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1. A niche is a microenvironment which has an influence on cell behavior. When we are talking about a niche, we are talking about the matrix, the soluble factors (growth factors) and other cells (microenvironment). Whatever happens in the microenvironment, will help determine which path the cell will go down and which way the cell will differentiate. "Loss of the niche can lead to loss of stem cells, indicating the reliance of stem cells on niche signals" (Li and Neaves). The feeder cell layer provides a niche for the cells. The niche provides signals for the cells while they grow. Using stem cells is still considered an obstacle because researchers are still trying to fully understand the signals, which turn on and off genes, which will then differentiate the stem cells down a specific pathway.
Some specific, concrete examples of stem cell niches include hematopoietic stem cells, human embryonic stem cells, and adult stem cells. In order for researchers to replicate the in vivo (living within the body or tissue) niche conditions in vitro (living outside the body), studies have to be conducted to understand the niche. This is because scientists want to be able to control the cell proliferation and differentiation in flasks or plates, so the proper quantity and type (having one type or multiple depending on desire) are produced before they are introduced back into the patient.
When talking about human embryonic stem cells, these are often grown in fetal bovine serum (FBS) that is supplemented with media. When using a flask, the bottom of the flask is considered the matrix, because it has the media (substrate) for the stem cells to grow on. This is considered to be the feeder layer of the cells, which is believed in helping retain the pluripotent characteristics (differentiate into cells derived from any of the three germ layers) which is why embryonic stem cells are used. Even if all the environmental surroundings are used, it may not truly imitate in vivo niche conditions properly because there are other factors as well that go into supporting embryonic stem cell growth, such as other conditions (matrices, soluble factors, and other cells).
Through an adult's life, the adult stem cells that are found throughout a person remain in a state that is not altered. "An adult stem cell is an undifferentiated cell found among differentiated cells in a tissue or organ, can renew itself, and can differentiate to yield the major specialized cell types of the tissue or organ" (Stem Cell Information). Once these cells are cultured in vitro, they undergo a process in which their whole morphology and shape changes and their ability to proliferate in a specific capacity are decreased. The adult stem cells role in the area of where they are found is to help repair and build back up what has been damaged. Another term associated with adult stem cells is also somatic cells. Even though the researchers and scientists know adult stem cells are found in organs and tissues, it is still unknown what adult stem cells do in mature tissues. When the adult stem cells are found, they are actually found in small numbers. The reason this is thought, is because they are quiescent (not dividing) until some sort of disease or injury happens in that specific area, which will then render them to start dividing. The adult stem cells also exhibit the ability to form specialized cell types of other tissues which is also known as plasticity.
Li, Linheng and William Neaves. "Normal Stem Cells and Cancer Stem Cells: The Niche Matters." American Association for Cancer Research (2005): 4553-4557.
"Stem Cell Information." National Institues of Health (2010).
2. Tensegrity helps in everyday life, with understanding how every day structures are built, such as how a cell's structure works to hold itself together. "A universal set of building rules seems to guide the design of organic structures - from simple carbon compounds to complex cells and tissues" (Ingber, 1). This can be seen in "an organism, from a bacterium to a monkey, as it develops through an incredibly complex series of interaction involving a vast number of different components" (Ingber). Somewhere in between the complex tensegrity structure to the proteins and molecules which help in the process of tensegrity are the cellular structures.
Cells contain an internal structure, or cytoskeleton, which are composed of three different sub unit types: known as microfilaments or actin (more dynamic because it is has more bending, tension and is assembled), intermediate filaments and microtubules (least dynamic). A cell's cytoskeleton can be changed or altered in many ways because of the balance of physical forces (tension and compression) across the cell surface pulling the membrane in different directions (Ingber). Tensegrity is an ongoing process within the cells of the body and the restructuring of the cells and cytoskeleton tells the cell what to do and how to act. Cells are made up of a cytoskeleton no matter if they are flat or round in shape. If they are flat then the cells will respond by reproducing to cover the surrounding substrate until they reach contact inhibition and once the cells start to round, this indicates to other cells that there are too many cells in the surrounding area and it will indicate that some may need to die in order to create space. A group of built up cells make up the tissues within the body. These cells are anchored to the environment, by integrins (proteins), which help connect cells to the surroundings. When transferring cell lines, these integrins, which can be made up of Ca2+, are cut by an enzyme called trypsin. After the proteins are cut, the tension on the cell is then lessened because the inner cytoskeleton is able to round up, which causes the nucleus to change. When the cells are rounded up, they can then be easily put into suspension and then passed to create more space within the substrate and allow other cells to bind to their surroundings.
