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 kidney's 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)-ß1, plasminogen activator inhibitor (PAI)-1, and fibronectin (FN) provided evidence that B2 cells attenuated canonical pathways of profibrotic extracellular matrix production.

Linear measurement of cell contraction in a capillary collagen gel system.

Three-dimensional collagen gel contraction is the standard assay utilized for functionally quantifying a variety of cell types, in particular smooth muscle cells (SMCs) and myofibroblasts. Here, we have developed a method to effectively reduce the three-dimensional parameters of the standard collagen gel into a single, linear measurement. Cell/collagen suspensions that are cast into glass capillary tubes provide several advantages over the well plate format, such as eliminating the need for digital imaging equipment and software to quantify the amount of cellular contraction. In addition, capillary tube gels require significantly fewer cells and far less reagents than standard methods.

Biomechanical and biologic effects of meniscus stabilization using triglycidyl amine.

The susceptibility of meniscus allografts to enzymatic degradation may be reduced through tissue stabilization. We have previously reported on an epoxide-based crosslinker, triglycidyl amine (TGA), which can be used alone or with a bisphosphonate (MABP) to stabilize heterograft heart valves and reduce their pathologic calcification. Our objective was to evaluate the effects of TGA and TGA-MABP pretreatment on an orthopedic allograft involving meniscus crosslinking, degradation, calcification, and compressive properties. Ovine menisci treated with TGA or TGA-MABP for up to seven days and glutaraldehyde crosslinked controls were examined in vitro for degree of crosslinking, resistance to degradation by collagenase, and material property changes. Likewise treated menisci were implanted in rats for eight weeks and examined for calcium content and biomechanical changes. TGA treatment for three days significantly reduced collagen loss by 88% and increased thermal denaturation temperatures (Ts) above 80 degrees C versus Ts of 70 degrees C or less for non-crosslinked meniscus. In vitro, TGA and TGA-MABP significantly increased aggregate modulus by 19% and 32% compared to native controls, respectively. TGA decreased permeability by 53% while TGA-MABP increased it by 303%. In vivo, TGA significantly reduced explant calcification by 42% compared to glutaraldehyde, and including MABP reduced it by 90%. Analyses revealed that TGA and TGA-MABP stabilized menisci had significantly lower modulus and permeability values than glutaraldehyde controls by at least 28% and 86%, respectively. It is concluded that TGA crosslinking of meniscus increases resistance to both collagenase degradation and pathologic calcification, while demonstrating comparable or improved biomechanical properties versus glutaraldehyde controls.

Transforming growth factor-beta1 mechanisms in aortic valve calcification: increased alkaline phosphatase and related events.

BACKGROUND: Aortic valve stenosis is the most frequent indication for valve replacement surgery, and is commonly associated with pathologic calcification. Previous investigations by our group have shown a strong association of transforming growth factor-beta1 (TGF-beta1)-related mechanisms with calcific aortic stenosis in both cell culture and clinical pathology studies. METHODS: In the present investigations we sought to investigate the sequence of events involved in TGF-beta1-initiated aortic valve interstitial cell calcification in cell culture, and to study related gene expression pattern differences comparing calcific aortic stenosis surgical specimens with normal aortic valve leaflets. RESULTS: Sheep aortic valve interstitial cells (SAVIC) in culture progressively calcified over 14 days after the addition of TGF-beta1 to a significantly greater extent than non-TGF-beta1 controls. The TGF-beta1-induced SAVIC calcification was associated with maximal levels of alkaline phosphatase by 72 hours. Annexin V positive apoptosis was increased in TGF-beta1-treated SAVIC cultures at 14 days compared with controls. Matrix metalloproteinase 9 per gel zymography was detectable only in SAVIC cultures treated with TGF-beta1 from seven days on. Matrix metalloproteinase 2 was present in all SAVIC cultures per gel zymograms, either with or without TGF-beta1, but the active form of matrix metalloproteinase 2 significantly increased over 14 days in response to TGF-beta1. Quantitative gene expression studies (re: RNA levels) of human aortic valve cusps obtained at cardiac surgery demonstrated a number of related trends, including upregulation of the expression of TGF-beta1, alkaline phosphatase, and matrix metalloproteinase 9 in calcified human aortic valves. CONCLUSIONS: Transforming growth factor-beta1 causes SAVIC to calcify due to an early maximal increase in alkaline phosphatase activity with associated apoptotic events and increased matrix metalloproteinase 9. These TGF-beta1-related mechanistic events may be of clinical relevance based upon the gene expression pattern changes observed in calcific aortic stenosis valve cusps.

Mechanisms of the in vivo inhibition of calcification of bioprosthetic porcine aortic valve cusps and aortic wall with triglycidylamine/mercapto bisphosphonate.

