Biomedical subjects

Cecilia M Giachelli

Publications and source records attributed to Cecilia M Giachelli.

At least 19 recordsLinked to original sources

PubMed2007

Mechanisms of vascular calcification.

Vascular calcification is highly prevalent and correlated with high rates of cardiovascular mortality in chronic kidney disease patients. Recent evidence suggests that mineral, hormonal, and metabolic imbalances that promote phenotype change in vascular cells as well as deficiencies in specific mineralization inhibitory pathways may be important contributory factors for vascular calcification in these patients. This article reviews current mechanisms proposed for the regulation of vascular calcification and data supporting their potential contribution to this process in chronic kidney disease.

Animals↗

PubMed2006

Molecular mediators of alphavbeta3-induced endothelial cell survival.

The alphavbeta3 integrin interaction with the extracellular matrix (ECM) plays an essential role in inhibiting apoptosis in endothelial cells. We have recently shown that alphavbeta3 ligation on rat aortic endothelial cells (RAECs) specifically activates the transcription factor nuclear factor kappaB (NF-kappaB) and promotes cell survival. Inhibiting NF-kappaB nuclear translocation abolished the protective effect of alphavbeta3 ligands. Here, we report that ligation of alphavbeta3 by its ligand, osteopontin (OPN), induces the phosphorylation and activation of inhibitory kappa B kinase beta IKKbeta and promotes the specific degradation of inhibitory kappa Balpha (IkappaBalpha) in RAECs. Overexpression of a dominant negative (DN) IKKbeta protein prevents IkappaBalpha phosphorylation, NF-kappaB activation, and inhibits the protective effects of OPN. The NF-kappaB-inducing kinase (NIK) has been shown to be one of the upstream kinases involved in IKK activation. OPN-mediated NF-kappaB activity is increased upon NIK wild-type (WT) overexpression and blocked following NIK DN overexpression. In addition, NIK-/-mouse embryonic fibroblasts (MEFs) plated on OPN display reduced NF-kappaB activity and decreased IkappaBalpha phosphorylation compared to NIK+/+MEFs. Finally, functional inhibition of integrin beta3-dependent NF-kappaB signaling decreases OPN-induced IkappaBalpha, IKKbeta and NIK phosphorylation. These studies for the first time show that the alphavbeta3-NF-kappaB-dependent endothelial survival pathway is dependent on IkappaBalpha, IKKbeta, and NIK.

Animals↗

PubMed2006

Osteoprotegerin inactivation accelerates advanced atherosclerotic lesion progression and calcification in older ApoE-/- mice.

OBJECTIVE: Osteoprotegerin (OPG), a member of the tumor necrosis factor (TNF) superfamily of proteins, plays an important role in bone remodeling and is expressed in both mouse and human atherosclerotic lesions. The current study was designed to assess whether OPG plays a role in the progression and calcification of advanced atherosclerotic lesions in apoE(-/-) mice. METHODS AND RESULTS: Atherosclerotic lesion area and composition and aortic calcium content were examined in mice deficient in both OPG and apolipoprotein E (OPG(-/-).apoE(-/-) mice) at 20, 40, and 60 weeks of age. Littermate OPG(+/+).apoE(-/-) mice were used as controls. The average cross-sectional area of lesions in the innominate arteries was increased in OPG(-/-).apoE(-/-) mice at 40 and 60 weeks of age. The increase in lesion area was coupled with a reduced cellularity and an increase in connective tissue including laminated layers of elastin. Sixty-week-old OPG(-/-).apoE(-/-) mice also had an increase in the area of calcification of the lesions. There were no differences in markers of plaque stability. In vitro, OPG induced matrix metalloproteinase-9 (MMP-9) activity in macrophages and smooth muscle cells and acted as a survival factor for serum-deprived smooth muscle cells. CONCLUSIONS: OPG inhibits advanced plaque progression by preventing an increase in lesion size and lesion calcification. OPG may act as a survival factor and may modulate MMP9 production in vascular cells.

Aging↗

PubMed2006

Role of the sodium-dependent phosphate cotransporter, Pit-1, in vascular smooth muscle cell calcification.

