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Cartilage damage typically starts at its surface, either due to wear or trauma. Treatment of these superficial defects is important in preventing degradation and osteoarthritis (OA). Biomaterials currently used for deep cartilage defects lack appropriate properties for this application. Therefore, we investigated photo-crosslinked methacrylamide-modified gelatin (gelMA) as a candidate for treatment of surface defects. It allows for liquid application, filling of surface defects and forming a protective layer after UV-crosslinking, thereby keeping therapeutic cells in place. GelMA and photo-initiator (Li-TPO) concentration were optimized for application as a carrier to create a favourable environment for human articular chondrocytes (hAC). Primary hAC were used in passages 3 and 5, encapsulated into two different gelMA concentrations (7.5 wt% (soft) and 10 wt% (stiff)) and cultivated for 3 weeks with TGF-β3 (0, 1 and 10 ng/mL). Higher TGF-β3 concentrations induced spherical cell morphology independent of gelMA stiffness, while low TGF-β3 concentrations only induced rounded morphology in stiff gelMA. Gene expression did not vary across gel stiffnesses. As a functional model gelMA was loaded with two different cell types (hAC and/or human adipose-derived stem cells (ASC/TERT1) and applied to human osteochondral osteoarthritic plugs. GelMA attached to the cartilage, smoothened the surface and retained cells in place. Resistance against shear forces was tested using a tribometer, simulating normal human gait and revealing maintained cell viability. In conclusion gelMA is a versatile, biocompatible material with good bonding capabilities to cartilage matrix, allowing sealing and smoothening of superficial cartilage defects while simultaneously delivering therapeutic cells for tissue regeneration. This article is protected by copyright. All rights reserved.

Several clinical studies have been conducted into the practicality and safety of regenerative therapy using hESC/iPSC-retinal pigment epithelium (RPE) as a treatment for the diseases including age-related macular degeneration. These studies used either suspensions of RPE cells or an RPE cell sheet. The cells can be injected using a minimally invasive procedure but the delivery of an intended number of cells at an exact target location is difficult; cell sheets take a longer time to prepare, and the surgical procedure is invasive but can be placed at the target area. In the research reported here, we combined the advantages of the two approaches by producing a quickly formed hiPSC-RPE strip in as short as 2 days. The strip readily expanded into a monolayer sheet on the plate, and after transplantation in nude rats, it showed a potency to partly expand with the correct apical/basal polarity in vivo, although limited in expansion area in the presence of healthy host RPE. The strip could be injected into a target area in animal eyes using a 24G canula tip.

Cell transplants in therapeutic studies do not preserve their long-term function inside the donor body. In mesenchymal stem cell (MSC) transplants, transplanted cells disperse through the body and are prone to degradation by immune cells after the transplant process. Various strategies, such as usage of the immunosuppressive drugs to eliminate allograft rejection, are designed to increase the efficiency of cell therapy. Another strategy is the construction of biomimetic encapsulates using polymeric materials, which isolate stem cells and protect them from environmental effects. In this study, fibroblasts (L929) and MSCs were investigated for their improved viability and functionality once encapsulated inside the alginate microbeads under in vitro conditions for up to 12 days of incubation. Thus, uniform and injectable (

Peripheral artery disease is a progressive, devastating disease that leads to critical limb ischemia (CLI). Therapeutic angiogenesis using stem cell therapy has emerged as a promising approach for its treatment; however, adapting cell-based therapy has been limited by poor cell survival and low treatment efficiency. To overcome unmet clinical needs, we developed a fibroblast growth factor 2 (FGF2)-immobilized matrix that enabled control of cell adhesion to the surface and exerted a priming effect on the cell. Human adipose-derived stem cells (hASCs) grown in this matrix formed a functionally enhanced cells spheroid (FECS-Ad) that secreted various angiogenic factors including interleukin-8 (IL-8). We demonstrated that IL-8 was upregulated by the FGF2-mediated priming effect during FECS-Ad formation. Immobilized FGF2 substrate induced stronger IL-8 expression than soluble FGF2 ligands, presumably through the FGFR1/JNK/NF-κB signaling cascade. In IL-8-silenced FECS-Ad, vascular endothelial growth factor (VEGF) expression was decreased and angiogenic potential was reduced. Intramuscular injection of FECS-Ad promoted angiogenesis and muscle regeneration in mouse ischemic tissue, while IL-8 silencing in FECS-Ad inhibited these effects. Taken together, our data demonstrate that IL-8 contributes to therapeutic angiogenesis and suggest that FECS-Ad generated using the MBP-FGF2 matrix might provide a reliable platform for developing therapeutic agents to treat CLI.

In orchestrating the wound healing process, the immune system plays a critical role. Hence, controlling the immune system to repair skin defects is an attractive approach. The highly complex immune system includes the coordinated actions of several immune cells, which can produce various inflammatory and antiinflammatory cytokines and affect the healing of skin wounds. This process can be optimized using biomaterials, bioactive molecules, and cell delivery. The present review discusses various immunomodulation strategies for supporting the healing of chronic wounds. In this regard, following the evolution of the immune system and its role in the wound healing mechanism, the interaction between the extracellular mechanism and immune cells for acceleration wound healing will be firstly investigated. Consequently, the immune-based chronic wounds will be briefly examined and the mechanism of progression, and conventional methods of their treatment are evaluated. In the following, various biomaterials-based immunomodulation strategies are introduced to stimulate and control the immune system to treat and regenerate skin defects. Other effective methods of controlling the immune system in wound healing which is the release of bioactive agents (such as antiinflammatory, antigens, and immunomodulators) and stem cell therapy at the site of injury are reviewed.

A significant barrier to implementation of cell-based therapies is providing adequate vascularization to provide oxygen and nutrients. Here we describe an approach for cell transplantation termed the Therapeutic Vascular Conduit (TVC), which uses an acellular vessel as a scaffold for a hydrogel sheath containing cells designed to secrete a therapeutic protein. The TVC can be directly anastomosed as a vascular graft. Modeling supports the concept that the TVC allows oxygenated blood to flow in close proximity to the transplanted cells to prevent hypoxia. As a proof-of-principle study, we used erythropoietin (EPO) as a model therapeutic protein. If implanted as an arteriovenous vascular graft, such a construct could serve a dual role as an EPO delivery platform and hemodialysis access for patients with end-stage renal disease. When implanted into nude rats, TVCs containing EPO-secreting fibroblasts were able to increase serum EPO and hemoglobin levels for up to 4 weeks. However, constitutive EPO expression resulted in macrophage infiltration and luminal obstruction of the TVC, thus limiting longer-term efficacy. Follow-up in vitro studies support the hypothesis that EPO also functions to recruit macrophages. The TVC is a promising approach to cell-based therapeutic delivery that has the potential to overcome the oxygenation barrier to large-scale cellular implantation and could thus be used for a myriad of clinical disorders. However, a complete understanding of the biological effects of the selected therapeutic is absolutely essential.

