Hydrogels and cell based therapies in spinal cord injury regeneration

Spinal cord injury (SCI) is a central nervous system- (CNS-) related disorder for which there is yet no successful treatment. Within the past several years, cell-based therapies have been explored for SCI repair, including the use of pluripotent human stem cells, and a number of adult-derived stem and mature cells such as mesenchymal stem cells, olfactory ensheathing cells, and Schwann cells. Although promising, cell transplantation is often overturned by the poor cell survival in the treatment of spinal cord injuries. Alternatively, the therapeutic role of different cells has been used in tissue engineering approaches by engrafting cells with biomaterials. The latter have the advantages of physically mimicking the CNS tissue, while promoting a more permissive environment for cell survival, growth, and differentiation. The roles of both cell- and biomaterial-based therapies as single therapeutic approaches for SCI repair will be discussed in this review. Moreover, as the multifactorial inhibitory environment of a SCI suggests that combinatorial approaches would be more effective, the importance of using biomaterials as cell carriers will be herein highlighted, as well as the recent advances and achievements of these promising tools for neural tissue regeneration.The authors would like to acknowledge the Portuguese Foundation for Science and Technology (Grant no. PTDC/SAU-BMA/114059/2009; IF Development Grant to António J. Salgado); Prémios Santa Casa Neurociências for funds attributed to António J. Salgado under the scope of the Prize Melo e Castro for Spinal Cord Injury Research; cofunded by Programa Operacional Regional do Norte (ON.2—O Novo Norte), ao abrigo do Quadro de Referência Estratégico Nacional (QREN), através do Fundo Europeu de Desenvolvimento Regional (FEDER)

Influence of different ECM-like hydrogels on neurite outgrowth induced by adipose tissue-derived stem cells

Mesenchymal stem cells (MSCs) have been proposed for spinal cord injury (SCI) applications due to their capacity to secrete growth factors and vesicles-secretome-that impacts important phenomena in SCI regeneration. To improve MSC survival into SCI sites, hydrogels have been used as transplantation vehicles. Herein, we hypothesized if different hydrogels could interact differently with adipose tissue-derived MSCs (ASCs). The efficacy of three natural hydrogels, gellan gum (functionalized with a fibronectin peptide), collagen, and a hydrogel rich in laminin epitopes (NVR-gel) in promoting neuritogenesis (alone and cocultured with ASCs), was evaluated in the present study. Their impact on ASC survival, metabolic activity, and gene expression was also evaluated. Our results indicated that all hydrogels supported ASC survival and viability, being this more evident for the functionalized GG hydrogels. Moreover, the presence of different ECM-derived biological cues within the hydrogels appears to differently affect the mRNA levels of growth factors involved in neuronal survival, differentiation, and axonal outgrowth. All the hydrogel-based systems supported axonal growth mediated by ASCs, but this effect was more robust in functionalized GG. The data herein presented highlights the importance of biological cues within hydrogel-based biomaterials as possible modulators of ASC secretome and its effects for SCI applications.This study is funded by Prémios Santa Casa Neurociências—Prize Melo e Castro for Spinal Cord Injury Research. This is also partially funded by EU-FP7-Health-2011-Collaborative Project 278612, Biohybrid—Templates for peripheral nerve regeneration, and Portuguese Foundation for Science and Technology (IF Development Grant to A. J. Salgado; postdoctoral fellowship to N. A. Silva—SFRH/BPD/97701/2013; PhD fellowships of R. C. Assunção-Silva and E. D. Gomes—PDE/BDE/113596/2015 and SFRH/BD/103075/2014, resp.). This article is a result of the project (NORTE-01-0145-FEDER-000013) supported by the Norte Portugal Regional Operational Programme (NORTE 2020), under the Portugal 2020 Partnership Agreement, through the European Regional Development Fund (ERDF); Cofinanciado pelo ProgramaOperacional Regional do Norte(ON.2 SR&TD Integrated Program—NORTE-07-0124-FEDER-000021), ao abrigo do Quadro de Referência Estratégico Nacional (QREN), através do Fundo Europeude Desenvolvimento Regional (FEDER); Projeto Estratégico—LA 26–2011-2012 and Projeto Estratégico—LA 26–2013-2014 cofinanciado por fundos nacionais, através da Fundação para a Ciência e a Tecnologia (PEst-C/SAU/LA0026/2011; PEst-C/SAU/LA0026/2013), e pelo Fundo Europeu de Desenvolvimento Regional (FEDER), através do COMPETE (FCOMP-01-0124-FEDER-022724; FCOMP-01-0124-FEDER-037298). The authors would like to thank Professor Jeffrey Gimble at the Tulane University Center for Stem Cell Research and Regenerative Medicine and LaCell LLC (New Orleans, Louisiana, USA) for kindly providing the ASCs used in this study.info:eu-repo/semantics/publishedVersio

