What exactly is an extracellular matrix?

The extracellular matrix (ECM) is a dynamic 3-dimensional network of proteins and molecules surrounding the cells that provides structural support, regulates cellular behavior, and influences processes like growth, healing, and communication. The ECM is found in tissues throughout the body and plays a crucial role in shaping how cells interact with their environment, helping to maintain the integrity and function of organs and tissues. It is composed of proteins like collagen, elastin, and glycoproteins, along with various carbohydrates. It influences essential processes such as cell adhesion, migration, differentiation, and wound healing. The ECM’s composition varies between tissue types, and its stiffness or elasticity can significantly affect how cells behave, impacting everything from tissue development to the progression of diseases like cancer. By controlling how cells interact with their surroundings, the ECM helps maintain tissue integrity, facilitates repair, and supports overall biological function. Its study is vital for fields like regenerative medicine and cancer biology, where understanding and manipulating the ECM can lead to breakthroughs in treatment strategies. Collagen fibers and proteoglycans within the ECM bind to integrin receptors on the cell membrane. These integrins, in turn, are linked to microfilaments of the cytoskeleton inside the cell, forming a structural and signaling bridge between the extracellular environment and the cell's internal framework. This interaction plays a critical role in cell adhesion, signaling, and mechanical stability.

Figure 1 shows the interaction between the extracellular matrix (ECM) and the cell through integrins. The collagen and proteoglycan fibers in the ECM are bound to integrins on the cell membrane, which are connected internally to microfilaments of the cytoskeleton. This structure helps transmit signals and mechanical forces between the ECM and the cell’s interior.

Components of the Extracellular Matrix:

1. Collagens: Formed as fibrils within the ECM (Types I, II, III, V and XI). It provides tensile strength and influences cell processes, for example, adhesion and migration. Collagen type I is often used as a coating on gel scaffolds to promote cell adhesion, in addition to its ability to stimulate myogenic and osteogenic differentiation of stem cells. As a natural material, it sometimes initiates an inflammatory response from the host.

   
   

2. Elastin: It is composed of single tropoelastin subunits cross-linked with an outer layer of fibrillin microfibrils making up an elastic fibre. It is closely linked to collagens and allows tissues such as the skin and tendon to recover. It is being investigated as a biomaterial for use in tissue engineering, particularly in vascular tissue engineering due to its importance in blood vessels.

   
   

3. Fibronectin: It is formed either by plasma (within blood) or cellular protein (created by fibroblasts) and is arranged into a mesh of fibrils similar to collagen which is linked to cell surface receptors (integrins). Found in the basement membrane of the ECM, it plays a critical role in cell adhesion, embryonic development and the healing process following wound injury. Importance of fibronectin in embryonic development and wound healing indicates that it needs to play a key role in tissue engineering applications; especially, the RGD sequence that is critical in ensuring cell adhesion, transforming substrates allowing for cell attachment.

   
   

4. Laminins: Laminins are another type of glycoprotein, with a trimeric structure. They are made up of three different chains, α, β and γ which exist in various genetically distinct forms. They reside in the basement membrane and are expressed by various tissue types including both muscle and epithelial cells. Laminins play a vital part in several cell processes including differentiation and migration via their integrins. Similarly to fibronectin, laminins have the capacity for cell binding and are another option to be used to enhance cell adhesion in culture conditions.

   
   

5. Tenascins: Tenascins are a group of ECM proteins and exist as five different manifestations, TN-C, TN-R, TN-W, TN-X and TN-Y. It is linked to mechanical activity. Tenascins are typically found within connective tissues where load bearing is required, although they also occur within the skin and brain. During embryonic development and tissue repair, TN-C is highly expressed, it is also typically found within stem cell niches. However, it prevents cell adhesion when used as a protein coating for cell culture substrates.

   
   

6. Growth Factors: Tied to the ECM through either heparan or heparan sulphate. They can be linked to tissues with their names. For example, vascular endothelial growth factor stimulates the formation of blood vesselsTriggered into action by a variety of processes (not necessarily in a soluble form) including wound healing and tissue remodelling. Multiple growth factors demonstrated as being crucial to development and differentiation of many tissues. Their use is being explored within tissue engineering, for example, improving wound healing of tendon tissue.

   
   

7. MMPs: Structure occurs as zinc-dependent endopeptidases. They are capable of disintegrating the ECM, associated with many different processes including angiogenesis and wound repair. MMPs are key modulators for tissue remodelling; their expression can be useful indicators of cellular behaviour for tissue engineering investigations.

