Programa de Pós-Graduação em Odontologia Área de concentração Dentística
Dissertação
Hipóxia pode influenciar na performace de hMSCs? Scoping review
Fernanda Müller Antunes
Fernanda Müller Antunes
Hipóxia pode influenciar na performance de hMSCs? Scoping review
Orientadora: Dra. Adriana Fernandes da Silva Coorientador: Dr. Rafael Guerra Lund
Pelotas, 2017
Dissertação apresentada ao Programa de Pós- Graduação em Odontologia da Faculdade de Odontologia da Universidade Federal de Pelotas, como requisito parcial à obtenção do título de Mestre em Odontologia, área de concentração Dentística.
Fernanda Müller Antunes
Hipóxia pode influenciar na performance de hMSCs? Scoping review
Dissertação apresentada, como requisito parcial, para obtenção do grau de Mestre em Odontologia área de concentração Dentística, Programa de Pós-Graduação em Odontologia, Faculdade de Odontologia, Universidade Federal de Pelotas.
Data da Defesa: 23 de fevereiro de 2017.
Banca examinadora:
Profa. Dra. Adriana Fernandes da Silva (Orientadora)
Doutora em Biologia Buco-Dental pela Universidade de Campinas
Profa. Dra. Fernanda Nedel
Doutora em Biotecnologia pela Universidade Federal de Pelotas
Profa.Dra .Marina Sousa Azevedo
Doutora em Odontologia pela Universidade Federal de Pelotas
Profa. Dra. Luciane Geanine Pena dos Santos (suplente)
Doutora em Odontologia pela Universidade Federal de Santa Catarina
Profa. Dra. Natália Pola (suplente)
Dedico este trabalho aos meus pais, irmãos e ao meu namorado.
Agradecimentos
Aos meus pais, Gertrud e Pedro, que sempre foram exemplo de perseverança e sucesso.
As minhas irmãs, Caroline e Francine, que foram companheiras e amigas durante essa caminhada.
Ao meu namorado, Mateus, que foi extremamente companheiro e presente nestes 2 anos, agradeço pelo incentivo e ajuda.
Ao Professor Rafael, que me acompanha desde a graduação, agradeço o incentivo e a amizade.
A Professora Adriana, que aceitou me orientar e me acompanhou durante estes 2 anos, dividindo seu tempo entre seus bebes e meu trabalho, tenho certeza que não foi fácil.
Ao Wellington, sempre atencioso e prestativo, fazendo seu melhor para poder me ajudar.
A Mariane e Carolina, que dividiram seu tempo com os afazeres deste trabalho.
"Se temos de esperar, que seja para colher a semente boa que lançamos hoje no solo da vida. Se for para semear, então que seja para produzir milhões de sorrisos, de solidariedade e de amizade."
Resumo
ANTUNES, Fernanda Müller. Hipóxia pode influenciar na
performance dehMSC? Scopingreview. 50f. Dissertação (Mestrado em Dentística) - Programa de Pós-Graduação em Odontologia,
Faculdade de Odontologia, Universidade Federal de Pelotas, Pelotas, 2017.
Este estudo teve como objetivo revisar sistematicamente a literatura como forte influência da hipóxia em células-tronco mesenquimais humanas (hMSCs). A revisão é relatada de acordo com a Declaração PRISMA. Dois revisores realizaram uma pesquisa bibliográfica de três bancos de dados: PubMed (Medline), Web of Science e Scopus. Todos os estudos que analisaram a condição de hipóxia no desenho do experimento foram incluídos. Os dados relativos ao autor, ao ano de publicação, ao tipo de célula, ao tipo de estudo do local do tecido, à concentração de oxigênio utilizada, aos métodos de avaliação, ao curso de indução e aos achados principais foram tabulados para avaliar os seguintes resultados de interesse: mudanças no comportamento celular (paracrino ou autocrino), diferenciação celular, viabilidade / proliferação, migração. Após a triagem de um total de 1537 documentos potencialmente relevantes, 139 estudos foram submetidos a análise qualitativa. Em 69% dos estudos, a modificação celular foi realizada. Adicionalmente, as modificações mais estudadas foram migração celular, proliferação / viabilidade, diferenciação e expressão de VEGF, HIF-1α. Em conclusão, há evidências na literatura de que o condicionamento na hipóxia melhora o metabolismo e a expressão de hMSCs, mas esse resultado não pode ser estendido a todos os tipos. Em conclusão, a maioria das células estaminais precisa da hipóxia como o ambiente natural em seus nichos, o que permitirá usa-la no seu máximo potencial e capacitar o desempenho em terapias regenerativas.
Palavras-chave: hipóxia celular; células tronco; células estromais mesenquimais, scoping review.
8
Abstract
ANTUNES, Fernanda Müller. Can hypoxia influence on the performance of the hMSCs? Scoping review. 50f. Dissertation (Master Degree em Dentística) - Programa de Pós-Graduação em Odontologia, Faculdade de Odontologia, Universidade Federal de Pelotas, Pelotas, 2017.
