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.
Organ and tissue loss through disease and injury motivate the development of therapies that can regenerate tissues and decrease reliance on transplantations. Regenerative medicine, an interdisciplinary field that applies engineering and life science principles to promote regeneration, can potentially restore diseased and injured tissues and whole organs. Since the inception of the field several decades ago, a number of regenerative medicine therapies, including those designed for wound healing and orthopedics applications, have received Food and Drug Administration (FDA) approval and are now commercially available. These therapies and other regenerative medicine approaches currently being studied in preclinical and clinical settings will be covered in this review. Specifically, developments in fabricating sophisticated grafts and tissue mimics and technologies for integrating grafts with host vasculature will be discussed. Enhancing the intrinsic regenerative capacity of the host by altering its environment, whether with cell injections or immune modulation, will be addressed, as well as methods for exploiting recently developed cell sources. Finally, we propose directions for current and future regenerative medicine therapies.
Natural biological processes are intricately controlled by the timing and spatial distribution of various cues. To mimic this precise level of control, the physical sizes of gold nanoparticles are utilized to sterically entrap them in hydrogel materials, where they are subsequently released only in response to ultrasound. These nanoparticles can transport bioactive factors to cells and direct cell behavior on-demand.
Alginate hydrogels are well-characterized, biologically inert materials that are used in many biomedical applications for the delivery of drugs, proteins, and cells. Unfortunately, canonical covalently crosslinked alginate hydrogels are formed using chemical strategies that can be biologically harmful due to their lack of chemoselectivity. In this work we introduce tetrazine and norbornene groups to alginate polymer chains and subsequently form covalently crosslinked click alginate hydrogels capable of encapsulating cells without damaging them. The rapid, bioorthogonal, and specific click reaction is irreversible and allows for easy incorporation of cells with high post-encapsulation viability. The swelling and mechanical properties of the click alginate hydrogel can be tuned via the total polymer concentration and the stoichiometric ratio of the complementary click functional groups. The click alginate hydrogel can be modified after gelation to display cell adhesion peptides for 2D cell culture using thiol-ene chemistry. Furthermore, click alginate hydrogels are minimally inflammatory, maintain structural integrity over several months, and reject cell infiltration when injected subcutaneously in mice. Click alginate hydrogels combine the numerous benefits of alginate hydrogels with powerful bioorthogonal click chemistry for use in tissue engineering applications involving the stable encapsulation or delivery of cells or bioactive molecules.
Delayed healing or nonhealing of bone is an important clinical concern. Although bone, one of the two tissues with scar-free healing capacity, heals in most cases, healing is delayed in more than 10% of clinical cases. Treatment of such delayed healing condition is often painful, risky, time consuming, and expensive. Tissue healing is a multistage regenerative process involving complex and well-orchestrated steps, which are initiated in response to injury. At best, these steps lead to scar-free tissue formation. At the onset of healing, during the inflammatory phase, stationary and attracted macrophages and other immune cells at the fracture site release cytokines in response to injury. This initial reaction to injury is followed by the recruitment, proliferation, and differentiation of mesenchymal stromal cells, synthesis of extracellular matrix proteins, angiogenesis, and finally tissue remodeling. Failure to heal is often associated with poor revascularization. Since blood vessels mediate the transport of circulating cells, oxygen, nutrients, and waste products, they appear essential for successful healing. The strategy of endogenous regeneration in a tissue such as bone is interesting to analyze since it may represent a blueprint of successful tissue formation. This review highlights the interdependency of the time cascades of inflammation, angiogenesis, and tissue regeneration. A better understanding of these inter-relations is mandatory to early identify patients at risk as well as to overcome critical clinical conditions that limit healing. Instead of purely tolerating the inflammatory phase, modulations of inflammation (immunomodulation) might represent a valid therapeutic strategy to enhance angiogenesis and foster later phases of tissue regeneration.
Targeting small molecules to diseased tissues as therapy or diagnosis is a significant challenge in drug delivery. Drug-eluting devices implanted during invasive surgery allow the controlled presentation of drugs at the disease site, but cannot be modified once the surgery is complete. We demonstrate that bioorthogonal click chemistry can be used to target circulating small molecules to hydrogels resident intramuscularly in diseased tissues. We also demonstrate that small molecules can be repeatedly targeted to the diseased area over the course of at least one month. Finally, two bioorthogonal reactions were used to segregate two small molecules injected as a mixture to two separate locations in a mouse disease model. These results demonstrate that click chemistry can be used for pharmacological drug delivery, and this concept is expected to have applications in refilling drug depots in cancer therapy, wound healing, and drug-eluting vascular grafts and stents.
Biomaterials may improve outcomes of endothelial progenitor-based therapies for the treatment of ischemic cardiovascular disease, due to their ability to direct cell behavior. We hypothesized that local, sustained delivery of exogenous vascular endothelial growth factor (VEGF) and stromal cell-derived factor (SDF) from alginate hydrogels could increase recruitment of systemically infused endothelial progenitors to ischemic tissue, and subsequent neovascularization. VEGF and SDF were found to enhance in vitro adhesion and migration of outgrowth endothelial cells (OECs) and circulating angiogenic cells (CACs), two populations of endothelial progenitors, by twofold to sixfold, and nearly doubled recruitment to both ischemic and nonischemic muscle tissue in vivo. Local delivery of VEGF and SDF to ischemic hind-limbs in combination with systemic CAC delivery significantly improved functional perfusion recovery over OEC delivery, or either treatment alone. Compared with OECs, CACs were more responsive to VEGF and SDF treatment, promoted in vitro endothelial sprout formation in a paracrine manner more potently, and demonstrated greater influence on infiltrating inflammatory cells in vivo. These studies demonstrate that accumulation of infused endothelial progenitors can be enriched using biomaterial-based delivery of VEGF and SDF, and emphasize the therapeutic benefit of using CACs for the treatment of ischemia.
Studies of cellular mechanotransduction have converged upon the idea that cells sense extracellular matrix (ECM) elasticity by gauging resistance to the traction forces they exert on the ECM. However, these studies typically utilize purely elastic materials as substrates, whereas physiological ECMs are viscoelastic, and exhibit stress relaxation, so that cellular traction forces exerted by cells remodel the ECM. Here we investigate the influence of ECM stress relaxation on cell behaviour through computational modelling and cellular experiments. Surprisingly, both our computational model and experiments find that spreading for cells cultured on soft substrates that exhibit stress relaxation is greater than cells spreading on elastic substrates of the same modulus, but similar to that of cells spreading on stiffer elastic substrates. These findings challenge the current view of how cells sense and respond to the ECM.