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 °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 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.     

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.     

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.     

A Molded Hyaluronic Acid Gel as a Micro-Template for Blood Capillaries

Abstract

Fabrication of blood capillaries in tissue-engineered tissue is necessary for creating thick three-dimensional (3D) tissue with a high cellular density. For inducing blood capillaries in the tissue in vitro, a molded hyaluronic acid (HA) capillary-shaped gel was made as a template for blood capillaries by photolithography and power free pumping techniques. The fabricated HA capillary-shaped gel was sandwiched between two cell sheets consisting of neonatal normal human dermal fibroblasts (NHDFs), human umbilical vein endothelial cells (HUVECs), or co-cultured NHDFs and HUVECs, and eventually covered with the cells. Although a slight degradation of the HA gel was observed in the sandwiched tissue with HUVEC or NHDF cell sheets, significant degradation of the HA gel was observed in the sandwiched tissue with co-cultured cell sheets. Moreover, by continuing to culture the co-cultured tissue with HA gel, a tube formation was observed at the HA gel site. A sandwiched HA capillary-shaped gel with two cell sheets has a potential for creating blood capillaries in vitro and fabricating vascularized artificial organs.     

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.     

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.     

Hyaluronic acid-based hydrogels as 3D matrices for in vitro evaluation of chemotherapeutic drugs using poorly adherent prostate cancer cells

The current investigation aimed to develop a biomimetic, three-dimensional (3D) culture system for poorly adherent bone metastatic prostate cancer cells (C4-2B) for use as an in vitro platform for anti-cancer drug screening. To this end, hyaluronic acid (HA) derivatives carrying complementary aldehyde (HAALD) and hydrazide (HAADH) groups were synthesized and characterized. In situ encapsulation of C4-2B cells was achieved by simple mixing of HAALD and HAADH in the presence of the cells. Unlike two-dimensional (2D) monolayer culture in which cells adopt an atypical spread morphology, cells residing in the HA matrix formed distinct clustered structures which grew and merged, reminiscent of real tumors. Anti-cancer drugs added to the media surrounding the cell/gel construct diffused into the gel and killed the embedded cells. The HA hydrogel system was used successfully to test the efficacy of anti-cancer drugs including camptothecin, docetaxel, and rapamycin, alone and in combination, including specificity, dose and time responses. Responses of cells to anti-neoplastics differed between the 3D HA hydrogel and 2D monolayer systems. We suggest that the data obtained from 3D HA systems is superior to that from conventional 2D monolayers as the 3D system better reflects the bone metastatic microenvironment of the cancer cells.     

Injectable in situ cross-linking hyaluronic acid/carboxymethyl cellulose based hydrogels for drug release

A series of injectable in situ cross-linking hyaluronic acid/carboxymethyl cellulose based hydrogels (HA/CMC) was prepared via disulfide bonds by the oxidation of dissolved oxygen. The results showed that HA/CMC hydrogels exhibited tunable gelling time, appropriate rheology properties, high swelling ratio, good stability, and sustained drug release ability. The gelling time of HA/CMC hydrogels ranged from 1.4 to 7.0 min, and the values of the storage modulus, complex shear modulus, dynamic viscosity, and yield stress of HA3/CMC3 hydrogel were about 5869 Pa, 5870 Pa, 587 Pa·s, and 1969 Pa, respectively. The degradation percentage of HA1/CMC1, HA2/CMC2, and HA3/CMC3 hydrogels were about 60, 49, and 41% after incubating 42 days, and the in vitro cumulative release percentage of BSA from HA1/CMC1, HA2/CMC2, and HA3/CMC3 drug-loaded hydrogels were about 99, 91, and 82% after 30 days. The series of injectable in situ cross-linking HA/CMC hydrogels exhibited good comprehensive performance, signifying that these hydrogels could be potentially used in the fields of short- and medium-term controlled drug release, cell encapsulation, regenerative medicine, and tissue engineering.     

Enzymatically cross-linked hyaluronic acid/graphene oxide nanocomposite hydrogel with pH-responsive release

Hyaluronic acid (HA) is made up of repeating disaccharide units (β-1,4-d-glucuronic acid and β-1,3-N-acetyl-d-glucosamine) and is a major constituent of the extracellular matrix. HA and its derivatives which possess excellent biocompatibility and physiochemical properties have been studied in drug delivery and tissue engineering applications. Tyramine-based HA hydrogel with good compatibility to cell and tissue has been reported recently. However, inferior mechanical property may limit the biomedical application of the HA hydrogel. In this study, HA/graphene oxide (GO) nanocomposite (NC) hydrogel was prepared through a horseradish peroxidase catalyzed in situ cross-linking process. As compared with pure HA hydrogels, incorporation of GO to the HA matrix could significantly enhance the mechanical properties (storage moduli 1800 Pa) of the hydrogel and prolong the release of rhodamine B (RB) as the model drug from the hydrogel (33?h) as well. In addition, due to the multiple interactions between GO and RB, the NC hydrogels showed excellent pH-responsive release behavior. The release of RB from the NC hydrogel was prolonged at low pH (pH 4.0) in the presence of GO, which could be attributed to the enhanced interactions between GO and HA as well as with RB. In situ three-dimensional encapsulation of mouse embryonic fibroblasts (BALB 3T3 cells) in the NC hydrogels and cytotoxicity results indicated the cytocompatibility of both the enzymatic cross-linking process and HA/GO NC hydrogels (cell viability 90.6 ± 4.25%). The enzymatically catalyzed fabrication of NC hydrogels proved to be an easy and mild approach, and had great potential in the construction of both tissue engineering scaffolds and stimuli-responsive drug release matrices.     