This study aimed to systematically review the literature as fort influence of the hypoxia on human mesenchymal stem cells (hMSCs). The review is reported in accordance with the PRISMA Statement. Two reviewers conducted a literature search of three databases: PubMed (Medline), Web of Science and Scopus, until May 2016. All studies that analyzed condition of hypoxia in the design experiment were included. Data regarding author, year of publication, cell type, tissue site type of study, oxygen concentration used, evaluation methods, induction time courseand main findings were tabulated to assess the following outcomes of interest: changes in (paracrine or autocrine effect) cell behavior(cell differentiation, viability/proliferation, migration). After screening a total of 1537 potentially relevant documents, 139 studies were subjected to qualitative analysis. In 69% of the studies, cellular modification was performed. Additionally, the most studied modifications were cell migration, proliferation/viability, differentiation and expression of VEGF, HIF-1α. In conclusion, there is evidence in the literature that the conditioning in hypoxia improves the metabolism and expression of hMSCs but this result cannot be extended to all type. In conclusion, most stem cells need of the hypoxia as the natural environment in their niches which will allowusing the at most and empower the performance in regenerative therapies.
Key-words: cell hypoxia; stem cells; mesenchymal stromal cells, scoping review.
Sumário 1 Introdução ...9 2 Capítulo 1 ...11 3 Considerações finais ...38 Referências ...39 Apêndices ...47
1 Introdução
Células tronco mesenquimais (MSC) são células multipotentes não hematopoiéticas que residem e podem ser isoladas de diferentes tecidos, como medula óssea, tecido adiposo, polpa dental, líquido amniótico, cordão umbilical entre outros (ERICES et al., 2000; MINGUELL et al., 2000; CAMPAGNOLI et al., 2001; KOGLER et al., 2004). O Comitê de Células Tronco Mesenquimais e Tecidos da Sociedade Internacional de Terapia Celular estabelece três critérios para que uma célula seja caracterizada como tronco, são eles: aderência ao plástico, expressão específica de antígenos de superfície e potencial de diferenciação multipotente (pelo menos três linhagens diferentes) (PROCKOP et al., 1997; PITTENGER et al., 2000). Estas células representam uma ótima alternativa por apresentarem alto potencial de expansão ex vivo, auto renovação, imunomodulação, somados com sua capacidade multipotente se mostram como alternativa para terapia celular e regeneração de tecidos (BARRY et al., 2004; JAVAZON et al., 2004; JORGENSEN et al., 2004)
A condição de hipóxia é caracterizada por baixa concentração de oxigênio, HOCHACHKA e colaboradores (1996) propuseram uma teoria na qual a resposta celular frente a hipóxia seria composta por duas fases: uma de defesa, onde os processos moleculares seriam reduzidos para que houvesse economia de energia e outra fase de resgate celular, onde haveria ativação do fator de transcrição induzido pela hipóxia 1α (HIF-1α) e supressão do metabolismo celular.
O HIF-1 é uma proteína expressa por diferentes tipos celulares (WANG e SEMENZA, 1995; WANG et al., 1995; JIANG et al., 1996) e sua regulação é responsável pela manutenção da homeostasia de oxigênio (HIROTA e SEMENZA, 2006). A expressão da subunidade HIF-1α aumenta em condições de hipóxia e é degradada após a reoxigenação (SEMENZA e WANG, 1992; WANG e SEMENZA, 1993; WANG et al., 1995; AMEMIYA et al., 2003).
Um estudo analisou a regulação da ativação de fatores angiogênicos VEGF, FGFb pelo HIF-1α em células tronco e fibroblastos, ambos provenientes de DPSC, e demonstraram que, quando estas células eram cultivadas em hipóxia (1% de O2) houve aumento da expressão do VEGF comparado com
células cultivadas em normóxia (21% de O2). Este estudo foi pioneiro na utilização de células tronco originárias de polpa de dentes permanentes sob a condição de hipóxia para avaliar a expressão de fatores angiogênicos (ARANHA et al., 2010).
A hipóxia está envolvida tanto em processos fisiológicos quanto patológicos, embora a hipóxia grave possa levar a morte celular, a exposição à hipóxia tem mostrado conferir benefícios citoprotetores, incluindo promoção da sobrevivência celular, aumento da expressão de fatores de crescimento e indução de angiogênese, ambos processos fundamentais para o sucesso de terapias celulares (MATSUSHITA et al., 2000; HAIDER & ASHRAF, 2010).
1.2 Objetivo
O objetivo da presente dissertação foi avaliar por meio de uma scoping review a influência do efeito parácrino e/ou autocrino da hipóxia usando células tronco mesenquimais humanas.
