Extracellular matrix particle-glycosaminoglycan composite hydrogels for regenerative medicine applications.

Tissue extracellular matrix (ECM) is a complex material made up of fibrous proteins and ground substance (glycosaminoglycans, GAGs) that are secreted by cells. ECM contains important biological cues that modulate cell behaviors, and it also serves as a structural scaffold to which cells can adhere. For clinical applications, where immune rejection is a constraint, ECM can be processed using decellularization methods intended to remove cells and donor antigens from tissue or organs, while preserving native biological cues essential for cell growth and differentiation. In this study, a decellularized ECM-based composite hydrogel was formulated by using modified GAGs that covalently bind tissue particles. These GAG-ECM composite hydrogels combine the advantages of solid decellularized ECM scaffolds and pepsin-digested ECM hydrogels by facilitating ECM hydrogel formation without a disruptive enzymatic digestion process. Additionally, engineered hydrogels can contain more than one type of ECM (from bone, fat, liver, lung, spleen, cartilage, or brain), at various concentrations. These hydrogels demonstrated tunable gelation kinetics and mechanical properties, offering the possibility of numerous in vivo and in vitro applications with different property requirements. Retained bioactivity of ECM particles crosslinked into this hydrogel platform was confirmed by the variable response of stem cells to different types of ECM particles with respect to osteogenic differentiation in vitro, and bone regeneration in vivo. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 147-159, 2018.

Chondroitin Sulfate-Based Biocompatible Crosslinker Restores Corneal Mechanics and Collagen Alignment.

Purpose: To evaluate the crosslinking effect of functionalized chondroitin sulfate (CS) in an ex vivo rabbit cornea model. Methods: Chondroitin sulfate molecules were chemically modified with the N-hydroxysuccinimide (NHS) group. Enucleated rabbit eyes were crosslinked with 2, 5, or 10 mg/mL CS-NHS solution for 30 or 60 minutes. The CS-NHS penetration, corneal swelling ratio, Young's modulus, and ultrastructure of the crosslinked corneas were characterized. In addition, rabbit corneas were further treated with a collagenase-chondroitinase solution to create an ex vivo keratoconus (KC)-like model. The KC model corneas were crosslinked with a standard riboflavin-ultraviolet (UV) method or alternatively with CS-NHS. Corneal mechanics, ultrastructure, and keratocyte gene expression were evaluated after UV and CS-NHS crosslinking. Results: CS-NHS effectively penetrated into the corneal stroma within 60 minutes of treatment initiation. CS-NHS crosslinking reduced the swelling ratio by 35%, increased Young's modulus by 20%, and increased collagen fibril diameter and density. CS-NHS crosslinking improved corneal mechanics of KC model corneas to levels comparable to those with UV crosslinking. Moreover, CS-NHS crosslinking demonstrated significant downregulation of proinflammatory gene expression of keratocytes, indicating a potential protective effect imparted by CS-NHS during crosslinking. Conclusions: Our results demonstrated that CS-NHS can reinforce normal and KC model corneal mechanics, and restore collagen density and alignment in KC model corneas without causing extensive keratocyte apoptosis and proinflammatory gene upregulation. Therefore, CS-NHS crosslinking can potentially provide an effective, safe, and biocompatible means of corneal reinforcement.

Metabolic variations in normal and fibrotic human laryngotracheal-derived fibroblasts: A Warburg-like effect.

