Applied Materials Today 12 (2018) 309–321 Contents lists available at ScienceDirect Applied Materials Today j ourna l h o mepage: www.elsevier.com/locate/apmt Injectable gellan-gum/hydroxyapatite-based bilayered hydrogel composites for osteochondral tissue regeneration D.R. Pereira a,b , R.F. Canadas a,b , J. Silva-Correia a,b , A. da Silva Morais a,b , M.B. Oliveira a,b , I.R. Dias a,b,d , J.F. Mano a,b , A.P. Marques a,b,c , R.L. Reis a,b,c , J.M. Oliveira a,b,c,∗ a 3B’s Research Group, I3Bs – Research Institute on Biomaterials, Biodegradables and Biomimetics, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, AvePark, Parque de Ciência e Tecnologia, Zona Industrial da Gandra, 4805-017 Barco, Guimarães, Portugal b ICVS/3B’s – PT Government Associate Laboratory, Braga/Guimarães, Portugal c The Discoveries Centre for Regenerative and Precision Medicine, Headquarters at University of Minho, Avepark, 4805-017 Barco, Guimarães, Portugal d Department of Veterinary Sciences, University of Trás-os-Montes e Alto Douro, Quinta de Prados, P.O. Box 1013, 5001-801 Vila Real, Portugal a r t i c l e i n f o Article history: Received 31 March 2018 Received in revised form 13 June 2018 Accepted 17 June 2018 Keywords: Injectable biomaterials Bilayered hydrogel composites Gellan-gum Hydroxyapatite Osteochondral regeneration Orthotopic knee model a b s t r a c t Multilayer systems capable of simultaneous dual tissue formation are crucial for regeneration of the osteochondral (OC) unit. Despite the tremendous effort in the field there is still no widely accepted system that stands out in terms of superior OC regeneration. Herein, we developed bilayered hydrogel composites (BHC) combining two structurally stratified layers fabricated from naturally derived and syn- thetic polymers, gellan-gum (GG) and hydroxyapatite (HAp), respectively. Two formulations were made from either low acyl GG (LAGG) alone or in combination with high acyl GG (HAGG) for the cartilage-like layer. Four bone-like layers were made of LAGG incorporating different ratios of hydroxyapatite (HAp). BHC were assembled in one single construct resulting in eight distinct bilayered constructs. Architectural observations by stereomicroscope and micro-CT (-CT) demonstrated a connected stratified structure with good ceramic dispersion within the bone-like layer. Swelling and degradation tests as well mechani- cal analyse showed a stable viscoelastic construct under dynamic forces. In-vitro studies by encapsulating rabbit’s chondrocytes and osteoblasts in the respective layers showed the cytocompatibility of the BHC. Further studies comprising subcutaneous implantation in mice displayed a weak immune response after four weeks. OC orthotopic defects in the rabbit’s knee were created and injected with the acellular BHC. OC tissue was regenerated four weeks after implantation as confirmed by cartilaginous and bony tissue formation assessed by histologic staining and -CT analysis. The successful fabrication of injectable BHC and their in-vitro and in-vivo performance may be seen as advanced engineered platforms to treat the challenging OC defects. © 2018 Elsevier Ltd. All rights reserved. 1. Introduction Osteochondral (OC) defects arise as a consequence of trauma, bone tumors, tissue resection, and metabolic diseases. When these affect the bone layer, a critical size lesion occurs and often leads to joint non-union. The OC unit is a multiphasic tissue comprising two distinct tissue types: the articular cartilage and the subchon- dral bone. Articular cartilage is a thin layer of soft anisotropic tissue responsible for protecting the surface of bones by reducing ∗ Corresponding author at: 3B’s Research Group, I3Bs – Research Institute on Biomaterials, Biodegradables and Biomimetics, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, AvePark, Parque de Ciência e Tecnologia, Zona Industrial da Gandra, 4805- 017 Barco, Guimarães, Portugal. E-mail address: miguel.oliveira@i3bs.uminho.pt (J.M. Oliveira). friction and facilitating joint movement and shock-absorbance, while the subchondral bone fulfils a crucial role in mechanical support [1]. The avascular nature, unique chemical and cellular composition make hyaline cartilage difficult to regenerate in lesions that exceed a critical size [2]. Although loose tissue forms during cartilage healing [3], the degradation of the repaired tissue, cartilage dehydration and tissue thinning contribute to the pro- gression of the lesion into deeper regions of the subchondral bone [4]. A wide range of clinical approaches has been exploited as pri- mary/secondary treatments for managing a full-thickness chondral defect. Autograft transplantation [5] and bone marrow stimu- lation techniques such as microfracture [6], and subchondral drilling [7] are the surgical procedures most often employed to treat lesions classified from grade II to IV by the Outerbridge classification [8–11]. Cell-based techniques such as autolo- gous chondrocyte implantation (ACI) [12] and matrix-associated https://doi.org/10.1016/j.apmt.2018.06.005 2352-9407/© 2018 Elsevier Ltd. All rights reserved.