Suzanne N. King

In vitro characterization of macrophage interaction with mesenchymal stromal cell‐hyaluronan hydrogel constructs

Macrophages play a critical role in mediating not only normal tissue healing, but also the host reaction against biomaterial scaffolds. There is increasing interest in regenerative medicine to combine mesenchymal stromal/stem cells (MSCs) with biomaterial scaffolds to modulate inflammatory response while restoring tissue architecture. The objective of the current study was to investigate the interaction between MSCs (derived from bone marrow, adipose or vocal fold tissue) encapsulated in hyaluronan-based hydrogel and differentiating macrophages as measured by extracellular matrix (ECM) gene expression and cytokine, chemokine, and growth factor concentrations. Gene expression was analyzed using real-time polymerase chain reaction from MSCs embedded in Carbylan-GSX after 7 days of coculture with or without CD14+ cells. Protein concentrations were measured using a Bio-plex assay from cell culture supernatants on days 3 and 7 for all conditions. Following 7 days, we identified upregulation of collagen-I, collagen-III, procollagen, and matrix metalloproteinase-9 genes compared to control conditions. We demonstrate increased concentrations of immunoregulatory cytokines [interleukin (IL)-1β, tumor necrosis factor-α, macrophage inflammatory protein-1α, IFN-γ, IL-12, and IL-10] and remodeling growth factors (vascular endothelial growth factor, hepatocyte growth factor) in MSC-3D constructs cocultured with macrophages compared to control conditions, with some temporal variation. Our results indicate an alteration of expression of ECM proteins important to tissue regeneration and cytokines critical to the inflammatory cascade when 3D constructs were cultured with differentiating macrophages.

Vocal fold fibroblasts immunoregulate activated macrophage phenotype

Recent evidence suggests that fibroblasts play a critical role in regulating inflammation during wound healing because they express several inflammatory mediators in response to bacteria. The objective of this study was to analyze the effects of lipopolysaccharide (LPS) on the immunomodulatory properties of vocal fold fibroblasts (VFFs) derived from polyps, scar and normal tissue co-cultured with macrophages, to provide insight into their interactions during the inflammatory process. Fibroblasts were co-cultured with CD14+ monocytes and after 7 days, wells were treated with LPS for 24 and 72 h. Culture supernatants were collected and concentrations of TNF-α, IL-6, IL-8, IL-10, IL-12, IL-1β and MCP-1 were quantified by ELISA. Normal VFF and CD14+ monocultures were used as controls. Twenty-four hours after LPS activation, macrophages co-cultured with polyp VFF had significantly increased expression of TNF-α, IL-1β, IL-12 and IL-10 compared to controls (p<0.0001). In contrast, macrophages co-cultured with scar VFF had significantly lower expression of TNF-α, IL-1β and IL-12 with significantly higher IL-10 compared to control (p<0.0001). After 72 h, macrophages co-cultured with polyp VFF increased expression of TNF-α, IL-1β, IL-10, IL-6, IL-8, MCP-1 and TGF-β (p<0.01) and macrophages co-cultured with scar VFF significantly decreased their expression of IL-1β and IL-12 compared to control (p<0.0001). Scar VFF at both time points produced significantly lower levels of IL-8, MCP-1, IL-6 and TGF-β compared to controls (p<0.05). Based on our findings, VFF and macrophages secrete several inflammatory mediators that modify their diverse functions. Polyp and scar VFF may play a role in regulating abnormal inflammatory responses, which could result in excessive ECM deposition that disrupts the function of the vocal folds.

Vocal fold myofibroblast profile of scarring

Vocal fold fibroblasts (VFF) are responsible for extracellular matrix synthesis supporting lamina propria in normal and diseased conditions. When tissue is injured, VFF become activated and differentiate into myofibroblasts to facilitate wound healing response. We investigated if vocal fold myofibroblasts can be utilized as surrogate cells for scarred VFF.

Characterization of the Leukocyte Response in Acute Vocal Fold Injury.

Macrophages location in the superficial layer of the vocal fold (VF) is not only at the first line of defense, but in a place of physiologic importance to voice quality. This study characterizes and compares macrophage function in two models of acute injury. Porcine VF injuries were created bilaterally by either surgical biopsy or lipopolysaccharide (LPS) (1.5μg/kg) injection. Animals were sacrificed at 1- or 5-day post LPS or 3-, 7-, or 23-days post-surgical injury (n = 3/time/ injury). Flow cytometry characterized immunophenotypes and RT-PCR quantified cytokine gene expression. Uninjured VF were used as controls. Post-surgical and LPS injury, SWC9+/SWC3- cells identified as hi SLA-DR+ (p<0.05) compared to controls along with hi CD16+ expression at 1-day and 3-days respectively compared to all other time points (p<0.05). Surgical injuries, SWC9+/SWC3- cells exhibited hi CD163+ (p<0.05) at 3-days along with upregulation in TNFα and TGFβ1 mRNA compared to 23-days (p<0.05). No measurable changes to IL–12, IFNγ, IL–10, IL–4 mRNA post-surgery. LPS injuries induced upregulation of TNFα, IL–12, IFNγ, IL–10, and IL–4 mRNA at 1- and 5-days compared to controls (p<0.05). Higher levels of IL–10 mRNA were found 1-day post-LPS compared to 5-days (p<0.05). No changes to CD163 or CD80/86 post-LPS were measured. Acute VF injuries revealed a paradigm of markers that appear to associate with each injury. LPS induced a regulatory phenotype indicated by prominent IL–10 mRNA expression. Surgical injury elicited a complex phenotype with early TNFα mRNA and CD163+ and persistent TGFβ1 transcript expression.

