Covalent binding of aminopropanehydroxydiphosphonate to glutaraldehyde residues in pericardial bioprosthetic tissue: Stability and calcification inhibition studies

Calcification has limited the clinical utility of bioprosthetic heart valves fabricated from either glutaraldehyde-pretreated bovine pericardium or porcine aortic valves. Aminopropanehydroxydiphosphonate (APDP), covalently bound to residual aldehyde groups in glutaraldehyde-treated pericardial bioprosthetic tissue, has been shown to inhibit cardiovascular calcification in the rat subdermal model. Using 3H-labeled glutaraldehyde (GLUT) at a concentration of 0.02 M and 0.14 M 14C-labeled APDP, we assessed the effects of GLUT incubation temperature (4[deg] or 25[deg]C) and pH of the GLUT incubation solution (pH 4.0, 7.4, or 10.0) on the GLUT incorporation step and subsequent APDP binding (24 hr 25[deg]C) into bioprosthetic valve (BPV) tissue (bovine pericardium). Increased incorporation of GLUT and APDP occurred at lower GLUT incubation temperature (GLUT, 346.05 +/- 1.9 nM/mg, 4[deg]C vs 259.76 +/- 1.39 nM/mg, 25[deg]C; APDP, 57.56 +/- 4.43 nM/mg, 4[deg]C vs 36.36 +/- 0.46 nM/mg, mean +/- standard error, at 25[deg]C). There also was a greater incorporation of GLUT but not APDP at the higher glutaraldehyde pretreatment pH (GLUT, pH 10.0, 213.73 +/- 73 nM/mg vs pH 4, 132.08 +/- 43 nM/mg; APDP, pH 10.0, 51.41 +/- 12 nM/mg vs pH 4.0, 49.97 +/- 6 nM/mg). In vivo studies revealed that all groups with treated BPV implanted for 21 days in male 3-week-old CD rats demonstrated a loss of both GLUT (12%-50%) and APDP (48%-64%) compared to preimplant content. BPV implant calcification was significantly inhibited in all groups treated with APDP compared to control Ca2+ (5.54 +/- 2.1-9.64 + 1.2 [mu]g/mg, APDP pretreated, vs 93.64 +/- 11.65 [mu]g/mg, control; P [les] 0.001) despite the progressive loss of both GLUT and APDP with time. It is concluded that preincubation of BPV tissue in GLUT at lower temperature (4[deg]C) and higher pH (10.0) enhanced BPV GLUT uptake and subsequent APDP covalent binding. In addition, in the rat subdermal model, BPV tissue calcification was markedly inhibited by APDP, despite a significant loss of bound drug.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/27918/1/0000341.pd

Scanning Electron Microscopy Methodology for Study of the Pathophysiology of Calcification in Bioprosthetic Heart Valves

Scanning electron microscope (SEM) morphologic analysis combined with energy dispersive characteristic X-ray (EDX) microprobe analysis provides insight into the mechanisms associated with disease-related crystal formation in biological materials. SEM and EDX were employed in analyzing specimens which were embedded in standard fashion in glycolmethacrylate (JB-4). The specimen surfaces under electron microscope investigation resulted from microtomy used in the preparation of reference light microscope histological sections; thus histology served as a direct reference for the SEM and EDX analyses. The particular application of these methods was in the study of bioprosthetic heart valve calcification, largely responsible for clinical failure of these heart valve substitutes. To simulate the clinically observed mineralization processes, glutaraldehyde-pretreated porcine heart valve leaflets were implanted subcutaneously in rats and subsequently removed at various time intervals from 1 to 56 days. Also, to address the hypothesis that the calcification process generates crystalline materials analogous to those in bone, EDX data obtained from pure hydroxyapatite were compared with the embedded tissue results. Further, EDX results were compared with data obtained by chemical analysis of the bulk specimens to assess the validity of the electron microscope technique

Effects of metallic ions and diphosphonates on inhibition of pericardial bioprosthetic tissue calcification and associated alkaline phosphatase activity

This study focused on the association of extrinsic alkaline phosphatase (AP) activity with both early and advanced calcification of glutaraldehyde-pretreated bovine pericardial bioprosthetic (GPBP) tissue, and the inhibition of both calcification and AP activity by pre-incubation in diphosphonates (sodium-ethanehydroxydiphosphonate [NaEHDP], aminopropanehydroxydiphosphonate [APD]) and metallic salts (FeCl3 Ga(NO3)3, AlCl3). GPBP specimens were implanted subcutaneously in 3 wk old male rats after ore-incubation. Following explantation of the tissue at 72 h and 21 d, calcification was assessed morphologically by light microscopy and chemically by atomic adsorption spectroscopy for calcium content and by molybdate complexation for phosphorus. AP activity was characterized by enzymatic hydrolysis of paranitrophenyl phosphate and by histochemical studies. In both control and pretreated groups, AP levels were greater in 72 h explants than 21 d retrievals, which demonstrated extensive calcification in control expiants. All pre-incubations that resulted in inhibition of calcification after 21 d, except for APD, were associated with 72 h AP content which was lower than control specimens. The typical time of initiation of calcification was 72 h, as determined by previous studies with this model system. Covalently bound APD inhibited calcification. Increased AP activity in the APD group may be due to the toxicity of this agent with resultant acute inflammation, or other incompletely understood effects of diphosphonates on calcification and AP. Furthermore, EHDP and Ga3+ incubations were also associated with decreased GPBP AP at 72 h compared to control, but were not effective for inhibiting calcification after 21 d. We concluded that inhibition of peak GPBP AP activity is not necessarily associated with the prevention of GPBP mineralization.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/30881/1/0000547.pd

