reported to have good biocompatibility and anti-bacterial activity. An osseo-inductive recombinant elastin-like biopolymer (P-HAP), that contains a peptide derived from the protein statherin, has been reported to induce biomineralization and osteoblast differentiation. In this study, chitosan/P-HAP bi-layers were built on a titanium surface using a layer-by-layer (LbL) assembly technique. The difference in the water contact angle between consecutive layers, the representative peaks in diffuse reflectance infrared Fourier transform spectroscopy (DRIFTS), X-ray photoelectron spectroscopy (XPS), and the changes in the topography between surfaces with a different number of bi-layers observed using atomic force microscopy (AFM), all indicated the successful establishment of chitosan/P-HAP LbL assembly on the titanium surface. The LbL-modified surfaces showed increased biomineralization, an appropriate mouse pre-osteoblastic cell response, and significant anti-bacterial activity against Streptococcus gordonii, a primary colonizer of tissues in the oral environment.

The morbidity of bone fractures and defects is steadily increasing due to changes in the age pyramid. As such, novel biomaterials that are able to promote the healing and regeneration of injured bones are needed to overcome the limitations of auto-, allo-, and xenografts, while providing a ready-to-use product that may help to minimize surgical invasiveness and duration. In this regard, recombinant biomaterials, such as elastin-like recombinamers (ELRs), are very promising as their design can be tailored by genetic engineering, thus allowing scalable production and batch-to-batch consistency, among others. Furthermore, they can self-assemble into physically crosslinked hydrogels above a certain transition temperature, in this case body temperature, but are injectable below this temperature, thereby markedly reducing surgical invasiveness. In this study, we have developed two bioactive hydrogel-forming ELRs, one including the osteogenic and osteoinductive bone morphogenetic protein-2 (BMP-2) and the other the Arg-Gly-Asp (RGD) cell adhesion motif. The combination of these two novel ELRs results in a BMP-2-loaded extracellular matrix-like hydrogel. Moreover, elastase-sensitive domains were included in both ELR molecules, thereby conferring biodegradation as a result of enzymatic cleavage and avoiding the need for scaffold removal after bone regeneration. Both ELRs and their combination showed excellent cytocompatibility, and the culture of cells on RGD-containing ELRs resulted in optimal cell adhesion. In addition, hydrogels based on a mixture of both ELRs were implanted in a pilot study involving a femoral bone injury model in New Zealand white rabbits, showing complete regeneration in six out of seven cases, with the other showing partial closure of the defect. Moreover, bone neoformation was confirmed using different techniques, such as radiography, computed tomography, and histology. This hydrogel system therefore displays significant potential in the regeneration of bone defects, promoting self-regeneration by the surrounding tissue with no involvement of stem cells or osteogenic factors other than BMP-2, which is released in a controlled manner by elastase-mediated cleavage from the ELR backbone.

Understanding the mechanisms responsible for generating different phases and morphologies of calcium phosphate by elastin-like recombinamers is supreme for bioengineering of advanced multifunctional materials. The generation of such multifunctional hybrid materials depends on the properties of their counterparts and the way in which they are assembled. The success of this assembly depends on the different approaches used, such as recombinant DNA technology and click chemistry. In the present work, an elastin-like recombinamer bearing lysine amino acids distributed along the recombinamer chain has been cross-linked via Huisgen [2 + 3] cycloaddition. The recombinamer contains the SN_A15 peptide domains inspired by salivary statherin, a peptide epitope known to specifically bind to and nucleate calcium phosphate. The benefit of using click chemistry is that the hybrid elastin-like-statherin recombinamers cross-link without losing their fibrillar structure. Mineralization of the resulting hybrid elastin-like-statherin recombinamer hydrogels with calcium phosphate is described. Thus, two different hydroxyapatite morphologies (cauliflower- and plate-like) have been formed. Overall, this study shows that crosslinking elastin-like recombinamers leads to interesting matrix materials for the generation of calcium phosphate composites with potential applications as biomaterials.

The ultrastructure of the bone provides a unique mechanical strength against compressive, torsional and tensional stresses. An elastin-like recombinamer (ELR) with a nucleation sequence for hydroxyapatite was incorporated into films prepared from a collagen – silk fibroin blend carrying microchannel patterns to stimulate anisotropic osteogenesis. SEM and fluorescence microscopy showed the alignment of adipose-derived stem cells (ADSCs) and the human osteoblasts (HOBs) on the ridges and in the grooves of microchannel patterned collagen-fibroin-ELR blend films. The Young's modulus and the ultimate tensile strength (UTS) of untreated films were 0.58 ± 0.13 MPa and 0.18 ± 0.05 MPa, respectively. After 28 days of cell culture, ADSC seeded film had a Young's modulus of 1.21 ± 0.42 MPa and UTS of 0.32 ± 0.15 MPa which were about 3 fold higher than HOB seeded films. The difference in Young's modulus was statistically significant (p: 0.02). ADSCs attached, proliferated and mineralized better than the HOBs. In the light of these results, ADSCs served as a better cell source than HOBs for bone tissue engineering of collagen-fibroin-ELR based constructs used in this study. We have thus shown the enhancement in the tensile mechanical properties of the bone tissue engineered scaffolds by using ADSCs.