2Capítulo 1
Can hypoxia influence the fate of the hMSCs? – A scoping review
Fernanda Müller Antunesa, Wellington Luiz de Oliveira da Rosaa, Rafael Guerra Lundb, Adriana Fernandes da Silvab*
Affiliations:
aDDS, Post-graduate Student, Faculty of Dentistry, Federal University of Pelotas, Pelotas, RS, Brazil
bDDS, PhD, Associate Professor, Departament of Restorative Dentistry, Faculty of Dentistry, Federal University of Pelotas, Pelotas, RS, Brazil
*Correspondingauthor
bDra. Adriana Fernandes da Silva
Federal University of Pelotas, Faculty of Dentistry
Department of Restorative Dentistry
Gonçalves Chaves St., 457/503
Zip Code: 96015-560, Pelotas, RS, Brazil. E-mail address: adriana@ufpel.edu.br
+55 53 32256741 (134)
____________________________________
1Artigo estruturado segundo as normas do periódico Stem Cells (FI 2017: 5.902)
Abstract
This study aimed to systematically review the literature as fort influence of the hypoxia under performance of human mesenchymal stem cells (hMSCs). The scoping review is reported in accordance with the PRISMA Statement. Two reviewers conducted a literature search of three databases: PubMed (Medline), Web of Science and Scopus, until May 2016. Search terms are “Mesenchymal stromal cell” and “cell hypoxia”. Eligibility criteria to inclusion studies was: studies in vitro and in vivo, stem cells from human connective tissue and studies that analyzed the autocrine or paracrine effect of the hypoxia using stem cells. All studies that analyzed condition of hypoxia in the design experiment were included. Data regarding author, year of publication, cell type, tissue site type of study, oxygen concentration used, evaluation methods, induction time courseand main findings were tabulated to assess the following outcomes of interest: changes in (paracrine and/or autocrine effect) cell behavior(cell differentiation, viability/proliferation, migration). To access the risk of bias, the following criteria were analyzed: selective reporting, description of passage utilized, presence of negative and positive control, description of the hypoxia procedure and evaluation of the cell behavior. After screening a total of 1537 potentially relevant documents, 139 studies were subjected to qualitative analysis. In 69% of the studies, cellular modification was performed. There is evidence in the literature that the conditioning in hypoxia improves the metabolism of hMSCs but this result cannot be extended to all type. The risk of bias remained close to 30%. In conclusion, most stem cells need of the hypoxia as the natural environment in their niches which will allowusing the at most and empower the performance in regenerative therapies.
Prospero registration: name “Can hypoxia influence the fate of the hMSCs? A systematic review.”, number CRD42017057148.
Keywords: cell hypoxia, stem cells, mesenchymal stromal cells, scoping review.
Introduction
Studies based on tissue engineering have been extensively developed, the aim are to use mesenchymal stem cells (MSCs) in various forms of therapy as well as to use as a tool to understand the mechanisms that lead to the repair and regeneration of tissues and organs(1). Advances in tissue culture have already been documented, such as the production of three-dimensional matrices (scaffolds) that are used in cell culture to provide an in vitro environment more similar to the environment in vivo(2). But tissue engineering encounters several limitations, such as loss of cell viability in scaffold, difficulty in developing angiogenesis, limited self-renewal among others(1).
Oxygen is an important biochemical signaling molecule in the body, exerts function on proliferation, migration, adhesion, survival, metabolism, secretion and differentiation of MSCs(3). It was proposed a theory in which the cellular response to hypoxia condition would be composed of two phases. The first of defense, where the molecular processes would be reduced for energy saving, and the second phase of cellular rescue, where there would be activation of the transcription factor induced by hypoxia 1 (HIF-1) and suppression of cellular metabolism (4). So, it is known that severe or prolonged hypoxia leads to apoptosis, however conditioning under hypoxia confers cytoprotective benefits, as it facilitates cell development, maintains pluripotency of MSC, induces differentiation, regulates signaling of several cascades such as angiogenesis (4).
Studies have shown that MSCs cultured under hypoxia secrete an increased amount of proteins, such as vascular endothelial growth factor (VEGF), fibroblast growth factor 2 (FGF2), transforming growth factor beta 1 (TGF-β1) and interleucin 6 (IL 6) which are involved in the inflammatory phase of wound healing (5), as well as the hypoxia-1α-induced transcription factor (HIF-1α). HIF-1α, is a mediator of the cellular response to hypoxia, acting to modulate gene expression and cellular metabolism for survival in a hypoxic environment. However, typically in normoxic conditions, HIF-1α is degraded.
Studies have shown that the oxygen tension in the tissue considered as normal (normoxia) is around 20-21%, but it is not found in the niches of stem cells in the body since the oxygen tension is shown low, varying around 5%. The hypoxia is necessary to keep the characteristics of hMSCs and use the maximum therapeuticefficacy due to the plurality of hMSCs applications of stimulating regeneration or repair (6,7).
The effects of hypoxic conditioning on hMSCs are still unclear as there are controversial data in the literature specially in different cell types. Due to these, the purpose of this scoping review was to evaluate the influence of different concentrations of hypoxia on fate of the hMSCs, know what the autocrine and the paracrine effect of hypoxia under hMSCs. The hypothesis tested was that hMSCs under hypoxia would improve paracrine and/or autocrine cellular behavior regarding cell viability, migration and differentiation when compared with cultured in normoxia.
