The impact of detergents on the tissue decellularization process: a ToF-SIMS study

Biologic scaffolds are derived from mammalian tissues, which must be decellularized to remove cellular antigens that would otherwise incite an adverse immune response. Although widely used clinically, the optimum balance between cell removal and the disruption of matrix architecture and surface ligand landscape remains a considerable challenge. Here we describe the use of time of flight secondary ion mass spectroscopy (ToF-SIMS) to provide sensitive, molecular specific, localized analysis of detergent decellularized biologic scaffolds. We detected residual detergent fragments, specifically from Triton X-100, sodium deoxycholate and sodium dodecyl sulphate (SDS) in decellularized scaffolds; increased SDS concentrations from 0.1% to 1.0% increased both the intensity of SDS fragments and adverse cell outcomes. We also identified cellular remnants, by detecting phosphate and phosphocholine ions in PAA and CHAPS decellularized scaffolds. The present study demonstrates ToF-SIMS is not only a powerful tool for characterization of biologic scaffold surface molecular functionality, but also enables sensitive assessment of decellularization efficacy

Extracellular matrix hydrogels from decellularized tissues: structure and function

Extracellular matrix (ECM) bioscaffolds prepared from decellularized tissues have been used to facilitate constructive and functional tissue remodeling in a variety of clinical applications. The discovery that these ECM materials could be solubilized and subsequently manipulated to form hydrogels expanded their potential in vitro and in vivo utility; i.e. as culture substrates comparable to collagen or Matrigel, and as injectable materials that fill irregularly-shaped defects. The mechanisms by which ECM hydrogels direct cell behavior and influence remodeling outcomes are only partially understood, but likely include structural and biological signals retained from the native source tissue. The present review describes the utility, formation, and physical and biological characterization of ECM hydrogels. Two examples of clinical application are presented to demonstrate in vivo utility of ECM hydrogels in different organ systems. Finally, new research directions and clinical translation of ECM hydrogels are discusse

Esophageal Extracellular Matrix Hydrogel to Restore Dysregulated Microenvironment-Cell Signaling for Treatment of Pre-Malignant and Neoplastic Esophageal Disease

Esophageal adenocarcinoma (EAC) has a 20% five-year survival rate, and the standard of care esophagectomy has a 20% mortality and 50% morbidity. EAC pathogenesis involves dysplastic and metaplastic changes within the mucosa in response to dysregulated microenvironmental cues, i.e., gastric reflux and chronic inflammation. Extracellular matrix (ECM) is a primary component of the microenvironment that influences cell phenotype through dynamic reciprocity. ECM bioscaffolds have been used to treat 14 EAC patients after mucosal resection resulting in preservation of esophageal function, restoration of a near normal mucosa, and no recurrence of cancer for 1-8 years. The implanted ECM bioscaffolds completely degraded within 2 weeks, suggesting that the ECM degradation products normalized cell phenotype in the post-cancer resection niche. ECM bioscaffolds can be digested by pepsin in vitro to simulate ECM degradation products, and furthermore these degradation products can be formulated as a hydrogel for clinical therapy. The objectives of the present dissertation were to determine the effects of solubilized non-malignant, metaplastic, and neoplastic esophageal ECM upon macrophage activation and epithelial cell function, to further understand the role of ECM-cell crosstalk in EAC progression. Solubilized, non-malignant ECM hydrogel was evaluated for its potential to mitigate diseased esophageal epithelial cells in vitro and in vivo. Metaplastic and neoplastic ECM contained distinctive matrix-bound nanovesicle (MBV) miRNA cargo and ECM protein signatures that activated macrophages toward a pro-inflammatory, TNFalpha+ state, and increased epithelial cell migration. Non-malignant esophageal ECM (eECM) hydrogel downregulated cancer cell proliferation and signaling pathways (e.g., PI3K-Akt, cell cycle/DNA replication), with a minimal effect on normal esophageal epithelial cells. eECM hydrogel treatment mitigated macroscopic esophagitis, regressed Barrett’s epithelium toward normal squamous epithelium, and downregulated the pro-inflammatory TNFalpha+ cell infiltrate in a pre-clinical, large animal model of Barrett’s esophagus. Similar results were seen with eECM hydrogel treatment in an in vivo rodent model of EAC. Finally, practical aspects of the clinical translation of eECM hydrogels were determined. eECM hydrogel mechanical properties can be tailored to facilitate oral, endoscopic, or submucosal injection delivery. Taken together, we conclude that eECM hydrogel is a suitable therapy for esophageal disease based upon the accepted concepts of cell-matrix interactions