The ability to guide molecular self-assembly at the nanoscale into complex macroscopic structures could enable the development of functional synthetic materials that exhibit properties of natural tissues such as hierarchy, adaptability, and self-healing. However, the stability and structural integrity of these kinds of materials remains a challenge for many practical applications. We have recently developed a dynamic biopolymer-peptide co-assembly system with the capacity to grow and undergo morphogenesis into complex shapes. Here we explored the potential of different synthetic (succinimidyl carboxymethyl ester, poly (ethylene glycol) ether tetrasuccinimidyl glutarate and glutaraldehyde) and natural (genipin) cross-linking agents to stabilize membranes made from these biopolymer-peptide co-assemblies. We investigated the cross-linking efficiency, resistance to enzymatic degradation, and mechanical properties of the different cross-linked membranes. We also compared their biocompatibility by assessing the metabolic activity and morphology of adipose-derived stem cells (ADSC) cultured on the different membranes. While all cross-linkers successfully stabilized the system under physiological conditions, membranes cross-linked with genipin exhibited better resistance in physiological environments, improved stability under enzymatic degradation, and a higher degree of in vitro cytocompatibility compared to the other cross-linking agents. The results demonstrated that genipin is an attractive candidate to provide functional structural stability to complex self-assembling structures for potential tissue engineering or in vitro model applications.

Statement of Significance

 Molecular self-assembly is widely used for the fabrication of complex functional biomaterials to replace and/or repair any tissue or organ in the body. However, maintaining the stability and corresponding functionality of these kinds of materials in physiological conditions remains a challenge. Chemical cross-linking strategies (natural or synthetic) have been used in an effort to improve their structural integrity. Here we investigate key performance parameters of different cross-linking strategies for stabilising self-assembled materials with potential biomedical applications using a novel protein-peptide co-assembling membrane as proof-of-concept. From the different cross-linkers tested, the natural cross-linker genipin exhibited the best performance. This cross-linker successfully enhanced the mechanical properties of the system enabling the maintenance of the structure in physiological conditions without compromising its bioactivity and biocompatibility. Altogether, we provide a systematic characterization of cross-linking alternatives for self-assembling materials focused on biocompatibility and stability and demonstrate that genipin is a promising alternative for the cross-linking of such materials with a wide variety of potential applications such as in tissue engineering and drug delivery.

Over the last decades, novel therapeutic tools for osteochondral regeneration have arisen from the combination of mesenchymal stromal cells (MSCs) and highly specialized smart biomaterials, such as hydrogel-forming elastin-like recombinamers (ELRs), which could serve as cell-carriers. Herein, we evaluate the delivery of xenogeneic human MSCs (hMSCs) within an injectable ELR-based hydrogel carrier for osteochondral regeneration in rabbits. First, a critical-size osteochondral defect was created in the femora of the animals and subsequently filled with the ELR-based hydrogel alone or with embedded hMSCs. Regeneration outcomes were evaluated after three months by gross assessment, magnetic resonance imaging and computed tomography, showing complete filling of the defect and the de novo formation of hyaline-like cartilage and subchondral bone in the hMSC-treated knees. Furthermore, histological sectioning and staining of every sample confirmed regeneration of the full cartilage thickness and early subchondral bone repair, which was more similar to the native cartilage in the case of the cell-loaded ELR-based hydrogel. Overall histological differences between the two groups were assessed semi-quantitatively using the Wakitani scale and found to be statistically significant (p < 0.05). Immunofluorescence against a human mitochondrial antibody three months post-implantation showed that the hMSCs were integrated into the de novo formed tissue, thus suggesting their ability to overcome the interspecies barrier. Hence, we conclude that the use of xenogeneic MSCs embedded in an ELR-based hydrogel leads to the successful regeneration of hyaline cartilage in osteochondral lesions.

Osteoporosis, a systemic skeletal disorder, occurs when bone turnover balance is disrupted. With the identification of the genes involved in the pathogenesis of the disease, studies on development of new treatments has intensified. Short interfering RNA (siRNA) is used to knockdown disease related gene expressions. Targeting siRNA in vivo is challenging. The maintenance of therapeutic plasma level is hampered by clearance of siRNA from the body. Targeted systems are useful in increasing the drug concentration at the target site and decreasing side effects. Aim of the present study was to develop an injectable siRNA delivery system to protect siRNA during systemic distribution and target the siRNA to bone tissue using a thermoresponsive, genetically engineered, elastin-like recombinamer (ELR), designed to interact with the mineral component of bone. The delivery system consisted of DNAoligo as a siRNA substitute complexed with the cationic polymer, polyethyleneimine (PEI), at N/P ratio of 20. The complex was encapsulated in poly(lactic acid-co-glycolic acid) (PLGA) nanocapsules. PLGA capsules were characterized by SEM, TEM and XPS. FTIR was used to show the preferential attachment of ELR to HAp. Encapsulation efficiency of the complex in PLGA nanocapsules was 48%. The release kinetics of the complex fits the Higuchi release kinetics.

Controlling molecular interactions between bioinspired molecules can enable the development of new materials with higher complexity and innovative properties. Here we report on a dynamic system that emerges from the conformational modification of an elastin-like protein by peptide amphiphiles and with the capacity to access, and be maintained in, non-equilibrium for substantial periods of time. The system enables the formation of a robust membrane that displays controlled assembly and disassembly capabilities, adhesion and sealing to surfaces, self-healing and the capability to undergo morphogenesis into tubular structures with high spatiotemporal control. We use advanced microscopy along with turbidity and spectroscopic measurements to investigate the mechanism of assembly and its relation to the distinctive membrane architecture and the resulting dynamic properties. Using cell-culture experiments with endothelial and adipose-derived stem cells, we demonstrate the potential of this system to generate complex bioactive scaffolds for applications such as tissue engineering.