Lipidic implants for controlled release of bioactive insulin: Effects on cartilage engineered in vitro

Abstract

Controlled release systems for growth factors and morphogens are potentially powerful tools for the engineering or the treatment of living tissues. However, due to possible instabilities of the protein during manufacture, storage, and release, in the development of new release systems it is paramount to investigate into the maintenance of bioactivity of the protein. Within this study, recently developed protein releasing lipid matrix cylinders of 2 mm diameter and 2 mm height made from glycerol tripalmitate were manufactured in a compression process without further additives. Insulin in different concentrations (0.2%, 1%, and 2%) served as model protein. The bioactivity of the protein released from the matrices was investigated in a long-term cartilage engineering culture for up to four weeks; additionally, the release profiles were determined using ELISA. Insulin released from the matrices increased the wet weights of the cartilaginous cell-polymer constructs (up to 3.2-fold), the amount of GAG and collagen in the constructs (up to 2.4-fold and 3.2-fold, respectively) and the GAG and collagen content per cell (1.8-fold and 2.5-fold, respectively), compared to the control. The dose-dependent effects on tissue development correlated well with release profiles from the matrices with different insulin loading. In conclusion, the lipid matrices, preserving the bioactivity of incorporated and released protein, are suggested as a suitable carrier system for use in tissue engineering or for the localized treatment of tissues with highly potent protein drugs such as used in the therapy of brain cancer or neurodegenerative CNS diseases.

Introduction

The field of tissue engineering (Langer and Vacanti, 1993) aims at the regeneration of mechanical and functional body tissue or organ defects that cannot be sufficiently cured by self-healing processes. One strategy in tissue engineering is to seed and culture cells on 3-D scaffold structures in vitro in order to generate tissue constructs for implantation. Cell proliferation and differentiation as well as the formation of an adequate extracellular matrix (ECM) in an in vitro culture largely depend on the supplementation of growth factors and other morphogens (Babensee et al., 2000). In addition growth factors can strongly improve the integration of the engineered tissue after implantation. These effects render growth factors an important tool for tissue engineering purposes, however, their efficacy is limited by their short half-lives and their potential toxicity at systemic levels (Babensee et al., 2000). To overcome these problems the use of protein carriers that ensure a sustained release and at the same time retain the biological activity of the growth factors is desirable (Tabata, 2003). Unfortunately, protein stability is easily compromised during the manufacture, storage, and drug release (Schwendeman et al., 1996). For example, for the well established biocopolymer poly(lactic-co-glycolic acid) (PLGA) it has been demonstrated that degradation products from the release matrix can influence protein stability due to changes in the microclimate of the microspheres during degradation, e.g., higher osmotic pressure or acidic environment (Lucke et al., 2002, Lucke and Goepferich, 2003). In order to overcome such problems, stabilizing additives were introduced such as Mg(OH)₂ (Zhu et al., 2000), Ca(OH)₂ (Zhu and Schwendeman, 2000), and, especially for insulin, zinc salts (Kim et al., 2001, Choi and Kim, 2003, Surendrakumar et al., 2003). The latter was additionally used to prolong the release of insulin (Brange and Langkjaer, 1992, Brange and Langkjaer, 1993, Cai et al., 2002). As an alternative approach, controlled release systems based on lipids as a matrix material have recently attracted increasing attention, as they avoid detrimental effects of breakdown products of the biomaterial (Thomas et al., 2004, Dellamary et al., 2004, Prego et al., 2005). However, the processes used for the production of a lipid matrix often include organic solvents likely resulting in organic–water interfaces, which in turn are known as destabilizing factor for proteins (Fu et al., 2000). We recently developed cylindrical matrices based on solid triglycerides, especially designed for the purpose of a long-term release (Vogelhuber et al., 2003). For the production of these protein-loaded matrices neither emulsions with organic solvents, surfactants nor ultrasonification are needed, which in other systems may lead to a loss of bioactivity of the incorporated proteins (Maschke et al., 2004). These matrices may not only be of major interest in the field of tissue engineering, but also can be loaded with proteins and other types of drugs for the local treatment of tissues such as needed in the treatment of brain cancer (Vogelhuber et al., 2003) or neurodegenerative CNS diseases.

Previously, we established a 3-D cartilage engineering culture that can be utilized as a test system for sustained-release carriers (Kellner et al., 2001). Readily available insulin is used as a model protein; insulin was demonstrated to have strong anabolic effects on engineered cartilaginous constructs similar to those of insulin-like growth factor-I (IGF-I). The model provides quantifiable data and responds sensitively to supplemented insulin in a dose-dependent manner over a cultivation period of several weeks (Kellner et al., 2001). Even if sustained-release carriers are typically applied in an in vivo situation, this 3-D culture offers the opportunity to evaluate newly developed release systems with regard to their effects within a defined tissue engineering setting.

In this study, insulin-loaded triglyceride matrices were manufactured in order to investigate the biological effects of released insulin in the 3-D cartilage engineering culture. The first specific aim was the determination of the release kinetics of matrices with varying amounts of incorporated insulin. Further specific aims were the analysis of the effects of released insulin on the tissue construct weight, cell number, and amounts of ECM components, namely glycosaminoglycans and collagen, within the engineered tissue.