OBJECTIVES/HYPOTHESIS: Laryngotracheal stenosis (LTS) is a chronic fibrotic disease characterized by fibroblast proliferation, collagen deposition, and matrix remodeling in the lamina propria of the larynx and/or trachea. Current medical therapies are limited by a poor understanding of the effector cell's (fibroblasts) cellular biology and metabolism. The purpose of this study was to compare cellular proliferation, function, and metabolism between normal and LTS-derived fibroblasts in vitro. We hypothesize that LTS-derived fibroblasts will demonstrate aberrant behavior with faster proliferation, increased collagen production, and altered metabolic allocation compared with normal fibroblasts. STUDY DESIGN: In vitro comparative analysis. METHODS: Human biopsies of normal and iatrogenic LTS tissue (n = 7) were obtained, and fibroblasts were isolated and cultured in vitro. Cellular proliferation, cellular histology, gene expression, and metabolic analyses were performed. Statistical analyses comparing normal and scar-derived fibroblasts were performed. RESULTS: LTS fibroblast proliferation rate, cellular surface area, and collagen-1 expression were increased compared to normal fibroblasts. Cellular metabolic analysis of LTS-derived fibroblasts demonstrated reduced oxidative phosphorylation and increased glycolysis/oxidative phosphorylation ratio compared with normal fibroblasts. CONCLUSIONS: Human iatrogenic LTS-derived fibroblasts demonstrated aberrant behavior when compared with normal fibroblasts. A Warburg-like effect was revealed, suggesting human iatrogenic LTS fibroblasts drive their proliferation with aerobic glycolysis. The distinct metabolism suggests metabolic inhibitors could reduce fibroblast hyperplasia and hypertrophy in LTS and fibrosis in general. LEVEL OF EVIDENCE: NA Laryngoscope, 127:E107-E113, 2017.

Metabolically Active Three-Dimensional Brown Adipose Tissue Engineered from White Adipose-Derived Stem Cells.

Brown adipose tissue (BAT) has a unique capacity to expend calories by decoupling energy expenditure from ATP production, therefore BAT could realize therapeutic potential to treat metabolic diseases such as obesity and type 2 diabetes. Recent studies have investigated markers and function of native BAT, however, successful therapies will rely on methods that supplement the small existing pool of brown adipocytes in adult humans. In this study, we engineered BAT from both human and rat adipose precursors and determined whether these ex vivo constructs could mimic in vivo tissue form and metabolic function. Adipose-derived stem cells (ASCs) were isolated from several sources, human white adipose tissue (WAT), rat WAT, and rat BAT, then differentiated toward both white and brown adipogenic lineages in two-dimensional and three-dimensional (3D) culture conditions. ASCs derived from WAT were successfully differentiated in 3D poly(ethylene glycol) hydrogels into mature adipocytes with BAT phenotype and function, including high uncoupling protein 1 (UCP1) mRNA and protein expression and increased metabolic activity (basal oxygen consumption, proton leak, and maximum respiration). By utilizing this "browning" process, the abundant and accessible WAT stem cell population can be engineered into 3D tissue constructs with the metabolic capacity of native BAT, ultimately for therapeutic intervention in vivo and as a tool for studying BAT and its metabolic properties.

Rapamycin inhibits human laryngotracheal stenosis-derived fibroblast proliferation, metabolism, and function in vitro.

OBJECTIVE: To determine if rapamycin inhibits the growth, function, and metabolism of human laryngotracheal stenosis (LTS)-derived fibroblasts. STUDY DESIGN: Controlled in vitro study. SETTING: Tertiary care hospital in a research university. SUBJECTS AND METHODS: Fibroblasts isolated from biopsies of 5 patients with laryngotracheal stenosis were cultured. Cell proliferation, histology, gene expression, and cellular metabolism of LTS-derived fibroblasts were assessed in 4 conditions: (1) fibroblast growth medium, (2) fibroblast growth medium with dimethylsulfoxide (DMSO), (3) fibroblast growth medium with 10(-10) M (low-dose) rapamycin dissolved in DMSO, and (4) fibroblast growth medium with 10(-9) M (high-dose) rapamycin dissolved in DMSO. RESULTS: The LTS fibroblast count and DNA concentration were reduced after treatment with high-dose rapamycin compared to DMSO (P = .0007) and normal (P = .0007) controls. Collagen I expression decreased after treatment with high-dose rapamycin versus control (P = .0051) and DMSO (P = .0093) controls. Maximal respiration decreased to 68.6 pMoles of oxygen/min/10 mg/protein from 96.9 for DMSO (P = .0002) and 97.0 for normal (P = .0022) controls. Adenosine triphosphate (ATP) production decreased to 66.8 pMoles from 88.1 for DMSO (P = .0006) and 83.3 for normal (P = .0003) controls. Basal respiration decreased to 78.6 pMoles from 108 for DMSO (P = .0002) and 101 for normal (P = .0014) controls. CONCLUSIONS: Rapamycin demonstrated an anti-fibroblast effect by significantly reducing the proliferation, metabolism, and collagen deposition of human LTS fibroblast in vitro. Rapamycin significantly decreased oxidative phosphorylation of LTS fibroblasts, suggesting at a potential mechanism for the reduced proliferation and differentiation. Furthermore, rapamycin's anti-fibroblast effects indicate a promising adjuvant therapy for the treatment of laryngotracheal stenosis.

