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Free Radic Biol Med. Author manuscript; available in PMC 2023 August 01.
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Published in final edited form as:

Free Radic Biol Med. 2022 August 01; 188: 175–184. doi:10.1016/j.freeradbiomed.2022.06.223.

Extracellular Biomolecular Free Radical Formation During Injury

Madeline R. Hines1, Jessica E. Goetz1, Piedad C. Gomez-Contreras1, Samuel N. Rodman
III1, Suryamin Liman1, Elise L. Femino1, Paige N. Kluz2, Brett A. Wagner1, Garry R.
Buettner1, Eric E. Kelley3, Mitchell C. Coleman1
1The University of Iowa, Iowa City, IA
2University of Wisconsin-Madison, Madison, WI
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3West Virginia University, Morgantown, WV

Abstract
Objective: Determine if oxidative damage increases in articular cartilage as a result of injury and
matrix failure and whether modulation of the local redox environment influences this damage.

Osteoarthritis is an age associated disease with no current disease modifying approaches available.
Mechanisms of cartilage damage in vitro suggest tissue free radical production could be critical
to early degeneration, but these mechanisms have not been described in intact tissue. To assess
free radical production as a result of traumatic injury, we measured biomolecular free radical
generation via immuno-spin trapping (IST) of protein/proteoglycan/lipid free radicals after a 2
J/cm2 impact to swine articular cartilage explants. This technique allows visualization of free
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radical formation upon a wide variety of molecules using formalin-fixed, paraffin-embedded

approaches. Scoring of extracellular staining by trained, blinded scorers demonstrated significant
increases with impact injury, particularly at sites of cartilage cracking. Increases remain in
the absence of live chondrocytes but are diminished, thus appear to be a cell-dependent and
-independent feature of injury. We then modulated the extracellular environment with a pulse
of heparin to demonstrate the responsiveness of the IST signal to changes in cartilage biology.
Addition of heparin caused a distinct change in the distribution of protein/lipid free radicals at
sites of failure alongside a variety of pertinent redox changes related to osteoarthritis. This study
directly confirms the production of biomolecular free radicals from articular trauma, providing a
rigorous characterization of their formation by injury.

Graphical Abstract
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Mitchell Coleman – [email protected] -- 3193357550 – 25 South Grand Ave, 1182 Medical Laboratories, Iowa City Iowa,
52242.
Author contribution statement:
M.R.H. designed this study alongside M.C.C., conducted the experiments, and composed the first draft of the experiments, J.E.G.
designed the custom algorithm for image analysis with input from M.R.H. and M.C.C. and contributed to data interpretation and the
composition of the manuscript, P.C.G.C. preformed the western blot and contributed to the composition of the manuscript, S.N.R.
served as a blinded trained scorer, assisted in interpretation, and contributed to the final draft of the manuscript, S.L. served as a
blinded trained scorer, assisted in interpretation, and contributed to the final draft of the manuscript, E.L.F. served as a blinded trained
scorer, assisted in interpretation, and contributed to the final draft of the manuscript, P.N.K served as a blinded trained scorer, assisted
in interpretation, and contributed to the final draft of the manuscript, B.A.W. assisted in design and interpretation of the study as well
as with the final manuscript, G.R.B. assisted in design and interpretation of the study as well as with the final manuscript, E.E.K.
assisted in design and interpretation of the study as well as with the final manuscript, M.C.C designed this study alongside M.R.H and
directed interpretation and composition of the manuscript.
 Hines et al. Page 2
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Keywords
DMPO; Osteoarthritis; Posttraumatic; Trauma; Redox; SOD3; Cartilage; Free Radical; Radical;
Oxidative Stress; Oxidation; Injury
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INTRODUCTION:
Post traumatic osteoarthritis (PTOA) involves progressive erosion and destruction of
articular cartilage extracellular matrix (ECM) following a traumatic joint injury. Our group
has shown that intracellular reactive oxygen species (ROS) and mitochondrial dysfunction
increase in chondrocytes immediately after cartilage impact injury and initiate degenerative
joint disease in a swine model of intra-articular fracture[1–4]. A wide variety of studies have
demonstrated oxidation acutely in cartilage; however, most of these studies were directly
focused upon intracellular biochemistry. Thorough description of the sources and targets of
oxidation in the extracellular space of articular cartilage after injury is challenging given the
tissue’s strength and relative opacity. To explore the formation of extracellular free radicals
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after injury, we investigated the potential of a novel technique termed immuno-spin trapping
(IST), which permits visualization of free radicals in formalin-fixed, paraffin-embedded
tissue[5, 6].

