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Hyaluronic acid grafting mitigates calcification of glutaraldhyde-fixed bovine pericardium
Abstract
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.
... 3,7,10,11 Alternatively, glycosaminoglycans (GAGs) have been used to prevent calcium deposition in BP, preventing hydroxyapatite nucleation. 11,12 Hyaluronic acid (HA) is a linear GAG with repeating disaccharide units of D-glucuronic acid and N-acetyl-D-glucosamine. 12 Surface modification of BP fixed using GA occurs by grafting high molecular weight HA to free aldehyde groups present on the surface of the biomaterial. 12 To date, the mechanical properties of BP subjected to anticalcification treatment using HA derivative have not been well investigated. ...
... 3,7,10,11 Alternatively, glycosaminoglycans (GAGs) have been used to prevent calcium deposition in BP, preventing hydroxyapatite nucleation. 11,12 Hyaluronic acid (HA) is a linear GAG with repeating disaccharide units of D-glucuronic acid and N-acetyl-D-glucosamine. 12 Surface modification of BP fixed using GA occurs by grafting high molecular weight HA to free aldehyde groups present on the surface of the biomaterial. 12 To date, the mechanical properties of BP subjected to anticalcification treatment using HA derivative have not been well investigated. ...
... 11,12 Hyaluronic acid (HA) is a linear GAG with repeating disaccharide units of D-glucuronic acid and N-acetyl-D-glucosamine. 12 Surface modification of BP fixed using GA occurs by grafting high molecular weight HA to free aldehyde groups present on the surface of the biomaterial. 12 To date, the mechanical properties of BP subjected to anticalcification treatment using HA derivative have not been well investigated. ...
We studied the mechanical behavior of bovine pericardium (BP) after anticalcification treatment using hyaluronic acid (HA) derivative. To simulate the physiological environment and stimulate the calcification process, the BP samples were immersed into simulated body fluid solution. We conducted scanning electron microscopy with energy dispersive X‐ray spectrometry, and uniaxial mechanical tests of HA‐treated and non‐treated samples. Although our microstructural analyses indicated that the HA treatment actually prevents the formation of calcium phosphate deposits, the mechanical tests show significant increase of stiffness of the HA‐treated samples. Using data from our mechanical tests as input parameters, we performed finite element (FE) computer simulations to estimate how this increase in the BP stiffness affects the stress distribution in the bioprosthetic leaflet. Although the maximum stress observed during the closing phase of the membrane in vivo is below the experimental yield stress in all cases we analyzed, our FE results indicate that increase of BP stiffness due to HA anticalcification treatment results in higher risk of disruption and failure of the leaflets in bioprosthetic heart valves. Since our FE results indicate that the commissure and the fixed edge are the regions that withstand the highest mechanical stresses during the closing phase, new designs of the valve might be efficient to enhance the endurance of the prosthesis. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res B Part B, 2019.
... The anti-calcification property of GAG was confirmed of which HA reacts with the free aldehyde groups to form stable hydrazone bond. HA can also be used as a hydrogel on pericardium, by cross-linking the hydrazide bonds for a continuous anti-calcification effect [41,59]. In addition, space is created when the tissues are decellularized, which increases the possibility of calcium being deposited. ...
... Hence bisphosphonate, a stable analogue of pyrophosphate and a calcium-binding compound was synthesized [61]. It is known as crystal poison as it binds to hydroxyapatite crystals and inhibits further crystal growth [59]. Ethylenediaminetetraacetic acid (EDTA) Open Access 9 is a chelating agent which can be used to minimize calcification by sequestering metal ions like Ca 2+ and reducing the dimensions of the crystal. ...
... Ether and chitosan also enable detoxification of graft tissue. The details are tabulated in table 3 [15,[18][19][20][21][22][28][29][30]32,34,[37][38][39][40]46,52,54,56,[58][59][60][64][65][66][67][68][69][70][71][72][73]. ...
... It also constitutes the backbone of cartilage proteoglycan. Because of its unique physicochemical properties and various biological functions, HA and modified HA have been extensively investigated and widely used for pharmaceutical and medical applications, (Balazs and Denlinger, 1993;Illum et al., 1994;Hahn et al., 2004;Ohri et al., 2004) such as for arthritis treatment (Balazs and Denlinger, 1993), ophthalmic surgery (Bothner and Wik, 1987), drug development , and tissue engineering (West et al., 1985;Ohri et al., 2004). HA is known to be involved in wound healing, promoting cell motility and differentiation during development (Laurent, 1998). ...
... It also constitutes the backbone of cartilage proteoglycan. Because of its unique physicochemical properties and various biological functions, HA and modified HA have been extensively investigated and widely used for pharmaceutical and medical applications, (Balazs and Denlinger, 1993;Illum et al., 1994;Hahn et al., 2004;Ohri et al., 2004) such as for arthritis treatment (Balazs and Denlinger, 1993), ophthalmic surgery (Bothner and Wik, 1987), drug development , and tissue engineering (West et al., 1985;Ohri et al., 2004). HA is known to be involved in wound healing, promoting cell motility and differentiation during development (Laurent, 1998). ...
... HA is known to be involved in wound healing, promoting cell motility and differentiation during development (Laurent, 1998). A number of strategies for the chemical modification of HA for improving its physicochemical properties through carboxyl and hydroxyl groups have been developed (Balazs and Leshchiner, 1987;Kuo et al., 1991;Illum et al., 1994;Luo et al., 2000;Hahn et al., 2004;Ohri et al., 2004). ...
Hyaluronic acid (HA) is a biodegradable, biocompatible, non-toxic, non-immunogenic and non-inflam-matory linear polysaccharide, which has been used for various medical applications including arthritis treatment, wound healing, ocular surgery, and tissue augmentation. Because of its mucoadhesive property and safety, HA has received much attention as a tool for drug delivery system development. It has been used as a drug delivery carrier in both nonparenteral and parenteral routes. The nonparenteral application includes the ocular and nasal delivery systems. On the other hand, its use in parenteral systems has been considered important as in the case of sustained release formulation of protein drugs through subcutaneous injection. Particles and hydrogels by various methods using HA and HA derivatives as well as by con-jugation with other polymer have been the focus of many studies. Furthermore, the affinity of HA to the CD44 receptor which is overexpressed in various tumor cells makes HA an important means of cancer targeted drug delivery. Current trends and development of HA as a tool for drug delivery will be outlined in this review.
... Among successfully tested low-molecular-weight anticalcification agents one should mention amino-containing α-amino oleic acid (AOA) [15][16][17] and L-arginine [18], amino-containing [19] and other diphosphonates [20], urazole with sodium borohydride [21] as well as citric acid [22] and some surfactants [23]. Among tested polymer materials promising results were obtained with generally biocompatible poly(ethylene oxide) [24][25][26], other biocompatible polymers [27], amino-containing polyacrylamide [28] as well as with glycosaminoglycans such as hyaluronic acid [29] and heparin [30,31]. In general, according to subcutaneous implantation express-tests in rats, polymeric modifiers [24][25][26][27][28][29][30][31] demonstrated higher efficiency in the reduction of calcification in comparison with their low-molecular-weight counterparts [15][16][17][18][19][20][21][22][23]. ...
... Among tested polymer materials promising results were obtained with generally biocompatible poly(ethylene oxide) [24][25][26], other biocompatible polymers [27], amino-containing polyacrylamide [28] as well as with glycosaminoglycans such as hyaluronic acid [29] and heparin [30,31]. In general, according to subcutaneous implantation express-tests in rats, polymeric modifiers [24][25][26][27][28][29][30][31] demonstrated higher efficiency in the reduction of calcification in comparison with their low-molecular-weight counterparts [15][16][17][18][19][20][21][22][23]. Yet, the treatment procedure with polymers was typically rather complex and in some cases an additional step of a special chemical modification was required to ensure polymer grafting [24,25,29]. ...
... In general, according to subcutaneous implantation express-tests in rats, polymeric modifiers [24][25][26][27][28][29][30][31] demonstrated higher efficiency in the reduction of calcification in comparison with their low-molecular-weight counterparts [15][16][17][18][19][20][21][22][23]. Yet, the treatment procedure with polymers was typically rather complex and in some cases an additional step of a special chemical modification was required to ensure polymer grafting [24,25,29]. ...
Calcification of bovine pericardium dramatically shortens typical lifetimes of biological prosthetic heart valves and thus precludes their choice for younger patients. The aim of the present work is to demonstrate that the calcification is to be mitigated by means of treatment of bovine pericardiumin solutions of chitosan in carbonic acid, i.e. water saturated with carbon dioxide at high pressure. This acidic aqueous fluid unusually combines antimicrobial properties with absolute biocompatibility as far as at normal pressure it decomposes spontaneously and completely into H2O and CO2. Yet, at high pressures it can protonate and dissolve chitosan materials with different degrees of acetylation (in the range of 16–33%, at least) without any further pretreatment. Even exposure of the bovine pericardium in pure carbonic acid solution without chitosan already favours certain reduction in calcification, somewhat improved mechanical properties, complete biocompatibility and evident antimicrobial activity of the treated collagen tissue. The reason may be due to high extraction ability of this peculiar compressed fluidic mixture. Moreover, exposure of the bovine pericardiumin solutions of chitosan in carbonic acid introduces even better mechanical properties and highly pronounced antimicrobial activity of the modified collagen tissue against adherence and biofilm formation of relevant Gram-positive and Gram-negative strains. Yet, the most important achievement is the detected dramatic reduction in calcification for suchmodified collagen tissues in spite of the fact that the amount of the thus introduced chitosan is rather small (typically ca. 1 wt.%), which has been reliably detected using original tritiumlabelling method. We believe that these improved properties are achieved due to particularly deep and uniform impregnation of the collagen matrix with chitosan from its pressurised solutions in carbonic acid.
... A growing evidence has demonstrated the importance of glycosaminoglycan chains for the regulation of mineralization [33,34]. Previous studies have shown that oversulfated chondroitin sulfate promotes osteoblast differentiation and mineralization [35] and decorin glycosaminoglycan chain regulates human VSMC calcification [11]. ...
... An animal study has revealed that inhibition of HA synthesis by 4MU accelerates atherosclerosis in ApoE -/mice [36]. HA has been shown to inhibit osteoblast differentiation [18] and the calcification-mitigating role of the HA grafts on the biomaterial has been demonstrated [34]. In this study, we find that HMHA inhibits osteogenic differentiation and calcification of rat VSMCs and aterial calcification. ...
Vascular calcification is a highly regulated biological process similar to bone formation involving osteogenic differentiation of vascular smooth muscle cells (VSMCs). Hyaluronan (HA), a major structural component of the extracellular matrix in cartilage, has been shown to inhibit osteoblast differentiation. However, whether HA affects osteogenic differentiation and calcification of VSMCs remains unclear. In the present study, we used in vitro and ex vivo models of vascular calcification to investigate the role of HA in vascular calcification. Both high and low molecular weight HA treatment significantly reduced calcification of rat VSMCs in a dose-dependent manner, as detected by alizarin red staining and calcium content assay. Ex vivo study further confirmed the inhibitory effect of HA on vascular calcification. Similarly, HA treatment decreased ALP activity and expression of bone-related molecules including Runx2, BMP2 and Msx2. By contrast, inhibition of HA synthesis by 4-methylumbelliferone (4MU) promoted calcification of rat VSMCs. In addition, adenovirus-mediated overexpression of HA synthase 2 (HAS2), a major HA synthase in VSMCs, also inhibited calcification of VSMCs, whereas CRISPR/Cas9-mediated HAS2 knockout promoted calcification of rat A10 cells. Furthermore, we found that BMP2 signaling was inhibited in VSMCs after HA treatment. Recombinant BMP2 enhanced high calcium and phosphate-induced VSMC calcification, which can be blocked by HA treatment. Taken together, these findings suggest that HA inhibits vascular calcification involving BMP2 signaling.
... However, drawbacks of GA-crosslinked ABPs were revealed progressively during the past two decades including its structural degradation, calcification and immune responses after implantation, of which most were suggested to have probable connections to the use of GA [3] . Therefore, many researchers endeavoured to replace GA with other types of crosslinkers to stabilise the ABPs [4][5][6][7][8] , while others focused on applying post-treatments on GAcrosslinked ABPs to suppress adverse effects [9][10][11][12] . Only some researchers investigated the effects of using alternative GA crosslinking conditions, showing feasible routes to improve the properties when reducing the usage [13][14][15][16] . ...
Chemically crosslinked acellular bovine pericardium (ABP) has been widely used in clinical practice as bioprostheses. To ensure its consistency and durability, crosslinkers are used in excess, with stability guided by indicators including the hydrothermal denaturation temperature, the enzymatic resistance and the degree of crosslinking. Yet, understanding of the intermolecular structure in collagen fibrils which imparts the intrinsic stability of the ABPs is lacking, and the discrepancies in the stability criteria in varied conditions are poorly explained. In this study, synchrotron small-angle X-ray scattering (SAXS) in combination with thermal and colorimetric methods are employed to investigate the changes in the structure and the stability of ABPs during crosslinking using glutaraldehyde (GA) or 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) at different concentrations. Based on the findings, a mechanism is proposed to explicate the crosslinking effects on collagen structure and the relationship between the structure and each stability indicator. At low crosslinker concentrations, the telopeptidyl-helical linkages are preferred which cause rearrangements in the intermolecular structure of collagen, and efficiently contribute to its stabilities. Excess crosslinking is revealed by a revert trend in structural changes and the plateauing of the stabilities, assigning to the helical-helical linkages and monovalent bindings. The former would improve thermal stability but not collagenase resistance, whereas the latter have negligible effects. Overall, this study provides mechanistic understanding of the chemical crosslinking of ABPs which will contribute to the future development of more efficient and economically viable strategies to produce bioprostheses.
