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

Porous aligned ZnSr-doped β-TCP/silk fibroin scaffolds using ice-templating method for bone tissue engineering applications

The bone is a complex and dynamic structure subjected to constant stress and remodeling. Due to the worldwide incidence of bone disorders, engineered bone tissues have emerged as solution for bone grafting, which require sophisticated scaffolding architectures while keeping high mechanical performance. However, the conjugation of bone-like scaffold architecture with efficient mechanical properties is still a critical challenge for biomedical applications.  In this sense, the present study is focused on the development of silk fibroin (SF) scaffolds crosslinked with horseradish peroxidase and mixed with zinc (Zn) and strontium (Sr)-doped β-tricalcium phosphate (ZnSr.TCP) to mimic bone structures. The ZnSr.TCP-SF hydrogels were tuned in programmable ice-templating parameters, and further freeze-dried, to obtain 3D scaffolds with controlled pore orientation. The results showed interconnected channels in the ZnSr.TCP-SF scaffolds that mimic the porous network of the native subchondral bone. The architecture of the scaffolds was characterized by microCT and showing tunable pore size according to freezing temperatures (-196 ºC: ~80.2 ± 20.5 µm; -80 ºC: ~73.1 ± 20.5 µm; -20 ºC: ~104.7 ± 33.7 µm). The swelling ratio, weight loss, and rheological properties were also assessed, revealing that the scaffolds were able to keep their integrity and morphology after aqueous immersion. Thus, the ZnSr.TCP-SF scaffolds made of aligned porous structure were developed as affordable candidates for future applications in clinical osteoregeneration and in vitro bone tissue modelling.FCT for the financial support of the Hierarchitech project (M435 ERA.NET/0001/2014) and for the distinctions attributed to S.P. (CEECIND/03673/2017) and C. 436 G. (SFRH/BPD/94277/2013). R. F. Canadas is also thankful to FCT for the doctoral scholarship 437 (SFRH/BD/92565/2013), Fundo Europeu de Desenvolvimento Regional (FEDER), and Programa 438 Operacional Competitividade e Internacionalização (POCI) for funding the Young Researcher 439 Contract (B-Liver Project, PTDC/EMD-EMD/29139/2017

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

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

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

Vancomycin-Loaded, Nanohydroxyapatite-Based Scaffold for Osteomyelitis Treatment: In Vivo Rabbit Toxicological Tests and In Vivo Efficacy Tests in a Sheep Model

The treatment for osteomyelitis consists of surgical debridement, filling of the dead space, soft tissue coverage, and intravenous administration of antimicrobial (AM) agents for long periods. Biomaterials for local delivery of AM agents, while providing controllable antibiotic release rates and simultaneously acting as a bone scaffold, may be a valuable alternative; thus, avoiding systemic AM side effects. V-HEPHAPC is a heparinized nanohydroxyapatite (nHA)/collagen biocomposite loaded with vancomycin that has been previously studied and tested in vitro. It enables a vancomycin-releasing profile with an intense initial burst, followed by a sustained release with concentrations above the Minimum Inhibitory Concentration (MIC) for MRSA. In vitro results have also shown that cellular viability is not compromised, suggesting that V-HEPHAPC granules may be a promising alternative device for the treatment of osteomyelitis. In the present study, V-HEPHAPC (HEPHAPC with vancomycin) granules were used as a vancomycin carrier to treat MRSA osteomyelitis. First, in vivo Good Laboratory Practice (GLP) toxicological tests were performed in a rabbit model, assuring that HEPHAPC and V-HEPHAPC have no relevant side effects. Second, V-HEPHAPC proved to be an efficient drug carrier and bone substitute to control MRSA infection and simultaneously reconstruct the bone cavity in a sheep model.This work was financed by FEDER-Fundo Europeu de Desenvolvimento Regional funds through the COMPETE 2020-Operacional Programme for Competitiveness and Internationalisation (POCI), Portugal 2020; by Portuguese funds through FCT/MCTES in the framework of the project institute for Research and Innovation in Health Sciences (POCI-01-0145-FEDER-007274); by the Project Biotherapies (NORTE-01-0145-FEDER-000012); and by the project HEPHAPC Program RESOLVE, Norte 2020 (NORTE-01-0246-FEDER-000018). The authors would also like to acknowledge the technical support for histology and histochemical studies of Rui Fernandes and Rossana Correia and all the staff from HEMS/i3S, as well as the support of all the staff and students at the Hospital Veterinario-Universidade de Evora

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

Hierarchical HRP-crosslinked silk fibroin/ZnSr-doped TCP nancocomposites towards osteochondral tissue regeneration: Biomechanical performance and in vivo assessment

[Excerpt] Introduction. Recent investigations highlight promising regenerative strategies for osteochondral (OC) tissue treatment, such as hierarchical nanocomposite scaffolds containing ionic dopants.1,2 They allow cell infiltration and ECM formation throughout the engineered cartilage and subchondral tissues. The biomechanical behavior, antibacterial properties, and in vivo performance of hierarchical nanostructures combining enzymatically crosslinked silk fibroin (SF) and ZnSr-doped β-tricalcium phosphate (ZnSrTCP) for OC tissue regeneration is herein assessed. [...]Thanks to the Portuguese Foundation for Science and Technology for M-era-Net/0001/2014 project, and for the distinctions (IF/01285/2015) and (CEECIND/03673/2017)

Vancomycin-Loaded, Nanohydroxyapatite-Based Scaffold for Osteomyelitis Treatment: In Vivo Rabbit Toxicological Tests and In Vivo Efficacy Tests in a Sheep Model

The treatment for osteomyelitis consists of surgical debridement, filling of the dead space, soft tissue coverage, and intravenous administration of antimicrobial (AM) agents for long periods. Biomaterials for local delivery of AM agents, while providing controllable antibiotic release rates and simultaneously acting as a bone scaffold, may be a valuable alternative; thus, avoiding systemic AM side effects. V-HEPHAPC is a heparinized nanohydroxyapatite (nHA)/collagen biocomposite loaded with vancomycin that has been previously studied and tested in vitro. It enables a vancomycin-releasing profile with an intense initial burst, followed by a sustained release with concentrations above the Minimum Inhibitory Concentration (MIC) for MRSA. In vitro results have also shown that cellular viability is not compromised, suggesting that V-HEPHAPC granules may be a promising alternative device for the treatment of osteomyelitis. In the present study, V-HEPHAPC (HEPHAPC with vancomycin) granules were used as a vancomycin carrier to treat MRSA osteomyelitis. First, in vivo Good Laboratory Practice (GLP) toxicological tests were performed in a rabbit model, assuring that HEPHAPC and V-HEPHAPC have no relevant side effects. Second, V-HEPHAPC proved to be an efficient drug carrier and bone substitute to control MRSA infection and simultaneously reconstruct the bone cavity in a sheep model