Seeing the unseen: The role of bioimaging techniques for the diagnostic interventions in intervertebral disc degeneration

Abstract

Intervertebral Disc Degeneration is a pathophysiological condition that primarily affects the spinal discs, causing back pain and neurological deficits. It is caused by the contribution of several factors such as genetic predisposition, age-related degeneration, and lifestyle choices like obesity and physical activity. Even though there are medications to treat pain, there is a lack of medicines for a complete cure. The main difficulty lies in poor diagnosis of the morphological and functional changes in the disc. With the ever-increasing research on bioimaging techniques, new techniques are being developed and repurposed to evaluate disc shape and composition, and their defects like thinning or deformities on the disc, leading to the proper diagnostic intervention in intervertebral disc degeneration. In this review, we aim to present a comprehensive overview of the imaging techniques used in the pre-clinical and clinical stages for the diagnosis of intervertebral disc degeneration. First, we will discuss about patho-anatomy and the pathophysiology of degenerative disc disease with the significance and a brief description of various dyes and tracers utilized for bioimaging. Then we will shed light on the latest advancements in diagnostic modalities in intervertebral disc degeneration; concluded by an analysis of the repercussions of the methodologies and experimental systems employed in identifying mechanisms and developing therapeutic strategies in intervertebral disc degeneration.

Graphical abstract

Highlights

• •Intervertebral disc disease is a complex and progressive pathological cascade.
• •It is the most common contributor to the pathogenesis of low back pain.
• •Tracers and dyes play crucial roles in diagnostic applications of disc disease.
• •Bioimaging utilizes versatile technology to generate images of the discs.
• •MRI, CT, PET enable studying of structural, functional & molecular aspects of discs.

1. Introduction

Intervertebral disc degeneration (IVDD) refers to the gradual deterioration of the intervertebral discs in the spinal column. It is a primary cause of persistent back pain and impairment in the elderly population (Novais et al., 2021). Low back pain (LBP) is an important community health concern globally, showing a high proportion affecting people of all ages, from teenagers to the aged population (Wang et al., 2022a; Beyera et al., 2019). Nearly 40–85 % of the population has experienced an acute episode or a chronic illness at some point in their lives (Tagliaferri et al., 2020). Yearly costs of LBP impact an economic burden in healthcare of nearly US $ 100 billion (Mohd Isa et al., 2022a). IVDD is the most common contributor in the pathogenesis and progression of LBP, influenced by various risk factors like ageing, environmental and genetic, metabolic changes, frequent heavy lifting, smoking or tobacco intake, trauma, obesity, and hereditary susceptibility (Xia et al., 2022). IVDD can cause lower back pain via a variety of causes. The vertebrae may come into contact with one another, generating friction and inflammation. As the discs lose their capacity to absorb trauma, it can cause inflammation and pain receptor activation in the surrounding tissues (Molinos et al., 2015; Khan et al., 2017). When the discs deteriorate, they may expand or herniate, protruding from their natural position. This can strain adjacent nerves, causing sciatica (pain, numbness, or tingling sensations radiating down the legs) (Kulali and von Wild, 1996). Furthermore, disc height reduction owing to degeneration might cause spine instability. Because of this instability, the surrounding muscles must work harder to maintain normal alignment, which can result in muscular spasms further exacerbating LBP (Rahyussalim et al., 2020).

Most of the common treatments for IVDD management range from conservative measures such as analgesics, physical therapy, and anti-inflammatory drugs to interventional therapies and surgical alternatives that aid in relieving symptoms (Mahyudin et al., 2022). These approaches intend to lower the clinical symptoms of the disease, but none directly address the underlying pathophysiology or reverse the degenerative cascade. Hence, early detection of the impairment is necessary due to the non-regenerative nature of the disc, ensuring a better lifestyle for the patient.

Conventional imaging techniques like magnetic resonance imaging (MRI), computed tomography (CT), positron emission tomography (PET), fluorescence microscopy, optical imaging techniques, and confocal microscopy are some of the key imaging modalities being utilized for diagnostic purposes, to evaluate disc shape and composition, and their defects like thinning or deformities on the disc leading to the proper diagnostic intervention in IVDD for both in preclinical and clinical stages (Lotz et al., 2012; da Costa et al., 2020a). Furthermore, with the advancement in research, new techniques are being developed to improve the diagnosis process.

2. Anatomy and functions of the intervertebral disc

Intervertebral discs (IVDs) are essential to the human skeletal system, serving as shock-absorbing cushions between the vertebral column to absorb and distribute the mechanical loads and stresses that occur during activities such as walking, running, lifting, and jumping, preventing damage to the vertebrae and the spinal cord. This allows flexibility and movement of the spine, maintenance of the proper spacing between adjacent vertebrae and protection of nerve roots (Zhou et al., 2014; Mohd Isa et al., 2022b). They are located in the spinal column, sandwiched between each vertebrae from the neck down to the tailbone. These major spinal column joints account for roughly one-third of the height of the spine (Karpiński et al., 2019). It works as a ligament that structurally supports the spinal vertebrae, a shock absorber, and a biomechanical pivot point, which enables the spine to bend, rotate, and twist (Navani and Chrystal, 2022). It also provides flexibility to the spinal canal and is essential in accommodating mechanical stress. IVD's function is to sustain and transmit axial stresses, maintaining mobility and stability throughout everyday activities (Borem et al., 2021). The normal human IVD comprises three different basic regions: annulus fibrosus (AF), nucleus pulposus (NP), and cartilaginous end plate (CEP) (Gan et al., 2021). NP is a mostly hydrated gelatinous structure made of large proteoglycans (PGs) like aggrecan, perlecan, versican and type I and II collagens. Together they maintain the structural stability and biomechanical balance of the IVD (Lai et al., 2021; Adams and Roughley, 2006). The biomechanical features of the disc heavily rely on the AF (Chu et al., 2018). It contains 15–25 concentric lamellae of collagen-rich fibrocartilaginous layers, which stabilize and enclose the NP cell, giving hard encapsulation and maintaining physiological intradiscal pressure under mechanical loading (Gkantsinikoudis et al., 2022; Harmon et al., 2021). Between the vertebrae and the disc, the CEP is composed of cartilage tissue, mainly hyaline cartilage, and vertebral endplate, which serves as a mechanical barrier, helps to supply nutrients, and supports the disc tissue integrity (Borem et al., 2021; Fujiwara et al., 2019). Together, these components allow the intervertebral discs to function as shock absorbers, maintain spinal flexibility, provide spacing between vertebrae, and protect spinal nerves from compression (Fig. 1). (See Table 1.)

Fig. 1.

Different components of the intervertebral disc.

Image depicting the components of the healthy intervertebral disc, the outer covering is made up of annulus fibrosus, and the central core contains nucleus pulposus, along with type I and II collagen, aggrecan, hyaluronan, elastin, and small leucine-rich proteoglycans. A healthy and intact cartilaginous endplate adjacent to the vertebral body is shown at the superior and anterior parts of the disc. The blood vessels and nerve fibres remain non-innervated in healthy discs.

Table 1.

Different diagnostic modalities in the diagnostic intervention of intervertebral disc disease.

