Animal models of regenerative medicine for biological treatment approaches of degenerative disc diseases

Degenerative disc disease (DDD) is a painful, chronic and progressive disease, which is characterized by inflammation, structural and biological deterioration of the intervertebral disc (IVD) tissues. DDD is specified as cell-, age-, and genetic-dependent degenerative process that can be accelerated by environmental factors. It is one of the major causes of chronic back pain and disability affecting millions of people globally. Current treatment options, such as physical rehabilitation, pain management, and surgical intervention, can provide only temporary pain relief. Different animal models have been used to study the process of IVD degeneration and develop therapeutic options that may restore the structure and function of degenerative discs. Several research works have depicted considerable progress in understanding the biological basis of disc degeneration and the therapeutic potentials of cell transplantation, gene therapy, applications of supporting biomaterials and bioactive factors, or a combination thereof. Since animal models play increasingly significant roles in treatment approaches of DDD, we conducted an electronic database search on Medline through June 2020 to identify, compare, and discuss publications regarding biological therapeutic approaches of DDD that based on intradiscal treatment strategies. We provide an up-to-date overview of biological treatment strategies in animal models including mouse, rat, rabbit, porcine, bovine, ovine, caprine, canine, and primate models. Although no animal model could profoundly reproduce the clinical conditions in humans; animal models have played important roles in specifying our knowledge about the pathophysiology of DDD. They are crucial for developing new therapy approaches for clinical applications.     

Intervertebral disc biology, degeneration and novel tissue engineering and regenerative medicine therapies

Degeneration of the intervertebral disc (IVD) is a major cause of low back pain affecting a large percentage of the population at some point in their lives. Consequently IVD degeneration and its associated low back pain has a huge socio-economic impact and places a burden on health services world-wide. Current treatments remove the symptoms without treating the underlying problem and can result in reoccurrence in the same or adjacent discs. Tissue engineering offers hope that new therapies can be developed which can regenerate the IVD. Combined with this, development of novel biomaterials and an increased understanding of mesenchymal stem cell and IVD cell biology mean that tissue engineering of the IVD may soon become a reality. However for any regenerative medicine approach to be successful there must first be an understanding of the biology of the tissue and the pathophysiology of the disease process. This review covers these key areas and gives an overview of the recent developments in the fields of biomaterials, cell biology and tissue engineering of the IVD.     

Cellular mechanobiology of the intervertebral disc: New directions and approaches

The more we learn about the intervertebral disc (IVD), the more we come to appreciate the intricacies involved in transmission of forces through the ECM to the cell, and in the biological determinants of its response to mechanical stress. This review highlights recent developments in our knowledge of IVD physiology and examines their impact on cellular mechanobiology. Discussion centers around the continually evolving cellular and microstructural anatomy of the nucleus pulposus (NP) and the annulus fibrosus (AF) in response to complex stresses generated in support of axial load and spinal motion. Particular attention has been given to cells from the immature NP and the interlamellar AF, and assessment of their potential mechanobiologic contributions to the health and function of the IVD. In addition, several innovative approaches that have been brought to bear on studying the interplay between disc cells and their micromechanical environment are discussed. Techniques for “engineering” cellular function and technologies for fabricating more structurally defined biomaterial scaffolds have recently been employed in disc research. Such tools can be used to elucidate the biological and physical mechanisms by which different IVD cell populations are regulated by mechanical stress, and contribute to advancement of preventative and therapeutic measures.     

The influence of serum,glucose and oxygen on intervertebral disc cell growth <Emphasis Type="Italic">in vitro</Emphasis>: implications for degenerative disc disease

Introduction  

The avascular nature of the human intervertebral disc (IVD) is thought to play a major role in disc pathophysiology by limiting nutrient supply to resident IVD cells. In the human IVD, the central IVD cells at maturity are normally chondrocytic in phenotype. However, abnormal cell phenotypes have been associated with degenerative disc diseases, including cell proliferation and cluster formation, cell death, stellate morphologies, and cell senescence. Therefore, we have examined the relative influence of possible blood-borne factors on the growth characteristics of IVD cells in vitro.     