Methods
This scoping review is reported according to the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA Statement)(8). The focused questionswas proposed by following the PICO principle (Population, Intervention, Comparison, and Outcome): “Which autocrine effect of hypoxia under hMSCs?" and "Which paracrine effect of hypoxia under hMSCs?” Prospero registration CRD42017057148.
Search strategies
The literature search was performed by two reviewers in three databases - PubMed (Medline), Web of Science and Scopus - until May 2016 using the search strategy initially developed for PubMed (Medline) and adapted for use in the other databases. The terms related to mesenchymal stem cells and cell hypoxia were crossed to optimize the retrieval of relevant documents (Table 1). The references cited in the included papers were also hand searched. All of the studies were imported into Endnote X7.4 software (Thompson Reuters, New York, NY, USA) to remove duplicates.
Study selection
Two reviewers assessed the titles and abstracts of all of the documents. Thus, our review included in vitro and in vivo experiments with no data limits. The eligibility criteria for inclusion and exclusion studies are in Table 2.
Full copies of all of the potentially relevant studies were identified. Two reviewers assessed the full-text papers. Any disagreement regarding study inclusion was resolved through discussion and consensus or by a third reviewer.
Data extraction
The reviewers tabulated the following data of all of the included studies: author; year of publication; cell type; tissue site; type of study; concentration of oxygen in hypoxia and normoxia; evaluation methods and main findings. The following outcomes of interest were assessed: cell behavior (viability/proliferation, migration and differentiation in different cell lines). If there was any information missing, the authors of the included papers were contacted via e-mail to retrieve these data.
Quality assessment
The studies were evaluated to provide a framework for judging the studies methodological quality according to the descriptions of the following information: selective reporting (reporting bias), descrition of passage the cell utilized, presence of positive and negative control, description of the hypoxia procedure and evaluation ofcell behavior. Each component was graded as having a low, unclear or high risk of bias using RevMan software, version 5.2 (The Cochrane Collaboration, Denmark). To low risk of bias were: have no selective reporting, use up to 10 passage, presence of a positive and negative control, description of device and methodology of hypoxia and evaluation of cell migration, differentiation and proliferation/viability.
Results
Search strategy
A total of 1.537 potentially relevant records were identified from all of the databases (Figure 1). 311 duplicates were removed. After the title and abstract examination, 731 documents remained for detailed review. 592 studies could not be included: in 193 studies the isolated cells were from unhealthy patients, 168 studies did not use isolated hypoxia, 104 had missing data, 80 studies used animal stem cells, 27 performed chemical hypoxia, 15 did not measure or reported the concentration of hypoxia during the experiment and 5 does not compare the results obtained under hypoxia with those obtained under normoxia. A total of 139 studies fulfilled all of the selection criteria and were included in the qualitative analysis.
Descriptive analysis
One hundred thirty nine studies evaluated the effects of hypoxia in human mesenchymal stem cells. The year of publication of the articles selected varied between 2004 and 2016. The cells sites used in the experiments were from teeth (3%), placenta (8%), umbilical cord blood (12%), adipose tissue (18%), bone marrow (54%) and others (5%) (Figure 2B). The most commonly used cell passages ranged from 3 to 7 passages. The main oxygen concentrations for hypoxia conditioning were 1 and 5%, respectively (Figure 3B) with induction time varying from 24h to 72h in most experiments (Figure 2A). The most used hypoxia conditioning device was hypoxia chamber and incubator (Figure 2C), using preconditioning under hypoxia, continuous hypoxia and intermittent hypoxia (Figure 3A), where hypoxia precondition is characterized by exposure to hypoxia for a given time prior to the experiment, intermittent hypoxia occurs when the cells are placed under hypoxia, withdrawn to another medium exchange, for example, and relocated under hypoxia for the experiment and continues hypoxia occurs when both time of conditioning and experiment are done under hypoxia without moments of normoxia.
. The most used assays for the evaluation of the cellular behavior were: cell differentiation, viability and migrationand the difference between autocrine and paracrine effect of hypoxia (Figure 5A, 5B). Besides, the cells
differentiations analysed were angiogenesis, osteogenesis (involves osteogenesis, dentinogenesis and mineralization), condrogenesis, adipogenesis, neurogenesis and myogenesis according to cells sites (Figure 4). The results of the selected articles demonstrated that hypoxia clearly stimulates cell migration, especially from teeth, adipose tissue (ADMSC) and bone marrow (BMSC) sites, both in relation to the autocrine and paracrine effect of hypoxia (Figure 5A, 5B). The proliferation/viability results varied according to the cell sites, where in autocrine effect adipose tissue was stimulated (Figure 5A) and in paracrine stimulated umbilical cord blood and teeth (Figure 5B). With respect to differentiation, hypoxia stimulates bone marrow and adipose tissue in autocrine effect (Figure 5A) and teeth, umbilical cord blood, adipose tissue, bone marrow and other in paracrine effect (Figure 5B). The proliferation/viability and differentiation experiments were performed at different times, they were not both induced in the same experiment since the cells either proliferate or differentiate.
