Engineering Pro-Regenerative Hydrogels for Scarless Wound Healing.

Skin and skin appendages protect the body from harmful environment and prevent internal organs from dehydration. Superficial epidermal wounds usually heal without scarring, however, deep dermal wound healing commonly ends up with nonfunctioning scar formation with substantial loss of skin appendage. Wound healing is one of the most complex dynamic biological processes, during which a cascade of biomolecules combine with stem cell influx and matrix synthesis and synergistically contribute to wound healing at all levels. Although many approaches have been investigated to restore complete skin, the clinically effective therapy is still unavailable and the regeneration of perfect skin still remains a significant challenge. The complete mechanism behind scarless skin regeneration still requires further investigation. Fortunately, recent advancement in regenerative medicine empowers us more than ever to restore tissue in a regenerative manner. Many studies have elucidated and reviewed the contribution of stem cells and growth factors to scarless wound healing. This article focuses on recent advances in scarless wound healing, especially strategies to engineer pro-regenerative scaffolds to restore damaged skin in a regenerative manner.

Engineered human vascularized constructs accelerate diabetic wound healing.

Stem cell-based therapy is emerging as a promising approach for chronic diabetic wounds, but strategies for optimizing both cellular differentiation and delivery remain as major obstacles. Here, we study bioengineered vascularized constructs as a therapeutic modality for diabetic wound healing. We developed a wound model in immunodeficient rodent and treated it with engineered vascularized constructs from endothelial progenitors or early vascular cells-derived from human induced pluripotent stem cells (hiPSCs) reprogrammed either from healthy donor or type-1 diabetic patient. We found that all vascularized constructs expedited wound closure and reperfusion, with endothelial progenitor constructs having the earliest maximum closure rate followed closely by healthy and diabetic hiPSC-derivative constructs. This was accompanied by rapid granulation layer formation and regression in all vascularized construct groups. Macrophage infiltration into the hydrogel matrix occurred during early stages of healing, seeming to facilitate rapid neovascularization of the wound that could then better persist in the vascularized constructs. Blood perfusion of the human vasculature could be detected after three days, indicating rapid integration with the host vasculature. Overall, we propose a potential therapeutic strategy using allograft or autologous vascularized constructs to treat type-1 diabetic wounds. This approach highlights the unprecedented prospects of designing patient-specific stem cell therapy.

Acellular Hydrogels for Regenerative Burn Wound Healing: Translation from a Porcine Model.

Currently available skin grafts and skin substitutes for healing following third-degree burn injuries are fraught with complications, often resulting in long-term physical and psychological sequelae. Synthetic treatment that can promote wound healing in a regenerative manner would provide an off-the-shelf, non-immunogenic strategy to improve clinical care of severe burn wounds. Here, we demonstrate the vulnerary efficacy and accelerated healing mechanism of a dextran-based hydrogel in a third-degree porcine burn model. The model was optimized to allow examination of the hydrogel treatment for clinical translation and its regenerative response mechanisms. Hydrogel treatment accelerated third-degree burn wound healing by rapid wound closure, improved re-epithelialization, enhanced extracellular matrix remodeling, and greater nerve reinnervation, compared with the dressing-treated group. These effects appear to be mediated through the ability of the hydrogel to facilitate a rapid but brief initial inflammatory response that coherently stimulates neovascularization within the granulation tissue during the first week of treatment, followed by an efficient vascular regression to promote a regenerative healing process. Our results suggest that the dextran-based hydrogels may substantially improve healing quality and reduce skin grafting incidents and thus pave the way for clinical studies to improve the care of severe burn injury patients.

Recapitulating physiological and pathological shear stress and oxygen to model vasculature in health and disease.

Studying human vascular disease in conventional cell cultures and in animal models does not effectively mimic the complex vascular microenvironment and may not accurately predict vascular responses in humans. We utilized a microfluidic device to recapitulate both shear stress and O2 levels in health and disease, establishing a microfluidic vascular model (µVM). Maintaining human endothelial cells (ECs) in healthy-mimicking conditions resulted in conversion to a physiological phenotype namely cell elongation, reduced proliferation, lowered angiogenic gene expression and formation of actin cortical rim and continuous barrier. We next examined the responses of the healthy µVM to a vasotoxic cancer drug, 5-Fluorouracil (5-FU), in comparison with an in vivo mouse model. We found that 5-FU does not induce apoptosis rather vascular hyperpermeability, which can be alleviated by Resveratrol treatment. This effect was confirmed by in vivo findings identifying a vasoprotecting strategy by the adjunct therapy of 5-FU with Resveratrol. The µVM of ischemic disease demonstrated the transition of ECs from a quiescent to an activated state, with higher proliferation rate, upregulation of angiogenic genes, and impaired barrier integrity. The µVM offers opportunities to study and predict human ECs with physiologically relevant phenotypes in healthy, pathological and drug-treated environments.

Hyaluronic acid hydrogel stiffness and oxygen tension affect cancer cell fate and endothelial sprouting.