Induction of neurite outgrowth in 3D hydrogel-based environments

The ability of peripheral nervous system (PNS) axons to regenerate and re-innervate their targets after an injury has been widely recognized. However, despite the considerable advances made in microsurgical techniques, complete functional recovery is rarely achieved, especially for severe peripheral nerve injuries (PNIs). Therefore, alternative therapies that can successfully repair peripheral nerves are still essential. In recent years the use of biodegradable hydrogels enriched with growth-supporting and guidance cues, cell transplantation, and biomolecular therapies have been explored for the treatment of PNIs. Bearing this in mind, the aim of this study was to assess whether Gly-Arg-Gly-Asp-Ser synthetic peptide (GRGDS)-modified gellan gum (GG) based hydrogels could foster an amenable environment for neurite/axonal growth. Additionally, strategies to further improve the rate of neurite outgrowth were also tested, namely the use of adipose tissue derived stem cells (ASCs), as well as the glial derived neurotrophic factor (GDNF). In order to increase its stability and enhance its bioactivity, the GDNF was conjugated covalently to iron oxide nanoparticles (IONPs). The impact of hydrogel modification as well as the effect of the GDNF-IONPs on ASC behavior was also screened. The results revealed that the GRGDS-GG hydrogel was able to support dorsal root ganglia (DRG)-based neurite outgrowth, which was not observed for non-modified hydrogels. Moreover, the modified hydrogels were also able to support ASCs attachment. In contrast, the presence of the GDNF-IONPs had no positive or negative impact on ASC behavior. Further experiments revealed that the presence of ASCs in the hydrogel improved axonal growth. On the other hand, GDNF-IONPs alone or combined with ASCs significantly increased neurite outgrowth from DRGs, suggesting a beneficial role of the proposed strategy for future applications in PNI regenerative medicineEU-FP7-Health-2011-collaborative project 278612, Biohybrid—Templates for peripheral nerve regeneration; Prémios Santa Casa Neurociências—Prize Melo e Castro for Spinal Cord Injury Research; Portuguese Foundation for Science and Technology (IF Development Grant to A J Salgado; Post-Doctoral fellowship to N A Silva — SFRH/BPD/97701/2013); co-funded by Programa Operacional Regional do Norte (ON.2—O Novo Norte), ao abrigo do Quadro de Referência Estratégico Nacional (QREN), através do Fundo Europeu de Desenvolvimento Regional (FEDER); Professor Jeffrey Gimble at the Tulane University Center for Stem Cell Research and Regenerative Medicine and LaCell LLC (New Orleans, Louisiana, USA) for kindly providing the ASCs used in this studyinfo:eu-repo/semantics/publishedVersio

Exploiting the impact of the secretome of MSCs isolated from different tissue sources on neuronal differentiation and axonal growth