Vascular calcification is associated with cardiovascular morbidity and mortality. Hyperphosphatemia is an important contributor to vascular calcification. Our previous studies demonstrated that elevated phosphate induces calcification of smooth muscle cells (SMC) in vitro. Inhibition of phosphate transport by phosphonoformic acid blocked phosphate-induced calcification, implicating sodium-dependent phosphate cotransporters in this process. In the present study, we have investigated the role of the type III sodium-dependent phosphate cotransporter, Pit-1, in SMC calcification in vitro. Human SMC stably expressing Pit-1 small interfering double-stranded RNA (SMC-iRNA) were established using a retroviral system. SMC-iRNA had decreased Pit-1 mRNA and protein levels and sodium-dependent phosphate transport activity compared with the control transduced cells (SMC-CT) (2.9 versus 9.78 nmol/mg protein per 30 minutes, respectively). Furthermore, phosphate-induced SMC calcification was significantly inhibited in SMC-iRNA compared with SMC-CT at all time points examined. Overexpression of Pit-1 restored phosphate uptake and phosphate-induced calcification in Pit-1 deficient cells. Mechanistically, although Pit-1-mediated SMC calcification was not associated with apoptosis or cell-derived vesicles, inhibition of phosphate uptake in Pit-1 knockdown cells blocked the induction of the osteogenic markers Cbfa-1 and osteopontin. Our results indicate that phosphate uptake through Pit-1 is essential for SMC calcification and phenotypic modulation in response to elevated phosphate.

Aorta↗

PubMed2006

Mimicking cell-cell interactions at the biomaterial-cell interface for control of stem cell differentiation.

The ability to regulate stem cell proliferation and differentiation has relevance in numerous medical applications, including medical devices, tissue engineering, and regenerative medicine. To control cellular behavior at the biomaterial or scaffold interface, many studies have employed surface modifications that mimic the extracellular matrix. Strikingly absent is the immobilization of cell-surface ligands to the biomaterial surface. One cell-to-cell signaling pathway that has been shown to regulate tissue development and stem cell fate is the Notch pathway. Recently, the Notch signaling pathway was identified as a key regulator of epithelial differentiation. Utilizing this knowledge, we applied an affinity immobilization scheme designed to attach and orient the Notch ligand, Jagged-1, in an active conformation on a biomaterial surface. When epithelial stem cells were plated on the bound ligand, the Notch/CBF-1 signaling pathway was stimulated and the cells upregulated both intermediate- and late-stage differentiation markers. In addition, the ligand promoted tight clustering and extensive stratification. Soluble Jagged-1 showed no Notch/CBF-1 signaling and very little, if any, cell differentiating activity. The high potency of bound Jagged-1 suggests that modification of a surface with a Notch ligand presents a powerful method to control stem cell differentiation at the cell-biomaterial interface.

Animals↗

PubMed2006

Development of an esophagus acellular matrix tissue scaffold.

A cell-extraction protocol yielding an esophagus acellular matrix (EAM) scaffold for use in tissue engineering of an esophagus, including hypotonic lysis, multiple detergent cell extraction steps, and nucleic acid digestion, was developed in a rat model. Histological techniques, burst pressure studies, in vitro esophageal epithelial cell seeding, and in vivo implantation were used to assess cell extraction, extracellular matrix (ECM) preservation, and biocompatibility. Microscopy demonstrated that cell extraction protocols using sodium dodecyl sulfate (SDS) (0.5%, wt/vol) as a detergent resulted in cell-free EAM with retained ECM protein collagen, elastin, laminin, and fibronectin. Burst pressure studies indicated a loss of tensile strength in EAMs, but at intraluminal pressures that were unlikely to affect in vivo application. In vitro cell seeding studies exhibited epithelial cell proliferation with stratification similar to native esophagi after 11 days, and subcutaneously implanted EAMs displayed neovascularization and a minimal inflammatory response after 30 days of implantation. This study presents an esophagus acellular matrix tissue scaffold with preserved ECM proteins, biomechanical properties, and the ability to support esophageal cell proliferation to serve as the foundation for a tissue-engineered esophagus.

Animals↗

PubMed2005

Controlling osteopontin orientation on surfaces to modulate endothelial cell adhesion.