Though the existing literature on the topic is limited, thorough benchtop studies
of extracellular free radical formation have focused on individual, specific chemical
mechanisms of ROS-mediated cartilage damage like superoxide (O2•-)[7, 8]. These in vitro
studies demonstrated collagen fiber cleavage at proline residues by O2•- levels as low as 1
nM via xanthine/xanthine oxidase (a well characterized source of O2•-) and indirectly by
hydroxyl radical (HO•) through transition metal reactions[7, 9]. HO• is highly reactive and
known to cleave collagen fibers[10] in proximity of generation[11]. Extracellular superoxide
dismutase (SOD3), a key antioxidant enzyme present in the extracellular compartment of
articular cartilage, inhibits this in vitro collagen cleavage[7]. These studies have established
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that extracellular free radical chemistry can influence collagen fiber damage but have not
defined key loci for these reactions.

Detailed mechanistic descriptions of extracellular redox chemistry in vivo after trauma have
not been made, though nitric oxide has otherwise been an extensive focus of the field and
some focus on SOD3 is present within the literature. It has been shown that loss of the
SOD3 heparin binding domain, causing widespread loss of cartilage SOD3 content, led

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to decreased proteoglycan as well as increased cartilage erosion at the articular surface

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after prolonged exercise in a murine model[12]. In larger tissue, with more expansive
ECM, human samples obtained from total knee arthroplasties show decreased SOD3 and
increased 3-nitrotyrosine-modified protein (3-NT) in the extracellular space relative to
healthy controls, suggesting the possibility of increases in O2•- concentration and nitric
oxide in the ECM of diseased cartilage[13]. Thus, SOD3 and free radical chemistry are
crucial to extracellular cartilage function, but the sources and locations of extracellular free
radicals in cartilage remain unclear.

Rather than concentrate on the generation of specific radical species, we chose to apply
the IST method targeting any radical formed directly on proteins/proteoglycans/lipids
of the tissue. Prior studies of non-cartilage tissues have described how biomolecular
free radical formation provides a valuable indication of oxidative distress, since these
larger biomolecules are often protected by the intracellular antioxidants[5]. In most other
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tissues, the presence of these free radicals could be rigorously interrogated using electron
paramagnetic resonance (EPR); however, the stiffness and high water content of cartilage
preclude applying this technique. In lieu of this, we decided to investigate biomolecular
free radical formation in the ECM of injured cartilage using the IST technique that detects
labeled biomolecular free radicals in histological sections of paraffin embedded tissue[5, 6,
14–17].

IST uses 5, 5-dimethyl-1-pyrroline N-oxide (DMPO), a common EPR reagent used to

prolong free radical signals through stable, covalent bonds with protein/lipid radicals in vivo
or in vitro[5, 6, 15]. DMPO adducts on protein/lipid radicals form stable nitrones which are
retained though formalin fixation. These nitrones can then be identified using an antibody
raised against DMPO adducts that binds to the nitrone structure, normally a structure not
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found in vivo [16]. Thus, IST is an ideal means of exploring specific loci of extracellular
free radical production and oxidative injury in articular cartilage after mechanical trauma.
We hypothesized that a controlled and well-characterized impact injury to articular cartilage
would increase formation of biomolecular free radicals at sites of ECM failure as indicated
by increased DMPO staining. We then tested a prototypic modulation of biomolecular free
radical formation from injury by adding heparin to the articular cartilage of skeletally mature
Yucatan minipigs. This induced a variety of biological changes reminiscent of arthritic tissue
and demonstrates the responsiveness of this signal to changes in cartilage biochemistry[12].
Our results demonstrate that free radical formation is associated with ECM failure and that
this signal arises from both cell-dependent and -independent mechanisms. Finally, changes
in free radical formation at sites of failure after heparin treatment suggest there may be
distinct responses to injury in normal and unhealthy tissue. This novel application of a
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variety of custom-developed and interdisciplinary techniques demonstrates the value of a

broader view of free radical biochemistry for exploring redox biology within challenging
physiological niches.

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METHODS:
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Tissue Culture
This study used two sources of swine stifle (knee) joints. One source was from agricultural
animals obtained fresh from a local abattoir (Bud’s Custom Meats, Riverside, IA).
The agricultural animals’ sexes were unknown and assumed random. The other source
was Yucatan minipig stifles obtained postmortem from an ongoing collaboration in the
Department of Orthopedics and Rehabilitation at the University of Iowa. The Yucatan
minipigs were an even mix of males and females greater than 2 years of age and thus
skeletally mature. From both groups, cylindrical osteochondral plugs, 8 mm diameter, were
taken from the load bearing portion of the medial and lateral femoral condyle. Medial tibias
of agricultural swine were cut into two separate 1 cm3 explants. After harvest, the explants
and plugs were placed in normal growth medium (45% Dulbecco’s modified Eagle’s
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medium, 45% F-12, 10% Fetal Bovine Serum (FBS) (Gibco), with penicillin-streptomycin
and amphotericin B). Media was stored in a humidified chamber at 5% oxygen (O2) and
5% carbon dioxide at 37°C to maintain a low O2 concentration. Media and all samples were
stored in the humidified chamber during the duration of the experiment.

