Diabetic foot ulcers (DFU) are a major, debilitating complication of diabetes mellitus. Unfortunately, many DFUs are refractory to existing treatments and frequently lead to amputation. The development of more effective therapies has been hampered by the lack of predictive in vitro methods to investigate the mechanisms underlying impaired healing. To address this need for realistic wound-healing models, we established patient-derived fibroblasts from DFUs and site-matched controls and used them to construct three-dimensional (3D) models of chronic wound healing. Incorporation of DFU-derived fibroblasts into these models accurately recapitulated the following key aspects of chronic ulcers: reduced stimulation of angiogenesis, increased keratinocyte proliferation, decreased re-epithelialization, and impaired extracellular matrix deposition. In addition to reflecting clinical attributes of DFUs, the wound-healing potential of DFU fibroblasts demonstrated in this suite of models correlated with in vivo wound closure in mice. Thus, the reported panel of 3D DFU models provides a more biologically relevant platform for elucidating the cell-cell and cell-matrix-related mechanisms responsible for chronic wound pathogenesis and may improve translation of in vitro findings into efficacious clinical applications.
Immunotherapy is a promising treatment modality for cancer as it can promote specific and durable anti-cancer responses. However, limitations to current approaches remain. Therapeutics administered as soluble injections often require high doses and frequent re-dosing, which can result in systemic toxicities. Soluble bolus-based vaccine formulations typically elicit weak cellular immune responses, limiting their use for cancer. Current methods for ex vivo T cell expansion for adoptive T cell therapies are suboptimal, and achieving high T cell persistence and sustained functionality with limited systemic toxicity following transfer remains challenging. Biomaterials can play important roles in addressing some of these limitations. For example, nanomaterials can be employed as vehicles to deliver immune modulating payloads to specific tissues, cells, and cellular compartments with minimal off-target toxicity, or to co-deliver antigen and danger signal in therapeutic vaccine formulations. Alternatively, micro-to macroscale materials can be employed as devices for controlled molecular and cellular delivery, or as engineered microenvironments for recruiting and programming immune cells in situ. Recent work has demonstrated the potential for combining cancer immunotherapy and biomaterials, and the application of biomaterials to cancer immunotherapy is likely to enable the development of effective next-generation platforms. This review discusses the application of engineered materials for the delivery of immune modulating agents to the tumor microenvironment, therapeutic cancer vaccination, and adoptive T cell therapy.
Implantable 3D engineered vascular tissue constructs can be formed by co-culturing endothelial and fibroblast cells on macroporous scaffolds. Here we show that these constructs can be used for studying the dynamics of neovascular formation in-vitro by a combination of live confocal imaging and an array of image processing and analysis tools, revealing multiple distinct stages of morphogenesis. We show that this process involves both vasculogenic and angiogenic elements, including an initial endothelial multicellular cluster formation followed by rapid extensive sprouting, ultimately resulting in a stable interconnected endothelial network morphology. This vascular morphogenesis is time-correlated with the deposition and formation of an extensive extra-cellular matrix environment. We further show that endothelial network junctions are formed by two separate morphogenic mechanisms of anastomosis and cluster thinning.
Diabetic foot ulcers (DFU) represent a severe health problem and an unmet clinical challenge. In this study, we tested the efficacy of novel biomaterials in improving wound healing in mouse models of diabetes mellitus (DM). The biomaterials are composed of alginate- and deoxyribonucleic acid (DNA)-based gels that allow incorporation of effector cells, such as outgrowth endothelial cells (OEC), and provide sustained release of bioactive factors, such as neuropeptides and growth factors, which have been previously validated in experimental models of DM wound healing or hind limb ischemia. We tested these biomaterials in mice and demonstrate that they are biocompatible and can be injected into the wound margins without major adverse effects. In addition, we show that the combination of OEC and the neuropeptide Substance P has a better healing outcome than the delivery of OEC alone, while subtherapeutic doses of vascular endothelial growth factor (VEGF) are required for the transplanted cells to exert their beneficial effects in wound healing. In summary, alginate and DNA scaffolds could serve as potential delivery systems for the next-generation DFU therapies.
