Biodegradable Tyramine Functional Gelatin/6 Arms-PLA Inks Compatible with 3D Two Photon-Polymerization Printing and Meniscus Tissue Regeneration.

The meniscus regeneration can present major challenges such as mimicking tissue microstructuration or triggering cell regeneration. In the case of lesions that require a personalized approach, photoprinting offers the possibility of designing resolutive biomaterial structures. The photo-cross-linkable ink composition determines the process ease and the final network properties. In this study, we designed a range of hybrid inks composed of gelatin(G) and 6-PLA arms(P) that were photo-cross-linked using tyramine groups. The photo-cross-linking efficiency, mechanical properties, degradation, and biological interactions of inks with different G/P mass ratios were studied. The G50P50 network properties were suitable for meniscus regeneration, with Young's modulus of 6.5 MPa, degradation in 2 months, and good cell proliferation. We then confirmed the potential of these inks to produce high-resolution microstructures by printing well-defined microstructures using two-photon polymerization. These hybrid inks offer new perspectives for biocompatible, degradable, and microstructured tissue engineering scaffold creation.

Development of hybrid bioactive nanofibers composed of star Poly(lactic acid) and gelatin by sol-gel crosslinking during the electrospinning process.

The design of a biomimetic scaffold is a major challenge in tissue engineering to promote tissue reconstruction. The use of synthetic polymer nanofibers is widely described as they provide biocompatible matrices whose topography mimics natural extracellular matrix (ECM). To closely match the biochemical composition of the ECM, bioactive molecules such as gelatin are added to the nanofibers to enhance cell adhesion and proliferation. To overcome the rapid solubilization of gelatin in biological fluids and to allow a lasting biological effect, the covalent crosslinking of this macromolecule in the network is crucial. The sol-gel route offers the possibility of gentle crosslinking during shaping but is rarely combined with electrospinning. In this study, we present the creation of Poly(lactic acid)/Gelatin hybrid nanofibers by sol-gel route during electrospinning. To enable sol-gel crosslinking, we synthesized star-shaped PLA and functionalized it with silane groups; then we functionalized gelatin with the same groups for their subsequent reaction with the polymer and thus the creation of the hybrid nanonetwork. We evaluated the impact of the presence of gelatin in Poly(lactic acid)/Gelatin hybrid nanofibers at different percentages on the mechanical properties, nanonetwork crosslinking, degradation and biological properties of the hybrid nanofibers. The addition of gelatin modulated nanonetwork crosslinking that impacted the stiffness of the nanofibers, resulting in softer materials for the cells. Moreover, these hybrid nanofibers also showed a significant improvement in fibroblast proliferation and present a degradation rate suitable for tissue reconstruction. Finally, the bioactive hybrid nanofibers possess versatile properties, interesting for various potential applications in tissue reconstruction.

Design of Hybrid Polymer Nanofiber/Collagen Patches Releasing IGF and HGF to Promote Cardiac Regeneration.

Cardiovascular diseases are the leading cause of death globally. Myocardial infarction in particular leads to a high rate of mortality, and in the case of survival, to a loss of myocardial functionality due to post-infarction necrosis. This functionality can be restored by cell therapy or biomaterial implantation, and the need for a rapid regeneration has led to the development of bioactive patches, in particular through the incorporation of growth factors (GF). In this work, we designed hybrid patches composed of polymer nanofibers loaded with HGF and IGF and associated with a collagen membrane. Among the different copolymers studied, the polymers and their porogens PLA-Pluronic-PLA + PEG and PCL + Pluronic were selected to encapsulate HGF and IGF. While 89 and 92% of IGF were released in 2 days, HGF was released up to 58% and 50% in 35 days from PLA-Pluronic-PLA + PEG and PCL + Pluronic nanofibers, respectively. We also compared two ways of association for the loaded nanofibers and the collagen membrane, namely a direct deposition of the nanofibers on a moisturized collagen membrane (wet association), or entrapment between collagen layers (sandwich association). The interfacial cohesion and the degradation properties of the patches were evaluated. We also show that the sandwich association decreases the burst release of HGF while increasing the release efficiency. Finally, we show that the patches are cytocompatible and that the presence of collagen and IGF promotes the proliferation of C2C12 myoblast cells for 11 days. Taken together, these results show that these hybrid patches are of interest for cardiac muscle regeneration.