Cell therapies for the treatment of skin disorders could benefit from simple, safe and efficient technology for the transdermal delivery of therapeutic cells. Conventional cell delivery by hypodermic-needle injection is associated with poor patient compliance, requires trained personnel, generates waste and has non-negligible risks of injury and infection. Here, we report the design and proof-of-concept application of cryogenic microneedle patches for the transdermal delivery of living cells. The microneedles are fabricated by stepwise cryogenic micromoulding of cryogenic medium with pre-suspended cells, and can be easily inserted into porcine skin and dissolve after deployment of the cells. In mice, cells delivered by the cryomicroneedles retained their viability and proliferative capability. In mice with subcutaneous melanoma tumours, the delivery of ovalbumin-pulsed dendritic cells via the cryomicroneedles elicited higher antigen-specific immune responses and led to slower tumour growth than intravenous and subcutaneous injections of the cells. Biocompatible cryomicroneedles may facilitate minimally invasive cell delivery for a range of cell therapies. Cryogenic microneedle patches can deliver mammalian cells before dissolving into the skin, as shown with the transdermal delivery of ovalbumin-pulsed dendritic cells in mice with subcutaneous tumours.

To effectively apply microwell array cell delivery devices their biodegradation rate must be tailored towards their intended use and implantation location. Two microwell array devices with distinct degradation profiles, either suitable for the fabrication of retrievable systems in the case of slow degradation, or cell delivery systems capable of extensive remodeling using a fast degrading polymer, were compared in this study. Thin films of a poly(ethylene glycol)‐poly(butylene terephthalate) (PEOT‐PBT) and a poly(ester urethane) were evaluated for their in vitro degradation profiles over 34 weeks incubation in PBS at different pH values. The PEOT‐PBT films showed minimal in vitro degradation over time, while the poly(ester urethane) films showed extensive degradation and fragmentation over time. Subsequently, microwell array cell delivery devices were fabricated from these polymers and intraperitoneally implanted in Albino Oxford rats to study their biocompatibility over a 12‐week period. The PEOT‐PBT implants shown to be capable to maintain the microwell structure over time. Implants provoked a foreign body response resulting in multilayer fibrosis that integrated into the surrounding tissue. The poly(ester urethane) implants showed a loss of the microwell structures over time, as well as a fibrotic response until the onset of fragmentation, at least 4 weeks post implantation. It was concluded that the PEOT‐PBT implants could be used as retrievable cell delivery devices while the poly(ester urethane) implants could be used for cell delivery devices that require remodeling within a 4–12 week period.

Prevascularized 3D microtissues have been shown to be an effective cell delivery vehicle for cardiac repair. To this end, our lab has explored the development of self-organizing, prevascularized human cardiac organoids by co-seeding human cardiomyocytes with cardiac fibroblasts, endothelial cells, and stromal cells into agarose microwells. We hypothesized that this prevascularization process is facilitated by the endogenous upregulation of hypoxia-inducible factor (HIF) pathway in the avascular 3D microtissues. In this study, we used Molidustat, a selective PHD (prolyl hydroxylase domain enzymes) inhibitor that stabilizes HIF-α, to treat human cardiac organoids, which resulted in 150 ± 61% improvement in endothelial expression (CD31) and 220 ± 20% improvement in the number of lumens per organoids. We hypothesized that the improved endothelial expression seen in Molidustat treated human cardiac organoids was dependent upon upregulation of VEGF, a well-known downstream target of HIF pathway. Through the use of immunofluorescent staining and ELISA assays, we determined that Molidustat treatment improved VEGF expression of non-endothelial cells and resulted in improved co-localization of supporting cell types and endothelial structures. We further demonstrated that Molidustat treated human cardiac organoids maintain cardiac functionality. Lastly, we showed that Molidustat treatment improves survival of cardiac organoids when exposed to both hypoxic and ischemic conditions in vitro. For the first time, we demonstrate that targeted HIF-α stabilization provides a robust strategy to improve endothelial expression and lumen formation in cardiac microtissues, which will provide a powerful framework for prevascularization of various microtissues in developing successful cell transplantation therapies.

We report a particulate cell delivery platform, toroidal spiral particles (TSPs), for continuous cell activation, expansion, and local sustained release. Biocompatible TSPs, generated by a self-assembly process of polymeric droplet sedimentation in an aqueous solution and subsequent polymer solidification, possess many engineering design flexibilities to manipulate the microenvironment of the cells to control cell proliferation, migration, and release kinetics. These millimeter-size particles with desired mechanical and physicochemical properties may be potentially used for adoptive cellular therapy (ACT) delivery by a minimally invasive procedure to the tumor mass.

Self-healing injectable hydrogel biomaterials uniquely enable precise therapeutic deposition and deployment at specific bodily locations through versatile and minimally invasive processes that can preserve cargo integrity and cell viability. Despite the distinct advantages that injectable hydrogels offer in tissue engineering and therapeutic delivery, exceptionally few have been created using components naturally present in the cellular niche. In this work, we introduce a shear-thinning hydrogel based on guest-host complexation of gelatin. As a biocompatible, biodegradable, and non-immunogenic biopolymer derived from the most abundant extracellular matrix protein (collagen), gelatin offers great utility as the structural component of biomaterials. Taking advantage of reversible guest-host interactions between β-cyclodextrin (CD) and adamantane (AD) on modified gelatins, we report the first strategy to afford a self-healing material based solely on a functionalized extracellular matrix (ECM) protein. By varying the initial material formulation, hydrogels were synthesized with variable moduli and shear-thinability across a broad range. Gels were demonstrated to exhibit shear-thinning and self-healing properties, supporting protection of clinically relevant stem-cell-derived cardiomyocytes during injection. These materials are expected to expand clinical opportunities in cell delivery for in vivo tissue regeneration.

For tissue regeneration or repair, a suitable temporary scaffold needs to be constructed for delivering regenerative cells to damaged or diseased tissue. Scaffold types are currently categorised in...

Inadequate oxygenation is a major challenge in cell encapsulation, a therapy which holds potential to treat many diseases including type I diabetes. In such systems, cellular oxygen (O2) delivery is limited to slow passive diffusion from transplantation sites through the poorly O2-soluble encapsulating matrix, usually a hydrogel. This constrains the maximum permitted distance between the encapsulated cells and host site to within a few hundred micrometers to ensure cellular function. Inspired by the natural gas-phase tracheal O2 delivery system of insects, we present herein the design of a biomimetic scaffold featuring internal continuous air channels endowed with 10,000-fold higher O2 diffusivity than hydrogels. We incorporate the scaffold into a bulk hydrogel containing cells, which facilitates rapid O2 transport through the whole system to cells several millimeters away from the device-host boundary. A computational model, validated by in vitro analysis, predicts that cells and islets maintain high viability even in a thick (6.6 mm) device. Finally, the therapeutic potential of the device is demonstrated through the correction of diabetes in immunocompetent mice using rat islets for over 6 months., Cell encapsulation into biomaterials for transplantation is currently limited by inadequate oxygenation. Here the authors present a biomimetic scaffold featuring internal continuous air channels endowed with 10,000-fold higher O2 diffusivity than hydrogels and demonstrate correction of diabetes in immunocompetent mice using rat islets for over 6 months.

