Alginate as a chondrocyte-delivery substance in combination with a non-woven scaffold for cartilage tissue engineering☆
Introduction
Hyaline cartilage defects still pose a major problem to orthopaedic and facial reconstructive surgeons, since these defects do not heal spontaneously with hyaline cartilage. The use of tissue-engineering techniques could be an appropriate way to generate hyaline cartilage grafts for the treatment of cartilage defects in the future. These techniques combine isolated cells with biodegradable scaffolds in order to stimulate production of new cartilage tissue with the typical characteristics of native hyaline cartilage. Autologous cells are generally preferred to avoid risks of immunological rejection and transmission of infectious diseases.
The ideal biodegradable scaffold should provide a preformed three-dimensional shape and initial mechanical strength. In addition it should make uniform cell spreading possible, stimulate the chondrogenic phenotype of the transplanted chondrocytes and prevent cells from floating out of the defect. Combinations of polylactic and polyglycolic acid are frequently used as cell carrier [1], [2], [3]. In previous studies, we obtained promising results using Ethisorb 210, a non-woven fleece composed of a polyglycolic–polylactic-copolymer punctually glued with polydioxanon. (Ethicon, Norderstedt, Germany) [4], [5], [6].
Although isolated chondrocytes can be seeded directly into the scaffold, one could also use a carrier gel for cell-seeding. Nowadays, alginate is frequently applied as cell-carrying gel, both in vitro and in vivo [6], [7], [8], [9], [10], [11], [12]. The use of alginate may facilitate a uniform distribution of chondrocytes in the relatively wide pores of the scaffold and prevent cells from floating out. Moreover, in vitro experiments showed that alginate can stimulate expression of the chondrogenic phenotype [13]. Whereas alginate has been used in humans for transplantation of pancreatic-islets cells without negative effects [14], alginate or its degradation products could possibly influence chondrocyte matrix neosynthesis.
In this study, we investigated the effect of the use of alginate as a chondrocyte-delivery substance for the construction of a cartilage graft. E210 was used as a scaffold and implanted in athymic mice, with and without alginate and with and without differentiated bovine articular chondrocytes.
Section snippets
Chondrocyte isolation
Full-thickness cartilage slices were harvested under sterile conditions from the metacarpophalangeal joints of 12–18 months old bovine steers, obtained from a local slaughter house, within eight hours after death. Chondrocytes were pooled from four different steers to exclude interindividual differences. The cartilage was washed with sterile physiological saline and incubated with protease XIV (2 mg/ml; Sigma, St. Louis, MO) for 2 h followed by an overnight incubation with collagenase B (1.5
Macroscopic analysis
All constructs were retrieved. When handling, the constructs without chondrocytes (construct F&FA) appeared very weak. In particular the ‘bare’ fleeces (construct F) were too weak to cut in four pieces for separate analysis without destroying the whole construct. The structure of the constructs with chondrocytes without alginate (construct FC) was firm. However, they had shrunk and warped. The constructs with chondrocytes in alginate (construct FAC) had retained their shape, were more elastic
Discussion
For clinical application, tissue-engineered cartilage should meet certain demands. The mechanical properties of the graft should allow surgical handling and in the long run, the shape of the graft should be retained. In this study, macroscopically the constructs with chondrocytes were all firm. However, the constructs with chondrocytes but without alginate were warped and appeared less elastic than the constructs with alginate. These two types of constructs did not differ in the amount of GAG,
Acknowledgements
This study was supported by the Sophia Foundation, the Foundation ‘Vereniging Trustfonds Erasmus University Rotterdam’ in the Netherlands and the Technology Foundation, applied science division of NWO and the technology programme of the ministry of economic affairs the Netherlands. The monoclonal antibodies II-II 6B3 and M38 were obtained from the Developmental Studies Hybridoma Bank, maintained by the Department of Pharmacology and Molecular Sciences, John Hopkins University School of
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This work was carried out at the combined cartilage research laboratory of the departments of Orthopaedics and Otorhinolaryngology, Erasmus University Medical Center, Rotterdam, The Netherlands.