Synthesis and characterization of a novel hyaluronic acid hydrogel

Hyaluronic acid (hyaluronan, HA) has many medical applications as a biomaterial. To enhance its biostability, a novel hydrogel of cross-linked hyaluronic acid was prepared using a double cross-linking process, which involves building cross-linkages between hydroxyl group pairs and carboxyl group pairs. The present study explored a number of cross-linking processes in order to obtain different degrees of cross-linking, which were evaluated by the measurement of water absorption capacity as an index of the gel network density. To gain a better understanding of the stability of the gel, the chemical structure and particularly the rheological behaviour of the cross-linked HA, which included the influences of factors, such as degree of cross-linking, HA concentration and gel particle size, were investigated. The in vitro biostability against hyaluronidase and free radical degradation was tested to show that the cross-linked hydrogel had improved resistance to in vitro hyaluronidase and free radical degradation.     

Preparation of cross-linked hyaluronic acid film using 2-chloro-1-methylpyridinium iodide or water-soluble 1-ethyl-(3,3-dimethylaminopropyl)carbodiimide

In order to obtain much slower biodegradable films, which are often required for biomedical applications, we have developed a series of studies on heterogeneous cross-linking of hyaluronic acid (HA) films by using 2-chloro-1-methylpyridinium iodide (CMPI) or 1-ethyl-(3,3-dimethylaminopropyl)carbodiimide (EDC) as cross-linking reagents. From the in vitro degradation rate, we found that EDC cross-linked HA films completely dissolved in PBS at 37 degrees C during the period of 4-6 days. However, CMPI cross-linked HA films showed only a low percentage of weight loss over 30 days. This phenomenon could be explained from the mechanism of reaction between carboxyl group of HA and EDC. The latter reacted with carboxyl group to form an unstable intermediate O-acylurea, which showed a relatively low reactivity and quickly rearranged to form a stable N-acylurea. Thus, most of the EDC-activated carboxyl groups in HA were chemically transferred into N-acylurea or left as unreactive O-acylurea, and only a few of cross-linking bonds were formed between HA. On the other hand, the intermediate obtained from the reaction between carboxyl group and CMPI showed a relatively high reactivity and reacted with the hydroxyl group of the same and/or different molecules of HA to form an inter- and intramolecular esterification. Apparently, CMPI cross-linked HA films have a much higher cross-linking density and constructed a more rigid three-dimensional network. Therefore, it produced HA films, which dramatically increased its enzymatic stability in aqueous solution of hyaluronidase. The obtained results from elemental analyses, FT-IR spectra and NMR spectra also indicate that acylurea groups were introduced into EDC-cross-linked HA films.     

Preparation of cross-linked hyaluronic acid film using 2-chloro-1-methylpyridinium iodide or water-soluble 1-ethyl-(3,3-dimethylaminopropyl)carbodiimide

In order to obtain much slower biodegradable films, which are often required for biomedical applications, we have developed a series of studies on heterogeneous cross-linking of hyaluronic acid (HA) films by using 2-chloro-1-methylpyridinium iodide (CMPI) or 1-ethyl-(3,3- dimethylaminopropyl)carbodiimide (EDC) as cross-linking reagents. From the in vitro degradation rate, we found that EDC cross-linked HA films completely dissolved in PBS at 37 °C during the period of 4-6 days. However, CMPI cross-linked HA films showed only a low percentage of weight loss over 30 days. This phenomenon could be explained from the mechanism of reaction between carboxyl group of HA and EDC. The latter reacted with carboxyl group to form an unstable intermediate O-acylurea, which showed a relatively low reactivity and quickly rearranged to form a stable N-acylurea. Thus, most of the EDC-activated carboxyl groups in HA were chemically transferred into N-acylurea or left as unreactive O-acylurea, and only a few of cross-linking bonds were formed between HA. On the other hand, the intermediate obtained from the reaction between carboxyl group and CMPI showed a relatively high reactivity and reacted with the hydroxyl group of the same and/or different molecules of HA to form an inter- and intramolecular esterification. Apparently, CMPI cross-linked HA films have a much higher cross-linking density and constructed a more rigid three-dimensional network. Therefore, it produced HA films, which dramatically increased its enzymatic stability in aqueous solution of hyaluronidase. The obtained results from elemental analyses, FT-IR spectra and NMR spectra also indicate that acylurea groups were introduced into EDC-cross-linked HA films.     

