Cartilage and bone regeneration: how close are we to bedside?

The treatment/regeneration of bone and cartilage diseases or defects, whether induced by rheumatism, joint dysplasia, trauma, or surgery presents great challenges that have not been fully solved by the current therapies. In the last few years, tissue engineering and regenerative medicine have been proposing advanced tools and technologies for bone and cartilage tissue regeneration, and some of which have successfully reached the market. Beyond the source of cells, the creation of superior structures for replacing defective bone and cartilage requires strong research in biomechanical signaling and synthesis of advanced biomaterials to mimic human tissues at the most varied levels. Natural and synthetic polymers, bioresorbable inorganic materials, and composites have been investigated for its potential as scaffolding materials with enhanced mechanical and biological properties. Porous scaffolds, hydrogels, and fibers are the most commonly biomimetic structures used for bone and cartilage tissue engineering. Herein, the concepts and current treatment strategies for bone and cartilage repair, as well as biomimetic strategies for bone and cartilage tissue engineering are overviewed. A global review of the ongoing clinical trials and of the scaffolds commercially available for the repair of osteochondral tissue is also presented.(undefined

Novel bilayered Gellan gum/Gellan gum hydroxyapatite scaffolds for osteochondral tissue engineering applications

Osteoarthritis is a major cause of disability during aging. By the age of 60, close to 100% of the population will have histologic changes of degeneration in their knee cartilage (Loeser, 2000). Because of its avascular nature, cartilage has little capacity to self-regenerate. Despite the progress already achieved in osteochondral regeneration, some limitations have to be overcome. The formation of fibrocartilage has to be avoided and the innervation has to be improved. Further, one main feature to be promoted is the induction of vascularization in the bony part but not in the cartilage part and to avoid de-differentiation processes. A promising strategy could pass through the development and optimization of novel culture systems. The ideal approach could integrate scaffolds presenting regions with different physical characteristics, combined with different growth factors to support different stem cells fates, regarding the complex tissue physiology to be regenerate. This work aims to develop novel bilayered gellan gum (GG)/gellan gumhydroxyapatite (HAp) hydrogels based structures for osteochondral tissue engineering applications. Bilayered GG/GG-HAp hydrogels were produced by joining both solutions of GG 2% (w/v) with and without HAp (20% wt.) for bony and cartilage parts, respectively. The solutions were introduced into a silicone mould with 20:10 mm (height and diameter, respectively). Gelation of GG was promoted by immersion in PBS solution for 24 h. The architecture of the bilayered scaffolds was investigated by micro-computed tomography. Results have shown that the freeze-dried bilayered scaffolds composed by low acyl GG(2%(w/ v)/low acyl GG(2%(w/v)-HAp20%(w/w) possess a porosity of 83.4 ± 0.8%, pore size of 279.3 ± 38.6 lm and interconnectivity of 62.2 ± 5.4%. Degradability assays are being performed with the intent to use this system to culture human adipose derived stem cells inducing cell co-differentiation into chondrocytes and osteoblasts. Ultimately, the developed bilayered scaffolds will provide new therapeutic possibilities for the regeneration of osteochondral defects

Bioengineered nanoparticles loaded-hydrogels to target TNF Alpha in inflammatory diseases

Rheumatoid Arthritis (RA) is an incurable autoimmune disease that promotes the chronic impairment of patientsâ mobility. For this reason, it is vital to develop therapies that target early inflammatory symptoms and act before permanent articular damage. The present study offers two novel therapies based in advanced drug delivery systems for RA treatment: encapsulated chondroitin sulfate modified poly(amidoamine) dendrimer nanoparticles (NPs) covalently bonded to monoclonal anti-TNF α antibody in both Tyramine-Gellan Gum and Tyramine-Gellan Gum/Silk Fibroin hydrogels. Using pro-inflammatory THP-1 (i.e., human monocytic cell line), the therapy was tested in an inflammation in vitro model under both static and dynamic conditions. Firstly, we demonstrated effective NP-antibody functionalization and TNF-α capture. Upon encapsulation, the NPs were released steadily over 21 days. Moreover, in static conditions, the approaches presented good anti-inflammatory activity over time, enabling the retainment of a high percentage of TNF α. To mimic the physiological conditions of the human body, the hydrogels were evaluated in a dual-chamber bioreactor. Dynamic in vitro studies showed absent cytotoxicity in THP-1 cells and a significant reduction of TNF-α in suspension over 14 days for both hydrogels. Thus, the developed approach showed potential for use as personalized medicine to obtain better therapeutic outcomes and decreased adverse effects.The authors thank the financial support provided under the Norte2020 project (NORTE-08-5369-FSE000044). D.C.F. acknowledges the Portuguese Foundation for Science and Technology (FCT) for her PhD scholarship (PD/BD/143081/2018) and F.R.M. for her contract under the Transitional Rule DL 57/2016 (CTTI-57/18-I3BS(5)). The FCT distinction attributed to J.M.O. under the Investigator FCT program (number IF/01285/2015) is also greatly acknowledged