The angiogenesis was analyzed in all cells sites, osteogenesis was analyzed especially in teeth, adipose tissue and bone marrow, condrogenesis was analyzed in adipose tissue and bone marrow, neurogenesis in teeth, umbilical cord blood and bone marrow and myogenesis in umbilical cord blood and bone marrow (Figure 4).
Risk of bias
Regarding the assessment of risk of bias, Figure 6 summarizes the information used to assess the methodological quality of the studies. For selective reporting and presence of positive and negative control most of 75% of studies presented low risk of bias. Meanwhile, for description of passage utilized, description of the hypoxia procedure and evaluation of cell behavior the low risk of bias were reported among 50% and 75% of all included studies. In general, the studies presented low risk of bias in all parameters analyzed.
Discussion
The hypothesis tested was accepted, the conditioning of hMSCs under hypoxia improved and its dependent of cell site. The cell migration was increase in stem cells from teeth, umbilical cord blood, adipose tissue, bone marrow and
in only one study of placenta. To cell viability/proliferation, the results for stimulated hypoxia to similar effect between hypoxia and normoxia were similar for the most cells sites. To adipose tissue, in the autocrine effect showed better stimulates under hypoxia and to teeth cells, in the paracrine effect showed better effect under hypoxia. Regarding the differentiation, there was best performance for the teeth cells under paracrine effect, for adipose tissue and bone marrow there was stimulation in autocrine effect.
Cell site
The bone marrow is the most well-known source of mesenchymal stem cells and is still extensively studied, which was confirmed by this scoping review, since more than 50% of the selected studies used hMSCs isolated from bone marrow. Other sources are also being used, the adipose tissue was the second most used source, since it is an easy to obtain tissue, since it is rejected after removal in aesthetic procedures, for example (1). In the case of placenta and umbilical cord, which are isolated from tissues that would be discarded, therefore they are safely and non-invasively removed, are found in abundance and there are no ethical problems, as well as facilitating the storage process in tissue bank (9). Stem cells of dental origin and their attachments are still poorly studied, perhaps because of the difficulty of isolation, a study(10) demonstrated that the stem cells are approximately 0.2% of the total cells that reside inside the dental pulp.
Hypoxia
Oxygen tension is a key factor in progenitor cell proliferation and differentiation. The oxygen tension varies significantly between the tissues of the body. It has been reported that in the bone marrow the concentration of oxygen varies from 1-7%, while in the umbilical cord blood it varies between 10 and 20%, in the lungs and liver it revolves around 13%, in dental pulp is close to 5% and 2-8% close to vascular structures (11). This variability may explain why the same oxygen concentration is capable of stimulating distinct behavior in one cell type and not stimulating in another cell type. Based on Fotia et al. (2015) (12) suggest that oxygen tension can act as a regulator of the maintenance of MSCs in an undifferentiated state and their proliferation can be regulated by
oxygen tension. With those data, it is seen that the concentration hypoxic of oxygen actually would be normoxic and would still vary according to the tissue site.
In our findings, the oxygen concentration used in the experiments varied from less than 1% to more than 5%, but 1%, 2% and 5% were the most used concentrations. Concentrations close to 1% are considered as real hypoxia for tissues, since it is the concentration closest to anoxia (13), already 5% is considered to be normoxia depending on the tissue site. This concentration was more used in experiments with hMSCs from the bone marrow, which is already considered the normal concentration for the stem cell niches come from this tissue (11) (Figure 3B).
Cell behavior
There is no longer any discussion about the fact that hypoxia empower the proliferation of a variety of cell lines, including hMSCs (14), as confirmed by this scoping review. This effect was demonstrated by increased cellular mobilization in vivo under hypoxia (15), which may be directly related to the role of this cellular type in tissue repair since wounds typically constitute hypoxic environments (16,17). A study (18) showed that incubation of hMSCs for 48h or more in 1% oxygen induces metabolic changes that promote MSC survival both in vitro and in vivo (19). In addition, it has also been demonstrated that MSCs respond to hypoxia by altering their secretome (20). Other study (21) reported that the recruitment of progenitor cells to the regeneration was regulated by different hypoxia gradients via HIF-1 induction of SDF-1. The higher viability/proliferative effect under hypoxia was related to better cell adhesion and extracellular matrix formation in the presence of higher amount of growth factors(22). Hypoxia induces positive regulation of transcriptional activity, which stimulates proliferation, survival and migration(23), as observed by the studies evaluated. Proliferation/viability was different for cell types, hypoxia induced better effect on paracrine cells as well as for umbilical cord. Bone marrow cells have shown to be improved for hypoxia proliferation/viability in both autocrine and paracrine forms. Already adipose tissue cells showed only improvement under hypoxia for autocrine use.