Impact
Samples were leveled and potted using polycaprolactone (Sigma Aldrich, St. Louis) then
impacted using a stainless steel flat impermeable platen. A previously characterized custom
drop tower was used to deliver an impact of 2 J/cm2, and explants were returned to the
incubator for 24 h[18–21]. Impact experiments were performed in dim lighting to limit free
radical production by photochemistry[22].

DMPO
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DMPO stock solution was prepared by diluting DMPO (Dojindo, D048, Kumamoto) to 1
M using distilled water bubbled with argon, then aliquoted and stored at −20°C. For use,
the 1 M aliquoted solution was diluted in low O2 medium to the desired concentration. To
determine the appropriate concentration of DMPO in articular cartilage, we evaluated the
DMPO staining of porcine plugs pre-exposure with a range of DMPO concentrations (5 mM
−100 mM). Three serial sections from each sample were assessed for reproducible DMPO
staining and each group was compared to determine if the change in DMPO concentration
caused a change in staining. DMPO concentrations of 50 mM and below had little to no
DMPO staining. DMPO concentrations above 50 mM DMPO staining was present, but the
increased concentration of DMPO did not reproduce in appearance until 70 mM and above,
Supplemental figure 1.
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The reason for using such an elevated concentration of DMPO is due to a direct competition
between the spin trap, DMPO, and molecular O2 for free radicals formed on lipids and
proteins/proteoglycan subsequent to reaction with reactive species. O2 will react with
these protein and lipid radicals at very fast rates (~108 mol/s) whereas DMPO’s affinity
for carbon, sulfur and nitrogen-centered biomolecular free radicals are significantly less
(~105-107 mol/s)[11, 17]. As such, considerably greater concentrations of DMPO compared
to O2 enables DMPO to effectively compete with O2 for these biomolecular free radicals

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before they form hydroperoxides (ROOH) and are no longer reactive with the spin trap.
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After this determination of the optimum DMPO concentration (70 mM), we treated the
remaining porcine osteochondral samples for 1 h prior to impact with medium containing 70
mM or 0 mM DMPO and cultured in DMPO medium for 24 h after impact, unless otherwise
specified.

To generate a positive control, samples were incubated in low O2 media with or without 100
μM each of myoglobin, iron sulfate, and hydrogen peroxide (H2O2). Myoglobin generates
a flux of hydrogen peroxyl radical and excess iron facilitates Fenton chemistry and the
generation of the HO•. This creates a strong enough flux of free radicals to lead to
macromolecule free radical formation. DMPO was added to this solution and samples were
co-incubated in that solution for 24 h, prior to fixation and histological processing.

Western Blot
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Samples from the femur of agricultural swine were cultured in low O2 media supplemented
with FastGro synthetic, animal free chemically defined FBS (MPbio, 2640049), instead
of FBS. This was to prevent FBS for obscuring proteins from the media and dominating
the signal observed. The samples were incubated with DMPO and impacted as described
above. The samples were cultured for 24 h after impact in groups of six samples per
experimental group. We included negative control (impact, no DMPO), positive control
(100 μM myoglobin/H2O2/iron sulfate), impact DMPO, no impact DMPO. After incubation,
supernatants from impacted plugs were collected, total proteins were quantified by BCA
method (Thermo, 23221). The protein samples were denatured and reduced by addition
of LDS sample buffer (Invitrogen NP0007) and DTT reducing agent (Invitrogen, NP0009)
and heated at 70°C by 10 min. The amount of 50 μg of protein was loaded per well and
electrophoresed through a 10% Bis-Tris NuPage acrylamide gel (Invitrogen, NP0315), at
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120 V constant by 2 h with MES-SDS running buffer (Invitrogen, NP0002). After the
electrophoresis, the gel proteins were transferred to a 0.45 μM nitrocellulose membrane
(BioRad, 1620145), at 300 mAmp constant by 2 h with 20% methanol 0.1% SDS transfer
buffer (Invitrogen, NP0006–1). The blotted membrane was stained using Ponceau S, as a
loading and protein quality control. The membrane was blocked by 30 min with a goat
serum-gelatin solution 5%. Poly clonal chicken anti-DMPO primary antibody (1:1000), a
generous gift from Dr. Ronald Mason (NIEHS), diluted in goat serum-gelatin 2.5%, was
incubated by 1 h RT, secondary ab was an HRP Goat anti Chicken (1:6000) diluted in goat
serum-gelatin 2.5% incubated by 1h RT (Abcam). After rinsing with TBST, the protein
signal was developed and visualized using Super Signal West Femto (Thermo, 34095) and
Amercham Hyperfilm ECL system (GE Healthcare, 28906839) film.
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Non-articular Collagen Failure

Rat tails were used as a source of type I collagen to provide a comparison with distinct
anatomy from articular cartilage. The tails were degloved and incubated for 1 h in media
with either 0 mM DMPO or 70 mM DMPO. Following incubation, rat tails were either cut
cleanly via scalpel or torn, manually, then returned to media for 24 h. After 24 h incubation
in DMPO media samples were fixed and processed as described below.