After researching cells and their internal structure, they found out the cells do get their figure and overall structure from tensegrity. After some researchers had modeled what tensegrity can do, it was found not only do they get their structure from the inner cytoskeleton, which is made up of microfilaments, intermediate filaments and microtubules, they also get it from the environment (what the cell is in), which anchors the cell to the surface (Ingber). The microfilaments, which are a key element of the cytoskeleton, extend throughout the cell, where it is attached to both the cell's nucleus and the cell's membrane and exerts tension on the whole cell by pulling the membrane towards the nucleus. As this is pulling on the cell and causing tension, there are two other forces (both inside and outside the cell) which are acting in opposition to this, which causes the cell to exhibit tensegrity. Cells that are growing in vitro display tensegrity because this helps them grow and flourish in an environment that they aren't accustomed to.
Ingber, Donald. "The Architecture of Life." Scientific American (1997): 48-57.
3. The nature of stem cells including the current nomenclature includes "naming" which would contain the three main types of stem cells. Embryonic stem cells, fetal stem cells, and adult stem cells which are considered the main types but there are also germ stem cells. Any type of differentiation of the stem cells includes specialization. The differentiation is a specialization of a cell to perform specific functions in the body. The stem cells go through a degree of specialization, where the embryonic stem cells are on the one end (not having any degree of specialization) and on the other end of the spectrum are the differentiated adult cells, where they are considered complete. The degree of specialization is considered a continuum, where it has plasticity and is able to change. The stem cells provide a basic model of all cellular development.
Any type of cellular interactions includes tissue and organ development. The development of tissues or organs, is a change in the total number of cells that produce the structures of the body. In order to develop the eleven systems within a human, the cells which make up the tissues must develop into specialized functional subunits. The growth of the stem cells includes cell cycling. The cell cycling can produce two types of division. The stem cell characteristics include the stem cell going through a specific type of division. The divisions which can occur are synchronous division or asynchronous division. The synchronous division is dealing with the stem cell reproducing itself. The asynchronous division deals with one daughter cell replacing the stem cell.
There are relative relationships among the stem cells. As one cell is produced, down the line, another stem cell will be produced based off of how it differentiates. The egg and sperm are both highly specialized and will then form the embryonic stem cell. The embryonic stem cells are not specialized, are able to develop into any kind of cell based off of signals going on in the human body and it generates the three embryonic "germ layers"; it is also considered totipotent which means the cells are produced from the fusion of an egg and sperm cell. The cells produced by the first few separations of the fertilized egg are also considered totipotent.
The embryonic stem cell will then differentiate into fetal stem cells. The fetal stem cells help establish organ rudiments, expand and build organ tissues, help in supporting maturation into adult form, are closer to the embryonic stem cells on the degree of specialization scale, and have many ethical questions associated with them. There are continuous signals that are sent between cells in the culture, to differentiate which particular pathway embryonic cells will develop. So, researchers have been trying to understand the niche (environment/surroundings) a cell lives in, in order to understand the environmental cues better and the signals that are sent between them. Once the cells choose their specific path to go down, there is normally no turning back to differentiate into another type of cell, which means it is turning into its final shape or will reach terminal differentiation. The goal of using stem cells is to hopefully one day replace or regenerate failing body parts and to also hopefully cure those diseases which still remain unknown (Lanza and Rosenthal).
From the fetal stem cells, it can produce progenitor cells. The progenitor cells are considered the most differentiated. "In contrast, progenitors have some ability to renew themselves during proliferation, but they are restricted by an internal counting mechanism to a finite number of cell divisions. With increasing differentiation, the ability of the progenitors' offspring to multiply declines steadily" (Clarke and Becker). Out of all the stem cells, the progenitor cell is also considered the normal stem cell or mature cell and it undergoes mutagenesis (multiple "hits" over time).
Then the adult stem cells help in generating differentiated somatic cells, and help in rebuilding and maintaining the certain tissues/organs. The adult stem cells are considered to be an undifferentiated cell but can be found among differentiated cells throughout the body, commonly found within specific tissues and organs within a person.
