Belinda J. Wagner

Early Cellular and Stromal Responses in Regeneration Versus Repair of a Mammalian Bladder Using Autologous Cell and Biodegradable Scaffold Technologies

PURPOSE Internal organ regeneration holds promise for changing medical technology and decreasing organ shortages. Current medical treatment for internal organ failure is largely limited to organ transplantation. A construct composed of synthetic biopolymer with autologous cells has shown long-term clinical benefit in patients undergoing augmentation cystoplasty. However, to our knowledge early cellular and stromal events during bladder regeneration have not been elucidated. MATERIALS AND METHODS In situ cellular responses to 2 biopolymer implants, including a poly(lactic-co-glycolic acid) (Sigma-Aldrich) based biodegradable mesh scaffold with autologous urothelial and smooth muscle cells (construct) and a poly(lactic-co-glycolic acid) based biodegradable mesh scaffold alone without cells (scaffold), were compared in a canine model of augmentation cystoplasty. Healing events were correlated with urodynamic assessments. RESULTS Construct implants regenerated baseline urodynamics as early as 4 months after implantation. Urodynamics following scaffold implantation failed to return to baseline by study termination at 9 months. Functional differences elicited by construct and scaffold implants correlated with structural differences in the neotissues. Construct stroma had greater vascularization with gently folded, interwoven connective tissue elements. Scaffold stroma was dense, haphazardly organized connective tissue. Urothelium regenerated in response to construct and scaffold implantation. However, only construct had normal stroma, well developed detrusor and abundant alpha-smooth muscle actin (Vector Laboratories, Burlingame, California) cell staining at early time points, leading to a structurally and functionally complete regenerated bladder wall at 9 months. CONCLUSIONS Early cellular and stromal events distinguish healing processes that lead to bladder wall regeneration or repair. Construct implants containing cells elicit early healing processes that culminate with the regeneration of complete mucosal and muscular components, whereas the response to scaffold implantation is consistent with reparative healing, that is with mucosal growth but incomplete tissue layer development.

Long-term durability, tissue regeneration and neo-organ growth during skeletal maturation with a neo-bladder augmentation construct

AIMS To comparatively evaluate bladder regeneration following 80% cystectomy and augmentation using a synthetic biopolymer with autologous urothelial and smooth muscle cells (autologous neo-bladder augmentation construct [construct]) or autotransplantation of native bladder (reimplanted native urinary bladder [reimplant]) in canines. MATERIALS & METHODS Voiding function, urodynamic assessment and neo-organ capacity-to-body-weight ratio (C:BW) were assessed longitudinally for a total of 24 months following trigone-sparing augmentation cystoplasty in juvenile canines. RESULTS Within 30 days postimplantation, hematology and urinalysis returned to baseline. Constructs and reimplants yielded neo-organs with statistically equivalent urodynamics and histology. Linear regression analysis of C:BW showed that constructs regained baseline slope and continued to adapt with animal growth. CONCLUSIONS Constructs and reimplants regained and maintained native bladder histology by 3 months, capacity at 3-6 months and compliance by 12-24 months. Furthermore, construct C:BW demonstrated the ability of regenerated bladder to respond to growth regulation.

Tubular cell-enriched subpopulation of primary renal cells improves survival and augments kidney function in rodent model of chronic kidney disease

Established chronic kidney disease (CKD) may be identified by severely impaired renal filtration that ultimately leads to the need for dialysis or kidney transplant. Dialysis addresses only some of the sequelae of CKD, and a significant gap persists between patients needing transplant and available organs, providing impetus for development of new CKD treatment modalities. Some postulate that CKD develops from a progressive imbalance between tissue damage and the kidneys intrinsic repair and regeneration processes. In this study we evaluated the effect of kidney cells, delivered orthotopically by intraparenchymal injection to rodents 4-7 wk after CKD was established by two-step 5/6 renal mass reduction (NX), on the regeneration of kidney function and architecture as assessed by physiological, tissue, and molecular markers. A proof of concept for the model, cell delivery, and systemic effect was demonstrated with a heterogeneous population of renal cells (UNFX) that contained cells from all major compartments of the kidney. Tubular cells are known contributors to kidney regeneration in situ following acute injury. Initially tested as a control, a tubular cell-enriched subpopulation of UNFX (B2) surprisingly outperformed UNFX. Two independent studies (3 and 6 mo in duration) with B2 confirmed that B2 significantly extended survival and improved renal filtration (serum creatinine and blood urea nitrogen). The specificity of B2 effects was verified by direct comparison to cell-free vehicle controls and an equivalent dose of non-B2 cells. Quantitative histological evaluation of kidneys at 6 mo after treatment confirmed that B2 treatment reduced severity of kidney tissue pathology. Treatment-associated reduction of transforming growth factor (TGF)-β1, plasminogen activator inhibitor (PAI)-1, and fibronectin (FN) provided evidence that B2 cells attenuated canonical pathways of profibrotic extracellular matrix production.