   
   

The Role of the Extracellular Matrix in aging and diseases

Over the past two centuries, human life expectancy has more than doubled, rising from under 30 years to 72 years by 2015, primarily due to improvements in public health, hygiene, nutrition, and medical advances like antibiotics and vaccines. However, age-dependent diseases such as cancer, diabetes, cardiovascular, and neurodegenerative illnesses are now the leading causes of death. Since aging is the main risk factor for these diseases, understanding the aging process has become critical. Treating individual age-related diseases in isolation provides limited benefits, as increased survival leads to a higher risk of developing other age-dependent illnesses. The ECM is a very essential component in all the organs and tissues of the human body. It plays a critical role in providing structure and physical scaffold for cells and is responsible for regulating many cellular processes. The ECM has the ability to create spaces, control growth and influence cell differentiation. Recent research shows that the extracellular matrix is one of the very important regulators of plasticity, learning, and memory and might also potentially contribute to several neurological disorders like epilepsy, schizophrenia, addiction and dementia. Identifying molecules and mechanisms that regulate these processes is essential for understanding brain function, neurological disorders, and developing targeted therapies. The extracellular matrix (ECM), especially the perineuronal net (PNN), surrounds neurons and plays a key role in brain development. It helps neurons grow, connect, and store signals for migration and synaptic changes. The PNN also controls when synaptic plasticity occurs. Problems with the ECM, particularly the PNN, are linked to disorders like autism, schizophrenia, bipolar disorder, Fragile X syndrome, and epilepsy.

Mood disorders and anxiety caused by environmental stress are major concerns today. A recent review article explained how the extracellular matrix (ECM) influences brain function during development and adulthood, highlighting stress effects on both diffuse and structured ECM and their impact on emotion, learning, and memory. Another research article conducted research using a rat spinal cord injury model, studied changes in the ECM, particularly the NG2 proteoglycan. They found increased NG2 levels near the injury site, potentially affecting axonal growth and synaptic recovery. Another article analyzed ECM proteoglycans, Brevican and Neurocan, in cerebrospinal fluid and serum from neurological patients. They suggested that tracking neurocan fragments could serve as biomarkers for brain ECM integrity and aid in diagnosing neurological disorders. Glial cells, especially astrocytes, are key players in shaping the brain's extracellular matrix (ECM) throughout development, adulthood, aging, and in diseases. They not only produce ECM molecules but also release enzymes that remodel the ECM and perineuronal nets (PNNs). A review published on the topic, highlights how glial cells drive ECM remodeling in both healthy and diseased brains, emphasizing their central role. This insight opens up exciting possibilities for glia-focused treatments alongside neuron-centered approaches to address brain disorders more effectively.

Techniques to analyze ECM

There are several techniques to analyze the extracellular matrix. These approaches allow for the composition, structure and bio-mechanical properties of the ECM to be examined. Some of the techniques are briefly explained below: 

1. Atomic Force Microscopy: Researchers have used atomic force microscopy or AFM in the past few years as a reliable method to measure the mechanical properties in terms of stiffness (Young’s modulus) of a matrix. The technique uses two main parts: a sensor and a detector. researchers confirmed AFM as a valid method to monitor differentiation of stem cells into tenocytes, by measuring the changes that occurred in elastic modulus at a cellular level. The sensor is a cantilever beam that bends in response to forces, and the detector measures this bending. The AFM operates in three modes: contact, non-contact, and tapping. One of the greatest advantages of using AFM in ECM analysis include the ability to take measurements on three separate axis (x, y and z) providing 3D images of a sample surface.

   
   

2. Immunostaining: Immunostaining of the ECM can be done through immunohistochemical (IHC) or immunocytochemical (ICC) staining, both of which detect specific ECM proteins using antibody-antigen interactions with a visualization tag. IHC requires tissue samples to be embedded in resin or paraffin and sliced into thin sections before staining. IHC staining has been commonly used in tendon fibroblasts to determine the expression levels of proliferation cell nuclear antigen (PCNA) and α-smooth muscle actin (α-SMA), which are released by healing tendon fibroblasts. The fibroblasts are responsible for synthesising the ECM during the wound healing process. IHC was able to measure the increasing levels of PCNA and α-SMA post injury.

   
   

3. Confocal Microscopy: Confocal microscopy, combined with immunostaining, is a powerful tool for studying the 3D structure of the extracellular matrix (ECM). By stacking 2D images, it provides sharp, high-resolution visuals of protein organization and distribution, such as fibronectin, collagen type VI, and elastin. This technique not only reveals ECM composition but also highlights changes in its architecture, offering crucial insights into how aging impacts the ECM environment.

   
   

4. Gelatin substrate zymography: Gelatin substrate zymography (GSZ) is a common method for detecting MMP-2 and MMP-9 which are the enzymes that degrade the extracellular matrix (ECM). By separating proteins based on molecular weight using SDS-PAGE, this technique identifies MMPs and distinguishes their active and latent forms. Gelatin is the preferred substrate for observing these enzymes. Despite its lengthy two-day protocol, GSZ is widely used to study MMP activity in biological samples and for drug screening.

   
   

Transmission electron microscopy (TEM), Scanning electron microscopy (SEM), Tandem mass spectroscopy (MS), Raman Spectroscopy and SHGM are also some really good techniques that are used to analyze the extracellular matrix from various perspectives.

Conclusion

Understanding the aging effects on the extracellular matrix (ECM) reveals its critical role in processes like wound healing and regenerative medicine. Age-related changes, such as altered collagen content and increased tissue stiffness, influence healing outcomes, transitioning from scarless to scarred phenotypes. Advanced analytical techniques, such as those measuring matrix stiffness and cellular interactions, have been instrumental in uncovering these dynamics. The ECM’s potential as a scaffold for regenerative healing holds promise, but much remains to be explored. As research continues, the ECM will undoubtedly remain a focal point for innovations in tissue repair and regenerative therapies.