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Background: Bone formation in the ovary, with the exception of developing in the setting of mature cystic teratoma, is exceedingly rare., Case: A 46-year-old woman with a history of endometriosis and chronic pelvic pain underwent total abdominal hysterectomy and bilateral salpingo-oophorectomy. A 3 cm solid heavily calcified mass with a stony hard consistency was detected within the right ovary. Microscopic examination revealed extensive calcification of the right ovarian stroma with formation of abundant mature bone, adjacent to small foci of endometriosis., Conclusions: Endometriosis can be associated with ovarian ossification, forming an extensively calcified adnexal mass. Conservative treatment with close follow-up may be adequate in patients with a history of endometriosis who present with a small heavily calcified ovarian mass and wish to preserve their fertility.

Calcification of polyurethane cardiovascular implants is an important disease process that has the potential to compromise the long-term function of devices such as polymer heart valves and ventricular assist systems. In this study we report the successful formulation and characterization of bisphosphonate-derivatized polyurethanes, hypothesized to resist implant calcification based on the pharmacologic activity of the immobilized bisphosphonate. Fully polymerized polyurethanes (a polyurea-polyurethane and a polycarbonate polyurethane) were modified (post-polymerization) with bromoalkylation of the hard segments followed by attachment of a bisphosphonate group at the bromine site. These bisphosphonate-polyurethanes resisted calcification in rat 60 day subdermal implants compared to nonmodified control polyurethane implants, that calcify. Bisphosphonates-modified polyurethanes were also studied in circulatory implants using a pulmonary valve cusp replacement model in sheep. Polyurethane cusps modified with bisphosphonate did not calcify in 90 day implants. compared to control polyurethane cusps implants, that demonstrated nodular surface oriented calcific deposits. It is concluded that bisphosphonate modified polyurethanes resist calcification both in subdermal implants and in the circulation. This novel biomaterial approach offers great promise for long-term blood stream implantation with calcification resistance.

We previously showed that the expression of tenascin (TN-C), an extracellular matrix glycoprotein found in developing bone and atherosclerotic plaque, and matrix metalloproteinase-2 (MMP-2) are coordinated and interdependent in cultured vascular smooth muscle cells. In this study, we hypothesized that TN-C and MMP-2 are mechanistically involved in the pathobiology of calcific aortic stenosis. Human calcific aortic stenosis cusps demonstrated immunohistochemically prominent deposition of TN-C, MMP-2, and alkaline phosphatase activity, as well as MMP-2 gelatinolytic activity. Although far lesser amounts of TN-C were noted in several of the grossly non-calcified valve cusps, MMP-2 and AP were never detected. Further, when aortic valve interstitial cells (both sheep and human) were cultivated on collagen supplemented with TN-C, both MMP-2 mRNA expression and MMP-2 gelatinolytic activity (both pro and active forms), were up-regulated compared to control. These observations support the view that accumulation of first TN-C and then MMP-2 are associated with progression of calcification. The residual presence of these proteins in severe calcifications is indicative of their involvement in the pathogenesis.

Elastin, a major extracellular matrix protein present in arterial walls provides elastic recoil and resilience to arteries. Elastin is prone to calcification in a number of cardiovascular diseases including atherosclerosis and bioprosthetic heart valve mineralization. We have recently shown that purified elastin when implanted subdermally in rats undergoes severe calcification. In the present study, we used this elastin implant model to investigate the molecular mechanisms underlying elastin calcification. Intense matrix metalloproteinase (MMP-2) and tenascin-C (TN-C) expression were seen in the proximity of the initial cal-cific deposits at 7 days. Gelatin zymography studies showed both MMP-2 (latent and active form) and MMP-9 expression within the implants. To investigate the role of MMPs in calcification, rats were administered a MMP inhibitor, (2S:-allyl-N:-hydroxy-3R:-isobutyl-N:-(1S:-methylcarbamoyl-2-ph enylet hyl)-succinamide (BB-1101) by daily injection, either systemically or at the implant site. The site-specific BB-1101 administration almost completely suppressed TN-C expression, as shown by immunohistochemical staining, within the implants. The systemic BB-1101 injections also significantly reduced TN-C expression within the elastin implants. Moreover, calcification of elastin implants was significantly reduced in the site-specific administration group (5.43 +/- 1.03 microg/mg Ca for BB-1101 group versus 21.71 +/- 1.19 for control group, P: < 0.001). Alizarin Red staining clearly showed that the elastin fibers were heavily calcified in the control group, whereas in BB-1101 group the calcification was scarce with few fibers showing initial calcification deposits. The systemic administration of BB-1101 also significantly reduced elastin calcification (28.07 +/- 5.81 control versus 16.92 +/- 2.56 in the BB-1101 group, P: < 0.05), although less than the site-specific administration. Thus, the present studies indicate that MMPs and TN-C play a role in elastin-oriented calcification.

Background and Aim of the Study: Calcification is a major cause of failure of bioprosthetic heart valves derived from glutaraldehyde-crosslinked bovine pericardium or porcine aortic valve (PAV) cusps. Recently, we have shown that ethanol pretreatment of PAV cusps prevents calcification in animal models., Methods and Results: In this study we showed that ethanol pretreatment was equally effective in preventing calcification of glutaraldehyde-crosslinked bovine pericardium (control Ca2+ = 121.16+/-7.49 microg/mg tissue; ethanol-pretreated Ca2+ = 2.95+/-0.78 microg/mg). Furthermore, other low-molecular weight alcohols such as methanol and isopropanol were also effective in mitigating calcification of PAV cusps. Storage of ethanol-pretreated cusps in glutaraldehyde before implantation allowed partial return of calcification, suggesting a role for ethanol-glutaraldehyde interactions in preventing calcification. However, when ethanol-pretreated cusps were stored in ethanolic glutaraldehyde up to one month, the anti-calcification effect of ethanol persisted. The conditions whereby PAV cusps were crosslinked in pure, non-aqueous, alcoholic glutaraldehyde solutions were also examined. The crosslinking was equivalent to the standard aqueous glutaraldehyde crosslinking as indicated by thermal denaturation temperatures (Td) obtained by differential scanning calorimetry (DSC) and resistance to collagenase digestion. However, these cusps had lower water content and showed a marked decrease in spin-lattice relaxation times (T1) obtained by solid-state proton nuclear magnetic resonance (NMR). Moreover, these cusps calcified heavily in the 21-day rat subdermal implants. Thus, alcohol treatment during glutaraldehyde crosslinking was not useful., Conclusion: Glutaraldehyde storage after ethanol pretreatment aggravates calcification; moreover, alcoholic-glutaraldehyde crosslinking solutions are not beneficial for anti-calcification. Ethanol pretreatment of glutaraldehyde-pretreated bovine pericardium prevents its calcification.

