ARTICLE IN PRESS

The Spine Journal 000 (2019) 1−12

Inflammaging determines health and disease in lumbar

discs—evidence from differing proteomic signatures of
healthy, aging, and degenerating discs
S. Rajasekaran, PhD, FRCS, MCha,*, Chitraa Tangavel, PhDb,
Sri Vijay Anand K.S., MSa,
Dilip Chand Raja Soundararajan, MS, DNB, FNBa,
Sharon Miracle Nayagam, MScb, Monica Steffi Matchado, MTechb,
M Raveendran, PhDc, Ajoy Prasad Shetty, MS, DNBa,
Rishi Mugesh Kanna, MS, MRCS, FNB Spinea, K. Dharmalingam, PhDd
a
Department of Spine Surgery, Ganga Hospital, 313, Mettupalayam Rd, Coimbatore, India
b
Ganga Research Centre, No 91, Mettupalayam Rd, Coimbatore 641030, India
c
Department of Plant Biotechnology, Tamil Nadu Agricultural University, Coimbatore 641003, India
d
Aravind Medical Research Foundation, Madurai 625020, India
Received 5 February 2019; revised 26 April 2019; accepted 26 April 2019

Abstract BACKGROUND CONTEXT: The true understanding of aging and disc degeneration (DD) is still
elusive. MRI has not helped our attempts to understand the health and disease status of the discs as it
reflects mainly the end morphologic changes and not the changes at a molecular level. Understanding
degeneration at a molecular level through proteomics might allow differentiation from normal aging
and also aid in the development of biomarkers for early diagnosis and preventive therapies.
PURPOSE: To utilize proteomics to understand the molecular basis of healthy, aging, and degen-
erating discs and conclusively differentiate normal aging and degeneration.
STUDY DESIGN: Proteomic analysis of human intervertebral disc samples.
METHODS: L4−L5 disc samples from three groups were acquired and subjected to proteomic anal-
ysis. Samples from individuals aged in the second, third, and fourth decades were used to represent
young healthy discs (Group A). Those from MRI normal donors aged in the fifth, sixth, and seventh
decades represented normal aging (Group B). Five degenerated discs obtained from patients at sur-
gery represented degeneration (Group C). The entire proteome map and alteration in protein expres-
sions were further analyzed using bioinformatics analysis. This was a self-funded project.
RESULTS: There were 84 common proteins. Specific proteins numbered 225 in A, 315 in B, and
283 in C. By gene ontology biological process identification, Group A predominated with extracel-
lular matrix organization, cytoskeletal structural and normal metabolic proteins. Group B differed
in having additional basal expression of immune response, complement inhibitors, and senescence
proteins. Group C was different, with upregulation of proteins associated with oxidative stress
response, positive regulators of apoptosis, innate immune response, complement activation and
defense response to gram positive bacteria indicating ongoing inflammaging.
CONCLUSIONS: Our study documented diverse proteome signatures between the young, aging
and degenerating discs. Inflammaging was the main differentiator between normal biological aging
and DD.

FDA device/drug status: Not applicable.
Author disclosures: SR: Nothing to disclose. CT: Nothing to disclose.
AKSSV: Nothing to disclose. DCRS: Nothing to disclose. SMN: Nothing
to disclose. MSM: Nothing to disclose. MR: Nothing to disclose. APS:
Nothing to disclose. RMK: Nothing to disclose. KD: Nothing to disclose.
Funding: The project was funded by Ganga Orthopaedic Research and
Education Foundation (GOREF-06-2018), Coimbatore.
IRB approval: The study was performed only after approval of the IRB
committee.
* Corresponding author. Department of Spine Surgery, Ganga Hospital,
313, Mettupalayam Rd, Coimbatore, India. Tel.: +91-9843022325;
fax: +91-422-4383863.
E-mail addresses: rajasekaran.orth@gmail.com,
sr@gangahospital.com (S. Rajasekaran).

https://doi.org/10.1016/j.spinee.2019.04.023
1529-9430/© 2019 Elsevier Inc. All rights reserved.
 ARTICLE IN PRESS
2 S. Rajasekaran et al. / The Spine Journal 00 (2019) 1−12
CLINICAL SIGNIFICANCE: Multiple inflammatory molecules unique to DD were identified,
allowing the possibility of developing specific biomarkers for early diagnosis and thereby provide
evidence-based metrics for preventive measures rather than surgical intervention and also to moni-
tor progress of the disease. © 2019 Elsevier Inc. All rights reserved.

