Elastin-like Recombinamer Hydrogels as Platforms for Breast Cancer Modeling.

The involvement of the extracellular matrix (ECM) in tumor progression has motivated the development of biomaterials mimicking the tumor ECM to develop more predictive cancer models. Particularly, polypeptides based on elastin could be an interesting approach to mimic the ECM due to their tunable properties. Here, we demonstrated that elastin-like recombinamer (ELR) hydrogels can be suitable biomaterials to develop breast cancer models. This hydrogel was formed by two ELR polypeptides, one containing sequences biodegradable by matrix metalloproteinase and cyclooctyne and the other carrying arginylglycylaspartic acid and azide groups to allow cell adhesion, biodegradability, and suitable stiffness through "click-chemistry" cross-linking. Our findings show that breast cancer or nontumorigenic breast cells showed high viability and cell proliferation for up to 7 days. MCF7 and MCF10A formed spheroids whereas MDA-MB-231 formed cell networks, with the expression of ECM and high drug resistance in all cases, evidencing that ELR hydrogels are a promising biomaterial for breast cancer modeling.

Influence of the Thermodynamic and Kinetic Control of Self-Assembly on the Microstructure Evolution of Silk-Elastin-Like Recombinamer Hydrogels.

Complex recombinant biomaterials that merge the self-assembling properties of different (poly)peptides provide a powerful tool for the achievement of specific structures, such as hydrogel networks, by tuning the thermodynamics and kinetics of the system through a tailored molecular design. In this work, elastin-like (EL) and silk-like (SL) polypeptides are combined to obtain a silk-elastin-like recombinamer (SELR) with dual self-assembly. First, EL domains force the molecule to undergo a phase transition above a precise temperature, which is driven by entropy and occurs very fast. Then, SL motifs interact through the slow formation of ß-sheets, stabilized by H-bonds, creating an energy barrier that opposes phase separation. Both events lead to the development of a dynamic microstructure that evolves over time (until a pore size of 49.9 ± 12.7 µm) and to a delayed hydrogel formation (obtained after 2.6 h). Eventually, the network is arrested due to an increase in ß-sheet secondary structures (up to 71.8 ± 0.8%) within SL motifs. This gives a high bond strength that prevents the complete segregation of the SELR from water, which results in a fixed metastable microarchitecture. These porous hydrogels are preliminarily tested as biomimetic niches for the isolation of cells in 3D cultures.

Förster Resonance Energy Transfer-Paired Hydrogel Forming Silk-Elastin-Like Recombinamers by Recombinant Conjugation of Fluorescent Proteins.

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 coerulescens enhanced 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.

Regeneration of hyaline cartilage promoted by xenogeneic mesenchymal stromal cells embedded within elastin-like recombinamer-based bioactive hydrogels.

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.

Nanotechnological Approaches to Therapeutic Delivery Using Elastin-Like Recombinamers.

This Review discusses the use of elastin-like polymers and their recombinant version, elastin-like recombinamers, in drug-delivery systems. These macromolecules exhibit a number of interesting properties that are rarely found together in any other family of materials, especially extremely high biocompatibility, high bioactivity and functionality, complex yet fully controlled composition, and stimuli responsiveness. Appropriate design of these molecules opens up a broad range of different possibilities for their use in new therapeutic platforms. The first of these described herein is the use of ELRs in single-molecule devices as therapeutic entities in their own right. Subsequently, we describe how the self-assembly properties of these materials can be exploited to create nanocarriers and, eventually, microcarriers that are able to temporally and spatially control and direct the release of their drug load. Intracellular drug-delivery devices and nanocarriers for treating cancer are among the uses described in that section. Finally, the use of ELRs as base materials for implantable drug depots, in the form of hydrogels, is discussed.

Effects of hypoxia on bronchial and alveolar epithelial cells linked to pathogenesis in chronic lung disorders.