Clarke, Michael and Michael Becker. "Stem Cells: The Real Culprits in Cancer?" Scientific American (2006): 53-59.
Lanza, Robert and Nadia Rosenthal. "The Stem Cell Challenge." Scientific American (2004): 93-99.
4. "Stem-cell populations are established in 'niches' - specific anatomic locations that regulate how they participate in tissue generation, maintenance and repair. The niche saves stem cells from depletion, while protecting the host from over-exuberant stem-cell proliferation" (Scadden, 1075).
"Coordinated interactions with soluble factors, other cells, and extracellular matrices define a local biochemical and mechanical niche with complex and dynamic regulation that stem cells sense" (Discher). When we talk about the niche, we are talking about three factors, the matrix, soluble factors and other cells which make up the microenvironment. The niche is the microenvironment surrounding the cells, which help regulate, support and influence overall cell behavior. Researchers have been trying to understand the niche (environment/surroundings) a cell lives in, in order to understand the environmental cues better and the signals that are sent between them. A specific stem cell niche (such as a hematopoietic stem cell niche) has certain cells (blood cells) that are found in a particular location (bone marrow) in the body to help maintain overall function of the stem cells from day to day (Li). A niche refers to either in vivo (living within the body or tissue) or in vitro (cells living outside the body) stem cell microenvironment. "The simple location of stem cells is not sufficient to define a niche. The niche must have both anatomic and functional dimensions" (Scadden, 1075).
One of the first factors that contribute to the concept of the niche includes the matrix. There are two main types of matrices: natural and synthetic. The natural matrix is dealing with extracellular matrices, such as fibers with collagen like factors. The synthetic matrix is more defined and quasisynthetic. "The niche is a physical anchoring site for stem cells, and adhesion molecules are involved in the interaction between stem cells and the niche and between stem cells and the extracellular matrix" (Li). "Some types of matrixes involve decellularized tissue matrices and synthetic polymer matrices" (Discher).
The second factor that contributes to the concept of the niche includes soluble factors, also known as the "growth factors". Some of the growth factors include FBS (fetal bovine serum), PBS, trypsin, complete media (which includes DMEM, P/S, and FBS) and any other type of soluble factors which help in the growth of cells. The media is used in cell culture preparation; the media is used as an environment for the cells to take up the nutrients and chemicals to grow in the culture. The chemicals and nutrients in the medium help the cell to build new proteins and other components which help the multiple cells grow and function while in vitro (Ryan). Using only the medium will not help support cell growth but must also be supplemented with an animal serum such as fetal bovine serum (FBS) or any other type of serum that is out there. "The niche generates extrinsic factors that control stem cell number, proliferation, and the fate determination. Many developmental regulatory signal moleculesâ€¦ have been shown to play roles in controlling stem cell self-renewal and in regulating lineage fate in different systems" (Li).
The third factor that contributes to the concept of the niche includes cells. The cells include the specific environments such as hematopoietic stem cells, which are found in bone marrow, embryonic stem cells, which are cells that are able to develop into any kind of cell based off of signals going on in the human body, germ stem cell, and adult stem cells, which are undifferentiated stem cells that are found in tissues and organs. These are all found in different areas throughout a person and have different functions throughout a person's life.
Discher, Dennis and et al. "Growth Factors, Matrices, and Forces Combine and Control Stem Cells." Science (2009): 1673 - 1677.
"Fundamental Techniques in Cell Culture." 2005. Sigma-Aldrich Co. 2010.
Li, Linheng and William Neaves. "Normal Stem Cells and Cancer Stem Cells: The Niche Matters." American Association for Cancer Research (2005): 4553-4557.
Ryan, John. "Introduction to Animal Cell Culture." Corning Life Sciences (2008): 1-8.
Scadden, David. "The stem-cell niche as an entity of action." Nature (2006): 1075-1079.
5. "Stem cell research is at the center of a raging controversy due to its ethical implications. Although few debate the potential marvels that mastering stem cells could provide by way of medical advancements in the treatment and prevention of life threatening diseases, many object strenuously to the measures being taken to reach that goal" (International Society for Complexity, Information, and Design).