Cartilage derived morphogenetic protein 2 – A potential therapy for intervertebral disc regeneration?

Low back pain is amongst the top ten risk factors that contribute to disability, ranking higher than diabetes and mental health disease globally as a contributor to years lost to disability (YLD), and escalating as Western societies age. Abundant evidence suggests that intervertebral disc (IVD) damage is central to the origin of pain in the spine. IVD degeneration involves the progressive deterioration of the highly organized disc tissue extracellular matrix, losing its elasticity and hence its' cushioning ability for the spine.Cartilage derived morphogenetic protein-2 (CDMP2) is a small peptide morphogen. Naturally occurring mutations segregate with skeletal defects in IVD development. CDMP2 signalling influences chondrogenic tissue determination, retards osteogenic tissue development and is crucial to early dorso–ventral axis defining events in zebrafish and Xenopus laevis.The potential of biological treatments to offer cutting edge early intervention, tissue regeneration and to preserve spinal motion segments shows great promise. The unique qualities of CDMP2 in IVD tissue formation, delineating discal matrix from vertebral bone, may prove adaptable in therapeutic applications to early discal degeneration.Here we explore the prevalence and origin of backache, the biology of CDMP2 and its potential application as an early intervention to arrest the disc degeneration sequelae.     

Modeling the role of IGF-1 on extracellular matrix biosynthesis and cellularity in intervertebral disc

The insulin-like growth factor-1 (IGF-1) is a well-known anabolic agent for intervertebral disc (IVD), promoting both proteoglycan (PG) biosynthesis and cell proliferation. Accordingly, it is believed that IGF-1 may play a central role in IVD homeostasis. Furthermore, the exogenous administration of IGF-1 has been proposed as a possible therapeutic strategy for disc degeneration. The objectives of this study were to develop a new computational framework for describing the mechanisms regulating IGF-mediated homeostasis in IVD, and to apply this numerical tool for investigating the effectiveness of exogenous administration of IGF-1 for curing disc degeneration. A diffusive–reactive model was developed for describing competitive binding of IGF-1 to its binding proteins and cell surface receptors, with the latter reaction initiating the intracellular signaling mechanism leading to PG production and cell proliferation. Because PG production increases cell metabolic rate, and cell proliferation increases nutritional demand, nutrients transport and metabolism were also included into the model, and co-regulated, together with IGF-1, IVD cellularity. The sustainability and the effectiveness of IGF-mediated anabolism were investigated for conditions of pathologically insufficient nutrient supply, and for the case of exogenous administration of IGF-1 to degenerated IVD. Results showed that pathological nutrients deprivation, by decreasing cellularity, caused a reduction of PG biosynthesis. Also, exogenous administration of IGF-1 was only beneficial in well-nourished regions of IVD, and exacerbated cell mortality in malnourished regions. These findings remark the central role of nutrition in IVD health, and suggest that adequate nutritional supply is paramount for achieving a successful IGF-based therapy for disc degeneration.     

Programmed cell death in intervertebral disc degeneration

Intervertebral disc (IVD) degeneration is largely a process of destruction and failure of the extracellular matrix (ECM), and symptomatic IVD degeneration is thought to be one of the leading causes of morbidity or life quality deterioration in the elderly. To date, however, the mechanism of IVD degeneration is still not fully understood. Cellular loss from cell death in the process of IVD degeneration has long been confirmed and considered to contribute to ECM degradation, but the causes and the manners of IVD cell death remain unclear. Programmed cell death (PCD) is executed by an active cellular process and is extensively involved in many physiological and pathological processes, including embryonic development and human degenerative diseases. Thus, the relationship between PCD and IVD degeneration has become a new research focus of interest in recent years. By reviewing the available literature concentrated on PCD in IVD and discussing the methodology of detecting PCD in IVD cells, its inducing factors, the relationship of cell death to ECM degradation, and the potential therapy for IVD degeneration by modulation of PCD, we conclude that IVD cells undergo PCD via different signal transduction pathways in response to different stimuli, that PCD may play a role in the process of IVD degeneration, and that modulation of PCD might be a potential therapeutic strategy for IVD degeneration.     