An injectable vehicle for nucleus pulposus cell-based therapy

An injectable hydrogel, acting as a reservoir for cell delivery and mimicking the native environment, offers promise for nucleus pulposus (NP) repair and regeneration. Herein, the potential of a stabilised type II collagen hydrogel using poly(ethylene glycol) ether tetrasuccinimidyl glutarate (4S-StarPEG) cross-linker, enriched with hyaluronic acid (HA) was investigated. The optimally stabilised type II collagen hydrogel was determined by assessing free amine groups, resistance to enzymatic degradation, gel point. The potential toxicity of the cross-linker was initially assessed against adipose-derived stem cells (ADSCs). After addition of HA (molar ratio type II collagen:HA 9:0, 9:1, 9:4.5, 9:9) within the hydrogel, the behaviour of the encapsulated NP cells was evaluated using cell proliferation assay, gene expression analysis, cell distribution and cell morphology. A significant decrease (p < 0.05) in the free amine groups of collagen was observed, confirming successful cross-linking. Gelation was independent of the concentration of 4S-StarPEG (8 min at 37 °C). The 1 mm cross-linked hydrogel yielded the most stable after enzymatic degradation (p < 0.05). No toxicity of the 4S-StarPEG was noted for the ADSCs. NP cell viability was high regardless of the concentration of HA (>80%). A cell proliferation was not seen after 14 days in its presence. At a gene expression level, HA did not influence NP cells phenotype after seven days in culture. After seven days in culture, the type I collagen mRNA expression was maintained (p > 0.05). The optimally stabilised and functionalised type II collagen/HA hydrogel system developed in this study shows promise as an injectable reservoir system for intervertebral disc regeneration.     

Carbodiimide cross-linked hyaluronic acid hydrogels as cell sheet delivery vehicles: characterization and interaction with corneal endothelial cells

It was reported that cell-adhesive gelatin discs have been successfully used as delivery vehicles for intraocular grafting of bioengineered corneal endothelial cell sheets. Development of alternative biomaterials to bovine-based gelatin vehicles can potentially eliminate the risk of bovine spongiform encephalopathy. In the present work, to investigate whether it was appropriate for use as cell sheet delivery vehicles, 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC) cross-linked hyaluronic acid (HA) hydrogels were studied by determinations of morphological characteristic, mechanical and thermal property, water content, in vitro degradability and cytocompatibility. Glutaraldehyde (GTA) cross-linked HA samples were used for comparison. It was found that HA discs after cross-linking significantly increased its tensile stress but reduced its tensile strain, water uptake and enzymatic degradability. The results of differential scanning calorimetry demonstrated that cross-linking could lead to the alteration of polymer structure. In addition, the EDC-cross-linked HA discs had a smoother surface structure, a faster degradation rate and a relatively lower cytotoxicity as compared to the GTA cross-linked counterparts. It is concluded that EDC can be successfully applied for HA cross-linking to fabricate structurally stable, mechanically reinforced, readily deformable, transparent and cytocompatible HA hydrogel discs with the potential to be applied as delivery vehicles for corneal endothelial cell therapy.     