Creation of a Stable Nanofibrillar Scaffold Composed of Star-Shaped PLA Network Using Sol-Gel Process during Electrospinning.

PLA nanofibers are of great interest in tissue engineering due to their biocompatibility and morphology; moreover, their physical properties can be tailored for long-lasting applications. One of the common and efficient methods to improve polymer properties and slow down their degradation is sol-gel covalent crosslinking. However, this method usually results in the formation of gels or films, which undervalues the advantages of nanofibers. Here, we describe a dual process sol-gel/electrospinning to improve the mechanical properties and stabilize the degradation of PLA scaffolds. For this purpose, we synthesized star-shaped PLAs and functionalized them with triethoxysilylpropyl groups (StarPLA-PTES) to covalently react during nanofibers formation. To achieve this, we evaluated the use of (1) a polymer diluent and (2) different molecular weights of StarPLA on electrospinnability, StarPLA-PTES condensation time and crosslinking efficiency. Our results show that the diluent allowed the fiber formation and reduced the condensation time, while the addition of low-molecular-weight StarPLA-PTES improved the crosslinking degree, resulting in stable matrices even after 6 months of degradation. Additionally, these materials showed biocompatibility and allowed the proliferation of fibroblasts. Overall, these results open the door to the fabrication of scaffolds with enhanced stability and prospective long-term applications.

Electrospun microstructured PLA-based scaffolds featuring relevant anisotropic, mechanical and degradation characteristics for soft tissue engineering.

Electrospun scaffolds combine suitable structural characteristics that make them strong candidates for their use in tissue engineering. These features can be tailored to optimize other physiologically relevant attributes (e.g. mechanical anisotropy and cellular affinity) while ensuring adequate degradation rates of the biomaterial. Here, we present the fabrication of microstructured scaffolds by using a combination of micropatterned electrospinning collectors (honeycomb- or square-patterned) and poly(lactic acid) (PLA)-based copolymers (linear or star-shaped). The resulting materials showed appropriate macropore size and fiber alignment that were key parameters to enhance their anisotropic properties in protraction. Moreover, their elastic modulus, which was initially similar to that of soft tissues, gradually changed in hydrolytic conditions, matching the degradation profile in a 2- to 3-month period. Finally, honeycomb-structured scaffolds exhibited enhanced cellular proliferation compared to standard electrospun mats, while cell colonization was shown to be guided by the macropore contour. Taking together, these results provide new insight into the rational design of microstructured materials that can mimic the progressive evolution of properties in soft tissue regeneration.

Biomimicking Fiber Platform with Tunable Stiffness to Study Mechanotransduction Reveals Stiffness Enhances Oligodendrocyte Differentiation but Impedes Myelination through YAP-Dependent Regulation.

A key hallmark of many diseases, especially those in the central nervous system (CNS), is the change in tissue stiffness due to inflammation and scarring. However, how such changes in microenvironment affect the regenerative process remains poorly understood. Here, a biomimicking fiber platform that provides independent variation of fiber structural and intrinsic stiffness is reported. To demonstrate the functionality of these constructs as a mechanotransduction study platform, these substrates are utilized as artificial axons and the effects of axon structural versus intrinsic stiffness on CNS myelination are independently analyzed. While studies have shown that substrate stiffness affects oligodendrocyte differentiation, the effects of mechanical stiffness on the final functional state of oligodendrocyte (i.e., myelination) has not been shown prior to this. Here, it is demonstrated that a stiff mechanical microenvironment impedes oligodendrocyte myelination, independently and distinctively from oligodendrocyte differentiation. Yes-associated protein is identified to be involved in influencing oligodendrocyte myelination through mechanotransduction. The opposing effects on oligodendrocyte differentiation and myelination provide important implications for current work screening for promyelinating drugs, since these efforts have focused mainly on promoting oligodendrocyte differentiation. Thus, the platform may have considerable utility as part of a drug discovery program in identifying molecules that promote both differentiation and myelination.