Mesenchymal stem cell tracking in the intervertebral disc

Low back pain is a common clinical problem, which leads to significant social, economic and public health costs. Intervertebral disc (IVD) degeneration is accepted as a common cause of low back pain. Initially, this is characterized by a loss of proteoglycans from the nucleus pulposus resulting in loss of tissue hydration and hydrostatic pressure. Conservative management, including analgesia and physiotherapy often fails and surgical treatment, such as spinal fusion, is required. Stem cells offer an exciting possible regenerative approach to IVD disease. Preclinical research has demonstrated promising biochemical, histological and radiological results in restoring degenerate IVDs. Cell tracking provides an opportunity to develop an in-depth understanding of stem cell survival, differentiation and migration, enabling optimization of stem cell treatment. Magnetic Resonance Imaging (MRI) is a non-invasive, non-ionizing imaging modality with high spatial resolution, ideally suited for stem cell tracking. Furthermore, novel MRI sequences have the potential to quantitatively assess IVD disease, providing an improved method to review response to biological treatment. Superparamagnetic iron oxide nanoparticles have been extensively researched for the purpose of cell tracking. These particles are biocompatible, non-toxic and act as excellent MRI contrast agents. This review will explore recent advances and issues in stem cell tracking and molecular imaging in relation to the IVD.     

Expression of cartilage-derived morphogenetic protein in human intervertebral discs and its effect on matrix synthesis in degenerate human nucleus pulposus cells

Introduction  

Loss of intervertebral disc (IVD) matrix and ultimately disc height as a result of 'degeneration' has been implicated as a major cause of low back pain (LBP). The use of anabolic growth factors as therapies to regenerate IVD matrix, hence restoring disc height and thus reversing degenerative disc disease, has been suggested. Cartilage-derived morphogenetic protein (CDMP) is a growth factor which stimulates proteoglycan production in chondrocyte-like cells and thus could be a useful growth factor for LBP therapies. However, little is known about the expression of CDMP or its receptor in human IVD, nor its effects on human disc cells.     

3D finite element analysis of nutrient distributions and cell viability in the intervertebral disc: effects of deformation and degeneration

The intervertebral disc (IVD) receives important nutrients, such as glucose, from surrounding blood vessels. Poor nutritional supply is believed to play a key role in disc degeneration. Several investigators have presented finite element models of the IVD to investigate disc nutrition; however, none has predicted nutrient levels and cell viability in the disc with a realistic 3D geometry and tissue properties coupled to mechanical deformation. Understanding how degeneration and loading affect nutrition and cell viability is necessary for elucidating the mechanisms of disc degeneration and low back pain. The objective of this study was to analyze the effects of disc degeneration and static deformation on glucose distributions and cell viability in the IVD using finite element analysis. A realistic 3D finite element model of the IVD was developed based on mechano-electrochemical mixture theory. In the model, the cellular metabolic activities and viability were related to nutrient concentrations, and transport properties of nutrients were dependent on tissue deformation. The effects of disc degeneration and mechanical compression on glucose concentrations and cell density distributions in the IVD were investigated. To examine effects of disc degeneration, tissue properties were altered to reflect those of degenerated tissue, including reduced water content, fixed charge density, height, and endplate permeability. Two mechanical loading conditions were also investigated: a reference (undeformed) case and a 10% static deformation case. In general, nutrient levels decreased moving away from the nutritional supply at the disc periphery. Minimum glucose levels were at the interface between the nucleus and annulus regions of the disc. Deformation caused a 6.2% decrease in the minimum glucose concentration in the normal IVD, while degeneration resulted in an 80% decrease. Although cell density was not affected in the undeformed normal disc, there was a decrease in cell viability in the degenerated case, in which averaged cell density fell 11% compared with the normal case. This effect was further exacerbated by deformation of the degenerated IVD. Both deformation and disc degeneration altered the glucose distribution in the IVD. For the degenerated case, glucose levels fell below levels necessary for maintaining cell viability, and cell density decreased. This study provides important insight into nutrition-related mechanisms of disc degeneration. Moreover, our model may serve as a powerful tool in the development of new treatments for low back pain.     