Elastin, an abundant structural protein present in the arterial wall, is prone to calcification in a number of disease processes including porcine bioprosthetic heart valve calcification and atherosclerosis. The mechanisms of elastin calcification are not completely elucidated. In the present work, we demonstrated calcification of purified elastin in rat subdermal implants (Ca(2+) = 89.73 +/- 9.84 microgram/mg after 21 days versus control, unimplanted Ca(2+) = 0.16 +/- 0.04 microgram/mg). X-ray diffraction analysis along with resolution enhanced FTIR spectroscopy demonstrated the mineral phase to be a poorly crystalline hydroxyapatite. We investigated the time course of calcification, the effect of glutaraldehyde crosslinking on calcification, and mechanisms of inhibition of elastin calcification by pretreatment with aluminum chloride (AlCl(3)). Glutaraldehyde pretreatment did not affect calcification (Ca(2+) = 89.06 +/- 17.93 microgram/mg for glutaraldehyde crosslinked elastin versus Ca(2+) = 89.73 +/- 9.84 microgram/mg for uncrosslinked elastin). This may be explained by radioactive ((3)H) glutaraldehyde studies showing very low reactivity between glutaraldehyde and elastin. Our results further demonstrated that AlCl(3) pretreatment of elastin led to complete inhibition of elastin calcification using 21-day rat subdermal implants, irrespective of glutaraldehyde crosslinking (Ca(2+) = 0.73-2.15 microgram/mg for AlCl(3) pretreated elastin versus 89.73 +/- 9.84 for untreated elastin). The AlCl(3) pretreatment caused irreversible binding of aluminum ions to elastin, as assessed by atomic emission spectroscopy. Moreover, aluminum ion binding altered the spatial configuration of elastin as shown by circular dichroism (CD), Fourier transform infrared (FTIR), and (13)C nuclear magnetic resonance (NMR) spectroscopy studies, suggesting a net structural change including a reduction in the extent of beta sheet structures and an increase in coil-turn conformations. Thus, it is concluded that purified elastin calcifies in rat subdermal implants, and that the AlCl(3)-pretreated elastin completely resists calcification due to irreversible aluminum ion binding and subsequent structural alterations caused by AlCl(3).

Background and Aim of the Study: Cardiac valve calcification is the predominant pathology in patients needing valve replacement. The aim of this study was to determine if aortic valve cells calcify spontaneously and, if so, to characterize the nodular complex and response to growth factors., Methods: Aortic valves were obtained from humans undergoing surgical valve replacement, and from female dogs. The valvular endothelium was removed and explants cultured in medium., Results: A population of valvular interstitial cells spontaneously formed distinct calcified nodules containing hydroxyapatite within two to three weeks in canine and within six weeks in human aortic valves. The nodules contained an inner ring of dead cells surrounded by an outer ring of living cells. Cells associated with nodules had osteoblast-like characteristics and stained positively for extracellular bone matrix proteins. Incubating canine cells with potential calcifying stimuli tested the stimulus for calcification. The rate of nodule formation was increased with transforming growth factor beta-1 (+25 nodules), 25-hydroxycholesterol (+9 nodules) and bone morphogenetic protein 2 (+4 nodules) as compared with vehicle control (+3 nodules) over 25 days., Conclusions: We identified a population of valvular interstitial cells with osteoblast-like characteristics that spontaneously form calcific nodules in cell culture. In addition, the rate of calcific nodule formation was increased with transforming growth factor beta-1 and 25-hydroxycholesterol. Further study of these 'calcifying valve cells' may yield a new in vitro model for testing therapy aimed at preventing calcific valve stenosis.

The effectiveness of ethanol pretreatment on preventing calcification of glutaraldehyde-fixed porcine aortic bioprosthetic heart valve (BPHV) cusps was previously demonstrated, and the mechanism of action of ethanol was attributed in part to both lipid removal and a specific collagen conformational change. In the present work, the effect of ethanol pretreatment on BPHV aortic wall calcification was investigated using both rat subdermal and sheep circulatory implants. Ethanol pretreatment significantly inhibited calcification of BPHV aortic wall, but with less than complete inhibition. The maximum inhibition of calcification of BPHV aortic wall was achieved using an 80% ethanol pretreatment; calcium levels were 71.80+/-8.45 microg/mg with 80% ethanol pretreatment compared to the control calcium level of 129.90+/-7.24 microg/mg (p = 0.001). Increasing the duration of ethanol exposure did not significantly improve the inhibitory effect of ethanol on aortic wall calcification. In the sheep circulatory implants, ethanol pretreatment partly prevented BPHV aortic wall calcification with a calcium level of 28.02+/-4.42 microg/mg compared to the control calcium level of 56.35+/-6.14 microg/mg (p = 0.004). Infrared spectroscopy (ATR-FTIR) studies of ethanol-pretreated BPHV aortic wall (vs. control) demonstrated a significant change in protein structure due to ethanol pretreatment. The water content of the aortic wall tissue and the spin-lattice relaxation times (T1) as assessed by proton nuclear magnetic resonance spectroscopy did not change significantly owing to ethanol pretreatment. The optimum condition of 80% ethanol pretreatment almost completely extracted both phospholipids and cholesterol from the aortic wall; despite this, significant calcification occurred. In conclusion, these results clearly demonstrate that ethanol pretreatment is significantly but only partially effective for inhibition of calcification of BPHV aortic wall and this effect may be due in part to lipid extraction and protein structure changes caused by ethanol. It is hypothesized that ethanol pretreatment may be of benefit for preventing bioprosthetic aortic wall calcification only in synergistic combination with another agent.

Clinical usage of bioprosthetic heart valves (BPHVs) fabricated from glutaraldehyde-pretreated porcine aortic valves is restricted due to calcification-related failure. We previously reported a highly efficacious ethanol pretreatment of BPHVs for the prevention of cuspal calcification. The aim of the present study is to extend our understanding of the material changes brought about by ethanol and the relationship of these material effects to the ethanol pretreatment anticalcification mechanism. Glutaraldehyde-crosslinked porcine aortic valve cusps (control and ethanol-pretreated) were studied for the effects of ethanol on tissue water content and for spin-lattice relaxation times (T1) using solid state proton NMR. Cusp samples were studied for protein conformational changes due to ethanol by ATR-FTIR spectroscopy. The changes in cuspal tissue-cholesterol (in vitro) interactions also were studied. Cusp material stability was assessed in terms of residual glutaraldehyde content and collagenase degradation. Water content of the cusp samples was decreased significantly due to ethanol pretreatment. The cuspal collagen conformational changes (per infrared spectroscopy) brought about by ethanol pretreatment were persistent even after rat subdermal implantation of cusp samples for 7 days. In vitro cholesterol uptake by cusps was greatly reduced as a result of ethanol pretreatment. Ethanol pretreatment of cusps also resulted in increased resistance to collagenase digestion. Cuspal glutaraldehyde content was not changed by ethanol pretreatment. We conclude that ethanol pretreatment of bioprosthetic heart valve cusps causes multi-component effects on the tissue/material and macromolecular characteristics, which partly may explain the ethanol-pretreatment anticalcification mechanism.

