Vascularization of the human intervertebral disc: A scoping review

Abstract Intervertebral discs (IVDs) are often referred to as the largest avascular structures of the human body, yet a collective resource characterizing the vascularization of the IVD does not exist. To address this gap, the objective of this study was to conduct a comprehensive search of the literature to review and summarize current knowledge of the prevalence and localization of blood supply in human IVDs, with a scoping review. A comprehensive search of peer‐reviewed publications on the topic of IVD vascularization in humans was conducted across six electronic databases: PubMed, EMBASE, MEDLINE, Scopus, Web of Science, and BIOSIS Previews. Studies of humans were included regardless of age, sex, ethnicity, and health status, with the exception of IVD herniation. Two independent reviewers screened titles and abstracts and full‐texts according to eligibility criteria. The review was conducted and reported according to Preferred Reporting Items for Systematic Reviews Extension for Scoping Reviews guidelines. Our search yielded 3122 articles, with 22 articles meeting the inclusion criteria. The study samples ranged in age from fetal to >90 years and included both sexes, various health statuses, and used different methodologies (eg, histology, medical imaging, and gross dissection) to assess vasculature. Overall, consistent observations were that (a) the nucleus pulposus of the IVD is avascular throughout life, (b) both the cartilage endplates and annulus fibrosus receive considerable blood supply early in life that diminishes over the lifespan, and (c) vascular ingrowth into the cartilage endplates and inner layers of the annulus fibrosus is commonly associated with damaged or disrupted tissue, irrespective of age. Histology and immunohistochemistry are often used to report vascularization of the IVD. The body of the current literature suggests that the IVD should not be generalized as an avascular tissue. Instead, vascularization of the IVD differs based on the constituent tissues, their age, and state of degeneration or damage.


| INTRODUCTION
Human intervertebral discs (IVD) are complex fibrocartilaginous joints that are generally referred to as the largest avascular structures in the human body. IVDs are composed of three distinct but interdependent tissues. The nucleus pulposus is the innermost gelatinous tissue composed primarily of proteoglycans and water. 1 The nucleus pulposus has been shown to naturally inhibit endothelial cell migration through the secretion of an anti-angiogenic extracellular matrix composed of proteoglycans (eg, aggrecan) and sulfated glycosaminoglycans (eg, chondroitin sulfate). 2,3 The cartilage endplates are thin layers of hyaline cartilage that anchor the IVD to the adjacent vertebral bones and act as a selectively permeable barrier for diffusion of nutrients into the IVD. 4 The annulus fibrosus is a series of well-organized concentric lamellae of fibrocartilage that encompass the nucleus pulposus of the IVD. 5,6 Although the IVD is often described in general terms in the literature as being avascular, previous studies have reported blood vessels confined to the outer one-third of the annulus fibrosus. 7,8 Moreover, there is controversy regarding the specific localization of blood vessels within the IVD, most often associated with degeneration. These contradictory claims may be associated with divergent criteria and/or specificity used to identify regions of the IVD. Based on the methodologies used, the term "nucleus pulposus" can be used to indicate whatever tissue is found in the center of an IVD, or it can be defined as the histologically distinct tissue (comprising proteoglycans, type II collagen, and chondrocyte-like cells) found near the center of the IVD. The critical difference is that histologically distinct annulus fibrosus fibrocartilage can be found collapsed into the central region of a decompressed or severely degenerated IVD, and this tissue can contain blood vessels. 9 To date, a comprehensive overview of the current literature that characterizes the prevalence and localization of blood supply of the human IVD across the lifespan has not been conducted. Addressing this gap in our understanding of IVD vascularization has relevance to IVD biology, pathobiology, and development of regenerative strategies.
The primary pathway for nutritional supply to IVD cells is through passive diffusion from the richly vascularized vertebral bony endplate through the cartilage endplate. 4 Previous studies have shown oxygen concentration and pH differences of the IVD, with low oxygen and pH levels in the center of the nucleus pulposus. 4 Further, oxygen and glucose concentrations are higher in the annulus fibrosus compared to the nucleus pulposus. 4 Of note, the gradient of oxygen and glucose concentrations across the IVD corresponds to a gradient of cell density, which is higher in the annulus fibrosus and lower in the nucleus pulposus. 4 This gradient leads to differences in cellular demand, which is dictated by the available supply of nutrients. 4 However, the extent of human IVD vascularization throughout life and the source of blood vessels directly supplying the IVD is currently unclear. Overall, an improved understanding of the vascularization of the IVD associated with physiological aging may further inform the mechanisms of nutrient delivery, which is relevant to better understanding age-related changes impacting cell survival, tissue repair, and IVD degeneration ultimately leading to back pain. 10 The association between vascular ingrowth and IVD disease status in humans is poorly understood. Clinical studies are limited to small sample sizes from severely diseased tissue retrieved from surgery and postmortem evaluations. Surgical tissues are often restricted to fragments of resected tissue, most commonly from symptomatic herniated IVDs. Moreover, postmortem tissues are often associated with advanced age and confounded by unknown comorbidities of donors. This has prevented a detailed characterization of the prevalence and spatial localization of blood vessels of the IVD, under nonpathological and pathological conditions.