Injured tissue secretes specific proteins that facilitate migration, adhesion, and infiltration of MSCs, similar to the mechanism seen in recruitment of leukocytes to sites of inflammation. The most extensively studied pathway of stem cell mobilization and migration to injury sites was SDF1/CXCR4 axis (24). In hypoxic tissue, SDF-1 and CXCR4 are important factors for cell migration. The damaged tissues secrete SDF-1 to attract CXCR4-expressed cells, particularly the therapeutic progenitors. Invitro culture of MSCs under hypoxic conditions showed benefits in maintaining cell self renewal, migration, vascular tube formation and release of paracrine factors for chemotactic and proangiogenic properties (25). A recent study evaluated the migration of hMSCs to the lesion area and demonstrated that under hypoxia condition there was an increase in migration of about 20% and the result indicated the involvement of Akt and NfkB signaling to regulate expression of the CXCR4 protein on migration of MSC (26). These studies confirm the findings of our review, hypoxia induces migration on all cells sites analyzed, with these studies was possible to observe that the paracrine effect of hypoxia on stimulates the expression of CXCR4, pAKT and SDF-1, so this may be the reason why there was increase in the migration. Regarding the autocrine and paracrine effects, the migration was stimulated in both.
Cell differentiation
According to the studies selected for this scoping review, cell differentiation (osteogenesis, angiogenesis, condrogenesis, neurogenesis) was shown to be stimulated to hypoxia, it being only the adipogenic differentiation shown to be stimulated by normoxia. Other study (28) stated that hypoxia maintains the potential for differentiation of adipose stem cells and states that hypoxia improves the differentiation potential of ADMSC (29). A study (27) only has demonstrated greater chondrogenic differentiation in hypoxia, which agrees with the scoping review studies, but it concludes that this result should be considered with caution.
A controversial aspect is the cellular differentiation under conditioning in hypoxia. Some studies have demonstrated that hypoxia has an inhibitory effect on the differentiation of progenitors (30). But in fact what is likely to occur is the loss of much of the differentiation capacity of hMSCs when cultured in vitro (31),
because recent studies have shown that cell differentiation is regulated by oxygen tension, as was demonstrated in the case of differentiation of endothelial progenitor cells (7). Osteogenesis is the most studied type of differentiation; it is already proven that this type of differentiation occurs under conditions of serum deprivation and hypoxia, which mimics unhealthy conditions, such as ischemia for example (32). According to the studies analyzed in this review, it can be concluded that osteogenesis also occurs in hypoxia only, and was the most observed type of differentiation (Figure 4). The expression can not be applied for adipogenesis, because although the presence of lipid vacuoles and adipocyte type phenotype has been recorded, such differentiation has not been proven under hypoxia, only under normoxia (33). Stem cells isolated from dental tissues have the capacity of multilineering differentiation like the others, but by their origin of the neural crest they also present capacity of neurogenic differentiation (34). However, few studies are already available neural differentiation and based on these studies it is possible to show that there is stimulus for neural differentiation under hypoxia, but this is a preliminary result, further studies are needed to confirm such finding. The differentiation was improved under hypoxia mainly in the paracrine effect, since many studies carried out only analysis of the proteins released to the culture medium where there was the induction of hypoxia on the cells.
Angiogenesis
Angiogenesis was evaluated by 18 studies, involving all cell sites. In most of them there was stimulation of angiogenesis under hypoxia, and this effect was seen indirectly by the growth factors and cytokines that were expressed by hMSCs, it is a paracrine effect. Of the total, five studies evaluated autocrine angiogenesis, were able to demonstrate the formation of tubular structures and pre vascularization in both plaques and scaffolds. Only two studies found a similar result between hypoxia and normoxia and a study with a better result of angiogenesis under normoxia.
A well-functioning vascular system is vital, as it ensures gas exchange, nutrient supply, and waste removal for all organs. At later developmental stages and in adult life, new blood vessels are mainly formed by angiogenesis, which is the sprouting of new vessels from existing ones. Both processes are controlled
by angiogenic growth factors. In ischemia therapy, angiogenesis is crucial. The effect of hypoxic culture conditions may decrease the cell expansion time and induce differentiation of BMSCs when compared with standard protocols and more angiogenesis-associated cytokines are released, including basic fibroblast growth factor (bFGF), VEGF, interleukin-6 (IL-6), IL-8 (35). Amongst all the molecules participating in angiogenesis, VEGF is particularly relevant since it modulates the function of vascular and non-vascular cells and promotes every step of angiogenesis, in both physiological and pathological condition (36). The activation of angiogenesis is a potential mechanism to counterbalance tissue hypoxia. In oxidative stress, angiogenesis is induced by VEGF signalling. The angiogenic action of VEGF involves additionally an anti-apoptotic effect that promotes cell survival, angiogenic potential assessed by endothelial tube formation was consistently superior, with well-developed endothelial tubes, branching points, and formation of interconnecting network (32).
Hypoxia can be considered a common driving force given that in both cases the growing tissue experiences a moment of oxygen deprivation as a result of inadequate diffusion. Hypoxic cells secrete proangiogenic factors that either act on neighboring endothelial cells to induce angiogenesis of engineered teeth or stimulate the invasion of endothelial cell precursors in the dental papilla during tooth development (37).