Sl.no.	Name of the technique	Nature of action	Mode of action in IVDD	Advantages	Disadvantages	Ref.
Preclinical stage
1.	Confocal Microscopy	Involves the use of an antibody for a targeted marker in the tissue and the use of a laser beam to illuminate at a specific focal wavelength to excite the antibody to visualize proteins.	1. Illuminate a specific focal plane within the disc. 2. Selectively captures fluorescence from a specific plane.	1. Evaluate cellular morphology, extracellular matrix organization, and the presence of specific markers. 2. Generates 3D images, enabling the visualization of complex structures and spatial relationships. 3. Produce real-time imaging and provide dynamic cellular changes.	1. Limited depth of penetration, making it less suitable for imaging of thick sections. 2. Time-consuming due to the scanning of multiple focal planes. 3. Intense laser lights can lead to photobleaching. 4. Limited to the detection of fluorescence within a specific spectral range.	(Elliott, 2020;Nwaneshiudu et al., 2012;Semwogerere and E.R.J.E.o.b. Weeks, 2005)
2.	Multiphoton microscopy	Based on the nonlinear excitation of fluorophores, where two or more photons are absorbed simultaneously by a fluorophore, using ultrafast intense pulsed laser light.	1. Provide detailed information about cellular morphology, extracellular matrix organization, and the presence of specific markers associated with IVDD. 2. Detailed insights into the cellular and molecular changes associated with disc degeneration.	1. Uses longer wavelength light, which allows for deeper tissue penetration and reduces phototoxicity. 2. Fluorophore excitation occurs through a nonlinear process, resulting in the emission of fluorescence only at the focal point of the laser beam. 3. Provides high-resolution images with reduced background noise and improved sectioning. 3. Generate 3-D images, which provide a comprehensive view and spatial relationships.	1. Can be expensive to acquire and maintain. 2. A smaller field of view compared to other imaging techniques, requiring multiple scans for large samples. 3. Requires specialized training and expertise, limiting its widespread use. 4. Slow processing compared to other imaging techniques.	(König, 2000; Hoover and Squier, 2013; Sriram et al., 2020)
3.	Raman spectroscopy	Based on the measuring of the spectrum of a shift in wavelength between the inelastic scattering of light by molecules of the tissue of the target.	1. The Raman scattered light exhibits a shift in energy corresponding to the vibrational energy level of the molecules in the disc. By analyzing the Raman shift, a Raman spectrum can be obtained, which provides information about the molecular composition and structure of the disc tissue. 2. Provides detailed insights into the biochemical changes associated with disc degeneration. 3. Detect the presence of inflammatory markers or other biochemical changes associated with disc degeneration. 4. Provides information about the molecular composition and structure of the disc tissue.	1. It is a non-destructive technique that allows for the analysis of samples without altering their integrity or composition. 2. Provides molecular-specific information, allowing for the identification and characterization of different compounds and functional groups within a sample 3. Can detect low concentrations of molecules, making it suitable for trace analysis and detection of subtle changes in sample composition.	1. Not suitable for all sample types, such as highly scattering or opaque samples, which can limit its applicability. 3. Typically limited to a specific spectral range, which may not cover all desired molecular vibrations in the tissue. 3. Can be expensive to acquire and maintain.	(Dodo et al., 2022; Lyon et al., 1998; Ember et al., 2017; Gong et al., 2013)
4.	Super-resolution-Microscopy	By surpassing the diffraction limit of light by blocking fluorophores outside the focal spot, resulting in improved resolution.	1. Allows for the visualization of nanoscale features, such as individual molecules or organelles, 2. Provide molecular mechanisms and cellular processes involved in disease progression. 2. Bring forth detailed information on cellular morphology, subcellular structures, and interactions within the disc.	1. Higher resolution level compared to conventional microscopy. 2. Allowing the visualization of fine structures and organelles with improved clarity and precision in live mode. 3. Simultaneous imaging of multiple fluorophores with distinct colours, enables to study the of complex biological interactions.	1. Require high numerical aperture objectives, sensitive detectors, and advanced imaging systems, which can be expensive. 2. Analysis and processing of data can be complex and time-consuming. 3. More prone to photobleaching and phototoxicity due to the higher intensity of excitation light.	(Schermelleh et al., 2019; Yamanaka et al., 2014; Huang et al., 2010)
Clinical stage
1.	Magnetic resonance imaging	By interaction of magnetic fields and radio waves with the body's tissues by aligning the protons (hydrogen atoms) in the body's tissues, particularly in water molecules.	1. Visualize the degeneration of the disc including, disc height, herniation, bulging and the presence of tears or fissures. 2. Structural information like hydration, and integrity of the discs. 3. Show changes in the surrounding tissues, such as inflammation, and spinal cord compression. 4. Guide treatment decisions. 5. Non-invasive and safe because of no radiation exposure.	1. Non-invasive. 2. Real-time imaging with no radiation exposure. 4. Accurate diagnosis, and versatility.	1. High cost. 2. Time-consuming. 3. Contraindications for patients with metal implants in the body. 4. Use of contrast agents carries a risk of allergic reactions in some individuals.	(Thompson et al., 2021; Fiani et al., 2020; Mwale et al., 2008; De Goyeneche et al., 2019)
2.	Computed tomography	By use of hurling X-rays from all planes (axial, sagittal, and coronal) to develop an intense targeted 2D image and computer processing to create detailed cross-sectional 3D images.	1. Provide detailed information about the extent and severity of degeneration like loss of disc height, bulging, herniation, and the presence of bony changes or osteophytes. 2. Show changes in the surrounding structures, such as the spinal canal, nerve roots, and facet joints.	1. Requires minimal time to perform. 2. Cost-effective. 3. Ability to use contrast agents to enhance the visibility of abnormalities. 4. Can be performed on patients with metal implants: Unlike MRI.	1. It involves exposure to ionizing radiation, which carries the risk of potential harm, especially with repeated scans. 2. Contrast agents may cause allergic reactions. 3. Do not provide functional imaging capabilities like fMRI, limiting their ability to assess certain disc functions.	(Taubmann et al., 2018; Power et al., 2016; Griffeth, 2005)
3.	Positron emission tomography	By use of radioactive tracers (glucose for brain activity or FDG for cancer detection etc.) emitting positrons binding to electrons in the body of the targeted tissue, releasing two gamma rays in opposite directions to be detected by detectors to generate an image.	1. Provide information about the metabolic activity and potential presence of inflammation in the intervertebral discs. 2. Ability to detect changes at the cellular level, which often occur before structural changes.	1. Non-invasive and safe. 2. Provides valuable information about the response to treatment and monitor disease progression.	1. Involve exposure to ionizing radiation due to the use of radioactive tracers, which carries the risk of potential harm. 2. Expensive compared to other imaging modalities. 3. Not suitable for patients with conditions such as pregnancy or severe kidney disease.	(Vitor et al., 2017; Ehman et al., 2017; Freedenberg et al., 2014; Kapoor and Kasi, 2024)
4.	Optical Coherence Tomography	Uses low-coherence interferometry to measure the echo time delay and intensity of backscattered or broad-spectrum reflected light by a super-luminescent diode or a swept-source laser.	1. To assess the microstructure and thickness of the annulus fibrosus and surrounding tissue. 2. Provide insights into disease progression and treatment response. 3. Provide microstructural changes, like collagen organization and fibre density, in the intervertebral disc by generating high-resolution cross-sectional images of the tissue.	1. Can be used for longitudinal monitoring of tissue changes over time. 2. Can be performed on living tissues. 3. Provides high-resolution imaging with micrometer-scale axial and lateral resolution of different layers and structures within tissues.	1. Cannot provide detailed tissue characterization. Compared to other imaging modalities like MRI or histopathology. 3. Affected by artefacts, like motion or signal attenuation, which can impact image quality and interpretation. 4. Less suitable for imaging non-transparent tissues with low reflectivity.	(Aumann et al., 2019; Podoleanu, 2012; Boone et al., 2012)
5.	Ultrasound Imaging	Involves the principle of ultrasound waves by transducer and analyzing the relaxation-absorption ratio of the wave by different tissues by converting them to electrical impulses.	1. Assess surrounding structures in the disc and provide complementary information in IVDD evaluation. 2. Provides information on the integrity and potential involvement of various soft tissues, such as muscles, ligaments, and blood vessels in IVDD.	1. Non-invasive procedure. 2. Allows immediate visualization of structures. 3. Can be used to guide injections or aspirations, ensuring accurate placement and minimizing complications. 6. Safe imaging for pregnant women and children.	1. Dense structures like bone or air-filled organs, limit the penetration, resulting in restricted visualization. 3. Affected by artefacts, such as shadowing, reverberation, or acoustic enhancement. 4. Has a limited field view, which may require multiple scans or repositioning to visualize the structures.	(Thiravit et al., 2021; Chan and Perlas, 2011; Champaneria et al., 2010)
6.	Photoacoustic Imaging	Deliver short laser pulses into the tissue, leading to rapid heating and subsequent expansion. Which generates ultrasound waves due to the photoacoustic effect that gets captured by detectors to provide images.	1. Vascularization and blood flow within the disc can be assessed, which can be indicative of inflammation or other pathological processes. 2. Help to assess the oxygenation levels and metabolic activity within the disc.	1. Offer high spatial resolution. 2. Allow detailed visualization of tissue structure. 2. Provides functional and molecular information. 3. Real-time imaging capabilities.	1. Imaging limitations with highly scattering or absorbing tissues. 3. Challenging to imagine moving structures or organs due to motion artefacts and the need for precise synchronization.	(Attia et al., 2019; Yang et al., 2009; Beard, 2011; Capart et al., 2022)
7.	X-RAY imaging	Based on the principle of using X-ray, the high-energy electromagnetic waves property to penetrate through the body and interact with different tissues and structures differently and detect and analyze the post-penetrating ray to create an image.	1. Can show changes in disc height, narrowing space, presence of osteophytes, and disc degeneration. 2. Useful for evaluating bony structures, such as the vertebral bodies and facet joints, which can be affected by IVDD.	2. Provide real-time images, allowing for immediate assessment and intervention during procedures. 3. Particularly effective for evaluating bony structures.	1. Involves ionizing radiation, which carries a risk of potential harm, especially with repeated or high-dose exposures. 2. Does not provide detailed contrast or resolution for soft tissues. 4. Can be affected by motion, metal, or overlapping structures, which can impact image interpretation.	(Martz et al., 2016; Russo, 2017; Gureyev et al., 2011; Schlüter et al., 2014)