| Research question
The overarching objective of this scoping review is to summarize the Joanna Briggs Institute. 13,14 Scoping reviews utilize similar methods as systematic reviews to identify and assess the available literature; however, scoping reviews target boarder research questions than systematic reviews. To maintain the quality and integrity of the scoping review, it was also conducted in accordance with the Preferred Reporting Items for Systematic Reviews Extension for Scoping Reviews (PRISMA-ScR). 15

| Eligibility criteria
This scoping review included only peer-reviewed articles that described the vascularization of the IVD in a human population. No restrictions were set based on age, sex, ethnicity, or health status (except for IVD herniation) in order to capture any descriptions of vascular structures within the IVD. Studies focused on IVD herniation were exclude since previous histological analysis of herniated IVD tissues from the lumbar spine has shown the invasion of small blood vessels, 16 which is likely the result of an extradiscal inflammatory response to the extruded material. Moreover, this scoping review considered articles that evaluated vascularization of the IVD through a variety of different methodologies including histology, gross dissection, and medical imaging. Importantly, the outcomes of these articles had to address the presence of vasculature in the IVD and describe its localization within the tissue. We integrated consultation with experts in the fields of vascularization and microanatomy throughout this scoping review to ensure the appropriateness of search terms and applicable methodologies as well as the accuracy of the scoping review results and interpretation of the data.

| Information sources
Quantitative and descriptive articles from peer-reviewed journals were considered for inclusion. Narrative reviews, letters, and editorials were

| Search strategy
A systematic three-step search strategy was followed based on the framework outlined by the Joanna Briggs Institute, 14 and implemented through consultations with experienced health sciences librarians at our institution. First, an initial search was performed in PubMed to identify seminal articles related to the review question. This preliminary search was followed by a comparable search in EMBASE, to explore words in the titles and abstracts, indexing nomenclature, keywords, and various spelling associated with our search topics of humans, IVD, and vasculature. Second, additional indexing terms and keywords were retrieved from seminal articles to form the comprehensive search strategy, which was again reviewed by experienced librarians for potential sources of error. This comprehensive search strategy was then modified to fit the requirements of each included information source. As per PRISMA-ScR guidelines, an example of a comprehensive search for one of the information sources can be found in Appendix S1. 15 Lastly, the reference lists of articles meeting full-text inclusion were screened for additional relevant articles that may have been missed in our search.

| Selection of sources of evidence
All retrieved article citations were imported into the reference manager software package to remove duplicates and distribute to secondary evaluators (EndNote VX.9, Clarivate Analytics: PA). A slight modification to the traditional two-step approach of reviewing titles and abstracts and then full-texts was used. 11 First, all titles and abstracts (n = 3122) were screened by one reviewer to exclude irrelevant articles, according to the following criteria: (a) animal studies; (b) topic not IVD; and (c) studies focused on surgical applications. Surgically oriented articles which focused on surgical techniques, medical devices, and case reports of clinical complications during surgery were excluded as they may describe the general anatomy but do not specifically characterize the prevalence or localization of the vascularization of the IVD. Second, screening of the remaining list of titles and abstracts (n = 718) was conducted independently by two reviewers to minimize selection bias. In addition to the criteria used in the initial screening, articles were excluded if the topic of the study was solely herniated IVD tissue without controls or characterized vasculature of vertebra but not the IVD. Articles were categorized as "include, exclude, or uncertain," and the screening results were compared between the two reviewers. During a consensus meeting, the uncertain articles and any other disagreements were resolved by discussion.
Third, the articles that met the inclusion criteria based on titles and abstracts were retrieved and assessed for full-text eligibility by two independent reviewers, and disagreements were resolved during another consensus meeting. At the end of analysis, the comprehensive search was rerun to identify articles published during the review process. Two independent reviewers screened these titles and abstracts using the same eligibility criteria and results were compared.

