Comparison of Decellularized and Gamma-Irradiated Septal and Costal Cartilage Allografts in a Rabbit Model.

Background: Traditional gamma-irradiated allograft costal cartilage used for cartilage-depleted rhinoplasty patients contains cellular remnants that are potentially responsible for immunological stimulation and graft resorption. Objective: To determine whether decelullarized and/or chemically crosslinked rabbit costal and nasal septal cartilage allografts reduce the risk of allograft resorption. Materials and Methods: In vitro and in vivo analyses of septal and costal cartilage grafts in New Zealand white rabbits were carried out. Irradiated, decellularized, and/or carbodiimide crosslinked cartilage grafts were compared with nontreated autografts and allografts controls. Gross analysis, biomechanical testing, DNA quantification, and histological analyses were performed. Results: All treated grafts had a similar "feel" to native cartilage except for crosslinked grafts, which were significantly stiffer with decreased maximum load and tensile strain. Decellularization effectively reduced DNA content. Biomechanics of explants were unchanged except in untreated allografts, which exhibited increased stiffness, decreased strain, and significant scarring/fibrosis. There was increased glycosaminoglycans retention and less resorption in crosslinked septal cartilage grafts. Conclusion: This study demonstrates the potential benefits of decellularization and crosslinking allograft cartilage and further refinements in crosslinking may improve resorption characteristics while maintaining suitable physical characteristics.

Characterization of a Cryopreserved Split-Thickness Human Skin Allograft-TheraSkin.

OBJECTIVE: The purpose of this study was to examine the characteristics of a cryopreserved split-thickness skin allograft produced from donated human skin and compare it with fresh, unprocessed human split-thickness skin. BACKGROUND: Cutaneous wound healing is a complex and organized process, where the body re-establishes the integrity of the injured tissue. However, chronic wounds, such as diabetic or venous stasis ulcers, are difficult to manage and often require advanced biologics to facilitate healing. An ideal wound care product is able to directly influence wound healing by introducing biocompatible extracellular matrices, growth factors, and viable cells to the wound bed. MATERIALS AND METHODS: TheraSkin (processed by LifeNet Health, Virginia Beach, Virginia, and distributed by Soluble Systems, Newport News, Virginia) is a minimally manipulated, cryopreserved split-thickness human skin allograft, which contains natural extracellular matrices, native growth factors, and viable cells. The authors characterized TheraSkin in terms of the collagen and growth factor composition using ELISA, percentage of apoptotic cells using TUNEL analysis, and cellular viability using alamarBlue assay (Thermo Fisher Scientific, Waltham, Massachusetts), and compared these characteristics with fresh, unprocessed human split-thickness skin. RESULTS: It was found that the amount of the type I and type III collagen, as well as the ratio of type I to type III collagen in TheraSkin, is equivalent to fresh unprocessed human split-thickness skin. Similar quantities of vascular endothelial growth factor, insulinlike growth factor 1, fibroblast growth factor 2, and transforming growth factor ß1 were detected in TheraSkin and fresh human skin. The average percent of apoptotic cells was 34.3% and 3.1% for TheraSkin and fresh skin, respectively. CONCLUSIONS: Cellular viability was demonstrated in both TheraSkin and fresh skin.

Constructing kidney-like tissues from cells based on programs for organ development: toward a method of in vitro tissue engineering of the kidney.

The plausibility of constructing vascularized three-dimensional (3D) kidney tissue from cells was investigated. The kidney develops from mutual inductive interactions between cells of the ureteric bud (UB), derived from the Wolffian duct (WD), and the metanephric mesenchyme (MM). We found that isolated MMs were capable of inducing branching morphogenesis of the WD (an epithelial tube) in recombination cultures; suggesting that the isolated MM retains inductive capacity for WD-derived epithelial tubule cells other than those from the UB. Hanging drop aggregates of embryonic and adult renal epithelial cells from UB and mouse inner medullary collecting duct cell (IMCD) lines, which are ultimately of WD origin, were capable of inducing MM epithelialization and tubulogenesis with apparent connections (UB cells) and collecting duct-like tubules with lumens (IMCD). This supports the view that the collecting system can be constructed from certain epithelial cells (those ultimately of WD origin) when stimulated by MM. Although the functions of the MM could not be replaced by cultured mesenchymal cells, primary MM cells and one MM-derived cell line (BSN) produced factors that stimulate UB branching morphogenesis, whereas another, rat inducible metanephric mesenchyme (RIMM-18), supported WD budding as a feeder layer. This indicates that some MM functions can be recapitulated by cells. Although engineering of a kidney-like tissue from cultured cells alone remains to be achieved, these results suggest the feasibility of such an approach following the normal developmental progression of the UB and MM. Consistent with this notion, implants of kidney-like tissues constructed in vitro from recombinations of the UB and MM survived for over 5 weeks and achieved an apparently host-derived glomerular vasculature. Lastly, we addressed the issue of optimal macro- and micro-patterning of kidney-like tissue, which might be necessary for function of an organ assembled using a tissue engineering approach. To identify suitable conditions, 3D reconstructions of HoxB7-green fluorescent protein mouse rudiments (E12) cultured on a filter or suspended in a collagen gel (type I or type IV) revealed that type IV collagen 3D culture supports the deepest tissue growth (600 +/- 8 microm) and the largest kidney volume (0.22 +/- 0.02 mm(3)), and enabled the development of an umbrella-shaped collecting system such as occurs in vivo. Taken together with prior work (Rosines et al., 2007; Steer et al., 2002), these results support the plausibility of a developmental strategy for constructing and propagating vascularized 3D kidney-like tissues from recombinations of cultured renal progenitor cells and/or primordial tissue.