Direct Synthesis of Peptide-Containing Silicones: A New Way to Bioactive Materials.

A simple and efficient way to synthesize peptide-containing silicone materials is described. Silicone oils containing a chosen ratio of bioactive peptide sequences were prepared by acid-catalyzed copolymerization of dichlorodimethylsilane, hybrid dichloromethyl peptidosilane, and Si(vinyl)- or SiH-functionalized monomers. Functionalized silicone oils were first obtained and then, after hydrosilylation cross-linking, bioactive polydimethylsiloxane (PDMS)-based materials were straightforwardly obtained. The introduction of an antibacterial peptide yielded PDMS materials showing activity against Staphylococcus aureus. PDMS containing RGD ligands showed improved cell-adhesion properties. This generic method was fully compatible with the stability of peptides and thus opened the way to the synthesis of a wide range of biologically active silicones.

Biomimicking Fiber Scaffold as an Effective In Vitro and In Vivo MicroRNA Screening Platform for Directing Tissue Regeneration.

MicroRNAs effectively modulate protein expression and cellular response. Unfortunately, the lack of robust nonviral delivery platforms has limited the therapeutic application of microRNAs. Additionally, there is a shortage of drug-screening platforms that are directly translatable from in vitro to in vivo. Here, a fiber substrate that provides nonviral delivery of microRNAs for in vitro and in vivo microRNA screening is introduced. As a proof of concept, difficult-to-transfect primary neurons are targeted and the efficacy of this system is evaluated in a rat spinal cord injury model. With this platform, enhanced gene-silencing is achieved in neurons as compared to conventional bolus delivery (p < 0.05). Thereafter, four well-recognized microRNAs (miR-21, miR-222, miR-132, and miR-431) and their cocktails are screened systematically. Regardless of age and origin of the neurons, similar trends are observed. Next, this fiber substrate is translated into a 3D system for direct in vivo microRNA screening. Robust nerve ingrowth is observed as early as two weeks after scaffold implantation. Nerve regeneration in response to the microRNA cocktails is similar to in vitro experiments. Altogether, the potential of the fiber platform is demonstrated in providing effective microRNA screening and direct translation into in vivo applications.

Scaffold-mediated sequential drug/gene delivery to promote nerve regeneration and remyelination following traumatic nerve injuries.

Neural tissue regeneration following traumatic injuries is often subpar. As a result, the field of neural tissue engineering has evolved to find therapeutic interventions and has seen promising outcomes. However, robust nerve and myelin regeneration remain elusive. One possible reason may be the fact that tissue regeneration often follows a complex sequence of events in a temporally-controlled manner. Although several other fields of tissue engineering have begun to recognise the importance of delivering two or more biomolecules sequentially for more complete tissue regeneration, such serial delivery of biomolecules in neural tissue engineering remains limited. This review aims to highlight the need for sequential delivery to enhance nerve regeneration and remyelination after traumatic injuries in the central nervous system, using spinal cord injuries as an example. In addition, possible methods to attain temporally-controlled drug/gene delivery are also discussed for effective neural tissue regeneration.

Scaffold-Mediated Sustained, Non-viral Delivery of miR-219/miR-338 Promotes CNS Remyelination.

