Gravity, Tissue Engineering, and the Missing Link

TIBTEC 1574 No. of Pages 4

Forum

Gravity, Tissue Engineering, and the Missing Link Raquel Costa-Almeida,1,2,@,@ Pedro L. Granja,3,4,5,6,@ and Manuela E. Gomes1,2,7,*,@,@ The influence of microgravity and hypergravity on living systems has attracted significant attention, but the use of these tools in tissue engineering (TE) remains relatively unexplored. This Forum article highlights an emerging field of research to uncover new potential applications at the interface between altered gravity and TE. 21st Century: The ‘Century of Space Travel’ Space exploration activities and spaceflight have been defying the limits of human physiology, arousing the curiosity of scientists from various research fields from physics to biology and medicine. Investigations under altered gravity are being spotlighted in the news, making dazzling headlines such as ‘Why are scientists shooting stem cells into space?’ or ‘Can organs grow in space?’. On Earth, gravity influences both physical and biological phenomena, impacting development, homeostasis, and evolution of living systems. Reduced gravitational forces in the space environment (microgravity, 1 g) [5], which can be simulated using centrifuges (MidiCAR, the large-diameter centrifuge, or even the human centrifuge), constitutes a hot topic of research under the hypothesis that it may be a way to rescue cell behavior and tissue function after exposure to near-weightlessness conditions [6]. Strikingly, microgravity and hypergravity can provide physical/ mechanical signals to modify cell behavior [7] or stem cell fate [8,9], with particular

Trends in Biotechnology, Month Year, Vol. xx, No. yy

1


(A)

interest in the field of TERM. Table 1 presents an overview of biological effects under altered gravity. Such alterations can be potentially explored as a way to manipulate cell fate in TERM strategies.

Microgravity as a tool in ssue engineering

Cellular self-assembly

(i)

Tissue engineering

Microgravity

Cell suspension (ii)

3D mulcellular spheroid

Scaffold-free microssue (ii)

In vitro stem cell expansion

Control over differenaon

Maintenance of stemness properes Oct-4

Fat

Ectodermal stem cell

Bone

Mesodermal stem cell

Nanog

Sox-2

Pluripotent stem cell

Endodermal stem cell

Carlage

Rex-1

(B)

Hypergravity as a tool in ssue engineering Modulang cell behaviour

(i)

Hypergravity loading

Endothelial

(ii) barrier formaon

(iii) Myoblast differenaon 5g

Control

20 g

10 g

++ Myosin

100 µm

2g

(iv) Osteogenic differenaon

Mineralizaon ++ Calcium deposits

2D cell culture Hypergravity

(C)

Tissue engineering tools – complex in vitro models

(i) 3D culture models

(ii) Topographies

(iii) Cellular complexity

Cell guidance • Different cell

types • Tissue•

Biomaterals (hydrogels etc.)

•

engineered constructs

At a very early stemness stage, real microgravity (Space Shuttle Discovery, NASA STS-131 mission) was shown to inhibit the differentiation of embryonic stem cells, preserving their stemness and suggesting impaired regenerative potential in the space environment [8]. Nonetheless, adipose stem cells (ASCs) previously cultured under microgravity exhibited increased proliferation and enhanced differentiation abilities in induction media (Figure 1Aii) compared with ASCs in monolayers [10]. These results hold great interest for prolonged stem cell expansion in TERM strategies. Intriguingly, altering the duration of exposure to simulated microgravity showed that the fate of mesenchymal stem cells can be directed toward either differentiation into soft tissue lineages (72 h, endothelial, neuronal and adipogenic differentiation) or osteogenic differentiation (10 days) [9]. These results raise several questions. Is the differentiation potential differently affected depending on the stemness stage (from early pluripotency to multipotency and eventually the progenitor cell stage)? Can cell fate be manipulated in vitro to generate potentially alternative cell sources for TE (Figure 1Aiii)?

Decellularized matrices

In turn, hypergravity can be used as a way of generating loading over cells Figure 1. Strategies for Bidirectional Crosstalk between Altered-Gravity Research and Tissue (Figure 1B), having positive effects particEngineering. (A) Microgravity as a tool in tissue engineering. (i) Cellular self-assembly process for microtissue ularly on musculoskeletal tissues (e.g., formation. Cells exposed to microgravity, either in cell suspension or detaching from 2D culture, tend to bone, tendons, cartilage) by fastening aggregate and form multicellular spheroids, which can be later combined and fused to form microtissues. (ii) Microgravity can be used for stem cell expansion and maintenance of stemness properties, including and increasing the deposition of extracelpluripotency markers and multilineage differentiation. The example provided uses adipose stem cells (ASCs). lular matrix components [5]. Strikingly, Reproduced, with permission, from [10]. (iii) Given the existence of pluripotent stem cell markers in ASCs, early culturing mesenchymal stem cells (MSCs) control over stem cell fate constitutes an interesting hypothesis to be further explored through the use of under simulated hypergravity conditions cultures under microgravity to potentially generate new cell sources for tissue engineering approaches. (B) Hypergravity as a tool in tissue engineering. (i) Exposing cells to hypergravity may constitute a way of simulating upregulated osteogenesis-related genes (over)loading and of modulating the behavior of various cell types, a potential strategy for tissue engineering. (ii) [11]. Hypergravity also enhanced myoExposing endothelial cells to hypergravity resulted in enhanced endothelial barrier formation and integrity. genic differentiation and myotube formaReproduced from [15]. (iii) Hypergravity exposure improved myoblast differentiation in a g-level-dependent tion [12]. Additionally, soft tissue lineages 2



