Clickable Dynamic Bioinks Enable Post-Printing Modifications of Construct Composition and Mechanical Properties Controlled over Time and Space.

Bioprinting is a booming technology, with numerous applications in tissue engineering and regenerative medicine. However, most biomaterials designed for bioprinting depend on the use of sacrificial baths and/or non-physiological stimuli. Printable biomaterials also often lack tunability in terms of their composition and mechanical properties. To address these challenges, the authors introduce a new biomaterial concept that they have termed "clickable dynamic bioinks". These bioinks use dynamic hydrogels that can be printed, as well as chemically modified via click reactions to fine-tune the physical and biochemical properties of printed objects after printing. Specifically, using hyaluronic acid (HA) as a polymer of interest, the authors investigate the use of a boronate ester-based crosslinking reaction to produce dynamic hydrogels that are printable and cytocompatible, allowing for bioprinting. The resulting dynamic bioinks are chemically modified with bioorthogonal click moieties to allow for a variety of post-printing modifications with molecules carrying the complementary click function. As proofs of concept, the authors perform various post-printing modifications, including adjusting polymer composition (e.g., HA, chondroitin sulfate, and gelatin) and stiffness, and promoting cell adhesion via adhesive peptide immobilization (i.e., RGD peptide). The results also demonstrate that these modifications can be controlled over time and space, paving the way for 4D bioprinting applications.

Harnessing cell-material interactions to control stem cell secretion for osteoarthritis treatment.

Osteoarthritis (OA) is the most common debilitating joint disease, yet there is no curative treatment for OA to date. Delivering mesenchymal stromal cells (MSCs) as therapeutic cells to mitigate the inflammatory symptoms associated with OA is attracting increasing attention. In principle, MSCs could respond to the pro-inflammatory microenvironment of an OA joint by the secretion of anti-inflammatory, anti-apoptotic, immunomodulatory and pro-regenerative factors, therefore limiting pain, as well as the disease development. However, the microenvironment of MSCs is known to greatly affect their survival and bioactivity, and using tailored biomaterial scaffolds could be key to the success of intra-articular MSC-based therapies. The aim of this review is to identify and discuss essential characteristics of biomaterial scaffolds to best promote MSC secretory functions in the context of OA. First, a brief introduction to the OA physiopathology is provided, followed by an overview of the MSC secretory functions, as well as the current limitations of MSC-based therapy. Then, we review the current knowledge on the effects of cell-material interactions on MSC secretion. These considerations allow us to define rational guidelines for next-generation biomaterial design to improve the MSC-based therapy of OA.

Click and bioorthogonal hyaluronic acid hydrogels as an ultra-tunable platform for the investigation of cell-material interactions.

The cellular microenvironment plays a major role in the biological functions of cells. Thus, biomaterials, especially hydrogels, which can be design to mimic the cellular microenvironment, are being increasingly used for cell encapsulation, delivery, and 3D culture, with the hope of controlling cell functions. Yet, much remains to be understood about the effects of cell-material interactions, and advanced synthetic strategies need to be developed to independently control the mechanical and biochemical properties of hydrogels. To address this challenge, we designed a new hyaluronic acid (HA)-based hydrogel platform using a click and bioorthogonal strain-promoted azide-alkyne cycloaddition (SPAAC) reaction. This approach facilitates the synthesis of hydrogels that are easy to synthesize and sterilize, have minimal swelling, are stable long term, and are cytocompatible. It provides bioorthogonal HA gels over an uncommonly large range of stiffness (0.5-45 kPa), all forming within 1-15 min. More importantly, our approach offers a versatile one-pot procedure to independently tune the hydrogel composition (e.g., polymer and adhesive peptides). Using this platform, we investigate the independent effects of polymer type, stiffness, and adhesion on the secretory properties of human adipose-derived stromal cells (hASCs) and demonstrate that HA can enhance the secretion of immunomodulatory factors by hASCs.

Material-Assisted Strategies for Osteochondral Defect Repair.

The osteochondral (OC) unit plays a pivotal role in joint lubrication and in the transmission of constraints to bones during movement. The OC unit does not spontaneously heal; therefore, OC defects are considered to be one of the major risk factors for developing long-term degenerative joint diseases such as osteoarthritis. Yet, there is currently no curative treatment for OC defects, and OC regeneration remains an unmet medical challenge. In this context, a plethora of tissue engineering strategies have been envisioned over the last two decades, such as combining cells, biological molecules, and/or biomaterials, yet with little evidence of successful clinical transfer to date. This striking observation must be put into perspective with the difficulty in comparing studies to identify overall key elements for success. This systematic review aims to provide a deeper insight into the field of material-assisted strategies for OC regeneration, with particular considerations for the therapeutic potential of the different approaches (with or without cells or biological molecules), and current OC regeneration evaluation methods. After a brief description of the biological complexity of the OC unit, the recent literature is thoroughly analyzed, and the major pitfalls, emerging key elements, and new paths to success are identified and discussed.