^⁎1IVDD- Intervertebral disc disease.

²2D-Two dimensional.

³3D- Three dimensional.

⁴MRI- Magnetic resonance imaging.

3. Pathophysiology of IVDD

IVDD is a complex pathophysiological process involving progressive structural and functional changes in the discs, which act as shock absorbers and facilitate spinal flexibility (Fiani et al., 2021). It often begins with a breakdown of the extracellular matrix (ECM) within the disc, primarily composed of collagen and proteoglycans. These structural alterations result from a combination of genetic predisposition, mechanical stress, and age-related factors. Reduced water content and impaired nutrient exchange contribute to disc dehydration, making them less resilient and prone to fissures (Feng et al., 2006). Consequently, microtears develop in AF, and as the NP loses its ability to withstand compression, it may herniate into the spinal canal, potentially impinging on nerves or causing inflammatory responses (Tenny and Gillis, 2024; De Cicco and Camino Willhuber, 2024). Inflammation and the release of catabolic enzymes further degrade the disc, leading to a loss of disc height, instability, and potentially osteophyte formation, resulting in pain, radiculopathy, and reduced spinal function. These complex processes collectively underlie the pathophysiology of IVDD, a significant contributor to back pain and spinal disorders (Khan et al., 2017) (Fig. 2).

Fig. 2.

A comparative profile of the physiologic microenvironment of healthy and degenerated discs.

In healthy discs, depicted by green on the left-hand side, CTGF and Shh results help in the orderly arrangement of the extracellular matrix; Aggrecan is the large proteoglycan that inhibits VEGF, and its inhibition maintains the avascularity of the Intervertebral disc. Further, the hypoxic environment leads to elevated expression of HIF, which stimulates glycolytic activity and results in nutritional enrichment of the disc. The HIF also inhibits catabolic enzymes like MMPs, and ADAMTs to maintain ECM homeostasis. On the right-hand side, the pathological microenvironment of the disc is represented as red. Numerous morphological and structural abnormalities occur. The elevated expression of pro-inflammatory cytokines brought upon by immune cells during blood vessel innervation inhibits the TIMPs, which are responsible for the inhibition of MMPs and ADAMTs. This inhibition elevates the catabolic activity, followed by loss of ECM content. Also, the hypoxic environment gets inhibited by the immune cells. All these consequences cause the loss of the boundaries between NP-AF and diminished disc homeostasis. CTGF, connective tissue growth factor; Shh, Sonic hedgehog; ECM, extracellular matrix; VEGF, Vascular endothelial growth factor; HIF, Hypoxia-inducible factor; MMPs: Matrix metalloproteinases; ADAMTs, A disintegrin, and metalloproteinase with thrombospondin motifs; TIMPs, Tissue inhibitors of metalloproteinases; Col-I, Collagen I; Col-II, Collagen II; SLRPs, Small leucine-rich proteoglycans; HA; Hyaluronic acid.

4. Introduction to bioimaging: role of dyes and tracers

Bioimaging is a multidisciplinary field that involves the visualization and analysis of biological structures and processes using various imaging techniques. It plays a crucial role in advancing our understanding of living organisms at the cellular and molecular levels. It enables researchers and clinicians to observe and study biological phenomena in real-time, providing valuable insights into the mechanisms of diseases, cellular functions, and developmental processes. Bioimaging is fundamental in biomedical research, diagnostics, and treatment monitoring, contributing to the continual progress of life sciences and medicine (Goyal and Kumar, 2023).

Tracers and dyes play a crucial role in diagnosis and research applications in IVDD. These substances are employed to trace and visualize specific biological processes, such as the migration of cells or the integrity of the ECM within the discs (Henriksson et al., 2019). Introducing tracers or dyes into the disc provides insights into the dynamic aspects of disc biology, including the assessment of cell viability, matrix composition, and the progression of degenerative changes (Klibanov et al., 2005). This enables a more precise understanding of the mechanisms underlying IVDD, aiding in the development of targeted interventions and therapeutic strategies for this condition. Here is a list of tracers and diagnostic dyes commonly used in the study of intervertebral discs (Fig. 3).

Fig. 3.

Role of different dyes, tracers and sensors in bioimaging.

Dyes, tracers and sensors play pivotal roles in advancing bioimaging technologies, enabling researchers and clinicians to visualize and understand complex biological processes at the molecular and cellular levels. Fluorescent dyes, emit light when exposed to specific wavelengths, aiding in the visualization of cellular structures and functions with high precision. Some dyes like calcein-AM are present in a non-fluorescent inactive form which upon entering the cell goes to modification (hydrolysis) to produce the active fluorescent form calcein. Other dyes like propidium iodide can only be uptaken selectively by dead cells allowing them to be a perfect marker for apoptosis. Tracers, often labelled with radioactive or fluorescent markers, can be employed to track specific molecules or cells within living organisms, providing valuable insights into pathways, interactions, and dynamic events. like 99Tc white blood labelled cells and annexin-V labelled cells are used to detect inflammation and apoptosis respectively. Sensors, enhance bioimaging by selectively detecting and reporting changes in biological parameters such as pH, ion concentrations, or enzymatic activities. like methylene blue which is degraded by live cell’s enzyme to become non-fluorescent, while in dead cells it remains fluorescent allowing it to be a perfect marker to detect cell death in bioimaging. These tools collectively contribute to the non-invasive and real-time monitoring of biological phenomena, advancing our understanding of health, disease, and the effectiveness of therapeutic interventions. WBC; White blood corpuscles.

4.1. Fluorescent dyes

4.1.1. Calcein acetoxymethyl (Calcein-AM)

Calcein acetoxymethyl (Calcein-AM) is a cell-permeant green fluorescent dye commonly used in live cell imaging, to assess the viability and proliferative properties of cells within discs and to evaluate the cytotoxic effects of different substances (Xia et al., 2023; Uggeri et al., 2000). This can be important for understanding how certain factors contribute to cell death or dysfunction within the disc (Pereira et al., 2011).Also, it plays an important role in the assessment of matrix remodelling to understand the dynamic processes involved in maintaining or altering the ECM. This is particularly relevant when studying the effects of various treatments, drugs, or conditions on the health of disc cells (Penolazzi et al., 2020).

