LUMBAR INTERVERTEBRAL DISC (IVD)

INTRADISCAL CELLS

International Publicized Data

GMCD Instructional  Course Lectures

Author :  

Dr. med. Guy M.C. Declerck MD (GMCD)

Medical FRCS-, FRCS Ed Orth-, M Ch Orth-, PhD-studies

Spinal Surgical and Research Fellow, Perth, Western Australia

Spinal Orthopaedic Surgeon and Surgical Instructor

Consultant R&D Innovative & Restorative Spinal Technologies

President International Association of Andullation Technology (IAAT)

Copywriter / Translator:   Filip Vanhaecke PhD

Illustrative expertise:     Lennart Benoot, Mincko, Halle, Flanders, Belgium

Artistic illustrations:    Alonso Ríos Vanegas, sculptor and writer, Medellín, Colombia

Page layout:   Lennart Benoot, Mincko, Halle, Flanders, Belgium

Review scientific literature:   Medical Consulting Advice, Ostend, Flanders, Belgium

Support:   International Association of Andullation Therapy (IAAT)

Legal advice:   Anthony De Zutter, kornukopia.be

Dedication to the Colombian Family Gloria Rúa Meneses and her sons Andres David and Juan Camilo Hinestroza, both members of the National Colombian Waterpolo Team. For their friendship and hospitality. December 2014.

Acknowledgements

Relying on the work of giants is the lifeblood of scientific research. Indeed, if I have seen further, it is by standing on the shoulders of giants. One might even say that I have always depended on the kindness of strangers in this regard (*).

The continuous support by professor BA Kakulas (Neuropathology), professor JR Taylor (Spinal Anatomy and Human Biology), and Sir George M Bedbrook (Spinal Orthopaedic and Rehabilitation Surgeon) made it possible to analyse 23539 post-mortem human spines, normal and pathological, in the Department of Neuropathology, Royal Perth Hospital/University Western Australia, Perth.

Note: in order not to disturb easy reading of the underneath scientifically based chapters, only a few authors are mentioned in the text where dr. Guy considered it essential. Their names are placed between brackets. Further information on their individual research can be read in the last chapter ‘Literature Encyclopedia’.

         (*) Mirsky Steve. Technology is making it harder for word thieves to earn outrageous fortunes. Scientific American, February 2014, p. 64

Contents

1. Cells in the human body: number, function and aging

2. Lumbar intervertebral discs contain the lowest cellularity of all human organs

3. Lumbar intervertebral discs are the largest non-vascular organs in the human body

4. The notochordal and chondrocytic-like cells in the nucleus pulposus

5. All disc cells show an intense relationship between mechanical functions and metabolic activities

6. The intervertebral disc matures when it begins to sense mechanical responsibilities

7. The decreasing number of notochordal cells initiates the aging processes

8. The endplates contain chondrocytic-like cells

9. The annulus fibrosus contains fibrochondrocytic-like cells

10. Notochordal cells are capable of repairing intervertebral disc tissue

11. Mechanotransduction: cells receive physical information from the environment to function

12. Mechanobiology: biologic response of the disc cells and their matrices to mechanical stimuli

13. What happens to the cells in the aging and degenerating intervertebral disc?

14. Cells in the ‘herniating’ intervertebral discs

15. Therapeutic implications

16. Literature Encyclopaedia

1. Cells in human body: number, fate and aging

The adult human body is a complex multicellular organism. It is estimated that the human organism is build up by approximately forty thousand billion cells (40.000 x 1.000.000.000). All cells in the different organs (brain, heart, lungs, etc …) can remain healthy if they receive sufficient blood supply. To continue their routine duties and enjoy reproducing new cells,  every cell needs to sense various functional incentives. When cells no longer are stimulated, they become non-active and totally unnecessary. Some break down and become necrotic. Other cells initiate self-destructing – apoptotic – processes and disappear unnoticed. A few survive and become senescent. The senescent cells cease dividing and undergo distinctive phenotypic alterations. However, these senescent cells continue their metabolic activities at slower rate.

