Porous methacrylate scaffolds: supercritical fluid fabrication and in vitro chondrocyte responses
Introduction
The repair of damaged cartilage remains one of the major challenges of orthopaedics. Once damaged, cartilage has only limited potential to repair itself, because of the low mitotic activity of chondrocytes and the absence of vascularisation and innervation in cartilage. Current therapies for treatment of articular cartilage have varying degrees of success because they are aimed at diminishing the pain or joint swelling but cannot restore the original hyaline cartilage [1].
The last 10 years have seen the emergence of a novel approach that liberates chondrocytes from their matrix [2], [3]. The chondrocytes are cultured and expanded prior to being transplanted into cartilaginous defects. This however requires a method of delivery and stabilising the cells in the defects. Chondrocytes when cultured in a two-dimensional (2-D) monolayer culture lose their rounded morphology and assume a more fibroblastic appearance [4], [5]. This change in morphology is indicative of the loss of chondrocyte phenotype and is evidenced by changes in the production of extracellular matrix (ECM) proteins. The cells switch from production of cartilage specific type II collagen to the production of type I collagen and switch production of aggrecan to low molecular weight proteoglycan [2], [6]. For this purpose, a 3-D scaffold structure is used to aid chondrocyte delivery, attachment and proliferation.
Many 3-D scaffolds are being evaluated for a possible role in cartilage repair. These include: (a) biological scaffolds such as types I and II collagen, proteoglycans, hyaluronan and fibrin clots, (b) degradable materials such as poly(glycolic acid) and poly(lactic acid), and (c) non-degradable materials made from cellophane, silicone rubber, carbon fibres, PTFE (Teflon), polyester (Dacron), poly(HEMA) sponges, poly(vinyl alcohol) sponges. Many of these have shown success, with the repair tissue resembling normal hyaline cartilage [7], [8].
This paper describes the fabrication of porous 3-D scaffolds using a non-degradable polymer system consisting of poly(ethyl methacrylate)/tetrahydrofurfuryl methacrylate (PEMA/THFMA). Although non-biodegradable, the advantage of using this material is that it has been demonstrated to support the repair of full-thickness defects in vivo. Rabbits with osteochondral defects had the material implanted just below the level of the subchondral bone and after 8 months the defect had completely filled with dense cartilaginous tissue that was integrated into the surrounding normal cartilage [9], [10], [11]. In vitro studies, also using the non-porous PEMA/THFMA, have demonstrated that the material supported bovine chondrocyte growth and differentiation [8], [12], [13].
The ability of PEMA/THFMA to support such growth has been attributed to both surface and bulk properties of the polymer system. McFarland et al. [14] have demonstrated that the material appears to present adsorbed fibronectin in a more favourable conformation to support cell adhesion when compared to other polymers. Whilst such adsorption facilitated cell attachment, chondrocyte phenotype and morphology was maintained over extended periods [13]. The surface wettability of the polymer system may play a role here [15]. It has also been suggested that the water uptake properties of the material may contribute to the cartilage repair observed in vivo, by localising soluble factors from the surrounding environment to stimulate repair [9].
Many novel technologies have been applied to the manufacture of 3-D scaffolds. These include: mechanical stretching, fibre extrusion and bonding, template synthesis, phase separation and the use of gases and solvents as porogens (for review see Hutmacher [16]). Carbon dioxide gas above a critical temperature (Tc=31.1°C) and pressure (Pc=73.8 bar) functions as a porogen. In this state, the carbon dioxide is said to be supercritical (scCO2), and it is a unique processing medium with the properties of both gas and liquid. It is capable of diffusing into materials easily and extracting scCO2 soluble residues [17], [18]. Importantly some polymers when treated with scCO2 swell, creating porous foams examples include poly(methyl methacrylate) (PMMA), polystyrene, polycarbonate and poly(ethelyeneterephthalate). More recently the biomedical polymers poly(d,l-lactide) and poly(d,l-lactide-co-glycolide) have been foamed using scCO2 [19], [20], [21], [22], [23]. The foaming of these polymers relies on the following principles: (a) the polymer is saturated with carbon dioxide at high pressure, (b) the polymer/gas mixture is quenched into a supersaturated state by reducing the pressure, and (c) nucleation and growth of gas bubbles dispersed throughout the sample evolves until all thermodynamic forces driving mass transport vanish and we are left with a porous structure [24].