Table 1. Examples of Biological Effects under Altered-Gravity Conditionsa[160_TD$IF] Cell type

Gravity conditions

Biological effect

Refs

Real microgravity, Space Shuttle, 15 days

Inhibition of multilineage differentiation potential Increased expression of stemness markers on return to

[8]

Clinostat, 3 days and subsequent culture at 1 g

Downregulation of genes involved in MAP kinase and focal

Clinostat, 72 h or 10 days

Initial remodeling of actin filaments (diffuse) after 72 h and

Microgravity (real and simulated) Mouse embryonic stem cells

Earth’s gravity

Rat MSCs

[14]

adhesion signaling pathways Immediate downregulation of genes involved in cardiac morphogenesis Inhibition of cardiomyocyte-specific genes after 10 days at 1 g (post-clinorotation) Decreased beating activity of embryoid bodies at day 10 (post-clinorotation) [9]

reestablishment of actin filaments after 10 days (more microfilaments) Involvement of RhoA[16_TD$IF]-associated pathway: initial decrease of RhoA activity after 72 h; significant increase after 10 days On induction: MSCs exposed for 72 h underwent endothelial, neuronal, and adipogenic differentiation; MSCs exposed for 10 days differentiated into osteoblasts Human ASCs

Microgravity bioreactor (spinner flask)

Spontaneous formation of 3D spheroids Upregulation of pluripotency markers; improvement of

[10]

multipotent differentiation capacity HUVECs

[162_TD$IF]RPM, 0.001 g, 15-min intervals

Decreased endothelial barrier integrity

[15]

LDC, 20 g for 3 h

Stretched cells with parallel actin filaments and increased

[11]

Simulated hypergravity Rat MSCs

cell area Upregulation of osteogenesis-related gene Runx2[163_TD$IF] and RhoA Higher expression of collagen type I at the protein level PC12 neuron-like cells (rat cell line)

Mouse myoblasts (C2C12 cell line)

HUVECs

[159_TD$IF]a

50 g and 150 g for 1 h using a bench centrifuge

No differences in cell morphology; increased metabolic

[13]

activity Upregulation of Ina 164_TD$IF][ Longer b3-tubulin-positive neurites for cells at 150 g

LDC, 5 g, 10 g, or 20 g for 2h

Increased cell numbers on 10-g and 20-g stimulation and

[162_TD$IF]LDC, 2 g or 4 g, 15-min intervals

Enhanced cell–cell integrity: increased expression of VE-

LDC, 3 g for 4 h or 16 h and 10 g for 16 h

Reduced formation of capillary-like structures in 3D

[12]

protein synthesis (significant at 20 g) Thicker actin filaments with increasing g level Reduced expression of myogenin accompanied by increased formation of myosin-positive myotubes [15]

cadherin and peripheral F-actin [7]

Matrigel assay in a g-level- and time-dependent manner

Abbreviations: RhoA, ras homolog family member A, small protein involved in cytoskeleton regulation and osteogenesis; HUVECS, human umbilical vein endothelial cells; Runx2, runt-related transcription factor 2; Ina, neurofilament-66.

manner. Reproduced, with permission, from [12]. (iv) Hypergravity is well known to influence osteogenic differentiation and increase mineralization. (C) Tissue engineering tools can provide more complex and physiologically relevant in vitro models to be used in altered-gravity research, including: (i) 3D culture systems; (ii) surfaces and 3D materials with topographical cues; and (iii) increasing cellular complexity.


3


also benefited from hypergravity conditions, as demonstrated by the formation of longer neurites [13]. Hence, can altered gravity be used to pre-stimulate cells in tissue-engineered constructs? The functional behavior of endothelial cells in a 3D environment has been briefly studied and the main outcomes have shown reduced formation of capillary-like structures [7]. Nonetheless, the effects of exposing cells embedded in 3D matrices remain relatively unexplored, constituting promising avenues in an emerging line of research. Overall, simulated altered-gravity conditions appear to be good candidates in TE to control stem cell fate and modulate or accelerate cell behavior, but additional research is still needed at a more fundamental level.

rendering 3D assemblies the ideal platform for studying the effect of altered physical forces on cell and tissue biology. Thus, tissue engineers can create in vitrorelevant models (e.g., biomaterials, decellularized matrices) and can take cellular complexity to a blue-sky level by combining different cell types in a single construct (Figure 1C). Conversely, applying alteredgravity conditions can potentially lead to advanced tissue constructs by accelerating and/or modifying cell fate. Nonetheless, the lack of communication between tissue engineers and space researchers, as well as limited awareness of this interfacial research, means the field moves slowly, but studies are emerging and open questions will hopefully drive research advances in the next years. Acknowledgments