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.     

Controlled release of vascular endothelial growth factor by use of collagen hydrogels

In vivo profile of vascular endothelial growth factor (VEGF) release from collagen hydrogels was investigated comparing that of hydrogel degradation while angiogenesis induced by the released VEGF was assessed. Collagen sponges were chemically cross-linked with different amounts of glutaraldehyde for various time periods. When ¹²⁵I-labeled collagen hydrogels incorporating VEGF were subcutaneously implanted into the back subcutis of mice, the hydrogel radioactivity decreased with time, the decrement profile depending on the cross-linking conditions. The radioactivity was retained for longer time periods as the glutaraldehyde concentration and cross-linking time increased. Implantation study of collagen hydrogels incorporating ¹²⁵I-labeled VEGF revealed that the remaining VEGF radioactivity decreased with time and the retention period was prolonged with the decreased hydrogel biodegradation. The slower the hydrogel degradation, the longer the period of VEGF retention. The collagen hydrogel incorporating VEGF induced significant angiogenesis around the implanted hydrogel, in marked contrast to VEGF in the solution form and VEGF-free empty hydrogel. The retention period of angiogenesis became longer with a decrease of the in vivo degradation rate of hydrogels. It is possible that the slower degraded hydrogel achieves a longer period of VEGF release, resulting in prolonged angiogenetic effect. We concluded that in our hydrogel system, biologically active VEGF was released as a result of in vivo degradation of the hydrogel.     

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.     

Chemically crosslinkable thermosensitive polyphosphazene gels as injectable materials for biomedical applications

Chemically crosslinkable and thermosensitive poly(organophosphazenes) containing multiple thiol (–SH) groups along with hydrophobic isoleucine ethyl ester and hydrophilic α-amino-ω-methoxy-poly(ethylene glycol) of the molecular weight 550 have been synthesized and characterized as an injectable biomaterial. The aqueous solutions of these polymers were transformed into hydrogel with desired gel strength at body temperature via hydrophobic interactions, and the gel strength was further improved by the cross-linking of thiol groups with crosslinkers, divinyl sulfone (VS) and PEG divinyl sulfone (PEGVS) under physiological conditions. The kinetics of cross-linking behavior of polymer thiol groups with crosslinkers was studied in both in vitro and in vivo conditions. Field Emission-Scanning Electron Microscopy (FE-SEM), swelling experiments, and rheology study of present polymers revealed that the inner three-dimensional hydrogel networks depended on the degree of thiol units in the polymer network. From the in vivo (in mice) degradation studies, the dual cross-linked gels showed to have a controlled degradation. These results demonstrate that the inner network of the hydrogels can be tuned, gel strength and degradation rate can be controlled, and the chemically crosslinkable and thermosensitive poly(organophosphazenes) hold promises for uses as injectable systems for biomedical applications including tissue engineering and protein delivery.     

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.     

Modulated degradation of hydrogels with thermo-responsive network in relation to their swelling behavior

Hydrogels consisting of poly(N-isopropylacrylamide-co-N,N-dimethylacrylamide-co-butyl methacrylate) and a novel biodegradable cross-linker were synthesized with different concentrations of ammonium peroxodisulfate (APS) as a free radical initiator by photo-induced radical reaction. The swelling ratio of the hydrogels decreased rapidly in proportion to temperature. In vitro enzymatic degradation of the hydrogels was examined in buffer solution containing papain at different temperatures. The enzymatic degradation of the hydrogel prepared with higher APS concentration was observed to proceed at 30°C, however, the hydrogel was not degraded above 35°C. Such an on-mloff switching degradation in response to temperature was not observed for hydrogels prepared with lower APS concentration. It is considered that modulated degradation of hydrogels with a thermo-responsive network is achieved by controlling the cross-link density and eliminating the entanglement of polymeric main chains.     

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.     

Synthesis of PAMAM dendrimer-based fast cross-linking hydrogel for biofabrication

Hydrogels possess great potential in biofabrication because they allow cell encapsulation and proliferation in a highly hydrated three-dimensional environment, and they provide biologically relevant chemical and physical signals. However, development of hydrogel systems that mimic the complexity of natural extracellular matrix remains a challenge. In this study, we report the development of a binary hydrogel system containing a synthetic poly(amido amine) (PAMAM) dendrimer and a natural polymer, i.e., hyaluronic acid (HA), to form a fast cross-linking hydrogel. Live cell staining experiment and cell viability assay of bone marrow stem cells demonstrated that cells were viable and proliferating in the in situ formed PAMAM/HA hydrogel system. Furthermore, introduction of a Arginylglycylaspartic acid (RGD) peptide into the hydrogel system significantly improved the cell viability, proliferation, and attachment. Therefore, this PAMAM/HA hydrogel system could be a promising platform for various applications in biofabrication.     

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.