The loss of oligodendrocytes (OLs) and subsequently myelin sheaths following injuries or pathologies in the CNS leads to debilitating functional deficits. Unfortunately, effective methods of remyelination remain limited. Here, we present a scaffolding system that enables sustained non-viral delivery of microRNAs (miRs) to direct OL differentiation, maturation, and myelination. We show that miR-219/miR-338 promoted primary rat OL differentiation and myelination in vitro. Using spinal cord injury as a proof-of-concept, we further demonstrate that miR-219/miR-338 could also be delivered non-virally in vivo using an aligned fiber-hydrogel scaffold to enhance remyelination after a hemi-incision injury at C5 level of Sprague-Dawley rats. Specifically, miR-219/miR-338 mimics were incorporated as complexes with the carrier, TransIT-TKO (TKO), together with neurotrophin-3 (NT-3) within hybrid scaffolds that comprised poly(caprolactone-co-ethyl ethylene phosphate) (PCLEEP)-aligned fibers and collagen hydrogel. After 1, 2, and 4 weeks post-treatment, animals that received NT-3 and miR-219/miR-338 treatment preserved a higher number of Olig2+ oligodendroglial lineage cells as compared with those treated with NT-3 and negative scrambled miRs (Neg miRs; p < 0.001). Additionally, miR-219/miR-338 increased the rate and extent of differentiation of OLs. At the host-implant interface, more compact myelin sheaths were observed when animals received miR-219/miR-338. Similarly within the scaffolds, miR-219/miR-338 samples contained significantly more myelin basic protein (MBP) signals (p < 0.01) and higher myelination index (p < 0.05) than Neg miR samples. These findings highlight the potential of this platform to promote remyelination within the CNS.

Sustained delivery of siRNA/mesoporous silica nanoparticle complexes from nanofiber scaffolds for long-term gene silencing.

A low toxicity and efficient delivery system is needed to deliver small interfering RNAs (siRNA) in vitro and in vivo. The use of mesoporous silica nanoparticles (MSN) is becoming increasingly common due to its biocompatibility, tunable pore size and customizable properties. However, bolus delivery of siRNA/MSN complexes remains suboptimal, especially when a sustained and long-term administration is required. Here, we utilized electrospun scaffolds for sustained delivery of siRNA/MSN-PEI through surface adsorption and nanofiber encapsulation. As a proof-of-concept, we targeted collagen type I expression to modulate fibrous capsule formation. Surface adsorption of siRNA/MSN-PEI provided sustained availability of siRNA for at least 30 days in vitro. As compared to conventional bolus delivery, such scaffold-mediated transfection provided more effective gene silencing (p < 0.05). On the contrary, a longer sustained release was attained (at least 5 months) when siRNA/MSN-PEI complexes were encapsulated within the electrospun fibers. In vivo subcutaneous implantation and biodistribution analysis of these scaffolds revealed that siRNA remained localized up to ∼290 µm from the implants. Finally, a fibrous capsule reduction of ∼45.8% was observed after 4 weeks in vivo as compared to negative scrambled siRNA treatment. Taken together, these results demonstrate the efficacy of scaffold-mediated sustained delivery of siRNA/MSN-PEI for long-term non-viral gene silencing applications. STATEMENT OF SIGNIFICANCE: The bolus delivery of siRNA/mesoporous silica nanoparticles (MSN) complexes shows high efficiency to silence protein agonists of tumoral processes as cancer treatments. However, in tissue engineering area, scaffold mediated delivery is desired to achieve a local and sustained release of therapeutics. We showed the feasibility and the efficacy of siRNA/MSN delivered from electrospun scaffolds through surface adsorption and nanofiber encapsulation. We showed that this method enhances siRNA transfection efficiency and sustained targeted proteins silencing in vitro and in vivo. As a proof of concept, in this study, we targeted collagen type I expression to modulate fibrous capsule formation. However this platform can be applied to the release and transfection of siRNA or miRNA in cancer and tissue engineering applications.

Site-specific grafting on titanium surfaces with hybrid temporin antibacterial peptides.