Statement of significance
Chemical crosslinking imparts suitable properties to acellular bovine pericardium (ABP) for clinical applications, yet the understanding is lacking on the structure-stability relationship especially under different crosslinking conditions. Structural evidence in this study differentiates the binding sites during crosslinking in collagen fibrils at different crosslinker concentrations, highlighting the excess usage in the conventional crosslinking treatments. The mechanism based on the structure of collagen also successfully explains the dissimilarity in hydrothermal and enzymatic stabilities with varied crosslinking conditions. Future researches focusing on developing biomaterials via chemical crosslinking of ABPs would benefit from this study, for its contribution to the better understanding of the relationship of collagen structure and functions.
... 20 HA is a ubiquitous natural polysaccharide in the body with excellent physicochemical properties such as biodegradable, biocompatible, nontoxic, and nonimmunogenic characteristics. Accordingly, HA has been widely used for various medical applications such as arthritis treatment 21 , ophthalmic surgery 22 , drug delivery, 23,24 and tissue engineering, 25 So we did not set up a group for CIA treated with 0.5% CMC or the blank hydrogel in the study. The total duration of drug administration was 21 days. ...
Background:
Iguratimod (IGUR) is a novel disease-modifying antirheumatic drug used for treating rheumatoid arthritis (RA). To improve its bioavailability and to alleviate gastrointestinal side effects, we changed the formulation into nanoiguratimod-loaded hydrogel (NanoIGUR-loaded hydrogel) composites for sustained release of therapeutics.
Methods:
IGUR was first encapsulated in biodegradable polyvinyl alcohol micelle by liquid antisolvent precipitation (LAP) technology, and then loaded into an in situ injectable hyaluronic acid hydrogel, which was cross-linked by PEG (Thiol)2 (HS-PEG-SH) through Michael addition reaction. In vitro, the biological effects (proliferation, migration, and invasion) of NanoIGUR-loaded hydrogel on fibroblast-like synoviocytes (RA-FLS) from RA patients were evaluated. In vivo, the pharmacokinetics of NanoIGUR-loaded hydrogel was assessed and the efficacy of NanoIGUR-loaded hydrogel in treating collagen-induced arthritis (CIA) rats was evaluated.
Results:
By the LAP technique, we acquired the amorphous form nanoiguratimod, with an average size of 458 nm, which had higher dissolution rates and higher stability. The release of IGUR from hydrogel composite in PBS was gradual and sustained for up to 72 hrs compared with nanoiguratimod. Different concentrations of NanoIGUR-loaded hydrogel inhibited the proliferation, migration, and invasion of RA-FLS. The pharmacokinetic parameters showed better bioavailability and longer half-life time with NanoIGUR-loaded hydrogel by subcutaneous administration than oral raw iguratimod. Animal experiments confirmed that subcutaneous injection of NanoIGUR-loaded hydrogel (10 mg/kg every 3 days) and oral raw iguratimod (10mg/kg daily) showed similar efficacy in decreasing arthritis index score, pathological score, and expression of inflammatory cytokines.
Conclusion:
Overall, we demonstrate that NanoIGUR-loaded hydrogel provides a new route of administration and extends the administration interval. It could be a promising drug-delivery approach in the management of RA.
... Removal of GAGs from pericardial tissues increased calcification [30]. The addition of exogenous GAGs reduced calcification [31]. Hyaluronic acid and chondroitin sulfate are the two primary GAG types present in heart valves [23]. ...
Glutaraldehyde (GLUT) crosslinked bioprosthetic heart valves (BHVs) might fail due to progressive degradation and calcification. GLUT cannot stabilize glycosaminoglycans (GAGs), which are important for BHVs’ life time. In this current study we developed a new BHVs preparation strategy using exogenous hyaluronic acid (HA)/chondroitin sulfate (CS) supplement and sodium trimetaphosphate (STP) crosslinking method. Exogenous HA and CS provide additional GAGs for pericardiums. STP could link two GAGs by reacting with hydroxyl groups in GAGs’ repeating polysaccharides units. The feeding ratios of HA/CS were optimized. The GAGs content and long-term stability in vitro, biocompatibility, the in vivo GAGs stability and anti-calcification potential of GLUT/HA/CS and STP treated pericardiums were characterized. We demonstrated that GLUT/HA/CS and STP treated pericardiums had sufficiently increased GAGs’ amount and stability and decreased calcification. This new exogenous hyaluronic acid/chondroitin sulfate supplement and sodium trimetaphosphate crosslinking strategy would be a promising method to make BHVs with better structural stability and anti-calcification properties.
... Herrero et al showed that removal of GAGs from pericardial tissues increased calcification [17]. Later studies by Ohri et al showed that the addition of exogenous GAGs reduced calcification [18]. Multiple treatments have been developed to improve GAGs stability including the addition of neomycin trisulfate (NT) [16,[19][20][21][22] and periodate oxidation [23,24]. ...
Glutaraldehyde (GLUT) crosslinked bioprosthetic heart valves (BHVs) might fail within ten years due to progressive degradation and calcification. GLUT cannot stabilize glycosaminoglycans (GAGs), one important component of BHVs. In this current study we developed a new BHVs preparation strategy named as 'HPA/NT/HRP' treatment that utilized 3,4-hydroxyphenylpropionic acid (HPA)/tyramine (TRA)/neomycin trisulfate (NT) conjugated pericardiums and horseradish peroxidase (HRP)/H2O2 enzyme-oxidative-polymerization method. HPA/TRA-pericardium and HPA-NT conjugation would provide extra phenol groups for enzymatic crosslinking. HPA/TRA conjugated pericardium could be crosslinked by HRP/H2O2 enzyme-oxidative-polymerization. The feeding ratio of HPA-NT was optimized. The GAGs content, collagenase and elastase degradation in vitro, the in vivo GAGs stability and anti-calcification potential of HPA/NT/HRP treated pericardiums were characterized. We demonstrated that HPA/NT/HRP treated pericardiums had sufficiently increased GAGs stabilization and decreased calcification. This new HPA/NT/HRP enzyme-oxidative-polymerization strategy would be a promising method to make BHVs with better GAGs stability and anti-calcification properties.
... Several treatments that could prevent xenograft calcification were under investigation in animal models [14][15][16][17][19][20][21][22][23][24][25]. Among different approaches, chitosan emerges as a highly promising candidate due to its versatility, natural origin, and biocompatibility [18,26,27]. ...
Vascular xenografts are widely used in cardiovascular surgery as an alternative to autologous vessels and vascular allografts. Calcification is one of the main drawbacks of vascular grafts, especially among young patients and children. Among different anticalcification approaches, chitosan emerges as a highly promising candidate due to its versatility, natural origin, and biocompatibility. We investigated the anticalcification efficacy of globular chitosan ("Chitozol") as it demonstrated the improved rate of water solubility as compared with conventional linear macromolecules of chitosan. In addition, we supposed that compact globular form of "Chitozol" molecules could provide effective penetration of extracellular matrix of bovine jugular veins (BJVs). Our results revealed that "Chitozol" treatment mitigated calcification in the experimental groups as compared to the control groups (without any treatment, conventional treatment with glutaraldehyde, and commercially available Contegra conduit). Different concentrations of "Chitozol" (0.3% and 3%), as well as different incubation times (15 and 30min), were equally effective in the prevention of calcification. In addition, "Chitozol" treatment with decellularization of BJVs demonstrated slightly improved stress-strain properties of unimplanted samples. Thus, the filling of fresh BJV with globular chitosan is proposed as a promising emerging treatment for the mitigation of calcific degeneration in BJVs xenografts.
... Recent evidence suggests that HA incorporated into valve scaffolds renders them less susceptible to calcification and also promote cell endothelialization [101,102]. Surface modification of bioprosthetic heart valves obtained from with HA derivatives can reduce the calcification [103,104]. HA however exhibits significant swelling in hydrated environments, making control of both TEHV geometry and mechanics difficult. ...
... HA have been used in various fields of medical applications such as drug carrier and biological materials [17,18]. Its biological advantages have been reported such as bioresorbable and less immune responsible materials. ...
The aim of this study was to evaluate the biocompatibility and resorption pattern in three-layer poly (lactide-co-glycolide) (PLGA) membrane according to the concentrations of hyaluronic acid (HA) hydrogel in rabbit calvarial defect model. Four standardized circular defects with 8 mm diameter were created on the four rabbit calvarium. Three-layer PLGA membranes (5% and 10% HA gel) were used as the test groups, both collagen membrane and monolayer PLGA membrane as the control groups.
After sacrificing the animals after 4 and 8 weeks, block sections were harvested and histological observation was performed. Pus formation was observed in a site on the three-layer PLGA membranes (with 10% HA gel) of 4 weeks group and initial inflammatory responses were observed on the three-layer PLGA membrane group. However, when compared to both the monolayer PLGA membrane group and collagen membrane group, the HA gel-reinforced three-layer PLGA membrane showed improved cell occlusion and retention period, showing the formation of the capsule-like structure. There was no definite difference between the results of the membranes fabricated with either 5% or 10% HA hydrogel.
The HA reinforced three-layer PGLA membrane was retained longer than control group and showed good property in cell occlusion. Future study is under process to improve the inflammatory response of the three layer PLGA membranes, which were observed in this study.
... Atualmente na literatura podemos encontrar inúmeros trabalhos que sugerem a destoxificação do pericárdio bovino após tratamento com glutaraldeído com ácido alfa amino oléico (Gulbins, 2006), ácido deoxicólico (da Costa, 2004), triton (Graussa R.W., 2005); descelularização pela ação da tripsina (Graussa R.W, 2005); e mesmo a utilização de outros componentes químicos para recobrimento do pericárdio tratado como a ácido hialurônico (Ohri R., 2004) e fibronectina (Trantina-Yates AE, 2001); e mesmo a substituição do glutaraldeído como reticulante por triglicidilamina (Connolly J.M, 2005).Uma proposta para diminuição da toxicidade do PB foi a liofilização, apresentando resultados promissores (Maizato et al., 2003). ...
Resumo. Próteses cardíacas com enxerto de tecidos biológicos têm sido utilizados desde os anos 60 como alternativas as próteses cardíacas mecânicas. Desde 1974 o pericárdio bovino (PB) tornou-se um dos materiais mais utilizados para a preparação de bioproteses. Para melhora das propriedades mecânicas, foram desenvolvidos vários processos químicos e físicos de reticulação. Atualmente o tratamento mais usual para as bioproteses de pericárdio bovino é a reticulação com glutaraldeído (GA), que pode induzir a calcificação in vivo, o que leva a uma insuficiência valvar e a necessidade de substituição da prótese. Em um trabalho prévio, o grupo demonstrou que a irradiação em feixe de elétrons (25kGy a 4,67kGy/h) aplicada ao PB liofilizado na ausência de oxigênio, promove a reticulação entre as fibras de colágeno do pericárdio bovino. Neste trabalho, foi estudada a incorporação de fibroína de seda (SF), quiosana (CHIT) e suas misturas (1:1, 1:3 e 3:1) no PB. Após os tratamentos do PB, as amostras foram irradiadas e então analisadas quanto a sua citotoxicidade e a capacidade de adesão e crescimento das células endoteliais. Após a análise foi verificado que todas as amostras apresentaram citotoxicidade nos seus extratos, devido aos resíduos de tratamento do PB (ácido acético e etanol). Entretanto, após algumas lavagens foram removidos os resíduos tóxicos do biomaterial, tornando-o próprio para cultura celular. O teste de biofuncionalidade mostrou que as amostras modificadas com SF/CHIT (todas as proporções) e irradiadas favoreceram a adesão e o crescimento das células endoteliais no tecido. Palavras-chave: Pericárdio bovino, Quitosana, Fibroína de seda, Citotoxicidade, Adesão de células endoteliais.
... Recently, HA polymers have become a topic of interest for developing sustained drug delivery systems of peptide and protein drugs in subcutaneous formulations. A number of strategies for chemical modification of HA have been developed to improve its physicochemical properties [13][14][15][16][17][18][19]. Indeed, HA uptake in cells is known to be mediated through endocytosis with specific receptors, such as cluster determinant 44 (CD44) and receptor for hyaluronatemediated motility (RHAMM) [14,16]. ...
In spite of severe side effects, chemotherapy is widely used as a major anticancer treatment in non-small cell lung cancer (NSCLC). In order to enhance the therapeutic properties and reduce side effects, enormous efforts have been devoted to direct anticancer agents specifically to tumor tissues by the use of nanoparticles, or cancer cell marker attached drugs. However, cell-specific chemotherapy is still in its infancy and is not applicable to all types of cancers due to the complexity of the cancer occurrence and progress. In this study, we demonstrate that hyaluronan (HA)-conjugated cisplatin has highly selective and sensitive anticancer effects in NSCLC cells that overexpress the trans-membrane receptor, CD44, as HA is a specific ligand for CD44. In proliferation experiments, HA-conjugated cisplatin showed dramatic cell viability decreases (from 100% to 42.31%) in H1299 cells, which overexpress CD44, whereas no such change was observed in A549 and HFL1, which have little to no expression of CD44. More importantly, conjugation with HA decreased the dosage concentration of cisplatin by 50-fold for both cytotoxic and anti-metastatic effects. In addition, HA-cisplatin conjugate treatment selectively decreased migration (from 100% to 7.8%) and invasiveness (from 100% to 21.4%, respectively) of H1299. Based on our experimental results, we strongly believe that HA-cisplatin conjugate is a potential anticancer chemo-agent, which target CD44 overexpression in NSCLC, with minimal side effects and high therapeutic properties.
... HA derivatives are currently used in a various number of biomedical applications: arthritis treatment [4], ophthalmic surgery [5], tissue engineering [6], wound healing [7] and drug delivery [8,9]. ...
... In three-dimensional in vitro cultures of cartilage slices, the addition of hyaluronidase promoted increased mineral accumulation [26]. HA itself appears to have a complicated relationship with calcification, with some reports suggesting that HA promotes the late osteogenic differentiation of cells [38] while others have integrated HA into biomaterial scaffolds and found either enhancement [15] or prevention [22,25] of mineralization. Although there are no published investigations of the HA synthases in adult valves, the signaling pathways regulating the activity of HAS-1, HAS-2, and HAS-3 represent therapeutic targets for atherosclerosis [17] and potentially for CAVD as well. ...