Background: Aminooleic acid treatment has been demonstrated to prevent porcine valve calcification and to protect valvular hemodynamic function. Initial enthusiasm was tempered by histologic studies of these AOA valves, which showed cuspal hematomas, structural loosening, and surface roughening. This prompted a systematic review of the AOA treatment process. Unsolubilized particles of alpha aminooleic acid present in the treatment solution were identified as the cause of mechanical abrasion of valve cusps during processing. These particles were eliminated with a revamped protocol, which included filtration of the AOA solution before valve preparation., Methods: Porcine aortic valve cusps treated with this modified AOA protocol (AOA II) were studied in a rat subdermal implant model of mineralization. A juvenile sheep trial was then used to confirm the antimineralization effects of AOA II on glutaraldehyde-fixed porcine aortic roots in a circulatory model of accelerated calcification., Results: Retrieved AOA II-treated cusps from the subdermal model were markedly less calcified than control cusps (AOA II, 1 +/- 0, 17 +/- 4, 23 +/- 6, and 17 +/- 10 versus control, 189 +/- 14, 251 +/- 16, 250 +/- 14, and 265 +/- 10 mg calcium/mg sample at 4, 8, 12, and 16 weeks, respectively; p < 0.0001). Morphologic examination of the AOA II cusps of the valves retrieved from the sheep demonstrated freedom from the structural loosening, surface roughening, and hematoma formation that had limited the utility of the original AOA preparation technique. Cusps from AOA II-treated porcine roots had significantly less calcium than control cusps (AOA II, 5.5 +/- 3.0 mg/g; control, 91.2 +/- 19.5 mg/g; p = 0.0004). The aortic walls had similar levels of calcification (AOA II, 156 +/- 73 mg/g; control, 159 +/- 10 mg/g; p = not significant)., Conclusions: These data suggest that the modified AOA technique warrants further evaluation as an antimineralization treatment for glutaraldehyde-fixed porcine bioprostheses.

In this study, we examined separately calcification of cusps (C) and associated aortic wall (AW) of 38 (13 aortic and 25 mitral) porcine bioprosthetic heart valves explanted from 37 patients (ages 25-80 years, mean 59) for structural dysfunction, following 54-210 months (mean 125 months aortic, 119 months mitral). Valves were sectioned into C and corresponding AW components; calcification was assessed by atomic absorption spectroscopy for calcium and histologic examination. Overall, AW calcification was half that of C (33.3 +/- 5.4 vs. 65.9 +/- 6.3 microg/mg, mean +/- standard error of the mean respectively, p = 0.002). Correlation of calcification in individual C/AW pairs was weak (r2 = 0.34). Calcification in C was nodular, largely in the valve fibrosa, but AW calcification predominated in the cells between elastic lamellae; large nodules were sparse. We conclude that since AW calcification in these failed porcine valves was neither prominent nor clinically significant, this process should rarely if ever be a limiting factor in the function of stented porcine valves, and that development of anticalcification therapies directed toward the AW of stented valves should be of low priority. However, in stent-free valves, the AW is not covered by prosthetic material, and the level of calcification could be greater and more likely to cause clinical problems through stiffening, embolism, and/or protrusion into the lumen of calcific masses.

Postimplant calcific degeneration is a frequent cause of clinical failure of glutaraldehyde crosslinked porcine aortic valve bioprostheses. We demonstrated previously in rat subdermal and circulatory implants that alpha-amino oleic acid used as a bioprosthesis pretreatment was highly effective in mitigating aortic valve cusp but not aortic wall calcification. In this study we investigated the feasibility of synergistically applying two proven anticalcification agents (alpha-amino oleic acid and FeCl3) as pretreatments for mitigating both bioprosthetic cusp and aortic wall calcification. alpha-Amino oleic acid is hypothesized to prevent calcification by disrupting calcium phosphate formation kinetics, whereas suppression of alkaline phosphatase activity and ferric-phosphate complexation at a cellular membrane initiation sites may be important factors in ferric ion's inhibition of calcification. In vivo implant studies (21-day rat subdermal model) indicated that individually FeCl3 (0.01 or 0.1 M for 24 h) or alpha-amino oleic acid (saturated solution) treatments were equally effective in mitigating cuspal calcification (tissue calcium levels: 30.2 +/- 10.2, 29.8 +/- 2.7, and 31.6 +/- 7.8 micrograms/mg tissue, respectively). However, sequential application of first alpha-amino oleic acid and then FeCl3 synergistically reduced aortic wall calcification more effectively than either of the agents alone. The benefit of a synergistic application of two anticalcification treatments, alpha-amino oleic acid and FeCl3, was demonstrated. However, the synergistic effect was observed on aortic wall only at a higher FeCl3 concentration. (i.e., 0.1 M).

Background: Calcification of the cusps of bioprosthetic heart valves fabricated from either glutaraldehyde cross-linked porcine aortic valves or bovine pericardium frequently causes the clinical failure of these devices. Our investigations studied ethanol pretreatment of glutaraldehyde cross-linked porcine aortic valves as a new approach to prevent cuspal calcification. The hypothesis governing this approach holds that ethanol pretreatment inhibits calcification resulting from protein structural alterations and lipid extraction., Methods and Results: Results demonstrated complete inhibition of calcification of glutaraldehyde-pretreated porcine bioprosthetic aortic valve cusps by 80.0% ethanol in rat subdermal implants (60-day ethanol-pretreated calcium level, 1.87 +/- 0.29 micrograms/mg tissue compared with control calcium level, 236.00 +/- 6.10 micrograms/mg tissue) and in sheep mitral valve replacements (ethanol-pretreated calcium level, 5.22 +/- 2.94 micrograms/mg tissue; control calcium level, 32.50 +/- 11.50 micrograms/mg tissue). The mechanism of ethanol inhibition may be explained by several observations: ethanol pretreatment resulted in an irreversible alteration in the amide I band noted in the infrared spectra for both purified type I collagen and glutaraldehyde cross-linked porcine aortic leaflets. Ethanol pretreatment also resulted in nearly complete extraction of leaflet cholesterol and phospholipid., Conclusions: Ethanol pretreatment of glutaraldehyde cross-linked porcine aortic valve bioprostheses represents a highly efficacious and mechanistically based approach and may prevent calcific bioprosthetic heart valve failure.

Calcification complicates the use of the polymer polyurethane in cardiovascular implants. To date only costly experimental circulatory animal models have been useful for investigating this disease process. In this paper we report that polyurethane calcification in rat subdermal implants is enhanced by overdosing with a vitamin-D analog. The calcification-prone state, known as calciphylaxis, was induced in 4-week old rats by oral administration of a vitamin-D analog, dihydrotachysterol. We studied two commercially available polyurethanes (Biomer and Mitrathane) and two proprietary polyurethanes (PEU-2000 and PEU-100). PEU-100 is unique because it is derivatized with ethanehydroxy-bisphosphonate (EHBP) for calcification resistance. Polyurethane calcium and phosphate levels and morphological changes due to calciphylaxis were compared with those of control rat subdermal explants in 60-day studies. Increased polyurethane mineralization was observed due to calciphylaxis with 60-day rat subdermal explants of Biomer, Mitrathane, and PEU-2000 (calcium levels, respectively, 4.13 +/- 0.56, 18.61 +/- 2.73, and 3.37 +/- 0.22 microgram/mg, mean +/- standard error) as compared to control explants (calcium levels, respectively, 1.22 +/- 0.1, 12.57 +/- 0.86, and 0.20 +/- 0.86 microgram/mg). The study also demonstrated that with 60-day implants calciphylaxis had no side effects on somatic growth and serum calcium levels. Explant surface morphology of these polyurethane explants examined by scanning electron microscopy, back scattering electron imaging coupled with energy dispersive X-ray spectroscopy, and light microscopy demonstrated the presence of predominantly surface-oriented calcification. PEU-100, derivatized with 100 n.moles/ mg of EHBP, resisted calcification with explant calcium levels 0.51 +/- 0.01 (calciphylaxis) and 0.38 +/- 0.01 (control) microgram/mg. It is concluded that calciphylaxis enhances superficial polyurethane calcification in rat subdermal implants and that an EHBP-modified polyurethane resists calcification despite calciphylaxis. Rat subdermal implants using calciphylaxis may be generally useful for evaluating the calcification potential of various biomedical polymers.