Stem cell-based therapy appears as a promising strategy to induce regeneration of damaged and diseased tissues. However, low survival, poor engraftment and a lack of site-specificity are major drawbacks. Polysaccharide hydrogels can address these issues and offer several advantages as cell delivery vehicles. They have become very popular due to their unique properties such as high-water content, biocompatibility, biodegradability and flexibility. Polysaccharide polymers can be physically or chemically crosslinked to construct biomimetic hydrogels. Their resemblance to living tissues mimics the native three-dimensional extracellular matrix and supports stem cell survival, proliferation and differentiation. Given the intricate nature of communication between hydrogels and stem cells, understanding their interaction is crucial. Cells are incorporated with polysaccharide hydrogels using various microencapsulation techniques, allowing generation of more relevant models and further enhancement of stem cell therapies. This paper provides a comprehensive review of human stem cells and polysaccharide hydrogels most used in regenerative medicine. The recent and advanced stem cell microencapsulation techniques, which include extrusion, emulsion, lithography, microfluidics, superhydrophobic surfaces and bioprinting, are described. This review also discusses current progress in clinical translation of stem-cell encapsulated polysaccharide hydrogels for cell delivery and disease modeling (drug testing and discovery) with focuses on musculoskeletal, nervous, cardiac and cancerous tissues.

Cell delivery through spray instruments is a promising and effective method in tissue engineering and regenerative medicine. It is used for treating different acute and chronic wounds, including burns with different etiologies, chronic diabetic or venous wounds, postcancer surgery, and hypopigmentation disorders. Cell spray can decrease the needed donor site area compared with conventional autologous skin grafting. Keratinocytes, fibroblasts, melanocytes, and mesenchymal stem cells are promising cell sources for cell spray procedures. Different spray instruments are designed and utilized to deliver the cells to the intended skin area. In an efficient spray instrument, cell viability and wound coverage are two determining parameters influenced by various physical and biological factors such as air pressure, spraying distance, viscosity of suspension, stiffness of the wound surface, and velocity of impact. Besides, to improve cell delivery by spray instruments, some matrices and growth factors can be added to cell suspensions. This review focuses on the different types of cells and spray instruments used in cell delivery procedures. It also discusses physical and biological parameters associated with cell viability and wound coverage in spray instruments. Moreover, the recent advances in codelivery of cells with biological glues and growth factors, as well as clinical translation of cell spraying, have been reviewed. Impact statement Skin wounds are a group of prevalent injuries that can lead to life-threatening complexities. As a focus of interest, stem cell therapy and spray-based cell delivery have effectively decreased associated morbidity and mortality. This review summarizes a broad scope of recent evidence related to spray-based cell therapy, instruments, and approaches adopted to make the process more efficient in treating skin wounds. An overview including utilized cell types, clinical cases, and current challenges is also provided.

Soft materials that facilitate the three-dimensional (3D) encapsulation, proliferation, and facile local delivery of cells to targeted tissues will aid cell-based therapies, especially those that depend on the local engraftment of implanted cells. Herein, we develop a negatively charged fibrillar hydrogel based on the de novo-designed self-assembling peptide AcVES3-RGDV. Cells are easily encapsulated during the triggered self-assembly of the peptide leading to gel formation. Self-assembly is induced by adjusting the ionic strength and/or temperature of the solution, while avoiding large changes in pH. The AcVES3-RGDV gel allows cell–material attachment enabling both two-dimensional and 3D cell culture of adherent cells. Gel–cell constructs display shear-thin/recovery rheological properties enabling their syringe-based delivery. In vivo cellular fluorescence as well as tissue resection experiments show that the gel supports the long-term engraftment of cells delivered subcutaneously into mice.

Graphical abstract, Cell-based therapies have shown significant promise in tissue engineering with one key challenge being the delivery and retention of cells. As a result, significant efforts have been made in the past decade to design injectable biomaterials to host and deliver cells at injury sites. Intervertebral disc (IVD) degeneration, a major cause of back pain, is a particularly relevant example where a minimally-invasive cellular therapy could bring significant benefits specifically at the early stages of the disease, when a cell-driven process starts in the gelatinous core of the IVD, the nucleus pulposus (NP). In this present study we explore the use of graphene oxide (GO) as nano-filler for the reinforcement of FEFKFEFK (β-sheet forming self-assembling peptide) hydrogels. Our results confirm the presence of strong interactions between FEFKFEFK and GO flakes with the peptide coating and forming short thin fibrils on the surface of the flakes. These strong interactions were found to affect the bulk properties of hybrid hydrogels. At pH 4 electrostatic interactions between the peptide fibres and the peptide-coated GO flakes are thought to govern the final bulk properties of the hydrogels while at pH 7, after conditioning with cell culture media, electrostatic interactions are removed leaving the hydrophobic interactions to govern hydrogel final properties. The GO-F820 hybrid hydrogel, with mechanical properties similar to the NP, was shown to promote high cell viability and retained cell metabolic activity in 3D over the 7 days of culture and therefore shown to harbour significant potential as an injectable hydrogel scaffold for the in-vivo delivery of NP cells. Statement of Significance Short self-assembling peptide hydrogels (SAPHs) have attracted significant interest in recent years as they can mimic the natural extra-cellular matrix, holding significant promise for the ab initio design of cells’ microenvironments. Recently the design of hybrid hydrogels for biomedical applications has been explored through the incorporation of specific nanofillers. In this study we exploited graphene oxide (GO) as nanofiller to design hybrid injectable 3Dscaffolds for the delivery of nucleus pulposus cells (NPCs) for intervertebral disc regeneration. Our work clearly shows the presence of strong interactions between peptide and GO, mimicking the mechanical properties of the NP tissue and promoting high cell viability and metabolic activity. These hybrid hydrogels therefore harbour significant potential as injectable scaffolds for the in vivo delivery of NPCs.

Combining stem cells with biomaterial scaffolds serves as a promising strategy for engineering tissues for both in vitro and in vivo applications. This updated review details commonly used biomaterial scaffolds for engineering tissues from stem cells. We first define the different types of stem cells and their relevant properties and commonly used scaffold formulations. Next, we discuss natural and synthetic scaffold materials typically used when engineering tissues, along with their associated advantages and drawbacks and gives examples of target applications. New approaches to engineering tissues, such as 3D bioprinting, are described as they provide exciting opportunities for future work along with current challenges that must be addressed. Thus, this review provides an overview of the available biomaterials for directing stem cell differentiation as a means of producing replacements for diseased or damaged tissues.