Synergistic inhibition of the calcification of glutaraldehyde pretreated bovine pericardium in a rat subdermal model by FeCl3 and ethanehydroxydiphosphonate: pre-incubation and polymeric controlled release studies

Calcification is a frequent cause of the clinical failure of bioprosthetic heart valves fabricated from glutaraldehyde-pretreated porcine aortic valves or glutaraldehyde-pretreated bovine pericardium (GPBP). We investigated the hypothesis that ferric chloride (FeCl3) and sodiumethanehydroxydiphosphonate (EHDP) may act synergistically to prevent bioprosthetic tissue calcification. Pre-incubations and controlled release systems were studied individually. FeCl3-EHDP polymeric controlled release matrices were formulated using silicone rubber and evaluated for in vitro release kinetics at pH 7.4 and 37[deg]C. The effects of Fe-EHDP synergism on GPBP calcification were investigated with 21 d subdermal implants in 3 wk-old male rats. Results demonstrated that levels of Fe3+ and EHDP uptake, measured in GPBP tissues pre-incubated first in an FeCl3 solution (10-5 ) followed by an EHDP solution (0.1 ), were higher than in the reverse order of incubation. In the first series of rat implants, GPBP was pre-incubated in either FeCl3 or Na2EHDP solutions, or sequential pre-incubations of first FeCl3 and then Na2EHDP solutions, or the reverse. The inhibition of calcification was greatest when FeCl3 (first preincubation, 10-5 ) was combined with Na2EHDP (second pre-incubation, 0.1 ) (1.78 +/- 0.2 [mu]g of Ca2+/mg of dried tissue) compared with the other pre-incubation groups: EHDP (first preincubation) combined with FeCl3 (second pre-incubation) (21.7 +/- 6.4), FeCl3 solution alone at 10-5 (27.9 +/- 10.7), Na2EHDP solution alone at 0.1 (52.3 +/- 11.9) and the control group (72.3 +/- 10.2). In a second series of implants, GPBP specimens were co-implanted with individual controlled release systems containing one of the following formulations (weight percentage in silicone rubber): 1% FeCl3, 20% CaEHDP, 20% protamine sulphate, 1% FeCl3-20% CaEHDP, and 1% FeCl3-20% protamine sulphate. The 1% FeCl3-20% CaEHDP silicone-rubber matrices were the most effective for inhibiting GPBP mineralization (13.7 +/- 3.0 [mu]g Ca2+/mg of dried tissue) compared with non-drug silicone co-implant controls (74.7 +/- 5.58 [mu]g Ca2+/mg of dried tissue) and other polymeric treatment groups (32.3 +/- 2.3-80.0 +/- 19.7). No adverse effects on bone or overall growth of any treatment protocols were noted. Thus, combinations of FeCl3 and EHDP, using either pre-incubations or polymeric controlled release, were synergistic for inhibiting GPBP calcification.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/30723/1/0000370.pd

Pathological considerations in replacement cardiac valves

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/30262/1/0000663.pd

Biocompatibility and tissue regenerating capacity of crosslinked dermal sheep collagen

The biocompatibility and tissue regenerating capacity of four crosslinked dermal sheep collagens (DSC) was studied. In vitro, the four DSC versions were found to be noncytotoxic or very low in cytoxicity. After subcutaneous implantation in rats, hexamethylenediisocyanatecrcrosslinked DSC (HDSC) seldom induced an increased infiltration of neutrophils or macrophages, as compared with normal wound healing; whereas new formation of collagen was observed. DSC crosslinked with glutaraldehyde (GDSC) followed by reaction with NaBH4 shortly after implantation showed an increased infiltration of neutrophils with a deviant morphology. Furthermore, a high incidence of calcification was observed, which may explain the minor ingrowth of giant cells and fibroblasts, and the poor formation of new rat collagen. Acyl azide-crosslinked DSC (AaDSC) first induced an increased infiltration of macrophages, and then of giant cells, both with high lipid formation. AaDSC degraded at least twice as slowly as HDSC and GDSC, finally leaving a matrix of newly formed rat collagen. Samples crosslinked with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide (ENDSC) induced the same mild cellular reaction as HDSC; whereas, similar to AaDSC, the degradation rate was slow and an optimal rat collagen matrix was formed. Of the crosslinked DSC samples, ENDSC seems most promising for tissue regeneration