Gellan gum-based hydrogel bilayered scaffolds for osteochondral tissue engineering

It has been shown that hydrogel bilayered scaffolds combining cartilage- and bone-like layers are most advantageous for treating osteochondral defects. In this study, it is proposed the use of low acyl gellan gum (LAGG) for developing bilayered hydrogel scaffolds for osteochondral tissue engineering. The cartilage-like layer of the GG-based bilayered hydrogel scaffolds is composed of LAGG (2 wt%). By adding a 2 wt% LAGG aqueous solution to different amounts of HAp (5-20 wt%) it was possible to produce the bone-like layer. In vitro bioactivity tests were performed by means of soaking the LAGG/LAGG-HAp hydrogel scaffolds in a simulated body fluid solution up to 14 days. Scanning electron microscopy, Fourier transform infra-red spectroscopy and X-ray diffraction analyses demonstrated that apatite formation is limited to the bone-like layer of the LAGG/LAGG-HAp bilayered hydrogel scaffolds

3DICE coding matrix multidirectional macro-architecture modulates cell organization, shape, and co-cultures endothelization network

Natural extracellular matrix governs cells providing biomechanical and biofunctional outstanding properties, despite being porous and mostly made of soft materials. Among organs, specific tissues present specialized macro-architectures. For instance, hepatic lobules present radial organization, while vascular sinusoids are branched from vertical veins, providing specific biofunctional features. Therefore, it is imperative to mimic such structures while modeling tissues. So far, there is limited capability of coupling oriented macro-structures with interconnected micro-channels in programmable long-range vertical and radial sequential orientations. Herein, a three-directional ice crystal elongation (3DICE) system is presented to code geometries in cryogels. Using 3DICE, guided ice crystals growth templates vertical and radial pores through bulky cryogels. Translucent isotropic and anisotropic architectures of radial or vertical pores are fabricated with tunable mechanical response. Furthermore, 3D combinations of vertical and radial pore orientations are coded at the centimeter scale. Cell morphological response to macro-architectures is demonstrated. The formation of endothelial segments, CYP450 activity, and osteopontin expression, as liver fibrosis biomarkers, present direct response and specific cellular organization within radial, linear, and random architectures. These results unlock the potential of ice-templating demonstrating the relevance of macro-architectures to model tissues, and broad possibilities for drug testing, tissue engineering, and regenerative medicine.The authors are grateful for the Portuguese Foundation for Science and Technology (FCT) distinction attributed to R. F. Canadas (SFRH/ BD/92565/2013), and to J. M. Oliveira (IF/00423/2012, IF/01285/ 2015). R. F. Canadas is also thankful to FCT, Fundo Europeu de Desenvolvimento Regional (FEDER), and Programa Operacional Competitividade e Internacionalizaç˜ao (POCI) for funding the B-Liver Project (PTDC/EMD-EMD/29139/2017). The authors are also thankful to FCT for supporting the project Hierarchitech (M-ERA-NET/0001/2014) and for the funds provided under the 3 BioMeD project (JICAM/0001/2017). The authors acknowledge that this material and collaboration is based in part upon work supported by Luso-American Development Foundation (FLAD), 2016/CON15/CAN6). U. Demirci is also grateful for the Canary Center at Stanford for Cancer Early Detection Seed Award. The authors are also grateful for the support provided by Diana Bicho and Nicolas Cristini on scaffold characterization and cell culture, respectively

Posterior talar process as a suitable cell source for treatment of cartilage and osteochondral defects of the talus

