Neutrophil Granulopoiesis Optimized Through Ex Vivo Expansion of Hematopoietic Progenitors in Engineered 3D Gelatin Methacrylate Hydrogels.

Neutrophils are the first line of defense of the innate immune system. In response to methicillin-resistant Staphylococcus aureus infection in the skin, hematopoietic stem, and progenitor cells (HSPCs) traffic to wounds and undergo extramedullary granulopoiesis, producing neutrophils necessary to resolve the infection. This prompted the engineering of a gelatin methacrylate (GelMA) hydrogel that encapsulates HSPCs within a matrix amenable to subcutaneous delivery. The authors study the influence of hydrogel mechanical properties to produce an artificial niche for granulocyte-monocyte progenitors (GMPs) to efficiently expand into functional neutrophils that can populate infected tissue. Lin-cKIT+ HSPCs, harvested from fluorescent neutrophil reporter mice, are encapsulated in GelMA hydrogels of varying polymer concentration and UV-crosslinked to produce HSPC-laden gels of specific stiffness and mesh sizes. Softer 5% GelMA gels yield the most viable progenitors and effective cell-matrix interactions. Compared to suspension culture, 5% GelMA results in a twofold expansion of mature neutrophils that retain antimicrobial functions including degranulation, phagocytosis, and ROS production. When implanted dermally in C57BL/6J mice, luciferase-expressing neutrophils expanded in GelMA hydrogels are visualized at the site of implantation for over 5 days. They demonstrate the potential of GelMA hydrogels for delivering HSPCs directly to the site of skin infection to promote local granulopoiesis.

The critical impact of traumatic muscle loss on fracture healing: Basic science and clinical aspects.

Musculoskeletal trauma, specifically fractures, is a leading cause of patient morbidity and disability worldwide. In approximately 20% of cases with fracture and related traumatic muscle loss, bone healing is impaired leading to fracture nonunion. Over the past few years, several studies have demonstrated that bone and the surrounding muscle tissue interact not only anatomically and mechanically but also through biochemical pathways and mediators. Severe damage to the surrounding musculature at the fracture site causes an insufficiency in muscle-derived osteoprogenitor cells that are crucial for fracture healing. As an endocrine tissue, skeletal muscle produces many myokines that act on different bone cells, such as osteoblasts, osteoclasts, osteocytes, and mesenchymal stem cells. Investigating how muscle influences fracture healing at cellular, molecular, and hormonal levels provides translational therapeutic solutions to this clinical challenge. This review provides an overview about the contributions of surrounding muscle tissue in directing fracture healing. The focus of the review is on describing the interactions between bone and muscle in both healthy and fractured environments. We discuss current progress in identifying the bone-muscle molecular pathways and strategies to harness these pathways as cues for accelerating fracture healing. In addition, we review the existing challenges and research opportunities in the field.

The Effects of Adipose-Derived Stem Cells Differentiated Into Endothelial Cells and Osteoblasts on Healing of Critical Size Calvarial Defects.

Delayed vascularization and resultant resorption limits the clinical use of tissue engineered bony constructs. The objective of this study is to develop a strategy to accelerate the neovascularization of tissue-engineered bony constructs using endothelial differentiated adipose-derived stem cells (ASC). The authors harvested ASC from inguinal fat pads of male Lewis rats (n = 5) and induced toward endothelial and osteoblastic lineages. The authors created critical size calvarial defects on male Lewis rats (n = 30) and randomized the animals into 4 groups. For the repair of the defects the authors used hydroxyapatite/poly(lactide-co-glycolide) [HA-PLG] scaffolds in group I, HA-PLG scaffolds seeded with ASC in group II, HA-PLG scaffolds seeded with ASC-derived endothelial cells in group III, and HA-PLG scaffolds seeded with ASC-derived osteoblasts in group IV. The authors evaluated the bone healing histologically and with micro-computed tomography (CT) scans 8 weeks later. Adipose-derived stem cells exhibited the characteristics of endothelial and osteogenic lineages, and attached on HA-PLG scaffolds after differentiation. Micro-CT analysis revealed that highest bone mineral density was in group IV (1.46 ± 0.01 g/cm) followed by groups III (1.43 ± 0.05 g/cm), I (1.42 ± 0.05 g/cm), and II (1.3 ± 0.1 g/cm). Hematoxylin-Eosin and Masson Trichrome staining revealed similar results with the highest bone regeneration in group IV followed by groups II, III, and I. Regenerated bone in group IV also had the highest vascular density, but none of these differences achieved statistical significance (P > 0.05). The ASC-derived endothelial cells and osteoblasts provide a limited increase in calvarial bone healing when combined with HA-PLG scaffolds.

Neurite outgrowth in fibrin gels is regulated by substrate stiffness.

Fibrin is a promising matrix for use in promoting nerve repair given its natural occurrence in peripheral nerve injuries, and the biophysical properties of this matrix can be regulated to modulate tissue regeneration. In this study, we examined the effect of physical and mechanical properties of fibrin gels on dorsal root ganglia (DRG) neurite extension. Increases in fibrinogen concentration increased the number of fibrin strands, resulting in decreased pore size and increased stiffness. Neurite extension was reduced when DRG explants were cultured within fibrin gels of increasing fibrinogen concentrations (from 9.5 to 141 mg/mL). The addition of NaCl also increased the number of fibrin strands, reducing fiber diameter and porosity, while increasing mechanical strength, and reductions in neurite extension correlated with increases in NaCl content. We determined that neurite extension within fibrin gels is dependent on fibrinolysis and is mediated by the secretion of serine proteases and matrix metalloproteinases by entrapped DRGs, as confirmed by culturing cells in the presence of inhibitors against these enzymes and real-time-polymerase chain reaction. Taken together, the results of this study provide new insight into the effect of fibrin gel biophysical properties on neurite extension and suggest new opportunities to improve the efficacy of these materials when used as nerve guidance conduits.