Keywords: Proteomics; Disc degeneration; Low back pain; Inflammaging; Aging disc; Apoptosis; Intervertebral disc

Introduction voluntary organ donors in true living condition and clinically

sterile environment of the operating room. This allowed doc-
The social and economic impact of low back pain is
umentation of the unique protein signatures of the three enti-
immense and demands an urgent development of novel and
ties: healthy young, healthy aged, and degenerated discs.
effective therapeutic strategies [1]. Attempts to develop
effective interventions have been thwarted by several diffi-
culties [2]. Although mechanical loading, trauma, genetics, Materials and methods
smoking, and even subclinical infection have been impli-
Institutional board approval was obtained for conducting
cated, there is ambiguity in many studies with no clear defi-
this research. Study population of the intervertebral disc
nition of the initiating or aggravating factors [3−6]. Even
samples included three groups. Control samples from
the basic differences between aging, senescence, and
asymptomatic brain dead voluntary organ donors aged in
degeneration remain unknown. A lack of correlation
the second, third, and fourth decades were taken to repre-
between clinical findings and magnetic resonance imaging
sent young healthy (Group A).Those from MRI normal
(MRI) have led to inaccurate decisions, increased surgical
donors aged in the fifth, sixth, and seventh decades were
procedures, and poor outcomes [7].
taken to represent normal aging (Group B) and five degen-
Clinicians and researchers have relied on MRI for dis-
erated discs (Pfirrmann grade 4) obtained during surgery
ease phenotyping which may often be misleading [8]. To
from patients represented disc degeneration (Group C)
overcome limitations of the current standards of care, the
(Fig. 1). Group C (degenerated discs) was obtained after
solutions may come from a thorough understanding of disc
informed consent from those patients undergoing micro dis-
degeneration (DD) at molecular level and developing bio-
cectomy for herniation and spinal fusion for degenerative
markers that are sensitive indicators of true pain and also
stenosis with instability. For Groups A and B, three seg-
diagnostic of degeneration at a very early stage before obvi-
ments of the lumbar spine were procured from brain dead
ous radiological and structural changes have occurred [9].
yet alive patients, after retrieval of vital organs for trans-
For both, the proteomic analysis may be the answer as
plant through a licensed organ donor program, where the
every physiological and pathologic process has its specific
first-degree relatives consented for procurement of the disc
protein finger print and proteomics can identify the altera-
material. The demographic details of donors and patients
tions at femtomole level (10
15) [10]. Capacity to identify
are given in (Table 1).
minute alterations will allow us the possibility to diagnose
the disease at its initiation and will lead to novel methods
of intervention with gene therapy and growth factors.
Chronic low-grade inflammation known as inflammaging
has been associated with a number of degenerative disor-
ders of human beings including degenerative disc disease.
Proteomics will also document inflammaging more accu-
rately and help to identify the different inflammatory pro-
teins associated with degeneration [11]. This will help to
identify potential biomarkers for early diagnosis and devel-
opment of patient-targeted therapies.
A hitherto universal challenge in studies on DD has been
the unavailability of healthy MRI normal disc tissues as con-
trols. Previous studies have utilized specimens from cadavers
and patients with trauma or scoliosis [12−14]. But mechani-
cal stresses in scoliosis and end plate damage in trauma will
result in proteomic alterations of disc thereby questioning
Fig. 1. Organ donor samples in Group A (young health) and Group B (nor-
the validity of these studies [13,15]. The uniqueness of the mal aging) were harvested in sterile operating room and MRI images were
present study is the utilization of true controls by harvesting taken immediately. Degenerate discs (Group C) were taken from patients
MRI normal disc samples from asymptomatic brain dead undergoing spinal fusion.
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S. Rajasekaran et al. / The Spine Journal 00 (2019) 1−12 3

