Utility of Telomerase-pot1 Fusion Protein in Vascular Tissue Engineering

Cell Transplantation. 2010  |  Pubmed ID: 19878625

While advances in regenerative medicine and vascular tissue engineering have been substantial in recent years, important stumbling blocks remain. In particular, the limited life span of differentiated cells that are harvested from elderly human donors is an important limitation in many areas of regenerative medicine. Recently, a mutant of the human telomerase reverse transcriptase enzyme (TERT) was described, which is highly processive and elongates telomeres more rapidly than conventional telomerase. This mutant, called pot1-TERT, is a chimeric fusion between the DNA binding protein pot1 and TERT. Because pot1-TERT is highly processive, it is possible that transient delivery of this transgene to cells that are utilized in regenerative medicine applications may elongate telomeres and extend cellular life span while avoiding risks that are associated with retroviral or lentiviral vectors. In the present study, adenoviral delivery of pot1-TERT resulted in transient reconstitution of telomerase activity in human smooth muscle cells, as demonstrated by telomeric repeat amplification protocol (TRAP). In addition, human engineered vessels that were cultured using pot1-TERT-expressing cells had greater collagen content and somewhat better performance in vivo than control grafts. Hence, transient delivery of pot1-TERT to elderly human cells may be useful for increasing cellular life span and improving the functional characteristics of resultant tissue-engineered constructs.

Tissue-engineered Lungs for in Vivo Implantation

Science (New York, N.Y.). Jul, 2010  |  Pubmed ID: 20576850

Because adult lung tissue has limited regeneration capacity, lung transplantation is the primary therapy for severely damaged lungs. To explore whether lung tissue can be regenerated in vitro, we treated lungs from adult rats using a procedure that removes cellular components but leaves behind a scaffold of extracellular matrix that retains the hierarchical branching structures of airways and vasculature. We then used a bioreactor to culture pulmonary epithelium and vascular endothelium on the acellular lung matrix. The seeded epithelium displayed remarkable hierarchical organization within the matrix, and the seeded endothelial cells efficiently repopulated the vascular compartment. In vitro, the mechanical characteristics of the engineered lungs were similar to those of native lung tissue, and when implanted into rats in vivo for short time intervals (45 to 120 minutes) the engineered lungs participated in gas exchange. Although representing only an initial step toward the ultimate goal of generating fully functional lungs in vitro, these results suggest that repopulation of lung matrix is a viable strategy for lung regeneration.

Bioreactor for the Long-term Culture of Lung Tissue

Cell Transplantation. 2011  |  Pubmed ID: 21092411

In this article we describe the design and validation of a bioreactor for the in vitro culture of whole rodent lung tissue. Many current systems only enable large segments of lung tissue to be studied ex vivo for up to a few hours in the laboratory. This limitation restricts the study of pulmonary biology in controlled laboratory settings, and also impacts the ability to reliably culture engineered lung tissues in the laboratory. Therefore, we designed, built, and validated a bioreactor intended to provide sufficient nutrient supply and mechanical stimulation to support cell survival and differentiation in cultured lung tissue. We also studied the effects of perfusion and ventilation on pulmonary cell survival and maintenance of cell differentiation state. The final bioreactor design described herein is capable of supporting the culture of whole native lung tissue for up to 1 week in the laboratory, and offers promise in the study of pulmonary biology and the development of engineered lung tissues in the laboratory.

Matrix Composition and Mechanics of Decellularized Lung Scaffolds

Cells, Tissues, Organs. Apr, 2011  |  Pubmed ID: 21502745

The utility of decellularized native tissues for tissue engineering has been widely demonstrated. Here, we examine the production of decellularized lung scaffolds from native rodent lung using two different techniques, principally defined by use of either the detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) or sodium dodecyl sulfate (SDS). All viable cellular material is removed, including at least 99% of DNA. Histochemical staining and mechanical testing indicate that collagen and elastin are retained in the decellularized matrices with CHAPS-based decellularization, while SDS-based decellularization leads to loss of collagen and decline in mechanical strength. Quantitative assays confirm that most collagen is retained with CHAPS treatment but that about 80% of collagen is lost with SDS treatment. In contrast, for both detergent methods, at least 60% of elastin content is lost along with about 95% of native proteoglycan content. Mechanical testing of the decellularized scaffolds indicates that they are mechanically similar to native lung using CHAPS decellularization, including retained tensile strength and elastic behavior, demonstrating the importance of collagen and elastin in lung mechanics. With SDS decellularization, the mechanical integrity of scaffolds is significantly diminished with some loss of elastic function as well. Finally, a simple theoretical model of peripheral lung matrix mechanics is consonant with our experimental findings. This work demonstrates the feasibility of producing a decellularized lung scaffold that can be used to study lung matrix biology and mechanics, independent of the effects of cellular components.

Lobular Endocervical Glandular Hyperplasia with Extensive Mucinous Differentiation of Endometrium and Endometrial Mucinous Adenocarcinoma in Situ: a Case Report and Review of Literature

Gynecologic Oncology. Sep, 2011  |  Pubmed ID: 21605894

Postmortem Detection of Isopropanol in Ketoacidosis

Journal of Forensic Sciences. Jan, 2012  |  Pubmed ID: 22268588

  Isopropanol (IPA) detected in deaths because of diabetic ketoacidosis (DKA) or alcoholic ketoacidosis (AKA) may cause concern for IPA poisoning. This study addressed this concern in a 15-year retrospective review of 260 deaths in which concentrations of acetone and IPA, as well as their ratios, were compared in DKA (175 cases), AKA (79 cases), and IPA intoxication (six cases). The results demonstrated the frequency of detecting IPA in ketoacidosis when there was no evidence of IPA ingestion. IPA was detectable in 77% of DKA cases with quantifiable concentrations averaging 15.1 ± 13.0 mg/dL; 52% of AKA cases with quantifiable concentrations averaging 18.5 ± 22.1 mg/dL; and in cases of IPA intoxication, averaging 326 ± 260 mg/dL. There was weak correlation of IPA production with postmortem interval in DKA only (r = -0.48). Although IPA concentrations were much higher with ingestion, potentially toxic concentrations were achievable in DKA without known ingestion.