Postimplant calcific degeneration is a frequent cause of clinical failures of glutaraldehyde cross-linked porcine bioprosthetic heart valves (BPHV). It was demonstrated previously that 2-amino oleic acid (AOA) used as a bioprosthesis treatment was highly effective in mitigating aortic valve cusp but not aortic wall calcification. Our main objective was to study the efficacy of various AOA exposure conditions for inhibiting calcification of both cusps and aortic wall tissues using rat subdermal implants. BPHV tissues were treated with a saturated AOA solution for different time intervals before experimental. Aortic wall AOA levels were consistently lower than that of the cusps after the same exposure times. The diffusion of calcium ion across both cusp and aortic wall tissues was evaluated, and the results demonstrated that there was an AOA exposure time-dependent retardation of calcium ion penetration for cusp but not aortic wall. An 8-month extraction study was performed to determine the stability of AOA binding. When Tween 80 was used as an extraction medium, cusp and aortic wall retained 12.9 and 48.7%, respectively, of their initial AOA levels. AOA inhibition of calcification in rat subdermal implants (60 days) was found to be exposure time-dependent with maximum treatment time (120 h), resulting in the lowest calcium levels (20.1 +/- 10.3 and 71.4 +/- 5.4 micrograms/mg of cusp and aortic wall, respectively) as compared with controls (219.1 +/- 6.8 and 104.9 +/- 8.5 micrograms/mg of cusp and aortic wall, respectively). The significance of AOA binding on BPHV tissue was determined by either blocking or reducing BPHV's (cusp and aortic wall) free aldehyde residues with lysine or NaBH4, respectively, before AOA treatment. For aortic cusps, the AOA contents after 72 h were 98.3 +/- 2.7, 34.2 +/- 3.6, and 54.1 +/- 3.0 nM/mg of tissue for AOA (control), lysine-pretreated (plus AOA) and NaBH4-pretreated (plus AOA) tissues, respectively. However, their calcium levels after 60 days of rat subdermal implant were all comparable (i.e., 48.1 +/- 6.2, 38.2 +/- 9.1, and 47.0 +/- 15.0 micrograms calcium per mg of tissue). Similar results were observed on BPHV aortic wall. It can thus be concluded that AOA inhibition of BPHV calcification is exposure time-dependent, but the efficacy of AOA for aortic wall is less than that noted for aortic cusps, perhaps because of lower AOA binding and differences in calcium diffusion kinetics.

The purpose of this study was to characterize the onset and progression of mineralization in porcine bioprosthetic valves implanted in sheep and to test the hypothesis that such valves simulate calcification that is observed clinically and in other experimental models. Hancock I porcine aortic bioprosthetic valves (Medtronic Heart Valve Division, Irvine, Calif.) were implanted as orthotopic mitral valve replacements in juvenile sheep, retrieved after 1 to 124 days, and analyzed as follows: gross inspection, radiography, light, transmission, and surface scanning electron microscopy, and calcium analysis by absorption spectroscopy. Mineralization increased with increasing time after implantation in both valve cusps and adjacent aortic wall. Mean cuspal calcification was 80 micrograms/mg in valves removed after 3 to 4 months. Nevertheless, considerable variability among valves was apparent in the level of calcification noted at specific time intervals. Virtually all aspects of the morphologic characteristics were identical to those previously noted for clinical explants and experimental specimens, both subcutaneous and circulatory. In particular, ultrastructural examination revealed that the earliest calcific deposits were associated with devitalized cuspal connective tissue cells and their fragments. Collagen calcification was sparse. Both surface scanning and transmission electron microscopy indicated a lack of endothelial or blood-derived cells on the valves at all sampling times. We conclude that porcine bioprosthetic valves implanted as mitral valves in sheep provide a useful calcification model, simulating morphologic and pathobiologic events that occur clinically and in noncirculatory models. However, sufficient specimen replicates must be done to overcome variability in calcification among valves and sampling sites.

Background: Calcification is a frequent cause of the clinical failures of glutaraldehyde-pretreated bioprosthetic heart valves (BPHV) fabricated from glutaraldehyde-cross-linked porcine aortic valves. 2-Amino oleic acid (AOA) has been shown in previous in vivo studies to be a promising anticalcification agent. Our objective was to investigate the mechanism of calcification inhibition mediated by AOA pretreatment of porcine aortic valve bioprostheses., Methods and Results: BPHV tissues were treated with an AOA solution for 72 hours before experimentation. The diffusion of AOA across both cusp and aortic wall was evaluated. The lag time for AOA to diffuse across the aortic wall was prolonged compared with that of the cusp. An extraction study was performed to determine the stability of AOA binding; the results indicated that the binding was relatively stable regardless of solvent extraction conditions. The interaction between ionic calcium and AOA on treated tissue also was investigated by evaluating the patterns of calcium diffusion across both treated and untreated tissues. The results showed that AOA significantly reduced the diffusion of calcium. AOA inhibition of aortic valve calcification (calcium level, 5.5 +/- 3.0 mg/g of tissue compared with control; calcium level, 91.2 +/- 19.5 mg/g of tissue) but not aortic wall (calcium level, 158.7 +/- 10.3 mg/g of tissue compared with control; calcium level, 157.5 +/- 7.9 mg/g of tissue) was demonstrated on representative specimens from valves implanted in left ventricular apicoaortic shunts explanted after 150 days., Conclusions: AOA covalently binds to glutaraldehyde-pretreated bioprosthetic heart valve tissue, presumably as the result of an aldehyde-amino reaction. Covalently bound AOA diminishes Ca2+ diffusion compared with non-AOA-pretreated bioprosthetic tissues. This may explain in part the anticalcification mechanism of AOA. Furthermore, AOA inhibits calcification of porcine BPHV cusps in the circulation.

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 nM/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 degrees 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 nM 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.

Calcification of bioprosthetic heart valves fabricated from glutaraldehyde pretreated bovine pericardium or porcine aortic valves (PAV) is a frequent cause of the failure of these devices. Of all strategies considered thus far, only detergent preincubations using compounds such as sodium dodecyl sulfate (SDS) inhibited PAV bioprosthetic mineralization in circulatory sheep bioprosthetic valve replacements. The present study sought to characterize the mechanism of action of SDS preinicubation. Results of transport and material characterization studies showed that SDS had a relatively high affinity for PAV, with a maximum uptake of 167.1 +/- 6.8 micrograms SDS/mg tissue over 24 h at 37 degrees C with a partition coefficient of 19.3. The PAV diffusion of SDS was 1.95 +/- 0.35 10(-6) cm2/sec. The principal effect of SDS on PAV was phospholipid extraction. The residual organic phosphate in the SDS pretreated tissue was 2.22 +/- 0.72 nmol/mg tissue compared to the control untreated group with 18.52 +/- 2.1 nmol/mg tissue. Incubations of PAV specimens in a 1% SDS solution for 24 h significantly inhibited calcification after 21 days in subdermal implants in 3-week-old male rats (PAV Ca2+ = 18.0 +/- 11.8 micrograms/mg) compared to control (177.8 +/- 6.0 micrograms/mg). In contrast, coimplants of 30% SDS silicone rubber polymers, for regional sustained SDS administration, did not impede PAV calcification in 21 day implants Ca2+ = 166.0 +/- 14.0 micrograms/mg compared to the nondrug silicone matrix controls, Ca2+ = 173.0 +/- 6.6 micrograms/mg). Thus, we conclude that the mechanisms of SDS inhibition of PAV calcification is due to material effects which occur during preincubation, and is not facilitated by sustained SDS administration.