The calcification process in aortic stenosis has garnered considerable interest but only limited investigation into selected signaling pathways. This study investigated mechanisms related to hypoxia, hyaluronan homeostasis, brown adipocytic differentiation, and ossification within calcified valves. Surgically explanted calcified aortic valves (n=14) were immunostained for markers relevant to these mechanisms and evaluated in the center (NodCtr) and edge (NodEdge) of the calcified nodule (NodCtr), tissue directly surrounding nodule (NodSurr); center and tissue surrounding small "prenodules" (PreNod, PreNodSurr); and normal fibrosa layer (CollFibr). Pearson correlations were determined between staining intensities of markers within regions. Ossification markers primarily localized to NodCtr and NodEdge, along with markers related to hyaluronan turnover and hypoxia. Markers of brown adipocytic differentiation were frequently co-localized with markers of hypoxia. In NodCtr and NodSurr, brown fat and ossification markers correlated with hyaluronidase-1, whereas these markers, as well as hypoxia, correlated with hyaluronan synthases in NodEdge. The protein product of tumor necrosis factor-α stimulated gene-6 strongly correlated with ossification markers and hyaluronidase in the regions surrounding the nodules (NodSurr, PreNodSurr). In conclusion, this study suggests roles for hyaluronan homeostasis and the promotion of hypoxia by cells demonstrating brown fat markers in calcific aortic valve disease.
... Both methods block side effects of GA residues and thus prevent calcification of the pericardium. Finally, a modified adipic dihydrazide hyaluronic acid has been proposed to be grafted on to glutaraldehyde-treated bovine pericardium ( Ohri et al., 2004). Calcifications decreased considerably with this post-treatment compared to the control group at two weeks following a subcutaneous implantation in mice. ...
During the last decades, salicylic acid (SA) and hyaluronic acid (HA) have been studied for a wide range of cosmetic and pharmaceutical applications. The current study investigated the drug loading potential of SA in HA-based crosslinked hydrogel films using a post-loading (osmosis) method of the unmedicated xerogels from saturated aqueous solutions of salicylic acid over a range of pH values. The films were characterized with Fourier-transform infra-red spectroscopy (FT-IR) and ultraviolet-visible (UV-Vis) spectrophotometry in order to elucidate the drug loading profile and the films’ integrity during the loading process. Additional studies on their weight loss (%), gel fraction (%), thickness increase (%) and swelling (%) were performed. Overall, the studies showed significant film disintegration at highly acidic and basic solutions. No drug loading occurred at neutral and basic pH, possibly due to the anionic repulsion between SA and HA, whereas at, pH 2.1, the drug loading was promising and could be detected via UV-Vis analysis of the medicated solutions, with the SA concentration in the xerogel films at 28% w/w.
Staplers are widely used in clinics; however, complications such as bleeding and leakage remain a challenge for surgeons. To tackle this issue, buttress materials are recommended to reinforce the staple line. This Review provides a systematic summary of the characteristics and applications of the buttress materials. First, the physical and chemical properties of synthetic polymer materials and extracellular matrix used for the buttress materials are introduced, as well as their pros and cons in clinical applications. Second, we review the clinical effects of reinforcement mesh in pneumonectomy, sleeve gastrectomy, pancreatectomy, and colorectal resection. Based on the analysis of numerous research data, we believe that buttress materials play a crucial role in increasing staple line strength and reducing the probability of complications, such as bleeding and leakage. However, considering the requirements of bioactivity, degradability, and biosafety, non-crosslinked small intestinal submucosa (SIS) matrix material is the preferred candidate. It has high research and application value, but further studies are required to confirm this. The aim of this Review is to provide comprehensive guidance on the selection of materials for staple line reinforcement.
Calcific nodules form in the fibrosa layer of the aortic valve in calcific aortic valve disease (CAVD). Glycosaminoglycans (GAGs), which are normally found in the valve spongiosa, are located local to calcific nodules. Previous work suggests that GAGs induce endothelial to mesenchymal transformation (EndMT), a phenomenon described by endothelial cells’ loss of the endothelial markers, gaining of migratory properties, and expression of mesenchymal markers such as alpha smooth muscle actin (α-SMA). EndMT is known to play roles in valvulogenesis and may provide a source of activated fibroblast with a potential role in CAVD progression. In this study, a 3D collagen hydrogel co-culture model of the aortic valve fibrosa was created to study the role of EndMT-derived activated valvular interstitial cell behavior in CAVD progression. Porcine aortic valve interstitial cells (PAVIC) and porcine aortic valve endothelial cells (PAVEC) were cultured within collagen I hydrogels containing the GAGs chondroitin sulfate (CS) or hyaluronic acid (HA). The model was used to study alkaline phosphatase (ALP) enzyme activity, cellular proliferation and matrix invasion, protein expression, and calcific nodule formation of the resident cell populations. CS and HA were found to alter ALP activity and increase cell proliferation. CS increased the formation of calcified nodules without the addition of osteogenic culture medium. This model has applications in the improvement of bioprosthetic valves by making replacements more micro-compositionally dynamic, as well as providing a platform for testing new pharmaceutical treatments of CAVD.
Bioprosthetic heart valves (BHVs) are important for transcatheter valve replacement. Current commercial BHVs on the market are basically porcine or bovine pericardium (BP) crosslinked with glutaraldehyde (GA). Simply applying GA to BHVs can enhance mechanical stability, but cannot alleviate in vivo calcification. In this work, we developed a two-step decellularization (TSD) strategy to modify this biomacromolecular network, in which BP was post-treated, as the second step of decellularization, with a mild biosurfactant n-dodecyl-β-D-maltoside in a mixture of isopropanol and phosphate-buffered saline after the first step of traditional decellularization and GA cross-linking. The TSD-treated BP exhibited not only low cytotoxicity and excellent mechanical properties in vitro, but also low immune responses and significant anticalcification in vivo. After 60 days of subcutaneous implantation in the back of Wistar rats, the calcium content was, as quantified with an inductively coupled plasma optical emission spectrometer, only 1.1 µg/mg compared to 138.6 µg/mg in the control group without the post-treatment. In addition, collagen fibrils were observed with field emitting scanning electron microscopy (SEM), and the morphology and composition of the calcified sites resulting from in vivo biomineralization were studied with SEM with energy dispersive spectroscopy and also X-ray diffraction. This study proposes a facile yet effective anticalcification strategy for the modification of the bovine pericardial bioprosthetic heart valve, a natural biomacromolecular network.
Bioprosthetic heart valve thrombosis (BHVT) is no longer considered a rare clinical entity in recent years and could cause valve motion abnormalities, which requires reoperation. Currently reported modifications of bioprosthetic heart valves (BHVs) mainly focused on improving the anti-calcification property. However, it was hard to improve their anti-thrombogenicity and crosslinking efficiency at the same time. Here we reported a method that combines improved hemocompatibility, crosslinking efficiency, cytocompatibility, and anti-calcification property for BHVs. Porcine pericardia (PPs) and 2-amino-4-pentanoic acid (APA) were first co-crosslinked with glutaraldehyde to introduce allyl moieties to the PPs, then poly(N, N-dimethylacrylamide) (PDMAA) was covalently immobilized on PPs through radical polymerization. In vitro and in vivo studies demonstrated that the structural stability of PDMAA modified PPs was much superior than that of traditional glutaraldehyde crosslinked PPs (GA). The PDMAA modified PPs exhibited enhanced antithrombogenic performance with a 75% decrease of platelet adhesion compared with GA. Moreover, the calcium content accumulated on the modified PPs after 90-day rat subcutaneous implantation decreased 93.5% compared with that on GA. The PDMAA modified PPs may alleviate BHVT and calcification, offering a potential option for BHVs fabrication in the future.
Valve replacement is the main therapy for valvular heart disease, in which a diseased valve is replaced by mechanical heart valve (MHV) or bioprosthetic heart valve (BHV). Since the 2000s, BHV surpassed MHV as the leading option of prosthetic valve substitute because of its excellent hemocompatible and hemodynamic properties. However, BHV is apt to structural valve degeneration (SVD), resulting in limited durability. Calcification is the most frequent presentation and the core pathophysiological process of SVD. Understanding the basic mechanisms of BHV calcification is an essential prerequisite to address the limited-durability issues. In this narrative review, we provide a comprehensive summary about the mechanisms of BHV calcification on 1) composition and site of calcifications; 2) material-associated mechanisms; 3) host-associated mechanisms, including immune response and foreign body reaction, oxidative stress, metabolic disorder, and thrombosis. Strategies that target these mechanisms may be explored for novel drug therapy to prevent or delay BHV calcification.
Heart valves have extraordinary fatigue resistance which beat ≈3 billion times in a lifetime. Bioprosthetic heart valves (BHVs) made from fixed heteroplasm that are incrementally used in heart valve replacement fail to sustain the expected durability due to thrombosis, poor endothelialization, inflammation, calcification, and especially mechanical damage induced biocompatibility change. No effective strategy has been reported to conserve the biological properties of BHV after long‐term fatigue test. Here, a double‐network tough hydrogel is introduced, which interpenetrate and anchor into the matrix of decellularized porcine pericardium (dCell‐PP) to form robust and stable conformal coatings and reduce immunogenicity. The ionic crosslinked hyaluronic acid (HA) network mimics the glycocalyx on endothelium which improves antithrombosis and accelerates endothelialization; the chemical crosslinked hydrophilic polyacrylamide (PAAm) network further enhances antifouling properties and strengthens the shielding hydrogels and their interaction with dCell‐PP. In vitro and rabbit ex vivo shunt assay demonstrate great hemocompatibility of polyacrylamide/HA hydrogel hybrid PP (P/H‐PP). Cell experiments and rat subcutaneous implantation confirm satisfactory endothelialization, biocompatibility, and anticalcification properties. For hydrodynamic experiment, P/H‐PP gains full mark at different flow conditions and sustains excellent biomechanical and biological properties after 200 000 000 cycles. P/H double‐network hydrogel armoring dCell‐PP is a promising progress to extend BHV durability for clinical implantation therapy.
Guided bone regeneration (GBR) is known to prevent the development of soft tissue on the defect sites as well as support the new bone formation on the other end. In the present study, we developed a multilayer biodegradable membrane for GBR applications. The multilayer membrane is primarily composed of β-tricalcium phosphate (TCP), polycaprolactone (PCL), and hyaluronic acid (HA), prepared by the spin-coating method. The triple layer system has PCL-TCP composite layer on top, a PCL layer in the middle, and PCL-HA as the bottom layer. The characterization of the PCL-TCP/PCL/PCL-HA by various techniques such as SEM, EDS, XRD, and FT-IR supported the uniform formation of the triple layers with an overall thickness of ∼72 µm. Multilayer composite membrane showed excellent physical parameters; neutral pH, high hydrophilicity, high swelling rate, low degradation rate, and high apatite formation after immersion in simulated body fluid (SBF) for 14 days. The multilayer membrane also exhibited biocompatibility which is evident by MTT assay and confocal images. The results suggested that the multilayer composite membrane has the potential for GBR applications.
Functional tissue engineered heart valves (TEHV) have been an elusive goal for nearly 30 years. Among the persistent challenges are the requirements for engineered valve leaflets that possess nonlinear elastic tissue biomechanical properties, support quiescent fibroblast phenotype, and resist osteogenic differentiation. Nanocellulose is an attractive tunable biological material that has not been employed to this application. In this study, we fabricated a series of photocrosslinkable composite hydrogels mNCC-MeGel (mNG) by conjugating TEMPO-modified nanocrystalline cellulose (mNCC) onto the backbone of methacrylated gelatin (MeGel). Their structures were characterized by FTIR, ¹HNMR and uniaxial compression testing. Human adipose-derived mesenchymal stem cells (HADMSC) were encapsulated within the material and evaluated for valve interstitial cell phenotypes over 14 days culture in both normal and osteogenic media. Compared to the MeGel control group, the HADMSC encapsulated within mNG showed decreased alpha smooth muscle actin (αSMA) expression and increased vimentin and aggrecan expression, suggesting the material supports a quiescent fibroblastic phenotype. Under osteogenic media conditions, HADMSC within mNG hydrogels showed lower expression of osteogenic genes, including Runx2 and osteocalcin, indicating resistance toward calcification. As a proof of principle, the mNG hydrogel, combined with a viscosity enhancing agent, was used to 3D bioprint a tall, self-standing tubular structure that sustained cell viability. Together, these results identify mNG as an attractive biomaterial for TEHV applications.
Injectable hydrogel possesses great application potential in disease treatment and tissue engineering, but damage to gel often occurs due to the squeezing pressure from injection devices and the mechanical forces from limb movement, and leads to the rapid degradation of gel matrix and the leakage of the load material. The self‐healing injectable hydrogels can overcome these drawbacks via automatically repairing gel structural defects and restoring gel function. The polysaccharide hydrogels constructed through the Schiff base reaction own advantages including simple fabrication, injectability, and self‐healing under physiological conditions, and therefore have drawn extensive attention and investigation recently. In this short review, the preparation and self‐healing properties of the polysaccharide hydrogels that is established on the Schiff base reaction are focused on and their biological applications in drug delivery and cell therapy are discussed. This review summarizes the recent advances in the injectable and self‐healing polysaccharide hydrogel based on the Schiff base reaction. It introduces the mechanism of their self‐repairing, their fabrication and their biomedical application in the delivery of drug and cell. The focus is on the modification strategies and methods of the polysaccharides applied in gel preparation.
The book describe the main cardiovascular biomaterials, biological and mechanical heart valve prostheses.
Also, the main surgical techniques used in valvular heart surgery are presented.