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Chondrocyte Viability
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To investigate the staining contributions of live cells, agricultural swine samples were placed
in 70% ethanol for 2 h. After the 2 h treatment, the lethality of ethanol was confirmed
using parallel samples stained using calcein AM (Thermofisher, 65-0853-39) for live cells
and ethidium homodimer (Thermofisher, E1169) for dead cells. The stained cartilage
was imaged using the Olympus FV1000 confocal laser scanning microscope (Olympus
America).

Heparin Treatment
Articular cartilage is an avascular tissue which is comprised mostly of ECM components
such as collagen and proteoglycans, leading to a dense and negatively charged tissue
with limited diffusion by size and charge[23, 24]. Evidence supports proteins greater
than 70 KDa have limited diffusion in the ECM, thus preventing direct supplementation
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with enzymes that might modulate the IST for this study[25, 26]. To modulate the
redox environment surrounding the injuries under study, we applied low molecular weight
heparin, which liberates proteins like SOD3 and others. We confirmed these changes via
immunohistochemistry (IHC). Heparin was prepared by dissolving the desired concentration
in low O2 (5% O2) media. Samples were treated with either a high concentration (2 mg/mL)
of heparin (Sigma Aldrich #H3149–10KU), a low concentration (1 mg/mL) of heparin, or
in media control. Samples were incubated overnight then co-incubated for 1 h with DMPO
prior to impact. After impact injury, the samples were returned to their respective treatment
media for 24 h incubation.

Fixation, Processing, and Embedding

Samples were fixed under vacuum for 4 h at 56 kPa in 0.3% Safranin-O (saf-O) in 10%
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neutral buffered formalin (NBF) to maintain chondrocyte and tissue morphology[27]. The
saf-O NBF solution was replaced with regular NBF after 4 h, and the samples were fixed
up to one week total. Samples were decalcified using daily changes of 5% formic acid until
decalcification was complete. Samples were bisected though the center of impact site to
reveal the central cross-sectional plane of the specimen, and then processed and embedded
in paraffin. Samples shown were sectioned at 5 μm for IHC.

Immunohistochemistry
Slides were deparaffinized at 55°C to remove moisture trapped in the sample. The slides
were then rehydrated via a series of washes: 100% xylene, 100% ethanol, 95% ethanol,
80% ethanol, finally distilled water. Slides were placed in citric acid antigen retrieval (0.1
M citric acid monohydrate (18 mL), 0.1 M trisodium citrate dehydrate (82 mL), deionized
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water (900 mL)) pH 6, overnight, up to 16 h, at 55°C. The samples were cooled to room
temperature, rinsed in distilled water, and endogenous peroxidases were quenched using
a 3% hydrogen peroxide solution. Samples were then blocked using normal goat serum
blocking solution without bovine serum albumin. The primary anti-DMPO antibody was
incubated overnight (1:150) at 4°C. The secondary antibody (1:200) was incubated 30–40
min at room temperature. While the secondary antibody was incubating, the ABC reagent
Elite Standard (Vector laboratories, Cat. # PK-6100) was mixed and left to incubate at room

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temperature for 30 min. Samples were then incubated in ABC reagent for 30 min. The
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samples were developed using DAB reagent (Vector laboratories, Cat. # SK-4100) which
was incubated for 5 min. No counterstain was used.

Assessment of biomolecular free radicals used polyclonal chicken anti-DMPO (1:150)

primary antibody as stated above[16]. To evaluate off target staining polyclonal chicken
IgY (R&D systems, AB-101-C) 1:150 isotype control was used and a no primary control.
The secondary antibody used was a goat anti-chicken HRP (Abcam, ab97135) (1:200).

The SOD3 content was evaluated using a mouse anti-SOD3 antibody (1:50) (Santa Cruz,
sc-101338) and goat anti-mouse secondary (1:200) (Thermofisher, 31430). Non-specific
binding was assessed using an isotype control (Abcam, ab37355) and a no primary control.
Immunofluorescence of 3-NT was done using a rabbit anti-nitrotyrosine antibody (Millipore,
06–284, 1:150) and goat anti-rabbit cy5 fluorescent secondary (Roche, 760–238) on a
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Discovery Ultra (Roche, Switzerland).