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 sodium-ethanehydroxydiphosphonate (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 degrees 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) M) followed by an EHDP solution (0.1 M), 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 pre-incubation, 10(-5) M) was combined with Na2EHDP (second pre-incubation, 0.1 M) (1.78 +/- 0.2 micrograms of Ca2+/mg of dried tissue) compared with the other pre-incubation groups: EHDP (first pre-incubation) combined with FeCl3 (second pre-incubation) (21.7 +/- 6.4), FeCl3 solution alone at 10(-5) M (27.9 +/- 10.7), Na2EHDP solution alone at 0.1 M (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 micrograms Ca2+/mg of dried tissue) compared with non-drug silicone co-implant controls (74.7 +/- 5.58 micrograms 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.

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, AICI3). GPBP specimens were implanted subcutaneously in 3 wk old male rats after pre-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 explants. 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.

Since calcification limits the durability of contemporary bioprosthetic heart valves, antimineralization treatments are being widely investigated. Potential antimineralization treatments must have sustained prevention of mineralization without adverse effects. The preclinical investigation of efficacy and safety of antimineralization treatments comprises four essential steps: (1) subcutaneous implantation in small animals, (2) in vitro biomechanical studies of hemodynamics and durability, (3) morphology of unimplanted valves, and (4) circulatory implants in large animals.

Aortic allograft conduits and valves frequently undergo calcific degeneration. To study this problem, a rat subdermal model of nonvalved aortic wall allograft calcification was characterized, and experimental studies were carried out to test the hypothesis that aortic allograft preincubation in either amino-propanehydroxydiphosphonate (APDP) or AlCl3 would inhibit calcification in a rat subdermal model. Fresh thoracic aortas were harvested under sterile conditions from male Sprague-Dawley rats (350-400 g). APDP aortas were preincubated immediately in either 4 x 10(-3) mol/l, 4 x 10(-4) mol/l, or 4 x 10(-5) mol/l [14C] APDP (37 degrees C, pH 7.4) and controls were incubated in 0.05 mol/l HEPES buffer (pH 7.4, 37 degrees C, 30 min). Al3+ aortas were preincubated in either 10(-1) mol/l, 10(-2) mol/l, or 10(-3) mol/l AlCl3. Pretreated aortas were next implanted subdermally in weanling rats (3-week-old, male, Sprague-Dawley, 50-60 g) and retrieved after 21 days. Control explants retrieved at intervals up to 21 days demonstrated progressive calcification with bulk aortic allograft Ca2+ levels increasing from a preimplant value of 0.8 +/- 0.1 micrograms/mg to 129.8 +/- 12.9 micrograms/mg by 21 days. Light microscopy revealed that much of the calcium deposition was associated with elastin. Calcification was significantly inhibited in the 4 x 10(-3) mol/l and 4 x 10(-4) mol/l APDP preincubated groups was observed (Ca2+ = 0.70 +/- 0.15 micrograms/mg, 36.6 +/- 19.8 micrograms/mg, respectively versus 117.2 +/- 24.3 micrograms/mg, control). Inhibition of calcification in the groups preincubated in the two most concentrated AlCl3 solutions (Ca2+ = 13.9 +/- 4.9 micrograms/mg [10(-2) mol/l AlCl3], 36.6 +/- 7.1 micrograms/mg [10(-3) mol/l AlCl3], 171.0 +/- 13.2 micrograms/mg [control]) was also demonstrated. No adverse effects of either pretreatment, APDP, or AlCl3 were noted on bone or overall somatic growth.

Clinical and experimental studies indicate that calcification of bioprosthetic valves depends on host, implant, and biomechanical factors. The earliest mineral deposits in both clinical and experimental bioprosthetic tissue are localize to transplanted connective tissue cells; collagen involvement occurs later. Passive calcium entry occurs unimpeded in cells modified by aldehyde cross-linking, but the mechanisms for calcium removal are dysfunctional; calcium influx contributes to apatite formation by reacting with compartmentalized, bound phosphorus. Specific strategies for prevention of bioprosthetic tissue mineralization involve modification of either valve preparation details or the local implant environment. Efficacy, mechanisms and safety must be demonstrated for any antimineralization strategy.

Calcification of cardiovascular prosthetic implants is a common and important problem. This review provides an update based upon the Conference on Cardiovascular Implant Calcification held as part of the 13th World Congress of the International Society for Heart Research, 1989. A variety of cardiovascular prostheses are affected clinically by calcification, including bioprosthetic heart valves, aortic homografts and trileaflet polymeric valve prostheses. In addition, experimental studies have demonstrated calcification of artificial heart devices in ventricular assist systems in long-term calf studies. The pathophysiology of this disease process is incompletely understood. A common element between the various types of cardiovascular implant calcification is the localization of calcific deposits to devitalized cells and membranous debris. Prevention of cardiovascular implant calcification by either biomaterial modifications or regional drug therapy (controlled release) is being investigated.

The principal cause of the clinical failure of bioprosthetic heart valves fabricated from glutaraldehyde-pretreated porcine aortic valves is calcification. Other prostheses composed of tissue-derived and polymeric biomaterials also are complicated by deposition of mineral. We have previously demonstrated that: (a) Failure due to calcification of clinical bioprosthetic valves can be simulated by either a large animal circulatory model or subdermal implants in rodents. (b) Calcification of bioprosthetic tissue has complex host, implant, and mechanical determinants. (c) The initial calcification event in the rat subdermal model is the mineral deposition in devitalized cells intrinsic to the bioprosthetic tissue within 48 to 72 h, followed later by collagen mineralization. (d) Initiation of bioprosthetic tissue mineralization, like that of physiological bone formation, has "matrix vesicles" as early nucleation sites. (e) Alkaline phosphatase (AP), an enzyme also associated with matrix vesicles involved in bone mineral nucleation, is present in both fresh and fixed bioprosthetic tissue at sites of initial mineralization. (f) Certain inhibitors of bioprosthetic tissue calcification (e.g., Al3+, Fe3+) are localized to the sites at which alkaline phosphatase is present. On the basis of these results, we hypothesize that alkaline phosphatase is a key element in the pathogenesis of mineralization of bioprosthetic tissue. In the present studies, we focused on the relationship of AP to early events in calcification, and the inhibition of both calcification and AP activity by FeCl3 and AlCl3 preincubations. Subdermal implants of glutaraldehyde pretreated bovine pericardium (GPBP) were done in 3-week-old rats. AP was characterized by enzymatic hydrolysis of paranitrophenyl phosphate (pnpp), and by histochemical studies. Calcification was evaluated chemically (by atomic adsorption spectroscopy) and morphologically (by light microscopy). The results of these studies are as follows: (a) Extractable AP activity is present in fresh but not glutaraldehyde-pretreated bovine pericardial tissue. However, histochemical studies reveal active AP within the intrinsic devitalized cells of GPBP, despite extended glutaraldehyde incubation. (b) Extrinsic AP is rapidly adsorbed following implantation, with peak activity at 72 h (424 +/- 67.2 nm pnpp/mg protein/min enzyme activity [units]), but markedly lesser amounts at 21 days (96.8 +/- 3.9 units). (c) Simultaneously to the AP activity maximum, bulk calcification is initiated, with GPBP calcium levels rising from 1.2 +/- 0.1 (unimplanted) to 2.4 +/- 0.2 micrograms/mg at 72 h, to 55.6 +/- 3.1 micrograms/mg at 21 days, despite a marked decline in AP activity at this later time.(ABSTRACT TRUNCATED AT 400 WORDS)