Osteochondral defects of the ankle are common lesions affecting the talar cartilage and subchondral bone. Current treatments include cell-based therapies but are frequently associated with donor-site morbidity. Our objective is to characterize the posterior process of the talus (SP) and the os trigonum (OT) tissues and investigate its potential as a new source of viable cells for application in tissue engineering and regenerative medicine.SP and OT tissues obtained from six patients were characterized by micro-computed tomography, and histological, histomorphometric and immunohistochemical analyses. Isolated cells proliferation and viability were evaluated by MTS assay, DNA quantification and Live/Dead staining. The TUNEL assay was performed to evaluate cell death by apoptosis. Moreover, the production of extracellular matrix was evaluated by toluidine blue staining, whereas cells phenotype was investigated by flow cytometry. Ankle explants characterization showed the presence of a cartilage tissue layer in both SP and OT tissues, which represent, at least 20% in average of the explant. The presence of type II collagen was detected in the extracellular matrix. Isolated cells presented a round morphology typical of chondrocytes. In in vitro studies, cells were viable and proliferating up to 21 days of culturing. No signs of apoptosis were detected. Flow cytometry analysis revealed that isolated cells maintained the expression of several chondrocytic markers during culturing. The results indicate that the SP and OT tissues are a reliable source of viable chondrocytes, which can find promising applications in ACI/MACI strategies with minimal concerns regarding donor zone complications.Portuguese Foundation for Science and Technology (FCT) through the project OsteoCart (Grant No. PTDC/CTM-BPC/115977/2009), Fundación MAPFRE (Ayudas a la Investigación Ignacio H. de Larramendi, Prevención, Salud y Medio Ambiente, Spain) under the project 'Preventing the progression of the knee osteoarthritis: advanced therapies combining injectable hydrogels, autologous stem cells and PRP' (Grant No. BIL/13/SA/235). This study was also carried out with the support of Fundo Europeu de Desenvolvimento Regional (FEDER) through Programa Operacional do Norte through the project Articulate (Grant No. 23189

Bioreactors and microfluidics for osteochondral interface maturation

The cell culture techniques are in the base of any biology-based science. The standard techniques are commonly static platforms as Petri dishes, tissue culture well plates, T-flasks, or well plates designed for spheroids formation. These systems faced a paradigm change from 2D to 3D over the current decade driven by the tissue engineering (TE) field. However, 3D static culture approaches usually suffer from several issues as poor homogenization of the formed tissues and development of a necrotic center which limits the size of in vitro tissues to hundreds of micrometers. Furthermore, for complex tissues as osteochondral (OC), more than recovering a 3D environment, an interface needs to be replicated. Although 3D cell culture is already the reality adopted by a newborn market, a technological revolution on cell culture devices needs a further step from static to dynamic already considering 3D interfaces with dramatic importance for broad fields such as biomedical, TE, and drug development. In this book chapter, we revised the existing approaches for dynamic 3D cell culture, focusing on bioreactors and microfluidic systems, and the future directions and challenges to be faced were discussed. Basic principles, advantages, and challenges of each technology were described. The reported systems for OC 3D TE were focused herein.The research leading to this work has received funding from the European Union’s Seventh Framework Program (FP7/2007-2013) under grant agreement n° REGPOT-CT2012-316331-POLARIS, and from QREN (ON.2 – NORTE-01-0124-FEDER-000016) co-financed by North Portugal Regional Operational Program (ON.2 – O Novo Norte), under the National Strategic Reference Framework (NSRF), through the European Regional Development Fund (ERDF). Thanks are also due to the Portuguese Foundation for Science and Technology (FCT) for the project PEst-C/SAU/LA0026/201 and for the distinction attributed to J.M. Oliveira under the Investigator FCT program (IF/01285/2015). The authors also thank FCT for the Ph.D. scholarship provided to R. F. Canadas (SFRH/BD/92565/2013)info:eu-repo/semantics/publishedVersio

A cellular bilayered scaffolds for osteochondral tissue regeneration

Last decade 9.6% of the men and 18% of the woman with more than 60 years presented symptomatic Osteoarthritis [1]. Despite the progress already achieved in osteochondral (OC) repair, some limitations still needs to be overcome. A promising strategy includes a scaffold presenting two regions with different physical characteristics. The use of an acellular structure capable of recruiting the cells during the in vivo OC repair could be a faster and more efficient approach to translate into the clinics. This work aims to develop bilayered gellan gum (GG)/gellan gum-hydroxyapatite (HAp) freeze-dried scaffolds for OC regeneration

