Flowable Polyethylene Glycol Hydrogels Support the in Vitro Survival and Proliferation of Dermal Progenitor Cells in a Mechanically Dependent Manner.

Cell-based therapies have garnered considerable interest largely because of their potential utility for tissue regeneration in a variety of organs, including skin. Designing vehicles that enable optimal delivery and purposeful integration of donor cells within tissues will be critical for their success. Here, we investigate the utility of an injectable, self-polymerizing, fully synthetic hydrogel in supporting the survival, proliferation, and function of cultured adult dermal progenitor cells (DPCs) which may serve as a source of renewable cells to repair severe skin injuries or restore hair growth. We show that modifying the stiffness of these transglutaminase cross-linked poly(ethylene glycol) (TG-PEG) hydrogels significantly alters DPC behavior and phenotype; increasing stiffness promotes their differentiation and migration whereas softer gels maintained them in a proliferative state. We found that 2-3% TG-PEG was optimal to promote cell expansion and survival. Unexpectedly, DPCs grown in all conditions maintained their inductive function and thus generated de novo hair follicles. Our data suggests that TG-PEG hydrogels may be a versatile platform for stem and progenitor cell transplantation and fate specification while maintaining functional competence.

Mechanically resilient injectable scaffolds for intramuscular stem cell delivery and cytokine release.

A promising strategy for treating peripheral ischemia involves the delivery of stem cells to promote angiogenesis through paracrine signaling. Treatment success depends on cell localization, retention, and survival within the mechanically dynamic intramuscular (IM) environment. Herein we describe an injectable, in situ-gelling hydrogel for the IM delivery of adipose-derived stem/stromal cells (ASCs), specifically designed to withstand the dynamic loading conditions of the lower limb and facilitate cytokine release from encapsulated cells. Copolymers of poly(trimethylene carbonate)-b-poly(ethylene glycol)-b-poly(trimethylene carbonate) diacrylate were used to modulate the properties of methacrylated glycol chitosan hydrogels crosslinked by thermally-initiated polymerization using ammonium persulfate and N,N,N',N'-tetramethylethylenediamine. The scaffolds had an ultimate compressive strain over 75% and maintained mechanical properties during compressive fatigue testing at physiological levels. Rapid crosslinking (<3 min) was achieved at low initiator concentration (5 mM). Following injection and crosslinking within the scaffolds, human ASCs demonstrated high viability (>90%) over two weeks in culture under both normoxia and hypoxia. Release of angiogenic and chemotactic cytokines was enhanced from encapsulated cells under sustained hypoxia, in comparison to normoxic and tissue culture polystyrene controls. When delivered by IM injection in a mouse model of hindlimb ischemia, human ASCs were well retained in the scaffold over 28 days and significantly increased the IM vascular density compared to untreated controls.

Biocomposite nanofiber matrices to support ECM remodeling by human dermal progenitors and enhanced wound closure.

Cell-based therapies have recently been the focus of much research to enhance skin wound healing. An important challenge will be to develop vehicles for cell delivery that promote survival and uniform distribution of cells across the wound bed. These systems should be stiff enough to facilitate handling, whilst soft enough to limit damage to newly synthesized wound tissue and minimize patient discomfort. Herein, we developed several novel modifiable nanofibre scaffolds comprised of Poly (ε-caprolactone) (PCL) and gelatin (GE). We asked whether they could be used as a functional receptacle for adult human Skin-derived Precursor Cells (hSKPs) and how naked scaffolds impact endogenous skin wound healing. PCL and GE were electrospun in a single facile solvent to create composite scaffolds and displayed unique morphological and mechanical properties. After seeding with adult hSKPs, deposition of extracellular matrix proteins and sulphated glycosaminoglycans was found to be enhanced in composite grafts. Moreover, composite scaffolds exhibited significantly higher cell proliferation, greater cell spreading and integration within the nanofiber mats. Transplantation of acellular scaffolds into wounds revealed scaffolds exhibited improvement in dermal-epidermal thickness, axonal density and collagen deposition. These results demonstrate that PCL-based nanofiber scaffolds show promise as a cell delivery system for wound healing.

Tough, Semisynthetic Hydrogels for Adipose Derived Stem Cell Delivery for Chondral Defect Repair.

Cell-based therapies have great potential to regenerate and repair injured articular cartilage, and a range of synthetic and natural polymer-based hydrogels have been used in combination with stem cells and growth factors for this purpose. Although the hydrogel scaffolds developed to date possess many favorable characteristics, achieving the required mechanical properties has remained a challenge. A hydrogel system with tunable mechanical properties, composed of a mixture of natural and synthetic polymers, and its use for the encapsulation of adipose derived stem/stromal cells (ASCs) is described. Solutions of methacrylated chondroitin sulfate (MCS) are mixed with solutions of acrylate-poly(trimethylene carbonate)-b-poly(ethylene glycol)-b-poly(trimethylene carbonate)-acrylate (PEG-(PTMC-A)2 ) in phosphate buffered saline and crosslinked via thermally initiated free radical polymerization. The hydrogel compressive equilibrium moduli and toughness are readily tailored by varying the concentration of the pre-polymers, as well as the molecular weight of the PEG used to prepare the PEG-(PTMC-A)2 . Two peptide sequences, GVOGEA and GGGGRGDS, are individually conjugated to the MCS to facilitate cell binding. The presence of the peptide ligands yields high ASC viability and long term metabolic activity following encapsulation in hydrogels prepared using the thermal initiator system. Overall, these hydrogels show promise as a minimally invasive ASC delivery strategy for chondral defect repair.

Enzyme responsive GAG-based natural-synthetic hybrid hydrogel for tunable growth factor delivery and stem cell differentiation.

We describe an enzymatically formed chondroitin sulfate (CS) and poly(ethylene glycol) (PEG) based hybrid hydrogel system, which by tuning the architecture and composition of modular building blocks, allows the application-specific tailoring of growth factor delivery and cellular responses. CS, a negatively charged sulfate-rich glycosaminoglycan of the extracellular matrix (ECM), known for its growth factor binding and stem cell regulatory functions, is used as a starting material for the engineering of this biomimetic materials platform. The functionalization of CS with transglutaminase factor XIII specific substrate sequences is utilized to allow cross-linking of CS with previously described fibrin-mimetic TG-PEG hydrogel precursors. We show that the hydrogel network properties can be tuned by varying the degree of functionalization of CS as well as the ratio and concentrations of PEG and CS precursors. Taking advantage of TG-PEG hydrogel, compatible tagged bio-functional building blocks, including RGD peptides or matrix metalloproteinase sensitive domains, can be incorporated on demand allowing the three-dimensional culture and expansion of human bone marrow mesenchymal stem cells (BM-MSCs). The binding of bone morphogenetic protein-2 (BMP-2) in a CS concentration dependent manner and the BMP-2 release mediated osteogenic differentiation of BM-MSCs indicate the potential of CS-PEG hybrid hydrogels to promote regeneration of bone tissue. Their modular design allows facile incorporation of additional signaling elements, rendering CS-PEG hydrogels a highly flexible platform with potential for multiple biomedical applications.