Critical aspects and challenges for intervertebral disc repair and regeneration—Harnessing advances in tissue engineering

Low back pain represents the highest burden of musculoskeletal diseases worldwide and intervertebral disc degeneration is frequently associated with this painful condition. Even though it remains challenging to clearly recognize generators of discogenic pain, tissue regeneration has been accepted as an effective treatment option with significant potential. Tissue engineering and regenerative medicine offer a plethora of exploratory pathways for functional repair or prevention of tissue breakdown. However, the intervertebral disc has extraordinary biological and mechanical demands that must be met to assure sustained success. This concise perspective review highlights the role of the disc microenvironment, mechanical and clinical design considerations, function vs mimicry in biomaterial‐based and cell engineering strategies, and potential constraints for clinical translation of regenerative therapies for the intervertebral disc.

patients with lumbar (33% increase) and cervical (42% increase) spinal disorders in the Medicare database; however, there were also decreases in both lumbar and cervical surgical and nonoperative treatments. 11 Discordance between diagnosis and treatment trends in the elderly points to a strong need to develop and optimize treatments for spinal care, particularly for the elderly. The burden of back pain affects both young and old patients, highlighting a demand for novel treatment strategies that reduce pain and improve quality of life for all back pain sufferers.
Tissue engineering and regenerative medicine strategies have the potential to address axial back pain and herniation. The complexities of diagnosis and patient selection in back and neck pain conditions highlight a strong need to develop safe and minimally invasive treatments that can repair IVDs and/or prevent painful conditions. In the case of axial back pain, the challenges of identifying a specific source of pain highlights a need for a safe and injectable treatment. As injectable treatments are developed, however, these strategies must also minimize the annulus fibrosus (AF) damage and comorbidities known to occur from IVD puncture, injection, and discography. 7,12 Current strategies for axial back pain are conservative treatment, physical therapy and oral analgesics which have limited efficacy for many patients. IVD herniation is a specific cause of back and leg pain and disability where discectomy procedures have improved outcomes compared to nonoperative controls. 5 Discectomy is an effective treatment for IVD herniation, yet even in the case of successful herniation procedures, long-term complications can include reherniation and recurrent back pain. 13 Tissue engineering and regenerative medicine treatments offer tremendous potential to repair and regenerate IVD tissues and potentially alter the course from painful to nonpainful conditions for axial back pain and herniation patients. Treating these varied conditions requires development of biomaterials for AF repair, and nucleus pulposus (NP) repair and regeneration. For final repair and regeneration strategies, the IVD tissue has tremendous biological and mechanical demands which must be addressed to achieve successful outcomes. Varying diagnoses also necessitate the development of multiple repair and regeneration strategies that focus on function and mimicry. This narrative review has four objectives, namely to describe: (1) the role of the host/disc microenvironment, (2) mechanical and clinical design constraints, (3) biomaterials and cell engineering for function vs mimicry in the IVD, and (4) challenges of clinical translation for these regenerative repair strategies.

| THE ROLE OF THE DISC MICROENVIRONMENT
Degeneration of the IVD occurs over many years and is influenced to an extent by genetic, environmental and physicochemical effects.
However, for normal cellular function and successful tissue regeneration, the local physicochemical microenvironment that is experienced by implanted cells is critical. The degenerated microenvironment of the human IVD is characterized by altered oxygen, 14 reduced glucose, 15,16 increased matrix acidity 14 and elevated levels of proinflammatory cytokines 17 thus presenting a challenging FIGURE 1 Variations in intervertebral disc (IVD) structure and composition with aging vs degeneration. Picrosirius red/alcian blue (PR/AB) staining of mid-sagittal sections of four different human IVDs. PR/AB highlights the differences between IVD aging and IVD degeneration. Column 1. Aging: Aging IVDs show subtle changes in structure and composition with retention of overall annulus fibrosus (AF) structural integrity.