Osteopontin (OPN) is an important extracellular matrix protein that has been shown to impact wound healing, inflammation, and the foreign body reaction, and has been identified as a potential surface component for engineered biomaterials. OPN contains the arginine-glycine-aspartic acid (RGD) moiety that has been shown to mediate cell adhesion through interactions with integrins. In its preferred orientation and conformation on a surface, the functional domains of OPN will be presented to cells to the greatest extent. However, control of protein orientation and conformation is still challenging. In this work, we investigated OPN adsorption and cell adhesion to the OPN layer on self-assembled monolayers (SAMs) of alkanethiols terminated with various functional groups and on a gold surface. The four SAM terminal groups studied were --CH3, --OH, --NH2, and --COOH, representing hydrophobic, hydrophilic but neutral, positively charged, and negatively charged surfaces, respectively. Surface plasmon resonance biosensor and atomic force microscopy were used to characterize the adsorption of OPN on these surfaces. An in vitro cell adhesion assay of bovine aortic endothelial cells was performed to test the functionality of OPN on various SAMs. Surface plasmon resonance results showed that the amount of protein adsorbed on the --NH2 surface is close to a monolayer and similar to that on the --COOH surface, consistent with the atomic force microscopy results. However, based on cell adhesion experiments, both cell count and average cell spreading area on the --NH2 surface are much higher than those on the --COOH surface. From these results, it is suggested that the orientation and conformation of OPN on a positively charged --NH2 surface is more favorable for cell adhesion and spreading than on a negatively charged --COOH surface. The surface coverage of bovine aortic endothelial cells on the surfaces studied decreased in the following order: --NH2 > Au > --CH3 > --COOH > --OH whereas the mean cell spreading area decreased in the following order: --NH2 > Au > --CH3 > --COOH. Our studies show that surface properties will alter OPN behavior on surfaces, thus influencing cell interactions.

Adsorption↗

PubMed2005

Smooth muscle cells deficient in osteopontin have enhanced susceptibility to calcification in vitro.

OBJECTIVE: Vascular calcification is an actively regulated process, correlating with cardiovascular morbidity and mortality especially in patients with diabetes and chronic renal diseases. Osteopontin (OPN) is abundantly expressed in human calcified arteries and inhibits vascular calcification in vitro and in vivo. How OPN functions in vascular calcification, however, is less clear. METHODS: Smooth muscle cells (SMCs) were isolated from aortas of OPN knock-out (OPN-/-) and wild type (OPN+/+) mice. RESULTS: OPN-/- SMCs were identical to OPN+/+ SMCs in morphology and stained positively for SM lineage proteins, desmin, smooth muscle alpha-actin and SM22alpha. No spontaneous calcification was observed in OPN-/- SMCs under normal culture conditions or in medium containing 1%, 3%, or 5% fetal bovine serum. However, when cultured in medium containing elevated concentrations of inorganic phosphate, an inducer of vascular calcification, a significantly higher calcification was observed in OPN-/- SMCs compared to OPN+/+ SMCs that, in response to elevated phosphate, synthesized and secreted OPN into the culture. Finally, retroviral transduction of mouse OPN cDNA into OPN-/- SMCs rescued the calcification phenotype of the cells. CONCLUSION: These results are the first to demonstrate an inhibitory role of endogenously produced OPN on SMC calcification, suggesting a novel feedback mechanism where OPN produced locally by the SMCs may serve as an important inducible inhibitor of vascular calcification.

Animals↗

PubMed2005

The role of osteopontin in foreign body giant cell formation.

Foreign body giant cells (FBGCs) are a hallmark of the foreign body reaction caused by biomaterial implantation and are thought to contribute to biomaterial degradation and the duration of the response. Osteopontin (OPN) is a secreted, acidic matricellular protein with multiple phosphorylation sites that is highly expressed at sites of inflammation. OPN wildtype and knockout mice were implanted with poly(vinyl alcohol) sponges and explanted at 14 days. OPN knockout mice had more foreign body giant cells but fewer macrophages surrounding the implants than their wildtype counterparts. In an in vitro human FBGC assay, addition of soluble OPN was found to reduce macrophage fusion to giant cells. These are the first studies to show a direct inhibitory role of OPN in FBGC formation in response to implantation.

Animals↗

PubMed2005

Calcification of advanced atherosclerotic lesions in the innominate arteries of ApoE-deficient mice: potential role of chondrocyte-like cells.