Ischemic heart disease is the leading cause of death globally by creating irreversible loss of cardiomyocytes and subsequent deterioration of cardiac function. To help repair the injured myocardium, promising approaches in regenerative medicine focus on rejuvenating the damaged heart. In this review, we highlight recent progress in molecular imaging applications for cardiac regenerative medicine in vivo including tracking cell delivery and retention, monitoring cell fates, evaluating therapeutic efficacy, and assessing side effects. We also discuss future directions to address two major challenges in this field, namely, improving the sensitivity of stem cell detection and advancing functional imaging of cell fates in vivo. We conclude that molecular imaging is crucial for facilitating regenerative medicine. Rapid progress in molecular imaging makes it a valuable tool to achieve the ultimate goal of improving cardiovascular outcomes.

Microgels have recently gained exposure as a potential scaffold either alone or in combination with other scaffold for bone tissue engineering. Microgel has been defined as a small size particle of...

Recent statistics report that more than 3.7 million new cases of cancer occur in Europe yearly, and the disease accounts for approximately 20% of all deaths. High-throughput screening of cancer cell cultures has dominated the search for novel, effective anticancer therapies in the past decades. Recently, functional assays with patient-derived ex vivo 3D cell culture have gained importance for drug discovery and precision medicine. We recently evaluated the major advancements and needs for the 3D cell culture screening, and concluded that strictly standardized and robust sample preparation is the most desired development. Here we propose an artificial intelligence-guided low-cost 3D cell culture delivery system. It consists of a light microscope, a micromanipulator, a syringe pump, and a controller computer. The system performs morphology-based feature analysis on spheroids and can select uniform sized or shaped spheroids to transfer them between various sample holders. It can select the samples from standard sample holders, including Petri dishes and microwell plates, and then transfer them to a variety of holders up to 384 well plates. The device performs reliable semi- and fully automated spheroid transfer. This results in highly controlled experimental conditions and eliminates non-trivial side effects of sample variability that is a key aspect towards next-generation precision medicine.

For bone tissue engineering, stem cell-based therapy has become a promising option. Recently, cell transplantation supported by polymeric carriers has been increasingly evaluated. Herein, we encapsulated human olfactory ectomesenchymal stem cells (OE-MSC) in the collagen hydrogel system, and their osteogenic potential was assessed in vitro and in vivo conditions. Collagen type I was composed of four different concentrations of (4 mg/mL, 5 mg/mL, 6 mg/mL, 7 mg/mL). SDS-Page, FTIR, rheologic test, resazurin assay, live/dead assay, and SEM were used to characterize collagen hydrogels. OE-MSCs encapsulated in the optimum concentration of collagen hydrogel and transplanted in rat calvarial defects. The tissue samples were harvested after 4- and 8-weeks post-transplantation and assessed by optical imaging, micro CT, and H&amp, E staining methods. The highest porosity and biocompatibility were confirmed in all scaffolds. The collagen hydrogel with 7 mg/mL concentration was presented as optimal mechanical properties close to the naïve bone. Furthermore, the same concentration illustrated high osteogenic differentiation confirmed by real-time PCR and alizarin red S methods. Bone healing has significantly occurred in defects treated with OE-MSCs encapsulated hydrogels in vivo. As a result, OE-MSCs with suitable carriers could be used as an appropriate cell source to address clinical bone complications.

Hydrogels have been widely used for cell delivery to enhance cell-based therapies for cartilage tissue regeneration. To better support cartilage deposition, it is imperative to determine hydrogel formulation with physical and biochemical cues that are optimized for different cell populations. Previous attempts to identify optimized hydrogels rely mostly on testing hydrogel formulations with discrete properties, which are time-consuming and require large amounts of cells and materials. Gradient hydrogels encompass a range of continuous changes in niche properties, therefore offering a promising solution for screening a wide range of cell-niche interactions using less materials and time. However, harnessing gradient hydrogels to assess how matrix stiffness modulates cartilage formation by different cell types in vivo have never been investigated before. The goal of this study is to fabricate gradient hydrogels for screening the effects of varying hydrogel stiffness on cartilage formation by mesenchymal stem cells (MSCs) and chondrocytes, respectively, the two most commonly used cell populations for cartilage regeneration. We fabricated stiffness gradient hydrogels with tunable dimensions that support homogeneous cell encapsulation. Using gradient hydrogels with tunable stiffness range, we found MSCs and chondrocytes exhibit opposite trend in cartilage deposition in response to stiffness changes in vitro. Specifically, MSCs require soft hydrogels with Young's modulus less than 5 kPa to support faster cartilage deposition, as shown by type II collagen and sulfated glycosaminoglycan staining. In contrast, chondrocytes produce cartilage more effectively in stiffer matrix (>20 kPa). We chose optimal ranges of stiffness for each cell population for further testing in vivo using a mouse subcutaneous model. Our results further validated that soft matrix (Young's modulus 20 kPa) is more desirable for supporting chondrocyte-based cartilage deposition. Our results show the importance of optimizing niche cues in a cell-type-specific manner and validate the potential of using gradient hydrogels for optimizing niche cues to support cartilage regeneration in vitro and in vivo. IMPACT STATEMENT: The present study validates the utility of gradient hydrogels for determining optimal hydrogel stiffness for supporting cartilage regeneration using both chondrocytes and stem cells. We demonstrate that such gradient hydrogels can be used for fast optimizing matrix stiffness for specific cell type to support optimal cartilage regeneration. To our knowledge, this is the first demonstration of applying gradient hydrogels for assessing optimal niche cues that support tissue regeneration in vivo and may be used for assessing optimal niche cues for different cell types to regeneration of different tissues.

Adipose-derived stem cells (ADSCs) have been widely researched and used as a drug therapy in many fields like disease treatment and tissue engineering. However, ADSCs are susceptible to the surrounding environment. The emergence of acellular extracellular matrix provides a solution, which can serve as biomaterial scaffold as well as original ecological niche for the stem cells. Therefore, we propose the hypothesis that human decellularized adipose tissues (hDAT) are processed into injectable hydrogels and then mixed evenly with ADSCs. So that the ADSCs embedded-hydrogels could directly carry the stem cells to the appropriate sites. The hDAT hydrogel could provide microenvironmental protection for ADSCs. In this study, we successfully made human decellularized adipose tissue hydrogel (hDAT-gel), which was temperature-sensitive, liquid at 4°C and semi-solid at 37°C. When the ADSCs were embedded in hDAT-gel, they survived well and continued to grow well in layers. When the pre-gel containing ADSCs was injected subcutaneously into nude mice, the sample results after 15 min showed gelation occurred in situ. These results suggested that hDAT-gel could provide a culture platform for ADSCs delivery.

Cell-based scaffold-free therapies seek to develop in vitro organotypic three-dimensional (3D) tissue-like surrogates, capitalising upon the inherent capacity of cells to create tissues with efficiency and sophistication that is still unparalleled by human-made devices. Although automation systems have been realised and (some) success stories have been witnessed over the years in clinical and commercial arenas, in vitro organogenesis is far from becoming a standard way of care. This limited technology transfer is largely attributed to scalability-associated costs, considering that the development of a borderline 3D implantable device requires very high number of functional cells and prolonged ex vivo culture periods. Herein, we critically discuss advancements and shortfalls of scaffold-free cell-based tissue engineering strategies, along with pioneering concepts that have the potential to transform regenerative and reparative medicine.