| Data items and charting
Data were aggregated from all full-text articles included. An electronic data extraction chart was created to capture general citation information, as well as information on the study population (eg, source of tissue, age, sex, health status) and vascularization measurements (eg, prevalence and localization) related to the research question. 14,15 The data extraction chart was tested by two reviewers, using three randomly selected included articles, before one reviewer independently assessed and charted the remaining included articles. The data extraction chart was modified and revised as necessary, during the iterative process of data extraction.

| Synthesis of results
Articles were grouped by anatomical features, such as the region of the vertebral column or the specific IVD tissue studied. Articles were also grouped by the methodology used to assess vasculature and, when possible, by the characteristics of the study population (eg, age and health status).

| Selection of sources of evidence
After duplicates were removed, a total of 3122 articles were identified from our comprehensive search, of which 2404 were clearly irrelevant to the research question and excluded during preliminary screening. Of the remaining 718 articles, another 624 articles were excluded following independent screening of their titles and abstracts by two reviewers, leaving 94 articles to be retrieved for full-text review to determine eligibility. Using IBM SPSS Statistics (Version 26.0, Armonk, NY), a Cohen's kappa statistic of 0.55 was found between reviewers. Landis and Koch suggest that Kappa statistics from 0.41 to 0.60 indicate moderate agreement. 17 Based on our study design, all discrepancies were discussed during a consensus meeting to ensure eligible articles were included in the analysis. Of the full-texts evaluated, 20 were published in a non-English language and were not able to be translated; 5 were unavailable; and 47 were excluded following full-text review. Ultimately, 22 articles were considered eligible for this scoping review ( Figure 1).
The comprehensive search was rerun on 06 April 2020 to ensure recently published articles during screening and analysis were not missed. A total of 130 articles were captured in this search and the titles and abstracts were independently screened by two reviewers. No new articles were considered eligible for inclusion.

| Results of individual sources of evidence
The general citation information of the included articles, study sample, and methodology used are presented in Table 1. The articles included were either descriptive (45%, n = 10/22) or case-control study designs (55%, n = 12/22). Typically, case-control studies compared IVD tissues retrieved from surgery to postmortem tissues without a known medical history of back pain, radiculopathy, or myelopathy. For several of the articles, only the specimens from study's control group met the eligibility criteria for inclusion for this scoping review.

| Study samples
Of the 22 studies included, 10 (45%) had a total sample size of <50 donors, 6 (27%) had 50 to 100 donors, and the remaining 6 (27%) had >100 donors. Individual group sample sizes ranged from 4 to 198 donors (median = 24). Most studies examined more than one spinal motion segment (IVD and the adjacent vertebra on either side) from each donor. The age of the study samples ranged from fetal/stillborn to greater than 90 years of age. While most studies reported the age range of donors; in studies that did not report age or used nonspecific descriptors, data were not assumed to represent any specific age group.
In general, there were more male donors than female. However, the sex of the donors was reported in only half of the articles from which data F I G U R E 1 PRISMA flow diagram of the article selection process of information sources T A B L E 1 Details of included articles: information and study sample characteristics

| Methodologies
The methods used to study the IVD included histology (n

| Synthesis of results
The primary findings from each included article have been grouped based on the three interdependent tissue types of the IVD in Tables 2-4 and summarized in a schematic in Figure 2. As a result of inconsistencies in the annotation of sex and the preferential focus on lumbar spine, differences based on sex or anatomical region could not be elucidated in the current scoping review.

| Nucleus pulposus
There was a lack of conclusive evidence that blood vessels were detected within the nucleus pulposus at any age, irrespective of methodology used to detect vascular structures ( Table 2). A recent study reported the presence of blood vessels along the nucleus pulposusannulus fibrosus transition zone and the "abnormal" localization of blood vessels within the inner IVD tissues associated with degeneration; however, the analysis of abnormal localization did not differentiate between the nucleus pulposus and inner annulus fibrosus. 36