When specifically studying adult stem cells, this is not the area that is hugely debated, since when they are harvested they are not causing any harm to anybody or anything. However, when people start talking about harvesting the embryonic stem cells, this is another whole situation all together, since this destroys the early stage embryos, known as blastocysts. The blastocysts possess the inner cell mass, which in turn forms the embryo. The reason the blastocyst is argued, is because some say that it only contains "a cluster of 150 cells and does not possess even the nervous system required to biologically qualify as a human being" (International Society for Complexity, Information, and Design). Others argue that the blastocysts are maintained from in vitro fertilization (a blastocyst that is living outside the body) clinics but only with permission from patients (those who donate the eggs).
"However, for the people whose moral beliefs state that human life begins at the moment of conception, embryonic research is simply unacceptableâ€¦" (International Society for Complexity, Information, and Design). The people, who believe in this, are generally considered "pro-lifers" who believe in saving an existing life. These are the people that believe the destruction of the blastocyst (a laboratory-fertilized human egg, which has been given permission from the patients who have donated it) is equivalent to murdering another human being. The individuals believe that it is not right (under any circumstance) to destroy an existing embryo, to help save or reduce other suffering that exists out in the world.
The reason scientists use embryonic stem cells, is because there is an endless amount of research that can be done on stem cells in helping to understand the overall human development and in the growth and treatment of all the diseases that are out in this world. Embryonic research will help in many cures that are years down the road. There are millions of Americans that suffer from diseases each and every day that will hopefully one day be treated more effectively with embryonic stem cell therapy.
The adult stem cells that are out there have surprisingly, already been used to help cure many of the diseases that are out in the world. New and emerging research is being used in the potential for dealing with umbilical cord blood for stem cell research. "Embryonic stem cells are thought by most scientists and researchers to hold potential cures for spinal cord injuries, multiple sclerosis, diabetes, Parkinson's disease, cancer, Alzheimer's disease, heart disease, hundreds of rare immune system and genetic disorders and much more" (White). Yet, there have not yet been any cures that have been made by embryonic stem cell therapy.
Even though people throughout the world may have issues with the decisions that are made by the medical professionals (such as scientists and researchers) and the women who donate the eggs, all of them carefully consider all aspects of the ethical and moral implications with every different circumstance/situation that arises. These people also believe that more funding could be put towards the adult stem cell research and not towards the human embryos.
International Society for Complexity, Information, and Design. 2005. 28 April 2010 <http://www.iscid.org/encyclopedia/Ethical_Implications_of_Stem_Cell_Research>.
White, Deborah. "Pros & Cons of Embryonic Stem Cell Research." 2010. About.com. 28 April 2010 <http://usliberals.about.com/od/stemcellresearch/i/StemCell1_2.htm>.
6. What defines a stem cell? "All stem cells-regardless of their source-have three general properties: they are capable of dividing and renewing themselves for long periods; they are unspecialized; and they can give rise to specialized cell types" (Stem Cell Information). The differentiation (into a specific type of cell) and the development of a cell make, it a stem cell. The differentiation is the specialization of a cell to perform specific functions in the body along with the development which changes the number of cells that produce the specific structures within the body. Cells do not differentiate by themselves but they use the cell signaling which happens in the niche. High cell turnover for stem cells occur mostly in the gut and the skin (Clarke and Becker).
A stem cell is a gradient/continuum. It has plasticity, which means it has the ability to change. "The capacity of stem cells to re-create themselves through self-renewal is their most important defining property. It gives them alone the potential for unlimited life span and future proliferation" (Clarke and Becker). Potency
(totipotent, pluripotent, multipotent, and unipotent) helps specify the differentiation potential of a stem cell or other cell. There are three main types of stem cells which include the embryonic stem cells, fetal stem cells and adult stem cells. One other stem cell which can also be considered is germ stem cells (found in adults). These are the precursors for sperm and eggs and are set aside at the blastocyst stage. All of these types of stem cells are not the same, and each of them have unique characteristics, which makes each of them have different functions. Stem cells provide a basic model of all cellular development. They go through growth, which deals with the cell cycling, through differentiation which is dealing with the degree of specialization, and different cellular interactions which are dealing with the tissue and organ development based off of the surrounding niche.