Dysregulated Macrophages Are Present in Bleomycin-Induced Murine Laryngotracheal Stenosis.

OBJECTIVE: To define the inflammatory cell infiltrate preceding fibrosis in a laryngotracheal stenosis (LTS) murine model. STUDY DESIGN: Prospective controlled murine study. SETTING: Laboratory. SUBJECTS AND METHODS: Chemomechanical injury mice (n = 44) sustained bleomycin-coated wire-brush injury to the laryngotracheal complex while mechanical injury controls (n = 42) underwent phosphate-buffered saline (PBS)-coated wire-brush injury. Mock surgery controls (n = 34) underwent anterior transcervical tracheal exposure only. Inflammatory and fibrosis protein and gene expression were assessed in each condition. Immunohistochemistry served as a secondary outcome. RESULTS: In chemomechanical injury mice, there was an upregulation of collagen I (P < .0001, P < .0001), Tgf-ß (P = .0023, P = .0008), and elastin (P < .0001, P < .0001) on day 7; acute inflammatory gene Il1ß (P = .0027, P = .0008) on day 1; and macrophage gene CD11b (P = .0026, P = .0033) on day 1 vs mechanical and mock controls, respectively. M1 marker inducible nitric oxide synthase (iNOS) expression decreased (P = .0014) while M2 marker Arg1 (P = .0002) increased on day 7 compared with mechanical controls. Flow cytometry demonstrated increased macrophages (P = .0058, day 4) and M1 macrophages (P = .0148, day 4; P = .0343, day 7; P = .0229, day 10) compared to mock controls. There were similarities between chemomechanical and mechanical injury mice with an increase in M2 macrophages at day 10 (P = .0196). CONCLUSIONS: The bleomycin-induced LTS mouse model demonstrated increased macrophages involved with the development of fibrosis. Macrophage immunophenotype suggested that dysregulated M2 macrophages have a role in abnormal laryngotracheal wound healing. These data delineate inflammatory cells and signaling pathways in LTS that may potentially be modulated to lessen fibroblast proliferation and collagen deposition.

Controlled release of vascular endothelial growth factor enhances intestinal adaptation in rats with extensive small intestinal resection.

BACKGROUND: Vascular endothelial growth factor (VEGF) has been shown to be an essential factor in the intestinal adaption after extensive bowel resection. The present study investigates the controlled release of VEGF as a way to improve intestinal adaptation. MATERIALS AND METHODS: Biodegradable microspheres with or without VEGF were made using a double emulsion technique. Approximately 80% of the small intestine was removed, leaving the entire duodenum, 10 cm of jejunum, and 7 cm of ileum. The microspheres were distributed on the anastomosed small bowel. The animals were sacrificed after 14 days, and the adapted jejunum and ileum were analyzed for sucrase activity and histologic parameters. RESULTS: The average villus lengths of the adapted jejunum and ileum were 680 and 350 µm in rats that received blank microspheres, compared to 810 and 720 µm in rats that received VEGF microspheres. The average sucrase activity of the adapted jejunum and ileum was 0.0202 and 0.0073 µmol/mg protein/min in rats that received blank microspheres, compared with 0.0236 and 0.0232 µmmol/mg protein/min in rats that received VEGF microspheres. CONCLUSION: The data suggest that the controlled delivery of VEGF from microspheres enhances the adaptation of the small intestine in rats. The controlled delivery of VEGF over time can potentially be used in patients following extensive intestinal resection.