Crosslinked hyaluronic acid hydrogels: a strategy to functionalize and pattern

The physiological activity of hyaluronic acid (HA) polymers and oligomers makes it a promising material for a variety of applications. The development of HA-hydrogel scaffolds with improved mechanical stability against degradation and biochemical functionality may enhance their application to tissue engineering. In this report, a crosslinking strategy targeting the alcohol groups via a poly(ethylene glycol) diepoxide crosslinker was investigated for the generation of degradable HA hydrogels. To provide support for cell adhesion in vitro, collagen was incorporated into the HA solution prior to the crosslinking process. The hydrogels have a continuous exterior and a porous interior, with pore diameters ranging from 6 to 9 microm. HA and HA-collagen hydrogels degrade in the presence of hyaluronidase and collagenase enzymes, indicating that the chemical modification does not prevent biodegradation. Complete degradation of the hydrogels occurred within 14 days in hyaluronidase (100 U/ml) and 3 days in collagenase (66 U/ml). Pattern transfer was employed to introduce a surface topography onto the hydrogel, which was able to orient cell growth. Furthermore, the hydrogels could be functionalized with the biomolecule neutravidin by incorporation of biotin along the HA backbone. This biotinylation approach may allow attachment of bioactive molecules that are conjugated to avidin.     

Control of the molecular degradation of hyaluronic acid hydrogels for tissue augmentation

A novel protocol to control the molecular degradation of hyaluronic acid (HA) hydrogels was successfully developed for tissue augmentation applications. HA has a different conformational structure in water and organic solvent, and the carboxyl group of HA is known to be the recognition site of hyaluronidase and HA receptors. Based on these findings, HA was chemically modified by grafting adipic acid dihydrazide (ADH) to the carboxyl group of HA in the water to prepare HA-ADH(WATER) and in the mixed solvent of water and ethanol to prepare degradation-controlled HA-ADH(WATER/ETHANOL). Three kinds of HA hydrogels were prepared by the crosslinking of HA-ADH(WATER) or HA-ADH(WATER/ETHANOL) with bis(sulfosuccinimidyl) suberate, and by the crosslinking of HA-OH with divinyl sulfone (DVS). In vitro and in vivo degradation tests showed that HA-DVS hydrogels were degraded most rapidly, followed by HA-ADH(WATER) hydrogels and HA-ADH(WATER/ETHANOL) hydrogels. There was no adverse effect during and after in vivo degradation tests. All of the HA hydrogel samples appeared to be biocompatible, according to the histological analysis with hematoxylin-eosin and Alcian blue.     

Study on Chemical Cross-linking Modification of Hyaluronan and the Biocompatibility of its Derivatives

Objective: Prepare cross-linked HA gels with higher mechanical stability,lower degradation velocity and desirable biocompatibility,so as to extend the usage of HA.Method: 1.Test molecular weight of HA (MrHA) by viscosimetry;2.Prepare cross-linked HA gels by DVS,GTA,DEC;3.Discuss the cross-linking and degradation procedure;4,evaluate the biocompatibility of the best HA gels.Results: The mechanical stability and durability to degradation of HA-DVS gels are superior to those of other gels,and when HA :DVS = 40:1 (g/g),at 35℃ and in 0.2M NaOH solution,the HA-DVS gel shows the best mechanical stability,and its cytotoxicity reaches class I,hemolysis ratio is lower than 5 %.Conclusion:Our HADVS gel can be used to prepare biologic scaffolds.     

Synthesis and characterization of hyaluronic acid–poly(ethylene glycol) hydrogels via Michael addition: An injectable biomaterial for cartilage repair

