Encapsulation of ß-NGF in injectable microrods for localized delivery accelerates endochondral fracture repair.

Introduction: Currently, there are no non-surgical FDA-approved biological approaches to accelerate fracture repair. Injectable therapies designed to stimulate bone healing represent an exciting alternative to surgically implanted biologics, however, the translation of effective osteoinductive therapies remains challenging due to the need for safe and effective drug delivery. Hydrogel-based microparticle platforms may be a clinically relevant solution to create controlled and localized drug delivery to treat bone fractures. Here, we describe poly (ethylene glycol) dimethacrylate (PEGDMA)-based microparticles, in the shape of microrods, loaded with beta nerve growth factor (ß-NGF) for the purpose of promoting fracture repair. Methods: Herein, PEGDMA microrods were fabricated through photolithography. PEGDMA microrods were loaded with ß-NGF and in vitro release was examined. Subsequently, bioactivity assays were evaluated in vitro using the TF-1 tyrosine receptor kinase A (Trk-A) expressing cell line. Finally, in vivo studies using our well-established murine tibia fracture model were performed and a single injection of the ß-NGF loaded PEGDMA microrods, non-loaded PEGDMA microrods, or soluble ß-NGF was administered to assess the extent of fracture healing using Micro-computed tomography (µCT) and histomorphometry. Results: In vitro release studies showed there is significant retention of protein within the polymer matrix over 168 hours through physiochemical interactions. Bioactivity of protein post-loading was confirmed with the TF-1 cell line. In vivo studies using our murine tibia fracture model show that PEGDMA microrods injected at the site of fracture remained adjacent to the callus for over 7 days. Importantly, a single injection of ß-NGF loaded PEGDMA microrods resulted in improved fracture healing as indicated by a significant increase in the percent bone in the fracture callus, trabecular connective density, and bone mineral density relative to soluble ß-NGF control indicating improved drug retention within the tissue. The concomitant decrease in cartilage fraction supports our prior work showing that ß-NGF promotes endochondral conversion of cartilage to bone to accelerate healing. Discussion: We demonstrate a novel and translational method wherein ß-NGF can be encapsulated within PEGDMA microrods for local delivery and that ß-NGF bioactivity is maintained resulting in improved bone fracture repair.

Local injections of ß-NGF accelerates endochondral fracture repair by promoting cartilage to bone conversion.

There are currently no pharmacological approaches in fracture healing designed to therapeutically stimulate endochondral ossification. In this study, we test nerve growth factor (NGF) as an understudied therapeutic for fracture repair. We first characterized endogenous expression of Ngf and its receptor tropomyosin receptor kinase A (TrkA) during tibial fracture repair, finding that they peak during the cartilaginous phase. We then tested two injection regimens and found that local ß-NGF injections during the endochondral/cartilaginous phase promoted osteogenic marker expression. Gene expression data from ß-NGF stimulated cartilage callus explants show a promotion in markers associated with endochondral ossification such as Ihh, Alpl, and Sdf-1. Gene ontology enrichment analysis revealed the promotion of genes associated with Wnt activation, PDGF- and integrin-binding. Subsequent histological analysis confirmed Wnt activation following local ß-NGF injections. Finally, we demonstrate functional improvements to bone healing following local ß-NGF injections which resulted in a decrease in cartilage and increase of bone volume. Moreover, the newly formed bone contained higher trabecular number, connective density, and bone mineral density. Collectively, we demonstrate ß-NGF's ability to promote endochondral repair in a murine model and uncover mechanisms that will serve to further understand the molecular switches that occur during cartilage to bone transformation.

Polymer scaffold architecture is a key determinant in mast cell inflammatory and angiogenic responses.

Implanted polymer scaffolds can induce inflammation leading to the foreign body response (FBR), fibrosis, and implant failure. Thus, it is important to understand how immune cells interact with scaffolds to mitigate inflammation and promote a regenerative response. We previously demonstrated that macrophage phenotype is modulated by fiber and pore diameters of an electrospun scaffold. However, it is unclear if this effect is consistent among other innate immune cells. Mast cells are inflammatory sentinels that play a vital role in the FBR of implanted biomaterials, as well as angiogenesis. We determined if altering electrospun scaffold architecture modulates mast cell responses, with the goal of promoting regenerative cell-scaffold interactions. Polydioxanone (PDO) scaffolds were made from 60 mg/mL or 140 mg/mL PDO solutions, yielding structures with divergent fiber and pore diameters. Mouse mast cells plated on these scaffolds were activated with IL-33 or lipopolysaccharide (LPS). Relative to the 60 mg/mL scaffold, 140 mg/mL scaffolds yielded less IL-6 and TNF, and greater VEGF secretion. Pores >4-6 µm elicited less IL-6 and TNF secretion. IL-33-induced VEGF regulation was more complex, showing effects of both pore size and fiber diameter. These data indicate parameters that can predict mast cell responses to scaffolds, informing biomaterial design to increase wound healing and diminish implant rejection. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 884-892, 2019.

Microenvironmental Regulation of Chondrocyte Plasticity in Endochondral Repair-A New Frontier for Developmental Engineering.

The majority of fractures heal through the process of endochondral ossification, in which a cartilage intermediate forms between the fractured bone ends and is gradually replaced with bone. Recent studies have provided genetic evidence demonstrating that a significant portion of callus chondrocytes transform into osteoblasts that derive the new bone. This evidence has opened a new field of research aimed at identifying the regulatory mechanisms that govern chondrocyte transformation in the hope of developing improved fracture therapies. In this article, we review known and candidate molecular pathways that may stimulate chondrocyte-to-osteoblast transformation during endochondral fracture repair. We also examine additional extrinsic factors that may play a role in modulating chondrocyte and osteoblast fate during fracture healing such as angiogenesis and mineralization of the extracellular matrix. Taken together the mechanisms reviewed here demonstrate the promising potential of using developmental engineering to design therapeutic approaches that activate endogenous healing pathways to stimulate fracture repair.

ADAM10 is required for SCF-induced mast cell migration.

A Disintegrin and Metalloproteinase (ADAM)-10 plays critical roles in neuronal migration and distribution. Recently, ADAM10 deletion was shown to disrupt myelopoiesis. We found that inducible deletion of ADAM10 using Mx1-driven Cre recombinase for a period of three weeks resulted in mast cell hyperplasia in the skin, intestine and spleen. Mast cells express surface ADAM10 in vitro and in vivo, at high levels compared to other immune cells tested. ADAM10 is important for mast cell migration, since ADAM10-deficiency reduced c-Kit-mediated migration. As with some mast cell proteases, ADAM10 expression could be altered by the cytokine microenvironment, being inhibited by IL-10 or TGFß1, but not by several other T cell-derived cytokines. Collectively these data show that the ADAM10 protease is an important factor in mast cell migration and tissue distribution, and can be manipulated by environmental cues.