In the present study, the authors investigated the mechanism by which Al3+ preincubations inhibited the pathologic calcification of glutaraldehyde-pretreated bovine pericardium (GPBP) implanted subdermally in rats. The concentration dependency of the Al3+ anticalcification effect was compared with that of other trivalent metal ions (Fe3+, Ga3+, La3+) known to interact with calcium phosphates. In vitro incubations of GPBP were carried out in AlCl3 (10(-3) mol/l [molar] to 10(-1) mol/l) to ascertain both the optimal conditions for uptake of Al3+ and the time course of Al3+ dissociation. Al3+ uptake by GPBP was concentration dependent and occurred rapidly, with tissue levels after 1 hour not differing significantly from those after 72 hours of incubation. Analyses of GPBP samples preincubated in AlCl3 (0.1 mol/l, 24 hours) showed that more than 75% of the Al3+ remained tightly bound after 60 days' in vitro release at 37 degrees C, pH 7.4. Preincubations of GPBP in AlCl3 significantly inhibited calcification after subdermal implantation in rats for 60 days (Ca++ = 5.1 +/- 0.9 microgram/mg, 11.5 +/- 4.6 micrograms/mg, 70.3 +/- 23.0 micrograms/mg, mean +/- standard error [SE], for 10(-1) mol/l, 10(-2) mol/l, 10(-3) mol/l AlCl3, respectively), compared with controls (Ca++ = 110.0 +/- 9.3 micrograms/mg). All animals were free of Al3(+)-mediated adverse effects on bone, as determined by light microscopic evaluation of femoral epiphyseal growth plates. Transmission electron microscopy coupled with electron energy loss spectroscopy (EELS) of GPBP incubated in 10(-1) mol l AlCl3 for 24 hours demonstrated discrete Al3+ localization in the sarcolemma and cytoplasmic and nuclear membranes of devitalized pericardial connective tissue cells at intracellular sites coincident with phosphorus loci. Similar intracellular localization remained prominent in explants removed after 60 days; no calcific deposits were noted in these specimens. Preincubations in Fe3+ but not Ga3+ and La3+ solutions yielded significant inhibition of GPBP calcification, which did not differ significantly from that provided by Al3- and had a comparable concentration dependency. Light microscopic examination (Prussian blue staining) and EELS of FeCl3-preincubated explants demonstrated Fe3+ localization within devitalized GPBP connective tissue cells. The authors conclude that Al3+ and Fe3+ significantly inhibit the pathologic mineralization of glutaraldehyde-pretreated bovine pericardium by mechanisms that are likely related to the high affinity of these cations for membrane associated and other intracellular phosphorus loci.

AI3+ preincubation has been shown in 21 and 60 day implants to inhibit calcification of glutaraldehyde preserved bovine pericardium (GPBP) in the rat subdermal model. This study was designed to assess the long-term anticalcification efficacy of GPBP AI3+ preincubation. Rat (50-60 gm, male, CD Sprague-Dawley) subdermal implants of GPBP, pretreated with 0.01 M or 0.1 M AICI3, were carried out for 21, 60, 90, and 120 days. Each explanted GPBP was analyzed for Ca2+ by atomic absorption spectroscopy and for AI3+ by atomic absorption spectroscopy or neutron activation irradiation. Results showed profound long-term (implant duration 120 days) inhibition of calcification after GPBP preincubation in 0.1 M AICI3 (Ca2+ = 21.8 +/- 12.6 micrograms/mg), compared to control (Ca2+ = 329.0 +/- 20.3 micrograms/mg). After preincubation in 0.1 M AICI3 and 21 day rat subdermal implantation, GPBP AI3+ levels declined from 9033 +/- 680 micrograms/g (control = 6.3 +/- 3.7 micrograms/g) to 2700 +/- 156 micrograms/g; however, there was no further significant decline in GPBP AI3+ levels after long-term implantation of 120 days (2618.4 +/- 349 micrograms/g). The authors conclude that GPBP preincubation in 0.1 M AICI3 markedly inhibited pathologic calcification to 7% of control values in the rat model after long-term (120 day) subdermal implantation.

Al+++ preincubation has been shown to inhibit calcification of glutaraldehyde pretreated bovine pericardium calcification in the rat subdermal model. This study was designed to assess in vitro the Al+++ (10(-1) M, 10(-2) M, 10(-3) M preincubations) binding characteristics of glutaraldehyde pretreated bovine pericardium. In vivo studies assessed the ability of AlCl3 (10(-1) M, 10(-2) M, 10(-3) M) preincubations to inhibit calcification after 21 and 60 days in the rat subdermal model. Other in vivo studies investigated the ability of other metallic cations (Fe+++, La+++, and Ga+++) used as a pretreatment to inhibit glutaraldehyde pretreated bovine pericardium calcification after a 21 day rat subdermal implant. Lyophylized glutaraldehyde pretreated bovine pericardium samples were analyzed for Al+++ by neutron activation. In vitro results showed the greatest glutaraldehyde pretreated bovine pericardium Al+++ uptake in specimens incubated in 0.1M AlCl3. Maximal Al+++ uptake occurred by 1 hour. Glutaraldehyde pretreated bovine pericardium Al+++ levels after incubation in 10(-1) M, 10(-2) M, and 10(-3) M AlCl3 for 1 hour were 351.4 +/- 15.8 nM/mg, 96.4 +/- 17.4 nM/mg, and 24.6 +/- 4.9 nM/mg, respectively versus 394.5 +/- 48.0 nM/mg, 105.3 +/- 2.3 nM/mg, 40.0 +/- 8.8 nM/mg, respectively for 3 days. In vivo 21 day rat (50-60 g, male, Sprague-Dawley) subdermal implants of 0.1 M AlCl3, FeCl3, FeCl3 plus citrate, LaCl3, and GaNO3 pretreated glutaraldehyde pretreated bovine pericardium showed calcification inhibition by Al+++, Fe+++, and Fe+++ plus citrate (Ca++ = 5.5 +/- 0.2 micrograms/mg, 13.6 +/- 4.6 micrograms/mg, 5.9 +/- 1.7 micrograms/mg, respectively) compared to control (Ca++ = 63.6 +/- 5.7 micrograms/mg).(ABSTRACT TRUNCATED AT 250 WORDS)