Disc cell senescence in intervertebral disc degeneration: Causes and molecular pathways

The accumulation of senescent disc cells in degenerative intervertebral disc (IVD) suggests the detrimental roles of cell senescence in the pathogenesis of intervertebral disc degeneration (IDD). Disc cell senescence decreased the number of functional cells in IVD. Moreover, the senescent disc cells were supposed to accelerate the process of IDD via their aberrant paracrine effects by which senescent cells cause the senescence of neighboring cells and enhance the matrix catabolism and inflammation in IVD. Thus, anti-senescence has been proposed as a novel therapeutic target for IDD. However, the development of anti-senescence therapy is based on our understanding of the molecular mechanism of disc cell senescence. In this review, we focused on the molecular mechanism of disc cell senescence, including the causes and various molecular pathways. We found that, during the process of IDD, age-related damages together with degenerative external stimuli activated both p53-p21-Rb and p16-Rb pathways to induce disc cell senescence. Meanwhile, disc cell senescence was regulated by multiple signaling pathways, suggesting the complex regulating network of disc cell senescence. To understand the mechanism of disc cell senescence better contributes to developing the anti-senescence-based therapies for IDD.     

An in vitro study investigating the survival and phenotype of mesenchymal stem cells following injection into nucleus pulposus tissue

Introduction  

The decreased disc height characteristic of intervertebral disc (IVD) degeneration has often been linked to low back pain, and thus regeneration strategies aimed at restoring the disc extracellular matrix and ultimately disc height have been proposed as potential treatments for IVD degeneration. One such therapy under investigation by a number of groups worldwide is the use of autologous mesenchymal stem cells (MSCs) to aid in the regeneration of the IVD extracellular matrix. To date, however, the optimum method of application of these cells for regeneration strategies for the IVD is unclear, and few studies have investigated the direct injection of MSCs alone into IVD tissues. In the present article, we investigated the survival and phenotype of human MSCs, sourced from aged individuals, following injection into nucleus pulposus (NP) tissue explant cultures.     

Computation models simulating notochordal cell extinction during early ageing of an intervertebral disc

Lower back pain due to intervertebral disc (IVD) degeneration is a prevalent problem which drastically affects the quality of life of millions of sufferers. Healthy IVDs begin with high populations of notochordal cells in the nucleus pulposus, while by the second stage of degeneration, these cells will be replaced by chondrocyte-like cells. Because the IVD is avascular, these cells rely on passive diffusion of nutrients to survive. It is thought that this transition in cell phenotype causes the shift of the IVD's physical properties, which impede the flow of nutrients. Our computational model of the IVD illustrates its ability to simulate the evolving chemical and mechanical environments occurring during the early ageing process. We demonstrate that, due to the insufficient nutrient supply and accompanying changes in physical properties of the IVD, there was a resultant exponential decay in the number of notochordal cells over time.     

Computation models simulating notochordal cell extinction during early ageing of an intervertebral disc

Lower back pain due to intervertebral disc (IVD) degeneration is a prevalent problem which drastically affects the quality of life of millions of sufferers. Healthy IVDs begin with high populations of notochordal cells in the nucleus pulposus, while by the second stage of degeneration, these cells will be replaced by chondrocyte-like cells. Because the IVD is avascular, these cells rely on passive diffusion of nutrients to survive. It is thought that this transition in cell phenotype causes the shift of the IVD's physical properties, which impede the flow of nutrients. Our computational model of the IVD illustrates its ability to simulate the evolving chemical and mechanical environments occurring during the early ageing process. We demonstrate that, due to the insufficient nutrient supply and accompanying changes in physical properties of the IVD, there was a resultant exponential decay in the number of notochordal cells over time.     