Table 1 Sample collection

The distribution of harvested L4−L5 disc samples subjected to proteomic
analysis Sample collection and preservation were performed in
the sterile operating room to minimize the risk of contami-
Group S. No Source of sample Age Sex Pfirrmann grade
nation. The samples were immediately transferred into ster-
A 1 OD 13 M 1 ile cryopreservation vials, snap frozen into liquid nitrogen,
2 OD 22 M 2 and later transported to the research laboratory. Protein
3 OD 32 F 2
extraction, precipitation, and liquid chromatography-tan-
B 4 OD 43 M 2
5 OD 55 F 2 dem mass spectrometry (LC-MS/MS) analysis were done
6 OD 67 M 2 using established standard methods and quality checks
C 1 DS 34 F 4 were performed frequently before subjecting the raw data
2 DS 52 F 4 to further analysis.
3 DL 57 F 4
4 DL 61 F 4
5 LL 72 F 5 Sample processing
OD, organ donor; DS, degenerative stenosis; DL, degenerative listhe-
sis; LL, lytic listhesis.
Around 100 mg of tissue from each of the six control
samples and five diseased discs were subjected for extrac-
tion of total proteins as described in the workflow and sub-
jected to electrospray ionization-liquid chromatography-
Experimental workflow
tandem mass spectrometry (ESI-LC-MS/MS) with condi-
The general workflow is displayed in Fig. 2. tions as described in our earlier report [5].

Fig. 2. Workflow for proteomic analysis: IVD tissue samples frozen in liquid nitrogen (100 mg) was processed for protein extraction as shown in the above
work flow. Tissues were harvested as described under the materials and method section of this study.
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4 S. Rajasekaran et al. / The Spine Journal 00 (2019) 1−12

Bioinformatics analysis
MS/MS raw data acquired from Orbitrap Velos Pro Mass
Spectrometer were analyzed by Proteome Discoverer v1.4
using Mascot (Matrix Science, London, UK; version
2.4.1.0) and inbuilt SequestHT search algorithm. The spec-
tral counts from SequestHT and Mascot were postprocessed
using the Percolator algorithm. The peptides with rank one
and having a q value <0.01 were considered for protein
identification. Gene Ontology and Pathway enrichment
analysis were carried out using The Database for annota-
tion, visualization, and integrated discovery (DAVID) ver-
sion 6.8.STRINGdatabase v10.5 was used for cluster
analysis.

Relative quantification by spectral count

Spectral counts (SpC) obtained by LC/MS-MS were fur-
ther subjected for normalization by normalized spectral
abundance factor (NSAF) method [16]. NSAF is calculated
as follows:

SpC
k
ðNSAFÞk ¼ PN L
SpC
L i
i¼1
SpC: Number of spectral counts; L: Protein Length; k: indi-
vidual protein
Statistical analysis
Statistical significance of detected proteins was analyzed
using one-way ANOVA followed by multiple t test (Holm-
Sidak’s test) using GraphPad Prism software 7.05 to com-
pare two groups. Results were expressed as mean§standard
error of the mean (SEM) in the box plot and p values <.05
were considered statistically significant.
Results
LC-MS/MS analysis documented 409 proteins in Group
A, 512 in Group B, and 412 in Group C. Eighty-four pro-
teins were common to three groups (Fig. 3). Number of pro-
teins identified specific to each group were—225 in A, 315
in B, and 283 in C. Gene symbols used to indicate proteins
in the manuscript have been listed along with abbreviations
(see appendix).
Proteins common to all three groups
The common proteins were grouped based on their bio-
logical processes (Fig. 4). The GO molecular functional
analysis (Pantherdb) of 409 total proteins in Group A
revealed 43% of the protein participation in extracellular
matrix (ECM) binding; 33% of proteins in catalytic activity,
and 28% constituted other functions: structural molecular
activity (5.5%); transporter activity (6%); antioxidant activ-
ity (1.5%); receptor activity (5.5%); signal transducer activ-
ity (3%); transregulator activity (0.7%); channel regulator
Fig. 3. Comparative analysis of total proteins between the three groups:
Venn diagram depicts the total proteins identified in three groups Group A
—healthy young discs; Group B—healthy aged discs; Group C—degener-
ated discs.
activity (0.4%). This was considered as true control to which
the proteins in Groups B and C were compared. Expression
pattern of certain specific proteins across the three groups is
shown in (Fig. 5).
Carrier proteins: Beta globin (HBB), Apolipoprotein A1
(APOA1); metabolic proteins—Phosphoglycerate kinase
1 (PGK1), Enolase 1 (ENO1); protease inhibitor—Alpha
2-antiplasmin (SERPINF2); Serine protease—Lactotrans-
ferrin (LTF), binding proteins—Annexin A2 (ANAX2),
Haptoglobin (HP) exhibited an upregulation trend in Group
B and showed highest expression in Group C.
Structural proteins: Keratin (KRT-2,5,10); ECM
linker proteins—Hyaluronan and proteoglycan link pro-
tein 1 (HAPLN1), heparan sulfate proteoglycan core pro-
tein (HSPG2), Sushi repeat-containing protein SRPX2
(SRPX2); ECM protein—Collagen alpha-1(II) chain
(COL2A); Proteoglycans—Decorin (DCN), Biglycan
(BGN); Metabolic protein—Triose-phosphate isomerase
(TPI); Metalloprotease—Melanotransferrin (MELTF) was
lowest in Group C when compared to Group A.
Proteins that accumulate during aging and which
decrease in degeneration: Proteoglycans: Aggrecan
(ACAN), Fibronectin (FN1), Prolargin (PRELP); Struc-
tural proteins—Histone H1.2 (HIST1H1C), Keratin
(KRT)
1,9,14,16,17; Glycoproteins—Fibromodulin
(FMOD), Lumican (LUM); ECM Link proteins—Carti-
lage intermediate layer protein 1 (CILP), Cartilage oligo-
meric matrix protein (COMP), Clusterin (CLU), Asporin
(ASPN), Osteoglycin (OGN), Chondroadherin (CHAD),
Chitinase-3-like protein 1 (CHI3L); Serine protease—Ser-
ine protease HTRA (HTRA); Protease inhibitor SERPIN
family -Alpha-1-antitrypsin (SERPINA1), Alpha 1-antichy-
motrypsin (SERPINA3), Plasma serine protease inhibitor
(SERPINA5), Plasma protease C1 inhibitor (SERPING1);
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S. Rajasekaran et al. / The Spine Journal 00 (2019) 1−12
Fig. 4. Distribution of biological process of the proteins common to all three groups showing mainly structural and metabolic processes representing disc
homeostatic events.
Defense proteins—Lysozyme (LYZ), Dermatopontin
(DPT), alpha-2-Macroglobulin (A2M); ECM proteins—
Annexin A5 (ANXA5), collagen type XI alpha 2 chain
(COL11A2); Binding proteins—Retinol binding protein
4 (RBP4), Fibroblast growth factor binding protein 2
(FGFBP2) and ABI family member 3 binding protein
(ABI3BP) were found to be upregulated in Group B and
downregulated in Group C.
Proteins involved in various stages of degradation of
ECM, such as thrombospondin 1 (THBS1), carbonic anhy-
drase (CA1), glyceraldehyde 3-phosphate dehydrogenase
(GAPDH), and vasoactive intestinal peptide (VIP), had the
highest expression in Group C.
Biological process participation by the proteins specific to
each group
The unique proteins specific to each group with higher
confidence (≥2 peptides) were subjected to GO functional
analysis using DAVID v6.8. The significant biological pro-
cess along with their gene clusters based on the p value is
listed in (Supplementary Table 1).
Group A had ECM, cytoskeletal, structural and normal
metabolic proteins required to maintain disc homeostasis.
Interestingly inflammatory, immune response and comple-
ment activation cascades were absent. In Group B, in addi-
tion to structural and metabolic proteins, the biological
process revealed the basal expression of proteins participat-
ing in: immune response; phagocytosis; complement regu-
lation, and senescence. Group C had a completely different
subset of proteins participating in distinct biological
5
processes which included: complement pathways; opsoni-
zation; oxidative stress proteins; detection of molecules of
bacterial origin; response to reactive oxygen species; cata-
bolic and apoptotic proteins as shown in (Supplementary
Table 1; Fig. 6). The details of the unique and upregulated
proteins in Group C and its function are elaborated in
(Table 2).
The differential expression of complement proteins and
its regulators across three groups were analyzed for its sta-
tistical significance and found to be significant (Fig. 7). The
upregulation of complement proteins and downregulation