Image Analysis and Statistics

Images from the IHC slides were scored in a blinded manner for the intensity of the
extracellular space on a scale from 0 (no staining) to 3 (high intensity staining) with graders
provided specific instructions to ignore intracellular staining. Using Graph Pad Prism 8, the
scores were compared, and a non-parametric one-way ANOVA (Kolmogorov-Smirnov test)
was used for statistical analysis with significance at p < 0.05 and post hoc analysis using
Mann-Whitney. Outliers were determined using ROUT (Q=1%)[28]. Finally, the interrater
reliability was evaluated by calculating the Fleiss’ kappa (κ)[29]. Impacted samples without
ECM failure were excluded from the study.
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To determine the staining intensity along sites of ECM failure in the heparin treated groups
in greater detail and with more precise quantitation, we applied a custom designed algorithm
to IST images. Three serial sections from each sample were stained. Images were taken
from the same site of ECM failure and each crack was evaluated with replicate measures if
appearing in multiple sections. Cracks only appearing in one of the sections were analyzed
as a single data point. This resulted in algorithmic analysis of 17 cracks from 4 plugs in
the DMPO group and 27 cracks from 5 plugs in the heparin DMPO group. The cracks
were traced manually in MATLAB to generate masks. From these masks the intensity was
assessed in 5 μm intervals along a perpendicular ray from the crack, yielding an average
intensity at each distance grouping of 0–5, 5–10, 10–15, and 15–20 μm outward from a
given crack. Maximum intensity in these distance groups was also determined. Analysis was
restricted to the tissue, and slide background regions such as above the articular surface
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and wide fissures were not included. From these values an ANOVA was run to determine
statistical significance between the groups, p < 0.05, outliers were removed using ROUT
(Q=1%) with post-hoc t-test.

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RESULTS:
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Injury Increases Extracellular Biomolecular Free Radical Formation

To first demonstrate the anti-DMPO antibody specificity, the DMPO and antibody controls
were evaluated. To control for DMPO in the sample, an impacted sample receiving no
DMPO was stained with an anti-DMPO antibody. To assess non-specific staining, sections
were taken from a DMPO exposed sample with impact injury. One slide was used as a
no primary control and the other was stained with anti-DMPO. The no DMPO and the
no primary had little to no staining compared to the anti-DMPO sample, Figure 1a. As a
positive control for free radical damage to the ECM, we used media containing DMPO with
either 100 μM myoglobin and H2O2 or 100 μM each of myoglobin, H2O2, and iron sulfate.
Iron sulfate was added to enable a greater flux through the Fenton reaction, which generates
HO•. By increasing the flux of possible free radicals generated with this highly reactive
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primary radical, we were able to cause biomolecular free radical formation on the ECM
without impact, Figure 1b.

To validate the reproducibility of IST resulting from impact injury, serial sections from the
same injured specimen were stained. Following a 70 mM DMPO pre-exposure, 2 J/cm2
impact injury, and return to DMPO media for 24 h incubation, the DMPO staining pattern
along sites of ECM damage was remarkably consistent between serial sections, Figure 1c.
This supports that positive staining is not a product of artifactual stain retention at damaged
sites. Throughout the impacted specimens, we observed increased DMPO staining indicative
of increased biomolecular free radical formation at sites of visible cracks or tears in the
ECM, Figure 1d. While cartilage from both the femur and the tibia showed increased DMPO
staining after 2 J/cm2 impact, representative images and scoring support more intense and
extensive staining in the ECM failure in the impacted femur than the impacted tibia, Figure
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1e. DMPO staining was reproduced after injury in rat tails, abundant in type I collagen,
Figure 1f. These increases in DMPO staining after injury support the hypothesis that ECM
damage increases biomolecular free radical production in the extracellular space in tissue.

To assess how widespread free radical formation might be among different extracellular
proteins after injury, culture media was collected 24 h after impact and analyzed via Western
blotting for anti-DMPO reactive, IST products. Media from six osteochondral plugs per
group were pooled in the following groups: impacted no DMPO, impact DMPO myoglobin
(myo)/H2O2/iron sulfate, impact DMPO, and no impact DMPO. All groups had similar
amounts of protein visualized by Ponceau S staining, Figure 2a. The anti-DMPO western
blot showed little-to-no staining in the impact without DMPO lane supporting the selectivity
of the antibody for DMPO. The impact DMPO myoglobin (myo)/H2O2/iron sulfate lane had
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strong staining as expected. The impact DMPO also demonstrated much darker staining than
the no impact DMPO, Figure 2a. This signal from a variety of proteins in the culture media
24 h after injury supports the hypothesis that protein radicals are formed by cartilage injury.

To determine if biomolecular free radicals are produced at the time of impact injury or
in the subsequent 24 h, we treated samples with DMPO prior to impact or immediately
following impact, Figure 2b. By pre-exposing samples, the DMPO is available to react
with free radicals generated at the time of impact, whereas exposure after impact captures

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free radicals formed in the subsequent 24 h. Increased DMPO staining was observed in
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the pre-exposed groups, 1 h and 24 h pre-exposure, Figure 2c and d, while post-exposure

produced staining similar to controls receiving no DMPO. This suggests that the impact
injury itself contributes the greatest proportion of the biomolecular free radicals DMPO
staining observed compared to contributions taking place after impact.