Background In recent years, adipose-derived stromal cells (ASCs) have been heavily studied for soft tissue regeneration, augmentation, and dermal wound healing. Methods In this review, we investigated the trends in injectable scaffolds for ASC delivery in the dermis, and injectable or implantable scaffolds for ASC delivery in the subcutis. A total of 547 articles were screened across three databases; of these, 22 studies were found to be eligible and were included. The scaffolds were subdivided and analyzed based on their tissue placement (dermis or subcutis), delivery method (injected or implanted), and by the origin of the materials (natural, synthetic, and combinatory). Results ASCs embedded in scaffolds generally showed improved viability. Neovascularization in the transplanted tissue was greater when undifferentiated ASCs were embedded in a combinatory scaffold or if differentiated ASCs were embedded in a natural scaffold. ASCs embedded in natural materials underwent more adipogenic differentiation than ASCs embedded in synthetic scaffolds, indicating an etiologically unknown difference that has yet to be described. Increased mechanical strength of the scaffold material correlated with improved outcome measurements in the investigated studies. Wound healing studies reported reduced healing time in all except one article due to contraction of the control wounds. Conclusions In future clinical trials, we recommend embedding ASCs in injectable and implantable scaffolds for enhanced protection, retained viability, and improved therapeutic effects. Trial registration This review was registered with PROSPERO: ID=CRD42020171534. Graphical abstract The use of scaffolds as a vehicle for ASC delivery generally improved cell viability, angiogenesis, and wound healing in vivo compared to utilizing ASCs alone. ASCs embedded in natural materials induced more adipogenesis than ASCs embedded in synthetic materials. Adipogenic-induced ASCs further increased this effect. The included studies indicate that the seeded scaffold material influences the differentiation of ASCs in vivo. All studies investigating the mechanical strength of ASC scaffolds reported improved outcome measurements with improved mechanical strength. The results suggest that scaffolds, in general, are favorable for ASC delivery. We recommend initiating clinical studies using scaffolds based on mechanical properties and tunability to improve ASC viability. For fat regeneration, natural scaffolds are recommended.

Emerging wearable and implantable biodevices have been significantly revolutionizing the diagnosis and treatment of disease. However, the geometrical mismatch between tissues and biodevices remains a great challenge for achieving optimal performances and functionalities for biodevices. Shape-adaptable biodevices enabling active compliance with human body tissues offer promising opportunities for addressing the challenge through programming their geometries on demand. This article reviews the design principles and control strategies for shape-adaptable biodevices with programmable shapes and actively compliant capabilities, which have offered innovative diagnostic/therapeutic tools and facilitated a variety of wearable and implantable applications. The state-of-the-art progress in applications of shape-adaptable biodevices in the fields of smart textiles, wound care, healthcare monitoring, drug and cell delivery, tissue repair and regeneration, nerve stimulation and recording, and biopsy and surgery, is highlighted. Despite the remarkable advances already made, shape-adaptable biodevices still confront many challenges on the road toward the clinic, such as enhanced intelligence for actively sensing and operating in response to physiological environments. Next-generation paradigms will shed light on future directions for extending the breadth and performance of shape-adaptable biodevices for wearable and implantable applications.

Discogenic back pain is a common condition without approved intervertebral disc (IVD) repair therapies. Cell delivery using injectable biomaterial carriers offers promise to restore disc height and biomechanical function, while providing a functional niche for delivered cells to repair degenerated tissues. This systematic review advances the injectable IVD cell delivery biomaterials field by characterising its current state and identifying themes of promising strategies. Preferred Reporting Items for Systematic Reviews and Meta- Analyses (PRISMA) guidelines were used to screen the literature and 183 manuscripts met the inclusion criteria. Cellular and biomaterial inputs, and biological and biomechanical outcomes were extracted from each study. Most identified studies targeted nucleus pulposus (NP) repair. No consensus exists on cell type or biomaterial carrier, yet most common strategies used mesenchymal stem cell (MSC) delivery with interpenetrating network/co-polymeric (IPN/CoP) biomaterials composed of natural biomaterials. All studies reported biological outcomes with about half the studies reporting biomechanical outcomes. Since the IVD is a load-bearing tissue, studies reporting compressive and shear moduli were analysed and two major themes were found. First, a competitive balance, or ‘seesaw’ effect, between biomechanical and biological performance was observed. Formulations with higher moduli had inferior cellular performance, and vice versa. Second, several low-modulus biomaterials had favourable biological performance and matured throughout culture duration with enhanced extracellular matrix synthesis and biomechanical moduli. Findings identify an opportunity to develop next-generation biomaterials that provide high initial biomechanical competence to stabilise and repair damaged IVDs with a capacity to promote cell function for long-term healing.

Therapeutic agents, such as drugs and cells, play an essential role in virtually every treatment of injury, illness, or disease. However, the conventional practices of drug delivery often result in undesirable side effects caused by drug overdose and off-target delivery. In the case of cell delivery, the survival rate of the transplanted cells is extremely low and difficulties with the administration route of cells remain a problem. Recently, magnetically actuated microrobots have started offering unique opportunities in targeted therapeutic delivery due to their tiny size and ability to access hard-to-reach lesions in a minimally invasive manner; considerable advances in this regard have been made over the past decade. Here, recent progress in magnetically actuated microrobots, developed for targeted drug/cell delivery, is presented, with a focus on their design features and mechanisms for controlled therapeutic release. Additionally, the practical challenges faced by the microrobots, and future research directions toward the swift bench-to-bedside translation of the microrobots are addressed.

Background: Hydrogels, a kind of three-dimensional (3-D) cross-linked polymer networks with higher water concentration, are receiving more and more attention in the recent years. Self-healing hydrogels, which can return to their original structure and function after physical damage, are especially attractive. Some self-healable hydrogels have several kinds of properties such as injectability, adhesiveness, conductivity, etc., which enable them to be used in the manufacture of drug/cell delivery vehicles, glues, electronic devices and so on. Main body: This review will focus on the self-healing hydrogel synthesis and applications s. Their repair mechanisms and potential applications in pharmaceutical, biomedical and others will be introduced. Conclusions: Self-healing hydrogels are used in various fields because of their ability to recover. Nowadays, new designs such as self-healing double-network (DN) hydrogels are developed to overcome the limitations of other soft materials, providing them with better mechanical properties. The prospect of self-healing hydrogels is promising and they may be further developed for more various applications.