(A) Forty-four-year-old male IVD retains healthy end plates with only slight irregularities, well-organized annular morphology, nearly normal nuclear tissue with only slight disorganization, and intense matrix staining. (B) Eighty-one-year-old female IVD shows only slight irregularities in the endplate. It maintains a well-organized annulus with only slight loss of annular-nuclear demarcation, and mild loss of nuclear staining intensity. This aged specimen also shows rounded end plates due to osteoporotic changes in underlying trabecular bone. Column 2. Degeneration: Degenerated IVDs show larger changes in structure that disrupt the gross integrity of the AF, the nucleus pulposus, and/or the end plates and changes in composition with loss of staining intensity. (C) Forty-seven-year-old female exhibits multiple irregularities in the endplate including thinning and focal breaks, a loss of boundary demarcation between the nucleus and annulus, and disorganized/fibrotic nuclear matrix and little AB staining. The IVD also displays horizontal fissures that extend into the annulus and disrupt its structure. (D) Eighty-five-year-old male IVD shows severe irregularities in the endplate, disorganization of the nucleus and complete rupture of the annulus. The faint staining shows nearly complete loss of matrix material, leading to collapse of the disc, bulging of the annulus, and areas of bone to bone contact. In this extreme case, there is complete loss of structural integrity of the IVD microenvironment for normal cell function. As the IVD is avascular, the surrounding blood vessels in the cartilage end plates (CEPs) and vertebral bodies supply vital nutrients to the disc primarily through diffusion. 16 Balance between nutrient transport and cellular consumption rates establishes a concentration gradient throughout the disc of these nutrients and metabolites which in turn markedly affect viability, proliferation and function of cells, and collectively will undoubtedly impact the degree of any subsequent regeneration.
Oxygen levels have been shown to vary considerably in human lumbar and thoracic discs, and do not appear to correlate with age, pathology or stage of degeneration. Concentrations decrease from the AF across the disc structure (19.5%-0.65%) with average physioxic concentrations in the central region of the NP of between 5% and 10%. 14 These gradient concentration profiles are dependent on the rate of oxygen transport through the CEP, cellular density and consumption rates. It is well established that cell viability of NP cells is diminished with low glucose but not low oxygen, highlighting the importance of glucose as a limiting nutrient for survival of disc cells.
Computational models have predicted a decrease in glucose concentrations from~5 mM at the disc boundaries to~0.8 mM in the center of healthy discs (uncalcified), 18 which can fall below critical levels with increasing calcification and as a function of static strain conditions. 16 Importantly, cell death has been shown to occur when subjected to glucose concentrations below 0.5 mM for more than 3 days 19 and in scoliotic discs, low cell viability was found to correlate with low glucose concentrations. 20 Another important factor is the pH microenvironment due to local lactic acid concentrations (typical range of 2-6 mM) as a result of glycolysis. 14 In vivo measurements reveal that pH varies from 5.7 to 7.5 (median, 7) 21 and can significantly influence cell survival, adversely affect matrix synthesis rates 19,22 and increase expression of proinflammatory cytokines and pain-related factors. 23 Importantly, energy metabolism rates are nonlinear coupled reactions and dependent on the local nutrient and acidic microenvironment. 24 Equally relevant from a regenerative medicine or tissue engineering perspective, oxygen concentrations appear to play a key role in regulating the phenotype and biosynthetic activity of cells intended for therapeutic applications, 25 while low glucose concentrations and low pH levels have been found to impair the survival and biological behavior of stem cells. [26][27][28] In addition, the IVD is subjected to biophysical forces in vivo, such as deformational strain and hydrostatic pressure. In vivo pressures appear to be task-dependent and vary significantly. Using micropressure transducers, it has been shown that pressures in the IVD (L4-L5 disc) range from 0.1 MPa when lying prone to 0.95 MPa during jogging to as high as 2.3 MPa when lifting a 20 kg object. 29 Furthermore, due to the presence of negatively charged proteoglycans, the IVD is an osmotic system, which has been shown to be a potent regulator of gene expression 30 and matrix synthesis by IVD cells. 31 Due to diurnal changes alone, the osmolarity can range from 450 to 550 mOsm, 32 which can also affect subsequent cellular response to biophysical stimulation. 33 A compounding issue occurs during aging; the CEPs become less permeable due to endplate calcification, which impedes the diffusion and nutrient exchange between the vertebral marrow and the disc itself. 34 Previous work has shown that occlusion of endplate openings correlates significantly with disc degeneration and is strongest for the endplate adjacent to the nuclear region, suggesting that endplate calcification may impair nutrient transport thereby leading to disc degeneration. 35 Marrow contact channel surface has been shown to be highest in the center of vertebral endplates compared to peripheral zones near the AF and strongly correlates with effective permeability measurements. 36 Alterations in mechanical stimuli have also been shown to alter the vascularization and the convective properties of the CEP, 36 38 and impeding nutrient transport.
Another important facet is the inflammatory milieu present in degenerated discs. Whether cytokines play a significant role in the initial pathology or if their production is stimulated by the altered physicochemical microenvironment has not been fully elucidated.