Skeletal muscles have a robust capacity to regenerate, but under compromised conditions, such as severe trauma, the loss of muscle functionality is inevitable. Research carried out in the field of skeletal muscle tissue engineering has elucidated multiple intrinsic mechanisms of skeletal muscle repair, and has thus sought to identify various types of cells and bioactive factors which play an important role during regeneration. In order to maximize the potential therapeutic effects of cells and growth factors, several biomaterial based strategies have been developed and successfully implemented in animal muscle injury models. A suitable biomaterial can be utilized as a template to guide tissue reorganization, as a matrix that provides optimum micro-environmental conditions to cells, as a delivery vehicle to carry bioactive factors which can be released in a controlled manner, and as local niches to orchestrate in situ tissue regeneration. A myriad of biomaterials, varying in geometrical structure, physical form, chemical properties, and biofunctionality have been investigated for skeletal muscle tissue engineering applications. In the current review, we present a detailed summary of studies where the use of biomaterials favorably influenced muscle repair. Biomaterials in the form of porous three-dimensional scaffolds, hydrogels, fibrous meshes, and patterned substrates with defined topographies, have each displayed unique benefits, and are discussed herein. Additionally, several biomaterial based approaches aimed specifically at stimulating vascularization, innervation, and inducing contractility in regenerating muscle tissues are also discussed. Finally, we outline promising future trends in the field of muscle regeneration involving a deeper understanding of the endogenous healing cascades and utilization of this knowledge for the development of multifunctional, hybrid, biomaterials which support and enable muscle regeneration under compromised conditions.
Interactions between cells and the extracellular matrix (ECM) are known to play critical roles in regulating cell phenotype. The identity of ECM ligands presented to mesenchymal stem cells (MSCs) has previously been shown to direct the cell fate commitment of these cells. To enhance osteogenic differentiation of MSCs, alginate hydrogels were prepared that present the DGEA ligand derived from collagen I. When presented from hydrogel surfaces in 2D, the DGEA ligand did not facilitate cell adhesion, while hydrogels presenting the RGD ligand derived from fibronectin did encourage cell adhesion and spreading. However, the osteogenic differentiation of MSCs encapsulated within alginate hydrogels presenting the DGEA ligand was enhanced when compared with unmodified alginate hydrogels and hydrogels presenting the RGD ligand. MSCs cultured in DGEA-presenting gels exhibited increased levels of osteocalcin production and mineral deposition. These data suggest that the presentation of the collagen I-derived DGEA ligand is a feasible approach for selectively inducing an osteogenic phenotype in encapsulated MSCs.
Embryonic skeletogenesis involves proliferation, condensation and subsequent chondrogenic differentiation of mesenchymal precursor cells, and the strains and stresses inherent to these processes have been hypothesized to influence skeletal development. The aim of this study was to determine the effect of growth-mimicking strain on the process of early skeletal development in vitro. To this end, we applied continuous uniaxial strain to embryonic skeletal precursor cells in micromass culture. Strain was applied at different times of culture to specifically address the effect of mechanical loading on the sequential stages of cellular proliferation, condensation and differentiation. We found that growth-mimicking strain at all three times did not affect proliferation or chondrogenic differentiation under the tested conditions. However, the timing of the applied strain did play a role in the density of mesenchymal condensations. This finding suggests that a mechanically dynamic environment, and specifically strain, can influence skeletal patterning. The growth-mimicking micromass model presented here may be a useful tool for further studies into the role of mechanical loading in early skeletal development.
Damage and degeneration of the skeletal elements due to disease, trauma, and aging lead to a significant health and economical burden. To reduce this burden, skeletal tissue engineering strategies aim to regenerate functional bone and cartilage in the adult body. However, challenges still exist. Such challenges involve the identification of the external cues that determine differentiation, how to control chondrocyte hypertrophy, and how to achieve specific tissue patterns and boundaries. To address these issues, it could be insightful to look at skeletal development, a robust morphogenetic process that takes place during embryonic development and is commonly modeled in vitro by the micromass assay. In this review, we investigate what the tissue engineering field can learn from this assay. By comparing embryonic skeletal precursor cells from different anatomic locations and developmental stages in micromass, the external cues that guide lineage commitment can be identified. The signaling pathways regulating chondrocyte hypertrophy, and the cues required for tissue patterning, can be elucidated by combining the micromass assay with genetic, molecular, and engineering tools. The lessons from the micromass assay are limited by two major differences between developmental and regenerative skeletogenesis: cell type and scale. We highlight an important difference between embryonic and adult skeletal progenitor cells, in that adult progenitors are not able to form mesenchymal condensations spontaneously. Also, the mechanisms of tissue patterning need to be adjusted to the larger tissue engineering constructs. In conclusion, mechanistic insights of skeletal tissue generation gained from the micromass model could lead to improved tissue engineering strategies and constructs.