This paper reports the preparation of porous PEMA/THFMA foams for use as scaffolds, their characterisation and the response of chondrocytes to these novel materials in vitro.
Section snippets
Preparation of PEMA/THFMA discs
PEMA/THFMA polymer discs were made by mixing 5 g of PEMA powder (Bonar Polymers Ltd., Newton Aycliffe, UK) and 3 ml of THFMA (Rohm Chemie, Darmstadt, Germany) monomer liquid. N,N-dimethyl-p-toluidine (DMPT) was added, (2.5% v/v) to effect polymerisation. This mixture was placed in a custom fabricated PTFE mould to cure, producing discs 10 mm in diameter and 10 mm in thickness. The discs were left overnight to polymerise.
Foaming was achieved as follows. Discs were placed into a 10 ml Thar extraction
Characterisation of the discs
On removal from the mould, the polymerised PEMA/THFMA discs had a glassy opaque appearance. After foaming with scCO2 there were two notable changes in the discs. First, the volume of the discs was increased by ca. 4.5 times. The second change that could be seen was the discs had lost their glassy opaque appearance and were now white. Closer inspection of the foamed discs revealed three distinct regions: an outer skin, a porous region under this and at the centre of the discs was a glassy region
Microscopy
Chondrocytes grown on the Thermanox discs initially had a rounded morphology. Within 2 days of culture, the cells had started to spread and become fibroblastic in appearance. By day 3 the proportion of cells that were spreading out had increased and on day 4, multilayering of the cells had occurred. It was observed that in the confluent lower layer the cells were fibroblastic in appearance (data not shown). This is typical of chondrocytes grown in monoculture and has been used to indicate
Conclusions
Carbon dioxide is both a rapid and clean method of processing polymer scaffolds. It is non-toxic and leaves no solvent residues in the polymer matrix. We have demonstrated that scCO2 may be used to produce foamed scaffolds of PEMA/THFMA. This study has demonstrated that the non-porous PEMA/THFMA supported the chondrogenic phenotype of bovine chondrocytes for longer than Thermanox controls, an observation in agreement with previous work [8], [12], [13]. Our results demonstrate that the change in
Acknowledgements
The authors would like to acknowledge the assistance of the following persons in undertaking this work, Ms. R. Butler, Dr. A.I. Cooper (University of Liverpool) and Professor K.M. Shakesheff (University of Nottingham). In addition Ms. B. Sim, Mr. P. Fields, Mr. R. Wilson, and Mr. J.M. Whalley for their technical help and advice. Funding for this project was provided by grants from the BBSRC (JJAB) and EPSRC (HSG).
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2020, Methods in Cell BiologyCitation Excerpt :At this point, co-solvents can be added in small amounts to modify its polarity as well as the solvent power (Herrero, Cifuentes, & Ibañez, 2006). All these unique properties make scCO2 a suitable choice in many applications such as extraction of plant seeds (Herrero et al., 2006; McHugh & Krukonis, 2013; Santana, Jesus, & Larrayoz, 2012), carrying of drugs in the pharmaceutical field (Davies et al., 2008; Temtem et al., 2009; York, Kompella, & Shekunov, 2004), inactivation of viruses (Fages et al., 1998), sterilization of biomaterials and medical devices (Chang, Chen, Chen, Chen, & Yu, 2011), and diverse tissue engineering protocols (Quirk, France, Shakesheff, & Howdle, 2004; Woods, Silva, Nouvel, Shakesheff, & Howdle, 2004) such as polymer synthesis (Kazarian, 2000; Nalawade, Picchioni, & Janssen, 2006), porous scaffold fabrication (Barry, Gidda, Scotchford, & Howdle, 2004), nanoparticle formation (Atila, Yıldız, & Çalımlı, 2010; Byrappa, Ohara, & Adschiri, 2008; Fages, Lochard, Letourneau, Sauceau, & Rodier, 2004; Yeo & Kiran, 2005) and aerogel preparation (García-González, Camino-Rey, Alnaief, Zetzl, & Smirnova, 2012; Ramsey, Qiubai, Zhang, Zhang, & Wei, 2009). Recently, several scCO2 assisted decellularized tissues and natural matrices have been reported.