Cell death in intervertebral disc degeneration

Degeneration of intervertebral disc (IVD) is mainly a chronic process of excessive destruction of the extracellular matrix (ECM), and also is thought to be the primary cause of low back pain. Presently, however, the underlying mechanism of IVD degeneration is still not elucidated. Cellular loss from cell death has been believed to contribute to the degradation of ECM and plays an important role in the process of IVD degeneration, but the mechanisms of cell death in degenerated IVD remain unclear. Apoptosis, a very important type of IVD cell death, has been considered to play a crucial role in the process of degeneration. Autophagy, a non-apoptosis death type of programmed cell death, has been considered extensively involved in many pathological and physiological processes, including the degenerative diseases. Thus, the research on cell death in IVD degeneration has become a new focus recently. In this review, by analyzing the available literature pertaining to cell death in IVD and discussing the inducing factors of IVD degeneration, NP cells and ECM in IVD degeneration, apoptotic signal transduction pathways involved in IVD cell death, the relationship of cell death with IVD degeneration and potential therapeutic strategy for IVD degeneration by regulating cell death, we conclude that different stimuli induce cell death in IVD via various signal transduction pathways, and that cell death may play a key role in the degenerative process of IVD. Regulation of cell death could be a potential and attractive therapeutic strategy for IVD degeneration.     

Effect of Cartilage Endplate on Cell Based Disc Regeneration: A Finite Element Analysis

This study examines the effects of cartilage endplate (CEP) calcification and the injection of intervertebral disc (IVD) cells on the nutrition distributions inside the human IVD under physiological loading conditions using multiphasic finite element modeling. The human disc was modeled as an inhomogeneous mixture consisting of a charged elastic solid, water, ions (Na⁺ and Cl⁻), and nutrient solute(oxygen,glucose and lactate) phases. The effect of the endplate calcification was simulated by a reduction of the tissue porosity (i.e., water volume faction) from 0.60 to 0.48. The effect of cell injection was simulated by increasing the cell density in the nucleus pulposus (NP) region by 50%, 100%, and 150%. Strain-dependent transport properties(e.g., hydraulic permeability and solute diffusivities) were considered to couple the solute transport and the mechanical loading. The simulation results showed that nutrient solute distribution inside the discis maintained at a stable state during the day and night. The physiological diurnal cyclic loading does not change the nutrient environment in the human IVD. The cartilage endplate plays a significant role in the nutrient supply to human IVD. Calcification of the cartilage endplate significantly reduces the nutrient levels in human IVD. Therefore, in cell based therapy for IVD regeneration, theincreased nutrient demand as a result of cell injection needs to be addressed. Excessive numbers of injected cells may cause further deterioration of the nutrient environment in the degenerated disc. This study is important for understanding the pathology of IVD degeneration and providing new insights into cell based therapies for low back pain.     

椎间盘微环境对干细胞疗法修复椎间盘的影响的研究概况

椎间盘(IVD)退变是一种常见的病理状态,保守治疗往往失败,IVD变性的患者最后往往需要手术干预。已经提出的几种治疗方案中,只有椎间盘切除术和关节融合术被证明可以达到预料的效果。生物治疗的目的是预防和控制椎间盘变性,改善椎间盘的功能、髓核和纤维环细胞的合成代谢和修复能力,并抑制基质降解。目前,临床应用仍处于起步阶段。间充质干细胞和基因治疗在预防和治疗IVD变性的作用还需要进一步的研究。最近的研究向我们展示了一种新的保护椎间盘的结构和功能的治疗策略:间充质干细胞(MSCs)移植,尤其是骨髓间充质干细胞(BM-MSCs)。而了解MSCs是否可以以及如何在有排拆性的退化的椎间盘中存活并繁殖是十分重要的。因此,本文着重讨论内源性蛋白酶、细胞因子、低氧、低营养、机械负荷及渗透压的调节对移植的MSCs的影响。     