MERS versus Standard Surgical Approaches for Porcine Vocal Fold Scarring with Adipose Stem Cell Constructs

Objective Cells, scaffold, and surgical approaches are important for regeneration of the lamina propria of the scarred vocal fold (VF). Microendoscopy of Reinke’s space (MERS) is a surgical approach used to access the lamina propria. The present study evaluated MERS in the treatment of VF scarring as compared with standardized approaches for the treatment of VF scarring with adipose stem cell constructs. Study Design Animal study. Setting Academic center. Subjects and Methods VF injury was performed bilaterally to induce scarring in 20 pigs. Eight weeks after injury, pigs were classified into no treatment, minithyrotomy, VF injection, VF incision/dissection, and MERS. All groups (except control) were implanted with adipose stem cell and hyaluronan. Four weeks after treatment, histology for collagen, hyaluronan, and fibronectin; mRNA expression for α-smooth muscle actin, tumor growth factor β1, collagen 1α1, collagen 3α1, matrix metalloproteinase 2, basic fibroblast growth factor, and hepatocyte growth factor; and tissue rheology were evaluated. Results Differences were measured among surgical approaches for protein levels of collagen, hyaluronan, and fibronectin (P = .0133, P < .0001, and P = .0025, respectively). Fibroblast growth factor, collagen 1α1, and matrix metalloproteinase 2 transcript levels were different among treatment groups (P = .003, P = .0086, and P = .014, respectively), while no differences were measured for α-smooth muscle actin, tumor growth factor β1, hepatocyte growth factor, and collagen 3α1. Rheologically, significant differences were not measured between groups. Conclusion MERS is a promising surgical approach for the treatment of VF scarring, optimizing the placement of implanted biomaterials.

Macrophage Response to Allogeneic Adipose Tissue-Derived Stromal Cells in Hyaluronan-Based Hydrogel in a Porcine Vocal Fold Injury Model:

Objective: Adipose tissue-derived stromal cells (ASC) embedded in hyaluronan scaffold is a beneficial prophylactic treatment for vocal fold (VF) surgical scar. Here, we investigated the macrophage inflammatory response to allogeneic ASC-constructs and identified changes in lamina propria extracellular matrix. Method: Pig ASC were characterized and transfected with GFP+ lentivirus. Thirty-three pigs underwent VF biopsies, and after 3 days, gel alone, gel+pASC, placebo, or pASC alone was injected into wound bed. Animals were sacrificed 3, 7, or 26 days post-injection. Flow cytometry; qPCR for NF-α, TGFβ, IL-10, IL-4, IFNγ, IL-12, FGF2, Col1A1, and HGF; and immunohistochemistry for collagen, elastin, HA, and fibronectin were performed to characterize macrophage phenotype, quantify cytokine transcription, analyze extracellular matrix remodeling, and track GFP+ cells. Results: No significant differences were found in SWC3+/SWC9+ phenotype or mRNA expression between cells+gel, gel, or placebo. The ASC alone exhibited significantly greater collagen, gel alone resulted in significantly less hyaluronan, and gel+pASC significantly more fibronectin (all P < .05). The pASC-GFP+ were detected 26 days post-injection. Conclusions: The ASC-constructs were biocompatible; they did not influence the macrophage inflammatory response or provoke increases in collagen expression. Long-term engraftment was confirmed.

Adipose Stem Cell Engineering: Characterization and Current Application in Otolaryngology

The clinical potential for adipose-derived stem cell (ASC) is abundant and their capacity to differentiate into multiple lineages demonstrates the impact they can provide to regenerative therapies in otolaryngology – head and neck surgery. Adipose tissues have sufficient amounts of ASCs that can be derived from a single sample and harvested with minimally invasive procedures. Furthermore, ASCs can prevent the incidence of graft versus host disease by suppressing the immune response. Tissue engineering strategies can combine cell delivery with appropriate scaffold biomaterials that can mimic the unique physiological and biomechanical properties of ear, nose and throat. This chapter presents a review of the literature regarding the use of ASCs in otolaryngology head and neck surgery related research, illustrating their current and potential use in the field.