However, it is evident that inflammatory cascades are potentiated in disc disease and that a multitude of cytokines and inflammatory molecules are involved in these processes which influence cell survival, differentiation and function. Among these, interleukin 1 (IL-1) and tumor necrosis factor-alpha (TNF-α) have received the greatest attention. 39 Evidence suggests the release of factors from degenerating discs such as nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), inflammatory and nociceptive factors also play a vital role in the cross-talk responsible for activation and recruitment of immune cells as well as promoting neo-innervation. 9,40 Additionally, low pH can stimulate the production of both NGF and BDNF 23 and has been implicated in pain perception. 41 Therefore, inflammatory cytokines, through their precipitation of neurotrophines, may indeed act as noxious stimulation which sensitizes nerves, and/or initiates in-growth of nerve fibers into the degenerate disc, thereby exacerbating back pain. 23 Minimally invasive delivery of cells into the disc space to regenerate matrix, and/or to positively alter the microenvironment and restore functionality may hold significant promise for disc regenera- For noninvasive characterization, the gold standard to date has been the Pfirrmann disc degeneration grading system based on signal intensity from T2-weighted magnetic resonance imaging (MRI) to estimate water content with morphological parameters. 8 While the Pfirrmann grading scale can classify disc degeneration from Grade I to V, it is primarily based on disc structure rather than the biochemical microenvironment. Enhanced imaging modalities and identification of key biomarkers at an earlier stage are required to deliver improved regenerative outcomes for disc regeneration. Recent studies have emerged utilizing the many facets of MRI to garner information on the biochemical state of the degenerated microenvironment. Quantitative MRI using the relaxation times T1 and T2, the magnetization transfer ratio, and the apparent diffusion coefficient facilitates noninvasive assessment and diagnosis of changes including disc matrix composition (water, proteoglycan, and collagen), integrity (percent collagen denaturation), 42   methods are required to identify and stratify suitable patient cohorts that will benefit from cell-based therapies ( Figure 2). Advances in imaging modalities to quantify the local structural and biochemical microenvironment that is amenable to repair procedures, coupled with biomarkers for tracking repair are obvious targets to address and may provide a suitable strategy and assist in developing personalized and effective treatments for disc degeneration.

| THE IMPORTANCE OF MECHANICAL COMPATIBILITY
As mentioned earlier, IVD herniation is a common cause of back and leg pain, and disability where discectomy procedures have improved outcomes as compared to nonoperative controls. 5 In the case of successful herniation procedures, herniated IVD tissues are removed to reduce the neuropathy condition. However, the IVD remains unrepaired with risk of long-term complications including reherniation and recurrent back pain, 13 whereby reherniation rates after discectomy are reported to be 5% to 25%. 13 Figure 3). Screening tests for optimization are designed to evaluate priority parameters to rapidly assess if the biomaterial will meet required design parameters. Importantly, they are intended to be adaptable for high throughput testing and include adhesion testing, material property determination and cytocompatibility assessments. 51 To date, many of these optimization tests are performed on isolated tissue samples with more free-boundaries than would be found in situ which creates high shear stresses. 54 As a result, the adhesion strength and material property parameters obtained from a screening test are  In situ biomechanical testing for advanced screening can include six degrees of freedom testing to evaluate spine biomechanical properties. These biomechanical properties can characterize the neutral zone as well as the stiffness and hysteresis. In situ biomechanical validation tests can also include acute and fatigue failure simulation Physiological NP matrix contains 70% to 90% water, while its dry weight consists of 20% collagen, mainly type II, and 30% to 50% proteoglycan. 67 For NP regeneration, it is crucial to address the water content since tissue dehydration is a major hallmark of the degenerative cascade. Hydrophilic materials such as hydrogels have widely been investigated to compensate for the water uptake properties of the NP. Injectable hydrogels represent an attractive minimally invasive approach. They can also be combined with cells to establish and maintain tissue homeostasis in cases where endogenous cells are deficient, abnormally functioning or inactive. 68 Natural-origin hydrogels can be processed but do not need to be synthesized, which reduces production costs. Furthermore, they are generally cytocompatible, bioactive, and participate in the physiological turnover process. 69 Of note, the IVD matrix metabolism is extremely slow, so the degradation rate of an ideal material should be compatible with the natural processes.