Osteoarthritis: From upcoming treatments to treatments yet to come.

Osteoarthritis affects hundreds of millions of people worldwide, and its prevalence is constantly increasing. While there is currently no treatment that can alter the course of the disease, promising therapeutic strategies and novel targets are being investigated. Innovative cell therapies are already reaching clinical trials, and recent progress in our understanding of the disease is opening new routes for gene therapy. In the long term, the development of new biofabrication tools, such as 3D bioprinting, may pave the way for personalized mini-joint models that could be used to screen drugs and to personalize treatments. This review provides an overview of the most promising therapeutic approaches in the field of osteoarthritis, from upcoming treatments to those that are yet to be discovered.

In Situ Forming, Silanized Hyaluronic Acid Hydrogels with Fine Control Over Mechanical Properties and In Vivo Degradation for Tissue Engineering Applications.

In situ forming hydrogels that can be injected into tissues in a minimally-invasive fashion are appealing as delivery vehicles for tissue engineering applications. Ideally, these hydrogels should have mechanical properties matching those of the host tissue, and a rate of degradation adapted for neo-tissue formation. Here, the development of in situ forming hyaluronic acid hydrogels based on the pH-triggered condensation of silicon alkoxide precursors into siloxanes is reported. Upon solubilization and pH adjustment, the low-viscosity precursor solutions are easily injectable through fine-gauge needles prior to in situ gelation. Tunable mechanical properties (stiffness from 1 to 40 kPa) and associated tunable degradability (from 4 days to more than 3 weeks in vivo) are obtained by varying the degree of silanization (from 4.3% to 57.7%) and molecular weight (120 and 267 kDa) of the hyaluronic acid component. Following cell encapsulation, high cell viability (> 80%) is obtained for at least 7 days. Finally, the in vivo biocompatibility of silanized hyaluronic acid gels is verified in a subcutaneous mouse model and a relationship between the inflammatory response and the crosslink density is observed. Silanized hyaluronic acid hydrogels constitute a tunable hydrogel platform for material-assisted cell therapies and tissue engineering applications.

Nanoparticle delivery of a pH-sensitive prodrug of doxorubicin and a mitochondrial targeting VES-H8R8 synergistically kill multi-drug resistant breast cancer cells.

Multi-drug resistance (MDR) remains a major obstacle in cancer treatment while being heavily dependent on mitochondrial activity and drug efflux. We previously demonstrated that cationic lipids, such as the vitamin E succinate modified octahistidine-octaarginine (VES-H8R8) conjugate, target mitochondria, resulting in depolarized mitochondria and inhibited drug efflux in MDR breast cancer cells. We hypothesized that the effective cell uptake, efflux inhibition, and mitochondrial depolarization properties of VES-H8R8 would synergistically enhance the toxicity of a pH-sensitive prodrug of doxorubicin (pDox) when co-encapsulated in nanoparticles (NPs). pDox was successfully synthesized and validated for pH-sensitive release from NPs under lysosome-mimicking, acidic conditions. The synergistic effect of VES-H8R8 and pDox was confirmed against MDR breast cancer cells in vitro. Importantly, synergism was only observed when VES-H8R8 and pDox were co-encapsulated in a single nanoparticulate system. The synergistic mechanism was investigated, confirming superior pDox uptake and retention, Pgp efflux inhibition, mitochondrial depolarization, and enhanced induction of ROS, and apoptosis. This work demonstrates the translational potential of doubly-loaded NPs co-encapsulating pDox with VES-H8R8 to synergistically kill MDR breast cancer cells.

Inverse Electron-Demand Diels-Alder Methylcellulose Hydrogels Enable the Co-delivery of Chondroitinase ABC and Neural Progenitor Cells.