The instructive role of metanephric mesenchyme in ureteric bud patterning, sculpting, and maturation and its potential ability to buffer ureteric bud branching defects.

Kidney organogenesis depends on reciprocal interactions between the ureteric bud (UB) and the metanephric mesenchyme (MM) to form the UB-derived collecting system and MM-derived nephron. With the advent of in vitro systems, it is clear that UB branching can occur independently of MM contact; however, little has been done to detail the role of MM cellular contact in this process. Here, a model system in which the cultured isolated UB is recombined with uninduced MM is used to isolate the effects of the MM progenitor tissue on the development and maturation of the collecting system. By morphometrics, we demonstrate that cellular contact with the MM is required for vectorial elongation of stalks and tapering of luminal caliber of UB-derived tubules. Expression analysis of developmentally significant genes indicates the cocultured tissue is most similar to an embryonic day 19 (E19) kidney. The likely major contributor to this is the functional maturation of the collecting duct and proximal nephron segments in the UB-induced MM, as measured by quantitative PCR, of the collecting duct-specific arginine vasopressin receptor and the nephron tubule segment-specific organic anion transporter OAT1, Na-P(i) type 2 cotransporter, and Tamm-Horsfall protein gene expressions. However, expression of aquaporin-2 is upregulated similarly in isolated UB and cocultured tissue, suggesting that some aspects of functional maturation can occur independently of MM cellular contact. In addition to its sculpting effects, the MM normalized a "branchless" UB morphology induced by FGF7 or heregulin in isolated UB culture. The morphological changes induced by the MM were accompanied by a reassignment of GFRalpha1 (a receptor for GDNF) to tips. Such "quality control" by the MM of UB morphology may provide resiliency to the branching program. This may help to explain a number of knockout phenotypes in which branching and/or cystic defects are less impressive than expected. A second hit in the MM may thus be necessary to make these defects fully apparent.

The effect of hyaluronic acid size and concentration on branching morphogenesis and tubule differentiation in developing kidney culture systems: potential applications to engineering of renal tissues.

Hyaluronic acid (HA) is a glycosaminoglycan of tissue engineering importance that plays a vital role in mammalian development. In vitro kidney culture methods were utilized to investigate the importance of HA during renal organogenesis. We found that HA has the ability to simultaneously modulate ureteric bud (UB) branching, promote mesenchymal-to-epithelial transformation, and promote differentiation of both metanephric mesenchyme (MM) and the UB depending on the concentration and molecular weight (MW) of HA. Hyaluronidase inhibited branching morphogenesis in both isolated UB and whole kidney cultures, suggesting endogenous HA is required for branching morphogenesis. HA exhibited morphogen-like properties, stimulating branching morphogenesis at low concentrations (0.1%) and low MW (6.55 kDa), but inhibiting at high concentrations (3.75%) and high MW (234.4 kDa). Furthermore, HA of every MW tested promoted collecting duct differentiation as measured by AQP-2 expression. E-cadherin immunostaining and qPCR of nephron differentiation markers (OAT-1, NaP(i)-2, AQP-1, and THP) demonstrated that HA of a variety of MWs strongly promotes mesenchymal epithelialization and nephron differentiation in a concentration-dependent manner. Since the HA synthesis and degradation genes, has-2 and hyal-2, are highly expressed during kidney development, this data suggests that specific sizes and concentrations of HA may act to independently regulate UB branching and promote tubular maturation, representing a potential switch for ending branching morphogenesis, as well as initiating nephron differentiation. In addition, the ability of HA to promote in vitro embryonic kidney growth and maturation, together with the biocompatibility and crosslinking capability of HA, suggests a potential use of HA for both creating an instructive, 3D scaffold for in vitro kidney engineering from developmental tissues, as well as promoting tubule regeneration in injured or cryopreserved kidneys.