Aging is characterized by a decreasing number of metabolically active cells. Aging processes proceedsilently, do not cause inflammatory reactions and are no direct source of pain because the intracellular matrix degrading enzymes (see topic Extracellular Matrix) are not released in the extracellular matrix. A very well-known example is osteoporosis, a disease caused by irreversible, progressively but, indeed, silently evolving cellular involutions.

2. Lumbar intervertebral discs: lowest cellularity

Compared to all other human organs, the amount of cells present in healthy and mature lumbar intervertebral discs (IVDs) only comprises 0.25 % to 1 % of their total disc tissue volume. In addition to their small amount (Table 2), the cells in the nucleus pulposus, endplates (EP) and annulus fibrosus (AF) have a slow ongoing cell proliferation. But because of their higher amount of cells (+/- 9 × 106 cells/cm3) the peripheral zones, the EP and the AF, can repair more easily their degenerative or traumatic fissures and tears.

Structure

Number of cells/cm3

Endplates (EP)

  9.000.000

Annulus fibrosus (AF)

  9.000.000

Nucleus pulposus (NP)

  4.000.000

Hyaline cartilage in peripheral joints (hip, shoulder, knee, …)

14.000.000

Table 2. Small number of cells in the different zones of the lumbar IVD

3. Lumbar disc: largest non-vascular organ

The central nucleus pulposus never contains blood vessels. The very small amount of nuclear cells (+/- 4 x 106/cm³) (Table 2) have to adapt to conditions of low oxygen and diminished transport of nutrients and metabolites. Therefore they live in an acid environment. To survive, the cells necessarily have no choice but to produce and preserve a large amount of surrounding extracellular matrix (ECM). These harsh conditions explain why the nuclear cells are unable to repair internal disruptive lesions caused by aging, degenerative or traumatic processes.

Initially, the EPs and the AF of the IVD possess a superior vascularisation (Fig. 3a). The young and immature endplates receive blood supply from the adjacent and well-vascularised vertebral bone tissue. However, within the first decade of life and during further maturation of the IVD, the human spinal column develops into an upright and weight-bearing structure and the lumbar IVDs become subjected to very complex mechanical forces at all times. Because of the increasing loading of the spine, calcification and ossification of the endplates intensify and progress which result in the blood supply becoming compromised and finally interrupted. The outer part of the AF always receives a small vascular supply but it declines with age (Fig. 3b).

Lack of blood supply and oxygen cause multiple well-known medical conditions. Whatever the reasons, occlusion of blood vessels lead to infarcts of lung, spleen, bone tissue, muscles, etc …  . Myocardial or cerebral infarcts are major reasons for dying. The mainly avascular intervertebral disc react differently.

 

Fig. 3a. Left: the endplates (EP) and the outer annulus fibrosus (AF) in the healthy and young/adolescent IVD are vascularized. The NP always remains without blood supply.

Fig. 3b. Right: in the adult IVD, the EPs become calcified and ossified and all blood vessels occlude. The outer AF becomes more and more avascular.

4. Nucleus pulposus: notochordal cells and chondrocytic-like cells

During the embryonic, fetal and young infantile stages remnants of the embryonic notochord, the notochordal cells, construct the central NP of the IVD. Not subjected to weight bearing circumstances, the notochordal cells remain very active and synthesize huge amounts of proteoglycans which attract and bind high quantities of water molecules. Dispersed into their extensive extracellular matrix, the young notochordal cells evolve to large and round entities.

The notochordal cells are responsible for the initial nature of the NP. The high concentration ofproteoglycans not only makes the young NP highly hydrophilic but inhibits the ingrowth of blood vessels. The immature NP is a wonderful structure: non-vascular, translucent, semi-liquid (+/- 80 % water) and gel-like (Fig.4).