Three-dimensional (3D) tissue culture models may recapitulate aspects of the tumorigenic microenvironment in vivo, enabling the study of cancer progression in vitro. Both hypoxia and matrix stiffness are known to regulate tumor growth. Using a modular culture system employing an acrylated hyaluronic acid (AHA) hydrogel, three hydrogel matrices with distinctive degrees of viscoelasticity - soft (78±16 Pa), medium (309± 57 Pa), and stiff (596± 73 Pa) - were generated using the same concentration of adhesion ligands. Oxygen levels within the hydrogel in atmospheric (21 %), hypoxic (5 %), and severely hypoxic (1 %) conditions were assessed with a mathematical model. HT1080 fibrosarcoma cells, encapsulated within the AHA hydrogels in high densities, generated nonuniform oxygen distributions, while lower cell densities resulted in more uniform oxygen distributions in the atmospheric and hypoxic environments. When we examined how varying viscoelasticity in atmospheric and hypoxic environments affects cell cycles and the expression of BNIP3 and BNIP3L (autophagy and apoptosis genes), and GLUT-1 (a glucose transport gene), we observed that HT1080 cells in 3D hydrogel adapted better to hypoxic conditions than those in a Petri dish, with no obvious correlation to matrix viscoelasticity, by recovering rapidly from possible autophagy/apoptotic events and alternating metabolism mechanisms. Further, we examined how HT1080 cells cultured in varying viscoelasticity and oxygen tension conditions affected endothelial sprouting and invasion. We observed that increased matrix stiffness reduced endothelial sprouting and invasion in atmospheric conditions; however, we observed increased endothelial sprouting and invasion under hypoxia at all levels of matrix stiffness with the upregulation of vascular endothelial growth factor (VEGF) and angiopoeitin-1 (ANG-1). Overall, HT1080 cells encapsulated in the AHA hydrogels under hypoxic stress recovered better from apoptosis and demonstrated greater angiogenic induction. Thus, we propose that oxygen tension more profoundly influences cell fate and the angiogenic potential of 3D cultured HT1080 fibrosarcoma cells than does matrix stiffness.

Self-organized vascular networks from human pluripotent stem cells in a synthetic matrix.

The success of tissue regenerative therapies is contingent on functional and multicellular vasculature within the redeveloping tissue. Although endothelial cells (ECs), which compose the vasculature's inner lining, are intrinsically able to form nascent networks, these structures regress without the recruitment of pericytes, supporting cells that surround microvessel endothelium. Reconstruction of typical in vivo microvascular architecture traditionally has been done using distinct cell sources of ECs and pericytes within naturally occurring matrices; however, the limited sources of clinically relevant human cells and the inherent chemical and physical properties of natural materials hamper the translational potential of these approaches. Here we derived a bicellular vascular population from human pluripotent stem cells (hPSCs) that undergoes morphogenesis and assembly in a synthetic matrix. We found that hPSCs can be induced to codifferentiate into early vascular cells (EVCs) in a clinically relevant strategy amenable to multiple hPSC lines. These EVCs can mature into ECs and pericytes, and can self-organize to form microvascular networks in an engineered matrix. These engineered human vascular networks survive implantation, integrate with the host vasculature, and establish blood flow. This integrated approach, in which a derived bicellular population is exploited for its intrinsic self-assembly capability to create microvasculature in a deliverable matrix, has vast ramifications for vascular construction and regenerative medicine.

Integration and regression of implanted engineered human vascular networks during deep wound healing.

The ability of vascularized constructs to integrate with tissues may depend on the kinetics and stability of vascular structure development. This study assessed the functionality and durability of engineered human vasculatures from endothelial progenitors when implanted in a mouse deep burn-wound model. Human vascular networks, derived from endothelial colony-forming cells in hyaluronic acid hydrogels, were transplanted into third-degree burns. On day 3 following transplantation, macrophages rapidly degraded the hydrogel during a period of inflammation; through the transitions from inflammation to proliferation (days 5-7), the host's vasculatures infiltrated the construct, connecting with the human vessels within the wound area. The growth of mouse vessels near the wound area supported further integration with the implanted human vasculatures. During this period, the majority of the vessels (∼60%) in the treated wound area were human. Although no increase in the density of human vessels was detected during the proliferative phase, they temporarily increased in size. This growth peaked at day 7, the middle of the proliferation stage, and then decreased by the end of the proliferation stage. As the wound reached the remodeling period during the second week after transplantation, the vasculatures including the transplanted human vessels generally regressed, and few microvessels, wrapped by mouse smooth muscle cells and with a vessel area less than 200 µm² (including the human ones), remained in the healed wound. Overall, this study offers useful insights for the development of vascularization strategies for wound healing and ischemic conditions, for tissue-engineered constructs, and for tissue regeneration.

Dextran hydrogel scaffolds enhance angiogenic responses and promote complete skin regeneration during burn wound healing.