4.1.2. Propidium iodide (PI)

PI is a fluorescent dye impermeable to living cells but may pass through the plasma membrane of dead or injured cells, where it intercalates with nucleic acids and becomes luminous. This enables to identification of dead cells in a population. It can be employed in a cell viability experiment to differentiate between live and dead cells (Shi et al., 2007). This can assist in determining the effect of degenerative processes, therapies, and experimental situations on cell survival or death inside the disc. Assessing cell death detects and measures cell death within the disc, which is critical for understanding the process of degeneration and the variables that contribute to cell loss. This allows and describes the spatial distribution of dead cells via fluorescent microscopy (Rannou et al., 2004).

4.2. Contrast agents

Various contrast agents are also employed in the bioimaging of IVDD. Like gadolinium-based contrast agents and superparamagnetic iron oxide nanoparticles used in MRI to enhance visualization of soft tissue and its abnormalities (Handley et al., 2015; Da Costa et al., 2020b). Diffusion Tensor Imaging (DTI) measures the diffusion of water molecules (Wang et al., 2018). Iohexol an iodine-based contrast agent is employed in CT for detailed anatomical imaging (da Costa et al., 2002). Whereas in SPECT and PET, technetium-99 (⁹⁹Tc) and fluorine-18 fluorodeoxyglucose (¹⁸F-FDG) are used to assess metabolic activity associated with inflammation respectively (Greco et al., 2023).

4.3. Biomechanical tracers and sensors

To measure the biomechanical alterations, like mechanical deformation in response to load and changes in intra-discal pressure strain gauges are being used (Rapoff et al., 1997). While, fibre-optic sensors are used to monitor biochemical processes like pH, and oxygen levels within the disc (Dennison et al., 2008). Degeneration-specific tracers like methylene blue and alcian blue trace proteoglycans especially glycosaminoglycan content aid in the visualization of degenerated disc (Li et al., 2018). Various inflammatory tracers like ⁹⁹Tc-labelled white blood cells indicate inflammation in nuclear medicine scans (Escalhão et al., 2017). Last but not least annexin V and caspase activity probes highlight cellular apoptosis (Yang et al., 2020).

5. Bioimaging: an emerging tool for diagnostic intervention in IVDD

Bioimaging refers to using various imaging techniques to visualize and study biological structures and processes at different levels, ranging from molecules and cells to tissues and whole organisms. It is crucial in biomedical research, clinical diagnosis, and treatment monitoring (Lahoti and Jogdand, 2022). Bioimaging techniques utilize versatile physical principles and technologies to generate images of biological samples. Bioimaging techniques have proven to be valuable tools in studying IVDD. They allow researchers and clinicians to visualize and assess the structural and functional changes within the discs, aiding in diagnosis, treatment planning, and monitoring of the condition (Kushchayev et al., 2018). Various imaging modalities are employed in the study of IVDD in the preclinical stages, Confocal microscopy, fluorescence microscopy, multiphoton microscopy, Raman spectroscopy, super-resolution microscopy; and in the clinical stage, MRI, CT, PET, MRI Optical coherence tomography, Ultrasound imaging, Photoacoustic imaging, X-ray are used (Fig. 4).

Fig. 4.

Different imaging techniques for the diagnostic intervention of intervertebral disc degeneration.

A comprehensive summary of the various techniques used in the diagnostic management of IVDD. It illustrates key imaging modalities like MRI, CT, PET and X-ray imaging, and various optical bioimaging techniques that are being used for both structural/molecular evaluation of the disc structure in 2D and 3D planes. They help assess the detection of tissue structure and arrangement of the extracellular matrix, detection of vascular and neuronal integrity around the disc, understanding of the cellular and metabolic activities of the cells of the disc, visualise any disc impairment in real timescale and also checking the effectiveness of a drug molecule for the therapy; ultimately improving outcomes by tailoring treatment strategies to the specific diagnostic findings. MRI, Magnetic resonance imaging; CT, Computed tomography; PET, positron emission tomography; 2D, Two dimensional; 3D, Three dimensional.

5.1. Bioimaging techniques for the preclinical stage

Preclinical bioimaging approaches are critical in IVDD research. It offers a non-invasive way of visualizing, monitoring and measuring the disease progression. It bridges the gap between animal models and clinical practice by facilitating the development of targeted treatments and the discovery of biomarkers, ultimately offering hope for improved diagnostics and interventions in the management of IVDD.

5.1.1. Confocal microscopy

Confocal microscopy is a technique that uses a laser beam that is precisely focused on a single point within the sample. The optical radiation emanating from this location is gathered using an objective lens. By introducing a pinhole into the optical pathway, extraneous light that is not in focus is obstructed, producing a clear and finely detailed image. A comprehensive image can be generated by traversing the laser beam across the specimen (Elliott, 2020; Nwaneshiudu et al., 2012; Semwogerere and E.R.J.E.o.b. Weeks, 2005). It is useful in IVDD pathophysiology and to check the biodistribution and efficacy of drugs. Alterations in the orientation of collagenous fibres may impact the mechanical characteristics of the IVDs and thereby play a role in their degenerative process (Yang and O'Connell, 2017). Researchers can use confocal microscopy to examine the arrangement and distribution of collagen fibres within the AF. The collagen fibres can be visualized, and their organization and alignment can be studied by incorporating fluorescent markers to label them. It is also applicable in the examination of disc cells, and also immune cells that may infiltrate the disc due to inflammatory responses. Using fluorescent markers, scientists can examine the dispersion, structure, and actions of these cells. This can yield significant insights into the underlying cellular mechanisms implicated in IVDD (Altendorf et al., 2012; Kandel et al., 2014). Furthermore, it is a viable technique for examining the spatial arrangement of proteoglycans and other molecules within the IVD (Poole and Mostaço-Guidolin, 2021). It is noteworthy that this methodology is predominantly still confined to research environments. Various factors, including expenses, accessibility, and the requirement for specialized apparatus and expertise could constrain its application in clinical settings.

5.1.2. Fluorescence microscopy

Fluorescence microscopy involves using fluorescent dyes or proteins to stain the sample, which subsequently emits light upon excitation by a specific wavelength of light. Using an objective lens to collect the released light and a filter to separate it from the excitation light, it is possible to observe different structures or molecules within the specimen (Ishikawa-Ankerhold et al., 2012). Fluorescence microscopy allows researchers to investigate the cellular and molecular processes involved in IVDD. Using specific fluorescent dyes or antibodies, researchers can label and visualize various components within the disc, such as ECM proteins, cells, and inflammatory markers (Penolazzi et al., 2020; Heo et al., 2023).

One of the key advantages of fluorescence microscopy is its ability to provide high-resolution imaging. This enables researchers to examine the fine details of cellular and molecular structures within the IVDs. Microscopy can visualize changes in the distribution and organization of collagen fibres, which are essential components of the disc's ECM (Poole and Mostaço-Guidolin, 2021; Hickey et al., 2021). It also allows for quantifying specific molecules or structures within the disc. By analyzing the fluorescence intensity or the number of labelled cells, researchers can assess the levels of various proteins or cell types associated with disc degeneration. This quantitative information can provide valuable insights into the progression and severity of the degenerative process (Verdaasdonk et al., 2014). In addition to visualizing static structures, this technique can also be used to study dynamic processes within the disc. For instance, researchers can track the migration and behaviour of cells involved in disc degeneration using time-lapse imaging. This can help elucidate the mechanisms underlying cell-mediated tissue remodelling and inflammation (Oichi et al., 2020). Furthermore, fluorescence microscopy can be combined with other techniques to gain a more comprehensive understanding of IVDD. For example, it can be used in conjunction with immunofluorescence staining to identify specific cell types or markers within the disc. It can also be combined with live-cell imaging techniques like fluoroscopy to study cellular processes in real-time (Hobson and Aaron, 2022). Fluoroscopy gives us live video imaging to evaluate and diagnose properly in different stages. It is a powerful tool to assess disc degeneration in modern times, ensuring early diagnosis and cure the patients.

