Anti-calcification of bovine pericardium for bioprosthetic heart valves after surface modification with hyaluronic acid derivatives

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.     

Enzymatic degradation of hyaluronan hydrogels with different capacity for in situ bio‐mineralization

In situ cross‐linked hyaluronan (HA) hydrogels with different capacities for biomineralization were prepared and their enzymatic degradation was monitored. Covalent incorporation of bisphosphonates (BPs) into HA hydrogel results in the increased stiffness of the hydrogel in comparison with the unmodified HA hydrogel of the same cross‐linking density. The rate of enzymatic degradation of HABP hydrogel was significantly lower than the rate of degradation of control HA hydrogel in vitro. This effect is observed only in the presence of calcium ions that strongly bind to the matrix‐anchored BP groups and promote further mineralization of the matrix. The degradation of the hydrogels was followed by noninvasive fluorescence measurements enabled after mild and chemoselective labeling of cross‐linkable HA derivatives with a fluorescent tag.     

Facile synthesis and characterization of disulfide-cross-linked hyaluronic acid hydrogels for protein delivery and cell encapsulation

Injectable hyaluronic acid (HA) hydrogels cross-linked via disulfide bond are synthesized using a thiol-disulfide exchange reaction. The production of small-molecule reaction product, pyridine-2-thione, allows the hydrogel formation process to be monitored quantitatively in real-time by UV spectroscopy. Rheological tests show that the hydrogels formed within minutes at 37 °C. Mechanical properties and equilibrium swelling degree of the hydrogels can be controlled by varying the ratio of HA pyridyl disulfide and macro-cross-linker PEG-dithiol. Degradation of the hydrogels was achieved both enzymatically and chemically by disulfide reduction with distinctly different kinetics and profiles. In the presence of hyaluronidase, hydrogel mass loss over time was linear and the degradation was faster at higher enzyme concentrations, suggesting surface-limited degradation. The kinetics of hydrogel erosion by glutathione was not linear, nor did the erosion rate correlate linearly with glutathione concentration, suggesting a bulk erosion mechanism. A cysteine-containing chemokine, stromal cell-derived factor 1α, was successfully encapsulated in the hydrogel and released in vitro without chemical alteration. Several different cell types, including fibroblasts, endothelial cells, and mesenchymal stem cells, were successfully encapsulated in the hydrogels with high cell viability during and after the encapsulation process. Substantial cell viability in the hydrogels was maintained up to 7 days in culture despite the lack of adhesion between the HA matrix and the cells. The facile synthesis of disulfide-cross-linked, dual-responsive degradable HA hydrogels may enable further development of bioactive matrices potentially suitable for tissue engineering and drug delivery applications.     

Hydrolytically degradable hyaluronic acid hydrogels with controlled temporal structures

Polysaccharides are being processed into biomaterials for numerous biological applications due to their native source in numerous tissues and biological functions. For instance, hyaluronic acid (HA) is found abundantly in the body, interacts with cells through surface receptors, and can regulate cellular behavior (e.g., proliferation, migration). HA was previously modified with reactive groups to form hydrogels that are degraded by hyaluronidases, either added exogenously or produced by cells. However, these hydrogels may be inhibitory and their applications are limited if the appropriate enzymes are not present. Here, for the first time, we synthesized HA macromers and hydrogels that are both hydrolytically (via ester group hydrolysis) and enzymatically degradable. The hydrogel degradation and growth factor release was tailored through the hydrogel cross-linking density (i.e., macromer concentration) and copolymerization with purely enzymatically degradable macromers. When mesenchymal stem cells (MSCs) were encapsulated in the hydrogels, cellular organization and tissue distribution was influenced by the copolymer concentration. Importantly, the distribution of released extracellular matrix molecules (e.g., chondroitin sulfate) was improved with increasing amounts of the hydrolytically degradable component. Overall, this new macromer allows for enhanced control over the structural evolution of the HA hydrogels toward applications as biomaterials.     