Introduction: Chronic lung disorders involve pathological alterations in the lung tissue with hypoxia as a consequence. Hypoxia may influence the release of inflammatory mediators and growth factors including vascular endothelial growth factor (VEGF) and prostaglandin (PG)E2. The aim of this work was to investigate how hypoxia affects human lung epithelial cells in combination with profibrotic stimuli and its correlation to pathogenesis. Methods: Human bronchial (BEAS-2B) and alveolar (hAELVi) epithelial cells were exposed to either hypoxia (1% O2) or normoxia (21% O2) during 24 h, with or without transforming growth factor (TGF)-ß1. mRNA expression of genes and proteins related to disease pathology were analysed with qPCR, ELISA or immunocytochemistry. Alterations in cell viability and metabolic activity were determined. Results: In BEAS-2B and hAELVi, hypoxia significantly dowregulated genes related to fibrosis, mitochondrial stress, oxidative stress, apoptosis and inflammation whereas VEGF receptor 2 increased. Hypoxia increased the expression of Tenascin-C, whereas both hypoxia and TGF-ß1 stimuli increased the release of VEGF, IL-6, IL-8 and MCP-1 in BEAS-2B. In hAELVi, hypoxia reduced the release of fibroblast growth factor, epidermal growth factor, PGE2, IL-6 and IL-8, whereas TGF-ß1 stimulus significantly increased the release of PGE2 and IL-6. TGF-ß1 stimulated BEAS-2B cells showed a decreased release of VEGF-A and IL-8, while TGF-ß1 stimulated hAELVi cells showed a decreased release of PGE2 and IL-8 during hypoxia compared to normoxia. Metabolic activity was significantly increased by hypoxia in both epithelial cell types. Discussion: In conclusion, our data indicate that bronchial and alveolar epithelial cells respond differently to hypoxia and profibrotic stimuli. The bronchial epithelium appears more responsive to changes in oxygen levels and remodelling processes compared to the alveoli, suggesting that hypoxia may be a driver of pathogenesis in chronic lung disorders.

hLMSC Secretome Affects Macrophage Activity Differentially Depending on Lung-Mimetic Environments.

Mesenchymal stromal cell (MSC)-based therapies for inflammatory diseases rely mainly on the paracrine ability to modulate the activity of macrophages. Despite recent advances, there is scarce information regarding changes of the secretome content attributed to physiomimetic cultures and, especially, how secretome content influence on macrophage activity for therapy. hLMSCs from human donors were cultured on devices developed in house that enabled lung-mimetic strain. hLMSC secretome was analyzed for typical cytokines, chemokines and growth factors. RNA was analyzed for the gene expression of CTGF and CYR61. Human monocytes were differentiated to macrophages and assessed for their phagocytic capacity and for M1/M2 subtypes by the analysis of typical cell surface markers in the presence of hLMSC secretome. CTGF and CYR61 displayed a marked reduction when cultured in lung-derived hydrogels (L-Hydrogels). The secretome showed that lung-derived scaffolds had a distinct secretion while there was a large overlap between L-Hydrogel and the conventionally (2D) cultured samples. Additionally, secretome from L-Scaffold showed an HGF increase, while IL-6 and TNF-α decreased in lung-mimetic environments. Similarly, phagocytosis decreased in a lung-mimetic environment. L-Scaffold showed a decrease of M1 population while stretch upregulated M2b subpopulations. In summary, mechanical features of the lung ECM and stretch orchestrate anti-inflammatory and immunosuppressive outcomes of hLMSCs.

A tunable physiomimetic stretch system evaluated with precision cut lung slices and recellularized human lung scaffolds.

Breathing exposes lung cells to continual mechanical stimuli, which is part of the microenvironmental signals directing cellular functions together with the extracellular matrix (ECM). Therefore, developing systems that incorporate both stimuli is urgent to fully understand cell behavior. This study aims to introduce a novel in vitro culture methodology combining a cyclic stretch that simulates in vivo breathing with 3D cell culture platforms in the form of decellularized lung slices (DLS) and precision cut lung slices (PCLS). To this end, we have constructed a device that mimics the amplitudes and frequencies of distensions seen in the breathing human lung. For its validation, we cultured H441 lung epithelial cells in human DLS exposed to 16 stretch cycles per minute with a 10% stretch amplitude. Cell viability (resazurin reduction), proliferation (Ki-67) and YAP1 activation were evaluated at 24 and 96 h by immunohistochemistry, while the expression of SFTPB, COL3A1, COL4A3 and LAMA5 was evaluated by qPCR. Cyclic stretch induced an increase in SFTPB expression after 24 h without a concomitant increase in the stretch responsive gene YAP1. Moreover, the ECM milieu lowered the expression of the basement membrane protein genes COL4A3 and LAMA5 compared to tissue culture plastic control cultures, but no effect was observed by the mechanical stimuli. The device also confirmed good compatibility with PCLS culture, showing preserved morphology and metabolism in rat PCLS after 72 h of mechanical stretch. Thus, we present a novel device and methodology for the easy assembling and study of lung tissue slice cultures subjected to physiomimetic mechanical stimuli, which shows promise for future studies of cell and tissue function in a lung ECM milieu with physiological or pathological mechanical stimuli.

Mesenchymal Stromal Cells Combined With Elastin-Like Recombinamers Increase Angiogenesis In Vivo After Hindlimb Ischemia.