There are certain stem cell characteristics. A stem cell can have synchronous division, which means it is a stem cell reproducing itself and asynchronous division, which is a daughter cell replacing the stem cell. In an article on stem cells, it says, "to begin producing new tissues, a stem cell divides in two, but only one of the resulting daughter cells might proceed down a path toward increasing specificity. "The other daughter [cell] may instead retain the stem cell identity" (Clarke and Becker).
The human embryonic stem cells occur spontaneously. "Stem cells' ability to differentiatie into a broad range of cell types allows them to form all the different elements of an organ or tissue system" (Clarke and Becker). This can also lead to tumors, which will have a negative effect on a person if it is malignant. "A dark side of stem cells - their potential to turn malignant - is at the root of a handful of cancers and may be the cause of many more" (Clarke and Becker).
Clarke, Michael and Michael Becker. "Stem Cells: The Real Culprits in Cancer?" Scientific American (2006): 53-59.
"Stem Cell Information." National Institues of Health (2010).
7. To harvest embryonic stem cells from an original natural source, takes many precise steps in order to not contaminate the embryo or whatever one is working with. One of the first steps includes retrieving all the materials that will be necessary in the harvesting of the embryonic stem cells from an original natural source. These materials can include retrieving the embryos (in this case zebra fish embryos), petri dish with alcohol, syringes, needles, 6 well plate, pipet pump, pipet, pasteur pipet, centrifuge, 0.1% bleach, 50ml conical tube, PBS, stereo microscope, and 1x trypsin. After all the materials are collected these should be brought back to the work area (in the horizontal hood). This lab requires individuals to be careful with the instruments because you are dealing with sharp objects, such as the syringe. The horizontal hood should be prepared before all the materials are brought back by cleaning (wiping down) the work area with 70% ethanol. When everything is in its place (in the hood), the embryos (zebrafish embryos) should be picked up from the source and placed into a 50ml conical tube. As the embryos are being picked up, the forceps and razor blades should be getting cleaned in the petri dish that is filled with ethanol. This will allow these objects to be cleaned of any other contaminants that are already on them. Once the embryos are brought back, they should be washed 3 times with 0.1% bleach solution for 1 minute. You don't want the zebra fish embryos to be in the bleach much longer then that or less because you can harm the embryos. From there they should be rinsed with 10ml of PBS, and once they settle the PBS should be poured off.
Half of the embryos will be digested and the others will be in fragments. The first embryos that we will be dealing with, should have the outer protective shell removed from them. Take half of the embryos that you have, place them into a conical tube and 5ml of 1x trypsin added to it. These should be incubated at room temperature for 30 minutes with agitation (inverting tube) every 5 minutes, which will lead these embryos to become digested at some point. The embryos are transferred to a conical tube and pelleted. Any extra supernatant is poured off and the fragments should be resuspended and transferred to a bead containing flask and shake for 20 minutes. The contents should be taken out and added into each well.
The rest of the embryos will be made into fragments. The rest of the outer protective shells need to be removed from the remaining embryos and the embryos need to be dissected into small pieces/fragments. These fragments will then be transferred into a well and must be evenly transferred to fill up a 6 well plate.
After the embryo fragment is added into each of the wells, complete medium needs to be added to each well containing a zebra fish embryo piece. The complete media will help to provide the necessary nutrients to the zebrafish embryos. Once they are harvested, observations should be made at least once each week if not more.
Refer to Lab 4: Zebrafish Emrbyonal (Stem) Cell, ZEC Laboratory
8. "Embryonic stem (ES) cells are derived from the inner cell mass of blastocyst stage embryos and have the unique capacity to proliferate extensively while maintaining pluripotency" (Yamanaka). Potency specifies the differentiation or the ability to separate into different cell types of a stem or other cell. The different types of potency include: totipotent, pluripotent, multipotent, and unipotent.
The combination of an egg and sperm cell, produce totipotent cells. Both the sperm and egg alone are both highly specialized, which is why totipotent is considered to not be specialized like the embryonic stem cell is. Any of the cells that are also produced from the first few distributions of the fertilized egg (fusion of the sperm and egg) are also considered totipotent. These specific cells can differentiate into embryonic and extraembryonic cell types. They have the ability (since they are forming from egg and sperm) to turn into any type of cell in the human body.
