Cross-linked gelatin microspheres with continuously tunable degradation profiles for renal tissue regeneration.

Collagen and gelatin-based biomaterials are widely used in tissue engineering applications. Various methods have been reported for the cross-linking of these macromolecules for the purpose of delaying their biodegradation to prolong their in vivo residence (in tissue engineering applications) or tailoring their drug releasing capacity (when used as drug carriers). In this study, a carbodiimide-based cross-linking method, also used in the production of United States Food and Drug Administration-approved products, was employed to obtain differentially cross-linked gelatin beads. The colorimetric determination of the in vitro enzymatic susceptibility of the beads indicated that the resistance to degradation linearly correlated with the concentration of carbodiimide used for the cross-linking reaction. This result was also confirmed in vivo by the histological evaluation of the residence time of orthotopically injected cell-seeded beads. These data would indicate that the production of gelatin-based microbeads with tunable degradation profiles might be applicable toward the development of products that catalyze regeneration of kidney and other solid organs.

Isolation of smooth muscle cells from bladder for generation of engineered urologic organs.

The isolation of smooth muscle cells from bladder tissue is a valuable technique used in cell biology research and tissue engineering. Smooth muscle cells can be used for analysis in many areas including, but not limited to, cell function and genotype experimentation. Smooth muscle cells can also be used in tissue engineering applications for research and/or regenerative medicine. Replacement tissue or tissue for augmentation can be created to stem or remediate problems in the urologic system.

Bioreactor design considerations for hollow organs.

There are many important considerations in the design, construction, and use of a bioreactor for growing hollow organs such as vessels, gastrointestinal tissue, esophagus, and others. The growth of new organs requires a specialized container that provides sterility and an environment conducive to cell-seeding and attachment onto a three-dimensional bioabsorbable porous scaffold, incubation, maturation, and shipping for implantation. The materials' selection, dimensions, manufacturing, testing, and use of the bioreactor are all factors that should be considered in designing a bioreactor for the development of hollow organs.

Formulation of selected renal cells for implantation into a kidney.

Delivery of cells to organs has primarily relied on formulating the cells in a nonviscous liquid carrier. We have developed a methodology to isolate selected renal cells (SRC) that have provided functional stability to damaged kidneys in preclinical models (Kelley et al. Poster presentation at 71st scientific sessions of American diabetes association , 2011; Kelley et al. Oral presentation given at Tissue Engineering and Regenerative Medicine International Society (TERMIS)-North America annual conference, 2010; Presnell et al. Tissue Eng Part C Methods 17:261-273, 2011; Kelley et al. Am J Physiol Renal Physiol 299:F1026-F1039, 2010). In order to facilitate SRC injection into the kidney of patients who have chronic kidney disease, we have developed a strategy to immobilize the cells in a hydrogel matrix. This hydrogel (gelatin) supports cells by maintaining them in a three-dimensional state during storage and shipment (both at cold temperatures) while facilitating the delivery of cells by liquefying when engrafting into the kidney. This chapter will define a method for the formulation of the kidney epithelial cells within a hydrogel.

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-ß signaling pathways related to tubulo-interstitial fibrosis associated with CKD progression was evaluated. Conditioned medium was observed to attenuate TGF-ß-induced epithelial-mesenchymal transition (EMT) in vitro in a human proximal tubular cell line (HK2).

Allogeneic cell therapy for the treatment of liver disease.

The scarcity of human organs available for transplantation is clearly evident. Efforts to maximize the use of available organs and to increase the number of donors have increased the number of transplantations performed, but at a rate that remains far behind the rate of growth of the waiting list. Thus, the likelihood of a patient with severe liver disease receiving a liver replacement is decreasing. In order to offer treatment to most patients with liver disease, alternatives to whole-organ replacement must be found. Cell-based treatments, in which suspensions of liver cells are injected into patients with liver failure and reconstitute the patient's liver functions, may be that alternative. Here, we report on a regulatory-compliant process for the production of a cryopreserved cell therapy product that yields viable, metabolically active hepatocytes that can be infused directly into patients with the goal of reconstituting liver function.

A chicken mRNA similar to heterogeneous nuclear ribonucleoprotein H1.

Heterogeneous nuclear ribonucleoproteins are predominantly nuclear RNA-binding proteins that function in a variety of cellular activities. The objective of these experiments was to clone a cDNA for a chicken protein similar to other previously reported heterogeneous ribonucleoproteins for other species. The 5' and 3' ends of the chicken mRNA were cloned using Rapid Amplification of cDNA Ends (RACE). Subsequently, the expression of the mRNA sequence was confirmed via Northern analysis. The deduced amino acid sequence was approximately 86% identical to corresponding regions of human, mouse, or zebrafish proteins similar to heterogeneous nuclear ribonucleoprotein H1. The expression data confirmed the size of the predicted mRNA sequence. The newly identified sequence may be employed in future studies aimed at understanding the role of heterogeneous nuclear ribonucleoproteins in avian species.

Glyceraldehyde-3-phosphate dehydrogenase expression varies with age and nutrition status.

OBJECTIVE: Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a key enzyme in the glycolytic pathway, and it is a popular internal standard for northern blot analysis. We examined GAPDH expression early in life when feed is either provided or not provided to animals. METHODS: Male broiler chickens were provided a standard starter diet plus Oasis nutritional supplement (fed group; Novus International, St. Louis, MO, USA) or no feed (starved group) for the first 3 d posthatch. Subsequently, the standard starter diet was provided to all chickens between 3 and 7 d posthatch. RNA was extracted from the pectoralis thoracicus, and GAPDH expression was evaluated with quantitative northern analysis. RESULTS: GAPDH expression was significantly (P < 0.05) higher in the fed than in the starved group at 3 d posthatch, suggesting that nutritional manipulations can alter GAPDH transcription. Similarly, GAPDH mRNA levels were significantly (P < 0.05) higher at 7 d posthatch compared with all younger animals, suggesting that GAPDH is developmentally upregulated with advancing age. CONCLUSION: GAPDH expression changes with age and nutrition status in the early posthatch chick, suggesting that GAPDH is not a proper internal standard for muscle studies using quantitative northern analysis.

Early posthatch starvation induces myonuclear apoptosis in chickens.

The effect of early posthatch starvation on myonuclear apoptosis was examined in chickens. Male broiler chickens were or were not provided feed for the first 3-d posthatch. Subsequently, all chickens were provided feed for an additional 4-d posthatch. Chickens were killed at 3- and 7-d posthatch, and the pectoralis thoracicus was harvested, fixed and embedded in paraffin. Muscle sections were labeled with the terminal deoxynucleotidyl transferase histochemical staining technique to identify apoptotic nuclei. At 3- and 7-d posthatch, there was a significantly (P < 0.05) smaller myofiber cross-sectional area for the starved compared with the fed chickens. A larger proportion (P < 0.05) of apoptotic nuclei relative to total nuclei was observed in the starved compared to the fed chickens killed at 3-d posthatch, but the proportion of apoptotic nuclei relative to total nuclei did not differ (P > 0.05) between the starved and fed chickens killed at 7-d posthatch. It appears that apoptosis is a mechanism contributing to the smaller myofiber size observed when feed is not provided early posthatch.