Bioprosthetic materials based on mammalian pericardium tissue are the gold standard in reconstructive surgery. Their application range covers repair of rectovaginal septum defects, abdominoplastics, urethroplasty, duraplastics, maxillofacial, ophthalmic, thoracic and cardiovascular reconstruction, etc. However, a number of factors contribute to the success of their integration into the host tissue including structural organization, mechanical strength, biocompatibility, immunogenicity, surface chemistry, and biodegradability. In order to improve the material's properties, various strategies are developed, such as decellularization, crosslinking, and detoxification. In this review, the existing issues and long-term achievements in the development of bioprosthetic materials based on the mammalian pericardium tissue, aimed at a wide-spectrum application in reconstructive surgery are analyzed. The basic technical approaches to preparation of biocompatible forms providing continuous functioning, optimization of biomechanical and functional properties, and clinical applicability are described.
Abstract
The review describes the milestones in the development of bioprosthetic materials based on mammalian pericardium tissue. The basic technical approaches to preparation of biocompatible forms including decellularization, crosslinking, detoxification, and application range of pericardial biomeshes in reconstructive surgery are discussed.
The lack of pharmaceutical targets available to treat patients with calcific aortic valve disease (CAVD) necessitates further research into the specific mechanisms of the disease. The significant changes that occur to the aortic valves extracellular matrix (ECM) during the progression of CAVD suggests that these proteins may play an important role in calcification. Exploring the relationship between valve interstitial cells (VICs) and the ECM may lead to a better understand of CAVD mechanisms and potential pharmaceutical targets. In this study, we look at the effect of two ECM components, collagen and hyaluronic acid (HA), on the mineralization of VICs within the context of a two-dimensional, polyacrylamide (PAAM) model system. Using a novel, nondestructive imaging technique, we were able to track calcific nodule development in culture systems over a 3-wk time frame. We saw a significant increase in the size of the nodules grown on HA PAAM gels as compared with collagen PAAM gels, suggesting that HA has a direct effect on mineralization. Directly looking at the two known receptors of HA, CD44 and receptor for HA-mediated motility (RHAMM), and using siRNA knockdown revealed that a decrease in CD44 expression resulted in a reduction of calcification. A decrease in CD44, through siRNA knockdown, reduces mineralization on HA PAAM gels, suggesting a potential new target for CAVD treatment.
NEW & NOTEWORTHY Our in vitro model of calcific aortic valve disease shows an interaction between the hyaluronic acid binding protein CD44 with the osteogenic factor OPN as a potential mechanism of aortic valve calcification. Using siRNA knockdown of CD44, we show an upregulation of OPN expression with a decrease in overall mineralization.
Products made of biomaterials, such as heart valve prostheses, vascular grafts, and patches for vascular and intracardiac plastics, are currently used in cardiovascular surgery. The biological tissue used for prosthetics is the alternation of transverse and longitudinal layers of collagen fibers consisting of type I collagen (75%), elastin (<5%), cell elements, as well as glycoproteins, glycosaminoglycans, and other components of the cell matrix. Chemical modifications of components of a biological tissue allow for retention of its natural architectonics and stability of collagen structure over time, while simultaneously increasing the collagen resistance to enzymatic and mechanical destruction and preventing cellular and immune effects on the part of the recipient organism. Proteins in biological tissues are chemically modified (preserved) by the formation of intramolecular and intermolecular cross-links between the amino groups of amino acid residues in collagen molecules. However, cross-linking increases the calcification of biomaterial, making the tissue more rigid and leading to the rupture of the valve flaps, stenosis (reduced clearance), or insufficiency (a decrease in the closure function) of the heart valves. Calcification can also result from specific physiological features of recipient (the patient who received the artificial organ), the nature of the preserving agent, components of the dead cells, defects of collagen structure, cavities in tissues, and the presence of lipids, elastin fibers, glycosaminoglycans, and so on. The factors that induce calcification of the materials used for prosthetic repair and the corresponding methods for its prevention are reviewed. All methods are conventionally divided into three groups: chemical pretreatment of tissues, modification of the preservation method, and posttreatment of preserved tissues with chemical agents. The mechanisms of the processes underlying the effect of chemical agents on the structures of biological tissues are described. The results of their use in clinical practice and prospects for methods still under development and in preclinical trials are discussed, as well as the reasons why some methods have failed. The advantages and disadvantages of various types of treatments are considered. Variants of new methods for chemical modification of biological materials potentially effective in reducing the risk of calcification are proposed.
Rapidly increasing malignant neoplastic disease demands immediate attention. Several dietary compounds have recently emerged as strong anti-cancerous agents. Amongst, Bromelain (BL), a protease from pineapple plant, was used to enhance its anti-cancerous efficacy using nanotechnology. In lieu of this, hyaluronic acid (HA) grafted PLGA copolymer, having tumor targeting ability, was developed. BL was encapsulated in copolymer to obtain BL-copolymer nanoparticles (NPs) that ranged between 140 to 281nm in size. NPs exhibited higher cellular uptake and cytotoxicity in cells with high CD44 expression as compared with non-targeted NPs. In vivo results on tumor bearing mice showed that NPs were efficient in suppressing the tumor growth. Hence, the formulation could be used as a self-targeting drug delivery cargo for the remission of cancer.
Often in composite biomaterials, molecular interactions at various interfaces are known to have significant role on mechanical response of the composite system as well as biocompatibility of the biomaterials. The biomaterial surface elicits a response from tissue that is specific to the nature of the surface and several surface modification techniques are used to analyze the response. Currently, many physical and spectroscopic methods are available to characterize the nature of the biomaterial surface. This Chapter introduces various characterization techniques for characterizing biointerfaces and biosurfaces in biomaterials Design. The important characterization tools used by biomaterials researchers are outlined in the chapter and the fundamental principles governing these tools are elaborated. © 2009 Tsinghua University Press, Beijing and Springer-Verlag GmbH Berlin Heidelberg.
Several polymers of both natural and synthetic origin have been used for a variety of biomedical applications including pharmaceutical preparations, drug targeting, imaging, drug delivery, prosthetics, and tissue engineering scaffolds. Due to their reproducible characteristics in terms of their molecular weight, degradation and mechanical properties, synthetic polymers are attractive for a variety of the aforementioned applications. However, synthetic polymers from the biological standpoint, synthetic polymers often lack much-desired bioactivity and biocompatibility, which may translate into adverse side effects. Natural polymers on the other hand are abundant and resemble the components present in biological extracellular matrices. Thus, natural polymers are readily accepted by the body and possess high bioactivity and biocompatibility. Natural polymers can be divided into three major classes according to their chemical structure: (i) polysaccharides, (ii) proteins, and (iii) polyesters. This chapter presents an overview of the polysaccharide-based biomaterials, their structure property, and applications in the area of drug delivery and tissue engineering. Particular emphasis is given to polysaccharides such as (i) hyaluronic acid (HA), (ii) chondroitin sulfate, (iii) chitin and chitosan, (iv) alginates, and (v) cellulose. These polymers and their popular derivatives are also discussed in the context of their chemical and biological properties. Polysaccharides in their native form may not be able to provide all the desired properties for a particular biomedical application. Thus, the chapter also focuses on the polysaccharide derivatives and their blend with other polymers for a variety of biomedical applications.
Acellular bovine pericardium was treated with modified hyaluronic acid (HA-ADH) after glutaraldehyde fixation. The acellularization effect was demonstrated by H&E staining and SEM. The thermal stabilities of BP samples were characterized by measuring shrinkage temperature with DSC, and no significant deference was found between glutaraldehyde fixation bovine pericardium (GFBP) and glutaraldehyde fixation bovine pericardium treated with HA-ADH (GHBP). In vivo calcification tests of GHBP and GFBP were performed by implanted subdermally in male Wistar rats. The efficacy of GHBP for prevention of calcification was evaluated by SEM and ICP, and the results demonstrated that much less calcium deposition on GHBP than GFBP (0.2539±0.0771 comparing to 2.4371±0.54738 μgCa/ mg dry tissue after 8 weeks implantation). In addition, GHBP groups not only present a lower calcification degree, but also show lower Ca/P molar ratios, which correspond to amorphous calcium phosphates. The obtained results indicate that GHBP is a potential candidate for cardiac valve fabrication, since it can protecting them against calcification, and therefore, increase valve durability.
Periodontitis is an inflammatory disease of the gums which spreads and affects the supporting tooth structures possibly leading to the loosening and loss of the tooth. Periodontal tissue engineering is considered a relatively new technique for the stimulation of the periodontal tissue formation using the basics of regenerative medicine. In this method, biodegradable porous scaffolds are employed as a temporary substitution of the injured or lost tissues to facilitate the regeneration process. Scaffolds are usually made of natural or synthetic polymers and ceramics doped with various nanobiomaterials for an intended functionalization. The addition of nanoparticles into the scaffold structure not only enhances the biomineralization potential of the composite scaffolds, but also improves their mechanical properties. Nanosized ceramic particles are of special importance as they mimic the mineral crystal structure of the natural tissues. They have been demonstrated to induce a considerable enhancement in the protein absorption and the cell adhesion compared to their micro-sized counterparts. This chapter reviews different nanobiomaterials employed in periodontal tissue engineering for the effective regeneration of lost tissues and discuss their benefits and drawbacks.
The objective of this work was to evaluate the effect of decellularization and hyaluronic acid derivative on the improvement of anticalcification of glutaraldehyde fixed bovine pericardium (GFBP) using a rat subcutaneous implantation model A cell extraction process was employed to remove the cells and cellular components from bovine pericardium (BP), leaving a framework of largely insoluble collagen. Then acellular BP was cross-linked by glutaraldehyde solution and treated with hyaluronic acid derivative (HA-ADH) which was obtained by coupling adipic dihydrazide (ADH) on-COOH of hyaluronic acid (HA). The results of in vivo calcification tests showed that the calcium content was decreased dramatically by decellularization alone (from 28.07±18.87 to 2.44±0.55μg Ca/mg dry tissue after 8 weeks' implantation), and even less concentration was shown by the combination of HA derivative treatment and decellularization (GFaBP-HA group) (0.25±0.08μg Ca/mg dry tissue after 8 weeks' implantation). In addition, GFaBP-HA group not only presented a lower degree of calcification, but also showed lower ratios of Ca/P molar, which corresponded to amorphous calcium phosphates. The obtained results indicated that GFaBP-HA was a potential candidate for the manufacture of anticalcification bioprostheses.
This work has investigated the in vitro calcification of bovine pericardium (BP) treated with chitosan (C), silk fibroin (SF) and electron beam irradiation after its endothelization in vitro. For this purpose, freeze-dried BP membranes treated with mixtures of C and SF (1:3, 1:1 and 3:1) and then irradiated by electron beam irradiation were seeded with human umbilical vein endothelial cells (HUVEC) in vitro. After 3 weeks of cultivation these membranes were submitted to in vitro calcification tests using simulated body fluid as the calcifying agent. Control membranes were also studied (without endothelial cells exposure). The results have shown that the membrane compatibility with HUVECs in vitro prevent such biomaterial from calcifying, showing a potential application in biomaterial area, such as cardiac valves and repair patches.
Viscosity, asymmetric flow field-flow fractionation (AFFFF) methods, and dynamic light scattering (DLS) experiments were used to characterize the effect of pH on the behavior of dilute and semidilute aqueous buffered solutions of hyaluronic acid (HA). It is shown that degradation of HA occurs at pH < 4 and pH > 11, and in the domain 4 < pH < 11 virtually no disruption of the HA chains occurs. The pH-induced scission of HA is attributed to the cleavage of glycosidic bonds. In dilute solutions, intramolecular rupture of HA chains occurs and in the semidilute concentration regime network-fragmentation is observed at low and high pH values. The degree of degradation of HA is most marked at high pH. From the molecular weight and radius of gyration obtained from AFFFF at different pH values, it is clear that the degradation of HA starts at early times after preparation of the solution, and continues for a couple of days. The kinetics of degradation of HA is fastest at high pH.
Surface modification of glutaraldehyde fixed bovine pericardium (GFBP) was successfully carried out with hyaluronic acid (HA)
derivatives. At first, HA was chemically modified with adipic dihydrazide (ADH) to introduce hydrazide functional group into
the carboxyl group of HA backbone. Then, GFBP was surface modified by grafting HA-ADH to the free aldehyde groups on the tissue
and the subsequent HA-ADH hydrogel coating. HA-ADH hydrogels could be prepared through selective crosslinking at low pH between
hydrazide groups of HA-ADH and crosslinkers containing succinimmidyl moieties with minimized protein denaturation. When HA-ADH
hydrogels were prepared at low pH of 4.8 in the presence of erythropoietin (EPO) as a model protein, EPO release was continued
up to 85% of total amount of loaded EPO for 4 days. To the contrary, only 30% of EPO was released from HA-ADH hydrogels prepared
at pH=7.4, which might be due to the denaturation of EPO during the crosslinking reaction. Because the carboxyl groups on
the glucuronic acid residues are recognition sites for HA degradation by hyaluronidase, the HA-ADH hydrogels degraded more
slowly than HA hydrogels prepared by the crosslinking reaction of divinyl sulfone with hydroxyl groups of HA. Following a
two-week subcutaneous implantation in osteopontin-null mice, clinically significant levels of calcification were observed
for the positive controls without any surface modification. However, the calcification of surface modified GFBP with HA-ADH
and HA-ADH hydrogels was drastically reduced by more than 85% of the positive controls. The anti-calcification effect of HA
surface modification was also confirmed by microscopic analysis of explan ted tissue after staining with Alizarin Red S for
calcium, which followed the trend as observed with calcium quantification.