Calcification, the major cause of bioprosthetic heart valve failures, is a serious clinical problem with uncertain pathogenesis. The objectives of the present study were to define the progressive chemical and morphologic sequence of mineralization in glutaraldehyde-treated porcine aortic valve cusps implanted subcutaneously in rats and to compare the pathology and pathophysiology of calcification in subcutaneous implants with that of orthotopic valve replacements in calves. Cusps were implanted subcutaneously in 3-week-old rats for 24 hours to 18 weeks. Cuspal calcium was 114 +/- 18 micrograms/mg of dry weight (mean +/- SEM) at day 21 and 218 +/- 6 at day 56 of implantation and unchanged thereafter. The earliest mineral deposits, noted at 48 hours, were associated with devitalized porcine connective tissue cells, but by 7 days, mineral deposits also involved collagen bundles. Scanning electron microscopy with energy-dispersive x-ray analysis demonstrated predominant accumulation in the spongiosa with a spongiosa to fibrosa energy-dispersive x-ray analysis count ratio of calcium of 15 at 21 days. In stent-mounted glutaraldehyde-preserved porcine valves implanted in five calves as mitral replacements for 69 to 142 days, cuspal calcium was 86 micrograms/mg (mean) (range 47 to 128). Calf implants also had cell oriented and collagen calcification predominating in the valvar spongiosa. In both rat subcutaneous and calf mitral valve models, early diffuse calcific microcrystals evolved into confluent nodules that disrupted tissue architecture. It is concluded that calcification of glutaraldehyde-preserved porcine aortic valves implanted subcutaneously in rats begins within 48 hours, earliest deposits are localized to residual porcine connective tissue cells, but latter deposits also involve collagen fibrils, mineralization is most prominent in the spongiosa, the pathology of calcification in rat subcutaneous implants and calf mitral replacements is comparable, suggesting a common pathophysiology, and calcific nodule formation most likely initiates clinical features.

Calcium binding proteins containing gamma-carboxyglutamic acid (Gla) have previously been demonstrated to occur in calcified atherosclerotic plaque and calcified cardiac valves. Experiments were carried out to determine if one of the Gla-containing proteins in human cardiovascular calcifications is the vitamin K-dependent bone protein, osteocalcin. A radio-immunoassay for human osteocalcin was employed, and EDTA extractions of calcified atheromata, and aortic valves as well as relevant noncalcified material were analyzed. Tissue calcium levels were also determined, as were Gla levels as a measure of total vitamin K-dependent protein content. Osteocalcin was present at low levels in all calcified cardiovascular tissues (4.5-175.7 ng osteocalcin/mg protein) with trace levels or nondetectable amounts present in noncalcified tissue. Osteocalcin accounted for a small proportion of the total protein-bound Gla (0.01-0.05%). The relationship of osteocalcin to the other Gla-containing proteins of atherosclerotic plaque including atherocalcin, the principal extractable Gla-containing protein of calcified plaque, is discussed.

Calcification is the principal cause of failure of tissue-derived cardiac valve replacements pretreated with glutaraldehyde (GLUT). The objective of this study was to determine the role of GLUT-induced cross-links in bovine pericardial tissue calcification. Various levels of 3H-GLUT incorporation were obtained by varying incubation pH, and protein modification was determined by amino acid analysis and resistance to collagenase digestion. Calcification of cross-linked tissue was studied using subdermal implants in rats. Low GLUT uptake (less than 150 nm/mg) resulted in minimal calcification (Ca2+, 12.8 micrograms/mg) and stability (4% residual weight following digestion) due to a limited crosslinking (lysine + hydroxylysine = 26.1 residues/1000 amino acids [AA]). In contrast, higher GLUT uptake induced more cross-links (Lys + Hyl = 8.2 residues/1000 AA) and consequent higher stability (95% residual wt); such tissues calcified severely (Ca2+, 93.5 micrograms/mg). Incorporation of GLUT two to three times beyond a critical level did not further enhance calcification. It is concluded that the amount of GLUT incorporated controls the extent of cross-links, which in turn directly determines tissue stability and calcification.

Calcification frequently causes failure of bioprosthetic heart valves (BHV) fabricated from glutaraldehyde-pretreated porcine aortic valve or bovine pericardium. Systemic diphosphonate therapy inhibits this disease process, but with adverse effects on overall growth, bone development, and calcium metabolism. The present study was designed to examine the hypothesis that the immobilization of ethanehydroxydiphosphonate (EHDP) within BHV as the poorly soluble Ca2+ salt would inhibit calcification at drug levels insufficient to produce side effects. Glutaraldehyde-pretreated pericardial BHV tissue was exposed to a physiologic concentration of Na2EHDP (0.14 M in 0.05 M HEPES, pH 7.4) and subsequently washed in a NaCl or CaCl2 (0.14 M in 0.05 M HEPES, pH 7.4) solution to precipitate the Na or Ca2+ salts of EHDP on/within the tissue, respectively. Incorporation of CaEHDP into BHV ranged from 74.8 nM/mg (after 1 h, 37 degrees C) to 353 nM/mg (2 weeks, 22 degrees C). None of the Na2EHDP was incorporated into BHV without exposure to CaCl2. In vitro release of CaEHDP from BHV into a physiologic buffer not containing Ca2+ was rapid, with greater than 95% removed after 4 d, while release of CaEHDP into buffer containing a physiologic concentration of Ca2+ ion (1.5 mM) was markedly reduced, with 30% of the precipitated CaEHDP remaining immobilized on or within the tissue matrix following 21 d.(ABSTRACT TRUNCATED AT 250 WORDS)

Calcification of glutaraldehyde-preserved bioprosthetic cardiac valves represents a serious clinical problem. Previous work from this laboratory has established the presence in clinical bioprosthetic valve calcifications of vitamin K-dependent calcium-binding proteins, which contain the calcium-binding amino acid gamma-carboxyglutamic acid; no proteins containing gamma-carboxyglutamic acid are present in nonmineralized valves. The purpose of the present study was to examine a series of bovine circulatory bioprosthetic valve explants for calcification and proteins containing gamma-carboxyglutamic acid. Biochemical analyses of explanted bioprosthetic valves from calves demonstrated proteins with gamma-carboxyglutamic acid accumulating in calcified valves during both the onset and progression of valve calcification; calcium levels in the explanted calf bioprostheses were in the same range as those noted in clinical material. Accumulation of calcium and protein with gamma-carboxyglutamic acid occurred simultaneously and progressively, beginning 2 months after implantation. Small amounts of osteocalcin, the bone-derived protein containing gamma-carboxyglutamic acid, were present in both human and bovine bioprosthetic valve calcifications at comparable levels. No osteocalcin was detectable in non-mineralized valve tissue. Warfarin anticoagulant therapy did not prevent calcification or accumulation of protein with gamma-carboxyglutamic acid. It is concluded that proteins containing gamma-carboxyglutamic acid are involved in both the onset and progression of bioprosthetic valve calcification, and that conventional means of vitamin K antagonism do not alter this association or the course of bioprosthetic valve mineralization.