Despite the extensive literature on the use of natural hydrogels for NP therapy, the range of core materials appears restricted. Most often described hydrogels include hyaluronic acid, collagen type I or II, fibrin, gelatin, alginate, chitosan, and gellan gum. 50,68 Synthetic hydrogels offer a valid alternative, as they can be produced in a standardized and reproducible way and can be tuned towards the desired mechanical and degradation properties. Examples of hydrogels described for NP tissue engineering applications include polyethylene glycol, polyvinyl alcohol, polyvinyl-pyrrolidone, polyurethane, and cellulosic. 50,68,70 Another molecular approach involves the design of injectable sulfonate-containing hydrogels with high fixed charge density and swelling pressure, which may serve as biomimetic glycosaminoglycan analogs. 71 Such materials that swell in situ to form a nondegradable gel as NP replacement aim at restoring disc height, though the cellular response and long-term tissue integration are also essential arguments to be considered. 72 While pure components are typically characterized by a low risk of cytotoxicity, their swelling behavior, durability, and the host tissue reaction need to be carefully considered to prevent subsidence or extreme overload, potentially causing CEP/EP fracture. 73,74 Mimicking the natural NP characteristics is essential for both immediate tissue restoration and cellular activation. The phenotype of the cells embedded in or exposed to a material is highly influenced by FIGURE 5 Nucleus pulposus (NP) mimics may consist of natural or synthetic hydrogels, decellularized matrix, specific adhesion proteins or osmo-responsive molecules; while annulus fibrosus (AF) mimics may be realized with crosslinked hydrogels or fibers arranged in oriented angle-ply laminates. Challenges in reproducing the authentic tissue include: interfaces NP-AF-cartilaginous endplate (CEP)-vertebrae; integration with native structures; degradation and remodeling properties; regulation of osmotic pressure; complexity of natural matrix, glyco-pattern, small molecules; cell adhesion properties; cell phenotype regulation; in vitro-ex vivo-in vivo translation. Potential strategies for implant integration are displayed its physical, chemical, and mechanical quality. Despite the prevalence of type II collagen, type I collagen has more often been used due to the remarkably lower cost and superior availability. A crosslinked formulation of type II collagen showed improved stability compared to the noncrosslinked molecule with still favorable cell differentiation response. 75 Atelocollagen is a low-immunogenic derivative of collagen that has been successfully used in vivo. 76 although their in vivo effect remains to be explored. 83 IVD-specific ECM-mimics that address the cellular response may prove more sustainable. Recently, a panel of laminin mimetic peptides conjugated to polyacrylamide gels were reported to promote an immature healthy NP phenotype after culture on soft peptide gels. 84 The results demonstrated that cell-matrix interactions play a crucial role in gaining and maintaining a regenerative phenotype and activity; thus, mimicking the ECM structure alone may not be sufficient without mimicking its functional cellular microenvironment. An attractive approach consists in the generation of decellularized matrices from healthy NP. Decellularization processes obtain biomaterials that represent the native tissue microstructure and biochemistry, supporting cellular adaptation. 85,86 Further preclinical studies are warranted to compare such matrices with other synthetic or natural biomaterials. Recently reported studies are only at the beginning of identifying the molecular patterns that determine the NP cell phenotype. [87][88][89] The AF is a multilamellar structure composed of 70% collagen, primarily type I, and 10% proteoglycan in dry weight. 67 One of the challenges of AF tissue engineering is the gradual transformation of structure and biochemistry from the outer AF to the inner AF and the NP that cannot easily be reproduced ex vivo. Various biomaterials have been suggested as a basis for AF repair, including collagen, atelocollagen, silk fibroin, poly-lactic-co-glycolic acid, and polycaprolactone (PCL). 7,68 Electrospun fibers generated from PCL are highly anisotropic and closely replicate the AF structural hierarchy; when seeded with mesenchymal stromal cells, these scaffolds promoted the deposition of an organized collagen-rich ECM that approached the angle-ply multilamellar architecture of native AF. 52 Porous silk scaffolds and their derivatives have also shown promising characteristics for AF tissue engineering, supporting AF-like matrix production of seeded cells. 90 Atelocollagen scaffolds seeded with autologous AF cells were effective in treating small AF defects in a rabbit in vivo model. 91 Nevertheless, it is still uncertain whether such hydrogels and fibers maintain the strength and robustness required to integrate with the adjacent AF and bony tissues long term.