A hydrogel that can deliver both proteins and cells enables the local microenvironment of transplanted cells to be manipulated with a single injection. Toward this goal, we designed a hydrogel suitable for the co-delivery of neural stem cells and chondroitinase ABC (ChABC), a potent enzyme that degrades the glial scar that forms after central nervous system (CNS) injury. We leveraged the inverse electron-demand Diels-Alder reaction between norbornene and methylphenyltetrazine to form rapidly gelling (<15 min) crosslinked methylcellulose (MC) hydrogels at physiological temperature and pH, with Young's modulus similar to that of brain tissue (1-3 kPa), and degradable, disulfide-containing crosslinkers. To achieve tunable, affinity-controlled release of a ChABC-Src homology 3 (SH3) fusion protein, we immobilized norbornene-functionalized SH3-binding peptides onto MC-methylphenyltetrazine and observed release of bioactive ChABC-SH3 over 4 days. We confirmed cytocompatibility by evaluating neural progenitor cell survival and proliferation. The combined encapsulation of neural stem cells and chondroitinase ABC from one hydrogel lays the framework for future in vivo studies to treat CNS injuries.

Lessons learned from intervertebral disc pathophysiology to guide rational design of sequential delivery systems for therapeutic biological factors.

Intervertebral disc (IVD) degeneration has been associated with low back pain, which is a major musculoskeletal disorder and socio-economic problem that affects as many as 600 million patients worldwide. Here, we first review the current knowledge of IVD physiology and physiopathological processes in terms of homeostasis regulation and consecutive events that lead to tissue degeneration. Recent progress with IVD restoration by anti-catabolic or pro-anabolic approaches are then analyzed, as are the design of macro-, micro-, and nano-platforms to control the delivery of such therapeutic agents. Finally, we hypothesize that a sequential delivery strategy that i) firstly targets the inflammatory, pro-catabolic microenvironment with release of anti-inflammatory or anti-catabolic cytokines; ii) secondly increases cell density in the less hostile microenvironment by endogenous cell recruitment or exogenous cell injection, and finally iii) enhances cellular synthesis of extracellular matrix with release of pro-anabolic factors, would constitute an innovative yet challenging approach to IVD regeneration.

Cationic block amphiphiles show anti-mitochondrial activity in multi-drug resistant breast cancer cells.

Currently, there are limited treatment options for multi-drug resistant breast cancer. Lipid-modified cationic peptides have the potential to reach the mitochondria, which are attractive targets for the treatment of multi-drug resistant (MDR) breast cancer; yet, little is known about their mitochondrial targeting and anti-cancer activity. Interestingly, lipid-modified cationic peptides, typically used as gene transfection agents, exhibit similar structural features to mitochondrial targeted peptides. Using octahistidine-octaarginine (H8R8) as a model cationic peptide for cell penetration and endosomal escape, we explored the anti-cancer potential of lipid-modified cationic peptides as a function of amphiphilicity, biodegradability and lipid structure. We found that cationic peptides modified with a lipid that is at least 12 carbons in length exhibit potent anti-cancer activity in the low micromolar range in both EMT6/P and EMT6/AR-1 breast cancer cells. Comparing degradable and non-degradable linkers, as well as L- and D-amino acid sequences, we found that the anti-cancer activity is mostly independent of the biodegradation of the lipid-modified cationic peptides. Two candidates, stearyl-H8R8 (Str-H8R8) and vitamin E succinate-H8R8 (VES-H8R8) were cytotoxic to cancer cells by mitochondria depolarization. We observed increased reactive oxygen species (ROS) production, reduced cell bioenergetics and drug efflux, triggering apoptosis and G1 cell cycle arrest. Compared to Str-H8R8, VES-H8R8 showed enhanced cancer cell selectivity and drug efflux inhibition, thereby serving as a potential novel therapeutic agent. This study deepens our understanding of lipid-modified cationic peptides and uncovers their potential in multi-drug resistant breast cancer.

Modeling of Photoreceptor Donor-Host Interaction Following Transplantation Reveals a Role for Crx, Müller Glia, and Rho/ROCK Signaling in Neurite Outgrowth.

The goal of photoreceptor transplantation is to establish functional synaptic connectivity between donor cells and second-order neurons in the host retina. There is, however, limited evidence of donor-host photoreceptor connectivity post-transplant. In this report, we investigated the effect of the host retinal environment on donor photoreceptor neurite outgrowth in vivo and identified a neurite outgrowth-promoting effect of host Crx(-/-) retinas following transplantation of purified photoreceptors expressing green fluorescent protein (GFP). To investigate the noncell autonomous factors that influence donor cell neurite outgrowth in vitro, we established a donor-host coculture system using postnatal retinal aggregates. Retinal cell aggregation is sensitive to several factors, including plate coating substrate, cell density, and the presence of Müller glia. Donor photoreceptors exhibit motility in aggregate cultures and can engraft into established aggregate structures. The neurite outgrowth-promoting phenotype observed in Crx(-/-) recipients in vivo is recapitulated in donor-host aggregate cocultures, demonstrating the utility of this surrogate in vitro approach. The removal of Müller glia from host aggregates reduced donor cell neurite outgrowth, identifying a role for this cell type in donor-host signaling. Although disruption of chondroitin sulfate proteoglycans in aggregates had no effect on the neurite outgrowth of donor photoreceptors, disruption of Rho/ROCK signaling enhanced outgrowth. Collectively, these data show a novel role of Crx, Müller glia, and Rho/ROCK signaling in controlling neurite outgrowth and provide an accessible in vitro model that can be used to screen for factors that regulate donor-host connectivity. Stem Cells 2019;37:529-541.