Challenges and Envisioned Future Opportunities

The authors acknowledge financial support from the

The field of altered-gravity and space research faces several limitations, including: (i) the limited number and high cost of space missions; and (ii) the fact that, with the exception of microgravity-induced microtissue formation, the majority of studies under altered gravity have been conducted in 2D cell cultures. The multidisciplinary field of TE can take studies under altered gravity ‘to infinity and beyond’. Crosstalk between these highly specialized fields would bring great bidirectional benefits. Combining TE techniques with ground-based platforms will create innovative and groundbreaking pathways for space physiology and ageing research and advance the design of novel tissue-engineered constructs. TE tools can generate physiologically relevant structures or tissue substitutes,

scholarship SFRH/BD/96593/2013 (R.C-A.) and for

4


Fundação para a Ciência e a Tecnologia (FCT) for PhD Career Consolidation Grant IF/00593/2015 (M.E.G.). 3B’s Research Group – Biomaterials, Biodegradables and Biomimetics, University of Minho, Headquarters of

*Correspondence: [email protected] (M.E. Gomes). URL: http://3bs.uminho.pt. https://doi.org/10.1016/j.tibtech.2017.10.017 References 1. White, R.J. and Averner, M. (2001) Humans in space. Nature 409, 1115–1118 2. Hides, J. et al. (2017) Parallels between astronauts and terrestrial patients – taking physiotherapy rehabilitation “To infinity and beyond”. Musculoskelet. Sci. Pract. 27, S32– S37 3. Aleshcheva, G. et al. (2016) Scaffold-free tissue formation under real and simulated microgravity conditions. Basic Clin. Pharmacol. Toxicol. 119, 26–33 4. Laschke, M.W. and Menger, M.D. (2017) Life is 3D: boosting spheroid function for tissue engineering. Trends Biotechnol. 35, 133–144 5. Genchi, G.G. et al. (2016) Hypergravity as a tool for cell stimulation: implications in biomedicine. Front. Astron. Space Sci. 3, 26 6. Tajino, J. et al. (2015) Intermittent application of hypergravity by centrifugation attenuates disruption of rat gait induced by 2 weeks of simulated microgravity. Behav. Brain Res. 287, 276–284 7. Costa-Almeida, R. et al. (2016) Effects of hypergravity on the angiogenic potential of endothelial cells. J. R. Soc. Interface Published online November 9, 2016. http://dx. doi.org/10.1098/rsif.2016.0688 8. Blaber, E.A. et al. (2015) Microgravity reduces the differentiation and regenerative potential of embryonic stem cells. Stem Cells Dev. 24, 2605–2621 9. Xue, L. et al. (2017) Duration of simulated microgravity affects the differentiation of mesenchymal stem cells. Mol. Med. Rep. 15, 3011–3018

1

the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, Avepark – Parque de Ciência e Tecnologia, Zona Industrial da Gandra, 4805-017 Barco, Guimarães, Portugal 2 ICVS/3B’s – PT Government Associate Laboratory, Guimarães, Portugal i3S – Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Rua Alfredo Allen, 208, 4200-135 Porto, Portugal 4 INEB – Instituto de Engenharia Biomédica, Universidade do Porto, Porto, Portugal 5 Faculdade de Engenharia, Universidade do Porto, Rua 3

Dr. Roberto Frias, 4200-465 Porto, Portugal ICBAS – Instituto de Ciências Biomédicas Abel Salazar, Universidade do Porto, Porto, Portugal 7 The Discoveries Centre for Regenerative and Precision Medicine, Headquarters at University of Minho, Avepark, 4805-017 Barco, Guimarães, Portugal @ Twitters: 6

@raquelccalmeida, @3bsuminho, @plgranja, @ManuelaEGomes

10. Zhang, S. et al. (2015) The effects of spheroid formation of adipose-derived stem cells in a microgravity bioreactor on stemness properties and therapeutic potential. Biomaterials 41, 15–25 11. Rocca, A. et al. (2015) Barium titanate nanoparticles and hypergravity stimulation improve differentiation of mesenchymal stem cells into osteoblasts. Int. J. Nanomed. 10, 433–445 12. Ciofani, G. et al. (2012) Hypergravity effects on myoblast proliferation and differentiation. J. Biosci. Bioeng. 113, 258–261 13. Genchi, G.G. et al. (2015) Hypergravity stimulation enhances PC12 neuron-like cell differentiation. BioMed Res. Int. 2015, 748121 14. Shinde, V. et al. (2016) Simulated microgravity modulates differentiation processes of embryonic stem cells. Cell. Physiol. Biochem. 38, 1483–1499 15. Szulcek, R. et al. (2015) Transient intervals of hyper-gravity enhance endothelial barrier integrity: impact of mechanical and gravitational forces measured electrically. PLoS One 10, e0144269