Injectable hydrogels based on hyaluronic acid (HA) and poly(ethylene glycol) (PEG) were designed as biodegradable matrices for cartilage tissue engineering. Solutions of HA conjugates containing thiol functional groups (HA-SH) and PEG vinylsulfone (PEG-VS) macromers were cross-linked via Michael addition to form a three-dimensional network under physiological conditions. Gelation times varied from 14 min to less than 1 min, depending on the molecular weights of HA-SH and PEG-VS, degree of substitution (DS) of HA-SH and total polymer concentration. When the polymer concentration was increased from 2% to 6% (w/v) in the presence of 100 U ml^?1 hyaluronidase the degradation time increased from 3 to 15 days. Hydrogels with a homogeneous distribution of cells were obtained when chondrocytes were mixed with the precursor solutions. Culturing cell–hydrogel constructs prepared from HA185k-SH with a DS of 28 and cross-linked with PEG5k-4VS for 3 weeks in vitro revealed that the cells were viable and that cell division took place. Gel–cell matrices degraded in approximately 3 weeks, as shown by a significant decrease in dry gel mass. At day 21 glycosaminoglycans and collagen type II were found to have accumulated in hydrogels. These results indicate that these injectable hydrogels have a high potential for cartilage tissue engineering.     

Bone regeneration using hyaluronic acid-based hydrogel with bone morphogenic protein-2 and human mesenchymal stem cells

Acrylated hyaluronic acid (HA) was used as a scaffold for bone morphogenic protein-2 (BMP-2) and human mesenchymal stem cells (hMSCs) for rat calvarial defect regeneration. HA was acrylated by two-step reactions: (1) introduction of an amine group using adipic acid dihydrazide (ADH); (2) acrylation by N-acryloxysuccinimide. Tetrathiolated poly(ethylene) glycol (PEG-SH(4)) was used as a cross-linker by a Michael-type addition reaction and the hydrogel was formed within 10min under physiological conditions. This hydrogel is degraded completely by 100U/ml hyaluronidase in vitro. hMSCs and/or BMP-2 was added during gelation. Cellular viability in vitro was increased up to 55% in the hydrogels with BMP-2 compared with the control. For in vivo calvarial defect regeneration, five different samples (i.e., control, hydrogel, hydrogel with BMP-2, hydrogel with MSCs, and hydrogel with BMP-2 and MSCs) were implanted for 4 weeks. The histological results demonstrated that the hydrogels with BMP-2 and MSCs had the highest expression of osteocalcin and mature bone formation with vascular markers, such as CD31 and vascular endothelial growth factors, compared with the other samples. This study demonstrated that HA base hydrogel can be used for cell and growth factor carriers for tissue regeneration.     

An Injectable Oxidated Hyaluronic Acid/Adipic Acid Dihydrazide Hydrogel as a Vitreous Substitute

Vitrectomy is a common procedure for treating ocular-related diseases. The surgery involves removing the vitreous humor from the center of the eye, and vitreous substitutes are needed to replace the vitreous humor after vitrectomy. In the present study, we developed a colorless, transparent and injectable hydrogel with appropriate refractive index as a vitreous substitute. The hydrogel is formed by oxidated hyaluronic acid (oxi-HA) cross-linked with adipic acid dihydrazide (ADH). Hyaluronic acid (HA) was oxidized by sodium periodate to create aldehyde functional groups, which could be cross-linked by ADH. The refractive index of this hydrogel ranged between 1.3420 and 1.3442, which is quite similar to human vitreous humor (1.3345). The degradation tests demonstrated that the hydrogel could maintain the gel matrix over 35 days, depending on the ADH concentration. In addition, the cytotoxicity was evaluated on retina pigmented epithelium (RPE) cells cultivated following the ISO standard (tests for in vitro cytotoxicity), and the hydrogel was found to be non-toxic. In a preliminary animal study, the oxi-HA/ADH hydrogel was injected into the vitreous cavity of rabbit eyes. The evaluations of slit-lamp observation, intraocular pressure, cornea thickness and histological examination showed no significant abnormal biological reactions for 3 weeks. This study suggests that the injectable oxi-HA/ADH hydrogel should be a potential vitreous substitute.     