Heart valve replacements fabricated from glutaraldehyde (Glut)-crosslinked heterograft materials, porcine aortic valves or bovine pericardium, have been widely used in cardiac surgery to treat heart valve disease. However, these bioprosthetic heart valves often fail in long-term clinical implants due to pathologic calcification of the bioprosthetic leaflets, and for stentless porcine aortic valve bioprostheses, bioprosthetic aortic wall calcification also typically occurs. Previous use of the epoxide-based crosslinker, triglycidyl amine (TGA), on cardiac bioprosthetic valve materials demonstrated superior biocompatibility, mechanics, and calcification resistance for porcine aortic valve cusps (but not porcine aortic wall) and bovine pericardium, vs. Glut-prepared controls. However, TGA preparation did not completely prevent long-term calcification of cusps or pericardium. Herein we report further mechanistic investigations of an added therapeutic component to this system, 2-mercaptoethylidene-1,1-bisphosphonic acid (MABP), a custom synthesized thiol bisphosphonate, which has previously been shown in a preliminary report to prevent bioprosthetic heterograft biomaterial calcification when used in combination with initial TGA crosslinking for 7 days. In the present studies, we have further investigated the effectiveness of MABP in experiments that examined: (1) The use of MABP after optimal TGA crosslinking, in order to avoid any competitive interference of MABP-reactions with TGA during crosslinking; (2) Furthermore, recognizing the importance of alkaline phosphatase (ALP) in the formation of dystrophic calcific nodules, we have investigated the hypothesis that the mechanism by which MABP primarily functions is through the reduction of ALP activity. Results from cell-free model systems, cell culture studies, and rat subcutaneous implants, show that materials functionalized with MABP after TGA crosslinking have reduced ALP activity, and in vivo have no significant calcification in long-term implant studies. It is concluded that bioprosthetic heart valves prepared in this fashion are compelling alternatives for Glut-prepared bioprostheses.

Reversibly labile, sclerotization-induced elastic properties in a keratin analog from marine snails: whelk egg capsule biopolymer (WECB).

Egg capsules from two caenogastropod whelks, Busycon canaliculatum and Kelletia kelletii, were studied to investigate the genesis of mechanical properties of nascent capsules and to formulate a biomechanical model of this material. Scanning electron microscopy revealed that the capsules possess fibrous hierarchical arrangements at all stages during processing while the mechanical integrity is developing. This suggests that an as yet uncharacterized sclerotization mechanism occurring in the ventral pedal gland primarily binds these fibrous components together. Decomposing the mechanical behavior of WECB through various physical and chemical treatments led us to develop a model for the structure and mechanical properties of this material that supports its designation as a keratin analog. Keratin mechanical models were applied to WECB in its representation as an intermediate state between matrix-free intermediate filament (IF)-type proteins and the more complex composite materials incorporating IFs such as keratin.

Structure and function of tuna tail tendons.

The caudal tendons in tunas and other scombrid fish link myotomal muscle directly to the caudal fin rays, and thus serve to transfer muscle power to the hydrofoil-like tail during swimming. These robust collagenous tendons have structural and mechanical similarity to tendons found in other vertebrates, notably the leg tendons of terrestrial mammals. Biochemical studies indicate that tuna tendon collagen is composed of the (alpha1)(2),alpha2 heterotrimer that is typical of vertebrate Type I collagen, while tuna skin collagen has the unusual alpha1,alpha2,alpha3 trimer previously described in the skin of some other teleost species. Tuna collagen, like that of other fish, has high solubility due to the presence of an acid-labile intermolecular cross-link. Unlike collagen in mammalian tendons, no differences related to cross-link maturation were detected among tendons in tuna ranging from 0.05 to 72 kg (approx. 0.25-6 years). Tendons excised post-mortem were subjected to load cycling to determine the modulus of elasticity and resilience (mean of 1.3 GPa and 90%, respectively). These material properties compare closely to those of leg tendons from adult mammals that can function as effective biological springs in terrestrial locomotion, but the breaking strength is substantially lower. Peak tendon forces recorded during steady swimming appear to impose strains of much less than 1% of tendon length, and no more than 1.5% during bursts. Thus, the caudal tendons in tunas do not appear to function as elastic storage elements, even at maximal swimming effort.

Mechanical characterization of an unusual elastic biomaterial from the egg capsules of marine snails (Busycon spp.).

Egg capsule material serves as a putative protection mechanism for developing snail embryos facing the perils of the marine environment. We conducted the first quantitative study of this acellular structural protein with the goals of characterizing its chemical and mechanical properties and the relationship of these properties to its biological protective function. We have found that this protein polymer exhibits long-range elasticity with an interesting recoverable yield evidenced by an order of magnitude decrease in elastic modulus (apparent failure) that begins at 3%-5% strain. This material differs significantly from other common structural proteins such as collagen and elastin in mechanical response to strain. Qualitative similarities in stress/strain behavior to keratin, another common structural protein, are more than coincidental when composition and detailed mechanical quantification are considered. This suggests the possibility of alpha-helical structure and matrix organization that might be similar in these two proteins. Indeed, the egg capsule protein may be closely related to vertebrate keratins such as intermediate filaments. We conclude that while this material's bimodal tensile properties may serve as useful protection against the impact loading egg capsules encounter in the intertidal zone, the full biological importance of these capsules is not known.