Osteochondral tissue engineering and regenerative strategies

Series: Studies in mechanobiology, tissue engineering and biomaterials, ISSN 1868-2006, vol. 21The orthopedic field has been facing challenging difficulties when it comes to regeneration of large and/or complex defects as we come across in osteochondral (OC) cases of lesions grade 4. Autologous OC mosaicplasty has proven to be a valid therapeutic option but donor site morbidity and the lack of long-term functionality remain sources of concern. OC tissue engineering has shown an increasing development to provide suitable strategies for the regeneration of damaged cartilage and underlying subchondral bone tissue. The use of two scaffolds with optimized properties for bone and cartilage architectures combined at the time of implantation as a multilayered structure was one of the first approaches for OC large defects regeneration. Last decade strategies using a bony-like scaffold supporting a cell layer for cartilage phase were introduced. Beyond the approaches already mentioned, three other strategies were reported for OCD regeneration. One methodology was the use of two different layers with a compact interface to create an integrated bilayered scaffold before cell seeding. A second strategy was the use of a single continuous structure but with different features in each layer. The last one was the combination of hydrogel phases creating this way the possibility to have injectable systems. These promising strategies for the regeneration of complex OCDs comprise the use of different biomaterials, growth factors, and cells alone or in combination, but the ideal solution is still to be found. The interface’s mechanical properties have to be optimized. A different problem is related with the cell culture method within the 3D bilayered structures with heterogeneous properties. With the increasing demand of these stratified 3D structures new cell culture systems are required. Moreover these structures present the potential to be used as in vitro models, which is a need also because of the pressure resulting from the 3R’s principle implementation that is now occurring. Regarding this, adapted bioreactors are being developed, but more efforts are required to target this scientific demand.Portuguese Foundation for Science and Technology and POPH/FSE program - (SFRH/BD/92565/2013)Investigator FCT program (IF/00423/2012

Biochemical gradients to generate 3D heterotypic-like tissues with isotropic and anisotropic architectures

Anisotropic 3D tissue interfaces with functional gradients found in nature are replicated in vitro for drug development and tissue engineering. Even though different fabrication techniques, based on material science engineering and microfluidics, are used to generate such microenvironments, mimicking the native tissue gradient is still a challenge. Here, the fabrication of 3D structures are described with linear/random porosity and gradient distribution of hydroxyapatite microparticles which are combined with a gradient of growth factors generated by a dual chamber for the development of heterotypicâ like tissues. The hydroxyapatite gradient is formed by applying a thermal ramp from the first to the second gel layer, and the porous architecture is controlled through ice templating. A 3D osteochondral (OC) tissue model is developed by codifferentiating fat pad adiposeâ derived stem cells. Osteogenic and chondrogenic markers expression is spatially controlled, as it occurs in the native osteochondral unit. Additionally, a prevasculature is spatially induced by the perfusion of proangiogenic medium in the boneâ like region, as observed in the native subchondral bone. Thus, in this study, precise spatial control is developed over cell/tissue phenotype and formation of prevasculature which opens up possibilities for the study of complex tissues interfaces, with broader applications in drug testing and regenerative medicine.The authors are grateful for the Fundação para a Ciência e a Tecnologia (FCT) distinctions attributed to R. F. Canadas (SFRH/ BD/92565/2013), who was awarded a PhD scholarship and to J.M.O. (IF/00423/2012 and IF/01285/2015). The authors acknowledge that this material and collaboration is based in part upon work supported by Luso-American Development Foundation (FLAD). This work was supported in part by the European Research Council Grant agreement ERC-2012-ADG 20120216-321266 for project ComplexiTE. The authors would like to acknowledge the National Science Foundation under Award No. NSF 1547791 and by the Office of the Assistant Secretary of Defense for Health Affairs under Award No. W81XWH-15-1-0576. Opinions, interpretations, conclusions, and recommendations are those of the author and are not necessarily endorsed by the National Institutes of Health or the Department of Defense. The authors also thank SAR—Soluções de Automação e Robótica for the support with bioreactor development, to João Costa (3B’s Research Group) for the assistance with dynamic mechanical analysis, to I. F. Cengiz (3B’s Research Group) and Filipe Carvalho (3B’s Research Group) for their help with material shipment and micro-CT analysis, and to Alessandro Tocchio (BAMM Lab) for their helpful and inspiring discussions.info:eu-repo/semantics/publishedVersio