Relying on a membrane-disturbing mechanism of action and not on any intracellular target, antimicrobial peptides (AMP) are attractive compounds to be grafted on the surface of implantable materials such as silicone catheters or titanium surgical implants. AMP sequences often display numerous reactive functions (e.g. amine, carboxylic acid) on their side chains and straightforward conjugation chemistries could lead to uncontrolled covalent grafting, random orientation, and non-homogenous density. To achieve an easy and site specific covalent attachment of unprotected peptides on titanium surfaces, we designed hybrid silylated biomolecules based on the temporin-SHa amphipathic helical antimicrobial sequence. With the grafting reaction being chemoselective, we designed five analogues displaying the silane anchoring function at the N-ter, C-ter or at different positions inside the sequence to get an accurate control of the orientation. Grafting density calculations were performed by XPS and the influence of the orientation of the peptide on the surface was clearly demonstrated by the measure of antimicrobial activity. Temporin amphipathic helices are described to permeabilize the bacterial membrane by interacting in a parallel orientation with it. Our results move in the direction of this mechanism as the selective grafting of hybrid temporin 2 through a lysine placed at the center of the peptide sequence, resulted in better biofilm growth inhibition of E. coli and S. epidermis than substrates in which temporins were grafted via their C- or N-terminus.

[Biodegradable synthetic polymers for the design of implantable medical devices: the ligamentoplasty case]. / Polymères synthétiques dégradables pour la conception de dispositifs médicaux implantables - Le cas de la ligamentoplastie.

The sector of implantable medical devices is a growing sector of health products especially dynamic in the field of research. To improve the management of patients and to meet clinical requirements, researchers are developing new types of medical devices. They use different families of biomaterials presenting various chemical and physical characteristics in order for providing clinicians with health products optimized for biomedical applications. In this article, we aim to show how, starting from a family of biomaterials (degradable polymers), it is possible to design an implantable medical device for the therapeutic management of the failure of anterior cruciate ligament. The main steps leading to the design of a total ligament reinforcement are detailed. They range from the synthesis and characterization of degradable polymer to the shaping of the knitted implant, through the assessment of the study of the impact of sterilization on mechanical properties and checking cytocompatibility.

In vivo evaluation of hybrid patches composed of PLA based copolymers and collagen/chondroitin sulfate for ligament tissue regeneration.

Biomaterials for soft tissues regeneration should exhibit sufficient mechanical strength, demonstrating a mechanical behavior similar to natural tissues and should also promote tissues ingrowth. This study was aimed at developing new hybrid patches for ligament tissue regeneration by synergistic incorporation of a knitted structure of degradable polymer fibers to provide mechanical strength and of a biomimetic matrix to help injured tissues regeneration. PLA- Pluronic® (PLA-P) and PLA-Tetronic® (PLA-T) new copolymers were shaped as knitted patches and were associated with collagen I (Coll) and collagen I/chondroitine-sulfate (Coll CS) 3-dimensional matrices. In vitro study using ligamentocytes showed the beneficial effects of CS on ligamentocytes proliferation. Hybrid patches were then subcutaneously implanted in rats for 4 and 12 weeks. Despite degradation, patches retained strength to answer the mechanical physiological needs. Tissue integration capacity was assessed with histological studies. We showed that copolymers, associated with collagen and chondroitin sulfate sponge, exhibited very good tissue integration and allowed neotissue synthesis after 12 weeks in vivo. To conclude, PLA-P/CollCS and PLA-T/CollCS hybrid patches in terms of structure and composition give good hopes for tendon and ligament regeneration. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 1778-1788, 2017.

Rolled knitted scaffolds based on PLA-pluronic copolymers for anterior cruciate ligament reinforcement: A step by step conception.

The aim of this study was to prepare a new knitted scaffold from PLA-Pluronic block copolymers for anterior cruciate ligament reconstruction. The impact of sterilization methods (beta-ray and gamma-ray sterilization) on copolymers was first evaluated in order to take into account the possible damages due to the sterilization process. Beta-ray radiation did not significantly change mechanical properties in contrast to gamma-ray sterilization. It was shown that ACL cells proliferate onto these copolymers, demonstrating their cytocompatibility. Thirdly, in order to study the influence of shaping on mechanical properties, several shapes were created with copolymers yarns: braids, ropes and linear or rolled knitted scaffolds. The rolled knitted scaffold presented interesting mechanical characteristics, similar to native anterior cruciate ligament (ACL) with a 67 MPa Young's Modulus and a stress at failure of 22.5 MPa. These findings suggest that this three dimensional rolled knitted scaffold meet the mechanical properties of ligament tissues and could be suitable as a scaffold for ligament reconstruction. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 735-743, 2017.