Design of biomimetic cell-interactive substrates using hyaluronic acid hydrogels with tunable mechanical properties

Hyaluronic acid (HA) is a natural polysaccharide abundant in biological tissues with excellent potential for constructing synthetic extracellular matrix analogues. In this work, we established a simple and dependable approach to prepare hyaluronic acid-based hydrogels with controlled stiffness and cell recognition properties for use as cell-interactive substrates. This approach relied on a new procedure for the synthesis of methacrylate-modified HA macromers (HA-MA) and, on photorheometry allowing real time monitoring of gelation during photopolymerization. We showed in this way the ability to obtain gels that encompass the range of physiologically relevant elastic moduli while still maintaining the recognition properties of HA by specific cell surface receptors. These hydrogels were prepared from HA macromers having a degree of methacrylation <0.5, which allows to minimize compromising effects on the binding affinity of HA to its cell receptors due to high substitution on the one hand, and to achieve nearly 100% conversion of the methacrylate groups on the other. When the HA hydrogels were immobilized on glass substrates, it was observed that the attachment and the spreading of a variety of mammalian cells rely on CD44 and its coreceptor RHAMM. The attachment and spreading were also shown to be modulated by the elastic properties of the HA matrix. All together, these results highlight the biological potential of these HA hydrogel systems and the needs of controlling their chemical and physical properties for applications in cell culture and tissue engineering.     

Development of a biostable replacement for PEGDA hydrogels

The exceptional tunability of poly(ethylene glycol) (PEG) hydrogel chemical, mechanical, and biological properties enables their successful use in a wide range of biomedical applications. Although PEG diacrylate (PEGDA) hydrogels are often used as nondegradable controls in short-term in vitro studies, it is widely acknowledged that the hydrolytically labile esters formed upon acrylation of the PEG diol make them susceptible to slow degradation in vivo. A PEG hydrogel system that maintains the desirable properties of PEGDA while improving biostability would be valuable in preventing degradation-related failure of gel-based devices in long-term in vivo applications. To this end, PEG diacrylamide (PEGDAA) hydrogels were synthesized and characterized in quantitative comparison to traditional PEGDA hydrogels. It was found that PEGDAA hydrogel modulus and swelling can be tuned over a similar range and to comparable degrees as PEGDA hydrogels with changes in macromer molecular weight and concentration. Additionally, PEGDAA cytocompatibility, low cell adhesion, and capacity for incorporation of bioactivity were analogous to that of PEGDA. In vitro hydrolytic degradation studies showed that the amide-based PEGDAA had significantly increased biostability relative to PEGDA. Overall, these findings indicate that PEGDAA hydrogels are a suitable replacement for PEGDA hydrogels with enhanced hydrolytic resistance. In addition, these studies provide a quantitative measure of the hydrolytic degradation rate of PEGDA hydrogels which was previously lacking in the literature.     

Hyaluronic Acid Enhances the Mechanical Properties of Tissue-Engineered Cartilage Constructs

There is a need for materials that are well suited for cartilage tissue engineering. Hydrogels have emerged as promising biomaterials for cartilage repair, since, like cartilage, they have high water content, and they allow cells to be encapsulated within the material in a genuinely three-dimensional microenvironment. In this study, we investigated the mechanical properties of tissue-engineered cartilage constructs using in vitro culture models incorporating human chondrocytes from osteoarthritis patients. We evaluated hydrogels formed from mixtures of photocrosslinkable gelatin-methacrylamide (Gel-MA) and varying concentrations (0–2%) of hyaluronic acid methacrylate (HA-MA). Initially, only small differences in the stiffness of each hydrogel existed. After 4 weeks of culture, and to a greater extent 8 weeks of culture, HA-MA had striking and concentration dependent impact on the changes in mechanical properties. For example, the initial compressive moduli of cell-laden constructs with 0 and 1% HA-MA were 29 and 41 kPa, respectively. After 8 weeks of culture, the moduli of these constructs had increased to 66 and 147 kPa respectively, representing a net improvement of 69 kPa for gels with 1% HA-MA. Similarly the equilibrium modulus, dynamic modulus, failure strength and failure strain were all improved in constructs containing HA-MA. Differences in mechanical properties did not correlate with glycosaminoglycan content, which did not vary greatly between groups, yet there were clear differences in aggrecan intensity and distribution as assessed using immunostaining. Based on the functional development with time in culture using human chondrocytes, mixtures of Gel-MA and HA-MA are promising candidates for cartilage tissue-engineering applications.     