Injury Causes Extracellular Free Radical Formation by Cell-Dependent and -Independent

Mechanisms
To determine whether chondrocytes were contributing directly to the DMPO staining, some
specimens were incubated in 70% ethanol for 2 h to induce chondrocyte death. The ethanol
media was removed, and all the samples received fresh media and were incubated overnight
prior to injury to ensure no residual ethanol remained prior to impact. Chondrocyte death
was confirmed using confocal microscopy, Supplemental Figure 2. Specimens were pre-
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exposed with DMPO for 1 h prior to impact. As anticipated, viable chondrocytes (open
arrow Figure 3a) had DMPO staining whereas the ethanol treated lacked cellular staining.
The extracellular DMPO staining was evaluated in the control impact group compared to the
ethanol treated impact group, Figure 3a. Ethanol treatment decreased the DMPO staining by
50%, Figure 3b. These findings suggest that both cell-dependent and -independent sources
of biomolecular free radicals are present after injury to articular cartilage.

Biomolecular Free Radicals at Sites of ECM Failure are Responsive to Manipulations of the
Local Redox Environment
Because aging, obesity, and a large number of other factors are associated with modulation
of the response to injury and arthritis, we wanted to explore how a controlled modulation of
the local redox environment in a manner consistent with previously published observations
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might alter free radical formation after injury[12, 13]. We treated the culture media with
heparin, hypothesizing that a bolus addition of heparin would cause a variety of disruptions
in cartilage biology reminiscent of arthritis including: disruption of proteoglycan; liberation
of local growth factors and proteinases from the tissue; and disruption of the heparin binding
domain of SOD3. These changes are not meant to model the sum total of arthritis in
cartilage so much as demonstrate that modulating tissue redox environs will significantly
impact the free radical production observed.

To support that heparin was inducing relevant changes in redox biochemistry, IHC analysis
of SOD3 after heparin treatment revealed significantly decreased SOD3 compared to
controls, Figure 4a. To compare our manipulation with prior results[12, 13], we assessed
3-NT-modified protein staining similarly to the cited studies. Heparin treatment resulted in
3-NT staining of a distinct, localized, punctate pattern in the ECM of unimpacted specimens,
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Figure 4b compared to impacted tissue in Supplemental Figure 3. This pattern was very
similar to published results of SOD3 deficient OA patient samples[13]. After impact, the
heparin increased IST at sites of minor ECM damage and at the edges of the cracks
themselves, but the tissue further away from the cracks appeared less intensely stained
compared to controls, Figure 4c.

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To provide more direct quantitation of the specific extracellular changes observed at sites
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of cartilage failure, we applied custom-designed computer algorithms that rely upon crack
traces, Figure 5a, to determine the intensity profile along the length of each individual
crack identified in our experiment. IST images were inverted, Figure 5b, and the cells
near the cracks were masked to avoid cells contributing to the signal, arrows. From each
traced crack, perpendicular vectors were generated along the length to collect intensity
values within distance ranges of 0–5, 5–10, 10–15, and 15–20 μm, Figure 5c. Under normal
conditions, IST appears uniform across the different distances after injury but disruption of
SOD3 localization concentrates biomolecular free radical formation immediately adjacent
to cracks in the tissue, Figure 5d. While heparin is not a specific or direct manipulation
of specific free radical species, we note that this supports the hypotheses of prior authors
that SOD3 may have a role in preventing damage to cartilage, in this case by preventing
accumulation of free radical damage at sites of matrix failure.
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DISCUSSION:
Though previous research has focused on specific ROS reactions within cartilage, we have
chosen the broader indicator of oxidative damage, biomolecular free radical formation
evaluated by IST. Here, that lack of specificity to any one free radical is a strength insofar
as the large variety of biomolecular free radicals that might be generated by ECM failure
is too great to be captured by measuring an individual species. Further, each specific free
radical’s individual concentration may be too low to measure accurately. Visualized in
the western blot, impact increased a variety of biomolecular free radicals in the media
supporting the idea of broad free radical damage to macromolecules occurring from impact.
Thus, assessing the formation of all biomolecular free radicals via IST enables a wide view
of oxidative injury spatially in tissue after trauma which other means of detection lack.
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This study revealed that the mechanical trauma itself may be a direct initiator of oxidative
damage to tissue. Our results support the hypothesis that areas of increased ECM damage
have increased biomolecular free radical formation.