A cell encapsulation system generates oxygen from carbon dioxide for self-oxygenation., Cell encapsulation represents a promising therapeutic strategy for many hormone-deficient diseases such as type 1 diabetes (T1D). However, adequate oxygenation of the encapsulated cells remains a challenge, especially in the poorly oxygenated subcutaneous site. Here, we present an encapsulation system that generates oxygen (O2) for the cells from their own waste product, carbon dioxide (CO2), in a self-regulated (i.e., “inverse breathing”) way. We leveraged a gas-solid (CO2–lithium peroxide) reaction that was completely separated from the aqueous cellular environment by a gas permeable membrane. O2 measurements and imaging validated CO2-responsive O2 release, which improved cell survival in hypoxic conditions. Simulation-guided optimization yielded a device that restored normoglycemia of immunocompetent diabetic mice for over 3 months. Furthermore, functional islets were observed in scaled-up device implants in minipigs retrieved after 2 months. This inverse breathing device provides a potential system to support long-term cell function in the clinically attractive subcutaneous site.

Neurodegeneration gives rise to a wide range of disorders which represent a growing health burden to both western societies and developing countries. Whilst for many disorders such as Alzheimer's and Parkinson's disease the cause is unknown, gene therapy is becoming the forefront of novel potential therapies described in the literature and has entered clinical trials. Furthermore, although in somewhat an earlier stage, biomaterials offer means of enhancing gene therapy strategies either through new delivery methods or provision of support for genetically manipulated cells. This review outlines recent uses of biomaterials in the CNS and captures recent advances in non-viral gene delivery to the brain. Three dimensional scaffolding systems for ex vivo gene delivery to the brain are also discussed highlighting the progress of hydrogel mediated cell delivery. This review also addresses the difficulties and safety considerations of these approaches; illustrating the ability of biomaterial strategies to significantly improve outcomes of gene therapies for neurodegenerative disorders.

Skeletal muscle is an ideal target for cell therapy. The use of its potent stem cell population in the form of autologous intramuscular transplantation represents a tantalizing strategy to slow the progression of congenital muscle diseases (such as Duchenne Muscular Dystrophy) or regenerate injured tissue following trauma. The syncytial nature of skeletal muscle uniquely permits the engraftment of stem/progenitor cells to contribute to new myonuclei and restore the expression of genes mutated in myopathies. Historically however, the implementation of this approach has been significantly limited by the inability to expand undifferentiated muscle stem cells (MuSCs) in culture whilst maintaining transplantation potential. This is crucial, as MuSC expansion and/or genetic manipulation is likely necessary for therapeutic applications. In this article, we review recent studies that have provided a number of important breakthroughs to tackle this problem. Progress towards this goal has been achieved by exploiting biochemical, biophysical and developmental paradigms to construct innovative in vitro strategies that are guiding stem cell therapies for muscle repair towards the clinic.

Protein microparticles assisted by an emulsion droplet template have shown great promise in drug/cell delivery and tissue engineering, as well as diagnosis and treatment of diseases. However, the usage of non-aqueous solvents involved in the oil-containing emulsion and their single-component nature severely hamper their use in medical applications. To address these limitations, here we present a facile strategy to fabricate dual-component protein (DCP) microparticles via the microfluidic electrospray technique. Due to the affinity partitioning properties of the all-aqueous system (AAS), electrostatic complexation takes place between the two oppositely charged proteins to form dual-component protein–protein structures in the all-aqueous droplets. We demonstrate that hemoglobin–bovine serum albumin (Hb–BSA) DCP microparticles possess characteristics resembling those of natural red blood cells, including remarkable softness and the ability to pass through narrow channels. Moreover, in vitro results show that other hemoglobin–immunoglobulin (Hb–BSA) DCP microparticles exhibit good cytocompatibility towards human stem cells. Accordingly, this work provides a novel engineering strategy using all-aqueous droplets as a template to design new biocompatible DCP microparticles for biomedical applications.

Safe hydrogel delivery requires stiffness-matching with host tissues to avoid iatrogenic damage and reduce inflammatory reactions. Hydrogel-encapsulated cell delivery is a promising combinatorial approach to spinal cord injury therapy, but a lack of in vivo clinical spinal cord injury stiffness measurements is a barrier to their use in clinics. We demonstrate that ultrasound elastography – a non-invasive, clinically established tool – can be used to measure spinal cord stiffness intraoperatively in canines with spontaneous spinal cord injury. In line with recent experimental reports, our data show that injured spinal cord has lower stiffness than uninjured cord. We show that the stiffness of hydrogels encapsulating a clinically relevant transplant population (olfactory ensheathing cells) can also be measured by ultrasound elastography, enabling synthesis of hydrogels with comparable stiffness to canine spinal cord injury. We therefore demonstrate proof-of-principle of a novel approach to stiffness-matching hydrogel-olfactory ensheathing cell implants to ‘real-life’ spinal cord injury values; an approach applicable to multiple biomaterial implants for regenerative therapies.

The treatment of chronic and acute wounds represents a significant challenge in the healthcare field. To overcome the limitations of current treatments and to improve the outcomes for burn and chronic wounds patients, new cell and tissue engineered therapies have been developed. Hydrogels have particularly emerged as an attractive platform for delivery of extracellular matrix, antimicrobials bioactive materials, and cells. Pioneering efforts in hydrogels used for wound healing were primarily for delivery of collagen and water to the wound bed. More complexity was added by incorporating antimicrobials including silver nitrate and antibiotics. Still others have explored the addition of cytokine and growth factors to create a novel hydrogel shown to improve revascularization and epithelialization in healing wounds. Next, the myriad of hydrogel compositions can be used as vehicles for delivery of primary skin cells through spraying or bioprinting. Using these technologies, researchers can leverage engineered biomaterials and potent biological components such as stem cells or cytokines to produce effective therapies to treat patients. This technology has progressed to even greater opportunities, including the production of full-thickness skin for patients with skin wounds. This chapter discusses skin regeneration by controlled delivery of cells through bioprinting and enabling technologies employed for skin regeneration, including the biomaterials and matrices that are integral in facilitating integration, drug, growth factor, and cell delivery using these materials to obtain the desired therapeutic effect.

Severe skin wounds are often associated with large areas of damaged tissue, resulting in substantial loss of fluids containing electrolytes and proteins. The net result is a vulnerability clinically to skin infections. Therapies aiming to close these large openings are effective in reducing the complications of severe skin wounds. Recently, cell transplantation therapy showed the potential for rapid re-epithelialization of severe skin wounds. Here, we show the improved effects of cell transplantation therapy using a robust protocol of efficient expansion and delivery of epidermal cells for treatment of severe skin wounds. Human skin tissues were used to generate human epidermal organoids maintained under newly established culture conditions. The human epidermal organoids showed an improved capacity of passaging for at least 10 rounds, enabling organoids to expand to cell numbers required for clinical applications. A newly designed auto micro-atomization device (AMAD) was developed for delivery of human epidermal organoids onto the sites of severe skin wounds enhancing uniform and concentrated delivery of organoids, facilitating their engraftment and differentiation for skin reconstitution. With the optimal design and using pneumatic AMAD, both survival and functions of organoids were effectively protected during the spraying process. Cells in the sprayed human epidermal organoids participated in the regeneration of the epidermis at wound sites in a mouse model and accelerated wound healing significantly. The novel AMAD and out new protocol with enhanced effects with respect to both organoid expansion and efficient transplantation will be used for clincal treatments of complex, uneven, or large-area severe skin wounds.