OBJECTIVE: Advanced atherosclerotic lesions in the innominate arteries of chow-fed apolipoprotein E-deficient mice become highly calcified with 100% frequency by 75 weeks of age. The time course, cell types, and mechanism(s) associated with calcification were investigated. METHODS AND RESULTS: The deposition of hydroxyapatite is preceded by the formation of fibro-fatty nodules that are populated by cells that morphologically resemble chondrocytes. These cells are spatially associated with small deposits of hydroxyapatite in animals between 45 and 60 weeks of age. Immunocytochemical analyses with antibodies recognizing known chondrocyte proteins show that these cells express the same proteins as chondrocytes within developing bone. Histological and electron microscopic analyses of lesions from animals between 45 and 60 weeks of age show that the chondrocyte-like cells are surrounded by dense connective tissue that stains positive for type II collagen. Nanocrystals of hydroxyapatite can be seen within matrix vesicles derived from the chondrocyte-like cells. In mice between 75 and 104 weeks of age, the lesions have significantly reduced cellularity and contain large calcium deposits. The few remaining chondrocyte-like cells are located adjacent to or within the large areas of calcification. CONCLUSIONS: Calcification of advanced lesions in chow-fed apolipoprotein E-deficient mice occurs reproducibly in mice between 45 and 75 weeks of age. The deposition of hydroxyapatite is mediated by chondrocytes, which suggests that the mechanism of calcification may in part recapitulate the process of endochondral bone formation.

Alkaline Phosphatase↗

PubMed2005

Regulation of vascular calcification: roles of phosphate and osteopontin.

Vascular calcification is prevalent in aging as well as a number of pathological conditions, and it is now recognized as a strong predictor of cardiovascular events in the general population as well as diabetic and end-stage renal disease patients. Vascular calcification is a highly regulated process involving inductive and inhibitory mechanisms. This article focuses on two molecules, phosphate and osteopontin, that have been implicated in the induction or inhibition of vascular calcification, respectively. Elevated phosphate is of interest because hyperphosphatemia is recognized as a major nonconventional risk factor for cardiovascular disease mortality in end-stage renal disease patients. Studies to date suggest that elevated phosphate stimulates smooth muscle cell phenotypic transition and mineralization via the activity of a sodium-dependent phosphate cotransporter. Osteopontin, however, appears to block vascular calcification most likely by preventing calcium phosphate crystal growth and inducing cellular mineral resorption.

Animals↗

PubMed2005

Mitigation of ectopic calcification in osteopontin-deficient mice by exogenous osteopontin.

Ectopic calcification is a major cause of bioprosthetic heart valve failure. New therapeutic opportunities are offered by the growing understanding that ectopic calcification is an actively regulated process involving several key gene products. One of these products, osteopontin (OPN), is a glycosylated phosphoprotein previously shown to inhibit apatite crystal formation, induce carbonic anhydrase II, and promote mineral resorption. In this study, OPN-deficient mice (OPN-/-) were utilized as an in vivo model to stimulate the ectopic calcification of glutaraldehyde-fixed bovine pericardium (GFBP) tissue and to examine OPN delivery and structure-function relationships with respect to its anti-calcific activity. Significant calcification of GFBP tissue was obtained within 7 days of subcutaneous implantation in OPN-/- mice. Direct rescue of the calcification phenotype was achieved by the administration of exogenous recombinant rat, histidine-fused OPN (rat His-OPN) to the implant site via soluble injection (up to 72% mitigation achieved) or adsorption onto the implant materials (up to 91% mitigation achieved). Effects were specific, since neither fibronectin nor polyhistidine alone could mitigate calcification of GFBP. The maximum anti-calcific effect was achieved only when rat His-OPN was adequately phosphorylated and contained a functional arginine-glycine-aspartate (RGD) cell adhesive domain. Furthermore, CAII levels in host cells surrounding GFBP were greatest when phosphorylated, RGD-containing rat His-OPN was adsorbed. These data suggest that both physical inhibition, mediated by phosphorylation sites in OPN, as well as the induction of CAII and mineral regression, mediated by the RGD domain, contribute to the unique ability of OPN to mitigate ectopic calcification of bioprosthetic valve tissue.

Animals↗

PubMed2005

Free fatty acid impairment of nitric oxide production in endothelial cells is mediated by IKKbeta.