The descendants of totipotent cells include pluripotent cells and these pluripotent cells can differentiate into cells derived from any of the three germ layers. "Both human embryonic stem cells and induced pluripotent stem cells can self-renew indefinitely in vitro while maintaining the ability to differentiate into advanced derivatives of all three germ layers, features very useful for understanding the differentiation and function of human tissues, for drug screen and toxicity testing, and for cellular transplantation therapies" (Yu and Thomson). Any layer (ectoderm, endoderm, and mesoderm) can be produced from the pluripotent but not the trophoectoderm layer. The pluripotent cells make all of the cells of the entire body.
The multipotent cells can only produce cells of a certain type (family of cells), such as hematopoietic stem cells. Hematopoietic stem cells are able to differentiate into any type of blood cell such as red blood cells, white blood cells, granulocytes, platelets, etc. The hematopoietic stem cells are more specific (only looking at the blood cells), but tend to be derived from mesenchymal (cells of mesodermal origin). Multipotent is considered more of the ectoderm and endoderm but is restricted to one and not both of them.
The cells that are produced from only one type of cell are called unipotent but they do have the ability to undergo self-renewal which helps distinguish them from non-stem cells (such as muscle stem cells). These are used for stem cells (skeletal cell). They are fairly unusual and are not as common compared to the different types of potency. These tend to be more common with progenitor cells and not somatic cells.
Yamanaka, Shinya. "Pluripotency and nuclear reprogramming." Philosophical Transactions of the Royal Society (2008): 2079-2087.
Yu, Junying and James Thomson. "Pluripotent stem cell lines." Genes and Development (2009): 1987-1997.
9. Is tensegrity (tissue engineering field) building principles universal? Researchers still do not know if they can apply the tensegrity rules, concepts and knowledge to both types of structures that are both small and large. From my current readings, the current state of tissue engineering is still in the process of being researched and understood why things happen the way they do. "A universal set of building rules seems to guide the design of organic structures-from simple carbon compounds to complex cells and tissues" (Ingber), which is known as tensegrity or tissue engineering.
Tissue engineering involves the process of tensegrity. Tissue engineering involves four essential components: (stem) cells, a matrix or scaffold of some sort, a bioreactor, and cytokines (Van Winterswijk). In order for tissue engineering to occur, the cells have to become embedded/seeded in the scaffold. What would be most ideal is if the correct microenvironment (either in vitro or in vivo) is created and the cells will multiply which will fill the scaffold with tissue and will allow the cells to grow into the correct shape. This new scaffold will then be able to become implanted into a body, and the scaffold will then help support directing the cell proliferation to become what it needs to. The scaffold will start to disintegrate as the cells start to proliferate, which will allow any and all blood vessels and cytokines to make contact with the cells (Van Winterswijk). The cells will continue to proliferate and differentiate into the desired tissue, which will make the scaffold to continuously degrade. Once the scaffold dissolves completely, the new tissue will start to function in its new surroundings. (Van Winterswijk).
Based off of the research done by scientists in the article "The Architecture of Life", given certain constraints from the environment, tissue engineering (tensegrity) is an efficient way to build at all levels, including from the smallest level, at the molecular scale, all the way to the macroscopic scale (Ingber). The researchers say, through time "it is possible that fully triangulated tensegrity structures may have been selected through evolution because of their structural efficiency-their high mechanical strength using a minimum of materials" (Ingber, 56).
Over time as structures go through different stresses and tension from its surroundings, it is considered advantageous because it allows the structures to mold into different shapes over time allowing for more complex and integrated structures. If a molecule or cell would be able to adapt to new temperatures or pressures, then the cells will be able to evolve over time allowing the progress of tissue engineering to continue to expand and grow.
As time goes by, "different molecular collectives self-assembled to form the first structures with specialized functions-the forerunners of present-day organelles-which then combined with one another to create the first simple cells" (Ingber). After the cells were formed, it helped to produce proteins, which then assembled together to help form the ECM (extracellular matrix) scaffold which promotes its own multi-cellular tissues (Ingber). After the tissues have developed on the scaffold, the organs will then start to develop. All of these steps that form from the cells to the tissues back to the organs, are considered all the stages of "self assembly".
Overall researchers are still trying to understand the concept of tissue engineering. This is because as the environment around a cell, tissue, organ changes it puts stress/tension on it and it will form as needed. So, the researchers are now also trying to understand how and why cells signal between each other in order to create tensegrity.