Translation in an academic environment requires a support system--people, goals, models, partnerships, and infrastructures--that will push promising basic science and technology projects forward into the clinic.
Monodisperse alginate microgels (10-50 μm) are created via droplet-based microfluidics by a novel crosslinking procedure. Ionic crosslinking of alginate is induced by release of chelated calcium ions. The process separates droplet formation and gelation reaction enabling excellent control over size and homogeneity under mild reaction conditions. Living mesenchymal stem cells are encapsulated and cultured in the generated 3D microenvironments.
Polymeric scaffolds, which release growth factors in a temporally controlled manner, have successfully directed the differentiation of stem cells into monolithic tissues of a single lineage. However, engineering precise boundaries in multilineage functional tissues, such as the juxtaposed cartilaginous and osseous tissue present in articulated joints, often remains a challenge. This work demonstrates a precise materials system for in vitro reconstruction of the three-dimensional architecture of these types of human tissues. Multilayer poly(lactide-co-glycolide) (PLG) scaffolds were used to produce spatiotemporal gradients to direct the differentiation of an initially uniform population of mesenchymal stem cells (MSCs) into juxtaposed cartilage and bone. Specifically, growth factors (chondrogenic transforming growth factor-β3 and osteogenic bone morphogenetic protein-4) and their neutralizing antibodies were incorporated within distinct layers of the PLG scaffolds to create spatially segregated morphogen fields within the scaffold volume. The multilayer PLG scaffold designs were optimized by mathematical modeling, and generation of spatially segregated morphogen gradients was validated by assessing activity of luciferase reporter cell lines responsive to each growth factor. Scaffolds seeded with MSCs demonstrated production of juxtaposed cartilage and bone, as evaluated by biochemical staining and western blotting for tissue-specific matrix proteins. This work demonstrates a significant advance for the engineering of implantable constructs comprising tissues of multiple lineages, with potential applications in orthopedic regenerative medicine.
We investigate ionic partition of negatively charged molecular probes into also negatively charged, covalently crosslinked alginate hydrogels. The aim is to delimit the domain of validity of the major nanoelectrostatic models, and in particular to assess the influence of hydrogel chain mobility on ionic partition. We find that the widely used Gibbs-Donnan model greatly overestimates exclusion of the co-ion probes used. For low molecular weight probes, a much better fit is obtained by taking into account the electrostatics in the nanometric gel pores by means of the Poisson-Boltzmann framework; the fit is improved slightly when taking into account alginate chain mobility. For high molecular weight probes, we find it essential to take into account local gel deformation due to electrostatic repulsion between the flexible gel strands and the probe. This is achieved by combining Poisson-Boltzmann simulations with heterogeneous pore size distribution given by the Ogston model, or more simply and precisely, by applying a semi-empirical scaling law involving the ratio between Debye length and pore size.
Diabetic foot ulceration is a major complication of diabetes. Substance P (SP) is involved in wound healing, but its effect in diabetic skin wounds is unclear. We examined the effect of exogenous SP delivery on diabetic mouse and rabbit wounds. We also studied the impact of deficiency in SP or its receptor, neurokinin-1 receptor, on wound healing in mouse models. SP treatment improved wound healing in mice and rabbits, whereas the absence of SP or its receptor impaired wound progression in mice. Moreover, SP bioavailability in diabetic skin was reduced as SP gene expression was decreased, whereas the gene expression and protein levels of the enzyme that degrades SP, neutral endopeptidase, were increased. Diabetes and SP deficiency were associated with absence of an acute inflammatory response important for wound healing progression and instead revealed a persistent inflammation throughout the healing process. SP treatment induced an acute inflammatory response, which enabled the progression to the proliferative phase and modulated macrophage activation toward the M2 phenotype that promotes wound healing. In conclusion, SP treatment reverses the chronic proinflammatory state in diabetic skin and promotes healing of diabetic wounds.