Injectable hydrogels have the potential to enhance stem cell-based therapies by improving cell localization, retention, and survival after transplantation. The inflammatory response to both the hydrogel and the encapsulated cells is a critical aspect of this strategy, with macrophages being highly involved in the process of hydrogel remodeling, angiogenesis, and tissue regeneration. As a step toward the development of a cell-based strategy for therapeutic angiogenesis, this work compared the intramuscular injection of allogeneic rat adipose-derived stem/stromal cells (rASCs) in an in situ gelling hydrogel with the injection of the hydrogel alone and rASCs in saline in an immunocompetent rat model by immunohistochemical analysis over 4 weeks. rASCs delivered in the hydrogel were retained intramuscularly at significantly higher densities as compared with cells delivered in saline. The encapsulated rASCs modulated the inflammatory response, promoting CD68(+) macrophage recruitment, with the majority of infiltrating cells expressing the M1 marker CCR7, as well as a higher fraction of CD163(+) M2c macrophages surrounding the hydrogel. Furthermore, rASCs reduced the initial expression of inducible nitric oxide synthase and promoted arginase-1 expression in the infiltrating macrophages over time, consistent with a shift toward a more proregenerative phenotype. Coincident with the enhanced macrophage infiltration, significantly more CD31(+) lumens were observed surrounding and within the hydrogels with rASCs at 2 and 4 weeks as compared with the hydrogels alone. Overall, these results are a promising indication that encapsulated rASCs can have immunomodulatory effects and may enhance angiogenic processes after intramuscular injection, promoting a regenerative macrophage response and blood vessel formation.

Magnetic targeting is a cell delivery system using the magnetic labeling of cells and the magnetic field; it has been developed for minimally invasive cell transplantation. Cell transplantation with both minimal invasiveness and high efficacy on tissue repair can be achieved by this system. Magnetic targeting has been applied for the transplantation of bone marrow mesenchymal stem cells, blood CD133-positive cells, neural progenitor cells, and induced pluripotent stem cells, and for the regeneration of bone, cartilage, skeletal muscles, and the spinal cord. It enhances the accumulation and adhesion of locally injected cells, resulting in the improvement of tissue regeneration. It is a promising technique for minimally invasive and effective cell transplantation therapy. Keywords: Stem cell, Regeneration, Magnet, Cell delivery

This study aimed to develop porous microspheres with a suitable porous structure and mechanical property for cell delivery using a comparatively low molecular weight (MW) poly(lactide-co-glycolide) (PLGA) having a weak mechanical strength and fast degradation rate, which could be potentially used for treatment of corneal endothelial diseases. Porous microspheres of 30 kDa PLGA with different pore sizes were prepared by varying preparation conditions, and the microspheres with mean pore diameters approximately 0.5, 1, 2 and 3 times that of a single green fluorescent protein-expressing human embryonic kidney 293 cell, used as a model cell, were chosen for cell loading study. The microspheres with an average pore diameter two times greater than that of the single cell were found to be the most appropriate for efficient cell loading in the inner pore spaces, along with demonstrating a good mechanical property, injectability and biodegradability. To maximize the cell loading amount in the microspheres, the cell adhesive property of the microspheres and cell loading conditions were optimized, leading to approximately 4.2 times increase in the cell loading amount. The porous microspheres designed using the low MW PLGA hold promise as a delivery system of corneal endothelial cells for regeneration of the corneal endothelium.

The obtaining of microcarriers for the cell culture and delivery is an urgent task of tissue engineering and regenerative medicine. The novel method of surface modification of alginate microcarriers in the form of microspheres with a diameter of 200–300 μm was developed. The described method consists in covalent crosslinking between collagen and surface of alginate microcarriers. It was shown that the method makes it possible to completely modify the surface of the alginate microcarrier, which can be used to improve the biological properties of the microcarrier. Such microcarriers with improved biological properties can be considered as effective systems for cell delivery and culture.

Cardiovascular diseases are the main cause of death in the world and are often associated with the occurrence of arrhythmias due to disruption of myocardial electrical integrity. Pathologies involving dysfunction of the specialized cardiac excitatory/conductive tissue are also common and constitute an added source of morbidity and mortality since current standard therapies withstand a great number of limitations. As electrical integrity is essential for a well-functioning heart, innovative strategies have been bioengineered to improve heart conduction and/or promote myocardial repair, based on: (1) gene and/or cell delivery; or (2) conductive biomaterials as tools for cardiac tissue engineering. Herein we aim to review the state-of-art in the area, while briefly describing the biological principles underlying the heart electrical/conduction system and how this system can be disrupted in heart disease. Suggestions regarding targets for future studies are also presented.

Targeted cell delivery by a magnetically actuated microrobot with a porous structure is a promising technique to enhance the low targeting efficiency of mesenchymal stem cell (MSC) in tissue regeneration. However, the relevant research performed to date is only in its proof-of-concept stage. To use the microrobot in a clinical stage, biocompatibility and biodegradation materials should be considered in the microrobot, and its efficacy needs to be verified using an in vivo model. In this study, we propose a human adipose-derived MSC-based medical microrobot system for knee cartilage regeneration and present an in vivo trial to verify the efficacy of the microrobot using the cartilage defect model. The microrobot system consists of a microrobot body capable of supporting MSCs, an electromagnetic actuation system for three-dimensional targeting of the microrobot, and a magnet for fixation of the microrobot to the damaged cartilage. Each component was designed and fabricated considering the accessibility of the patient and medical staff, as well as clinical safety. The efficacy of the microrobot system was then assessed in the cartilage defect model of rabbit knee with the aim to obtain clinical trial approval.

Background: Since the cornea is responsible for transmitting and focusing light into the eye, injury or pathology affecting any layer of the cornea can cause a detrimental effect on visual acuity. Aging is also a reason for corneal degeneration. Depending on the level of the injury, conservative therapies and donor tissue transplantation are the most common treatments for corneal diseases. Not only is there a lack of donor tissue and risk of infection/rejection, but the inherent ability of corneal cells and layers to regenerate has led to research in regenerative approaches and treatments. Methods: In this review, we first discussed the anatomy of the cornea and the required properties for reconstructing layers of the cornea. Regenerative approaches are divided into two main categories; using direct cell/growth factor delivery or using scaffold-based cell delivery. It is expected delivered cells migrate and integrate into the host tissue and restore its structure and function to restore vision. Growth factor delivery also has shown promising results for corneal surface regeneration. Scaffold-based approaches are categorized based on the type of scaffold, since it has a significant impact on the efficiency of regeneration, into the hydrogel and non-hydrogel based scaffolds. Various types of cells, biomaterials, and techniques are well covered. Results: The most important characteristics to be considered for biomaterials in corneal regeneration are suitable mechanical properties, biocompatibility, biodegradability, and transparency. Moreover, a curved shape structure and spatial arrangement of the fibrils have been shown to mimic the corneal extracellular matrix for cells and enhance cell differentiation. Conclusion: Tissue engineering and regenerative medicine approaches showed to have promising outcomes for corneal regeneration. However, besides proper mechanical and optical properties, other factors such as appropriate sterilization method, storage, shelf life and etc. should be taken into account in order to develop an engineered cornea for clinical trials. Electronic supplementary material The online version of this article (10.1007/s13770-020-00262-8) contains supplementary material, which is available to authorized users.