In situ cross-linkable hyaluronic acid hydrogels prevent post-operative abdominal adhesions in a rabbit model

We studied the efficacy of an in situ cross-linked hyaluronic acid hydrogel (HAX) in preventing post-surgical peritoneal adhesions, using a rabbit sidewall defect-cecum abrasion model. Two cross-linkable precursors were prepared by modifying hyaluronic acid with adipic dihydrazide and aldehyde, respectively. The hydrogel precursors cross-linked to form a flexible hydrogel upon mixing. The hydrogel was biodegradable and provided a durable physical barrier, which was highly effective in reducing the formation of post-operative adhesions. Ten out of 12 animals in the untreated control group developed fibrous adhesions requiring sharp dissection, while only 2 out of 8 animals treated with HAX gels showed such adhesions, and those occurred in locations that were not covered by the hydrogel. We also studied means by which gel degradation time can be modulated by varying the precursor concentration and molecular weight.     

Anti-inflammatory function of an in situ cross-linkable conjugate hydrogel of hyaluronic acid and dexamethasone

Postoperative peritoneal adhesions cause pelvic pain, infertility, and potentially lethal bowel obstruction. We have designed and synthesized an injectable hydrogel composed of cross-linkable modified hyaluronic acids (HAs) conjugated to dexamethasone (HAX-DEX), and investigated its anti-inflammatory function. HAX-DEX formed a hydrogel in <1min by cross-linking reactions between aldehyde groups and hydrazide groups. The hydrogel degraded in media over 5 days, releasing dexamethasone slowly over that time, reducing TNF-alpha and IL-6 production from lipopolysaccharide-stimulated primary mouse macrophages in vitro. HAX-DEX was biocompatible on subcutaneous injection, and caused less inflammation than unmodified cross-linked HA.     

Injectable hyaluronic acid microhydrogels for controlled release formulation of erythropoietin

An injectable hyaluronic acid (HA) microhydrogel was successfully developed as a novel drug carrier for controlled release formulation of protein drugs. HA hydrogels were prepared by the disulfide bond formation of thiolated HA (HA-SH). EPO was loaded in situ during HA-SH hydrogel preparation using an accelerating agent of sodium tetrathionate. The gelation time was drastically reduced from a day to 30 min when sodium tetrathionate was added for HA-SH hydrogel preparation. In vitro release of EPO in PBS at 37 degrees C showed that EPO was rapidly released for 3 days with an initial burst and then slowly up to 9 days from HA-SH hydrogels. HA-SH microhydrogels were prepared by the reactive spray drying of diluted HA-SH precursor solution. The mean particle size was approximately 2.3 mum and the water content after spray drying was approximately 14%. Ellman's test showed that sodium tetrathionate contributed not only for rapid crosslinking reaction but also for the reduction of residual free thiol content in HA-SH microhydrogels after spray drying. EPO recovery from HA-SH microhydrogels after degradation with hyaluronidase SD was higher than 95%. The released EPO appeared to be intact from the analysis with RP-HPLC. According to in vivo release test of EPO from HA-SH microhydrogels in Sprague Dawley (SD) rats, elevated plasma concentration of EPO higher than 0.1 ng/mL, which is a critical minimal concentration for EPO efficacy, was maintained up to 7 days. There was no adverse effect during and after the in vivo tests.     

Modulation of mesenchymal stem cell chondrogenesis in a tunable hyaluronic acid hydrogel microenvironment