of proteoglycans in DD by twofold with respect to control
group (A+B) is shown in (Fig. 8).
To identify active signaling pathways specific to degen-
eration, Pantherdb pathway enrichment analysis was per-
formed for the unique proteins of Organ donors (Group A+
B) and compared with that of degenerate discs (Group C)
showed unique pathway proteins: oxidative stress; cytoskel-
etal reorganization by Rho-GTPase pathway; apoptosis sig-
naling pathway; inflammation mediated by chemokines,
and cytokines signaling pathway. The proteins representing
these pathways are enlisted in (Table 3).
Discussion
While aging and degeneration are ubiquitous, there is still
no clear understanding of the inter-relationship between
aging, senescence, and degeneration [17−19]. Controversy
still exist on whether degeneration is just accelerated aging
and also the exact role of initiating factors and the various
pathomechanisms involved in DD remain unknown [20−22].
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6 S. Rajasekaran et al. / The Spine Journal 00 (2019) 1−12
Fig. 5. Protein expression pattern across three groups: differential expression of proteins between decades was plotted based on normalized spectral counts as
described in methodology.
Reliance on MRI images has unfortunately focused
research on radiological phenotypes and current therapies
are focused on symptoms rather than prevention of the
underlying cause [23]. There is an urgent need to unravel
the molecular mechanisms involved in DD to curtail this
global malady [24].
This study is unique as MRI normal discs from clinically
asymptomatic voluntary organ donors are used as controls
for the first time. Discs from donors younger than 40 years
represented the young healthy; discs from fifth to seventh
decade donors represented aging, and the five degenerate
disc obtained during fusion surgery represented DD. Prote-
omic analysis of these groups allowed a thorough compari-
son of the protein signatures and the differing biological
pathways.
Contrasting protein signatures of the three groups
The proteome of the three groups were distinctly different
in both expression and biological process involving them.
Group A predominated in intact matrix proteins, normal
metabolic pathways and was characterized by the absence of
inflammatory markers. This represents the proteomic expres-
sion and their involvement in maintaining homeostasis in a
normal healthy disc. Group B representing aging showed
basal expression of immunoglobin and complements. How-
ever, this increase in complements was sufficiently counter-
balanced by presence of regulatory proteins which keep the
complement mediated inflammation in check. This results in
aging yet healthy disc as in Group B. However, Group C was
distinct with marked upregulation of proteins related to com-
plement pathway, catabolism, apoptosis, oxidative stress,
response to reactive oxygen species, defense response to bac-
terial, and fungus indicating that inflammaging is the key
pathomechanism in disc degeneration (Figs. 6−9).
Inflammaging: the key to disc degeneration
An imbalance between proinflammatory and anti-inflam-
matory mediators may result in a chronic low-grade persis-
tent tissue inflammation, termed as inflammaging (inflamm-
ageing) [22]. Inflammaging has been implicated in many
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S. Rajasekaran et al. / The Spine Journal 00 (2019) 1−12
Fig. 6. Protein−protein interaction for the unique proteins in Group C indicating clusters with significant p value: inflammatory response proteins (4.92E-11);
complement activation (1.00E-16); apoptotic proteins (1.53E-07).
age-related disorders such as osteoarthritis, etc., and also
recently in DD [25−27]. However, the documentation of
inflammaging in DD has been largely restricted to cytokine
analysis by quantitative PCR and no studies have docu-
mented the molecular mechanisms at the proteomic level
and the biological processes involved [28]. Also, the absence
of inflammaging in healthy disc has not been documented
due to lack of true control.
Our study clearly documents that aging and degeneration
are two entities that can be differentiated by presence or
absence of inflammaging. In Group B, there is an increased
inflammatory process compared to Group A, but is suffi-
ciently counterbalanced by the presence of regulatory pro-
teins like metalloproteinase inhibitor 3 (TIMP-3), CLU
(Figs. 5 and 7), CFH complement factor H and complement
factor I (CFI). This keeps the complement mediated inflam-
mation in check resulting in aging yet healthy status.
7
The degenerate discs (Group C) however have a clear
imbalance in this pro- and anti-inflammatory mechanisms
evidenced by a decrease in TIMP-3, CLU, CFH, CFI and a
marked elevation of complement proteins (Figs. 5 and 7).
Also many innate immune response proteins like VIP,
S100, neutrophil defensin proteins, C-reactive protein were
uniquely present in Group C indicating a proinflammatory
state in DD. This proinflammatory state further accentuates
other inflammatory cascades like platelet degranulation
process releasing histamine and serotonin which are poten-
tial mediators of inflammation and pain [29,30].
Other important biological processes detected in degen-
eration were increased oxidative stress and apoptosis. The
expression of hemoglobin beta subunit (p=.0001) showed
increasing trend across the groups indicating an oxidative
state of degenerated discs and hypoxic state of healthy discs
[31,32]. This is further strengthened by upregulation of
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8 S. Rajasekaran et al. / The Spine Journal 00 (2019) 1−12