5.1.3. Multiphoton microscopy

Multiphoton microscopy is a technique that operates on the same principles as fluorescence microscopy, but it employs longer-wavelength light and the concurrent absorption of two or more photons to stimulate the fluorescent molecules. The outcome of this phenomenon is a reduction in scattering and an increase in tissue penetration depth (Hobson and Aaron, 2022; König, 2000). Additionally, it mitigates phototoxicity because longer-wavelength light is less deleterious to biological tissues (Hoover and Squier, 2013). It's a cutting-edge imaging technique that allows high-resolution imaging of the disc, providing valuable insights into the structural and cellular changes associated with IVDD (Torre et al., 2018).

One of the key advantages of multiphoton microscopy in studying disc disease is its ability to image deep within the tissue without the need for invasive procedures. The technique uses infrared laser light, which can penetrate deep into the discs, allowing for imaging of the disc's internal structures and cells. This non-invasive nature of multiphoton microscopy is particularly advantageous in studying IVDD, as it minimizes tissue damage and preserves the natural environment of the disc (Disney et al., 2018).

An upgraded version known as nonlinear intravital multiphoton microscopy (iMPM) can provide detailed information about the ECM of the disc, which plays a crucial role in maintaining disc health. By using specific fluorescent dyes or genetically encoded fluorescent proteins, researchers can visualize and analyze the organization and composition of the ECM components, such as collagen and proteoglycans. This can help identify changes in the matrix associated with IVDD, such as collagen degradation or loss of proteoglycans, which are key indicators of disc degeneration (Dondossola et al., 2016). Furthermore, a subtype of multiphoton microscopy called two-photon microscopy enables the visualization of cellular processes within the disc. Researchers can label specific cell types or use genetically encoded fluorescent markers to track cell behaviour and interactions in real time. This allows for studying cell proliferation, migration, and cell-matrix interactions within the disc, providing insights into the cellular mechanisms underlying IVDD (Kawakami and Flügel, 2010). In addition to structural and cellular imaging, multiphoton microscopy can be combined with other techniques to study functional aspects of the IVDs. For example, researchers can use multiphoton microscopy in conjunction with fluorescence lifetime imaging microscopy (FLIM) to measure metabolic activity or assess the presence of oxidative stress within the disc. These functional measurements can provide valuable information about the metabolic changes associated with IVDD and help identify potential therapeutic targets (Yew et al., 2014; Provenzano et al., 2009).

While multiphoton microscopy holds great promise in studying IVDD, it is still a relatively new technique, and further research is needed to understand its potential and limitations fully. Additionally, the practical implementation of multiphoton microscopy into clinical practice for IVDD diagnosis and monitoring may require advancements in imaging technology and standardization of imaging protocols.

5.1.4. Raman spectroscopy

Raman spectroscopy is a technique of non-invasive nature that provides detailed, intricate details about the molecular composition of a sample by analyzing the scattering of light. This technique involves illuminating a sample with a monochromatic light source and then detecting and analyzing the light's inelastic scattering. The phenomenon of scattered light is characterized by a distinct alteration in wavelength relative to the incident light. This alteration in wavelength is used to extract valuable insights regarding the vibration modes of the molecules present in the sample, thereby facilitating their identification and characterization (Dodo et al., 2022; Lyon et al., 1998). Raman spectroscopy can identify and measure the aforementioned biochemical alterations. It has been used to observe the structural and functional changes in the NP in cervical and lumbar discs following herniation by IVDD. Through the examination of Raman spectra obtained from disc tissue, researchers can discern distinct molecular patterns that are indicative of the degeneration of the disc. For instance, alterations in the Raman spectral features associated with proteoglycans and collagen may indicate the depletion of these crucial constituents within the deteriorating IVD (Wang et al., 2020; Wang et al., 2022b).

Furthermore, it enables the monitoring of the temporal evolution of disc degeneration. Through the comparative analysis of Raman spectra obtained from disc tissue at various time intervals, researchers can evaluate alterations in the molecular composition of the disc and determine the rate at which the degenerative process is either accelerating or decelerating. This information can provide valuable insights for making treatment and management strategies decisions, including the choice between conservative therapies or surgical intervention (Haughton, 2005). Raman spectroscopy has potential applications within regenerative medicine, particularly in the context of disc degeneration. For instance, in the case where a patient is subjected to a therapeutic intervention to reinstate the structural and functional integrity of the IVD, techniques such as cell therapy or tissue engineering, Raman spectroscopy can be employed to observe and evaluate the impact of the treatment, as well as determine if it is inducing the intended alterations in the molecular composition of the disc (Ember et al., 2017; Pereira et al., 2017; Gong et al., 2013).

5.1.5. Super-resolution microscopy

Super-resolution microscopy refers to a collection of methodologies that enable the acquisition of high-resolution images of individual cells. An instance of a technique that can achieve super-resolution imaging is photoactivated localization microscopy (PALM). This method employs fluorescently labelled molecules that are individually localised to generate a high-resolution image that is composed of a collection of individual molecules within a cell or tissue (Schermelleh et al., 2019; Yamanaka et al., 2014). Super-resolution microscopy is a high-resolution imaging technique that can be used for studying IVDD at the cellular and subcellular levels. The mode of action of super-resolution microscopy involves surpassing the diffraction limit of light, which allows for the visualization of structures and details beyond what is achievable with conventional microscopy (Huang et al., 2010). Various super-resolution microscopy techniques, such as single-molecule localization microscopy (SMLM) structured illumination microscopy (SIM) and, stimulated emission depletion (STED), utilize different principles to achieve enhanced resolution (Wegel et al., 2016).

It can provide detailed images of the microstructure of the IVD including the AF and the NP. Changes in these structures, such as thinning of the disc or tears in the AF, can be visualized at a high resolution, providing insights into the structural changes associated with disc degeneration. It can also be used to visualize cells and molecules within the disc. For example, it can be used to image cells such as chondrocytes and fibroblasts, which play a role in the maintenance and repair of the disc. Additionally, it can be used to image molecules such as collagen and proteoglycans, which make up the ECM of the disc to monitor the effects of treatments for disc degeneration at a molecular and cellular level. It can also be used to monitor changes in cell number or activity, or changes in the composition or organization of the ECM, in response to treatment (Kadow et al., 2015; Xie et al., 2023). In conclusion, super-resolution microscopy is a powerful tool for the study of IVDD. It allows for the visualization of the microstructure of the disc at a high resolution, providing valuable insights into the mechanisms of disc degeneration and potential therapeutic targets. However, more research is needed to fully understand its potential and limitations in this field. A new version of staining-free multiphoton microscopy is now being evaluated as it delivers a significant advantage in IVDD detection. Staining-free multiphoton microscopy allows imaging of the IVD without the need for exogenous dyes or stains. This preserves the natural properties of the tissue, including its native structure, composition, and molecular interactions (Disney et al., 2017). By avoiding the use of stains, the imaging process does not introduce any potential artefacts or alter the biological characteristics of the disc. Staining-free multiphoton microscopy enables real-time imaging of live IVD tissue. This is particularly advantageous for studying dynamic processes, such as cell behaviour, matrix remodelling, and fluid flow within the disc. Researchers can observe these processes in their native state, providing valuable insights into the mechanisms underlying IVDD (Borile et al., 2021). It is a non-invasive imaging technique that does not require the introduction of exogenous contrast agents or dyes. This reduces the risk of tissue damage or adverse reactions associated with staining procedures. Non-invasive imaging also allows for longitudinal studies, where the same disc can be imaged over time to track disease progression or treatment effects (Sriram et al., 2020). Moreover, combined with two-photon auto-fluorescence lifetime imaging (2P-FLIM), it can simultaneously capture multiple parameters, such as autofluorescence, second harmonic generation, and fluorescence lifetime imaging. This multi-parameter imaging allows for the assessment of various aspects of IVDD, including changes in cellular metabolism, collagen organization, and molecular composition (Ranawat et al., 2019).

