Controlled release from PCL-alginate microspheres via secondary encapsulation using GelMA/HAMA hydrogel scaffolds.

Controlling the release of bioactive agents has important potential applications in tissue engineering. While microspheres have been investigated to manipulate release rates, the majority of these investigations have been based on delivery into aqueous media, whereas the cellular environment in tissue engineering is more typically a hydrogel scaffold. If drug-loaded microspheres are introduced within scaffolds to deliver biologically active substances in situ, it is crucial to understand how the release rate is influenced by interactions between the microspheres and the scaffold. Here, we report the fabrication and characterization of a biodegradable scaffold that contains composite microspheres and is suitable for biological applications. Our approach evaluates the influence on the release profile of a model drug (FITC-dextran sulfate) from alginate and PCL-alginate microspheres within a hydrogel construct forming a secondary encapsulation. Increasing the degree of crosslinking in the secondary encapsulation matrix led to a slower cumulative release from 36% to 15%, from the alginate microspheres, whereas a decrease from 26% to 6% was observed for the PCL-alginate microspheres. These results suggest that the release of bioactive molecules can be fine tuned by independently engineering the properties of the scaffold and microspheres.

A dynamically loaded ex vivo model to study neocartilage and integration in human cartilage repair.

Articular cartilage injuries in the knee can lead to post-traumatic osteoarthritis if untreated, causing debilitating problems later in life. Standard surgical treatments fail to ensure long lasting repair of damaged cartilage, often resulting in fibrotic tissue. While there is a vast amount of research into cartilage regeneration, integrating engineered implants with cartilage remains a challenge. As cartilage is a load bearing tissue, it is imperative to evaluate tissue repair strategies and their ability to integrate under mechanical loading. This work established a dynamically loaded ex vivo model of cartilage repair using human cartilage explants. The model was used to assess the efficacy of a stem cell therapy delivered in a bioadhesive hydrogel comprised of photocrosslinkable gelatin methacryloyl (GelMA) and microbial transglutaminase to repair the model defect. Extensive neocartilage production and integration were observed via histology and immunohistochemistry after 28 days chondrogenic culture. Analysis of culture media allowed monitoring of glycosaminoglycan and type II collagen production over time. A mechanical assessment of integration via a push out test showed a 15-fold increase in push out strength over the culture duration. The model was successful in exhibiting robust chondrogenesis with transglutaminase or without, and under both culture conditions. The work also highlights several limitations of ex vivo models and challenges of working with bioreactors that must be overcome to increase their utility. This ex vivo model has the potential to delay the need for costly pre-clinical studies and provide a more nuanced assessment of cartilage repair strategies than is possible in vivo.

Controlled oxygen delivery to power tissue regeneration.

Oxygen plays a crucial role in human embryogenesis, homeostasis, and tissue regeneration. Emerging engineered regenerative solutions call for novel oxygen delivery systems. To become a reality, these systems must consider physiological processes, oxygen release mechanisms and the target application. In this review, we explore the biological relevance of oxygen at both a cellular and tissue level, and the importance of its controlled delivery via engineered biomaterials and devices. Recent advances and upcoming trends in the field are also discussed with a focus on tissue-engineered constructs that could meet metabolic demands to facilitate regeneration.

The impact of electrical stimulation protocols on neuronal cell survival and proliferation using cell-laden GelMA/graphene oxide hydrogels.

The development of electroactive cell-laden hydrogels (bioscaffolds) has gained interest in neural tissue engineering research due to their inherent electrical properties that can induce the regulation of cell behaviour. Hydrogels combined with electrically conducting materials can respond to external applied electric fields, where these stimuli can promote electro-responsive cell growth and proliferation. A successful neural interface for electrical stimulation should present the desired stable electrical properties, such as high conductivity, low impedance, increased charge storage capacity and similar mechanical properties related to a target neural tissue. We report how different electrical stimulation protocols can impact neuronal cells' survival and proliferation when using cell-laden GelMA/GO hydrogels. The rat pheochromocytoma cell line, PC12s encapsulated into hydrogels showed an increased proliferation behaviour with increasing current amplitudes applied. Furthermore, the presence of GO in GelMA hydrogels enhanced the metabolic activity and DNA content of PC12s compared with GelMA alone. Similarly, hydrogels provided survival of encapsulated cells at higher current amplitudes when compared to cells seeded onto ITO flat surfaces, which expressed significant cell death at a current amplitude of 2.50 mA. Our findings provide new rational choices for electroactive hydrogels and electrical stimulation with broad potential applications in neural tissue engineering research.

Biodegradable bioelectronics for biomedical applications.