There are a variety of strengths and specific implications from this study demonstrating
free radical formation after trauma in cartilage. Serial sectioning revealed a consistent
DMPO staining pattern between adjacent sections, supporting the robustness and usefulness
of the technique. This demonstrates that the staining does not result from an artifact of
stain trapping, which is stochastic and does not reproduce between sections. Reproducible
staining increases with ECM failure, anatomic site, and in the presence or absence of
treatments suggest an important extracellular biochemistry may be at work to preserve
articular cartilage components after mechanical damage.
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Although DMPO adducts are increased after impact, the western blot demonstrates that a
variety of proteins are adducted to DMPO supporting the importance of not looking for
a single free radical target. ECM damage increased DMPO staining after impact, whereas
impact without failure of the tissue did not seem to increase extracellular DMPO staining.
Though we have not measured or addressed differences in energies of impact, the presence
or absence of ECM damage is likely to dominate extracellular IST signal and higher energy
impacts are much more likely to cause ECM failure in this model system. Among the variety

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of tissues impacted in our model, we noted intensity differences between the femur and tibia
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that could be driven by the differences in material properties of either site or other physical
properties of the tissue like water or proteoglycan content.

Ethanol treated articular cartilage showed decreased DMPO staining at sites of ECM
failure compared to the live tissue. The ethanol treated specimens still produced visible
DMPO staining, suggesting the impact itself can induce biomolecular free radical formation
independent of the presence of live cells. However, some noteworthy diminishment of the
signal by ethanol suggests a distinct cellular contribution to the extracellular free radical
biochemistry. We hypothesize that the physical breakage of collagen fibers themselves could
be the source for the initiation of events leading to cell-independent free radical formation of
ECM. This appears to then blend with intracellular redox biochemistry from cell-dependent
events to yield the high amounts of radical formation observed.
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Though we did not manipulate SOD3 directly, our exploration of disrupting normal
redox function during ECM failure is indirectly supportive of previous findings which
demonstrated that SOD3 plays a role in cartilage homeostasis. Large animal tissue
is required for these extracellular injuries and explorations, limiting our ability to
transgenically manipulate SOD3. Further, because of its density and charge, cartilage greatly
limits diffusion of large protein complexes like SOD enzymes. Available SOD mimetics
participate in other redox reactions and we are also unsure of how the high concentration of
DMPO might impact these reagents and determinations. Thus, we chose to use heparin as
a means of altering the cartilage environment to mimic features of OA observed by Regan
et al, such as decreased SOD3, increased 3-NT in the ECM, and decreases in proteoglycan,
Supplemental fig 4. Our results build upon existing chronic literature on SOD3 and imply
it could be important to mediating the acute extracellular redox biochemistry observed[12,
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13]. Conversely, samples without heparin treatment showed a more uniform distribution
of DMPO staining over a wider area, suggesting that oxidative damage may be spread
out over a larger area, away from cracks. Whether this change is directly dependent upon
specific species or not, our data suggest a concentration of oxidative damage immediately
adjacent to cracks in unhealthy tissue with redox characteristics similar to OA cartilage.
This high concentration of oxidation might cause accelerated local degeneration if it results
in more significant production of ECM breakdown products associated with pro-catabolic
cascades. Future studies will explore more specific manipulations of the extracellular redox
environment targeting O2•−. We speculate that SOD3 may be serving a protective function,
preventing accumulation of high levels of damage at one location.

Damage to the collagen network is slow to repair because collagen synthesis in humans
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slows down substantially after reaching skeletal maturity[30]. We hypothesize that the
damage created by cracking or injury might be a contributor to PTOA. This might
operate though damaged pieces of cartilage, creating fibronectin fragments and other
well-recognized sources of inflammation to contribute to disease. Chronically, recurring
or ongoing damage at any sites like those described here represent an additional contributor
to joint degeneration after injury. Further study is required to better understand the role
of damage-induced free radical formation in the pathology of OA; nonetheless, this study

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 Hines et al. Page 12

demonstrates a new form of molecular damage to cartilage from injury resulting in free
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radicals that can be directly measured in articular cartilage ECM.

Supplementary Material
Refer to Web version on PubMed Central for supplementary material.

ACKNOWLEDGEMENTS:
In addition to our funders at NIAMS, we would like to acknowledge the gift of the anti-DMPO antibody from
Ronald Mason, PhD, National Institute of Environmental Health Sciences. The University of Iowa Orthopedics
Department for ongoing support, and the Orthopedic Histology Service Center for their outstanding technical
support.

Funding Sources:
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This work was supported by the National Institute of Arthritis and Musculoskeletal and Skin (AR070914) and the
National Institutes of Health [R01 DK124510-01 and R01 HL153532-01A1].