Hyaluronic acid (HA) is a biodegradable, biocompatible, non-toxic, non-immunogenic and non-inflammatory linear polysaccharide, which has been used for various medical applications such as arthritis treatment, ocular surgery, tissue augmentation, and so on. In this review, the effect of chemical modification of HA on its distribution throughout the body was reported for target specific and long-acting delivery applications of protein, peptide, and nucleotide therapeutics. According to the real-time bio-imaging of HA derivatives using quantum dots (QDot), HA-QDot conjugates with 35mol% HA modification maintaining enough binding sites for HA receptors were mainly accumulated in the liver, while those with 68mol% HA modification losing much of HA characteristics were evenly distributed to the tissues in the body. The results are well matched with the fact that HA receptors are abundantly present in the liver with a high specificity to HA molecules. Accordingly, slightly modified HA derivatives were used for target specific intracellular delivery of nucleotide therapeutics and highly modified HA derivatives were used for long-acting conjugation of peptide and protein therapeutics. HA has been also used as a novel depot system in the forms of physically and chemically crosslinked hydrogels for various protein drug delivery. This review will give you a peer overview on novel HA derivatives and the latest advances in HA-based drug delivery systems of various biopharmaceuticals for further clinical development.
Grafts of biological tissues have been used since the 1960s as an alternative to the mechanical heart prostheses. Nowadays, the most consolidated treatment to bovine pericardial (BP) bioprostheses is the crosslinking with glutaraldehyde (GA), although GA may induce calcification in vivo. In previous work, our group demonstrated that electron beam irradiation applied to lyophilized BP in the absence of oxygen promoted crosslinks among collagen fibers of BP tissue. In this work, the incorporation of silk fibroin (SF) and chitosan (CHIT) in the BP not treated with GA was studied. The samples were irradiated and then analyzed for their cytotoxicity and the ability of adhesion and growth of endothelial cells. Initially, all samples showed cytotoxicity. However, after a few washing cycles, the cytotoxicity due to acetic acid and ethanol residues was removed from the biomaterial making it suitable for the biofunctional test. The samples modified with SF/CHIT and electron beam irradiated favored the adhesion and growth of endothelial cells throughout the tissue.
A proposal is herein examined for a novel yet simple design of a polymeric nanogel, with tumor targeting properties and a controllable phototoxicity, utilizing a low molecular weight-hyaluronic acid (HA(LM))/photosensitizer conjugate. HA(LM) was acetylated prior to being dissolved in DMSO (Ac-HA(LM)) and then was conjugated with different amounts of pheophorbide a (Pba), resulting in the formulation of self-organizing nanogels in aqueous solutions (Ac-HA-Pba 1, 2, and 3). The nanogels observed were below 200 nm in size, with a monodispersed size distribution. The nanogels displayed auto photoquenching qualities in PBS, while their fluorescent intensity strongly correlated with the amount of Pba in the organic solvent (DMSO or DMF). The critical self-quenching concentration (CQC) of the conjugates was found to have decreased as the content of Pba rose. Although Pba was conjugated with HA, the nanogel's photoactivity, in terms of fluorescent properties, singlet oxygen generation, and photocytotoxicity, was approximately maintained. Confocal imaging and FACS analysis showed that Ac-HA-Pba nanogels were rapidly internalized into HeLa cells via an HA-induced endocytosis mechanism, a process which could be blocked with the application of an excess of HA polymer. The results of the study indicate that HA-based nanogels can potentially be applied in photodynamic therapy (PDT).
Although silicones possess many useful properties as biomaterials, their hydrophobicity can be problematic. To a degree, this issue can be addressed by surface modification with hydrophilic polymers such as poly(ethylene glycol), but the resulting structures are usually not conducive to cell growth. In the present work, we describe the synthesis and characterization of covalently linked hyaluronic acid (HA) (35 kDa) to poly(dimethylsiloxane) (PDMS) elastomer surfaces. HA is of interest because of its known biological properties; its presence on a surface was expected to improve the biocompatibility of silicone materials for a wide range of bioapplications. HA was introduced with a coupling agent in two steps from high-density, tosyl-modified, poly(ethylene glycol) tethered silicone surfaces. All materials synthesized were characterized by water contact angle, ATR-FTIR, XPS and (13)C solid state NMR spectroscopy. Biological interactions with these modified silicone surfaces were assessed by examining interactions with fibrinogen as a model protein as well as determining the in vitro response of fibroblast (3T3) and human corneal epithelial cells relative to unmodified poly(dimethylsiloxane) controls. The results suggest that HA modification significantly enhances cell interactions while decreasing protein adsorption and may therefore be effective for improving biocompatibility of PDMS and other materials.
A novel, biocompatible, and nontoxic dermal filler using hyaluronic acid (HA) hydrogels was successfully developed for tissue augmentation applications. Instead of using highly reactive cross-linkers such as divinyl sulfone (DVS) for Hylaform, 1,4-butanediol diglycidyl ether (BDDE) for Restylane, and 1,2,7,8-diepoxyoctane (DEO) for Puragen, HA hydrogels were prepared by direct amide bond formation between the carboxyl groups of HA and hexamethylenediamine (HMDA) with an optimized carboxyl group modification for effective tissue augmentation. The HA-HMDA hydrogels could be prepared within 5 min by the addition of HMDA to HA solution activated with 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDC) and 1-hydroxybenzotriazole monohydrate (HOBt). Five kinds of samples, a normal control, a negative control, a positive control of Restylane, adipic acid dihydrazide grafted HA (HA-ADH) hydrogels, and HA-HMDA hydrogels, were subcutaneously injected to wrinkled model mice. According to the image analysis on dorsal skin augmentation, the HA-HMDA hydrogels exhibited the best tissue augmentation effect being stable longer than 3 months. Furthermore, histological analyses after hematoxylin-eosin (H&E) and Masson's trichrome staining revealed the excellent biocompatibility and safety of HA-HMDA hydrogels. The dermal thickness and the dermal collagen density in wrinkled mice after treatment with HA-HMDA hydrogels for 12 weeks were comparable to those of normal mice. Compared with HA-DVS hydrogels and Restylane, the excellent tissue augmentation by HA-HMDA hydrogels might be ascribed to the biocompatible residues of amine groups in the cross-linker of HMDA. The HA-HMDA hydrogels will be investigated further as a novel dermal filler for clinical applications.
In recent years, a variety of devices (drug-eluting stents, artificial organs, biosensors, catheters, scaffolds for tissue engineering, heart valves, etc.) have been developed for implantation into patients. However, when such devices are implanted into the body, the body can react to these in a number of different ways. These reactions can result in an unexpected risk for patients. Therefore, it is important to assess and optimize the biocompatibility of implantable devices. To date, numerous strategies have been investigated to overcome body reactions induced by the implantation of devices. This review focuses on the foreign body response and the approaches that have been taken to overcome this. The biological response following device implantation and the methods for biocompatibility evaluation are summarized. Then the risks of implantable devices and the challenges to overcome these problems are introduced. Specifically, the challenges used to overcome the functional loss of glucose sensors, restenosis after stent implantation, and calcification induced by implantable devices are discussed.
Osteopontin (OPN) is an arginine-glycine-aspartate (RGD)- containing glycoprotein encoded by the gene secreted phosphoprotein 1 (spp1). spp1 is expressed during embryogenesis, wound healing, and tumorigenesis; however, its in vivo functions are not well understood. Therefore, OPN null mutant mice were generated by targeted mutagenesis in embryonic stem cells. In OPN mutant mice, embryogenesis occurred normally, and mice were fertile. Since OPN shares receptors with vitronectin (VN), we tested for compensation by creating mice lacking both OPN and VN. The double mutants were also viable, suggesting that other RGD-containing ligands replace the embryonic loss of both proteins. We tested the healing of OPN mutants after skin incisions, where spp1 was upregulated as early as 6 h after wounding. Although the tensile properties of the wounds were unchanged, ultrastructural analysis showed a significantly decreased level of debridement, greater disorganization of matrix, and an alteration of collagen fibrillogenesis leading to small diameter collagen fibrils in the OPN mutant mice. These data indicate a role for OPN in tissue remodeling in vivo, and suggest physiological functions during matrix reorganization after injury.
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.
The relaxation rate R = pi Delta nu(1/2) of the quadrupolar (23)Na nucleus was measured at pH approximately 7 using a 200 MHz NMR spectrometer with a view to observe the interaction between hyaluronan and its natural counterion Na(+) and the bications Ca(++), Mg(++) and Cu(++). An interpretation of our results, by means of the "entropy of fluctuations" concept of Na(+), is presented. We show that Cu(++) ions are more effective than Ca(++) and Mg(++). A possible model of complexation of Cu(++) in a cage formed by the 1-4 glycosidic bond, the carboxylate side-chain and the acetoamide side-chain is proposed, according to electrostatic potential computations using the ZINDO1 quantum semi empirical method.
Bioconjugate Techniques, 2e provides highly detailed information on the chemistry, reagent systems, and practical applications for creating labeled or conjugate molecules. It also describes dozens of reactions with details on hundreds of commercially available reagents and the use of these reagents for modifying or crosslinking peptides and proteins, sugars and polysaccharides, nucleic acids and oligonucleotides, lipids, and synthetic polymers. *A one-stop source for proven methods and protocols for synthesizing bioconjugates in the lab, allowing for fast retrieval of tried and tested techniques for synthesizing bioconjugates. *Step-by-step presentation makes the book an ideal source for researchers who are less familiar with the synthesis of bioconjugates. *Includes more than 600 figures that visually describe the complex reactions associated with the synthesis of bioconjugates, giving researchers a better understanding of how the commercially available reagents facilitate the crosslinking of peptides and proteins, sugars and polysaccharides, nucleic acids and oligonucleotides, lipids, and synthetic polymers. *Includes entirely new chapters on the latest areas in the field of bioconjugation as follows: Microparticles and nanoparticles Silane coupling agents Dendrimers and dendrons Chemoselective ligation Quantum dots Lanthanide chelates Cyanine dyes Discrete PEG compounds Buckyballs, fullerenes, and carbon nanotubes Mass tags and isotope tags Bioconjugation in the study of protein interactions.
Ectopic calcification is defined as inappropriate biomineralization occurring in soft tissues. 1 Ectopic calcifications are typically composed of calcium phosphate salts, including hydroxyapatite, but can also consist of calcium oxalates and octacalcium phosphate as seen in kidney stones. 2 In uremic patients, a systemic mineral imbalance is associated with widespread ectopic calcification, referred to as metastatic calcification. 3 In the absence of a systemic mineral imbalance, ectopic calcification is typically termed dystrophic calcification. Often, these sites show evidence of tissue alteration and/or necrosis. Dystrophic mineralization is commonly observed in soft tissues as a result of injury, disease, and aging. Although most soft tissues can undergo calcification, skin, kidney, tendons, and cardiovascular tissues appear particularly prone to developing this pathology. 4 In addition, a number of prosthetic devices are prone to ectopic calcification, as discussed below. Recent insights into the mechanisms regulating ectopic calcification have come from studies of cardiovascular calcification, including that by Kim et al 5 in this issue of the Journal, and thus will be the major focus of this article. The reader is referred to other reviews for information about additional tissue-specific ectopic calcifications. 2,6,7
Substitute heart valves composed of human or animal tissues have been used since the early 1960s, when aortic valves obtained fresh from human cadavers were transplanted to other individuals as allografts. Today, tissue valves are used in 40% or more of valve replacements worldwide, predominantly as stented porcine aortic valves (PAV) and bovine pericardial valves (BPV) preserved by glutaraldehyde (GLUT) (collectively termed bioprostheses). The principal disadvantage of tissue valves is progressive calcific and noncalcific deterioration, limiting durability. Native heart valves (typified by the aortic valve) are cellular and layered, with regional specializations of the extracellular matrix (ECM). These elements facilitate marked repetitive changes in shape and dimension throughout the cardiac cycle, effective stress transfer to the adjacent aortic wall, and ongoing repair of injury incurred during normal function. Although GLUT bioprostheses mimic natural aortic valve structure (a) their cells are nonviable and thereby incapable of normal turnover or remodeling ECM proteins; (b) their cuspal microstructure is locked into a configuration which is at best characteristic of one phase of the cardiac cycle (usually diastole); and (c) their mechanical properties are markedly different from those of natural aortic valve cusps. Consequently, tissue valves suffer a high rate of progressive and age-dependent structural valve deterioration resulting in stenosis or regurgitation (>50% of PAV overall fail within 10–15 years; the failure rate is nearly 100% in 5 years in those <35 years old but only 10% in 10 years in those >65). Two distinct processes—intrinsic calcification and noncalcific degradation of the ECM—account for structural valve deterioration. Calcification is a direct consequence of the inability of the nonviable cells of the GLUT-preserved tissue to maintain normally low intracellular calcium. Consequently, nucleation of calcium-phosphate crystals occurs at the phospholipid-rich membranes and their remnants. Collagen and elastin also calcify. Tissue valve mineralization has complex host, implant, and mechanical determinants. Noncalcific degradation in the absence of physiological repair mechanisms of the valvular structural matrix is increasingly being appreciated as a critical yet independent mechanism of valve deterioration. These degradation mechanisms are largely rationalized on the basis of the changes to natural valves when they are fabricated into a tissue valve (mentioned above), and the subsequent interactions with the physiologic environment that are induced following implantation. The “Holy Grail” is a nonobstructive, nonthrombogenic tissue valve which will last the lifetime of the patient (and potentially grow in maturing recipients). There is considerable activity in basic research, industrial development, and clinical investigation to improve tissue valves. Particularly exciting in concept, yet early in practice is tissue engineering, a technique in which an anatomically appropriate construct containing cells seeded on a resorbable scaffold is fabricated in vitro, then implanted. Remodeling in vivo, stimulated and guided by appropriate biological signals incorporated into the construct, is intended to recapitulate normal functional architecture. © 1999 John Wiley & Sons, Inc. J Biomed Mater Res, 47, 439–465, 1999.