Hydrogels of dihydroxypropyl chitosan crosslinked with irradiation at paste-like condition

In present study, a series of environmentally friendly hydrogel films were prepared from dihydroxypropyl chitosan (DHP-chitosan) using irradiation technique without any bifunctional crosslinking compounds. DHP-chitosan irradiated at high concentrated solution state (more than 10%, paste-like state) was found to introduce crosslinking structure. Crosslinking behavior, mechanical property, morphology, and swelling behavior of the hydrogel films were studied. It has been found that a concentration of 40% solution is the most effective for crosslinking. The hydrogel films of DHP-chitosan exhibited controllable mechanical property and typically pH-sensitive character in their swelling behavior. A preliminary biodegradation study confirmed that DHP-chitosan hydrogels also undergo biodegradation by enzymatic degradation test.     

Glucose-specific poly(allylamine) hydrogels--a reassessment

Polymer hydrogels synthesized by crosslinking poly(allylamine hydrochloride) with (+/-)-epichlorohydrin in the presence of d-glucose-6-phosphate monobarium salt do not show imprinting on the molecular level. A series of hydrogels was prepared using the following five templates: d-glucose-6-phosphate monobarium salt, d-glucose, l-glucose, barium hydrogen phosphate (BaHPO(4)), and d-gluconamide; a hydrogel was also prepared in the absence of a template. For all six hydrogels, batch binding studies were conducted with d-glucose, l-glucose, d-fructose, and d-gluconamide. The extent of analyte sugar binding was determined using (1)H NMR. Each hydrogel shows approximately the same relative binding affinity for the different sugar derivatives, and none displays selectivity for either glucose enantiomer. The results of the binding studies correlate with the octanol-water partition coefficients of the sugars, indicative that differential solubilities in the bulk polymer account for the binding affinities observed. Thus, in contrast to templated hydrogels prepared using methacrylate- or acrylamide-based reagents, true imprinting does not occur in this novel, crosslinked-poly(allylamine hydrochloride) system.     

Synthesis and characterization of dendron cross-linked PEG hydrogels as corneal adhesives

In pursuit of a wound-specific corneal adhesive, hydrogels formed by the reaction of propionaldehyde, butyraldehyde, or 2-oxoethyl succinate-functionalized poly(ethylene glycol) (PEG) with a peptide-based dendritic cross-linker (Lys(3)Cys(4)) were characterized. These macromers react within minutes of mixing to form transparent and elastic hydrogels with in vitro degradation times that range from hours to months based on the type of bonds formed during the cross-linking reaction, either thiazolidine or pseudoproline. The mechanical properties of these materials, determined via parallel plate rheology, were dependent on the polymer concentration, as was the hydrogel adhesive strength, which was determined by lap shear adhesive testing. In addition, these hydrogels were efficacious in closing ex vivo 4.1 mm central corneal lacerations: wounds closed with these hydrogel adhesives were able to withstand intraocular pressure values equivalent to, or in excess of, those obtained by closing the wounds with suturing.     

Evaluation of Physical and Mechanical Properties of Porous Poly (Ethylene Glycol)-co-(L-Lactic Acid) Hydrogels during Degradation

Porous hydrogels of poly(ethylene glycol) (PEG) have been shown to facilitate vascularized tissue formation. However, PEG hydrogels exhibit limited degradation under physiological conditions which hinders their ultimate applicability for tissue engineering therapies. Introduction of poly(_L-lactic acid) (PLLA) chains into the PEG backbone results in copolymers that exhibit degradation via hydrolysis that can be controlled, in part, by the copolymer conditions. In this study, porous, PEG-PLLA hydrogels were generated by solvent casting/particulate leaching and photopolymerization. The influence of polymer conditions on hydrogel architecture, degradation and mechanical properties was investigated. Autofluorescence exhibited by the hydrogels allowed for three-dimensional, non-destructive monitoring of hydrogel structure under fully swelled conditions. The initial pore size depended on particulate size but not polymer concentration, while degradation time was dependent on polymer concentration. Compressive modulus was a function of polymer concentration and decreased as the hydrogels degraded. Interestingly, pore size did not vary during degradation contrary to what has been observed in other polymer systems. These results provide a technique for generating porous, degradable PEG-PLLA hydrogels and insight into how the degradation, structure, and mechanical properties depend on synthesis conditions.     