Hindlimb ischemia is an unmet medical need, especially for those patients unable to undergo vascular surgery. Cellular therapy, mainly through mesenchymal stromal cell (MSC) administration, may be a potentially attractive approach in this setting. In the current work, we aimed to assess the potential of the combination of MSCs with a proangiogenic elastin-like recombinamer (ELR)-based hydrogel in a hindlimb ischemia murine model. Human bone marrow MSCs were isolated from four healthy donors, while ELR biomaterials were genetically engineered. Hindlimb ischemia was induced through ligation of the right femoral artery, and mice were intramuscularly injected with ELR biomaterial, 0.5 × 106 MSCs or the combination, and also compared to untreated animals. Tissue perfusion was monitored using laser Doppler perfusion imaging. Histological analysis of hindlimbs was performed after hematoxylin and eosin staining. Immunofluorescence with anti-human mitochondria antibody was used for human MSC detection, and the biomaterial was detected by elastin staining. To analyze the capillary density, immunostaining with an anti-CD31 antibody was performed. Our results show that the injection of MSCs significantly improves tissue reperfusion from day 7 (p = 0.0044) to day 21 (p = 0.0216), similar to the infusion of MSC + ELR (p = 0.0038, p = 0.0014), without significant differences between both groups. After histological evaluation, ELR hydrogels induced minimal inflammation in the injection sites, showing biocompatibility. MSCs persisted with the biomaterial after 21 days, both in vitro and in vivo. Finally, we observed a higher blood vessel density when mice were treated with MSCs compared to control (p<0.0001), but this effect was maximized and significantly different to the remaining experimental conditions when mice were treated with the combination of MSCs and the ELR biomaterial (p < 0.0001). In summary, the combination of an ELR-based hydrogel with MSCs may improve the angiogenic effects of both strategies on revascularization of ischemic tissues.

Combining tunable proteolytic sequences and a VEGF-mimetic peptide for the spatiotemporal control of angiogenesis within Elastin-Like Recombinamer scaffolds.

One of the main challenges in regenerative medicine is the spatiotemporal control of angiogenesis, which is key for the successful repair of many tissues, and determines the proper integration of the implant through the generation of a functional vascular network. To this end, we have designed a three-dimensional (3D) model consisting of a coaxial binary elastin-like recombinamer (ELR) tubular construct. It displays fast and slow proteolytic hydrogels on its inner and outer part, respectively, both sensitive to the urokinase plasminogen activator protease. The ELRs used to build the scaffold included crosslinkable domains to stabilize the structure and a conjugated VEGF-derived peptide (QK) to induce angiogenesis. The mechanical and morphological evaluation of the ELR hydrogels proved their suitability for soft tissue regeneration. In addition, in vitro studies evidenced the effect of the QK peptide on endothelial cell spreading and anastomosis. Moreover, immunohistochemical analyses after subcutaneous implantation of the ELR hydrogels in mice showed the induction of a low macrophage response that resolved over time. The implantation of the 3D model constructs evidenced the ability of the fast proteolytic sequence and the QK peptide to guide cell infiltration and capillary formation in the pre-designed arrangement of the constructs. These results set the basis for the application of this type of scaffolds in regenerative medicine, where spatiotemporally controlled vascularization will help in the promotion of an optimal tissue repair. STATEMENT OF SIGNIFICANCE: Herein, we show the spatiotemporal control of angiogenesis in vivo by the combination of proteolytic sequences, with fast and slow degradation kinetics, and VEGF-mimetic peptide (QK) in a coaxial binary elastin-like recombinamer (ELR) tubular scaffold. These two bioactivities have been previously described for angiogenesis purposes, but have never been combined. This work demonstrates that the bioactivities act synergistically in promoting cell infiltration and subsequent vascularization, thus leading to a controlled evolution in space and time of the vascular microstructure within the hydrogel-like tubular scaffold. This effect has not been showed before and holds great potential for future vascular applications, which might be of great interest for a substantial part of Acta Biomaterialia readership.

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.

Elastin-Like Recombinamer Hydrogels for Improved Skeletal Muscle Healing Through Modulation of Macrophage Polarization.

Large skeletal muscle injuries, such as a volumetric muscle loss (VML), often result in an incomplete regeneration due to the formation of a non-contractile fibrotic scar tissue. This is, in part, due to the outbreak of an inflammatory response, which is not resolved over time, meaning that type-1 macrophages (M1, pro-inflammatory) involved in the initial stages of the process are not replaced by pro-regenerative type-2 macrophages (M2). Therefore, biomaterials that promote the shift from M1 to M2 are needed to achieve optimal regeneration in VML injuries. In this work, we used elastin-like recombinamers (ELRs) as biomaterials for the formation of non- (physical) and covalently (chemical) crosslinked bioactive and biodegradable hydrogels to fill the VML created in the tibialis anterior (TA) muscles of rats. These hydrogels promoted a higher infiltration of M2 within the site of injury in comparison to the non-treated control after 2 weeks (p<0.0001), indicating that the inflammatory response resolves faster in the presence of both types of ELR-based hydrogels. Moreover, there were not significant differences in the amount of collagen deposition between the samples treated with the chemical ELR hydrogel at 2 and 5 weeks, and this same result was found upon comparison of these samples with healthy tissue after 5 weeks, which implies that this treatment prevents fibrosis. The macrophage modulation also translated into the formation of myofibers that were morphologically more similar to those present in healthy muscle. Altogether, these results highlight that ELR hydrogels provide a friendly niche for infiltrating cells that biodegrades over time, leaving space to new muscle tissue. In addition, they orchestrate the shift of macrophage population toward M2, which resulted in the prevention of fibrosis in the case of the chemical hydrogel treatment and in a more healthy-like myofiber phenotype for both types of hydrogels. Further studies should focus in the assessment of the regeneration of skeletal muscle in larger animal models, where a more critical defect can be created and additional methods can be used to evaluate the functional recovery of skeletal muscle.