Bioprosthetic heart valves (BPHVs) derived from glutaraldehyde-crosslinked porcine aortic valves are frequently used in heart valve replacement surgeries. However, the majority of bioprostheses fail clinically because of calcification and degeneration. We have recently shown that glycosaminoglycan (GAG) loss may be in part responsible for degeneration of glutaraldehyde-crosslinked bioprostheses. In the present studies, we used a mild reaction of periodate-mediated crosslinking to stabilize glycosaminoglycans in the bioprosthetic tissue. We demonstrate the feasibility of periodate reaction by crosslinking major components of extracellular matrix of bioprosthetic heart valve tissue, namely type I collagen and hyaluronic acid (HA). Uronic acid assay of periodate-fixed HA-collagen matrices showed 48% of HA disaccharides were bound to collagen. Furthermore, we show that such reactions are also feasible to fix glycosaminoglycans present in the middle spongiosa layer of bioprosthetic heart valves. The periodate reactions were compatible with conventional glutaraldehyde crosslinking and showed adequate stabilization of extracellular matrix as demonstrated by thermal denaturation temperature and collagenase assays. Moreover, uronic acid assays of periodate-fixed BPHV cusps showed 36% reduction in the amount of unbound GAG disaccharides as compared with glutaraldehyde-crosslinked cusps. We also demonstrate that calcification of BPHV cusps was significantly reduced in the periodate-fixed group as compared with the glutaraldehyde-fixed group in 21-day rat subdermal calcification studies (periodate-fixed tissue Ca 72.01 ± 5.97 μg/mg, glutaraldehyde-fixed tissue Ca 107.25 ± 6.56 μg/mg). We conclude that periodate-mediated GAG fixation could reduce structural degeneration of BPHVs and may therefore increase the useful lifetime of these devices. © 2001 John Wiley & Sons, Inc. J Biomed Mater Res 56: 478–486, 2001
Background:
Glutaraldehyde has been said to be responsible in part for the calcification of glutaraldehyde-treated tissues after implantation in animals or humans. We investigated whether the origin of the tissue, autologous or heterologous, could have a more prominent role in the process of calcification.
Methods:
Three-month-old sheep received sheep pericardial samples (n = 133) and human pericardial samples (n = 123) implanted subcutaneously. Samples were treated with 0.6% glutaraldehyde for 5, 10, or 20 minutes or 7 days and then rinsed thoroughly before implantation. Samples were then retrieved after 3 months. Calcium content was assessed by spectrophometry.
Results:
The results show a low calcium content in the autologous group (mean 1.14+/-2.07) and a high calcium content in the heterologous group (mean 38.97+/-26). These results were the same regardless of the duration of the treatment.
Conclusions:
Glutaraldehyde treatment (0.6%) does not play a significant role in the calcification of glutaraldehyde-treated tissue regardless of the origin, autologous or heterologous, of the tissue. Glutaraldehyde-treated autologous tissues are associated with an incidence of calcification lower than heterologous tissues.
Calcification (CALC) is the most frequent cause of failure in bioprosthetic heart valves (BHV) fabricated from glutaraldehyde pretreated porcine aortic valve or bovine pericardium. Site specific controlled administration of ethanehydroxy diphosphonate (EHDP), using numerous carriers, has been successful in inhibiting CALC of BHV tissue in an accelerated rat subdermal model, without adverse effects on serum calcium, bone development, or overall somatic growth. The current study was designed to evaluate refillable reservoir devices fabricated from polyurethane (Biomer Woburn, MA) with regard to their transport properties relative to EHDP. The refillable reservoirs were evaluated for EHDP release in vitro at 22 degrees C (under perfect sink conditions) into both a physiologic receptor phase (N-2 hydroxyethylpiperazine-N'-2-ethanesulfonic acid buffer, pH = 7.4) with (1.5 mM) or without Ca2+ present. Transport parameters for EHDP diffusion through the polyurethane membranes used to fabricate the reservoirs were significantly different for the two receptor phases evaluated. The mean diffusion coefficients (D) for EHDP through the polyurethane membrane into each receptor phase were not significantly different. However, an approximately 3.5-fold reduction was observed in the mean value of the partition coefficient (K) for EHDP when EHDP was evaluated for release into a receptor phase that contained 1.5 mM Ca2+.(ABSTRACT TRUNCATED AT 250 WORDS)
Hyaluronic acid (HA) is a linear polysaccharide with repeating disaccharide units of glucuronic acid and N-acetylglucosamine and is found in the extracellular matrix of connective tissues. Reaction of high molecular weight sodium hyaluronate (NaHA, MW approximately 2 x 10(6] with EDC at pH 4.75, either in the presence or absence of a primary diamine, gave the N-acylurea and O-acylisourea as NaHA-carbodiimide adducts. None of the expected intermolecular coupling with the amine component was observed. On the basis of this new observation, this method for chemical modification of HA was used in conjunction with new synthetic carbodiimides to prepare HA derivatives bearing lipophilic, aromatic, cross-linked, and tethered functional groups. The degree of conversion to NaHA-acylurea products appears to depend upon both the characteristics of various carbodiimides and the conformational structure of NaHA.
Study of the calcification of cartilage in endochondral ossification has yielded two apparently contradictory views of the role of proteoglycan in this process. The ability of proteoglycan to act as a calcium-concentrating agent (Kalksalzfänger) in cartilage is consistent with the view that proteoglycans are promoters of calcification. However, study of their effect on hydroxyapatite formation in vitro suggests that proteoglycans are inhibitors of cartilage calcification. A resolution of this paradox is now proposed. Proteoglycans inhibit hydroxyapatite formation under in vitro conditions of limited calcium availability (in part) by binding calcium ions. However, under in vivo conditions of essentially infinite calcium availability, proteoglycans may promote hydroxyapatite formation, since binding of calcium to proteoglycan will not decrease the free calcium concentration, and the bound calcium may easily be displaced. Therefore, it is proposed that the role of proteoglycans in the calcification of cartilage is to function as a cation-exchanging calcium reservoir.
Calcification is the principal cause of the clinical failure of bioprosthetic heart valves fabricated from glutaraldehyde pretreated porcine aortic valves or bovine pericardium. The present study investigated controlled-release implants for prevention of the calcification of glutaraldehyde pretreated bovine pericardium in a rat subdermal model. Either Al3+ and Fe3+ (inhibitors of the growth and dissolution rate of hydroxyapatite crystals), levamisole (alkaline phosphatase inhibitor) or protamine sulphate (charge modifier) were individually incorporated into various polymeric carriers (either silicone rubber, polyurethane or silicone rubber-polyurethane copolymer). Polymeric implants were evaluated for in vitro release kinetics, which revealed that sustained drug release was obtained from 21 d to more than 90 d from various drug matrices. In vivo efficacy was studied by co-implanting the polymeric delivery systems with glutaraldehyde pretreated bovine pericardium for 21 d using a subdermal rat model; glutaraldehyde pretreated bovine pericardium calcium levels were quantitated by atomic absorption spectroscopy in the explanted tissues. Fe3+ and Al3+ polymeric implants were the most effective for inhibiting deposition of calcium mineral. Al3+ demonstrated 82% inhibition of calcification compared to controls and Fe3+ resulted in 80% inhibition of calcification. Specific histologic staining methods showed that Fe3+ and Al3+ were localized within the devitalized cells of the explanted glutaraldehyde pretreated bovine pericardium. No adverse effects on somatic growth or recipient bone morphology were noted following controlled-release drug administration. Controlled release of protamine sulphate or levamisole did not significantly inhibit glutaraldehyde pretreated bovine pericardium calcification. It is concluded that regional controlled release of Fe3+ or Al3+ inhibits glutaraldehyde pretreated bovine pericardium calcification in the rat subdermal model without adverse effects.
The provisional calcification of epiphyseal cartilage involves deposition of hydroxyapatite (calcium phosphate) crystals in an extracellular matrix consisting principally of Type II collagen and cartilage proteoglycan. A mechanism is now proposed to explain how epiphyseal cartilage calcification is initiated. Calcium exists at high concentration in cartilage, but is mainly bound to the anionic groups of proteoglycans, and thus is unavailable for precipitation. A local increase in phosphate concentration displaces calcium ions from proteoglycan by an ion-exchange effect, raising the Ca X PO4 product above the threshold for precipitation of hydroxyapatite. Evidence for this hypothesis has been derived from studies of the effect of phosphate on the binding of calcium to cartilage proteoglycan, and on hydroxyapatite formation in the presence of chondroitin sulfate.
Binding of calcium to the glycosaminoglycans (GAGs) heparin, chondroitin sulfate (CS), keratan sulfate (KS), and hyaluronic acid (HA) has been studied by equilibrium dialysis using exclusion of sulfate to correct for Gibbs-Donnan effects. Calcium binding occurs to all of these GAG species, suggesting that both sulfate and carboxylate groups are involved in cation binding. For all GAGs, the binding stoichiometry is consistent with a calcium-binding "site" consisting of two anionic groups. The order of calcium binding affinities is heparin greater than CS greater than KS greater than HA, and is critically dependent upon charge density; heparin binds calcium with 10-fold higher affinity than CS. The mode of calcium binding to GAGs is consistent with a recently proposed mechanism of growth plate calcification which states that cartilage proteoglycan functions as a reservoir of calcium for calcification of epiphyseal cartilage.
Calcification (CALC) is the most frequent cause of the clinical failure of bioprosthetic heart valves (BHVs). Controlled release of disodium ethanehydroxydiphosphonate (EHDP) has been demonstrated to inhibit subdermal BHV calcification at effective low local doses, avoiding adverse effects. However, the eventual circulatory use of controlled release EHDP necessitates addressing several critical issues that may affect efficacy. These include the effects of sterilization on EHDP release and the efficacy of sustained release matrices containing CaEHDP, which is less soluble than NaEHDP. The effects of CaEHDP-NaEHDP incorporation and steam sterilization on controlled release of EHDP from silicone-rubber matrices was studied both in vitro and in vivo using a rat subdermal model and sheep tricuspid valve replacements. Autoclaved EHDP matrices (20% wt/wt) released 88.9% +/- 7.84 of contained drug after 140 days in vitro, compared with control (87.6% +/- 10.3 cumulative release). Autoclaved EHDP matrices completely inhibited BHV CALC in 60 day rat subdermal implants (8.84 +/- 3.68 micrograms Ca++/mg tissue), comparable to nonsterilized EHDP-loaded matrices (7.06 +/- 2.00 micrograms Ca++/mg tissue). Nontreated CALC levels were 183 +/- 7.60 micrograms Ca++/mg tissue. Na-CaEHDP co-incorporation into silicone rubber matrices markedly prolonged controlled release with the 1:1 Na-CaEHDP mixture demonstrating an extrapolated release duration of approximately 20 years, assuming the total amount of dispersed drug was released. Data from tricuspid valve replacements in sheep demonstrate erratic control calcification (41.3 +/- 14.9 micrograms Ca++/mg tissue), but complete suppression of BHV calcification with Na2EHDP controlled release (5.74 +/- 1.35 micrograms Ca++/mg tissue) after 150 days.
The relation between bone mineralization and glycosaminoglycans (GAG) was investigated in newborn and mature rabbit diaphyseal bone. Using the density fractionation technique, the bone was separated into fractions of increasing density from 1.4 to 2.3 grams/ml. Each fraction was analyzed by X-ray diffraction to determine the average crystal size. The GAG content of each fraction and of the unfractionated bone was determined by direct extraction and on a few fractions by sequential extraction in guanidine hydrochloride and guanidine/EDTA. There was a decrease in the GAG content with animal age and with increasing fraction density in the newborn rabbit. In one overlapping fraction (2.0-2.1 grams/ml), the GAG content was twice as high in the newborn as in the mature animal. Finally, the crystal size substantially increased from newborn to mature rabbits. Therefore, calcification and maturation of bone is associated with a decrease in the proteoglycan content of the organic matrix.
Bioprosthetic valve calcification is usually assessed pathologically by gross inspection, radiographic studies, and histologic examination. Quantitation of mineral content by chemical assay has not been reported for failed clinical valves removed from adults. In this study, calcium determination by atomic absorption spectroscopy was done on 52 removed porcine valves after routine pathologic examination, including specimen radiography done by a standard technique. Specimens included 31 valves with calcific primary tissue failure, two calcified (but not overtly dysfunctional) valves removed simultaneously with failed valves, 14 nondeteriorated valves obtained at reoperation or autopsy after long-term implantation, and five valves removed within 1 month after insertion. Chemically determined mineral content varied widely among patients and duration of function. Valves with calcific failure had 113 +/- 68 micrograms/mg calcium overall (mean +/- SD) after 36 to 156 months (mean 87) of function. Almost all dysfunctional porcine valves with radiographically demonstrated calcific deposits had greater than 34 and 67 micrograms/mg calcium for mitral and aortic valves, respectively. Nondeteriorated valves (implanted 8 to 145 months, mean 57) had 5 +/- 6 micrograms/mg calcium. Failed aortic valves had more calcium than failed mitral valves and valves with calcific stenosis more than valves with regurgitation caused by calcification with tearing. Correlation of semiquantitative radiographic grading with chemically determined valve mineral was good, indicating that radiographic assessment of calcification may be used reliably for clinical comparisons between valves.
Bioprostheses fabricated from porcine aortic valves are widely used to replace diseased heart valves. Calcification is the
principal cause of the clinical failure of these devices. In the present study, inhibition of the calcification of bioprosthetic
heart valve cusps implanted subcutaneously in rats was achieved through the adjacent implantation of controlled-release matrices
containing the anticalcification agent ethanehydroxydiphosphonate dispersed in a copolymer of ethylene-vinyl acetate. Prevention
of calcification was virtually complete, without the adverse effects of retarded bone and somatic growth that accompany systemic
administration of ethanehydroxydiphosphonate.
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.
Native hyaluronic acid (HA) is reported to be a weak anticomplementary agent. However, the normal buffer systems used for complement tests incorporate gelatin, Ca2+ and Mg2+, which may bind to HA, influence its conformation and interfere with its anticomplementary reactions with complement components such as Cl. In this study, metal ions (Ca2+ and Mg2+), gelatin and fibronectin appeared to react with native HA preparations and block their anticomplementary effects on Cl. In previous studies, we obtained evidence for a relationship between reversible heat-induced HA conformational changes and a subsequent reversible increase in anticomplementary activity. The anticomplementary activity of heat-treated HA preparations was also reduced by gelatin.
