Donate, Ricardo

Liu, Chaozong

Monzón, Mario

Oliveira, J Miguel

Ribeiro, Viviana

Tamaddon, Maryam

English

UCL Discovery

 Translation through collaboration: practice applied in BAMOS project in in vivo testing of innovative osteochondral scaffolds  Running title: In vivo tests of osteochondral scaffolds in BAMOS  Ricardo Donate1,*, Maryam Tamaddon2, Viviana Ribeiro3,4, Mario Monzón1,*, J. Miguel Oliveira3,4,*, Chaozong Liu2,*  1 Departamento de Ingeniería Mecánica, Grupo de Investigación en Fabricación Integrada y Avanzada, Universidad de Las Palmas de Gran Canaria, Las Palmas, Spain; 2 Division of Surgery & Interventional Science, University College London, Royal National Orthopaedic Hospital, Stanmore, UK; 3 3B's Research Group, I3B's-Research Institute on Biomaterials, Biodegradables and Biomimetics, University of Minho, Barco, Guimarães, Portugal; 4 ICVS/3B's-PT Government Associated Laboratory, Braga, Guimarães, Portugal.  *Corresponding authors: Ricardo Donate, ricardo.donatelpgc.es; Mario Monzón, mario.monzon@ulpgc.es; J. Miguel Oliveira, miguel.oliveira@i3bs.uminho.pt; Chaozong Liu, chaozong.liu@ucl.ac.uk.  Key Words: bone; cartilage; in vivo evaluation; regenerative medicine; tissue engineering  Abstract Osteoarthritis is the most common chronic degenerative joint disease, recognized by the World Health Organization as a public health problem that affects millions of people worldwide. BAMOS project, funded under the frame of the Horizon 2020 RISE program, aims to delay or avoid the use of joint replacements by developing novel cost-effective osteochondral scaffold technology for early intervention of osteoarthritis. This project brings together five internationally leading research organisations (Universidad de Las Palmas de Gran Canaria, Spain; University of Minho, Portugal; University College London, UK; Xi’an Jiaotong University and Zhejiang University, China) and two healthcare providers (Royal National Orthopaedic Hospital, UK; and Saúde Atlântica - Gestão Hospitalar, S.A., Portugal) to work on the development, manufacturing and marketing of osteochondral scaffolds for the repair of large cartilage damages in osteoarthritis patients. The multidisciplinary consortium of BAMOS collaborates through research and innovation staff  exchanges. The project covers all the stages of the development before the clinical trials: design of scaffolds, biomaterials development, processability under additive manufacturing, in vitro test, and in vivo test. This paper reports the translational practice adopted in the project in in vivo assessment of the osteochondral scaffolds developed.   Graphical Abstract The main results of the in vivo evaluations carried out in BAMOS project, funded under Horizon 2020 RISE program, are summarized. Animal models of osteochondral defect have been used to assess the biological performance of the different multimaterial and multilayered scaffolds developed.       Osteoarthritis is mainly characterized by articular cartilage progressive loss, osteophyte formation, synovial membrane inflammation and thickening of the subchondral bone, which leads to the generation of osteochondral (OC) defects with limited self-healing capacity. OC scaffold is a tissue engineering approach aiming to repair a joint defect and restore its function to delay or eliminate the need for a joint replacement. Although they have been established and are promising for the  treatment of small OC defects, no products to date have demonstrated the appropriate biomechanical properties required to promote successful long-lasting regeneration of large OC defects. BAMOS project addresses this challenge in osteoarthritis treatment [1] with the following main objectives: a) Define clinical specifications for OC scaffolds, b) develop new OC scaffolds biomaterials, c) develop innovative additive manufacturing techniques to produce patient-tailored OC scaffolds, d) assess the OC scaffold in both in vitro and in vivo, and e) train early-stage researchers in the context of collaborative research.  