The expression of HIF-1α is subject to hydroxylation dependent on the variation of the oxygen tension, when there is hypoxia, HIF-1α is translocated to the nucleus, hydroxylation is inhibited due to oxygen deprivation and HIF-1α accumulates, mediating significant changes in cellular metabolism, such as the expression of genes involved in energy metabolism, angiogenesis and apoptosis, and is a regulator of the homeostatic response to hypoxia (38,39).It has been shown that HIF-1α transcription regulates the expression of various growth factors, such as vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), epidermal growth factor (EGF), transforming growth factors (TGFs), insulin-like growth factor (IGF), hepatocyte growth factor (HGF), platelet-derived growth factor (PDGF)(40,41), among them also Oct-4, Sox2 and Nanog, which are essential transcription factors for the maintenance of pluripotency (42,43). When new blood vessels are formed and oxygen homeostasis is restored, HIF-1 expression decreases, followed by an arrest of
angiogenesis. Hypoxia is thus a major regulator of angiogenesis, particularly by stimulating the paracrine angiogenic activity of dental pulp cells (37).
Hypoxia device
The methodology used for hypoxic conditioning involves the need to use of device capable of being hermetically sealed. Most of the studies used hypoxia-specific incubators or chambers, few studies were performed at hypoxic workstations, which allows all cell manipulation to be performed under hypoxia, which would be ideal to definitively evaluate the effect of hypoxia on hMSCs. The type of equipment used ends up determining the type of experimental hypoxia, the workstation is the most suitable equipment to induce continuous hypoxia when it is necessary several days of experiment, since the hypoxia chamber induces continuous hypoxia only when the experiment is of few days because it is It is necessary to open it to exchange culture medium there is already a break in the continuous hypoxia, it becomes intermittent, as for incubators and may still be the case to perform only prehypoxia, where the cells are held in hypoxia for a few hours or days and the experiment is performed under normoxia. In the continuous hypoxia is where we find the best stimuli under hypoxia, already for prehypoxia as for intermittent hypoxia we find the most varied results, yet still the hypoxia being stimulus for migration, differentiation and proliferation / viability, this result is possibly explained by this being the natural microenvironment for stem cells, as already mentioned (44).
Limitations
The main limitation of these studies is the control of hypoxia, since it has already been discussed that there are differences between maintaining hypoxia continuously or discontinuously, since different behaviors will be stimulated depending on the way the experiment takes place. The studies reported here are almost entirely made in vitro, only 8 out of the 139 studies were performed in vivo, which makes these findings still preliminary, having to be confirmed in vivo. The most current in vivo studies are using animal models with some systemic disease, such as diabetes or ischemia, to evaluate the behavior of hMSCs in tissues where there is hypoxia due to the disease. This was not the objective of this scoping review, since we want to know the natural potential of
stem cells for their behavior, so that from there we can analyze their potential in diseased tissues.
Applications
This scoping review becomes very useful to support the hypoxia experiments on hMSCs, since it describes the main cellular behaviors modified under hypoxia, which is the concentration of oxygen that most stimulates this cell type and demonstrates that the trunk cells actually reside in hypoxic niches when in Their tissue sites of origin. Therefore, from these findings it is possible to determine the right type of experimental methodology to achieve the best hypoxia stimulation under hMSCs and from there use them in regenerative therapy and tissue engineering.
However, it is natural that there is improvement of their metabolism under hypoxia. With these data, it is necessary to determine what concentrations are found in vivo so that it is possible to maintain the cells full time under the right oxygen concentration, so that there is no loss of their capacity or cellular aggression by the variation of oxygen, since today the cells are maintained between 20 and 21% oxygen when they are manipulated. From this, it will be possible to know all the capacity of using the stem cells.
Conclusions
In conclusion, hypoxia actually improves the cellular response to proliferation/viability, migration and some types of differentiation, but this result can not be extended to all stem cell metabolism.
Acknowledgments
National Council of Technological and Scientific Development: CNPq. (Project Universal #456972/2014-5).