OBJECTIVE: Free fatty acids (FFA) are commonly elevated in diabetes and obesity and have been shown to impair nitric oxide (NO) production by endothelial cells. However, the signaling pathways responsible for FFA impairment of NO production in endothelial cells have not been characterized. Insulin receptor substrate-1 (IRS-1) regulation is critical for activation of endothelial nitric oxide synthase (eNOS) in response to stimulation by insulin or fluid shear stress. METHODS AND RESULTS: We demonstrate that insulin-mediated tyrosine phosphorylation of IRS-1 and serine phosphorylation of Akt, eNOS, and NO production are significantly inhibited by treatment of bovine aortic endothelial cells with 100 micromol/L FFA composed of palmitic acid for 3 hours before stimulation with 100 nM insulin. This FFA preparation also increases, in a dose-dependent manner, IKKbeta activity, which regulates activation of NF- kappaB, a transcriptional factor associated with inflammation. Similarly, elevation of other common FFA such as oleic and linoleic acid also induce IKKbeta activation and inhibit insulin-mediated eNOS activation. Overexpression of a kinase inactive form of IKKbeta blocks the ability of FFA to inhibit insulin-dependent NO production, whereas overexpression of wild-type IKKbeta recapitulates the effect of FFA on insulin-dependent NO production. CONCLUSIONS: Elevated levels of common FFA found in human serum activate IKKbeta in endothelial cells leading to reduced NO production, and thus may serve to link pathways involved in inflammation and endothelial dysfunction.

Animals↗

PubMed2005

Src kinase activity is required for integrin alphaVbeta3-mediated activation of nuclear factor-kappaB.

Integrin adhesion to extracellular matrix proteins protects adhesion-dependent cells from suspension-induced apoptosis. Previous studies indicate that activation of the transcription factor nuclear factor-kappaB was necessary for the integrin alpha(v)beta3 ligand osteopontin to protect endothelial cells from apoptosis caused by serum withdrawal. In this study, beta3 integrins were overexpressed in smooth muscle cells. When plated on osteopontin, cells overexpressing wild-type beta3 had enhanced cell adhesion, cell spreading, and nuclear factor-kappaB activation compared with vector control. Removal of four amino acids (759X) from the C terminus of beta3 eliminated the ability of the integrin to promote these processes. Single amino acid substitutions indicated that phosphorylation at tyrosine 759 was not required for activation of the transcription factor, however this residue appeared to play a structural role, because mutation to alanine significantly inhibited nuclear factor-kappaB activation. The Src family of tyrosine kinases represents important transducers during integrin signaling, and the C terminus of beta3 has been implicated as the binding site for Src. Immunoprecipitations demonstrated that Src associated with wild-type beta3 integrins, but Src and integrins lacking the C terminus (759X) did not form a complex. Pharmacological inhibition with the Src inhibitor PP2 (4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine) or overexpression of kinase-dead c-Src blocked nuclear factor-kappaB activation. Mouse embryonic fibroblasts deficient for Src failed to activate nuclear factor-kappaB when plated on osteopontin, in contrast to control fibroblasts. Together, these experiments indicate that the C terminus of beta3 and Src activity are required for integrin alpha(v)beta3-mediated nuclear factor-kappaB activation.

Amino Acid Sequence↗

PubMed2005

Esophageal epithelial cell interaction with synthetic and natural scaffolds for tissue engineering.

As an initial step towards a tissue-engineered esophagus, rat esophageal epithelial cells (REEC) were isolated and characterized for epithelial identity, adhesion protein preference, and in vitro interaction with natural and synthetic scaffolds. The scaffolds consisted of AlloDerm (LifeCell Corporation, Branchburg, NJ), poly(L-lactic acid) (PLLA), poly(lactic-co-glycolic) acid (75:25) (PLGA75), poly(lactic-co-glycolic) acid (50:50) (PLGA50), and polycaprolactone/poly(L-lactic acid) (50:50) (PCL/PLLA). Various factors-including calcium concentration, scaffold composition, and pore size--were evaluated for their influence on epithelial growth and differentiation. By day 18, keratinocytes seeded on AlloDerm cultured under high Ca(++) (1.5mm) conditions showed a proliferating basal cell layer, epithelial stratification (5--6 layers) and a thick keratin layer. The synthetic scaffolds (PLGA, PLLA, PCL/PLLA) also showed complete surface coverage, regions of proliferating basal cells, and evidence of stratification (2--3 layers) and keratinization. The highly porous nature of the synthetic scaffolds, however, limited the formation of a continuous epithelial layer and resulted in a lack of overall spatially-defined differentiation. In conclusion, rat esophageal epithelial cells were successfully isolated and characterized, with cells seeded on AlloDerm showing superior epithelial organization and stratification compared to synthetic scaffolds. Modification of the synthetic scaffold's surface properties and pore size may be necessary to mimic epithelial behavior on natural scaffolds.