Simple and Specific Grafting of Antibacterial Peptides on Silicone Catheters.

To fight against nosocomial infection initiated by colonization of medical devices, a strategy enabling the direct and fast functionalization of silicone surfaces is proposed. This strategy proceeds in a site-specific way using original hybrid silylated antibacterial peptides. This safe and up-scalable method guarantees a covalent and robust immobilization with the correct orientation of the bioactive moiety. Importantly it also avoids multi-step chemical modifications of the surface or multi-layer polymer coatings. As proof of concept, antibacterial silicone catheter has been prepared whose immediate and long term efficiency is superior by comparison to similar silver-embedded materials.

PLA-poloxamer/poloxamine copolymers for ligament tissue engineering: sound macromolecular design for degradable scaffolds and MSC differentiation.

The treatment of anterior cruciate ligament (ACL) failures remains a current clinical challenge. The present study aims at providing suitable degradable scaffolds for ligament tissue engineering. First, we focus on the design and the evaluation of poly(lactide)/poloxamer or poly(lactide)/poloxamine multiblock copolymers selected and developed to have suitable degradation and mechanical properties to match ACL repair. In the second part, it is shown that the copolymers can be processed in the form of microfibers and scaffolds consisting of a combination of twisted/braided fibers to further modulate the mechanical properties and prepare scaffold prototypes suitable for ligament application. Finally, after assessment of their cytocompatibility, the polymer scaffolds are associated with mesenchymal stem cells (MSCs). MSC differentiation toward a ligament fibroblast phenotype is promoted by a dual stimulation including an inductive culture medium and cyclic mechanical loads. RT-qPCR analyses confirm the potential of our scaffolds and MSCs for ACL regeneration with upregulation of some differentiation markers including Scleraxis, Tenascin-C and Tenomodulin.

Turning peptides in comb silicone polymers.

We have recently reported on a new class of silicone-peptide' biopolymers obtained by polymerization of di-functionalized chlorodimethylsilyl hybrid peptides. Herein, we describe a related strategy based on dichloromethylsilane-derived peptides, which yield novel polymers with a polysiloxane backbone, comparable with a silicone-bearing pendent peptide chains. Interestingly, polymerization is chemoselective toward amino acids side-chains and proceeds in a single step in very mild conditions (neutral pH, water, and room temperature). As potential application, a cationic sequence was polymerized and used for antibacterial coating.

Investigation on the properties of linear PLA-poloxamer and star PLA-poloxamine copolymers for temporary biomedical applications.

The objective of this work was to develop and study new biodegradable thermoplastics with improved mechanical properties for potential use as temporary implantable biomaterials. Linear poloxamer and star-shaped poloxamine have been used as macroinitiators for the ring-opening polymerization (ROP) of lactide to yield high molecular weight PLA-based thermoplastic block copolymers. The influence of the nature of the macroinitiator, PLA crystallinity and initial molecular weight on the copolymers properties was investigated by performing a 7-week degradation test in PBS. The evaluation of water uptakes and molecular weights during the degradation pointed out an early hydrolytic degradation of the 100-kg∙mol(-1) copolymers compared to the 200-kg∙mol(-1) ones (molecular weight decrease of ca. 40% and 20%, respectively). A dramatic loss of tensile mechanical properties was also observed for the 100-kg∙mol(-1) copolymers, whereas the 200-kg∙mol(-1) copolymers showed stable or even slightly improved properties with Young's moduli around 500 MPa and yield strains around 3% to 4%. Finally, the cytocompatibility of the more stable 200 kg∙mol(-1) copolymers was confirmed by murine mesenchymal stem cells (MSCs) culture.