Limb buds of chicken embryos contain within the peripheral mesoderm an avascular zone that is rich in hyaluronic acid. Epithelial
tissues that synthesize large amounts of hyaluronic acid relative to other glycosaminoglycans caused avascularity when implanted
into normally vascular wing mesoderm. Epithelia that synthesize little hyaluronic acid did not cause avascularity. Elvax implants
containing hyaluronic acid caused the formation of avascular zones, whereas similar implants containing other glycosaminoglycans
did not give rise to avascular zones. Hyaluronic acid may thus play a role in determining the location of blood vessels in
the embryo.
Hyaluronic acid is a naturally occurring polysaccharide with distinct physicochemical properties which underlie its application as a viscoelastic tool in ophthalmological surgery. In cataract surgery the role of hyaluronic acid in facilitating procedures and protecting the corneal endothelium is well established. Some benefit has also been gained with the use of hyaluronic acid in penetrating keratoplasty, trabeculectomy, retinal reattachment and trauma surgery, although its efficacy in these indications is less well-defined in the published literature. In addition to its lubricating and cushioning properties, demonstration of some in vitro anti-inflammatory activity and a possible disease-modifying effect for hyaluronic acid in animals has prompted its investigation as a treatment in osteoarthritis and, to a much lesser extent, in rheumatoid arthritis. Hyaluronic acid 20 mg, as weekly intra-articular injections for 3 to 7 weeks, improved knee pain and joint motion in patients with osteoarthritis. Although this occurred to a greater degree than with placebo in most comparisons, the effects of hyaluronic acid was similar to those of placebo in the largest trial. In the few available comparisons with other agents, hyaluronic acid appeared equivalent to methylprednisolone 40 mg (for 3 weeks) and to a single injection of triamcinolone 40 mg. Hyaluronic acid was distinguished from other therapies by providing a sustained effect after treatment discontinuation. Together with its very good tolerability profile, these properties justify further study of hyaluronic acid in patients with osteoarthritis. Some limited evidence of improvement in patients with rheumatoid arthritis, and a possible healing effect of hyaluronic acid on tympanic membrane perforations, represent additional areas of interest for future investigation. In summary, hyaluronic acid is a well-established adjunct to cataract surgery and may prove to be a promising option in the treatment of patients with osteoarthritis. Its very good tolerability provides further impetus for examination of its potential role in an extended scope of arthritic and ophthalmological indications, and in wound healing.
We proposed that a similar mechanism for calcification exists for poly(ether)urethanes and glutaraldehyde stabilized tissue. The mechanism is based on the propensity of the polyether component of the materials to complex calcium and provide initiating sites for ultimate formation of calcific deposits. Data evaluating the role ether containing materials have on calcification demonstrate that the rate of mineralization of either tissue valves or polymer valves can be controlled by paying attention to the basic chemical mechanism of complexation occurring at the surface and within the bulk of the implant devices. The molecular models described above, point out that the driving force for complexation with either the polyethers of the polyurethane or the polyether of glutaraldehyde is very strong, therefore, controlling the driving force may lead to medical devices with longer term durability.
Oligosaccharides derived from hyaluronic acid (HA), a naturally occurring linear polysaccharide composed of repeating disaccharide units of N-acetyl-D-glucosamine and D-glucuronic acid, can be chemically modified to introduce a pendant amine-like functionality (patent application pending). Covalent attachment of steroidal and nonsteroidal antiinflammatory drugs to functionalized HA oligosaccharides was accomplished with the incorporation of hydrolytically labile bonds. Further derivatization of the pendant group with homobifunctional crosslinkers allowed the introduction of covalent crosslinks. Chemically-modified HA oligosaccharides were unambiguously characterized in solution by high-resolution 1H NMR spectroscopy.
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.
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.
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.
The effects of hyaluronic acid (HA) on macrophage function, in terms of the phagocytosis of latex beads and superoxide anion and hydrogen peroxide release stimulated by phorbol myristate acetate (PMA), were studied in guinea-pig peritoneal macrophages. Phagocytosis was inhibited in a dose- and molecular-weight-dependent manner by HA. The addition of PMA to the culture, at a dose of more than 10 ng ml-1, caused an increase in the release of active oxygens. The release of active oxygens was inhibited by high molecular weight HA (MW 2.02 x 10(6), HA-202) in a dose-dependent manner. In cell-free systems, HA-202 had a negligible effect in scavenging these active oxygens. Of the three molecular sizes of HA (MW: 0.28 x 10(6), 0.98 x 10(6) and 2.02 x 10(6)), HA-202 most strongly inhibited the active oxygen release. These results indicate that high-molecular-weight HA acts directly on macrophages to inhibit phagocytosis and active oxygen formation, which, in turn, ameliorates the progression of chronic inflammation.
Viscosupplementation is a new medical concept that has as its therapeutic goal the restoration of rheological homeostasis in pathological structures such as osteoarthritic joints. When the normal viscoelasticity of a solid tissue compartment or the elastoviscosity of a liquid tissue compartment is decreased under pathological conditions, normal function and regenerative processes are impaired. By introducing viscosupplementary devices, the normal rheological state of such compartments is restored or augmented. These devices stay in the tissue compartment for various periods of time, depending on the nature of the viscosupplement and the pathophysiology of the tissue compartment.
Osteoarthritis is a common joint disorder in humans. Although intra-articular injection of hyaluronic acid (HA) is in widespread clinical use, there are limited data on the effect of HA on degenerated cartilage. When bovine articular cartilage is degraded with interleukin-1, HA penetrates the cartilage and accumulates in the pericellular matrix of chondrocytes. HA also enhances proteoglycan synthesis that has been reduced by interleukin-1. Thus HA seems to have anabolic effects on degraded cartilage.
Calcification in necrosis has long been known. Of the tissue components, the cells are most vulnerable. Nevertheless, little attention has been paid to the role of cell death in calcification. This review attempts to update the mechanism of calcification with an emphasis on the role of apoptosis in calcification. A brief review on the basic sciences relevant to calcification is followed by a discussion of abnormal Ca2+ and Pi homeostasis in cell injury and apoptosis. Concomitant increases in Ca2+ and Pi in blebs (and matrix vesicles) formed by apoptotic and/or necrotic cells are apparently the primary mechanism of calcification. In addition, membranous cellular degradation products (CDP) resulting from cell disintegration in toto frequently serve as the nidus of calcification. Published data on physiological calcification are compared with findings in various dystrophic calcinoses. This led to the conclusion that apoptosis most likely underlies the mechanism of both physiological and pathological calcifications. It is concluded that calcification is an important function of apoptosis. The mechanism of calcification by CDP and morphology of the resultant calcific deposits are complex.
Fraser JRE, Laurent TC, Laurent UBG (Monash University, Clayton, Victoria, Australia; and University of Uppsala, Uppsala, Sweden). Hyaluronan: its nature, distribution, functions and turnover (Minisymposium: Hyaluronan). J Intern Med 1997; 242: 27–33.
Hyaluronan is a polysaccharide found in all tissues and body fluids of vertebrates as well as in some bacteria. It is a linear polymer of exceptional molecular weight, especially abundant in loose connective tissue. Hyaluronan is synthesized in the cellular plasma membrane. It exists as a pool associated with the cell surface, another bound to other matrix components, and a largely mobile pool. A number of proteins, the hyaladherins, specifically recognize the hyaluronan structure. Interactions of this kind bind hyaluronan with proteoglycans to stabilize the structure of the matrix, and with cell surfaces to modify cell behaviour. Because of the striking physicochemical properties of hyaluronan solutions, various physiological functions have been assigned to it, including lubrication, water homeostasis, filtering effects and regulation of plasma protein distribution. In animals and man, the half-life of hyaluronan in tissues ranges from less than 1 to several days. It is catabolized by receptor-mediated endocytosis and lysosomal degradation either locally or after transport by lymph to lymph nodes which degrade much of it. The remainder enters the general circulation and is removed from blood, with a half-life of 2–5 min, mainly by the endothelial cells of the liver sinuoids.
Hyaluronan (HA), an abundant nonsulfated glycosaminoglycan component of synovial fluid and the extracellular matrix, is an attractive building block for new biocompatible and biodegradable polymers that have applications in drug delivery, tissue engineering, and viscosupplementation. This review consists of three subtopics: (i) chemical modification of HA; (ii) physicochemical and biochemical characterization of HA derivatives; and (iii) in vitro and in vivo bioevaluation studies. Important new products have already reached the marketplace, and the approval and introduction of a variety of new medical applications of HA-based biomaterials can be anticipated in the next decade.
In recent years, research on new, biocompatible, degradable materials has seen the development of a series of modified natural polymers. Among these, a new class of materials consisting of different hyaluronan derivatives promises to be useful in a whole range of clinical applications thanks to their varied biological properties. These new materials are obtained by chemical modification of purified hyaluronan consisting of the partial or total esterification of the carboxyl groups of this natural polymer. This review on the properties of the new materials reports some of their biocompatibility and characterization aspects based on findings from studies conducted on the ethyl and benzyl hyaluronan esters, two representative members of this new class of compounds, and is intended to arouse interest in the potential of other, as yet unexplored derivatives. From the results of a number of investigations, the various derivatives appear to possess different physico-chemical properties, especially as far as the degree of hydration and polymer stability are concerned. In addition, the type of esterification and extent of chemical esterification of hyaluronan considerably affects the biological properties of these materials, offering a range of polymers either favouring or, conversely, inhibiting the adhesion of certain types of cell.
Calcification of vascular tissue is a common complication in aging, atherosclerosis, diabetes, renal failure, aortic stenosis, and prosthetic valve replacement. Osteopontin is a noncollagenous adhesive protein routinely found at sites of dystrophic calcification and synthesized at high levels by macrophages in calcified aortic valves and atherosclerotic plaques. In the present study, we have characterized the calcification of bovine aortic smooth muscle cell (BASMC) cultures in vitro and have studied the effects of exogenous osteopontin on mineral deposition. Induction of calcification in BASMC cultures was alkaline phosphatase-dependent and was characterized by a multilayer cell morphology. Mineral deposition occurred in the basal matrix of multilayered areas as indicated by von Kossa staining, and transmission electron microscopy and electron diffraction identified the mineral as apatite. Ultrastructural analysis of the cultures showed the presence of extracellular matrix vesicles, calcifying collagen fibrils, and nodular-type calcifications similar to those found in calcified heart valves and atherosclerotic plaques. Purified osteopontin (0.05 to 5 microgram/mL) dose dependently inhibited calcification of BASMC cultures, whereas vitronectin and fibronectin had no effect. In contrast to the inhibitory mechanism of levamisole on mineral deposition, osteopontin did not inhibit alkaline phosphatase activity or reduce phosphorus levels in the culture medium. Addition of calcium to the cultures overcame the inhibitory effect of osteopontin on BASMC culture calcification and resulted in decreased levels of calcium in the culture medium and increased levels in the cell layer. Moreover, using high-resolution, colloidal-gold immunocytochemistry, osteopontin was found intimately associated with growing apatite crystals. These data indicate that the effect of osteopontin, although calcium-dependent, was not mediated by simple calcium chelation but most likely by direct interaction of osteopontin with crystal surfaces. These studies suggest that BASMCs can be used to model vascular calcification in vitro and that soluble osteopontin released near sites of vascular calcification may represent an adaptive mechanism aimed at preventing vascular calcification.
Glutaraldehyde-treated porcine aortic valve xenografts frequently fail due to calcification. Calcification in the prostheses begins intracellularly. In a previous study, various types of cell injury to canine valvular fibroblasts, including glutaraldehyde treatment, led to calcification. An influx of extracellular Ca2+ into the phosphate-rich cytosol was theorized to be the mechanism of calcification. To test the Ca2+ influx theory, cytosolic Ca2+ and Pi concentrations were assessed in glutaraldehyde-treated porcine aortic valve fibroblasts, and their relationship to a subsequent calcification was studied. Glutaraldehyde caused an immediate and sustained massive cytosolic Ca2+ increase that was dose dependent and a several-fold increase in Pi. Calcification of cells followed within a week. The earliest calcification was observed in blebs formed on glutaraldehyde-treated cells. Live control cells or cells fixed with glutaraldehyde in Ca2+-free solution did not calcify under the same conditions. Concomitant increases in Ca2+ and Pi in glutaraldehyde-treated cells appear to underlie the mechanism of calcification, and the presence of extracellular Ca2+ during glutaraldehyde fixation promotes calcification.
Bioprosthetic heart valve (BPHV) degeneration, characterized by extracellular matrix deterioration, remodeling, and calcification, is an important clinical problem accounting for thousands of surgeries annually. Here we report for the first time, in a series of in vitro accelerated fatigue studies (5-500 million cycles) with glutaraldehyde fixed porcine aortic valve bioprostheses, that the mechanical function of cardiac valve cusps caused progressive damage to the molecular structure of type I collagen as assessed by Fourier transform IR spectroscopy (FTIR). The cyclic fatigue caused a progressive loss of helicity of the bioprosthetic cuspal collagen, which was evident from FTIR spectral changes in the amide I carbonyl stretching region. Furthermore, cardiac valve fatigue in these studies also led to loss of glycosaminoglycans (GAGs) from the cuspal extracellular matrix. The GAG levels in glutaraldehyde crosslinked porcine aortic valve cusps were 65.2 +/- 8.66 microg uronic acid/10 mg of dry weight for control and 7.91 +/- 1.1 microg uronic acid/10 mg of dry weight for 10-300 million cycled cusps. Together, these molecular changes contribute to a significant gradual decrease in cuspal bending strength as documented in a biomechanical bending assay measuring three point deformation. We conclude that fatigue-induced damage to type I collagen and loss of GAGs are major contributing factors to material degeneration in bioprosthetic cardiac valve deterioration.