Table 2
Significant biological process of proteins exclusively present in Group C (disease disc)

Biological process Gene symbol

Oxidative stress response Myeloperoxidase, apolipoprotein E
Defense response to bacteria Lipopolysaccharide binding protein, cathepsin G
Innate immunity Ig gamma chain C region, fibrinogen beta chain, complement C1s subcomponent, comple-
ment 4-B, complement C2b, polyubiquitin-C fragment
Aging Catalase
Receptor-mediated endocytosis Hemoglobin alpha-1 globin chain, vitronectin
Apoptosis 75 kDa regulated glucose protein, heat shock protein 70, MAP kinase 8 domain, caspase 7,
bag family molecular chaperone regulator 4
Proteins upregulated in Group C (diseased disc)
Biological process Gene symbol
Receptor-mediated endocytosis Hemoglobin subunit beta, hemoglobin subunit alpha, apolipoprotein A-1, haptoglobin, Ig
kappa chain V−IV region
Innate immunity Complement C3, serum amyloid P-component, pigment epithelium-derived factor
Antibacterial humor response Ig Mu chain C region
Antibacterial humor response Lactotransferrin

GAPDH an enzyme involved in sixth step of oxidative
phosphorylation and represents a regulatory hurdle in
anaerobic glycolysis [33]. Apart from metabolic functions,
the translocation of GAPDH to the nucleus acts as a signal-
ing mechanism for apoptosis [34]. The presence of GAPDH
is also associated with the synthesis of proapoptotic pro-
teins like Bcl-2-associated X protein (BAX), c-JUN and
GAPDH itself and has been implicated in neurodegenera-
tive disorders [35].
In our study, the proteins involved in oxidative stress
(Apolipoprotein E) and proteins in response to oxidative
stress (Peroxidasin, Myeloperoxidase, Peroxiredoxin -5,6,
Catalase) were found to be upregulated or uniquely present
in degenerative discs, adding evidence to the oxidative
stress-induced damage. The byproducts of cellular oxida-
tive metabolism produce reactive oxygen species like
superoxide anion, hydroxyl radical, hydrogen peroxide, and
nitric oxide which are implicated in numerous disorders
including disc degeneration [36−38]. Excessive reactive
oxygen species cause oxidative stress and activates various
apoptotic signaling pathways including NF-kB and MAPK
pathway (found in our study) and has been well docu-
mented in animal experiments [39,40].
We found apoptosis to play an important role in DD. The
presence of caspases 7,9 which are key effectors of apopto-
sis and other proteins (protein kinase C delta (PRKCD),
heat shock protein 70 (HSP70), endoplasmic reticulum
chaperone BiP (GRP78/BIP), mitogen-activated protein
kinase 8, MAP kinase-activating death domain protein,
BAG family molecular chaperone regulator 4 (BAG4))
found exclusively in degenerate discs ascertain the presence
of apoptotic pathways involved in degeneration [41,42].
The hallmark of disc degeneration is matrix disassem-
bly. Degenerate discs in our study showed a significant
presence of collagen catabolic process, proteolysis, fibrino-
lysis, ECM-disassembly processes [43−46]. There is a
marked reduction in structural and matrix proteins like
COL2A1, KRT, BGN, Versican (VCAN), and DCN in
degenerate discs which was significant (Fig. 8).

