Article
Natural/synthetic porous scaffold designs and properties for fibro-cartilaginous tissue engineering
Journal of Bioactive and Compatible Polymers 26(5) 437–451 ! The Author(s) 2011 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav DOI: 10.1177/0883911511420149 jbc.sagepub.com
A Borzacchiello1, A Gloria1, L Mayol2, Sally Dickinson3, S Miot4, I Martin4 and L Ambrosio1
Abstract The goal of this study was to produce and characterize the scaffolds by combining the advantages of both natural and synthetic polymers for engineering fibro-cartilaginous tissues. Porous threedimensional composite scaffolds were produced based on glycosaminoglycans and hyaluronic acid (HYAFF11) reinforced with polycaprolactone. The mechanical properties of scaffolds were evaluated as a function of time and compared with those of scaffolds seeded with human chondrocytes (constructs) and cultured in vitro up to 6 weeks. The composite scaffolds had a porosity of 68% with interconnected macropores with average pore sizes of 200 mm, an equilibrium swelling of 350%, and a predominant elastic behavior, typical of a macromolecular gel. The composite constructs maintained chondrocyte phenotype and degraded with the deposition of macromolecules synthesized by the cells. The scaffold presented mechanical properties and the ability to dissipate energy similar to the fibro-cartilaginous tissue. Keywords scaffolds, hyaluronic acid derivatives, PCL, mechanical properties, cartilage tissue engineering
Introduction Cartilage is an avascular tissue consisting of only one type of cells, namely chondrocytes, that are embedded in a matrix composed of collagen and proteoglycans.1 Adult cartilage 1 Institute of Composite and Biomedical Materials-C.N.R and Interdisciplinary Research Centre on Biomaterials-University of Naples ‘‘Federico II’’ Piazzale Tecchio 80, 80125 Naples, Italy 2 School of Biotechnological Sciences, Department of Pharmaceutical and Toxicological Chemistry, University of Naples, Federico II, Via D. Montesano 49, 80131 Naples, Italy 3 Department of Cellular & Molecular Medicine, University of Bristol, Bristol BS8 1TD, UK 4 Departments of Surgery and of Biomedicine, University Hospital Basel, Switzerland
Corresponding author: Assunta Borzacchiello, Institute of Composite and Biomedical Materials-C.N.R and Interdisciplinary Research Centre on Biomaterials-University of Naples ‘‘Federico II’’ Piazzale Tecchio 80, 80125 Naples, Italy Email:
[email protected]
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tissue has limited self-repair capacity due to the sparse distribution of highly differentiated, non-dividing chondrocytes, slow matrix turnover, low supply of progenitor cells, and lack of vascular supply.1,2 Innovative efforts to induce cartilage healing and regeneration are currently directed toward tissue engineering (TE) approaches.3 Despite significant advances in the development of innovative artificial matrices and perfusion systems, attempts to engineer cartilaginous tissues, such as articular cartilage and meniscus, have led to tissue with biochemical composition and mechanical properties inferior to those of the native tissue.4,5 A crucial role in this approach is played by the use of an appropriate scaffold.6 Numerous structural features are required for scaffolds in cartilage TE, such as high porosity, adequate pore sizes, and pore interconnectivity, which are necessary for cell seeding and nutrient diffusion. The scaffold material has to be noncytotoxic and should enable cell adhesion and proliferation.7 It has to provide adequate elasticity, controllable degradation, and resorption rates that match neo-tissue formation rates. Cartilage TE, both natural-based and synthetic polymers, have been used.8,9 Among the natural materials, hyaluronic acid, collagens, and proteoglycans have been incorporated into three-dimensional scaffolds. The mechanical properties of such scaffolds may be not sufficient for load-bearing applications.10 As to the synthetic scaffold, many polymers such as poly(glycolic acid), poly(lactic acid), and their copolymers have been used to produce three-dimensional (3D) scaffolds that possess the porosity, the degradation rate, and the mechanical properties suitable for these applications.11,12 The ideal scaffold for cartilage TE, besides being biocompatible and biodegradable, has been strong enough to withstand the load in a joint and to maintain structural integrity under loaded condition; moreover; it should have a degradation profile that allows in-growth of new tissue and remodeling under load.10 To achieve complete ingrowth and incorporation of partial or total meniscous prosthesis, the scaffold must possess interconnected macropores in the range 150–500 mm. As to mechanical properties, it has been suggested that an initial modulus of about 0.1 MPa, which is in the lower range of that of the native meniscus, positively affects the formation of fibro-cartilaginous tissue. One widely used synthetic polymer for biomedical and TE applications, based on its properties, such as biocompatibility and processability at low temperature and good mechanical properties, is poly("-caprolactone) (PCL).13–17 However synthetic polymers do not posses the biological cues that favor cell interaction and nor the level of biocompatibility of natural polymers. By combining the advantages of both natural and synthetic polymers, composite scaffolds made up of PCL and various natural polymers, such as elastin, hyaluronic acid (HA), chitosan, fibrin, and collagen type I were produced and tested for TE applications.18,19 Among the natural polymers, HA proved to be fundamentally better for cartilage homeostasis and chondrocyte microenvironment. However, unmodified HA has poor processability and handling properties and is severely hampered as a scaffold material for TE.20–23 To circumvent these limitations, HA was chemically modified by crosslinking or coupling reactions, which preserve its biological activity. A series of derivatives, called HYAFFÕ , were developed by the esterification of the carboxyl group of the glucuronic acid moiety of the polymer with linear or aromatic alcohol.24,25 The HA benzyl ester, HYAFF11Õ , is easier to process and is biocompatible in vitro and in vivo.26–29 In this study, novel porous 3D HYAFF11Õ /PCL scaffolds were produced for the reconstruction of fibro-cartilaginous tissue, such as the central part of the meniscus.
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Structural properties of the scaffolds were investigated by SEM analysis, microtomography, and mercury porosimetry. The swelling properties were determined by absorption tests and the mechanical properties evaluated both in compression and through small deformation shear tests. To determine the structural and assess the bio-functionality of the candidate scaffolds, the mechanical properties were evaluated as a function of time and compared to the scaffolds seeded with human chondrocytes (constructs) and cultured in vitro up to 6 weeks. The preliminary histological and biochemical properties of the constructs were also studied as a function of time.
Materials and methods Scaffold preparation Benzyl ester derivatives of hyaluronic acid powder, with 75% of carboxyl groups esterified (HYAFFÕ 11p75HE), referred as HYAFFÕ 11, were provided by FAB, Abano Terme. PCL pellets (294 mg/mL, Mw ¼ 65,000, Aldrich) were dissolved in a tetrahydrofuran/dimethyl sulfoxide 80/20 w/w solution, with stirring at 45 C. A granular mixture of 93.9% w/w NaCl (315–400 mm), 3.5% w/w NaHCO3, (140–300 mm), and 2.6% w/w citric acid (