5.2. Bioimaging techniques for the clinical stage

Preclinical bioimaging bridges the gap between animal models and clinical practice, facilitating targeted treatments and biomarker discovery, and offering hope for improved diagnostics and interventions in disease management.

5.2.1. Magnetic resonance imaging (MRI)

MRI is a sophisticated medical imaging technique used to examine the physio-pathological conditions of the human body. MRI uses strong magnetic fields and radio waves to obtain exact images as opposed to CT and X-rays, which generate radiation with ionizing capabilities. It produces incredibly accurate images of organs, soft tissues, and even neurological components (Thiravit et al., 2021). It is a secure and non-intrusive imaging technique; the fundamental concept of MRI is nuclear magnetic resonance (NMR). This physical phenomenon serves as the cornerstone for MRI (Thompson et al., 2021). When an object, such as the human body, is subjected to a strong magnetic field, the nuclei of some atoms, particularly hydrogen nuclei (protons), align with the magnetic field. A short radio frequency pulse throws off this alignment. The protons return to their starting state and release energy through radiofrequency signals when the pulse is withdrawn. Another idea is that MRI can use proton signals to store spatial information using gradient magnetic fields. Different body slices may be scanned by adjusting the gradients, enabling three-dimensional reconstruction of internal structures (Berger, 2002).

MRI has emerged as a valuable diagnostic tool for assessing IVDD. MRI provides detailed images of the spine by utilizing radio waves and a strong magnetic field, allowing healthcare professionals to visualize the extent of disc degeneration. This non-invasive technique enables the identification of various degenerative changes, such as disc height loss, disc herniation, and the presence of osteophytes (Fiani et al., 2020). Additionally, MRI can help differentiate between different stages of disc degeneration, aiding in developing appropriate treatment plans. With its ability to provide high-resolution images of the IVDs, MRI plays a crucial role in accurately diagnosing and monitoring disc degeneration, facilitating timely interventions, and improving patient outcomes (Mwale et al., 2008).

In MRI, the patient is placed on the MRI table, and the area of the disc is carefully positioned in the middle of the magnetic field. The patient must be appropriately positioned for accurate imaging of the IVDD. Then the specialized receiver coils are utilized to pick up radiofrequency signals that the body's protons release. To enhance signal detection and image quality, these coils are placed around the scanned bodily component. Once the patient is appropriately positioned, a radiofrequency pulse is applied to an area of interest, causing hydrogen protons to oscillate and align in the opposite direction. When the pulse is turned off, the protons relax and align with the magnetic field, emitting radiofrequency signals that the coils pick up. An MRI system generates images using gathered signals, and modern computer algorithms reconstruct spatial information to provide detailed cross-sectional images of the disc. The Fourier transform approach converts the original data into spatial domain information, with frequency and phase information stored in k-space. The data is then translated into cross-sectional pictures using image reconstruction techniques. Post-processing methods like filtering, noise reduction, and contrast modifications are used to improve image quality. The final reconstructed images are then viewed by healthcare professionals to identify anomalies or diseases (Da Costa et al., 2020b; De Goyeneche et al., 2019).

The IVD structures can be seen precisely using MRI (Fomin and Timoshenko, 2020). T2 weighed (T2W) imaging may reveal the NP and AF. A healthy disc's NP and inner AF are super intense on T2W imaging, appearing as a hyperintense ellipsoid region on sagittal images. The brightness of the NP and T2W signal collagen concentrations resonates with proteoglycan concentrations but not with water concentrations (Da Costa et al., 2020b). Because of biochemical alterations in the nucleus' ECM, T2W imaging of degenerative discs cannot discriminate between annular and nuclear disc material. Changes in the proteoglycan composition and reduced water content of the NP, resulted in a decrease in T2W imaging signal intensity, rendering nuclear and annular material isointense to one another (Lee et al., 2022). It is essential to underline that degeneration of the disc, with the rare exception of discogenic spinal pain, is a rather frequent disease clinically normal and does not automatically cause clinical symptoms. The Pfirrmann technique is the most used method in human medicine for detecting degeneration based on sagittal T2W MRI data. It is based on Thompson et al.'s technique for categorising gross pathogenic changes in IVDs, which is the most extensively used criterion-referenced standard in human medicine (Thompson et al., 2021). The Pfirrmann approach has been validated, with findings showing a strong correlation between ageing and chondrodystrophic conditions. Contrast chemicals can be administered intravenously to increase the visibility of certain tissues or lesions and offer additional diagnostic data (Yu et al., 2012).

Each of these techniques is required to provide high-quality MRI images that aid in precise diagnosis and treatment planning. MRI is a versatile imaging technology used in a variety of medical specialities and diseases including IVDD, due to its ability to provide complete information about the core and periphery of the tissue, structural deformity, and vessel innervation without the use of ionizing radiation (Hussain et al., 2023; Bergknut et al., 2011; Ogon et al., 2020a). While MRI is a powerful imaging tool, the machines and procedures can be expensive compared to other imaging modalities. Whereas, the whole scans may take longer time to acquire than other imaging techniques, which can be challenging for patients with claustrophobia or those who are unable to remain still. Also, it may not be suitable for patients with certain metal implants, pacemakers, or cochlear implants due to potential safety risks (Ling et al., 2008; Detiger et al., 2015).

5.2.2. Computed tomography (CT)

CT is a medical imaging modality that employs X-rays and computational algorithms to generate cross-sectional, highly detailed images of the human body (Taubmann et al., 2018). CT furnishes significant insights regarding the inner structures of an organism, encompassing bones, organs, and soft tissues. It is extensively employed in diagnosing and assessing diverse medical conditions, such as trauma, tumours, infections, and vascular diseases (Hussain et al., 2022; Brooks, 1993). The high level of detail in the images generated by CT aid healthcare practitioners in achieving precise diagnoses, devising treatment plans, and evaluating the efficacy of interventions. It is crucial to consider the possible hazards of radiation exposure while employing CT imaging (Power et al., 2016; Buzug, 2011). It plays a significant role in the diagnosis and evaluation of IVDD. CT provides detailed cross-sectional spine images, allowing healthcare professionals to visualize the affected IVDs, spinal canal, and surrounding structures. This helps in identifying the location and extent of disc herniation or degeneration. It is particularly useful in assessing the bony structures of the spine, such as the vertebral bodies, facet joints, and neural foramina. It can detect any bony abnormalities, such as osteophytes or fractures, which may contribute to IVDD symptoms (Kim et al., 2020). CT aids in surgical planning for IVDD cases that require surgical intervention. Surgeons can use detailed images to determine the optimal approach, identify the extent of disc herniation, and plan for decompression or fusion procedures. It can help differentiate between soft tissues, such as the spinal cord, nerve roots, and IVDs. This differentiation is crucial in determining the severity and location of nerve compression or impingement caused by degeneration (da Costa et al., 2020a). It can detect potential complications associated with IVDD, such as spinal stenosis, foraminal narrowing, or the presence of calcified disc material. These findings can guide treatment decisions and help manage the condition effectively. It's important to note that while CT provides detailed anatomical information, they involve exposure to ionizing radiation. Therefore, the decision to use CT imaging should be made based on the individual patient's clinical presentation and the potential benefits outweighing the risks (Wu et al., 2020).