Fig. 8. Comparison between healthy control (Group A+Group B) and

Fig. 7. Box-plot representation of complement and its regulatory protein degenerated disease sample (Group C). Fold variation expressed in log2
expression along with statistical significance. Standard error of the mean scale. Positive values indicate the proteins present at higher amount in the
(SEM) is represented as the error bar. diseased samples and vice versa for negative values.

Table 3
Significant pathway-specific proteins across the groups using PANTHER database vs 13.1
S. No Pathways
1 Apoptosis signaling pathway
2 Blood coagulation
3 Cytoskeletal regulation By
Rho Gtpase
4 Inflammation mediated By
chemokine and cytokine
signaling pathway
oxidative stress response
5 Wnt signaling pathway
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S. Rajasekaran et al. / The Spine Journal 00 (2019) 1−12 9
Group C Group A and Group B
78 Kda Glucose-regulated protein Transcription factor Relb
Protein kinase C delta type Apoptosis-inducing factor 1, mitochondrial
Caspase-7
Mitogen-activated protein kinase 8
Bag family molecular chaperone regulator 4
Map kinase-activating death domain protein
Caspase-9
Heat shock cognate 71 Kda protein
Coagulation factor Xi Von Willebrand factor
Integrin alpha-Iib Plasminogen activator inhibitor 1
Tissue factor Tissue-type plasminogen activator
Fibrinogen beta chain Alpha-2-antiplasmin
Prothrombin Alpha-1-antitrypsin
Protein Z-dependent Protease inhibitor
Plasminogen
Coagulation factor Xii
Plasma kallikrein
Beta-actin-like protein 2
Profilin-1
Rho-associated protein kinase 1
Myosin-9
Myosin-13
Actin, cytoplasmic 2
Rho-associated protein kinase 2
Actin, alpha cardiac muscle 1
Cofilin-1
Tubulin beta chain
Rho Gtpase-activating protein 1
Beta-actin-like protein 2 Collagen alpha-1(Xx) chain
Collagen alpha-1(Xii) chain Signal transducer and activator of transcrip-
tion 6
Collagen alpha-3(Vi) chain Von Willebrand factor
C-X-C chemokine receptor type 2 Collagen alpha-1(Xiv) chain
Rho-associated protein kinase 1 1-phosphatidylinositol 4,5-bisphosphate
phosphodiesterase gamma-1
Mitogen-activated protein Kinase 4 Collagen alpha-6(Vi) chain
1-phosphatidylinositol 4,5-bisphosphate phosphodies- Collagen alpha-1(Vi) chain
terase epsilon-1
Myosin-9 Guanine nucleotide-binding protein G(K)
subunit alpha
Myosin-13
Actin, cytoplasmic 2
Rho-associated protein kinase 2
Collagen alpha-1(Vi) chain
Dual specificity protein phosphatase 5
Serine/threonine/tyrosine-interacting protein
Mitogen-activated protein kinase 4
Mitogen-activated protein kinase 8
Protein kinase C delta type Protocadherin alpha-8
Putative Myc-like protein Myclp1 Protein L-Myc
At-rich interactive domain-containing protein 1a Casein kinase I
Inositol 1,4,5-trisphosphate receptor type 2 Adenomatous polyposis coli protein 2
Myosin-13 Frizzled-5
Adenomatous Polyposis coli protein 2 Protocadherin-15
Cadherin-1 B-Cell Cll/lymphoma 9 protein
Alpha-catulin Secreted frizzled-related protein 3
Protocadherin beta-9
Actin, alpha cardiac muscle 1
Frizzled-2
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Fig. 9. The proteomic signatures of the three groups revealed three different biological processes—Group A—maintenance of extracellular matrix, cytoskele-
tal structure, and normal metabolism, Group B—regulation of immune and complement proteins indicating ongoing reparative and senescence mechanisms,
Group C—complement activation, opsonization, oxidative response, platelet degranulation, and host defense response implicating inflammaging.
It was interesting to note that proteins representing
inflammaging and disintegration were completely absent in
aging discs of even the seventh decade sample proving that
aging and degeneration are two different pathways depend-
ing on presence or absence of inflammation (Fig. 9).