Coordination for the Improvement of Higher Education Personnel: CAPES. (Project BEX 5279/12-7)
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Tables
Table 1– Search strategy used in PubMed (MEDLINE) Search terms
#3 Search #1 AND #2
#2 “Mesenchymal stromal cell”[Mesh] OR “Cell, Mesenchymal Stromal” OR
“Cells, Mesenchymal Stromal” OR “Mesenchymal Stromal Cell” OR “Stromal Cell, Mesenchymal” OR “Stromal Cells, Mesenchymal” OR “Mesenchymal Stem Cells” OR “Cell, Mesenchymal Stem” OR “Cells, Mesenchymal Stem” OR “Stem Cell, Mesenchymal” OR “Mesenchymal Progenitor Cells” OR “Stem Cells, Mesenchymal” OR “Mesenchymal Stromal Cells, Multipotent” OR “Multipotent Mesenchymal Stromal Cells” OR “Mesenchymal Progenitor Cell” OR “Cell, Mesenchymal Progenitor” OR “Cells, Mesenchymal Progenitor” OR “Progenitor Cell, Mesenchymal” OR “Progenitor Cells, Mesenchymal” OR “Mesenchymal Stem Cell” OR “Bone Marrow Stromal Cells, Multipotent” OR “Multipotent Bone Marrow Stromal Cells” OR “Bone Marrow Stromal Cells” OR “Bone Marrow Stromal Cell” OR "Dental Pulp"[Mesh] OR "Dental Pulp" OR “Pulp, Dental” OR “Pulps, Dental” OR “Dental Pulps” OR "Tooth"[Mesh] OR "Tooth" OR “Teeth” OR "Tooth, Deciduous"[Mesh] OR “Deciduous Tooth” OR “Dentition, Deciduous” OR “Deciduous Dentition” OR “Deciduous Dentitions” OR “Dentitions, Deciduous” OR “Dentition, Primary” OR “Dentitions, Primary” OR “Primary Dentition” OR “Primary Dentitions” OR “Milk Tooth” OR “Tooth, Milk” OR “Primary Teeth” OR “Teeth, Deciduous” OR “Deciduous Teeth” OR “Teeth, Primary” OR “Tooth, Primary” OR “Milk Teeth” OR “Teeth, Milk” OR “Baby Teeth” OR “Teeth, Baby” OR “Baby Tooth” OR “Tooth, Baby” OR “Primary Tooth” OR "Molar, Third"[Mesh] OR “Molar, Third” OR “Molars, Third” OR “Third Molar” OR “Third Molars” OR “Tooth, Wisdom” OR “Wisdom Tooth” OR “Teeth, Wisdom” OR “Wisdom Teeth” OR "Stem Cells"[Mesh] OR “Stem Cells” OR “Cell, Stem” OR “Cells, Stem” OR “Stem Cell” OR “Progenitor Cells” OR “Cell, Progenitor” OR “Cells, Progenitor” OR “Progenitor Cell” OR “Mother Cells” OR “Cell, Mother” OR “Cells, Mother” OR “Mother Cell” OR “Dental pulp stem cells” OR “DPSC” OR “Stem cells exfoliated deciduous teeth” OR “SHED” OR “Dental pulp cells” OR “Dental pulp derived cells” OR “DPC”
#1 "Cell Hypoxia"[Mesh] OR “Cell Hypoxia” OR “Cell Hypoxias” OR “Hypoxia, Cell” OR
“Hypoxias, Cell” OR “Hypoxia, Cellular” OR “Cellular Hypoxia” OR “Cellular Hypoxias” OR “Hypoxias, Cellular” OR “Anoxia, Cellular” OR “Anoxias, Cellular” OR “Cellular Anoxia” OR “Cellular Anoxias” OR “Cell Anoxia” OR “Anoxia, Cell” OR “Anoxias, Cell” OR “Cell Anoxias” OR “Low oxygen”
Table 2 – Eligibility criteria.
Inclusion Criteria Exclusion Criteria
▪ In vitro and in vivo studies;
▪ Stem cells from human connective tissue;
▪ Studies that analyzed the autocrine or paracrine effect of the hypoxia using stem cells
▪ Reviews, case reports, thesis, dissertations, conference abstract;
▪ Studies published in a language other than English.
▪ Studies that do not report the concentration of oxygen tension.
▪ Species others than human;
▪ Stem cells come from the pathologic tissue or cells;
Figure 2 – Results of hypoxia induction time course data (A), human mesenchymal stem cell sites (B) and hypoxia device (C).
Figure 5 – Main findings according autocrine(A) and paracrine(B) effect of hypoxia using hMSCs.
Figure 6 - Authors' judgements about each risk of bias item presented as percentages across all included studies.
3 Considerações finais
Em odontologia e medicina, regeneração e recuperação funcional de tecidos é um grande desafio. É amplamente aceito que as células-tronco, scaffolds, fatores indutores de crescimento e diferenciação apresentam papéis cruciais nos processos de regeneração e engenharia tecidual.
Estudos encontrados na literatura já realizaram avaliação da resposta de células-tronco mesenquimais humanas frente a indução de hipóxia e posterior reoxigenação e demonstraram que existe uma resposta celular dinâmica frente a estas condições, pois a taxa de proliferação celular se mostrou mais alta no grupo em que foi induzida condição de hipóxia e após oxigenação esta taxa reduziu ao nível do grupo controle, onde as células foram cultivadas sob condições normais de oxigênio (condição atmosférica).
Neste estudo foi possível demonstrar que hipóxia estimula a proliferação celular, a migração e angiogênese, entre outros. Resultados similares entre normóxia e hipóxia foram encontrados para diferenciação celular, viabilidade e apoptose.
Mas estudos são necessários, relacionados aos nichos de células tronco teciduais, para entender seu comportamento molecular e celular e, com isso, otimizar a sua capacidade de atividade em prol de terapias teciduais, diminuindo ou eliminando os fatores que hoje são considerados limitantes na engenharia tecidual.
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