Controlled release strategy designed for intravitreal protein delivery to the retina.

Therapeutic protein delivery directly to the eye is a promising strategy to treat retinal degeneration; yet, the high risks of local drug overdose and cataracts associated with bolus injection have limited progress, requiring the development of sustained protein delivery strategies. Since the vitreous humor itself is a gel, hydrogel-based release systems are a sensible solution for sustained intravitreal protein delivery. Using ciliary neurotrophic factor (CNTF) as a model protein for ocular treatment, we investigated the use of an intravitreal, affinity-based release system for protein delivery. To sustain CNTF release, we took advantage of the affinity between Src homology 3 (SH3) and its peptide binding partners: CNTF was expressed as a fusion protein with SH3, and a thermogel of hyaluronan and methylcellulose (HAMC) was modified with SH3 binding peptides. Using a mathematical model, the hydrogel composition was successfully designed to release CNTF-SH3 over 7 days. The stability and bioactivity of the released protein were similar to those of commercial CNTF. Intravitreal injections of the bioengineered thermogel showed successful delivery of CNTF-SH3 to the mouse retina, with expected transient downregulation of phototransduction genes (e.g., rhodopsin, S-opsin, M-opsin, Gnat 1 and 2), upregulation of STAT1 and STAT3 expression, and upregulation of STAT3 phosphorylation. This constitutes the first demonstration of intravitreal protein release from a hydrogel. Immunohistochemical analysis of the retinal tissues of injected eyes confirmed the biocompatibility of the delivery vehicle, paving the way towards new intravitreal protein delivery strategies.

Radical Copolymerization of Vinyl Ethers and Cyclic Ketene Acetals as a Versatile Platform to Design Functional Polyesters.

Free-radical copolymerization of cyclic ketene acetals (CKAs) and vinyl ethers (VEs) was investigated as an efficient yet simple approach for the preparation of functional aliphatic polyesters. The copolymerization of CKA and VE was first predicted to be quasi-ideal by DFT calculations. The theoretical prediction was experimentally confirmed by the copolymerization of 2-methylene-1,3-dioxepane (MDO) and butyl vinyl ether (BVE), leading to rMDO =0.73 and rBVE =1.61. We then illustrated the versatility of this approach by preparing different functional polyesters: 1) copolymers functionalized by fluorescent probes; 2) amphiphilic copolymers grafted with poly(ethylene glycol) (PEG) side chains able to self-assemble into PEGylated nanoparticles; 3) antibacterial films active against Gram-positive and Gram-negative bacteria (including a multiresistant strain); and 4) cross-linked bioelastomers with suitable properties for tissue engineering applications.

Local Affinity Release.

The use of hydrogels for therapeutic delivery is a burgeoning area of investigation. These water-swollen polymer matrices are ideal platforms for localized drug delivery that can be further combined with specific ligands or nanotechnologies to advance the controlled release of small-molecule drugs and proteins. Due to the advantage of hydrophobic, electrostatic, or specific extracellular matrix interactions, affinity-based strategies can overcome burst release and challenges associated with encapsulation. Future studies will provide innovative binding tools, truly stimuli-responsive systems, and original combinations of emerging technologies to control the release of therapeutics spatially and temporally. Local drug delivery can be achieved by directly injecting a therapeutic to its site of action and is advantageous because off-target effects associated with systemic delivery can be minimized. For prolonged benefit, a vehicle that provides sustained drug release is required. Hydrogels are versatile platforms for localized drug release, owing to the large library of biocompatible building blocks from which they can be formed. Injectable hydrogel formulations that gel quickly in situ and provide sustained release of therapeutics are particularly advantageous to minimize invasiveness. The incorporation of polymers, ligands or nanoparticles that have an affinity for the therapeutic of interest improve control over the release of small-molecule drugs and proteins from hydrogels, enabling spatial and temporal control over the delivery. Such affinity-based strategies can overcome drug burst release and challenges associated with protein instability, allowing more effective therapeutic molecule delivery for a range of applications from therapeutic contact lenses to ischemic tissue regeneration.