Biodegradable polymers have been widely used in tissue engineering with the potential to be replaced by regenerative tissue. While conventional bionic interfaces are designed to be implanted in living tissue and organs permanently, biocompatible and biodegradable electronic materials are now progressing a paradigm shift towards transient and regenerative bionic engineering. For example, biodegradable bioelectronics can monitor physiologies in a body, transiently rehabilitate disease symptoms, and seamlessly form regenerative interfaces from synthetic electronic devices to tissues by reducing inflammatory foreign-body responses. Conventional electronic materials have not readily been considered biodegradable. However, several strategies have been adopted for designing electroactive and biodegradable materials systems: (1) conductive materials blended with biodegradable components, (2) molecularly engineered conjugated polymers with biodegradable moieties, (3) naturally derived conjugated biopolymers, and (4) aqueously dissolvable metals with encapsulating layers. In this review, we endeavor to present the technical bridges from electrically active and biodegradable material systems to edible and biodegradable electronics as well as transient bioelectronics with pre-clinical bio-instrumental applications, including biodegradable sensors, neural and tissue engineering, and intelligent drug delivery systems.

Active and passive drug release by self-assembled lubricin (PRG4) anti-fouling coatings.

Electroactive polymers (EAPs) have been investigated as materials for use in a range of biomedical applications, ranging from cell culture, electrical stimulation of cultured cells as well as controlled delivery of growth factors and drugs. Despite their excellent drug delivery ability, EAPs are susceptible to biofouling thus they often require surface functionalisation with antifouling coatings to limit unwanted non-specific protein adsorption. Here we demonstrate the surface modification of para toluene sulfonate (pTS) doped polypyrrole with the glycoprotein lubricin (LUB) to produce a self-assembled coating that both prevents surface biofouling while also serving as a high-capacity reservoir for cationic drugs which can then be released passively via diffusion or actively via an applied electrical potential. We carried out our investigation in two parts where we initially assessed the antifouling and cationic drug delivery ability of LUB tethered on a gold surface using quartz crystal microbalance with dissipation monitoring (QCM) to monitor molecular interactions occurring on a gold sensor surface. After confirming the ability of tethered LUB nano brush layers on a gold surface, we introduced an electrochemically grown EAP layer to act as the immobilisation surface for LUB before subsequently introducing the cationic drug doxorubicin hydrochloride (DOX). The release of cationic drug was then investigated under passive and electrochemically stimulated conditions. High-performance liquid chromatography (HPLC) was then carried out to quantify the amount of DOX released. It was shown that the amount of DOX released from nano brush layers of LUB tethered on gold and EAP surfaces could be increased by up to 30% per minute by applying a positive electrochemically stimulating pulse at 0.8 V for one minute. Using bovine serum albumin (BSA), we show that DOX loaded LUB tethered on para toluene sulfonic acid (pTS) doped polypyrrole retained antifouling ability of up to 75% when compared to unloaded tethered LUB. This work demonstrates the unique, novel ability of tethered LUB to actively participate in the delivery of cationic therapeutics on different substrate surfaces. This study could lead to the development of versatile multifunctional biomaterials for use in wide range of biomedical applications, such as dual drug delivery and lubricating coatings, dual drug delivery and antifouling coatings, cellular recording and stimulation.

Within or Without You? A Perspective Comparing In Situ and Ex Situ Tissue Engineering Strategies for Articular Cartilage Repair.

Human articular cartilage has a poor ability to self-repair, meaning small injuries often lead to osteoarthritis, a painful and debilitating condition which is a major contributor to the global burden of disease. Existing clinical strategies generally do not regenerate hyaline type cartilage, motivating research toward tissue engineering solutions. Prospective cartilage tissue engineering therapies can be placed into two broad categories: i) Ex situ strategies, where cartilage tissue constructs are engineered in the lab prior to implantation and ii) in situ strategies, where cells and/or a bioscaffold are delivered to the defect site to stimulate chondral repair directly. While commonalities exist between these two approaches, the core point of distinction-whether chondrogenesis primarily occurs "within" or "without" (outside) the body-can dictate many aspects of the treatment. This difference influences decisions around cell selection, the biomaterials formulation and the surgical implantation procedure, the processes of tissue integration and maturation, as well as, the prospects for regulatory clearance and clinical translation. Here, ex situ and in situ cartilage engineering strategies are compared: Highlighting their respective challenges, opportunities, and prospects on their translational pathways toward long term human cartilage repair.

Microencapsulation of growth factors by microfluidic system.

