Jin Schofield (10) || STAFF REPORTER, EDITORIAL EDITOR
Abstract/Overview
After injury, patients who have faced certain degrees of physical trauma often develop scars in replacement of original tissue. Aside from the cosmetic concerns of scarring, scarring can cause complications including a difficulty to move, infection, a loss of self-esteem, and much else. However, in human embryos, healing of injuries hardly ever results in scarring. Researchers have identified some of the reasons for why this occurs, and are trying to use these discoveries to improve the natural healing of adult humans. Some treatments being pursued by researchers include transplantation of stem cells, manipulation of growth factors, and use of reactive oxygen species. However, there are many issues that prevent researchers from being able to immediately implement these solutions on humans. These include that transplantation can result in a potentially even more harmful immune response, and that the immuno-suppressant drugs used to combat this can increase the risk of developing certain diseases. Overall, regenerative medicine and embryonic wound healing have the potential to drastically improve how humans deal with injury, but we are far from developing safe, clinically effective treatments.
Introduction
As the largest organ in the body, skin is essential to human health. Skin protects against fluid imbalance, infection, thermal dysregulation, and controls the flexibility of joints such as those in the hand. Loss of skin function can occur after acute trauma to the skin, increasing the risk of infection, thermal dysregulation, and fluid loss. Examples of this acute trauma can include burns or degloving. Healing of the skin can be lengthy and result in the formation of scar tissue – aspects of human skin healing that can be problematic for patients. In developed countries alone, one hundred million patients develop scars. 11 million of these scars are keloid scars – which is a result of the growth of extra scar tissue into hard, smooth growths (these are called keloids). Another 4 million of these scars are burn scars, 70% of which develop in children. Although most scars are not considerably harmful, some can result in physical deformity resulting in disability, a loss of self-esteem, anxiety, and depression. More extreme cases can result in severe pain, tenderness, itching, and sleep disturbance. When excess scar tissue grows deep within the body, arthrofibrosis can develop, which reduces the ability to move after surgery. Plastic surgery can be used to assess and treat scars, but researchers are looking into a way to prevent the formation of scars by changing the way the human body heals.
In human embryos, healing takes place very differently. In embryos that have not reached the third trimester, embryonic wounds heal very quickly and without the formation of scar tissue. This is known as embryonic wound healing, or regenerative healing. Researchers hope to copy embryonic wound healing to improve the healing processes of adults, and avoid the complications of scar formation.
How Embryonic Wound Healing and Adult Wound Healing Differ
Adult Wound Healing And Scarring
Regular wound healing has six stages:
- Inflammation – when the body increases blood flow, cytokine flow (which signal for immune cells) and white blood cell amounts in the area of injury, turning it red and swollen. Clotting also occurs.
- Proliferation – when collagen and an extracellular matrix are formed, constructing granulation tissue and blood vessels.
- Epithelialization – when growth factors cause the replication and movement of living epithelial cells to close the wound.
- Angiogenesis – the growth of blood vessels.
- Remodelling – a.k.a. Maturation; the taking part and reparation of dermal tissue so that they gain tensile strength, and the replacement of non-functional fibroblasts with functional fibroblasts
- Scarring – when new collagen fibers mend a wound; resulting tissue has a different texture and quality than the original tissue
As demonstrated by the final stage, adult wound healing almost always results in scarring. Scars are a replacement for the functional tissue that was harmed during an injury. It is effective in preventing infection and acting as a mechanical replacement to the previous tissue, but, in excess, can damage the function of the surrounding tissue or organ. In different areas of the body, different amounts of scarring occur because of differences in the intrinsic properties of their cells, their extracellular matrix (non-cellular scaffolding for cells in tissues and organs), cytokine signalling within the area, and the cell activity (e.g. areas of the body that tend to be subject to more damage over time heal more slowly).
Knowing this, what about embryonic wound healing causes it to skip scar formation and occur more quickly than adult wound healing?