Bilayered OC scaffolds: After a complete physicochemical and in vitro characterization, the different hierarchical OC scaffolds developed in the context of BAMOS project were evaluated using in vivo models. Thus, for example, a rabbit knee critical size OC defect model was used for assessing in vivo OC regeneration when implanting a horseradish peroxidase cross-linked silk fibroin-based (HRP-SF) scaffold [2]. These three-dimensional structures were prepared by combining two distinct layers: an HRP-SF cartilage-like layer, and a subchondral bone-like layer composed of HRP-SF and beta-tricalcium phosphate (β-TCP) particles doped with zinc and strontium (HRP-SF/ZnSr-β-TCP) [3]. After 8 weeks of implantation, the hierarchical scaffolds showed good tissue integration (with no signs of inflammatory reactions), cartilage tissue regeneration and calcified tissue formation. Formation of collagen type II and glycosaminoglycans in the HRP-SF articular cartilage-like layer was confirmed by histological analyses, while de novo bone ingrowth and blood vessels infiltration were observed in the HRP-SF/ZnSr-β-TCP bone-like layer [2]. These OC bilayered scaffolds have also shown to possess adequate structural integrity, memory-shape properties, and excellent mechanical and in vitro biological properties, even preventing bacterial biofilm formation [3], which in sum confirms the potential of these structures to be used in OC tissue engineering applications. Also using a rabbit model, bilayered scaffolds were implanted into OC defects created at the distal femoral trochlea and tested for 24 weeks [4]. These scaffolds were composed of a titanium (Ti) matrix that served as bone layer, and a collagen/poly(lactic-co-glycolic acid) layer intended for cartilage regeneration. We concluded that the mechanical support provided by the Ti layer promoted subchondral bone formation and new tissue integration, which led to better cartilage regeneration.  Trilayered OC scaffolds: Following a different approach, multi-material trilayered OC scaffolds were also developed in BAMOS by combining additive manufacturing and other conventional technologies [5]: • Casting and freeze-drying methods were used to obtain a collagen/poly(lactic-co-glycolic acid) composite layer to act as cartilage-like layer.  • Material extrusion of polymers from a heated nozzle (MEX-TRB/P), commonly referred as fused deposition modelling, was used to manufacture a polylactic acid-based two-part junction layer that served as calcified cartilage of the hierarchical scaffold. • Powder bed fusion of metal (PBF-LB/M) was used to produce a porous Ti matrix intended for bone regeneration. The 12-week in vivo evaluation of the proposed OC trilayered scaffold, carried out in a sheep stifle condyle model, showed a stable mechanical fixation of the three-dimensional structure on the implantation site with no adverse effects observed on the surrounding tissues [6]. Improved bone ingrowth into the Ti matrix, as well as enhanced formation of hyaline-like cartilage tissue, were reported after histological examinations. The up-regulation of the cartilage-related markers aggrecan and collagen type-II confirmed the capacity of the proposed three-dimensional structures to regenerate cartilage tissue. In summary, the results obtained showed the potential of these cell-free scaffolds to be applied in the treatment of large OC defects.  Other in vivo tests carried out in BAMOS: Interestingly, the incorporation of bone marrow concentrate into the developed trilayered scaffolds has led to a non-significant improvement in bone regeneration when treating OC defects [7]. In this case, an ovine stifle condyle model was used during a 6-month test. Despite obtaining no significantly higher quantity of newly formed bone when using the Ti-polylactic acidPLA-poly(lactic-co-glycolic acid)/collagen scaffold, the results suggested that enhanced bone homogeneity and biomechanical durability were obtained when implanting the trilayered scaffolds (seeded with bone marrow concentrate), thus producing a higher quality of new subchondral bone tissue. Similarly, no statistically significant differences in terms of OC regeneration were obtained between collagen/hydroxyapatite scaffolds with or without bone marrow concentrate when tested in vivo using an ovine femoral condyle model for 6 months [8]. An ovine condyle model was also used to validate a novel numerical model developed in the context of BAMOS project, which is intended to optimize the scaffold design and material properties but also to predict its final biological performance [9]. The simulated cell distribution in the scaffold matched well with the in vivo regenerated bone tissue distribution. Therefore, this model could help reduce the number of preliminary time- and cost-consuming in vivo and in vitro tests needed to optimize the scaffold design.   