Elsevier

Biomaterials

Volume 25, Issues 7–8, March–April 2004, Pages 1339-1348
Biomaterials

In situ crosslinkable hyaluronan hydrogels for tissue engineering

https://doi.org/10.1016/j.biomaterials.2003.08.014Get rights and content

Abstract

We describe the development of an injectable, cell-containing hydrogel that supports cell proliferation and growth to permit in vivo engineering of new tissues. Two thiolated hyaluronan (HA) derivatives were coupled to four α,β-unsaturated ester and amide derivatives of poly(ethylene glycol) (PEG) 3400. The relative chemical reactivity with cysteine decreased in the order PEG-diacrylate (PEGDA)⪢PEG-dimethacrylate>PEG-diacrylamide>PEG-dimethacrylamide. The 3-thiopropanoyl hydrazide derivative (HA-DTPH) was more reactive than the 4-thiobutanoyl hydrazide, HA-DTBH. The crosslinking of HA-DTPH with PEGDA in a molar ratio of 2:1 occurred in approximately 9 min, suitable for an in situ crosslinking applications. The in vitro cytocompatibility and in vivo biocompatibility were evaluated using T31 human tracheal scar fibroblasts, which were suspended in medium in HA-DTPH prior to addition of the PEGDA solution. The majority of cells survived crosslinking and the cell density increased tenfold during the 4-week culture period in vitro. Cell-loaded hydrogels were also implanted subcutaneously in the flanks of nude mice, and after immunohistochemistry showed that the encapsulated cells retained the fibroblast phenotype and secreted extracellular matrix in vivo. These results confirm the potential utility of the HA-DTPH-PEGDA hydrogel as an in situ crosslinkable, injectable material for tissue engineering.

Introduction

Hydrogels are crosslinked hydrophilic polymer networks, and may absorb more than 1000× their dry weight in water, giving them physical characteristics similar to soft tissues. These biocompatible materials can be ideal for clinical applications [1], since adverse reactions are minimized [2], [3], [4]. Swelling and hydration occur without dissolution of the polymer, since the process of crosslinking creates an insoluble network. In addition, hydrogels are highly permeable, which facilitates exchange of oxygen, nutrients, and other water-soluble metabolites. Over the past three decades, chemically and physically diverse hydrogels have become standard materials for drug delivery, contact lenses, corneal implants, and scaffolds for the regeneration of new skin, encapsulation of cells, and regeneration of tendons, and cartilage [5], [6], [7], [8].

A major limitation of most scaffold materials used for tissue engineering is the need for surgical implantation. For many clinical uses, injectable in situ crosslinkable hydrogels would be strongly preferred for three main reasons. First, an injectable material could be formed into any desired shape at the site of injury. Because the initial materials could be sols or moldable putties, the systems may be positioned in complex shapes and then subsequently crosslinked to conform to the required dimensions. Second, the crosslinkable polymer mixture would adhere to the tissue during gel formation, and the resulting mechanical interlocking that would arise from surface microroughness would strengthen the tissue-hydrogel interface. Third, introduction of an in situ crosslinkable hydrogel could be accomplished by injection or laparoscopic methods, thereby minimizing the invasiveness of the procedure. During the last decade, many potential applications have been examined to-date for natural and synthetic polymeric systems [9], [10], [11], [12], [13], [14], [15], [16], [17], [18], [19], [20], [21], [22], [23], [24], [25], [26].

Development of an injectable hydrogel for tissue repair or tissue regeneration also presents considerable challenges. The gelation conditions for in vivo use are limited to a narrow range of physiologically acceptable temperatures, and the crosslinking must occur with no byproducts in a sensitive aqueous environment. Reagents must be nontoxic reagents and tolerant of moist, oxygen-rich environments. Furthermore, gelation must occur at a sufficiently rapid rate for clinical use in an outpatient or operating suite setting, yet sufficiently slow that complete mixing occurs prior to gelation. Hydrogels formed from photopolymerization of α,β-unsaturated esters and amides of PEG meet most of these requirements, and are consequently commonly used in tissue engineering [4], [12], [17], [18], [20], [21], [22], [23], [27], [28], [29], [30], [31]. However, there are limitations: the reactions may be too exothermic, particularly during radical polymerization, and in most cases photoinitiators must be used. Even though transdermal photopolymerization had been developed as a clinical approach, in many cases photopolymerization has not proven suitable for injectable in vivo use, despite the development of a transdermal photoactivation methodology [32].