Cell transplantation using Mesenchymal stem cell (MSC) secretome have recently been presented as a possible free-based therapy for CNS related disorders. MSC secretome is rich in several bio-factors that act synergically towards the repair of damaged tissues, thus making it an ideal candidate for regenerative applications. Great effort is currently being made to map the molecules that compose the MSC secretome. Previous proteomic characterization of the secretome (in the form of conditioned media - CM) of MSCs derived from adipose tissue (ASC), bone-marrow (BMSC) and umbilical cord (HUCPVC) was performed by our group, where proteins relevant for neuroprotection, neurogenic, neurodifferentiation, axon guidance and growth functions were identified. Moreover, we have found significant differences among the expression of several molecules, which may indicate that their therapeutic outcome might be distinct. Having this in mind, in the present study, the neuroregulatory potential of ASC, BMSC and HUCPVC CM in promoting neurodifferentiation and axonal outgrowth was tested in vitro, using human telencephalon neuroprogenitor cells and dorsal root ganglion explants, respectively. The CM from the three MSC populations induced neuronal differentiation from human neural progenitor cells, as well as neurite outgrowth from dorsal root ganglion explants. Moreover, all the MSC populations promoted the same extent of neurodifferentiation, while ASC CM demonstrated higher potential in promoting axonal growth.The authors acknowledge the financial support by Premios Santa Casa Neurociencias - Prize Melo e Castro for Spinal Cord ^ Injury Research (MC-17-2013 and MC-04-2017); Portuguese Foundation for Science and Technology (Doctoral fellowships PDE/ BDE/113596/2015 and SFRH/BD/120124/2016 to R.C Assunçao Silva ~ and B. Mendes-Pinheiro, respectively; Post-doctoral fellowhip to F.G. Teixeira and Patrícia Patrício - SFRH/BPD/118408/2016 and SFRH/BPD/116249/2016; IF Starting Grant to L. Pinto and IF Development Grant to A. J. Salgado); Canada Research Chair in Biomedical Engineering (LAB). This work is funded by national funds through FCT under the scope of grante reference TUBITAK/0007/ 2014. This article has been developed under the scope of the project NORTE-01-0145-FEDER-000013, supported by the Northern Portugal Regional Operational Programme (NORTE 2020), under the Portugal 2020 Partnership Agreement, through the European Regional Development Fund (FEDER). This work has been funded by FEDER funds, through the Competitiveness Factors Operational Programme (COMPETE), and by National funds, through the Foundation for Science and Technology, under the scope of the project POCI-01-0145-FEDER-007038. HUCPVCs and ASCs were kindly provided by Prof. John E. Davies (University of Toronto, Canada) and Prof. Jeff Gimble (LaCell Inc, USA).info:eu-repo/semantics/publishedVersio

Bioengineered cell culture systems of central nervous system injury and disease

Cell culture systems, either 2D or explant based, have been pivotal to better understand the pathophysiology of several central nervous system (CNS) disorders. Recently, bioengineered cell culture systems have been proposed as an alternative to the traditional setups. These innovative systems often combine different cell populations in 3D environments that more closely recapitulate the different niches that exist within the developing or adult CNS. Given the importance of such systems for the future of CNS-related research, we discuss here the most recent advances in the field, particularly those dealing with neurodegeneration, neurodevelopmental disorders, and trauma.Financial support is acknowledged from Prémios Santa Casa Neurociências – Prize Melo e Castro for Spinal Cord Injury Research; Portuguese Foundation for Science and Technology [Doctoral fellowship (SFRH/BD/103075/2014) to E.D.G.; IF Development Grant to A.J.S.; Starting Grant to F. Marques; PostDoctoral fellowship SFRH/BPD/97701/2013 to N.A.S.]; this work was co-funded by Programa Operacional Regional do Norte (ON.2 – O Novo Norte), ao abrigo do Quadro de Referência Estratégico Nacional (QREN), através do Fundo Europeu de Desenvolvimento Regional (FEDER).info:eu-repo/semantics/publishedVersio

Influence of passage number on the impact of the secretome of adipose tissue stem cells on neural survival, neurodifferentiation and axonal growth

