ARTICLE IN PRESS
Biomaterials 24 (2003) 4273–4281
Poly(hydroxybutyrate-co-hydroxyhexanoate) promoted production of extracellular matrix of articular cartilage chondrocytes in vitro Ying Denga, Xing-Sun Linb, Zhong Zhenga, Jin-Guang Denga, Jin-Chun Chena, Hui Mab, Guo-Qiang Chena,* a
Department of Biological Sciences and Biotechnology, Tsinghua University, Beijing 100084, China b Department of Physics, Tsinghua University, Beijing 100084, China Received 25 November 2002; accepted 13 May 2003
Abstract The present investigation describes the production of extracellular matrix of rabbit articular cartilage chondrocytes grown on scaffolds of polyhydroxybutyrate (PHB) blended with poly(hydroxybutyrate-co-hydroxyhexanoate) (PHBHHx) for up to 7 days. The mRNA level of type II collagen of chondrocytes seeded on all scaffolds consisting of PHBHHx were obviously higher than that of PHB-only scaffold throughout the culture period, suggesting the positive effect of PHBHHx on extracellular matrix production. Second-harmonic generation (SHG) imaging technique, combined with confocal ﬂuorescence microscopy (CFM) revealed that PHBHHx in PHB scaffold provided better surface properties for anchoring type II collagen ﬁlaments and their penetration into internal layers of the scaffolds. Glycosaminoglycan (GAG), a major composition of extracellular matrix, showed a sharp increase in construct of 1:2 PHB/PHBHHx scaffold after 7 day cultivation, while only a small increase was observed in all other tested scaffolds. At the same time, total collagen contents in all scaffolds containing PHBHHx increased with time, with the maximum collagen production of 742.1799.2 mg/g dry weight observed in construct of 1:2 PHB/PHBHHx scaffold inoculated for 7 days, this was almost 4-fold higher than that in scaffold of PHB only. It appears that the presence of right proportion of PHBHHx in the composite system of PHB/PHBHHx highly favored the production of extracellular matrix of articular cartilage chondrocytes. r 2003 Elsevier Ltd. All rights reserved. Keywords: Polyhydroxyalkanoates; PHB; Type II Collagen; SHG; Chondrocytes
1. Introduction Tissue engineering of articular cartilage represents an exciting direction in the efforts to solve the complex problem of cartilage regeneration. To promote healing of cartilage defects, many researchers are turning toward a tissue engineering approach involving ex vivo fabrication of cartilage constructs by culturing cells on porous, resorbable scaffolds. Several candidate materials are available including glass [1,2], porous calcium ceramics [3,4], and biodegradable polymers. Biodegradable polymers are attractive for several reasons. They may be used to support cell growth in vitro [5–7], to conduct tissue growth in vivo [8–10]. In terms of bone regeneration, especially cartilage regeneration, numerous biode*Corresponding author. Tel: +86-10-62783844; fax: +86-1062788784. E-mail address: email@example.com (G.-Q. Chen). 0142-9612/03/$ - see front matter r 2003 Elsevier Ltd. All rights reserved. doi:10.1016/S0142-9612(03)00367-3
gradable polymers have been investigated for these purposes [11–13]. Polyhydroxybutyrate (PHB) is ﬁnding some interesting potential applications as an implant material due to its biocompatibility and resorbability [14,15]. PHB appears suitable for use as temporary stents, bone plates, patches, nails and screws [16,17]. In some cases, its brittle mechanical properties limit its use. For that reason, materials such as triethylcitrate have been used as a typical plasticizer . Blend copolymer of PHA consisting of 3-hydroxybutyrate and 3-hydroxyvalerate (P(HB-co-HV)) were reported to be widely used with its improved mechanical properties over that of PHB . Therefore, PHA with better elastomeric properties, such as poly(hydroxybutyrate-co-hydroxyhexanoate) (PHBHHx), is expected to meet special application as scaffolds for cartilage engineering . Our previous studies...
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