An injectable and biodegradable hydrogel system comprising hyaluronic acid-tyramine (HA-Tyr) conjugates can safely undergo covalent cross-linking in vivo by the addition of small amounts of peroxidase and hydrogen peroxide (H(2)O(2)), with the independent tuning of the gelation rate and degree of cross-linking. Such hydrogel networks with tunable mechanical and degradation properties may provide the additional level of control needed to enhance chondrogenesis and overall cartilage tissue formation in vitro and in vivo. In this study, HA-Tyr hydrogels were explored as biomimetic matrices for caprine mesenchymal stem cells (MSCs) in cartilage tissue engineering. The compressive modulus, equilibrium swelling and degradation rate could be controlled by varying the concentration of H(2)O(2) as the oxidant in the oxidative coupling reaction. Cellular condensation reflected by the increase in effective number density of rounded cells in lacunae was greater in softer hydrogel matrices with lower cross-linking that displayed enhanced scaffold contracture. Conversely, within higher cross-linked matrices, cells adopted a more elongated morphology, with a reduced degree of cellular condensation. Furthermore, the degree of hydrogel cross-linking also modulated matrix biosynthesis and cartilage tissue histogenesis. Lower cross-linked matrix enhanced chondrogenesis with increases in the percentage of cells with chondrocytic morphology; biosynthetic rates of glycosaminoglycan and type II collagen; and hyaline cartilage tissue formation. With increasing cross-linking degree and matrix stiffness, a shift in MSC differentiation toward fibrous phenotypes with the formation of fibrocartilage and fibrous tissues was observed. These findings suggest that the tunable three-dimensional microenvironment of the HA-Tyr hydrogels modulates cellular condensation during chondrogenesis and has a dramatic impact on spatial organization of cells, matrix biosynthesis, and overall cartilage tissue histogenesis.     

交联透明质素特性的研究

目的研究交联透明质素的特征和性能,为其应用于临床提供理论依据。方法采用扫描电镜-能谱仪分析交联透明质素的化学成分。采用傅立叶变换红外光谱法对交联透明质素结构进行分析。使用旋转流变仪测定交联透明质素凝胶的储能模量、耗能模量及粘度等流变学性能的动态变化。采用激光粒度仪测定凝胶中微粒的大小及分布。结果能谱分析显示交联透明质素中主要成分与天然透明质素相同,但多了硫原子,该元素来自交联剂。红外光谱测定显示,交联透明质素的吸收特征基本与未交联透明质素相似,只是在示硫砜键振动的吸收加强。交联透明质素凝胶的储能模量、耗能模量显著高于天然透明质素凝胶,与频率的依赖性不明显,表现为强凝胶弹性体特征,凝胶微粒平均粒径约为500μm。结论交联透明质素是天然透明质素与二乙烯基砜交联反应形成的多聚物,该多聚物溶胀于水后生成水不溶性凝胶。交联透明质素微粒凝胶制剂具有稳定性好和粘弹性高等优点,有利于进一步拓展其临床应用。     

Chemical cross-linking of proteins of the influenza virion

Purified influenza virus (A/FPV/Rostock/34;H7N1) was exposed briefly to pH 5 before returning to an alkaline pH. Virus was then reacted with one of three chemical cross-linking reagents [dimethyl suberimidate (DMS), tartryl diazide (TDA), or formaldehyde which span 11, 6, and 2A, respectively]. Cross-linked polypeptides were analysed by SDS-polyacrylamide gel electrophoresis under reducing conditions and identified with monospecific antisera against HA1, HA2, NP and M1. Acidification resulted in changes in the cross-linking patterns for both HA1 and HA2 which could be detected with all three reagents. Most notable were the data with formaldehyde: under alkaline conditions cross-linking gave only HA1:HA2 heteropolymers but after brief acidification none of these were formed and in their place was a novel HA1 homodimer, an HA2 homotrimer and an HA2 of Mr 50k cross-linked to form a homodimer with another HA2 or to a heterodimer with M1. Although cross-linking by formaldehyde was much more affected by acidification of the virus than cross-linking by DMS or TDA, over half the polymers cross-linked by DMS were no longer formed after acidification. The patterns of cross-linking of NP and M1 were unchanged by low pH treatment.     