Mesenchymal stem cells (MSCs), and within them adipose tissue derived stem cells (ASCs), have been shown to have therapeutic effects on central nervous system (CNS) cell populations. Such effects have been mostly attributed to soluble factors, as well as vesicles, present in their secretome. Yet, little is known about the impact that MSC passaging might have in the secretion therapeutic profile. Our aim was to show how human ASCs (hASCs) passage number influences the effect of their secretome in neuronal survival, differentiation and axonal growth. For this purpose, post-natal rat hippocampal primary cultures, human neural progenitor cell (hNPCs) cultures and dorsal root ganglia (DRGs) explants were incubated with secretome, collected as conditioned media (CM), obtained from hASCs in P3, P6, P9 and P12. Results showed no differences when comparing percentages of MAP-2 positive cells (a mature neuronal marker) in neuronal cultures or hNPCs, after incubation with hASCs secretome from different passages. The same was observed regarding DRG neurite outgrowth. In order to characterize the secretomes obtained from different passages, a proteomic analysis was performed, revealing that its composition did not vary significantly with passage number P3 to P12. Results allowed us to identify several key proteins, such as pigment epithelium derived factor (PEDF), DJ-1, interleucin-6 (IL-6) and galectin, all of which have already proven to play neuroprotective and neurodifferentiating roles. Proteins that promote neurite outgrowth were also found present, such as semaphorin 7A and glypican-1. We conclude that cellular passaging does not influence significantly hASCs's secretome properties especially their ability to support post-natal neuronal survival, induce neurodifferentiation and promote axonal growth.Prémios Santa Casa Neurociências - Prize Melo e Castro for Spinal Cord Injury Research (MC-17-2013 and MC-04-2017), Canada Research Chair in Biomedical Engineering (LAB), Northern Portugal Regional Operational Programme (NORTE 2020),, European Regional Development Fund (FEDER), Competitiveness Factors Operational Programme (COMPETE), National Mass Spectrometry Network (RNEM)info:eu-repo/semantics/publishedVersio

O secretoma de células estaminais mesenquimatosas como uma terapia para lesões vertebromedulares