Delivery strategies for treatment of age-related ocular diseases: From a biological understanding to biomaterial solutions.

Age-related ocular diseases, such as age-related macular degeneration (AMD), diabetic retinopathy, and glaucoma, result in life-long functional deficits and enormous global health care costs. As the worldwide population ages, vision loss has become a major concern for both economic and human health reasons. Due to recent research into biomaterials and nanotechnology major advances have been gained in the field of ocular delivery. This review provides a summary and discussion of the most recent strategies employed for the delivery of both drugs and cells to the eye to treat a variety of age-related diseases. It emphasizes the current challenges and limitations to ocular delivery and how the use of innovative materials can overcome these issues and ultimately provide treatment for age-related degeneration and regeneration of lost tissues. This review also provides critical considerations and an outlook for future studies in the field of ophthalmic delivery.

Degradable vinyl polymers for biomedical applications.

Vinyl polymers have been the focus of intensive research over the past few decades and are attractive materials owing to their ease of synthesis and their broad diversity of architectures, compositions and functionalities. Their carbon-carbon backbones are extremely resistant to degradation, however, and this property limits their uses. Degradable polymers are an important field of research in polymer science and have been used in a wide range of applications spanning from (nano)medicine to microelectronics and environmental protection. The development of synthetic strategies to enable complete or partial degradation of vinyl polymers is, therefore, of great importance because it will offer new opportunities for the application of these materials. This Review captures the most recent and promising approaches to the design of degradable vinyl polymers and discusses the potential of these materials for biomedical applications.

Correction: A ring to rule them all: a cyclic ketene acetal comonomer controls the nitroxide-mediated polymerization of methacrylates and confers tunable degradability.

Correction for 'A ring to rule them all: a cyclic ketene acetal comonomer controls the nitroxide-mediated polymerization of methacrylates and confers tunable degradability' by Vianney Delplace et al., Chem. Commun., 2015, DOI: 10.1039/c5cc04610f.

A ring to rule them all: a cyclic ketene acetal comonomer controls the nitroxide-mediated polymerization of methacrylates and confers tunable degradability.

2-Methylene-4-phenyl-1,3-dioxolane (MPDL) was successfully used as a controlling comonomer in NMP with oligo(ethylene glycol) methyl ether methacrylate (MeOEGMA) to prepare well-defined and degradable PEG-based P(MeOEGMA-co-MPDL) copolymers. The level of ester group incorporation is controlled, leading to reductions in molecular weight of up to 95% on hydrolysis. Neither the polymer nor its degradation products displayed cytoxicity. The method was also successfully applied to methyl methacrylate.

Nitroxide-mediated radical ring-opening copolymerization: chain-end investigation and block copolymer synthesis.

Well-defined, degradable copolymers are successfully prepared by nitroxide-mediated radical ring opening polymerization (NMrROP) of oligo(ethylene glycol) methyl ether methacrylate (OEGMA) or methyl methacrylate (MMA), a small amount of acrylonitrile (AN) and cyclic ketene acetals (CKAs) of different structures. Phosphorous nuclear magnetic resonance allows in-depth chain-end characterization and gives crucial insights into the nature of the copoly-mer terminal sequences and the living chain fractions. By using a small library of P(OEGMA-co-AN-co-CKA) and P(MMA-co-AN-co-CKA) as macroinitiators, chain extensions with styrene are performed to furnish (amphiphilic) block copolymers comprising a degradable segment.

RGD decoration of PEGylated polyester nanocapsules of perfluorooctyl bromide for tumor imaging: influence of pre or post-functionalization on capsule morphology.

PEGylated polyester nanocapsules of perfluorooctyl bromide (PFOB) were surface-decorated with a RGD (arginine-glycine-aspartic acid) peptide by either pre-functionalization or post-functionalization strategies using carbodiimide-assisted chemistry. Both strategies allowed successful linkage of RGD at the surface of nanocapsules with up to 600-950 peptide units per nanocapsule without modifying the encapsulation efficacy of PFOB used as the (19)F MRI imaging moiety. Cryo-Transmission Electron Microscopy images evidence that slight changes of the polymer used to form the capsule shell strongly influence nanocapsule morphology. While, the use of copolymer blends induces the formation of acorn morphologies, PLA-b-PEG-COOH leads to elongated and "tears of wine"-like nanoconstructs. In vivo evaluation in mice bearing CT26 tumors by (19)F MRI reveals no significant difference of accumulation between PEGylated and RGD-decorated nanocapsules obtained by the post-functionalization approach (highest RGD density/capsule).