Neovascularization is a critical determinant of wound-healing outcomes for deep burn injuries. We hypothesize that dextran-based hydrogels can serve as instructive scaffolds to promote neovascularization and skin regeneration in third-degree burn wounds. Dextran hydrogels are soft and pliable, offering opportunities to improve the management of burn wound treatment. We first developed a procedure to treat burn wounds on mice with dextran hydrogels. In this procedure, we followed clinical practice of wound excision to remove full-thickness burned skin, and then covered the wound with the dextran hydrogel and a dressing layer. Our procedure allows the hydrogel to remain intact and securely in place during the entire healing period, thus offering opportunities to simplify the management of burn wound treatment. A 3-week comparative study indicated that dextran hydrogel promoted dermal regeneration with complete skin appendages. The hydrogel scaffold facilitated early inflammatory cell infiltration that led to its rapid degradation, promoting the infiltration of angiogenic cells into the healing wounds. Endothelial cells homed into the hydrogel scaffolds to enable neovascularization by day 7, resulting in an increased blood flow significantly greater than treated and untreated controls. By day 21, burn wounds treated with hydrogel developed a mature epithelial structure with hair follicles and sebaceous glands. After 5 weeks of treatment, the hydrogel scaffolds promoted new hair growth and epidermal morphology and thickness similar to normal mouse skin. Collectively, our evidence shows that customized dextran-based hydrogel alone, with no additional growth factors, cytokines, or cells, promoted remarkable neovascularization and skin regeneration and may lead to novel treatments for dermal wounds.

Controlled activation of morphogenesis to generate a functional human microvasculature in a synthetic matrix.

Understanding the role of the extracellular matrix (ECM) in vascular morphogenesis has been possible using natural ECMs as in vitro models to study the underlying molecular mechanisms. However, little is known about vascular morphogenesis in synthetic matrices where properties can be tuned toward both the basic understanding of tubulogenesis in modular environments and as a clinically relevant alternative to natural materials for regenerative medicine. We investigated synthetic, tunable hyaluronic acid (HA) hydrogels and determined both the adhesion and degradation parameters that enable human endothelial colony-forming cells (ECFCs) to form efficient vascular networks. Entrapped ECFCs underwent tubulogenesis dependent on the cellular interactions with the HA hydrogel during each stage of vascular morphogenesis. Vacuole and lumen formed through integrins α(5)ß(1) and α(V)ß(3), while branching and sprouting were enabled by HA hydrogel degradation. Vascular networks formed within HA hydrogels containing ECFCs anastomosed with the host's circulation and supported blood flow in the hydrogel after transplantation. Collectively, we show that the signaling pathways of vascular morphogenesis of ECFCs can be precisely regulated in a synthetic matrix, resulting in a functional microvasculature useful for the study of 3-dimensional vascular biology and toward a range of vascular disorders and approaches in tissue regeneration.

Functional neovascularization of biodegradable dextran hydrogels with multiple angiogenic growth factors.

Slow vascularization of functional blood limits the transplantation of tissue constructs and the recovery of ischemic and wounded tissues. Despite the widespread investigation of polysaccharide-based hydrogel scaffolds for their therapeutic applications, blood vessel ingrowth into these hydrogel scaffolds remains a challenge. We hypothesized that modifying the properties of biodegradable hydrogel scaffolds with immobilization of multiple angiogenic growth factors (GFs) would induce a rapid proliferation of functional vasculature into the scaffolds. To this end, we remodeled the hydrogel structure by decreasing crosslinking density via reduced degree of substitution of crosslinking groups, which resulted in improved hydrogel properties including reduced rigidity, increased swelling, increased vascular endothelial GF (VEGF) release capability, and facilitated rapid hydrogel disintegration and tissue ingrowth. Immobilizing VEGF in the scaffolds promoted tissue ingrowth and expedited biodegradation. Furthermore, a synergistic effect of multiple angiogenic GFs was established; the coimmobilization of VEGF+ angiopoietin-1, and VEGF+ insulin-like GF+ stromal cell-derived factor-1 induced more and larger blood vessels than any individual GF, while the combination of all GFs dramatically increased the size and number of newly formed functional vessels. Altogether, our data demonstrate that rapid, efficient, and functional neovascularization can be achieved by precisely manipulating hydrogel scaffold properties and immobilizing defined angiogenic GFs.

Functional groups affect physical and biological properties of dextran-based hydrogels.

Modification of dextran backbone allows the development of a hydrogel with specific characteristics. To enhance their functionality for tissue-engineered scaffolds, a series of dextran-based macromers was synthesized by incorporating various functional groups, including allyl isocyanate (Dex-AI), ethylamine (Dex-AE), chloroacetic acid (Dex-AC), or maleic-anhydride (Dex-AM) into dextrans. The dextran-based biodegradable hybrid hydrogels are developed by integrating polyethylene glycol diacrylate (PEGDA). To explore the effect of different derivatives on hydrogel properties, three different ratios of Dex/PEGDA are examined: low (20/80), medium (40/60), and high (60/40). Differences in physical and biological properties of the hydrogels are found, including swelling, degradation rate, mechanics, crosslinking density, biocompatibility (in vitro and in vivo), and vascular endothelial growth factor release. The results also indicate that the incorporation of amine groups into dextran gives rise to hydrogels with better biocompatible and release properties. We, therefore, conclude that the incorporation of different functional groups affects the fundamental properties of a dextran-based hydrogel network, and that amine groups are preferred to generate hydrogels for biomedical use.