Animals↗

PubMed2005

Activation of IKKbeta by glucose is necessary and sufficient to impair insulin signaling and nitric oxide production in endothelial cells.

Hyperglycemic impairment of nitric oxide (NO) production by endothelial cells is implicated in the effect of diabetes to increase cardiovascular disease risk, but the molecular basis for this effect is unknown. In skeletal muscle, diabetes induces activation of inhibitor kappaB kinase (IKKbeta), a key cellular mediator of the response to inflammatory stimuli, and this impairs insulin signal transduction via the insulin receptor substrate-phosphatidylinositol 3-OH kinase (IRS-1/PI3-kinase) pathway. Since activation of endothelial nitric oxide synthase (eNOS) is dependent on IRS-1/PI3-kinase signaling, we hypothesized that activation of IKKbeta may contribute to the effect of glucose to impair NO production. Here, we show that exposure of bovine aortic endothelial cells to high glucose (25 mM) for 24 h impaired insulin-mediated tyrosine phosphorylation of IRS-1, serine phosphorylation of Akt, activation of eNOS, and production of NO. High glucose treatment also activated IKKbeta, and pretreatment with aspirin, a pharmacological inhibitor of IKKbeta, prevented both glucose-induced IKKbeta activation and the effect of high glucose to impair insulin-mediated NO production. These adverse responses to glucose were also blocked by selective inhibition of IKKbeta signaling via overexpression of a kinase-inactive form of the enzyme. Conversely, overexpression of wild-type IKKbeta recapitulated the deleterious effect of high glucose on insulin-mediated activation of eNOS. These data demonstrate that activation of IKKbeta plays a critical and novel role to mediate the deleterious effects of high glucose on endothelial cell function.

Animals↗

PubMed2005

Arteriosclerosis, calcium phosphate deposition and cardiovascular disease in uremia: current concepts at the bench.

PURPOSE OF REVIEW: Cardiovascular disease is the leading cause of death in patients with chronic kidney disease. A growing body of data points to nontraditional risk factors, including disturbances in mineral metabolism, as important determinants of the extremely high cardiovascular morbidity and mortality rates in these patients. Disturbances in mineral metabolism, especially elevated calcium and phosphate levels, have been linked to vascular and valvular calcification, both of which are associated with poor prognosis in chronic kidney disease patients. This review highlights important recent findings regarding the etiology of vascular calcification, with special emphasis on pathways that may be particularly relevant in chronic kidney disease patients. RECENT FINDINGS: New studies indicate that not only vascular intimal calcification (associated with atherosclerosis) but also vascular medial calcification are correlated with decreased survival in chronic kidney disease patients. With the relatively recent recognition of vascular calcification as an actively regulated process, a growing list of inducers (calcium, phosphate, inflammatory cytokines) and inhibitors (matrix Gla protein, fetuin, pyrophosphate, osteopontin) have been discovered. Interesting recent evidence suggests that they may contribute to the prevalence of this pathology in chronic kidney disease patients. SUMMARY: Vascular calcification is associated with decreased survival in chronic kidney disease patients. Understanding the causes and regulatory factors controlling vascular calcification will help refine therapeutic modalities currently in use, as well as develop novel therapeutics to abate and potentially reverse this deleterious process.

Animals↗

PubMed2004

Hyaluronic acid grafting mitigates calcification of glutaraldehyde-fixed bovine pericardium.

Pathologic calcification is the leading cause of the clinical failure of glutaraldehyde-fixed bovine pericardium used in bioprosthetic valves. A novel surface modification of glutaraldehyde fixed bovine pericardium was carried out with high molecular weight hyaluronic acid (HA). HA was chemically modified with adipic dihydrazide (ADH) to introduce hydrazide functional groups onto the HA backbone. Glutaraldehyde-fixed bovine pericardium (GFBP) was modified by grafting this HA to the free aldehyde groups on the tissue via the hydrazide groups. Following a 2-week subcutaneous implantation in osteopontin (OPN)-null mice, the calcification of HA-modified bovine pericardium was drastically reduced (by 84.5%) compared to positive controls (tissue without HA-modification) (p = 0.005). The calcification-mitigating effect of HA surface modification was also confirmed by microscopic analysis of explanted tissue stained with Alizarin Red S for calcium.

Animals↗