Varying Cell Types and Activity
Platelets
Platelets are the cells found in your bloodstream that form clots around wounds, thereby promoting hemostasis (stopping the flow of blood). Their role in your bloodstream is to ensure you do not bleed out (and die). These platelets, in adults, act as aggregation factors. Aggregation factors are proteins that bind tissues together by forming links between the cell membranes of cells. Platelets also promote adhesion to the extracellular matrix, creating fibrin clots to seal wounds. After the platelets do this, they secrete chemotactic factors (which are substances that stimulate the migration of cells) to provoke the movement of inflammatory cells. This is known as degranulation. These inflammatory cells trap and attack any harmful agent in the injury area, and also remove damaged tissue so that healing can occur.
In embryos, there is less platelet aggregation (which is caused by an increased amount of hyaluronic acid) and less degranulation. This leads to less inflammation. As the embryo is protected within a woman’s womb, and not exposed to infectious agents in the environment, the lack of inflammation does not threaten the embryo. This lack of inflammation could contribute to the speed of embryonic healing.
Immune Cells
Another possible contributing factor to the differences of embryonic wound healing is the immaturity of the immune system in embryos. After injury in an adult, the clot formed by platelets cause the arrival of neutrophils. These are white blood cells that kill microbes by “eating” bacteria and using enzymes to digest them. Neutrophils then trigger the arrival of macrophages, which are larger white blood cells that also “eat” microbes and signal for the arrival of other immune cells. Macrophages use degranulation and phagocytosis to kill bacteria and dead immune cells. This elimination of dead immune cells cause wound closure to occur more rapidly. As macrophages grow in numbers, they signal for the arrival of monocytes, which phagocytose more left over debris and produce proteases (which break down and remodel granulation tissue).
In embryos, there is less cell differentiation among immune cells and differences in the extracellular matrix and therefore less of an overall immune response. Additionally, the number of macrophages is less than in adult wound healing. The only thing that can attract more macrophages in embryos is burn cauterization. This is because burn cauterization can lead to necrosis (dying tissue), which is very dangerous for the embryo.
Fibroblasts and Myofibroblasts
Fibroblasts are cells that produce collagen, glycosaminoglycans, and proteoglycans, which all help construct the extracellular matrix. In adult wound healing, near the end of inflammation, fibroblasts begin the formation of granulation tissue. Granulation tissue is what leads to scarring. After being exposed to the adult extracellular matrix and TGF-β (a growth factor that will be spoken about later in the article), fibroblasts turn into myofibroblasts. There are two types of myofibroblasts – secretory and contractile. Secretory myofibroblasts secrete collagen. Contractile fibroblasts are like smooth muscle cells and allow wound contraction by using the extracellular matrix to create tension. These myofibroblasts use collagen type I, the most abundant type of collagen and a version that is essential to the composition of several tissue.
In embryos, this granulation tissue, which contains much more collagen than in adults, is remodelled. Fibroblasts become myofibroblasts at a much lower rate because of less exposure to TGF-β. These embryonic fibroblasts instead use collagen types III and IV. They have more receptors, being more sensitive to hyaluronic acid (which, once again, lead to less platelet aggregation and degranulation, and therefore less inflammation), and less sensitive to TGF-β1. Ultimately, in embryos, the existence of more fibroblasts, and their use of collagen types III and IV, lead to reduced scar formation. Embryonic fibroblasts that have been transplanted into adult wounds cause the formation of reticular collagen patterns that are like healthy skin after healing, as opposed to scar tissue.
Stem Cells
Stem cells are cells that can develop into other, different types of cells – they can be separated into embryonic stem cells and adult stem cells. Each stem cell in the adult body has the potential to become a different type of adult cell. For example, mesenchymal stem cells form in adult bone marrow and can produce factors that induce angiogenesis and work to counteract inflammatory cytokines. The role of stem cells in adults is to cause cell turnover and regenerate tissue.
The embryo, itself, is a conglomeration of stem cells. Within embryos that have developed past a blastocyst (a young embryo consisting of an inner cell mass (which will become fetal tissue) and a trophoblast (which will become the placenta)), there is a subpopulation of bone marrow stem cells that exist, known as “dot cells” or “small stem cells”. These small stem cells prompt less scarring and may explain why bone marrow cells can enhance wound healing in adults. Small stem cells are rare in adults, but when found in adults, induce embryo-like healing.