Trends in the design and use of elastin-like recombinamers as biomaterials.

Elastin-like recombinamers (ELRs), which derive from one of the repetitive domains found in natural elastin, have been intensively studied in the last few years from several points of view. In this mini review, we discuss all the recent works related to the investigation of ELRs, starting with those that define these polypeptides as model intrinsically disordered proteins or regions (IDPs or IDRs) and its relevance for some biomedical applications. Furthermore, we summarize the current knowledge on the development of drug, vaccine and gene delivery systems based on ELRs, while also emphasizing the use of ELR-based hydrogels in tissue engineering and regenerative medicine (TERM). Finally, we show different studies that explore applications in other fields, and several examples that describe biomaterial blends in which ELRs have a key role. This review aims to give an overview of the recent advances regarding ELRs and to encourage further investigation of their properties and applications.

Elastin-Like Recombinamers As Smart Drug Delivery Systems.

BACKGROUND: Drug delivery systems that are able to control the release of bioactive molecules and designed to carry drugs to target sites are of particular interest for tissue therapy. Moreover, systems comprising materials that can respond to environmental stimuli and promote self-assembly and higher order supramolecular organization are especially useful in the biomedical field. Objetive: This review focuses on biomaterials suitable for this purpose and that include elastin-like recombinamers (ELRs), a class of proteinaceous polymers bioinspired by natural elastin, designed using recombinant technologies. The self-assembly and thermoresponsive behaviour of these systems, along with their biodegradability, biocompatibility and well-defined composition as a result of their tailormade design, make them particularly attractive for controlled drug delivery. RESULTS: ELR-based delivery systems that allow targeted delivery are reviewed, especially ELR-drug recombinant fusion constructs, ELR-drug systems chemically bioconjugated in their monomeric and soluble forms, and drug encapsulation by nanoparticle-forming ELRs. Subsequently, the review focuses on those drug carriers in which smart release is triggered by pH or temperature with a particular focus on cancer treatments. Systems for controlled drug release based on depots and hydrogels that act as both a support and reservoir in which drugs can be stored will be described, and their applications in drug delivery discussed. Finally, smart drug-delivery systems not based on ELRs, including those comprising proteins, synthetic polymers and non-polymeric systems, will also be briefly discussed. CONCLUSION: Several different constructions based on ELRs are potential candidates for controlled drug delivery to be applied in advanced biomedical treatments.

Random and oriented electrospun fibers based on a multicomponent, in situ clickable elastin-like recombinamer system for dermal tissue engineering.

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 of two model elastin-like recombinamer-based hydrogels formed through physical or chemical cross-linking for various applications in tissue engineering and regenerative medicine.

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-1ß, 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.

* Bone Regeneration Mediated by a Bioactive and Biodegradable Extracellular Matrix-Like Hydrogel Based on Elastin-Like Recombinamers.

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.

Recombinant Technology in the Development of Materials and Systems for Soft-Tissue Repair.

The field of biomedicine is constantly investing significant research efforts in order to gain a more in-depth understanding of the mechanisms that govern the function of body compartments and to develop creative solutions for the repair and regeneration of damaged tissues. The main overall goal is to develop relatively simple systems that are able to mimic naturally occurring constructs and can therefore be used in regenerative medicine. Recombinant technology, which is widely used to obtain new tailored synthetic genes that express polymeric protein-based structures, now offers a broad range of advantages for that purpose by permitting the tuning of biological and mechanical properties depending on the intended application while simultaneously ensuring adequate biocompatibility and biodegradability of the scaffold formed by the polymers. This Progress Report is focused on recombinant protein-based materials that resemble naturally occurring proteins of interest for use in soft tissue repair. An overview of recombinant biomaterials derived from elastin, silk, collagen and resilin is given, along with a description of their characteristics and suggested applications. Current endeavors in this field are continuously providing more-improved materials in comparison with conventional ones. As such, a great effort is being made to put these materials through clinical trials in order to favor their future use.