5.2.3. Positron emission tomography (PET)

PET is a non-invasive imaging technique, which provides the function and metabolism of the organs and tissues in a detailed way to assess the deformity. It provides insights into the tissue's biochemical processes (Kropotov, 2016). The PET uses a radioactive tracer, typically FDG (Fluorodeoxyglucose), injected into the patient's bloodstream. It is tagged with a positron-emitting radionuclide, which emits a positron and two gamma photons when it encounters an electron. The PET scanner detects these photons to create a three-dimensional image of the tracer concentration.

One of the key advantages of PET is its ability to detect changes at the cellular level, which often occur before structural changes can be identified with other imaging processes. This early detection capability can significantly improve the prognosis for conditions like cancer, allowing for earlier intervention (Griffeth, 2005). The severity of degeneration can also be evaluated using this technique. PET provide particular information on tissue metabolic activity and its consequences on the surrounding structures of the disc. In the context of IVDD, it can detect increased metabolic activity in the afflicted IVDs; increased glucose absorption in the discs is a marker of active disease processes and can aid in the diagnosis and monitoring of inflammation. The areas of low metabolic activity can also be detected associated with degenerative or necrotic discs (Yitbarek and Dagnaw, 2022). By monitoring metabolic activity, researchers can yield valuable data about how spinal canal stenosis or nerve root compression can affect neighbouring structures. It is used to track the patient's reaction to treatment following therapeutic intervention might indicate efficacy. Similar to other imaging modalities, PET entail the introduction of a radioactive tracer, which generates positrons that the scanner detects. The operation should be carefully reviewed in light of the patient's condition, medical history, and clinical issues. The benefits of PET imaging should be evaluated against the risks of radiation exposure and any patient-specific contraindications (Griffeth, 2005; Vitor et al., 2017; Ehman et al., 2017) For a more comprehensive evaluation of IVDD, it is essential to remember that PET could only offer useful metabolic information for a more thorough examination it is frequently used with other imaging techniques. Alone, it provides functional and metabolic data while combining with MRI and CT can give a more holistic view of the condition (Deer et al., 2022; Lurie and Tomkins-Lane, 2016).

However, PET also have their limitations. For instance, they are less effective at providing detailed images of certain body areas, such as the bones. Additionally, the use of radioactive tracers means that there is a small risk of radiation exposure. Another limitation of the PET technique that is worth mentioning is that it has a much lower spatial resolution than conventional CT/MRI techniques. However, the benefits of a PET usually far outweigh these risks for most patients (Freedenberg et al., 2014).

5.2.3.1. Combined imaging techniques: PET-CT/ PET-MRI

In recent years, combined PET/CT and PET/MRI scanners have been developed, which allow for the simultaneous acquisition of metabolic and anatomic information. This provides a more comprehensive picture of the patient's condition, improving diagnostic accuracy and aiding in treatment planning. These combined imaging techniques can detect metabolic changes within the discs, such as inflammation or altered glucose metabolism, which are indicative of degeneration. Additionally, PET-MRI's high-resolution images can help in precisely locating degenerative changes and assessing the involvement of adjacent tissues, nerves, and bone marrow, making these modalities invaluable in the comprehensive evaluation and management of IVDD (Pollard et al., 2022; Zaidi and Del Guerra, 2011; Gamie and El-Maghraby, 2008).

5.2.4. Optical coherence tomography

Optical Coherence Tomography (OCT) is a non-invasive imaging technique that employs low-coherence light to obtain high-resolution cross-sectional images of a sample. The incident light is divided into two separate beams: the sample beam and the reference beam. The sample and reference beams' reflected lights are coherently combined, resulting in an interference pattern used to generate an image. OCT utilizes low-coherence light, thereby enabling the detection of reflections from a particular depth within the sample and facilitating the acquisition of cross-sectional images (Aumann et al., 2019; Podoleanu, 2012).

OCT can provide a visual representation of alterations in the structure of the disc. Its utilization enables the acquisition of high-resolution images that can unveil intricate details about the microstructure of the disc. This includes the assessment of the arrangement and soundness of the collagen fibres within the AF, as well as the evaluation of the state of the NP. This technique can facilitate the early identification of disc degeneration, potentially preceding the manifestation of symptoms (Acheson et al., 2018; Boone et al., 2012). It has the unique capability to monitor the temporal progression of disc degeneration. Clinicians can evaluate the structural changes of the disc and determine the rate at which degeneration progresses by conducting a comparative analysis of OCT images captured at various time intervals. This information can provide insights for making decisions regarding treatment and management, including the choice between conservative therapies or surgical intervention. This technology can offer a comprehensive analysis of the structural modifications taking place within the disc, facilitate early identification, track the advancement of diseases, and assess the efficacy of regenerative therapies (Han et al., 2016; Ohnishi et al., 2020). However, its application in this particular domain remains relatively nascent. Further investigation is imperative to grasp both its capabilities and constraints comprehensively.

5.2.5. Ultrasound imaging

Although ultrasonic (US) imaging is widely employed for diagnostic purposes in many medical fields (Chan and Perlas, 2011), it has a limited function in assessing IVDD compared to other imaging modalities like MRI or CT (Champaneria et al., 2010). The US can still give useful information in some cases of disease. It can be used to direct therapeutic injections into the discs or adjacent tissues to help accurately inject drugs like corticosteroids into degenerating discs to alleviate pain and inflammation (Da Costa et al., 2020b). Also, used to guide needle placement during discography (injecting contrast dye into the discs to analyze their anatomy and detect painful discs). The dynamic assessment of the spine and real-time imaging of the disc, patient's movement or specific maneuvers can be monitored (Liptak et al., 2002). It may be a quick screening technique to evaluate the spine and identify probable anomalies or disorders. When diagnosing IVDD, it is critical to remember the limitations of ultrasonography. Because of the disc's deep placement within the body, US imaging may be challenging due to overlapping bones and gas-filled structures (Krueger et al., 2016). Furthermore, it gives less soft tissue imaging than MRI or CT, making it less effective for determining the specific anatomy and degenerative changes associated with degeneration (Emery et al., 2018).

5.2.6. Photoacoustic imaging

Photoacoustic imaging involves the use of a brief laser pulse to induce thermal energy within a confined area of the specimen. The process of heating results in a swift thermal expansion, which leads to the production of an ultrasound wave that an ultrasound transducer can identify. The spatial and temporal characteristics of the ultrasound wave yield valuable insights into the anatomical and physiological attributes of the specimen (Attia et al., 2019).

The medical procedure of endoscopy involves using an endoscope, a pliable tube equipped with a light source that serves to illuminate a particular organ or tissue. The use of an endoscope is a viable method for investigating the aetiology of symptoms such as gastrointestinal bleeding, pain, or dysphagia by inserting it through the oral cavity and into the digestive cavity of a patient. Photoacoustic imaging with endoscopy provides better image resolution than using them both alone (Yang et al., 2009; Wang et al., 2021). Photoacoustic imaging can be used to detect specific molecules associated with disc degeneration. For example, it can detect increased levels of inflammatory molecules often present in degenerative discs. This can provide early warning of disc degeneration, even before structural changes become apparent (Chan et al., 2013). It can be used to monitor the effectiveness of treatments, providing real-time, detailed images of the disc to help doctors assess whether a treatment is working and adjust the treatment plan, if necessary, thus making it a promising tool for the detection and management of IVDD. It offers a non-invasive, real-time, and detailed view of the disc's structure and function, which can help doctors diagnose disc degeneration, monitor its progression, and assess the effectiveness of treatments (Beard, 2011; Capart et al., 2022).