Endothelial progenitor cells are being broadly explored for the treatment of ischemic cardiovascular diseases, but their response to molecules commonly used to promote the growth of new blood vessels has not been fully characterized. In this study, angiogenic sprout formation in a 3-dimensional, in vitro model by one type of endothelial progenitor, outgrowth endothelial cells (OECs), was characterized in response to exposure to stromal cell-derived factor (SDF) and vascular endothelial growth factor (VEGF) and then compared to mature endothelial cells. Exposure to SDF alone did not increase angiogenic sprouting in comparison to control media, while a combination of VEGF and SDF demonstrated greater potency than VEGF alone for all cell types. Together, VEGF and SDF reduced the sprout initiation time and maintained sprouting levels over time. In direct competition with mature endothelial cells, OECs preferentially localized to the tip cell position, suggesting an enhanced sprouting potential. Overall, these results reveal the impact of the combination of VEGF and SDF on endothelial cell sprouting, and support the enhanced potential of OECs, as opposed to mature endothelial cells, for treating ischemic diseases.
UNLABELLED: A critical challenge in tissue regeneration is to develop constructs that effectively integrate with the host tissue. Here, we describe a composite, laser micromachined, collagen-alginate construct containing human mesenchymal stem cells (hMSCs) for tissue repair applications. Collagen type I was fashioned into laminated collagen sheets to form a mechanically robust fascia that was subsequently laser micropatterned with pores of defined dimension and spatial distribution as a means to modulate mechanical behavior and promote tissue integration. Significantly, laser micromachined patterned constructs displayed both substantially greater compliance and suture retention strength than non-patterned constructs. hMSCs were loaded in an RGD-functionalized alginate gel modified to degrade in vivo. Over a 7 day observation period in vitro, high cell viability was observed with constant levels of VEGF, PDGF-β and MCP-1 protein expression. In a full thickness abdominal wall defect model, the composite construct prevented hernia recurrence in Wistar rats over an 8-week period with de novo tissue and vascular network formation and the absence of adhesions to underlying abdominal viscera. As compared to acellular constructs, constructs containing hMSCs displayed greater integration strength (cell seeded: 0.92 ± 0.19 N/mm vs. acellular: 0.59 ± 0.25 N/mm, p=0.01), increased vascularization (cell seeded: 2.7-2.1/hpf vs. acellular: 1.7-2.1/hpf, p<0.03), and increased infiltration of macrophages (cell seeded: 2021-3630 μm(2)/hpf vs. acellular: 1570-2530 μm(2)/hpf, p<0.05). A decrease in the ratio of M1 macrophages to total macrophages was also observed in hMSC-populated samples. Laser micromachined collagen-alginate composites containing hMSCs can be used to bridge soft tissue defects with the capacity for enhanced tissue repair and integration.
STATEMENT OF SIGNIFICANCE: Effective restoration of large soft tissue defects caused by trauma or treatment complications represents a critical challenge in the clinic. In this study, a novel composite construct was engineered and evaluated for stem cell delivery and tissue repair. Laser micromachining was used to fabricate patterned, microporous constructs designed with pores of defined size and distribution as a means to tune mechanical responses, accommodate and protect incorporated cells, and enhance tissue integration. The construct was embedded within an engineered alginate gel containing hMSCs. Upon repair of a full thickness abdominal wall defect in a rat model, the composite construct modulated host innate immunity towards a reparative phenotypic response, promoted neovascularization and associated matrix production, and increased the strength of tissue integration.
A biomaterial-based vaccination system that uses minimal extracorporeal manipulation could provide in situ enhancement of dendritic cell (DC) numbers, a physical space where DCs interface with transplanted tumour cells, and an immunogenic context. Here we encapsulate GM-CSF, serving as a DC enhancement factor, and CpG ODN, serving as a DC activating factor, into sponge-like macroporous cryogels. These cryogels are injected subcutaneously into mice to localize transplanted tumour cells and deliver immunomodulatory factors in a controlled spatio-temporal manner. These vaccines elicit local infiltrates composed of conventional and plasmacytoid DCs, with the subsequent induction of potent, durable and specific anti-tumour T-cell responses in a melanoma model. These cryogels can be delivered in a minimally invasive manner, bypass the need for genetic modification of transplanted cancer cells and provide sustained release of immunomodulators. Altogether, these findings indicate the potential for cryogels to serve as a platform for cancer cell vaccinations.