Although collagen-containing implants are widely used in various surgical applications, there has been relatively little attention paid to the possibility that this type of biomaterial may undergo pathologic calcification which could compromise its function. The present study reports for the first time the calcification of a series of implants of purified collagen sponges prepared with graded degrees of aldehyde-induced cross-linkages (assessed by shrinkage-temperature, wetting time, and collagenase digestibility). Type I collagen sponges were pretreated with either glutaraldehyde (0.1% to 2.0% aqueous solution, for 5-180 minutes) or formaldehyde (as vapors for 15 minutes to 15 hours), and implanted subcutaneously for 21 days in weanling rats. Although specimens not pretreated with either aldehyde reagent and the formaldehyde sponges pretreated for 15 minutes were resorbed without evidence of calcification, all other aldehyde-pretreated implants mineralized. The degree of calcification did not correlate with extent of cross-linking. Formaldehyde-pretreated implants calcified more extensively (Ca2+ = 87.8 +/- 2.8 micrograms/mg, mean +/- standard error of the mean; n = 58) than did glutaraldehyde-pretreated implants (Ca2+ = 40.9 +/- 1.4 micrograms/mg; n = 52). It is concluded that both glutaraldehyde- and formaldehyde-pretreated Type I collagen sponges calcify after subdermal implantation in young rats. Although aldehyde pretreatment of Type I collagen sponge implants is a prerequisite for their eventual mineralization, the threshold level of aldehyde-induced cross-linking required to potentiate their maximal pathologic calcification is low.

Calcification is frequently noted in valved and non-valved aortic homografts, predominantly in the aortic wall portion. This study was designed to assess the individual anticalcification effects of the trivalent cation, aluminum (AI ), and the compound aminopropanehydroxydiphosphonate (APDP) when used to pretreat aortic wall homograft tissue before subdermal implantation in the rat model. Thoracic aortas of male rats (CD, Sprague-Dawley, 350 to 400 g) were harvested distal to the aortic valve and incubated in the following solutions: 1) APDP (0.004 M), 2) APDP (0.004 M) + glutaraldehyde (GLUT) (0.02 M), 3) GLUT (0.02 M), 4) ALCL3 (0.01 M), 5) HCL (0.001 M), and 6) HEPES (0.05 M). The homograft tissue was then implanted subcutaneously for 21 days in weanling male rats (50 to 60 gm) of the same strain. Explant results showed that calcification (CALC) was markedly inhibited in the APDP and 0.01 M ALCL3 groups, compared to the control and GLUT pretreated groups. No diphosphonate or aluminum related side effects were seen.

Calcification is the principal mode of failure of bioprosthetic heart valves (BPHV) fabricated from glutaraldehyde-pretreated porcine aortic valves or bovine pericardium. Covalent binding of aminopropanehydroxy-diphosphonate (APDP) to residual glutaraldehyde in pericardial BPHV tissue was studied as an approach for the inhibition of calcification. BPHV tissue was preincubated in 0.14 M APDP at pH 7.4, 9.0, and 11.0 for various durations (1 hour to 8 days). The need for NaBH4 stabilization of the tissue-bound APDP was also examined in vitro. The bound APDP was determined using 14C-labeled APDP. APDP uptake was dependent on incubation duration and pH. Calcification of APDP-pretreated BPHV was studied using 21-day rat subdermal implants. Calcification inhibition was directly related to the amount of tissue APDP incorporation. Inhibition of calcification to less than 15% of control was achieved with a concentration of bound APDP of greater than or equal to 30 nM/mg dry tissue with more than 1 hour of incubation at pH 11.0 (bound APDP, 33.55 nM/mg; BPHV calcium content = 3.1 +/- 0.9 micrograms/mg). No adverse effects such as rat growth inhibition or disruption of bone architecture were observed after any treatment. Additionally, in vitro, NaBH4 stabilized tissue-bound APDP. In conclusion, APDP covalently bound to residual aldehyde functions markedly inhibited calcification of BPHV tissue. This inhibition was dependent on the amount of APDP incorporated. NaBH4 stabilized APDP-glutaraldehyde covalent bonds.

Calcification of glutaraldehyde-preserved porcine aortic valve bioprosthetic cusps limits their success as cardiac valve substitutes. Subcutaneous implants of porcine bioprostheses in rats and rabbits have provided a convenient experimental tool to study this calcification process. Previous clinical research has suggested that the host's immune response to the porcine xenograft tissue may contribute to the calcific degeneration. To investigate the possible contribution of the immune response, porcine bioprosthetic cusps implanted subcutaneously in congenitally athymic (nude) BALBc mice and normal controls were analyzed biochemically and histologically after retrieval at 21 days. Calcification was comparable in implants retrieved from athymic (calcium 95.5 +/- 24.5 micrograms/mg) and normal mice (calcium 102.3 +/- 4.66 micrograms/mg). Explants from nude mice demonstrated fewer adherent cells than those from normal animals, but the morphologic characteristics of the calcification were the same in both groups, with dystrophic mineralization of the spongiosa predominating. Thus, normal T-lymphocyte function is not necessary for porcine bioprosthetic calcification, and immunologic processes do not contribute to this process.

Calcification (CALC) is the most frequent cause of the clinical failure of bioprosthetic heart valves (BHV) fabricated from glutaraldehyde pretreated porcine aortic valves or bovine pericardium. The present investigation describes the formulation, characterization, and the in vivo efficacy of prolonged controlled-release silicone matrices containing the anticalcification agent disodium 1,1-hydroxyethylidene diphosphonate (Na2EHDP). Controlled release of EHDP was regulated by codispersions of Na2EHDP and the less soluble salt Ca2EHDP. Prolonged and constant release rates (zero-order) were obtained by coating silicone matrices with permeable silicone membranes, which were prepared by leaching with acetone pre-embedded polyethyleneglycol. All EHDP-containing matrices (co-implanted subdermally with BHV cusps in rats) significantly inhibited BHV CALC without detectable adverse effects on bone mineral and calcium metabolism. Matrices containing Na2EHDP:Ca2EHDP ratios of 10:90 or greater with respect to Na2EHDP completely inhibited CALC. Significant inhibition of BHV CALC was also observed with prereleased matrices (5 months in vitro), thus demonstrating prolonged efficacy. It is concluded that sustained release of effective anticalcification therapy without side effects was achieved by using codispersions of calcium and sodium EHDP salts, and that a delayed and/or constant release rate of EHDP was obtained by coating reservoir-type matrices with silicone membranes that were pre-embedded with polyethyleneglycol.

To date, the clinical usefulness of bioprosthetic heart valves (BPV) fabricated from glutaraldehyde pretreated porcine aortic valves or bovine pericardium has been limited because leaflet calcification (CALC) leads to valvular stenosis and/or regurgitation and subsequent valve failure. The present study was designed to test the hypothesis that preincubation of glutaraldehyde pretreated pericardial BPV leaflets in a solution containing the trivalent cation, AL , inhibits BPV calcification. BPV tissue was preincubated in 0.1 M ALCL3 for 24 hours (T = 25 degrees C), then subdermally implanted in rats (weanling, male, CD, Sprague-Dawley) for 21 days. Compared to the control group (tissue Ca++ = 143.81 +/- 25.49 micrograms/mg), calcification was markedly inhibited in the group pretreated with AL (tissue Ca++ = 4.1 +/- 1.03 micrograms/mg) after a 21 day implant. No adverse effects on rat growth or bone morphology were detected. It is concluded that the trivalent cation AL provided a marked anticalcification effect for BPV tissue implanted subdermally in the rat for 21 days.

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 degrees or 25 degrees 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 degrees 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 degrees C vs 259.76 +/- 1.39 nM/mg, 25 degrees C; APDP, 57.56 +/- 4.43 nM/mg, 4 degrees C vs 36.36 +/- 0.46 nM/mg, mean +/- standard error, at 25 degrees 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 micrograms/mg, APDP pretreated, vs 93.64 +/- 11.65 micrograms/mg, control; P less than or equal to 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 degrees 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.