The encapsulation of growth factors is an important component of tissue engineer- ing. Using microspheres is a convenient approach in which the dose of factors can be regulated by increasing or decreasing the number of encapsulated microspheres. Moreover, microspheres offer the possibility of delivering the growth factors directly to the target site. However, the fabrication of microspheres by traditional emulsion methods is largely variable due to the experimental procedure. We have developed a protocol using a commercially available microfluidic system that allows formation of tunable particle-size droplets loaded with growth factors. The methodology includes a guide for preparing an alginate-growth factors solution followed by the specific set-up needed for using the microfluidic system to form the microspheres. The pro- cedure also includes a unique post-crosslinking process without pH modification. These methods allow the preservation of integrity and bioactivity of the growth factors tested (BMP-6 and TGFß -3) and their subsequent sustained delivery.•The protocol can be tuned to form particles of various sizes.•The gentle post-crosslinking process allows conformational integrity of various bioactive molecules.

Formation of alginate microspheres prepared by optimized microfluidics parameters for high encapsulation of bioactive molecules.

Drug delivery systems such as microspheres have shown potential in releasing biologicals effectively for tissue engineering applications. Microfluidic systems are especially attractive for generating microspheres as they produce microspheres of controlled-size and in low volumes, using micro-emulsion processes. However, the flow rate dependency on the encapsulation of molecules at a microscale is poorly understood. In particular, the flow rate and pressure parameters might influence the droplet formation and drug encapsulation efficiency. We evaluated the parameters within a two-reagent flow focusing microfluidic chip under continuous formation of hydrogel particles using a flourinated oil and an ionic crosslinkable alginate hydrogel. Fluorescein isothiocyanate-dextran sulfate (FITC-dextran sulfate MW: 40 kDa) was used to evaluate the variation of the encapsulation efficiency with the flow parameters, optimizing droplets and microsphere formation. The ideal flow rates allowing for maximum encapsulation efficiency, were utilised to form bioactive microspheres by delivering transforming growth factor beta-3 (TGFß-3) in cell culture media. Finally, we evaluated the potential of microfluidic-formed microspheres to be included within biological environments. The biocompatibility of the microspheres was tested over 28 days using adult human mesenchymal stem cells (hMSCs). The release profile of the growth factors from microspheres showed a sustained release in media, after an initial burst, up to 30 days. The metabolic activity of the cells cultured in the presence of the microspheres was similar to controls, supporting the biocompatibility of this approach. The fine-tuned parameters for alginate hydrogel to form microspheres have potential in encapsulating and preserving functional structure of bioactive agents for future tissue engineering applications.

FLASH: Fluorescently LAbelled Sensitive Hydrogel to monitor bioscaffolds degradation during neocartilage generation.

Regenerative therapies based on photocrosslinkable hydrogels and stem cells are of growing interest in the field of cartilage repair. Cell-mediated degradation is critical for the successful clinical translation of implanted hydrogels. However, characterising cell-mediated degradation, while simultaneously monitoring the deposition of a distinct new matrix, remains a major challenge. In this study we generated a Fluorescently LAbelled Sensitive Hydrogel (FLASH) to correlate the degradation of a hydrogel bioscaffold with neocartilage formation. Gelatine Methacryloyl (GelMA) was covalently bound to the FITC fluorophore to generate FLASH and bioscaffolds were produced by casting different concentrations of FLASH GelMA, with and without human adipose-derived stem cells (hADSCs) undergoing chondrogenesis. The loss of fluorescence from FLASH bioscaffolds was correlated with changes in mechanical properties, expression of chondrogenic markers and accumulation of a cartilaginous extracellular matrix. The ability of the system to be used as a sensor to monitor bioscaffold degradability during chondrogenesis was evaluated in vitro, in a human ex vivo model of cartilage repair and in a full chondral defect in vivo rabbit model. This study represents a step towards the generation of a high throughput monitoring system to evaluate de novo cartilage formation in tissue engineering therapies.

Growth factor delivery: Defining the next generation platforms for tissue engineering.

Growth factors play a crucial role in tissue engineering by directing the fate of cells and allowing the formation of tissues. Understanding the key requirements for growth factor delivery can point the way towards novel tissue engineering platforms. Growth factor suffer from a tendency to lose their bioactivity upon environmental and physical changes. The need for biomaterials that can preserve the native form of growth factor has directed interest towards hydrogels (e.g. gelatin, alginate) and hydrophobic polymers (e.g. polycaprolactone). In recent advances, the use of composite materials such as PCL-chitosan (hydrogel-hydrophobic polymer) and gelatin-alginate (hydrogel-hydrogel polymers) has opened the possibility of fine tuning the delivery systems. Other current research is exploring new delivery strategies for growth factors such as the use of bioreactors and delivery by molecule-recognition. In particular, systems that allow the delivery of growth factors in a 3-dimensional manner are promising compared to conventional methods. This review analyses the biological considerations, material selection, and delivery strategies that have been established as the pivotal components for growth factor delivery and will support the next generation of tissue engineering platforms by providing a comprehensive landscape for the growth factor delivery field. The last part of the review discusses the current challenges and promising future directions for delivering growth factors.