Synthesis and evaluation of hydrolyzable hyaluronan-tethered bupivacaine delivery systems

Local anesthetics are useful for reducing acute pain, but their short duration precludes them from use in solely managing postoperative pain. To prolong the duration of local anesthesia, we conjugated bupivacaine to native hyaluronan (HA) and divinyl sulfone cross-linked Hylan A (Hylan B particles) using a hydrolyzable linker incorporating an imide. Bupivacaine was prepared for conjugation to HA by forming the acryl imide derivative. Separately, the carboxyl group of HA was reacted with nipsylethylamine (NEA) using carbodiimide-mediated coupling to provide HA-NEA that was subsequently reduced with tris(2-carboxyethylphosphine) hydrochloride to yield HA carrying a free sulfhydryl (HA-SH). The HA-bupivacaine conjugate was assembled by reacting HA-SH with acrylbupivacaine. Characterization of the conjugates showed 22% degree of modification by 1 mol of carboxyl. In vitro release studies comparing bupivacaine admixed in HA with bupivacaine conjugated to HA showed half-lives of 0.4 +/- 0.1 h, and 16.9 +/- 0.2 h, respectively, and the bupivacaine was released chemically unaltered as confirmed by LC-MS. In vivo studies to assess the duration of anesthetic activity were performed in a rat sciatic nerve blockade model. For these studies, bupivacaine was conjugated to Hylan B following a similar procedure, and the degree of modification obtained was 14%. Free bupivacaine (3 and 16 mg/kg) and free bupivacaine (3 mg/kg) admixed with Hylan B particles showed nerve block over 4, 9, and 6 h, respectively. Free bupivacaine (3 mg/kg) admixed with bupivacaine (13 mg/kg) conjugated to Hylan B particles showed a four to 5-fold longer impairment of motor function over the free bupivacaine formulations with a total block time of 19 h. Bupivacaine conjugated to Hylan B particles has the potential to prolong the duration of local anesthesia.     

In situ forming chitosan hydrogels prepared via ionic/covalent co-cross-linking

In situ forming chitosan hydrogels have been prepared via coupled ionic and covalent cross-linking. Thus, different amounts of genipin (0.05, 0.10, 0.15, and 0.20% (w/w)), used as a chemical cross-linker, were added to a solution of chitosan that was previously neutralized with a glycerol-phosphate complex (ionic cross-linker). In this way, it was possible to overcome the pH barrier of the chitosan solution, to preserve its thermosensitive character, and to enhance the extent of cross-linking in the matrix simultaneously. To investigate the contributions of the ionic cross-linking and the chemical cross-linking, separately, we prepared the hydrogels without the addition of either genipin or the glycerol-phosphate complex. The addition of genipin to the neutralized solution disturbs the ionic cross-linking process and the chemical cross-linking becomes the dominant process. Moreover, the genipin concentration was used to modulate the network structure and performance. The more promising formulations were fully characterized, in a hydrated state, with respect to any equilibrium swelling, the development of internal structure, the occurrence of in vitro degradability and cytotoxicity, and the creation of in vivo injectability. Each of the hydrogel systems exhibited a notably high equilibrium water content, arising from the fact that their internal structure (examined by conventional SEM, and environmental SEM) was highly porous with interconnecting pores. The porosity and the pore size distribution were quantified by mercury intrusion porosimetry. Although all gels became degraded in the presence of lysozyme, their degradation rate greatly depended on the genipin load. Through in vitro viability tests, the hydrogel-based formulations were shown to be nontoxic. The in vivo injection of a co-cross-linking formulation revealed that the gel was rapidly formed and localized at the injection site, remaining in position for at least 1 week.     