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Figure 1: Increased DMPO staining in ECM after injury suggests increased extracellular
biomolecular free radical formation after injury.
a) Representative sections from no DMPO sample stained with anti-DMPO antibody, and
sections from a DMPO sample with no primary antibody and with anti-DMPO antibody.
The samples without DMPO or without primary antibody had minimal staining compared
to the anti-DMPO stained sample. Scale 200 μm. b) Samples were co-incubated with
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DMPO, H2O2, and myoglobin alone or myoglobin and iron acetate. The open arrows
denote chondrocytes. The pink chondrocytes are negative for DMPO in the no primary
sample whereas the brown chondrocytes seen in the treated samples are positive for DMPO
(arrows). The myoglobin and iron treated samples generated ECM free radicals without
impact, a positive control of free radical damage to the ECM. Scale 50 μm. c) Assessment
of stain reproducibility by using serial sections of a 2 J/cm2 impacted femoral explant with
70 mM DMPO. Scale 100 μm. d) Representative images of extracellular DMPO staining
demonstrate increased DMPO staining after the 2 J/cm2 impact compared to non-impacted

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tissue. Scale 100 μm. e) Blinded scoring of IST images demonstrated increased extracellular
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DMPO staining between the impacted femur and tibia. IST stain scoring was higher in the
femur compared to the tibia. *p < 0.05 Kolmogorov-Smirnov test, κ = 0.59, n = 4. f) DMPO
staining is observed in ECM of collagen fibers from rat tails after injury. Collagen fibers
which were cut with a scalpel had increased staining compared to the uninjured control.
The samples which were manually torn had the most intense staining in all the groups.
These results support biomolecular free radical formation as a result of ECM failure is not
restricted to articular cartilage injury. Scale 200 μm.
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Figure 2: Extracellular biomolecular free radical production at the time of impact compared to
the subsequent 24 hours.
a) Anti-DMPO western blot of culture media 24 h after impact injuries. The impacted
DMPO treated samples had more staining than the unimpacted samples, supporting ECM
radical formation increases with impact injury. b) Schematic of how exposures were
arranged. By staggering the timing of DMPO incubation prior or post injury the timing
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for biomolecular free radical formation can be determined. c) DMPO incubation prior to
injury has increased DMPO staining at sites of ECM failure (black arrows) compared to
DMPO incubation after injury. Scale 200 μm. d) Semi-quantitative scoring demonstrated the
pre-exposed samples did have more DMPO staining than the control and post exposure. Due
to the low n value significance was not reached, p = 0.057. *p < 0.05 Kolmogorov-Smirnov
test, κ = 0.15 n = 4.

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Figure 3: Extracellular DMPO staining is associated with viable chondrocytes.

a) Representative images illustrate increased extracellular staining (black arrows) in the
viable chondrocyte sample compared to the ethanol-treated samples. This suggests that
viable chondrocytes (open arrows) contribute to DMPO staining at sites of ECM failure.
Scale 200 μm. b) Histological scoring of the images indicates viable chondrocytes contribute
to increased DMPO staining at sites of failure. The scores also suggest that since removing
the cells does not return the staining to baseline, the impact itself is also a contributor to the
DMPO staining intensity. *p < 0.05 Kolmogorov-Smirnov test, κ = 0.33, n = 4.
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Figure 4: Heparin treatment delocalizes SOD3, produces extracellular 3-NT, and increases
DMPO staining at site of failure.
a) Representative images demonstrate that SOD3 is present in the articular cartilage of
our swine samples (back arrows) and that heparin treatment decreased SOD3 localization
compared to untreated in the absence of injury. Scale 100 μm. b) Confirmation of
extracellular 3-NT punctate staining (yellow arrows) observed in the ECM of heparin
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treated samples without injury. Red indicates safranin O counterstain and pink indicates
anti-3NT staining. Scale 50 μm. c) The staining pattern surrounding ECM failure was
different between the DMPO and heparin DMPO treated samples. The staining appeared
sharper around the failure in the heparin DMPO group compared to the DMPO group. Scale
50 μm.

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Figure 5: Delocalization of SOD3 increases biomolecular free radical formation at the site of
failure but decreases the biomolecular free radical formation radiating from the failure.
a) Using a custom MATLAB program, the cracks were traced. b) The image was inverted.
The cells near the traced lines were masked (arrows) to avoid cellular contribution to
calculations of staining intensity. c) Vectors perpendicular to the traced crack were generated
along the length of the crack. Each vector was divided into distance ranges 0–5 (white),
5–10 (blue), 10–15 (red), 15–20 (pink) μm. Each vector had an average and maximum
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intensity reported. d) The difference in average intensity between the DMPO (blue) and
Heparin DMPO (red) treated groups were significantly different at distances greater 5 μm
from the crack. This suggests that the delocalization of SOD3 prevents biomolecular free
radicals’ formation farther into the tissue. This is supported by the maximum intensity being
highest within the first 5 μm and the maximum intensities decreasing farther from the crack.
p < 0.05 scale 50 μm. n = 17 DMPO, n = 27 Heparin DMPO.
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