| Cartilage endplate
The cartilage endplate is a tissue of the IVD that is vascularized during fetal development and in infants, but transitions to an avascular tissue with age ( Note: Citation information: First author listed, year of publication, location displayed as 3-letter country abbreviation, based on corresponding author's information. Study samples: Italicized are sample groups that did not meet the scoping reviews eligibility criteria; however, the comparison group did. Tissues retrieved from c, controls; pm, post-mortem; or s, surgery. Region describes the anatomical region of the spine studied: C, cervical; T, thoracic; L, lumbar; and S, sacral. Precise motion segments are displayed with original authors described. Age displayed in years and presented as ranges. '-' represents when information was not provided by the original authors. Abbreviation: IVD, intervertebral disc. a Methodology used in study design is histology. b Methodology used in study design is gross evaluation. c Methodology used in study design is imaging.
blood vessels and capillaries were identified and localized within these channels. The source of these vessels is likely the richly vascularized vertebral endplate of the adjacent vertebra. Sometime between birth and 10 years of age there is a drastic decrease in the presence of blood vessels and capillaries within these channels, which are completely obliterated in adults. 8,18,30 Focal obliteration of the cartilage endplate vessels was already identified in some infantile IVDs at 27 weeks to 2 years of age, as a possible first sign of vascular regression. 8 In tissues from adults, it was noted that the cartilage endplates were free of blood vessels except for instances of focal damage that resulted in the invasion of blood vessels into the cartilage endplates. 18,25,30

| Annulus fibrosus
In general, the annulus fibrosus is a vascularized tissue of the IVD but the localization of blood vessels is variable (Table 4). In fact, the spatial localization of blood vessels within the annulus fibrosus was poorly annotated. For example, two studies described the presence of blood vessels relative to concentric lamellae of the tissue 8,29 and another two studies provided a quantitative measure of distance from the periphery. 9,23 In general, early studies refer to the annulus fibrosus as a whole whereas more recent studies differentiate between the inner and outer layers of the tissue.
Nonetheless, the annulus fibrosus of fetal and infantile tissues was richly vascularized. Interestingly, one study using gross dissection reported blood vessels around the entire periphery of the annulus fibrosus in fetal and infantile tissues, and also reported blood vessels confined to the lateral positions of the IVD with age. 23 Another article using histology reported an abundance of blood vessels in infantile tissues from 27 weeks to 2 years of age in the outer layers of the annulus fibrosus, extending toward the inner layers of the annulus fibrosus. 8 The source artery or arteries supplying these blood vessels to the annulus fibrosus were not identified. Some authors noted the presence of blood vessels traveling through longitudinal ligaments and penetrating the outer layers of the annulus fibrosus. 8,9,18,21,27 Others reported a lack of blood vessels associated with the longitudinal ligaments. 19,23,24 Regardless, it is evident that blood vessels are present in the annulus fibrosus in fetal stages and in infants. Similar to the cartilage endplate, the detection of these blood vessels in the annulus fibrosus drastically decreases between 1 and 30 years of age. 8,20,21,27 In adults, the annulus fibrosus is less vascu- larized, yet blood vessels do persist in the outermost lamellar layers.
With advancing age (>50 years of age) blood vessels were more often identified in the inner layers of the annulus fibrosus than in younger adults (25-50 years of age), resembling the vascularization noted in fetal/infant tissues. 21  or the number of concentric lamellae reached. 8,9,23,29 Such methods of counting lamellae or measuring distance may not be reproducible based on differences in size and shape of IVDs across anatomical regions or between individuals and sexes. Moreover, referencing concentric lamellae may be difficult to apply to methods of contrast radiography where the soft tissues cannot be as easily differentiated, but may be applicable to magnetic resonance imaging investigations.
Overall, the variability in the annotation of the spatial localization of blood vessels within the annulus fibrosus is currently limited to the demonstrated vascular ingrowths localized near or within damaged tissue, independent of age. 8,9,32,36 It is likely that blood vessel ingrowth is stimulated as a healing response due to the reduction of anti-angiogenic factors (eg, proteoglycans) and the increased secretion of angiogenic growth factors and cytokines (eg, VEGF and IL-1β). 40 Moreover, within degenerative tissues, vascular ingrowths were often reported to be accompanied by neural ingrowths. 9,29,36 Interestingly, both the presence of blood vessels without nerves 29

| Limitations
There are several notable limitations of the evidence included in the review that should be considered in its collective interpretation. The

| CONCLUSIONS
In summary, the current scoping review detailed the current state of knowledge of the vascularization of the IVD in humans. Histology is the predominate method to study blood vessels of the IVD, capitalizing on the specificity of immunohistochemistry markers of endothelial cells (eg, CD31, CD34). Overall, the IVD is not entirely avascular, as often cited.
While it was confirmed that the nucleus pulposus is avascular throughout life, both the cartilage endplate and annulus fibrosus receive variable blood supplies across the lifespan, with the localization and prevalence of blood vessels varying by age and with tissue degeneration or damage. In