Synthesis and evaluation of novel biodegradable hydrogels based on poly(ethylene glycol) and sebacic acid as tissue engineering scaffolds

Novel biodegradable poly(ethylene glycol) (PEG) based hydrogels, namely, PEG sebacate diacrylate (PEGSDA) were synthesized, and their properties were evaluated. Chemical structures of these polymers were confirmed by Fourier transform infrared and proton nuclear magnetic resonance (1H NMR) spectroscopy. After photopolymerization, the dynamic shear modulus of the hydrogels was up to 0.2 MPa for 50% PEGSDA hydrogel, significantly higher than conventional hydrogels such as PEG diacrylate (PEGDA). The swelling ratios of these macromers were significantly lower than PEGDA. The in vitro degradation study demonstrated that these hydrogels were biodegradable with weight losses about 66% and 32% for 25% and 50% PEGSDA after 8 weeks of incubation in phosphate-buffered saline at 37 degrees C. In vitro biocompatibility was assessed using cultured rat bone marrow stromal cells (MSCs) in the presence of unreacted monomers or degradation products. Unlike conventional PEGDA hydrogels, PEGSDA hydrogel without RGD peptide modification induced MSC cell adhesion similar to tissue culture polystyrene. Finally, complex three-dimensional structures of PEGSDA hydrogels using solid free form technique were fabricated and their structure integrity was better maintained than PEGDA hydrogels. These hydrogels may find use as scaffolds for tissue engineering applications.     

Photopolymerized water-soluble chitosan-based hydrogel as potential use in tissue engineering

Novel biodegradable hydrogels by photocrosslinking macromers based on chitosan derivative are reported. Photocrosslinkable macromers, a water-soluble (methacryloyloxy) ethyl carboxyethyl chitosan were prepared by Michael-addition reaction between chitosan and ethylene glycol acrylate methacrylate. The macromers were characterized by Fourier transform infrared spectroscopy, (1)H NMR and (13)C NMR. Hydrogels were fabricated by exposing aqueous solutions of macromers with 0.1% (w/v) photoinitiator to UV light irradiation, and their swelling kinetics as well as degradation behaviors was evaluated. The results demonstrated that the degradation rates were affected strongly by crosslinking density. The hydrogel was compatible to Vero cells, not exhibiting significant cytotoxicity. Cell culture assay also demonstrated that the hydrogels were good in promoting the cell attachment and proliferation, showing their potential as tissue engineering scaffolds.     

Dextran and hyaluronan methacrylate based hydrogels as matrices for soft tissue reconstruction

Polysaccharide hydrogels have become increasingly studied as matrices in soft tissue engineering because of their known cytocompatibility. In this work cross-linkable dextran methacrylates and hyaluronan methacrylate were synthesized and their transformation into stable hydrogels was studied. The in vitro degradation behaviour of the formed hydrogels could be controlled by the polysaccharide structure and the cross-linking density. Under in vitro conditions, the formed gels had no cytotoxic effects against fibroblasts, but cells could adhere only inefficiently in long term experiments. The use of composite gels improved the adherence of cells. Different scaffold architectures were studied including porous structures and perforated gel layers. Selected hydrogels were examined in an in vivo pilot study using a rabbit model to evaluate their biocompatibility, stability and degradation. No signs of inflammation were seen and with prolonged duration the material was degraded and lacunas were formed by immigrating or ingrowing cells. Optimizing their mechanical properties, the formed hydrogels represent promising candidates as matrices for soft tissue reconstruction.     

Photo-cross-linked hydrogels with polysaccharide-poly(amino acid) structure: new biomaterials for pharmaceutical applications

The aim of this work has been the preparation and characterization of novel hydrogels with polysaccharide-poly(amino acid) structure having suitable physicochemical properties for pharmaceutical applications. In the first step, hyaluronic acid (HA) and alpha,beta-poly(N-2-hydroxyethyl)-DL-aspartamide (PHEA) have been derivatized with methacrylic anhydride (AMA), thus obtaining HA-AMA and PHM derivatives, respectively. In the second step, aqueous solutions of both these derivatives have been irradiated at 313 nm to obtain chemical hydrogels. The hydrogel obtained by irradiating for 15 min an aqueous solution containing 4% w/v of HA-AMA and 4% w/v of PHM resulted in the highest yield. Its swelling ability was dependent on the pH and nature of the external medium. Besides, this hydrogel undergoes a partial hydrolysis, especially in the presence of enzymes, such as esterase or hyaluronidase, but the entity of this degradation is lower than that observed for a hydrogel based on HA-AMA alone. The ability of this hydrogel to entrap drug molecules has been evaluated by using thrombin as a model drug. In vitro release studies and a platelet aggregation test demonstrated that the HA-AMA/PHM hydrogel is able to release thrombin in the active form, thus suggesting its suitability for the treatment of hemorrhages.     