Many AF repair materials have been developed as components of whole tissue-engineered IVDs. For example, cell-seeded composites consisting of crosslinked bone matrix gelatin acting as the AF and acellular cartilage matrix as the NP component was shown to promote the development of IVD-like tissue in an ectopic in vivo model. 92 An IVD construct based on contracted collagen AF and alginate NP, implanted in a canine cervical total discectomy model, was maintained over several months, although the long-term functionality of such tissue-engineered whole IVDs remains a challenge. 93 The role of an organized AF structure, compared to a scaffold without any lamellar pattern structure, and its importance for the success of the implant is still not well defined. While an oriented lamellar structure may not be required to meet the goal of adhesion or defect filling, the resistance to deformation and tensile circumferential strains clearly depends on the structural organization of the tissue-engineered implant. 94 Biphasic  cacy has yet to be demonstrated fully. 50 Ideal intraoperative AF and NP repair methods would prevent reherniation, seal the remaining defects, restore biomechanical function, and reduce the likelihood of recurrent pain. 124 Successful translation of a repair method for clinical application must address patient needs and be adaptable to the constraints of the clinical setting in addition to meeting the biological and biomechanical requirements described. The treatments must be easily delivered via injection, or implantable at the time of procedure when discectomy is being performed. Injectable biological therapy appears ideal for meeting this need following discectomy to fill the gap in the NP and repair AF fissures. Discectomy procedures are relatively short, and the material must remain in place following implantation. 51 Consequently, gelation time of an injectable gel is an important parameter to define feasibility of clinical translation. The repair also needs to be able to withstand loading immediately when the patient undergoes the procedure and/or recovers from anesthesia and is subjected to dynamic loading associated with normal activities of daily life.
Clinical patient selection is another important consideration.
Inclusion criteria for recently reported clinical trials for regenerative cell-based therapies involved persistent lumbar discogenic low back pain for more than 6 months, and degenerative changes in the IVD (Pfirrmann grade 3 and more, disc height loss over 50%, positive provocative discography). 125 While chronic low back pain patients can have multiple sources of pain that do not directly come from IVD pathology, it is notable that clinical trials on cell therapy injections into IVDs appear to have potential to improve painful conditions. 126 Furthermore, IVD repair techniques have potential to repair or reverse degenerative changes in IVDs but would not address degenerative changes in facet joints or other spinal deformity conditions that would likely require augmentation with instrumentation. Therefore, clinical patient selection is likely to be varied and to require multiple repair methods to select the most suitable device/technique for IVD repair.
For intraoperative repair methods for lumbar disc herniation patients during discectomy, NP and AF can be repaired with biomaterials as replacement. In this scenario, the purpose and function of the replacement biomaterial will be to prevent or delay the progression of lumbar spine degeneration, to prevent reherniation, and to reduce chronic painful conditions. On the other hand, when the disc repair/regenerative therapy is minimally invasive and to be applied via fluoroscopic injection, for example, the main purpose will be to promote regenerative changes of the IVD with reduced pain, recovery of biomechanical properties and reversal of the degenerative cascade. Injectable therapies have the potential to prevent patients from progression of IVD degeneration in the future and may perhaps be most suitable for patients with early stage disc degeneration. In comparison to more invasive surgery such as intraoperative repair/replacement, the added advantage of injectable delivery is the reduced volume of material; however, there needs to be a benefit over risk of possible progressive degeneration which has been observed after IVD puncture. 7,12 6 | CONCLUSIONS Several biomaterial strategies exist for tissue-engineered IVD repair, replacement, and regeneration. Successful IVD repair remains an unmet clinical need due to the biological, biomechanical and clinical challenges that the repair biomaterial must face. The unique and harsh biological microenvironment in the IVD limits cell matrix production and often requires a biomaterial to help protect and ensure containment of cells in situ, while promoting viability and maintaining the desired phenotype. Once a biomaterial is injected into the IVD, it risks extrusion and reherniation due to significant mechanical loads that persist in normal daily activities, which could exacerbate the clinical condition, and risk further complications. As a result, biomaterials must undergo robust and rigorous biomechanical testing to ensure biomechanical compatibility and reduce the risk of herniation or fatigue failure. Biomaterials in development need to focus on functional mimicry of the native IVD structure. Tissue-engineered implants must be compatible with the clinical environment and specifically selected to address the unique clinical condition of the patient. Tissue engineering and regenerative medicine continues to advance at an astounding rate and it is likely that engineered biomaterials and cells will be capable of overcoming the challenging biological, biomechanical and clinical constraints required for IVD repair to improve patient outcomes.