Concentration-dependent rheological properties of ECM hydrogel for intracerebral delivery to a stroke cavity

Biomaterials composed of mammalian extracellular matrix (ECM) promote constructive tissue remodeling with minimal scar tissue formation in many anatomical sites. However, the optimal shape and form of ECM scaffold for each clinical application can vary markedly. ECM hydrogels have been shown to promote chemotaxis and differentiation of neuronal stem cells, but minimally invasive delivery of such scaffold materials to the central nervous system (CNS) would require an injectable form. These ECM materials can be manufactured to exist in fluid phase at room temperature, while forming hydrogels at body temperature in a concentration-dependent fashion. Implantation into the lesion cavity after a stroke could hence provide a means to support endogenous repair mechanisms. Herein, we characterize the rheological properties of an ECM hydrogel composed of urinary bladder matrix (UBM) that influence its delivery and in vivo interaction with host tissue. There was a notable concentration-dependence in viscosity, stiffness, and elasticity; all characteristics important for minimally invasive intracerebral delivery. An efficient MRI-guided injection with drainage of fluid from the cavity is described to assess in situ hydrogel formation and ECM retention at different concentrations (0, 1, 2, 3, 4, and 8 mg/mL). Only ECM concentrations >3 mg/mL gelled within the stroke cavity. Lower concentrations were not retained within the cavity, but extensive permeation of the liquid phase ECM into the peri-infarct area was evident. The concentration of ECM hydrogel is hence an important factor affecting gelation, host-biomaterial interface, as well intra-lesion distribution

MicroRNA Signature Characterizes Primary Tumors That Metastasize in an Esophageal Adenocarcinoma Rat Model

<div><p>Objective</p><p>To establish a miRNA signature for metastasis in an animal model of esophageal adenocarcinoma (EAC).</p><p>Background</p><p>The incidence of esophageal adenocarcinoma (EAC) has dramatically increased and esophageal cancer is now the sixth leading cause of cancer deaths worldwide. Mortality rates remain high among patients with advanced stage disease and esophagectomy is associated with high complication rates. Hence, early identification of potentially metastatic disease would better guide treatment strategies.</p><p>Methods</p><p>The modified Levrat’s surgery was performed to induce EAC in Sprague-Dawley rats. Primary EAC and distant metastatic sites were confirmed via histology and immunofluorescence. miRNA profiling was performed on primary tumors with or without metastasis. A unique subset of miRNAs expressed in primary tumors and metastases was identified with Ingenuity Pathway Analysis (IPA) along with upstream and downstream targets. miRNA-linked gene expression analysis was performed on a secondary cohort of metastasis positive (n=5) and metastasis negative (n=28) primary tumors.</p><p>Results</p><p>The epithelial origin of distant metastasis was established by IF using villin (VIL1) and mucin 5AC (MUC5AC) antibodies. miRNome analysis identified four down-regulated miRNAs in metastasis positive primary tumors compared to metastasis negative tumors: miR-92a-3p (p=0.0001), miR-141-3p (p=0.0022), miR-451-1a (p=0.0181) and miR133a-3p (p=0.0304). Six target genes identified in the top scoring networks by IPA were validated as significantly, differentially expressed in metastasis positive primary tumors: Ago2, Akt1, Kras, Bcl2L11, CDKN1B and Zeb2.</p><p>Conclusion</p><p><i>In vivo</i> metastasis was confirmed in the modified Levrat’s model. Analysis of the primary tumor identified a distinctive miRNA signature for primary tumors that metastasized.</p></div

Eleven differentially expressed miRNAs specific to distant organs excluding stomach.

<p>A two-tailed, two-sample equal variance T-test was performed to obtain the p-values.</p><p>Eleven differentially expressed miRNAs specific to distant organs excluding stomach.</p

Cellular interactions between miRNAs and target genes.

<p>Green represents downregulation and red represents upregulation of gene expression. A two-tailed, two-sample equal variance T-test was performed to obtain the p-values.</p

Immunofluorescence staining of rat tissue with MUC5AC.

<p>Panel A to F show immunofluorescence staining for metastatic lung, metastatic liver, metastatic stomach, non-metastatic lung, non-metastatic liver and non-metastatic stomach, respectively for representative cases. Positive MUC5AC cytoplasmic staining was detected in all metastatic samples with the Alexa Fluor 488 secondary antibody, conjugated to a green fluorophore. Metastasis negative liver and lung show the absence of MUC5AC staining. However, metastasis negative stomach stains positive for MUC5AC as gastric mucin M1 antigen is found in mucus cells of gastric epithelium.</p