The effect of hyaluronic acid incorporation on fibroblast spreading and proliferation within PEG-diacrylate based semi-interpenetrating networks

The nanometer-scale mesh size of many synthetic crosslinked hydrogel networks restricts encapsulated cells to a rounded morphology that can inhibit cellular processes such as proliferation and migration that are essential for the early stages of remodeling and tissue formation. The objective of these studies was to investigate an approach for accelerating cellular remodeling based on the creation of semi-interpenetrating networks (IPNs) composed of hydrolytically degradable poly(ethylene glycol) (PEG) diacrylate macromers and native, enzymatically degradable extracellular matrix (ECM) components (collagen, gelatin and hyaluronic acid (HA)). Among the three ECM components investigated, addition of HA at concentrations of 0.12% w/v and greater supported fibroblast spreading throughout the three-dimensional network and significantly increased proliferation relative to control hydrogels without HA. Incorporation of HA resulted in relatively small changes in hydrogel physical/chemical properties such as swelling, degradation rate, and elastic modulus. Fibroblast spreading was eliminated by the addition of hyaluronidase inhibitors, demonstrating that cell-mediated enzymatic degradation of HA is a necessary mechanism responsible for the observed increases in fibroblast activity. By accelerating early cellular remodeling and growth, these semi-IPNs may be useful vehicles for cell transplantation in a variety of tissue engineering applications.     

Fabrication and characterization of porous hyaluronic acid-collagen composite scaffolds

Hyaluronic acid (HA) plays a vital role in many tissues, influencing water content and mechanical function, and has been shown to have positive biological effects on cell behavior in vitro. To begin to determine whether these benefits can be accessed if HA is incorporated into collagen-based scaffolds for tissue engineering, HA-collagen composite matrices were prepared and selected properties evaluated. HA-collagen scaffolds were cross-linked with carbodiimide and loss rates of HA in culture medium assessed. Scaffold pore structures were evaluated by light and electron microscopy. Adult canine chondrocytes were grown in selected HA-collagen scaffolds to assess the effects of HA on cell behavior. Homogenous HA-collagen slurries were achieved when polyionic complexes were suppressed. HA was uniformly distributed through the scaffolds, which demonstrated honeycomb-like pores with interconnectivity among pores increasing as HA content increased. Virtually all of the HA added to the collagen slurry was incorporated into the composite scaffolds that underwent a 7-day cross-linking protocol. After 5 days in culture medium, the HA content in the scaffolds was 5-7% regardless of initial HA loading. After only 2 weeks in culture cartilaginous tissue was found in the chondrocyte-seeded HA-collagen scaffolds. This study contributes to the understanding of the effects of HA content, pH, and cross-link treatment on pore characteristics and degradation behavior essential for the design of HA-collagen scaffolds. The demonstration that these scaffolds can be populated by chondrocytes and support in vitro formation of cartilaginous tissue warrants further investigation of this material system for tissue engineering.     

Controlled degradation of hydrogels using multi-functional cross-linking molecules

Hydrogels, chemically cross-linked or physically entangled, have found a number of applications as novel delivery vehicles of drugs and cells. However, the narrow ranges of degradation rates and mechanical strength currently available from many hydrogels limits their applications. We have hypothesized that utilization of multi-functional cross-linking molecules to form hydrogels could provide a wider range and tighter control over the degradation rates and mechanical stiffness of gels than bi-functional cross-linking molecules. To address the possibility, we isolated alpha-L-guluronate residues of sodium alginate, and oxidized them to prepare poly(aldehyde guluronate) (PAG). Hydrogels were formed with either poly(acrylamide-co-hydrazide) (PAH) as a multi-functional cross-linking molecule or adipic acid dihydrazide (AAD) as a bi-functional cross-linking molecule. The initial properties and degradation behavior of both PAG gel types were monitored. PAG/PAH hydrogels showed higher mechanical stiffness before degradation and degraded more slowly than PAG/AAD gels, at the same concentration of cross-linking functional groups. The enhanced mechanical stiffness and prolonged degradation behavior could be attributed to the multiple attachment points of PAH in the gel at the same concentration of functional groups. This approach to regulating gel properties with multifunctional cross-linking molecules could be broadly used in hydrogels.