Stem cell therapies have emerged as promising treatments for injuries and diseases in regenerative medicine. Yet, delivering stem cells therapeutically can be complicated by invasive administration techniques, heterogeneity in the injection media, and/or poor cell retention at the injection site. Despite these issues, traditional administration protocols using bolus injections in a saline solution or surgical implants of cell‐laden hydrogels have highlighted the promise of cell administration as a treatment strategy. To address these limitations, we have designed an injectable polymer–nanoparticle (PNP) hydrogel platform exploiting multivalent, noncovalent interactions between modified biopolymers and biodegradable nanoparticles for encapsulation and delivery of human mesenchymal stem cells (hMSCs). hMSC‐based therapies have shown promise due to their broad differentiation capacities and production of therapeutic paracrine signaling molecules. In this work, the fundamental hydrogel mechanical properties that enhance hMSC delivery processes are elucidated using basic in vitro models. Further, in vivo studies in immunocompetent mice reveal that PNP hydrogels enhance hMSC retention at the injection site and retain administered hMSCs locally for upwards of 2 weeks. Through both in vitro and in vivo experiments, we demonstrate a novel scalable, synthetic, and biodegradable hydrogel system that overcomes current limitations and enables effective cell delivery.

Here, we propose magnetically driven microrobots for targeted cell delivery that can efficiently swim in various physiological environments. The three-dimensional (3D) microrobots were sophisticatedly fabricated by using micro-electro-mechanical systems (MEMS) technologies such as 3D laser lithography and metal sputtering. The magnetic field generated by an electromagnetic coil system allows efficient wireless actuation of the microrobots, which was characterized in various physiological conditions (in vitro viscous fluid, ex vivo rat brain blood vessels and a live nude mouse intraperitoneal cavity (in vivo)). Especially, the microrobots demonstrated stem cell transportation inside the body cavity of the nude mouse as a proof of concept of targeted cell delivery. Through this study, the use of the proposed microrobots has shown the feasibility of future applications in precision medicine.

Cardiac tissue engineering is of particular importance in the combination of contracting cells with a biomaterial scaffold, which serves as a cell-delivery construct, to replace cardiomyocytes (CMs) that are lost as a result of an infarction, to restore heart function. However, most biomaterial scaffolds are nonconductive and may delay regional conduction, potentially causing arrhythmias. In this study, a conductive CM-delivery construct that consists of a gelatin-based gelfoam that is conjugated with a self-doped conductive polymer (poly-3-amino-4-methoxybenzoic acid, PAMB) is proposed as a cardiac patch (PAMB-Gel patch) to repair an infarcted heart. A nonconductive plain gelfoam (Gel patch) is used as a control. The electrical conductivity of the PAMB-Gel patch is approximately 30 times higher than that of the Gel patch; as a result, the conductive PAMB-Gel patch can substantially increase electrical conduction between distinct clusters of beating CMs, facilitating their synchronous contraction. In vivo epicardial implantation of the PAMB-Gel patch that is seeded with CMs (the bioengineered patch) in infarcted rat hearts can significantly enhance electrical activity in the fibrotic tissue, improving electrical impulse propagation and synchronizing CM contraction across the scar region, markedly reducing its susceptibility to cardiac arrhythmias. Echocardiography shows that the bioengineered conductive patch has an important role in the restoration of cardiac function, perhaps owing to the synergistic effects of its conductive construct and the synchronously beating CMs. These experimental results reveal that the as-proposed bioengineered conductive patch has great potential for repairing injured cardiac tissues.

Amongst the diversity of scaffolding systems, hydrogel remains a popular choice for tissue engineering applications. However, good structural support and adequate biocompatibility are no longer the only emphasis in scaffold design; new generation of scaffolds are also expected to play a role in developmental guidance of the regenerated tissues. Thus, a simple hydrogel system could not satisfy the current demand. Instead of carrying out chemical modifications to hydrogel, we proposed the incorporation of microspheres into hydrogel to develop a hydrogel/microspheres (GS) composite system. By employing the microspheres as cell-affinitive interfaces for cell attachment or degradable moieties within hydrogels, the microspheres act as the bioresponsive entity within the composite model. Accordingly, this dissertation presents three distinct GS composite systems to deliver different cell types for cell-based therapy purposes. Based on the type of cell to be delivered, modification to GS system could be made respectively to suit the target cell development. Thermally stable microspheres were employed as permanent cellular attachment site within the GS system for delivery of typical anchorage-dependent cells (ADCs) like osteoblasts and mesenchymal stem cells (MSCs). For delivery of another type of model cell, HepG2 cell, partially crosslinked microspheres were used in the second GS system. Upon introduction of collagenase, microspheres in the GS system degrade, creating micro-cavities in the hydrogel bulk for further cell expansion and development. This design serves to use microsphere as a transient cell delivery vehicle and a porogen to establish a micro-cavitary hydrogel (MCG) construct. The dual functionality of the microspheres in the GS system enables direct cell delivery to the cavities of the hydrogel and also permits formation of controlled size cellular aggregates. In all previous set ups, cells were delivered on the microspheres while in this last GS system proposed, cells were delivered in the hydrogel phase of the composite system. As the cells would be encapsulated and maintained their rounded morphology, primary chondrocytes, a typical non-ADC was selected. This third GS composite is designed to be a thermally responsive system where microspheres dissolved upon exposure to physiological temperature without the need for collagenase. The beneficial effects of MCG are further demonstrated in this study where a macroscopic scaffold-free living hyaline cartilaginous graft (LhCG) consisting of only chondrocytes and ECM proteins could be derived. Given the high purity of LhCG construct, it served as an excellent in vitro cartilaginous template for mimicking endochondral ossification process. It is shown that the LhCG construct, rich in ECM protein, favours osteoblast and MSC attachment. Osteogenesis of the seeded cells on LhCG was achieved based on the positive expression of bone markers and calcification. Besides the ability to derive a scaffold-free LhCG construct, the thermally responsive GS system was also employed for delivery and differentiation of stem cells. Murine embryonic stem cells (mESCs) and induced pluripotent stem cells (iPSCs) were encapsulated respectively in this third GS system. Our results indicate that this system is able to facilitate embryoid bodies (EBs) formation and also allow differentiations to take place. Spontaneous formation of EBs and subsequent endodermal and hepatic differentiation could all occur in the continuous MCG system. In summary, the studies in this dissertation proved that GS composite system is an easily customizable platform for delivery of various cell types – model cell lines, primary cells, and pluripotent stem cells. DOCTOR OF PHILOSOPHY (SCBE)