Fig. 4. Fully hydrated nucleus pulposus of the L4-L5 intervertebral disc (X83-478 – M – 1 day old). NP (nucleus pulposus), EP (endplate), AF (annulus fibrosus), VB (vertebral body)

(Left: X83-478, Declerck / Kakulas, Neuropathology, Perth, Western Australia)

(Right: detailed illustration of X83-478 by Colombian Sculptor Alonso Ríos, www.alonsoriosescultor.com)

5. Disc cells: intense relationship between mechanical functions and metabolic activities

The IVD cells are functionally stimulated by continuously receiving signals by varying mechanical loads during sitting, standing, walking, bending, etc. They all sense the compression, tension and/or shear stresses differently. The IVD cells react by producing varying amounts of extracellular matrix and by synthesizing different concentrations of proteoglycans and collagens (= the two major molecules in the IVD). In compliance to their distinct mechanosensitive duties and ensuing metabolic responses, the cells in the nucleus pulposus also present different morphologic appearances and different from those in the endplates and in the annulus fibrosus.

6. Intervertebral disc matures when it begins to sense mechanical responsibilities

Within the first decade of life, the human spinal column develops into an upright and weight-bearing structure. From now on, the lumbar IVDs become subjected to mechanical forces which are very complex at all times.

The immature notochordal cells in the NP cannot cope at all with mechanical stress. These cells synthesize far too little collagens to express some mechanical strength. Therefore, the notochordal cells are totally unable to withstand the ever increasing and varying loads imposed on them. As a result, the number of notochordal cells starts decreasing by necrosis or apoptosis (= programmed cell death).

Some of these notochordal cells will revolt and yet acquire responsiveness to these mechanical stress. They start transforming into chondrocytic-like cells around the age of 8 years (Fig. 6a) and as the IVD matures chondrocytic-like cells become the dominant cells. When deformed by compressive strain, these cells respond by synthesizing increasing amounts of collagen type II. These molecules are essential in resisting and transmitting the deforming compression forces to the annulus fibrosus.

Fig. 6a. Schematic illustration of the different morphologic IVD cell appearances. Left: large vacuolated notochordal cell in the immature nucleus pulposus.

Middle: round chondrocytic-like cell in the more mature nucleus pulposus and the endplates.

Right: elongated appearance of a fibrochondrocytic-like cell in outer annulus. The cell is longitudinally localised to two collagen fibers.

The aging processes in the IVD begin in the early teenage years. The amount of notochordal cells decreases and the proteoglycan concentration falls. The NP starts dehydrating markedly and discoloration follows. The center of the disc loses its translucent and gelatinous appearance and becomes white in the child and young adults. The more collagen is synthesized, the more the NP changes to a yellow-brown color (Fig. 6b).

 

Fig. 6b. L4-L5 intervertebral discs during maturation and aging. On the left (X89/1450) the white appearance of the nucleus pulposus in a young adult (M - 22  yrs). On the right (X90/1420) the yellow-brown color in an adult man (42 yrs). The discoloration is due to the decreasing synthesis of water-attracting and water-binding proteoglycans. (Declerck / Kakulas, Neuropathology, Perth, Western Australia)

7. Decreasing number of notochordal cells initiates aging processes

The more the IVD matures, the less notochordal cells remain present and the more collagen type II is laid down by the chondrocytic-like cells. The older and further dehydrating NP becomes, the firmer it becomes. Although the aging processes result in a more fibrotic NP, the IVD may continue to function quite well. But in the end, the chondrocytic-like cells gradually start being replaced by fibrochondrocytic-like cells. These cells synthesize type I collagen molecules which are essential in resisting mechanical tension forces as well. The disc tissue of the NP transforms into a fibrocollagenous tissue where the distinction between NP and annulus fibrosus dissolves (Fig. 7).

From the age of 40/50 years onwards, the aging processes may become accompanied by more disruptive degenerative processes.

 

Fig. 7. Evolving fibrocollagenous structure in the L5-S1 intervertebral disc in a 60 yr old man (A90/102).

Note: the subchondral osseous changes in the anterior parts of the L5 vertebral body and sacrum related to disruptive lesions and resorption of the endplates.