Advanced glycation endproducts (AGEs), which accumulate on long-lived proteins and protein deposits (amyloids), induce the expression of proinflammatory cytokines through NF-kappaB-dependent pathways. Hyaluronic acid with a molecular weight above 1.2 MDa (HMW-HA) inhibits the AGE-induced activation of the transcription factor NF-kappaB and the NF-kappaB-regulated cytokines interleukin-1alpha, interleukin-6 and tumor necrosis factor-alpha. Since the molecular weight of hyaluronic acid in humans decreases with age and under conditions of oxidative stress, it is likely that the protective effect of HMW-HA against AGE-induced cellular activation is lost at sites of chronic inflammation and in older age.
Biodegradable materials for spatially and temporally controlled delivery of bioactive agents such as drugs, growth factors, or cytokines are key to facilitating tissue repair. We have developed a versatile method for chemical crosslinking high-molecular-weight hyaluronic acid under physiological conditions yielding biocompatible and biodegradable hydrogels. The method is based on the introduction of functional groups onto hyaluronic acid by formation of an active ester at the carboxylate of the glucuronic acid moiety and subsequent substitution with a side chain containing a nucleophilic group on one end and a (protected) functional group on the other. We have formed hyaluronic acid with amino or aldehyde functionality, and subsequently hydrogels with these hyaluronic acid derivatives and bifunctional crosslinkers or mixtures of the hyaluronic acid derivatives carrying different functionalities using active ester- or aldehyde-mediated reactions. Size analysis of the hyaluronic acid derivatives showed that the chemical modification did not lead to fragmentation of the polysaccharide. Hydrogels formed with hyaluronic acid derivatized to a varying degree and crosslinked with low- or high-molecular-weight crosslinkers were evaluated for biodegradability by digestion with hyaluronidase and for biocompatibility and ectopic bone formation by subcutaneous implantation in rats. Several hydrogel formulations showed excellent cell infiltration and chondro-osseous differentiation when loaded with bone morphogenetic protein-2 (BMP-2). Synergistic action of insulin-like growth factor-1 with BMP-2 promoted cartilage formation in this model, while addition of transforming growth factor-beta and BMP-2 led to rapid replacement of the matrix by bone.
Substitute heart valves composed of human or animal tissues have been used since the early 1960s, when aortic valves obtained fresh from human cadavers were transplanted to other individuals as allografts. Today, tissue valves are used in 40% or more of valve replacements worldwide, predominantly as stented porcine aortic valves (PAV) and bovine pericardial valves (BPV) preserved by glutaraldehyde (GLUT) (collectively termed bioprostheses). The principal disadvantage of tissue valves is progressive calcific and noncalcific deterioration, limiting durability. Native heart valves (typified by the aortic valve) are cellular and layered, with regional specializations of the extracellular matrix (ECM). These elements facilitate marked repetitive changes in shape and dimension throughout the cardiac cycle, effective stress transfer to the adjacent aortic wall, and ongoing repair of injury incurred during normal function. Although GLUT bioprostheses mimic natural aortic valve structure (a) their cells are nonviable and thereby incapable of normal turnover or remodeling ECM proteins; (b) their cuspal microstructure is locked into a configuration which is at best characteristic of one phase of the cardiac cycle (usually diastole); and (c) their mechanical properties are markedly different from those of natural aortic valve cusps. Consequently, tissue valves suffer a high rate of progressive and age-dependent structural valve deterioration resulting in stenosis or regurgitation (>50% of PAV overall fail within 10-15 years; the failure rate is nearly 100% in 5 years in those <35 years old but only 10% in 10 years in those >65). Two distinct processes-intrinsic calcification and noncalcific degradation of the ECM-account for structural valve deterioration. Calcification is a direct consequence of the inability of the nonviable cells of the GLUT-preserved tissue to maintain normally low intracellular calcium. Consequently, nucleation of calcium-phosphate crystals occurs at the phospholipid-rich membranes and their remnants. Collagen and elastin also calcify. Tissue valve mineralization has complex host, implant, and mechanical determinants. Noncalcific degradation in the absence of physiological repair mechanisms of the valvular structural matrix is increasingly being appreciated as a critical yet independent mechanism of valve deterioration. These degradation mechanisms are largely rationalized on the basis of the changes to natural valves when they are fabricated into a tissue valve (mentioned above), and the subsequent interactions with the physiologic environment that are induced following implantation. The "Holy Grail" is a nonobstructive, nonthrombogenic tissue valve which will last the lifetime of the patient (and potentially grow in maturing recipients). There is considerable activity in basic research, industrial development, and clinical investigation to improve tissue valves. Particularly exciting in concept, yet early in practice is tissue engineering, a technique in which an anatomically appropriate construct containing cells seeded on a resorbable scaffold is fabricated in vitro, then implanted. Remodeling in vivo, stimulated and guided by appropriate biological signals incorporated into the construct, is intended to recapitulate normal functional architecture.
Enzymatic degradation of hyaluronan (HA) by testicular hyaluronidase (HAase, hyaluronate 4-glucanohydrolase) requires inclusion of mono- or divalent cations in the reaction mixture. Most divalent cations activated HAase with equal potency; however, Cu2+ suppressed degradation, and Ca2+ showed a concentration-dependent regulation of size of the oligosaccharide products. Careful selection of HAase assay parameters is critical for discovery of novel HAase inhibitors and for preparation of controlled-size oligosaccharide fragments.
A new hyaluronic acid (HA)-based hydrogel film was prepared and evaluated for use in drug delivery. This biocompatible material crosslinks and gels in minutes, and the dried film swells and rehydrates to a flexible hydrogel in seconds. HA was first converted to the adipic dihydrazide derivative and then crosslinked with the macromolecular homobifunctional reagent poly(ethylene glycol)-propiondialdehyde to give a polymer network. After gelation, a solvent casting method was used to obtain a HA hydrogel film. The dried film swelled sevenfold in volume in buffer, reaching equilibrium in less than 100 s. Scanning electron microscopy (SEM) of the hydrogel films showed a condensed and featureless structure before swelling, but a porous microstructure when hydrated. The thermal behavior of the hydrogel films was characterized by differential scanning calorimetry. The enzymatic degradation of the HA hydrogel films by hyaluronidase was studied using both SEM and a spectrophotometric assay. Drug release from the hydrogel film was evaluated in vitro using selected anti-bacterial and anti-inflammatory drugs. This novel biomaterial can be employed for controlled release of therapeutic agents at wound sites.
A novel chemical modification of biological tissues was developed by the direct coupling heparin to bovine pericardium (BP). The heparinization involves pretreatment of BP using GA and followed by grafting heparin to BP by the reaction of residual aldehyde and amine group of heparin. BP was modified by direct coupling of heparin and the effect of heparin coupling on calcification was evaluated in vitro and in vivo. Heparinized BP was characterized by measuring shrinkage temperature, mechanical properties, digestion resistance to collagenase enzyme, in vitro cytotoxicity, and in vivo calcification. Thermal and mechanical properties showed that the durability of heparin-treated tissue increased as compared with fresh tissue and GA-treated tissue. Resistance to collagenase digestion revealed that heparin-treated tissue has greater resistance to enzyme digestion than did fresh tissue and GA-treated tissue. Heparinized tissue had shown to be non-cytotoxic, however, relatively high cytotoxicity was observed in the GA-treated tissues due to the release of GA. In vivo calcification study demonstrated much less calcium deposition on heparin-treated BP than GA-treated one. Obtained results attest to the usefulness of heparinized BP for cardiovascular bioprostheses.
Osteopontin is a secreted glycoprotein with a multidomain structure and functions characteristic of a matricellular protein. Osteopontin interacts with cell surface receptors via arginine-glycine-aspartate (RGD)- and non-RGD containing adhesive domains, in addition to binding to components of the structural extracellular matrix. While normally expressed in bone and kidney, osteopontin levels are elevated during wound healing and inflammation in most tissues studied to date. Since 1986, over one thousand studies have been published on osteopontin, including recent experiments in osteopontin-deficient mice. These studies reveal osteopontin as a cell adhesive, signaling, migratory, and survival stimulus for various mesenchymal, epithelial, and inflammatory cells, in addition to being a potent regulator of osseous and ectopic calcification. Based on these reports, a general picture of osteopontin as an important regulator of inflammation and biomineralization is emerging. A common denominator in osteopontin function in these situations is its ability to regulate the function of macrophage and macrophage-derived cells (i.e. osteoclasts). While we have learned much about osteopontin and the processes it appears to regulate over the past decade, many questions regarding this important multifunctional protein remain unanswered and provide important directions for future studies.
Bioprosthetic heart valves (BPHVs) derived from glutaraldehyde-crosslinked porcine aortic valves are frequently used in heart valve replacement surgeries. However, the majority of bioprostheses fail clinically because of calcification and degeneration. We have recently shown that glycosaminoglycan (GAG) loss may be in part responsible for degeneration of glutaraldehyde-crosslinked bioprostheses. In the present studies, we used a mild reaction of periodate-mediated crosslinking to stabilize glycosaminoglycans in the bioprosthetic tissue. We demonstrate the feasibility of periodate reaction by crosslinking major components of extracellular matrix of bioprosthetic heart valve tissue, namely type I collagen and hyaluronic acid (HA). Uronic acid assay of periodate-fixed HA-collagen matrices showed 48% of HA disaccharides were bound to collagen. Furthermore, we show that such reactions are also feasible to fix glycosaminoglycans present in the middle spongiosa layer of bioprosthetic heart valves. The periodate reactions were compatible with conventional glutaraldehyde crosslinking and showed adequate stabilization of extracellular matrix as demonstrated by thermal denaturation temperature and collagenase assays. Moreover, uronic acid assays of periodate-fixed BPHV cusps showed 36% reduction in the amount of unbound GAG disaccharides as compared with glutaraldehyde-crosslinked cusps. We also demonstrate that calcification of BPHV cusps was significantly reduced in the periodate-fixed group as compared with the glutaraldehyde-fixed group in 21-day rat subdermal calcification studies (periodate-fixed tissue Ca 72.01 +/- 5.97 microg/mg, glutaraldehyde-fixed tissue Ca 107.25 +/- 6.56 microg/mg). We conclude that periodate-mediated GAG fixation could reduce structural degeneration of BPHVs and may therefore increase the useful lifetime of these devices.
Cardiac valve calcification (VC) has long been regarded as a consequence of aging and abnormal calcium-phosphate metabolism in uremic patients. In view of the recent recognition of association among inflammation, malnutrition, and atherosclerosis, the possible role of inflammation and malnutrition in VC was investigated. Inflammatory markers (including C-reactive protein [CRP], fibrinogen, and basal metabolic rate) and nutritional status (assessed using serum albumin, subjective global nutrition assessment, and handgrip strength) were examined, in addition to calcium phosphate parameters and other traditional cardiovascular risk factors, including gender, smoking habits, BP, and lipid profile, in relation to VC in 137 patients who were on continuous ambulatory peritoneal dialysis. Compared with patients with no VC, patients with VC not only were older (60 [10] versus 54 [12] yr; P = 0.005), had higher plasma phosphate (1.89 [0.52] versus 1.64 [0.41] mmol/L; P = 0.003), and had higher parathyroid hormone (83 [40, 145] versus 38 [16, 71] pmol/L; P = 0.001) but also had higher CRP (4.5 [0.1, 13.4] versus 0.2 [0.1, 4.4] mg/L; P = 0.004), had higher fibrinogen (6.6 [1.9] versus 5.7 [1.3] g/L; P = 0.002), and had lower serum albumin (26 [4] versus 29 [3] g/L; P = 0004). Twenty-three percent of patients with VC versus 17% of patients with no VC were moderately to severely malnourished according to subjective global nutrition assessment (P = 0.05). Even after adjustment for patients' age, duration of continuous ambulatory peritoneal dialysis, diabetes, and calcium x phosphate product, cardiac VC remained strongly associated with CRP (odds ratio, 1.05; P = 0.026) and albumin (odds ratio, 0.85; P = 0.01). The data suggest that VC not only is a passive degenerative process but also involves active inflammation, similar to that seen in atherosclerosis. The presence of uncontrolled hyperphosphatemia and hyperparathyroidism further accelerates the progression of calcification. The data also indicate that VC and atherosclerosis should be considered as associated syndromes, sharing similar pathogenic mechanisms, namely active inflammation.
In an attempt to avoid the destructive process of bioprosthetic heart-valve calcification associated with the use of glutaraldehyde, valves are today prepared using low concentrations of the crosslinking reagent. In this review, we summarize our findings and those of others that confirm that the immunogenicity of such tissue is not sufficiently masked and that a defined humoral response is indeed mounted against a repertoire of antigens unrelated to those associated with vascularized and non-cross-linked xenograft organs. We demonstrate the need for increased cross-linking of tissue to satisfactorily mitigate that response; furthermore, we examine the impact of increased cross-link density on the macrophage as antigen presenting cell with respect to its involvement in both tissue erosion and pannus overgrowth. Finally we present evidence for a role of circulating antibodies in bioprosthesis calcification.
Ectopic calcification, the abnormal calcification of soft tissues, can have severe clinical consequences especially when localized to vital organs such as heart valves, arteries, and kidneys. Recent observations suggest that ectopic calcification, like bone biomineralization, is an actively regulated process. These observations have led a search for molecular determinants of ectopic calcification. A candidate molecule is osteopontin (OPN), a secreted phosphoprotein invariantly associated with both normal and pathological mineral deposits. In the present study, OPN was found to be a natural inhibitor of ectopic calcification in vivo. Glutaraldehyde-fixed aortic valve leaflets showed accelerated and fourfold to fivefold greater calcification after subcutaneous implantation into OPN-null mice compared to wild-type mice. In vitro and in vivo studies suggest that OPN not only inhibits mineral deposition but also actively promotes its dissolution by physically blocking hydroxyapatite crystal growth and inducing expression of carbonic anhydrase II in monocytic cells and promoting acidification of the extracellular milieu. These findings suggest a novel mechanism of OPN action and potential therapeutic approach to the treatment of ectopic calcification.