Biodegradable superabsorbent hydrogels derived from cellulose by esterification crosslinking with 1,2,3,4-butanetetracarboxylic dianhydride

Superabsorbent hydrogels were prepared from native celluloses dissolved in lithium chloride and N-methyl-2-pyrrolidinone (LiCl/NMP) by esterification crosslinking with 1,2,3,4-butanetetracarboxylic dianhydride (BTCA). Subsequent conversion of the unreacted carboxyl groups to sodium carboxylates by the addition of aqueous NaOH was performed to enhance the water affinity of the gels. The absorbency of the products was strongly dependent on the amount of BTCA that was esterified to cellulose, and the highest absorbency was observed for the hydrogel composed of approximately 0.25 molecules of BTCA per anhydroglucose unit (AGU) of cellulose. Furthermore, it was confirmed that the absorbency was enhanced as the average degree of polymerization (DP) of the starting cellulose increased. The use of cotton cellulose with a high DP of about 2400 produced a hydrogel with an absorbency of 720 times its dry weight, which exceeded the absorbency of commercial crosslinked sodium polyacrylate superabsorbent hydrogel (SPA). The hydrogels exhibited good biodegradability, with a maximum degradation of 95% within 7 days using cellulase.     

Synthesis and selective cytotoxicity of a hyaluronic acid-antitumor bioconjugate.

A cell-targeted prodrug was developed for the anti-cancer drug Taxol, using hyaluronic acid (HA) as the drug carrier. HA-Taxol bioconjugates were synthesized by linking the Taxol 2'-OH via a succinate ester to adipic dihydrazide-modified HA (HA-ADH). The coupling of Taxol-NHS ester and HA-ADH provided several HA bioconjugates with different levels of ADH modification and different Taxol loadings. A fluorescent BODIPY-HA was also synthesized to illustrate cell targeting and uptake of chemically modified HA using confocal microscopy. HA-Taxol conjugates showed selective toxicity toward the human cancer cell lines (breast, colon, and ovarian) that are known to overexpress HA receptors, while no toxicity was observed toward a mouse fibroblast cell line at the same concentrations used with the cancer cells. The drug carrier HA-ADH was completely nontoxic. The selective cytotoxicity is consistent with the results from confocal microscopy, which demonstrated that BODIPY-HA only entered the cancer cell lines.     

Evaluation of the in vitro degradation of macroporous hydrogels using gravimetry,confined compression testing,and microcomputed tomography

This study investigated the in vitro degradation characteristics of macroporous hydrogels based on poly(propylene fumarate-co-ethylene glycol) (P(PF-co-EG)). Four formulations were fabricated to test the effect of porosity and cross-linking density on the degradation of the resulting macroporous hydrogels. Macroporosity was introduced by the addition of sodium bicarbonate and ascorbic acid, the precursors of the carbon dioxide porogen, in the initiation system for the hydrogel cross-linking. Macroporous hydrogels with porosities of 0.80 +/- 0.03 and 0.89 +/- 0.03 were synthesized by the addition of sodium bicarbonate of concentrations 40 and 80 mg/mL and ascorbic acid of concentrations 0.05 and 0.1 mol/L, respectively. Poly(ethylene glycol) diacrylate (PEG-DA) was utilized as a cross-linker. The molecular weight between cross-links had a significant effect on weight loss after 12 weeks, where samples with M(C) of 1,880 +/- 320 synthesized with a P(PF-co-EG):PEG-DA ratio of 3:1 had a significantly greater mass loss due to degradation than those with M(C) of 1,000 +/- 100 synthesized with a P(PF-co-EG):PEG-DA ratio of 1:1. In contrast, porosity played a minimal role in determining the weight loss. Mechanical testing of the hydrogels under confined compression showed a decrease in compressive modulus over the degradation time for all formulations. In addition, an increase in hydrogel equilibrium water content and pore wall thickness was observed with degradation time, whereas the hydrogel porosity and surface area density remained invariant. The results from microcomputed tomography corroborated with the rest of the measurements and indicated a bulk degradation mechanism of the macroporous hydrogels.