Hyaluronan (HA) is a major constituent of the extracellular matrix (ECM), and is the only non-sulfated glycosaminoglycan (GAG) [33]. This polyanionic GAG is biocompatible, and biodegradable, and performs important biological functions, such as stabilizing and organizing the ECM [34], [35], regulating cell adhesion and motility [35], [36], and mediating cell proliferation and differentiation [37]. Thus, HA and its derivatives are now widely used in medicine [38], [39], [40], [41], [42], [43], [44], [45], [46], [47], [48], [49]. Recently, new crosslinking strategies were developed in order to prepare HA-based hydrogels, including the disulfide crosslinking of thiolated HA derivatives [50]. The resulting disulfide-crosslinked materials were cytocompatible, and murine L-929 fibroblasts, which were encapsulated in situ during air-induced hydrogel formation, remained viable and proliferated in vitro. However, the disulfide crosslinking reaction was too slow for injectable cell delivery.

Herein, we describe a new methodology to obtain chemically novel hydrogels in which thiol-modified GAGs can be crosslinked in situ in a fashion suitable for both cell encapsulation and in vivo injection, with subsequent tissue production. First, thiolated GAGs were synthesized [50], [51] and then hydrogels were fabricated based on the conjugate addition of thiols to α,β-unsaturated esters and amides of PEG. The resulting injectable hydrogels were evaluated in vitro with cultured human tracheal scar fibroblasts and by growth of new fibrous tissue in nude mice in vivo from in situ crosslinkable hydrogels seeded with human fibroblasts.

Section snippets

Materials

Fermentation-derived hyaluronan (HA, sodium salt, Mw 1.5 MDa) was provided by Clear Solutions Biotechnology, Inc. (Stony Brook, NY). 1-Ethyl-3-[3-(dimethylamino)propyl]-carbodiimide (EDCI), PEG acrylate (Mw 375) and PEG (Mw 3400 Da) were purchased from Aldrich Chemical Co. (Milwaukee, WI). PEG diamine (Mw 3400 Da, percent of substitution: 100% (1H NMR) and 95% (HPLC)) was purchased from Shearwater Polymers (Huntsville, AL). Dulbecco's phosphate buffered saline (DPBS), cysteine, and bovine

Relative rates of conjugate addition reactions

The conjugate addition of thiols to α,β-unsaturated esters and amides of PEG was selected for in situ gelation. The reaction is rapid, not exothermic, and both the thiolated components and unsaturated crosslinkers were readily prepared. Moreover, byproducts are minimized since the reaction is highly thiol-selective; competing reactions of hydroxyl, carboxylate, phosphate, and amine nucleophiles occurred several orders of magnitude more slowly in an aqueous environment at RT for the pH values

Conclusions

We described the preparation and evaluation of novel in situ crosslinkable hydrogels as biointegrative materials for tissue engineering. The reaction of four electrophilic derivatives of PEG 3400 with cysteine and with two thiol-containing HA derivatives was measured. An optimal system, HA-DTPH-PEGDA in a molar ratio of 2:1, had a gelation time of 9 min and was selected for in situ cell encapsulation and to measure growth and proliferation of human tracheal scar fibroblasts. When cultured in

Acknowledgements

We thank The University of Utah and the NIH (Grant DC04663 to the late S.D. Gray and G.D.P.) for financial support. Valuable discussions with Dr. J. Shelby, Dr. S.E. Morris, Ms. K.R. Kirker, and S.D. Gray contributed to the design and evaluation strategies for these new materials.

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    1

    Current address: Dipartimento di Chimica e Tecnologie Farmaceutiche, Universita’ di Palermo, via Archirafi, 32 90123Palermo, Sicily, Italy.

    2

    Current address: Barr Laboratories Inc., 2 Quaker Road, P.O. Box 2900, Pomona, New York 10970, USA.

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