Isolation and Characterisation of a Recombinant Antibody Fragment That Binds NCAM1-Expressing Intervertebral Disc Cells

Degeneration of the intervertebral discs (IVD) is a leading cause of neck and low back pain. Degeneration begins in the central nucleus pulposus region, leading to loss of IVD osmotic properties. Regeneration approaches include administration of matrix-mimicking scaffolds, cells and/or therapeutic factors. Cell-targeting strategies are likely to improve delivery due to the low cell numbers in the IVD. Single-chain antibody fragments (scFvs) that bind IVD cells were isolated for potential delivery of therapeutics to degenerated IVD. The most cell-distal domain of neural cell adhesion molecule 1 (NCAM1) was cloned and expressed in Escherichia coli. Phage display technology was used to isolate a human scFv against the recombinant domain by panning a scFv library on the immobilised protein. The isolated scFv bound cultured rat astrocytes, as well as bovine nucleus pulposus and annulus fibrosus cells in immunocytochemical studies. The scFv also labelled cells in bovine spinal cord and six-month and two-year old bovine IVD sections by immunohistochemistry. Antibody fragments can provide cell-binding moieties at improved cost, time, yield and functionalisation potential over whole antibodies. The described scFv has potential application in delivery of therapeutics to NCAM1-expressing cells in degenerated IVD.     

Expression of semaphorin 3A and its receptors in the human intervertebral disc: potential role in regulating neural ingrowth in the degenerate intervertebral disc

Introduction  

Intervertebral disc (IVD) degeneration is considered a major underlying factor in the pathogenesis of chronic low back pain. Although the healthy IVD is both avascular and aneural, during degeneration there is ingrowth of nociceptive nerve fibres and blood vessels into proximal regions of the IVD, which may contribute to the pain. The mechanisms underlying neural ingrowth are, however, not fully understood. Semaphorin 3A (sema3A) is an axonal guidance molecule with the ability to repel nerves seeking their synaptic target. This study aimed to identify whether members of the Class 3 semaphorins were expressed by chondrocyte-like cells of the IVD addressing the hypothesis that they may play a role in repelling axons surrounding the healthy disc, thus maintaining its aneural condition.     

Effects of Tobacco Smoking on the Degeneration of the Intervertebral Disc: A Finite Element Study

Tobacco smoking is associated with numerous pathological conditions. Compelling experimental evidence associates smoking to the degeneration of the intervertebral disc (IVD). In particular, it has been shown that nicotine down-regulates both the proliferation rate and glycosaminoglycan (GAG) biosynthesis of disc cells. Moreover, tobacco smoking causes the constriction of the vascular network surrounding the IVD, thus reducing the exchange of nutrients and anabolic agents from the blood vessels to the disc. It has been hypothesized that both nicotine presence in the IVD and the reduced solute exchange are responsible for the degeneration of the disc due to tobacco smoking, but their effects on tissue homeostasis have never been quantified. In this study, a previously presented computational model describing the homeostasis of the IVD was deployed to investigate the effects of impaired solute supply and nicotine-mediated down-regulation of cell proliferation and biosynthetic activity on the health of the disc. We found that the nicotine-mediated down-regulation of cell anabolism mostly affected the GAG concentration at the cartilage endplate, reducing it up to 65% of the value attained in normal physiological conditions. In contrast, the reduction of solutes exchange between blood vessels and disc tissue mostly affected the nucleus pulposus, whose cell density and GAG levels were reduced up to 50% of their normal physiological levels. The effectiveness of quitting smoking on the regeneration of a degenerated IVD was also investigated, and showed to have limited benefit on the health of the disc. A cell-based therapy in conjunction with smoke cessation provided significant improvements in disc health, suggesting that, besides quitting smoking, additional treatments should be implemented in the attempt to recover the health of an IVD degenerated by tobacco smoking.