Functional Evaluation of Primary Renal Cell/Biomaterial Neo-Kidney Augment Prototypes for Renal Tissue Engineering

Development of a tissue-engineered neo-kidney augment (NKA) requires evaluation of defined, therapeutically relevant cell and cell/biomaterial composites (NKA constructs) for regenerative potential in mammalian kidney. Previous work identified primary renal cell populations that extended survival and improved renal function in a rodent model of chronic kidney disease (CKD). This study extends that work toward the goal of developing NKA by (i) screening in vivo inflammatory and fibrotic responses to acellular biomaterials delivered to healthy rodent renal parenchyma, (ii) evaluating the functionality of renal cell/biomaterial combinations in vitro, (iii) generating NKA constructs by combining therapeutically relevant cell populations with biocompatible biomaterial, and (iv) evaluating in vivo neokidney tissue development in response to NKA constructs delivered to healthy rodent renal parenchyma. Gelatin and hyaluronic acid (HA)-based hydrogels elicited the least inflammatory and fibrotic responses in renal parenchyma relative to polycaprolactone (PCL) and poly(lactic-co-glycolic acid) (PLGA) beads or particles and were associated with neovascularization and cellular infiltration by 4 weeks postimplantation. Renal cell populations seeded onto gelatin or HA-based hydrogels were viable and maintained a tubular epithelial functional phenotype during an in vitro maturation of 3 days as measured by transcriptomic, proteomic, secretomic, and confocal immunofluorescence assays. In vivo delivery of cell-seeded NKA constructs (bioactive renal cells + gelatin hydrogels) to healthy rodent renal parenchyma elicited neokidney tissue formation at 1 week postimplantation. To investigate a potential mechanism by which NKA constructs could impact a disease state, the effect of conditioned media on TGF-β signaling pathways related to tubulo-interstitial fibrosis associated with CKD progression was evaluated. Conditioned medium was observed to attenuate TGF-β-induced epithelial–mesenchymal transition (EMT) in vitro in a human proximal tubular cell line (HK2).

Loss of Heterozygosity

The most common molecular alteration observed in human cancers,1 loss of heterozygosity (LOH), is a significant mecha-nism by which critical genes involved in growth regulation and homeostasis become inactivated, or silenced, during disease evolution. This chapter provides a review of LOH and its implications in various cancers as well as a review of LOH in nonmalignant diseases. Only 0.08% of those base pairs within the entire human genome (3 billion base pairs) vary between any two humans, and only 0.02% of those variations actually result in an expressed protein with a different amino acid as a result of the change.2 Even more remarkable, 90% of those variations are changes that are common in the population and lead to normal variation in traits among individuals; eye color, for example.

Tissue Engineering and Regenerative Medicine: Role of Toxicologic Pathologists for an Emerging Medical Technology:

Tissue Engineering Regenerative Medical (TERM) products are a new technology currently in human clinical testing for a variety of unmet medical needs involving tissue and organ dysfunction and failure. Safety evaluation of TERM products overlaps 3 established product paradigms: pharmaceuticals (biologically active substances), transplantation (cells or tissue), and devices (biomaterials). As TERM products recapitulate organ or tissue structure and function with unique biological activity and characteristics, they require new preclinical paradigms to bring TERM products through to clinical trials. Establishing TERM-product safety programs requires broad-based knowledge of tissue and organ homeostasis, regenerative biology, and translational medicine to design new preclinical paradigms. Therefore, toxicologic pathologists have a compelling scientific role in evaluating TERM products, characterizing tissue responses, and helping distinguish optimal (regeneration) from deficient or incomplete outcomes indicative of substandard functionality (repair). As new-tissue engineering and regenerative medical technologies develop for tissue and organ regeneration, the toxicologic pathologist will be asked to develop novel testing, reevaluate established toxicologic diagnostic criteria, and reinterpret tissue responses that may extend beyond current standards.