The Differences in The Extracellular Matrix
Collagen
Within the extracellular matrix of adults, type I collagen is the most commonly occurring isoform of collagen. It forms large, organized fibers that creates a scaffold on top of which wound healing occurs. This scaffold is stiff, controlling the movement of cells and the transformation of fibroblasts into myofibroblasts.
Inside embryos, type III collagen is more common. It forms a finer, more reticular lattice that therefore causes less myofibroblast formation and less scarring.
Hyaluronic Acid
Hyaluronic Acid cushions and lubricates joints, acts as an osmotic buffer in the kidneys, and controls hydration in the skin. In adults, HA (an abbreviation for hyaluronic acid) is produced early on during the formation of granulation tissue. In adults, a type of HA that consists of lighter molecules also encourages angiogenesis and lymphocyte infiltration.
In embryonic wounds, heavier HA molecules are more common. This encourages the movement of fibroblasts and the production of collagen III. This heavier HA is fully hydrated, meaning it has binded to its maximum possible amount of water. This stops the movement of large proteins and bacteria into the area, and allows damaged cells to be repaired more quickly because of how the HA takes up more volume and therefore allows more contact between proliferating cells. These larger HA molecules also inhibit angiogenesis and lymphocyte infiltration, decreasing inflammation.
Tenascin-C and Fibronectin
Tenascin-C is a polymer consisting of six subunits that is part of a larger group of de-adhesive proteins. It prevents apoptosis (cell death), promotes the movement and differentiation of myofibroblasts, and allows for the organization of the extracellular matrix, which allows more cell movement into the wound.
Fibronectin is a glycoprotein (proteins that have carbohydrates covalently bonded to them) that acts as an adhesive during wound healing, allows for longer activity of growth factors such as TGF-β, and activates macrophages to contribute to a stronger inflammatory response.
In embryonic wound healing, these two proteins counteract one another – tenascin-C promoting motility, and fibronectin promoting attachment. These proteins are upregulated more in embryos than in adult wound healing. For example, tenascin-C is detected one hour after injury, and fibronectin is detected four hours after injury in embryos. Contrastingly, in adult wound healing, tenascin-C can be detected after 24 hours, and fibronectin after 12 hours. The order of the appearance of each protein is also reversed in adult wound healing to promote attachment before movement.
Growth Factors
TGF-β
This growth factor is a cytokine that is secreted from platelets, neutrophile, macrophages, and many other celle during the inflammatory response. There are many isoforms of TGF-β – these are different between embryonic wound healing and adult wound healing. In adults, TGF-β1 upregulates collagen synthesis and reduces MMP (which breaks down collagen) activity in fibroblasts. This leads to a net increase in collagen production. In embryos, fibroblasts do not react as strongly to TGF-β1, because of less TGF-β1 receptor density.
MMPs
MMPs (matrix metalloproteinases) are part of a group of peptidases (an enzyme that breaks down proteins into polypeptides, something known as proteolysis) that lead to the degradation of extracellular matrix proteins. MMP-9 activates TGF-β, which then decreases matrix degradation by inhibiting and counteracting MMP expression. They are expressed by macrophages and other cells less in adult tissues than in embryos. Additionally, MMP-1, 2, and 14 are increased in adults when healing wounds. However, in embryos, MMP-1 and MMP-9 are upregulated at even greater rates.
Wound-Closing Mechanisms
In adult wound healing, there are four stages for wound closing:
1. Acute hemostasis – when bleeding is halted with clotting
2. Inflammatory phase – which lasts for approximately three days; when inflammation occurs
3. Granulation phase – which can last from 2-10 days; when scar progenitor tissue differentiates
4. Scar Remodelling Phase – when scar tissue forms in place of the original tissue
In order to fully close the wound, keratinocytes climb over and close the opening of the exposed substratum.