5.2.7. X-ray imaging

X-ray imaging, a widely used diagnostic technique, utilizes X-rays to create images of internal structures. X-rays are electromagnetic radiation with shorter wavelengths and higher energy than visible light. They interact with tissues in the body, with different tissues having varying abilities to absorb X-rays. This absorption difference allows for the visualization of anatomical structures and the detection of abnormalities (Martz et al., 2016). X-ray imaging involves an X-ray machine, a patient, and a detector. The machine consists of an X-ray tube that generates X-rays and a collimator to control the X-ray beam's size and shape. The patient is placed between the detector and the machine, which catches the X-rays passing through the body and converts them into an image (Russo, 2017). Upon activation, a high voltage current is applied to the X-ray tube, causing electrons to accelerate towards a metal target. The collision of electrons with the target produces X-rays through bremsstrahlung radiation. These X-rays form a divergent beam that passes through the patient's body. As the X-ray beam interacts with tissues, less dense tissues, such as organs and muscles, allow more X-rays to pass through and appear darker while denser tissues like bones absorb more X-rays and appear white in the resulting image. This differential absorption creates contrast in the X-ray image, enabling the observation of anatomical structures and the detection of anomalies (Gureyev et al., 2011; Munro et al., 2010). The exiting X-ray beam is captured by a detector, which can be a film cassette or a digital sensor. In film-based radiography, the X-ray beam exposes a film that is later developed to produce a visible image. In digital radiography, a digital sensor converts the X-rays into an electronic signal, which is processed and displayed on a computer screen for immediate visualization and manipulation (Schlüter et al., 2014). In the context of IVDD, X-ray imaging provides valuable information about structural changes in the spine. It allows for the assessment of disc space narrowing, a common sign of IVDD, by visualizing the reduction in space between adjacent vertebrae (Kague et al., 2021). X-ray images can also reveal osteophytes, and irregular bony outgrowths along the edges of vertebral bodies, which are often associated with osteoarthritis and IVDD (Yang et al., 2020). Additionally, X-ray imaging can detect endplate sclerosis, the hardening and thickening of bony endplates surrounding IVDs, as areas of increased density or whiteness in the vertebral endplates (Lamb et al., 2002). However, X-ray imaging has limitations in directly visualizing the IVDs, which are composed of soft tissue. Therefore, the diagnosis of IVDD using X-ray imaging relies on indirect signs of disc degeneration like impairment of skeletal integrity around the disc. To obtain a comprehensive assessment, additional imaging modalities like MRI may be necessary to directly visualize the discs and assess their condition, including the presence of disc herniation or nerve compression (de Strobel, 2016; Russo et al., 2023).

In summary, X-ray imaging is a valuable tool in diagnosing and managing IVDD. It provides information about structural changes in the spine, such as disc space narrowing, osteophyte formation, and endplate sclerosis. However, it should be used in conjunction with other imaging modalities, like MRI (Russo et al., 2023; Ogon et al., 2020b).

• 5.3Other Imaging Techniques

These are various other techniques that have the potential to be used in IVDD diagnosis and management. One of them is Second Harmonic Generation Imaging Microscopy (SHG). It is a nonlinear optical microscopy technique that allows for the visualization of non-centrosymmetric structures, such as collagen fibres, without needing exogenous dyes or stains. The principle behind SHG imaging is based on second harmonic generation, which occurs when two photons of the same frequency interact with a nonlinear material and combine to generate a photon with twice the frequency (half the wavelength) and twice the energy (Campagnola, 2011). SHG imaging provides a better assessment of collagen alterations, visualization of collagen organization, and evaluation of collagen cross-linking to diagnose IVDD (Theodossiou et al., 2006). Combining SHG with such as two-photon excitation fluorescence (TPEF) a mode advanced, nonlinear intravital microscopy provides the best assessment of cellular and molecular interactions, provides the best real-time label-free fluorescence imaging of cellular processes, and helps in treatment monitoring in IVDD (Dondossola et al., 2016; Perry et al., 2012). Synchrotron tomography is another advanced imaging technique that can be utilized in the study of IVDD. It involves using synchrotron radiation, a high-intensity and highly collimated X-ray beam produced by a synchrotron particle accelerator. Synchrotron tomography provides high-resolution, three-dimensional, non-destructive multi-modal imaging with a better resolution to assess disc morphology (Disney et al., 2017; Giuliani et al., 2017). Some shortcomings Synchrotron radiation tomography is restricted to ex-vivo applications and it is available only at a limited number of specialized centres. Moreover, it's non-destructiveness depends upon the level of radiation applied. If too much radiation is applied, it can damage the disc itself. Nevertheless, this technique offers extremely high spatial resolution, making it a great choice for IVDD diagnosis (Zbik et al., 2008). (Table-1).

6. Conclusion and future perspective

In conclusion, bioimaging techniques have emerged as powerful tools for diagnostic interventions in intervertebral disc degeneration. Techniques, including molecular imaging, MRI, CT, ultrasound, diagnostic dyes and tracers, have provided valuable insights into the pathophysiology of disc degeneration and have facilitated the development of targeted therapies. Bioimaging techniques have improved the accuracy of diagnosis and monitoring of disc degeneration by enabling non-invasive visualization of disc morphology, composition, and function. Moreover, these techniques have played a crucial role in guiding the delivery of therapeutic agents to the intervertebral disc, thereby enhancing the efficacy of treatment strategies. Still, there are several hurdles associated with diagnostic approaches in IVDD, such as, the detection of causes and symptoms and standardising diagnostic techniques is hard. IVDD must be diagnosed at an early stage to select a desired therapeutic regimen based on severity. Diagnosing IVDD is tough as the disc cell biochemistry, ECM, and environment differ. The disc's diverse environment makes diagnostic procedures tough to design. Diagnosis of IVDD demands excellent drug delivery systems to overcome these hurdles and deliver therapeutic medications to the disc in a regulated and comprehensive way. Diagnostic IVDD therapies promote tissue repair and disc function while relieving symptoms. Diagnostic therapies cannot repair the spinal disc because it recovers slowly. Clinical diagnosis is hard to apply, and the intricacy of the disc, the necessity to show long-term effectiveness and safety, and regulatory regulations make potential diagnostic treatments difficult to adopt in clinical praxis. We require multidisciplinary partnerships, imaging, biomarker development, drug usage, and research to solve these problems. IVDD diagnostic drugs to improve detection, therapy, and patient outcomes need further investigation. Looking ahead, the role of bioimaging techniques in diagnostic interventions for intervertebral disc degeneration is expected to expand further. Advances in imaging technology, such as the development of high-resolution and real-time imaging modalities, will enable more precise and detailed visualization of disc degeneration processes. This will facilitate early detection and intervention, potentially preventing the progression of disc degeneration and the development of associated complications. Furthermore, integrating bioimaging techniques with other emerging technologies, such as regenerative medicine and nanotechnology, holds great promise for developing innovative diagnostic approaches. For instance, the combination of imaging-guided drug delivery systems with regenerative therapies, such as stem cell-based therapies or tissue engineering approaches, could provide synergistic effects for disc regeneration. Bioimaging techniques can help monitor the distribution and efficacy of these therapies, allowing for real-time adjustments and optimization of treatment protocols.

In addition, the application of artificial intelligence and machine learning algorithms to bioimaging data can enhance the accuracy and efficiency of diagnosis, prognosis, and treatment planning for intervertebral disc degeneration. These technologies can analyze large datasets and extract meaningful patterns and biomarkers, enabling personalized and targeted interventions. Overall, the continued advancement and integration of bioimaging techniques with other cutting-edge technologies will undoubtedly revolutionize the field of diagnostic interventions for intervertebral disc degeneration. Bioimaging techniques will play a pivotal role in improving patient outcomes and advancing the field of spine care by providing comprehensive and real-time information about the disc's structure, function, and response to treatment.

Ethical approval and consent to participate

Not applicable.

Funding/support

This study received no specific grant from any funding agencies in the public, commercial, or not-for-profit sector.

CRediT authorship contribution statement

Gyanoday Tripathi: Writing – review & editing, Writing – original draft. Lahanya Guha: Writing – review & editing, Writing – original draft. Hemant Kumar: Supervision, Project administration, Conceptualization.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this article.

Data availability

No data was used for the research described in the article.