(Declerck / Kakulas, Neuropathology, Perth, Western Australia)

8. Endplates contain chondrocytic-like cells

The tissues of the mature endplates are populated by chondrocytic-like cells. They have a small and round appearance (Fig. 6a).

Interestingly, stem cells derived from these cartilage endplate (CESCs) are similar to mesenchymal stem cells derived from bone marrow (BM-MSCs). Because CESCs already can be induced into chondrocytes (Liu 2011), innovative biological disc tissue ‘restoring’ techniques are presently researched using bone marrow tissue.

9. Annulus fibrosus: fibrochondrocytic-like cells

From the embryonic start till the fully developed degenerative state of the intervertebral disc, fibrochondrocytic-like cells are the only cells in the outer third of the AF. They nearly exclusively synthetize collagen type-I fibers. These fibers explain the main mechanical function of the annular cells. The AF has to resist tensile deformation during the human development to the upright bipedal position but subsequently as well in response to the daily tensile transmission of the compressive forces from the nucleus pulposus. The fibrochondrocytic-like cells are unable to respond directly to hydrostatic compressive pressures and do not synthesize proteoglycans.

The concentration of the fibrochondrocytic-like cells in the annulus always remains higher than the similar cell population in the later stages of the aging and degenerating nucleus pulposus.

Because they are aligned along the well oriented type-I collagen fibrils of the lamellae, the annular fibrochondrocytes have an elongated ellipsoid morphology (Fig. 6a).

10. Notochordal cells are capable of repairing intervertebral disc tissue

Although their number steadily decreases as the IVD matures, a very small amount of notochordal cells always will remain present in the NP throughout life. They become largely vacuolated and develop prominent intracellular glycogen deposits (= energy reserves) (Fig. 6a). These remaining notochordal cells lose their ability to divide and to form new cells but are the only cells having the means to synthesize proteoglycans.

As long as the NP remains rich in proteoglycans, the central part of the IVD imbibes water and assists in resisting compressive forces. The notochordal cells are the only cells having the continuous capability to protect the NP against aging processes and to repair the disruptive degenerative lesions. The survival of these cells is an essential element in the potential development of innovative treatments of the aging and painful degenerating disc (= 80 % of all chronic low back pain syndromes worldwide).

11. Mechanotransduction: cells receive physical information from the environment to function

A well-defined spatial organisation of IVD cells is required for its three structures to function day-to-day. Various receptors on the IVD cell membranes receive normal signals or perturbed information. The cell membranes serve as signaling interfaces  that allow cells to exchange information with their environment. All individual IVD cells are continuously sensitized by pressure, vibration, temperature, nutritional factors, fluctuations in biochemical processes, growth factors, genetics, etc. Cells have chemical strategies for coping with chemical and environmental stresses and can rapidly adapt their metabolic state. All extracellular triggers finally will decide about the fate of the IVD cells.

All signal-sending and all signal-receiving cells make direct cell-to-cell contacts through membrane extensions, named cytonemes. In this way they communicate directly with each other. As such, all daily mechanical forces on the intervertebral discs are transmitted to the chondrocytic-like cells and the physical signals orchestrate the behaviour of the IVD cells. Neurons liaise over long distances through axons and dendrites. Similar to the osteocytes in bone tissue, the nuclear and annular IVD cells remain interconnected by specialized anchoring fibrils, called tight junctions.

12. Mechanobiology: biologic response of disc cells and their matrices to mechanical stimuli

As long as the notochordal cells produce sufficient proteoglycans, the central portion of the IVD remains highly hydrated and will act hydrostatically. As water is incompressible, the healthy NP will transform the compressive pressures evenly to the peripheral AF. When the cells in the NP no longer are able to respond to the mechanical stresses, they no longer produce the essential substances for an adequate functional extracellular matrix. The collagen/proteoglycan rate increases. The hydrostatic properties of the cells and their matrices decrease. Degenerative structural disruptions may become evident.

13. What happens to cells in aging and degenerating intervertebral disc?

As in all locations of the human body, an increasing number of aging cells in the IVD will die by necrosis. Cell death is a feature of the normal life circle.