In embryos, wound closing is enacted by the epidermal cells constructing an actin cable (actin is a common protein that helps with cell structure, motility, and contraction) around the injury. Like a purse string, this cable closes the wound. To achieve this, actin-rich filopodia (“thin membrane protrusions”) protrude out of the surrounding cells. Cells use endocytosis to remove junctions between healthy and injured cells. This allows the healthy cells to close the wound.
Applications for Humans
The goal of tissue engineering is to “assemble functional constructs that restore, maintain, or improve damaged tissues or whole organs”. It is the type of engineering that repairs skin after scarring, and is responsible for repairing tissues after injury as well. However, the current tissue-engineering solutions for scar repair risk causing the formation of more scar tissue. Additionally, tissue engineering can sometimes not be plausible without causing complications, such as an immune response. The following are methods by which researchers are trying to improve adult wound healing by mimicking embryonic wound healing.
The Use of Stem Cells
Stem cells that have been derived from adipose tissue and treated with TGF-β1 creates a media that causes increased expression of type I collagen, MMP-1, and fibroblast migration. This media can be used to promote growth factors on scaffolds, and reprograms fibrocytes to remodel (which leads to less scarring). New techniques, such as bioprinting, electrospinning, and adding cell-matrix, cell-cell, mechanical cues, and soluble factors to scaffolds, are being considered to enhance stem cell behaviour for wound healing. However, using stem cells from embryonic tissue in adult tissue is difficult because the stem cells often die in the hypoxic environment of the adult tissue.
Manipulation of Growth Factors and Matricellular Proteins
An extracellular matrix derived from a human placenta can be used as a skin graft (if decellularized, homogenized, and made into a porous sheet) and placed on rat wounds to improve regeneration. Growth factors and proteins including collagen, TGF-β1, FGF, and PDGF were observed to contribute to the effectiveness of the graft. Additionally, targeting specific TGF-β signaling pathways can have anti-fibrotic effects.
Matricellular proteins (non-structural proteins that modulate cell function), such as thrombospondins, osteopontin, and periostin, can be manipulated to reduce inflammation, accelerate wound closure, and lead to less scarring.
Connexin 43
Connexin 43, also known as GJA1 is a protein that is a component of gap junctions. Connexin 43 expression can be reduced by antisense oligonucleotides (“short nucleic acid polymers”), leading to a reduction of inflammation, reduced scar tissue formation in wounds and burns, benefitted spinal cord and central nervous system injuries, and accelerated skin wound healing (even in diabetic models).
Reactive Oxygen Species
Reactive oxygen species, also known as ROS, create a signal that allows for the polarity of cell-cell junctions, which contributes to wound healing. Additionally, ROS promoted the accumulation of actin and myosin in the cell cytoskeleton and around the wound, which allows for the clearing of the injury site and reparation of the tissue.
However, there are challenges to implementing these solutions to repair tissue without forming scar tissues.
When introducing new biomaterials, scaffolds, and graft stem cells, there is always a risk of complications because of the potential of a patient immune response. Scar formation and fibrotic tissue, themselves, are a part of the patient’s immune response. To combat this, immune-suppressant drugs can be used. However, these drugs increase the risk of developing diabetes, hypertension, and skin cancer. Another method to combat this would be to use autologous cells derived from the patient themselves. Unfortunately, some cases still result in an immune response, and most grafts result in scarring regardless. iPSCs (induced progenitor cells) are stem cells that can be created from adult tissue, and that can produce differentiated, mature cells. These cells have been rejected in mice as well.
Conclusion
At the moment, tissue engineering can be greatly improved by discovering ways for treatments to mimick the human embryo’s own method of healing. Researchers will have to navigate and avoid the human immune response and other complications that could worsen the state of a patient’s tissue. Whether by manipulating growth factors, using small stem cells, or using inorganic molecules such as reactive oxygen species to lessen the development of scar tissue, research is slowly advancing to fulfill this need.
For the full version of this article, with in-text citations, please contact Jin Schofield at jinschofield@gmail.com.
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