Author contributions  MM: Resources, Writing—Original Draft, Project administration, Funding acquisition. Ricardo Donate: Writing—Original Draft, Visualization. Maryam Tamaddon: Writing—Review and Editing. Viviana Ribeiro: Writing—Review and Editing. Chaozong Liu: Resources, Writing—Review and Editing. J. Miguel Oliveira: Resources, Writing—Review and Editing. All authors approved the final version of this manuscript. Financial support The work was supported by the H2020‐MSCA‐RISE program, which is part of the developments carried out in BAMOS project, and the European Union's Horizon 2020 Research and Innovation Programme (No. 734156). Acknowledgement Not applicable. Conflicts of interests statement The authors have no competing interests to declare. Data sharing statement  This is an open access journal, and articles are distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 4.0 License, which allows others to remix, tweak, and build upon the work non-commercially, as long as appropriate credit is given and the new creations are licensed under the identical terms.   References 1. Monzón M. Biomaterials and additive manufacturing: osteochondral scaffold innovation applied to osteoarthritis (BAMOS project). J Zhejiang Univ A 2018;19:329–30. https://doi.org/10.1631/JZUS.A18NW001. 2. Ribeiro VP, Pina S, Canadas RF, da Silva Morais A, Vilela C, Vieira S, et al. In Vivo Performance of Hierarchical HRP-Crosslinked Silk Fibroin/β-TCP Scaffolds for Osteochondral Tissue Regeneration. Regen Med Front 2019;1:e190007. https://doi.org/10.20900/rmf20190007 3. Ribeiro VP, Pina S, Costa JB, Cengiz IF, García-Fernández L, Fernández-Gutiérrez MDM, et al. Enzymatically Cross-Linked Silk Fibroin-Based Hierarchical Scaffolds for Osteochondral Regeneration. ACS Appl Mater Interfaces 2019;11:3781–99. https://doi.org/10.1021/acsami.8b21259. 4. Yang T, Tamaddon M, Jiang L, Wang J, Liu Z, Liu Z, et al. Bilayered scaffold with 3D printed stiff subchondral bony compartment to provide constant mechanical support for long-term cartilage regeneration. J Orthop Transl 2021;30:112–21. https://doi.org/10.1016/j.jot.2021.09.001. 5. Blunn G, Liu C, Tamaddon M. Improved bone and cartilage regeneration in a rapid- manufactured functionally-graded osteochondral. Hangzhou International Conference on Biomaterials, Bio-Design and Manufacturing (BDMC2018). Aug. 26 - 28, 2018; Hangzhou (China). https://2020.aconf.org/conf_156776/abstract/77.html 6. Tamaddon M, Blunn G, Tan R, Yang P, Sun X, Chen SM, Luo J, Liu Z, Xu W, Lu X, Donate R, Monzón M, Liu C. In vivo evaluation of additively manufactured multi-layered scaffold for the repair of large osteochondral defects. Bio-Design Manuf 2022. https://doi.org/10.1007/s42242-021-00177-w 7. Flaherty T, Tamaddon M, Liu C. Micro-computed tomography analysis of subchondral bone regeneration using osteochondral scaffolds in an ovine condyle model. Appl Sci 2021;11:1–14. https://doi.org/10.3390/app11030891. 8. Tamaddon M, Blunn G, Xu W, Alemán Domínguez ME, Monzón M, Donaldson J, et al. Sheep condyle model evaluation of bone marrow cell concentrate combined with a scaffold for repair of large osteochondral defects. Bone Joint Res 2021;10:677–89. https://doi.org/10.1302/2046-3758.1010.BJR-2020-0504.R1. 9.  Liu Z, Tamaddon M, Chen SM, Wang H, San Cheong V, Gang F, et al. Determination of an Initial Stage of the Bone Tissue Ingrowth Into Titanium Matrix by Cell Adhesion Model. Front Bioeng Biotechnol 2021;9:1–13. https://doi.org/10.3389/fbioe.2021.7360 

Translation through collaboration: practice applied in BAMOS project in in vivo testing of innovative osteochondral scaffolds

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Translation through collaboration: practice applied in BAMOS project in in vivo testing of innovative osteochondral scaffolds

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