Phosphonated polyurethanes that resist calcification

Cardiovascular implant mineralization involving bioprosthetic materials, such as glutaraldehyde cross linked porcine aortic valves or synthetic materials such as polyurethanes, is an important problem that frequently leads to clinical failure of bioprosthetic heart valves, and complicates long-term experimental artificial heart device implants. Novel, proprietary, calcification resistant polyetherurethanes (PEU) as an alternative to bioprosthetic materials were the subject of these investigations. A series of PEU was derivatized through a proprietary reaction mechanism to achieve covalent binding of 100 to 500 n M /mg of bisphosphonate (2-hydroxyethane bisphosphonic acid, HEBP). The stability of HEBP (physically dispersed or covalently bound) verified by studying the release kinetics in physiological buffer (pH 7.4) at 37°C, demonstrated the covalent binding reaction to be stable, efficient, and permanent. Surface (FTIR-ATR, ESCA, SEM/EDX) and bulk (solubility, GPC) properties demonstrated that the covalent binding of HEBP occurs in the soft segment of the PEU, reduces surface degradation, and does not affect the original material properties of the PEU (prior to derivatization). In vitro calcium diffusion of the derivatized PEU showed a decrease in calcium permeation as the concentration of HEBP covalent binding was increased. In vivo properties of underivatized and derivatized PEU (containing 100 n M of covalently bound HEBP) were studied with rat subdermal implants for 60 days. Explants demonstrated calcification resistance due to the covalently bound HEBP without any side effects. It is concluded that a PEU containing HEBP might serve as a calcification resistant candidate material for the fabrication of a heart valve prosthesis and other implantable devices. © 1994 John Wiley & Sons, Inc.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/38568/1/770050109_ftp.pd

Controlled release of ethanehydroxy diphosphonate from polyurethane reservoirs to inhibit calcification of bovine pericardium used in bioprosthetic heart valves

Calcification (CALC) of bioprosthetic heart valves (BHVs) fabricated from either glutaraldehyde-pretreated bovine pericardial tissue or porcine aortic valves is the most frequent cause of clinical failure of these devices. Previous studies have demonstrated that calcification is inhibited by diphosphonate compounds released into the vicinity of bioprosthetic tissue implanted subcutaneously in rats. Controlled release of the anticalcification agent ethanehydroxy diphosphonate (EHDP), as a 1:1 mixture of Na2 EHDP and CaEHDP from cylindrical polyurethane (PU) reservoirs (o.d. = 0.36 cm i.d. = 0.33 cm, length = 4 cm) fabricated by solvent casting was assessed in vitro and in vivo. The diffusivity (D), determined independently using standard diffusion cells, for ionic EHDP diffusion across the PU membrane was 1.2 x 10 cm2/s. Volume influx of buffer into the reservoirs in vitro was observed experimentally to reach a maximum at 7.8 days (288 +/- 44 [mu]l) with a biexponential decline to 147 +/- 6 [mu]l at 70 days. The cumulative EHDP released in vitro after 70 days was 4.2 +/- 0.6% (4.8 +/- 0.7 mg) compared to 15.7 +/- 3.2% (18.1 +/- 3.7 mg) in vivo (subcutaneously in 3 week-old, male, CD rats) over 21 days. The release rate of EHDP from the reservoirs was not a zero-order process. Reservoir administration of EHDP effectively inhibited pericardial BHV-CALC in 21-day subdermal explants (Ca2+ = 4.5 +/- 1.4 [mu]g Ca2+/mg tissue; control, Ca2+ = 120 +/- 13 [mu]g Ca2+/mg tissue) without diphosphonate-related untoward effects at a dose of approx. 3 mg/kg per day.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/28671/1/0000488.pd

What Explains Divorced Women's Poorer Health? The Mediating Role of Health Insurance and Access to Health Care in a Rural I owan Sample

Economic restructuring in rural areas in recent decades has been accompanied by rising marital instability. To examine the implications of the increase in divorce for the health of rural women, we examine how marital status predicts adequacy of health insurance coverage and health care access, and whether these factors help to account for the documented association between divorce and later illness. Analyzing longitudinal data from a cohort of over 400 married and recently divorced rural I owan women, we decompose the total effect of divorce on physical illness a decade later using structural equation modeling. Divorced women are less likely to report adequate health insurance in the years following divorce, inhibiting their access to medical care and threatening their physical health. Full‐time employment acts as a buffer against insurance loss for divorced women. The growth of marital instability in rural areas has had significant ramifications for women's health; the decline of adequate health insurance coverage following divorce explains a component of the association between divorced status and poorer long‐term health outcomes.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/95231/1/ruso91.pd