Membranes of other IVD cells capture abnormal mechanical signals through special cell-membrane channels and receptors (death receptor 5) and may trigger otherwise silent but carefully programmed intracellular - apoptotic - suicidal mechanisms (by activation of self-destructing interleukin-1 converting proteases). For example, when cells are subjected to too much stress, they curl up their toes and die (Lu). Apoptosis is responsible for the regulation of the number of functional cells in a tissue and their quality.

Remaining aging IVD cells become senescent, lose their ability to divide and are unable to replace other cells lost by necrosis and apoptosis. To increase their viability they form cell clusters.

The cell membranes channels (the so called pannexin 1 of PANX1 channels) of necrotic and apoptotic IVD cells produce molecular ‘find-me’ and ‘eat-me’ signals (phosphatidylserine - PtdSer – molecules) which transform some of the still functioning IVD cells into phagocytes or release signals (adenosine triphosphate ATP) to attract phagocytes to digest the disintegrating cells.

Senescent cells are armed with a self-elimination program that proceeds by attracting T cells, lymphocytes, macrophages and natural killer cells. This occurs because the senescent cells continuously produce and secrete proteases and proinflammatory cytokines and chemokines (IL-1, IL-6, IL-8, macrophage inflammatory proteins, TGF-beta) inducing local inflammation. Some senescent cells alter their gene expression and produce increased amounts of degradative matrix metalloproteinase enzymes (MMPs) which increase catabolic and decrease anabolic metabolism. Together with matrix metalloproteinases, the proinflammatory components perturb  and alter tissue structural organisation and function. As such, senescent cells help repairing degenerative disc tissues by stimulating the production of tissue fibrosis (IL-6 and IL-8) (Adams; Chekeni; Coppé; Elliott; Freund; Gregory; Poon; Van Duersen).

The overall disappearance of cells, the progressive loss of proteoglycan matrix (with dehydration at the same time), the production of an afunctional matrix (more collagen), and the increased amount of MMPs gradually create a weaker biomechanical intervertebral construction no longer able to resist compression, tension and shear forces. The nucleus pulposus cannot longer distribute mechanical stress evenly, which lead to internal nuclear disruptions and may initiate the occurrence of degenerative disruptions in the endplates and annulus fibrosus.

All these phenomena are at the origin of signs and symptoms of the degenerative discogenic syndrome responsible for approximately 80 % of all chronic low back pain.

14. Cells in ‘herniating’ intervertebral discs

Bulging, protruding, and extruding herniating IVDs are extremely common. The herniating pathways mostly are incidental radiological or postmortem findings and may or may not cause symptoms.

Nucleus pulposus fragments of herniating IVDs contain large amounts of necrotic, apoptotic, and senescent cells. Because of their herniating pathway, these cells experience higher than normal levels of blood supply, higher than normal levels of oxygen but respond adversely to the loading forces.

The changing environmental factors are responsible for the apoptotic cells synthesizing increased amounts of intracellular suicidal proteases, for the senescent cells producing more degradative matrix metalloproteinase enzymes (MMPs), and for making more ‘eat-me’ PtdSer signals. The combination of these mechanisms stimulates the phagocytes, which are made available as well by the local blood supply in the central spinal canal, to gradually resorb these herniating fragments. It needs approximately 3 months for digesting the fragments in about 80 % to 90 % of ‘herniating’ cases. This physiologic process clearly is evidenced by the radiological disappearance of extruded or sequestrated fragments on successive evaluations by MRI. This explains why the different expressions of the herniating pathway (protrusion, extrusion and sequestration) are not at all primary pathologies but secondary consequences of underlying aging and degenerative processes in the IVD.

15. Therapeutic implications

It is evident that further improvement in understanding the cellular (and molecular) mechanisms in regulating the functions of the intervertebral disc cells will result in novel therapeutic approaches to manage the signs and symptoms of the degenerative discogenic syndrome (DDS). However, in the author’s opinion, these approaches will not succeed if the abnormal daily loading patterns are not simultaneously stabilised or neutralised.

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