Could subclinical infection play a role in inflammaging?
Recently subclinical infection has been proposed as an
initiator of disc disease raising considerable interest and con-
troversy [47−49]. While the proinflammatory state of degen-
erated disc is evident, what perpetuates this inflammation
remains to be debated. The classical complement pathway
which is usually induced by antigen−antibody complex is
intimately associated with the phagocytic process and in nor-
mal host defense [50]. Once activated, the complement path-
ways can induce a self-perpetuating inflammatory cascade
leading to inflammaging and degeneration [51]. The increased
expression of complements and involvement of both classical
and alternative pathways in biologic process in degenerated
discs evokes suspicion of an infective etiology [52]. Further,
evidence for this hypothesis was found in our study by pres-
ence of host defense response proteins to bacteria and fungi,
defensin, lipopolysaccharide binding protein, S 100 calcium
binding protein A8, myeloperoxidase & cathepsin G in degen-
erative discs.
Clinical perspective of our study and implications for
practice
Clinicians rely on patient history, signs, and symptoms
and more importantly MRI to make decisions. The over reli-
ance on MRI for clinical diagnosis and staging of the disease
has unfortunately led to delayed diagnosis and a mechanistic
understanding of spinal pathologies. Therefore, mechanical
solutions are usually offered to patients frequently leading to
surgeries, not always with a good outcome. If improvement
of current results has to occur, understanding of DD has to
progress beyond radiological imaging on the lines of inflam-
mation and specifically inflammaging. This will have a
strong potential to change clinical practice through bio-
markers and preventive therapies. An early diagnosis before
structural changes will allow novel therapies like neutraliza-
tion of specific cytokines. Therapies that modulate inflamma-
tion and even those that restore ECM may be developed.
Gene therapy or genome editing along with growth factors
will attain center stage focus slowly depleting the current
importance of surgery for low back pain. The results of our
study and the undisputable documentation of inflammaging
in DD indicate that this will be the future pathway of treat-
ment for back pain.
Limitations
Our study documented more than 80 biological processes
and variations between the groups. However, it was beyond
the scope of this paper to elaborate on every single process.
All the discs analyzed were restricted to L4−L5 level to
ensure uniformity of mechanical stress and future study
should involve more number of samples at all levels of the
lumbar spine also. A larger study would also allow clinical
correlation of pain to the proteomic changes and presence of
inflammation. A future study should also integrate metage-
nomics to document the nature of bacterial presence.
Conclusions
Our study has established that young healthy, aging, and
degenerated are three biological states that have distinct prote-
ome signature and different biological process. Inflammaging
 ARTICLE IN PRESS
S. Rajasekaran et al. / The Spine Journal 00 (2019) 1−12
is the key differentiator between aging and degeneration.
We have identified numerous specific proteins unique to
degeneration that may serve as valuable biomarkers for
DD. These markers will help in early diagnosis, reduce dis-
ease clinical heterogeneity, and also serve to target patient
subsets with specific therapies.
Acknowledgments
The authors thank Ms. Alishya Maria Jose for her help in
data collection and maintenance.
Supplementary materials
Supplementary material associated with this article can
be found in the online version at https://doi.org/10.1016/j.
spinee.2019.04.023.
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