Biomaterials-based vaccines have emerged as a powerful method to evoke potent immune responses directly in vivo, without the need for ex vivo cell manipulation, and modulating dendritic cell (DC) responses in a noninflammatory context could enable the development of tolerogenic vaccines to treat autoimmunity. This study describes the development of a noninflammatory, injectable hydrogel system to locally enrich DCs in vivo without inducing their maturation or activation, as a first step toward this goal. Alginate hydrogels that form pores in situ are characterized and used as a physical scaffold for cell infiltration. These gels are also adapted to control the release of granulocyte-macrophage colony stimulating factor (GM-CSF), a potent inducer of DC recruitment and proliferation. In vivo, sustained release of GM-CSF from the pore-forming gels leads to the accumulation of millions of cells in the material. These cells are highly enriched in CD11b(+) CD11c(+) DCs, and further analysis of cell surface marker expression indicates these DCs are immature. This study demonstrates that a polymeric delivery system can mediate the accumulation of a high number and percentage of immature DCs, and may provide the basis for further development of materials-based, therapeutic vaccines.
A congenital or iatrogenic tissue defect often requires closure by open surgery or metallic components that can erode tissue. Biodegradable, hydrophobic light-activated adhesives represent an attractive alternative to sutures, but lack a specifically designed minimally invasive delivery tool, which limits their clinical translation. We developed a multifunctional, catheter-based technology with no implantable rigid components that functions by unfolding an adhesive-loaded elastic patch and deploying a double-balloon design to stabilize and apply pressure to the patch against the tissue defect site. The device uses a fiber-optic system and reflective metallic coating to uniformly disperse ultraviolet light for adhesive activation. Using this device, we demonstrate closure on the distal side of a defect in porcine abdominal wall, stomach, and heart tissue ex vivo. The catheter was further evaluated as a potential tool for tissue closure in vivo in rat heart and abdomen and as a perventricular tool for closure of a challenging cardiac septal defect in a large animal (porcine) model. Patches attached to the heart and abdominal wall with the device showed similar inflammatory response as sutures, with 100% small animal survival, indicating safety. In the large animal model, a ventricular septal defect in a beating heart was reduced to <1.6 mm. This new therapeutic platform has utility in a range of clinical scenarios that warrant minimally invasive and atraumatic repair of hard-to-reach defects.
The effectiveness of stem cell therapies has been hampered by cell death and limited control over fate. These problems can be partially circumvented by using macroporous biomaterials that improve the survival of transplanted stem cells and provide molecular cues to direct cell phenotype. Stem cell behaviour can also be controlled in vitro by manipulating the elasticity of both porous and non-porous materials, yet translation to therapeutic processes in vivo remains elusive. Here, by developing injectable, void-forming hydrogels that decouple pore formation from elasticity, we show that mesenchymal stem cell (MSC) osteogenesis in vitro, and cell deployment in vitro and in vivo, can be controlled by modifying, respectively, the hydrogel's elastic modulus or its chemistry. When the hydrogels were used to transplant MSCs, the hydrogel's elasticity regulated bone regeneration, with optimal bone formation at 60 kPa. Our findings show that biophysical cues can be harnessed to direct therapeutic stem cell behaviours in situ.
Stimuli-responsive polymeric depots capable of on-demand release of therapeutics promise a substantial improvement in the treatment of many local diseases. These systems have the advantage of controlling local dosing so that payload is released at a time and with a dose chosen by a physician or patient, and the dose can be varied as disease progresses or healing occurs. Macroscale drug depot can be induced to release therapeutics through the action of physical stimuli such as ultrasound, electric and magnetic fields and light as well as through the addition of pharmacological stimuli such as nucleic acids and small molecules. In this review, we highlight recent advances in the development of polymeric systems engineered for releasing therapeutic molecules through physical and pharmacological stimulation.