Tese de doutoramento em Ciências da SaúdeAs lesões vertebro-medulares (LVM) resultam de um trauma na espinal medula, seguido da perda parcial ou completa da função motora e sensorial abaixo do nível de lesão. A transplantação celular tem estado na vanguarda de muitas estratégias, mas apresentam uma taxa de implantação e sobrevivência baixas perante o ambiente de lesão nocivo. Como alternativa, estratégias baseadas no uso do secretoma de células estaminais têm sido muito exploradas. O secretoma de células estaminais mesenquimatosas (MSCs) têm mostrado efeitos benéficos através de citoquinas anti-inflamatórias e fatores de crescimento e regenerativos. Uma expressão diferencial destas moléculas por MSCs derivadas do tecido adiposo (ASCs), medula óssea (BMSCs) e do cordão umbilical (HUCPVCs) sugerem um impacto terapêutico distinto. Esta hipótese foi confirmada por experiências in vitro, onde o secretoma de ASCs promoveu níveis de crescimento axonal superiores aos das outras populações celulares. De seguida, o potencial terapêutico do secretoma de ASCs foi avaliado num modelo de LVM em Xenopus Laevis. A sua administração na espinal medula dos Xenopus após transeção completa aumentou a regeneração axonal e crescimento neuronal no local de lesão. Os animais tratados apresentaram formação de uma ponte axonal na zona lesionada, assim como uma melhoria da função motora. Finalmente, o potencial terapêutico do secretoma de ASCs foi avaliado num modelo de LVM em ratinho. O secretoma foi administrado por via intravenosa após transeção completa torácica da espinal medula. Os animais tratados demonstraram melhorias das funções motoras e sensoriais, acompanhado por uma redução evidente do número de células inflamatórias no local de lesão, o que sugere uma ação anti-inflamatória do secretoma das ASCs. Foi também observado crescimento e regeneração axonal após tratamento, assim como uma diminuição da cavidade de lesão. Em resumo, os resultados aqui apresentados providenciam evidências do potencial terapêutico do secretoma de ASCs após LVM, considerando os seus efeitos positivos ao nível da inflamação neuronal e crescimento e regeneração axonal, observado nos modelos in vitro e in vivo aqui estudados, e que estão associados à recuperação motora dos Xenopus Laevis e do ratinho.Spinal cord injury (SCI) results from a mechanical trauma to the spinal cord, followed by partial or complete loss of motor and sensory function below the level of injury. Cell transplantation has been in the forefront of regenerative medicine strategies, but often presents low engraftment and survival rate within the aggressive environment of SCI. Alternatively, cell-free based strategies using the secretome of stem cells has been highly explored. Mesenchymal stem cells (MSCs) secretome has been showing beneficial effects through anti-inflammatory cytokines and regenerating- and growth-permissive factors. A differential expression of these molecules by adipose tissue (ASCs)-, bone-marrow (BMSCs)- and umbilical cord (HUCPVCs)-derived MSCs suggested a distinct therapeutic outcome. Indeed, this was confirmed in in vitro experiments, where ASCs secretome promoted significantly higher levels of axonal growth, when compared to the other cell populations. Following this, the therapeutic potential of ASC secretome was evaluated in a Xenopus laevis model of SCI. ASC secretome delivery into a transected Xenopus spinal cord increased axonal regeneration and neuronal regrowth at the lesion site. Treated animals showed ablation gap closure and axonal bridge formation between the two spinal cord stumps, as well as an improved motor function. Finally, the therapeutic potential of ASC secretome was evaluated in a mice model of SCI. ASC secretome was intravenously administered into mice spinal cord after complete thoracic transection. Treated animals showed improved motor and sensorial function, accompanied by a marked reduction on the number of inflammatory cells at the lesion site, suggesting an anti-inflammatory action of ASC secretome. Axonal outgrowth and regeneration through the injury was also observed upon ASC secretome treatment, as well as decreased lesion cavities. Altogether, these results provide evidences of the therapeutic potential of ASC secretome after SCI, supported by indications on the positive effects exerted on neuroinflammation, and axonal outgrowth and regeneration, observed for in vitro and in vivo models herein studied, and that were associated to locomotor recovery to both Xenopus laevis and mice.Financial support from Science Foundation Ireland (SFI) and the European Regional Development Fund (Grant Number 13/RC/2073). Financial support was provided from Prémios Santa Casa Neurociências - Prize Melo e Castro for Spinal Cord Injury Research (MC-04/17); Portuguese Foundation of Science and Technology [Doctoral Fellowship to R. C. Assunção-Silva (PDE/BDE/113596/2015)]; funded by FEDER, through the Competitiveness Internationalization Operational Programme (POCI), and by National funds, through the Foundation for Science and Technology (FCT), under the scope of the projects POCI-01-0145- FEDER-007038; TUBITAK/0007/2014; PTDC/DTP-FTO/5109/2014; POCI-01-0145-FEDER-029206; POCI-01-0145-FEDER-031392; PTDC/MED-NEU/31417/2017 and NORTE-01-0145-FEDER-029968. This work has also been developed under the scope of the project NORTE-01-0145-FEDER-000013, supported by the Northern Portugal Regional Operational Programme (NORTE 2020), under the Portugal 2020 Partnership Agreement, through the European Regional Development Fund (FEDER)

Characterisation of microbial attack on archaeological bone

As part of an EU funded project to investigate the factors influencing bone preservation in the archaeological record, more than 250 bones from 41 archaeological sites in five countries spanning four climatic regions were studied for diagenetic alteration. Sites were selected to cover a range of environmental conditions and archaeological contexts. Microscopic and physical (mercury intrusion porosimetry) analyses of these bones revealed that the majority (68%) had suffered microbial attack. Furthermore, significant differences were found between animal and human bone in both the state of preservation and the type of microbial attack present. These differences in preservation might result from differences in early taphonomy of the bones. © 2003 Elsevier Science Ltd. All rights reserved