Mesenchymal Stem Cell Secretome Ameliorates Hyperalgesia in a Post-operative Pain Model

Background The treatment and management of post-operative pain remain a signicant challenge. There is a signicant unmet need that requires novel therapeutics. The aim of this study was to investigate the effects of the mesenchymal stem cell secretome (MSC-S) in a rat incisional post-operative pain model. Methods A post-operative pain model in rats was used to assess the human adipose-derived MSC-S therapeutic benets. Male Sprague Dawley (SD) rats (n=40) underwent surgery whereby a 1 cm longitudinal incision was made over the plantar surface of the right hind paw where the plantaris muscle was incised longitudinally. The incision was closed with two stitches. At 1-hour post-surgery, test and control articles were administered topically for 15 minutes to the incision site. Von Frey and hotplate testing were conducted to assess mechanical and thermal pain responses, respectively. Statistical analysis of thermal and mechanical withdrawal thresholds was undertaken using an ordinary 2-way analysis of variance (ANOVA) utilising uncorrected Fishers LSD test with individual variances computed for each comparison. of thermal and mechanical withdrawal thresholds was undertaken using a repeated measures 2-way analysis of variance (ANOVA) with the Geiser-Greenhouse correction utilising Dunnett’s Multiple comparison test with individual variances computed for each


Abstract Background
The treatment and management of post-operative pain remain a signi cant challenge. There is a signi cant unmet need that requires novel therapeutics. The aim of this study was to investigate the effects of the mesenchymal stem cell secretome (MSC-S) in a rat incisional post-operative pain model.

Methods
A post-operative pain model in rats was used to assess the human adipose-derived MSC-S therapeutic bene ts. Male Sprague Dawley (SD) rats (n=40) underwent surgery whereby a 1 cm longitudinal incision was made over the plantar surface of the right hind paw where the plantaris muscle was incised longitudinally. The incision was closed with two stitches. At 1-hour post-surgery, test and control articles were administered topically for 15 minutes to the incision site. Von Frey and hotplate testing were conducted to assess mechanical and thermal pain responses, respectively. Statistical analysis of thermal and mechanical withdrawal thresholds was undertaken using an ordinary 2-way analysis of variance (ANOVA) utilising uncorrected Fishers LSD test with individual variances computed for each comparison.

Results
A statistically signi cant increase in thermal nociceptive threshold (analgesia) was observed in the high dose MSC-S group when compared to vehicle at 1.5 hours (p<0.0001) and 3 hours post-surgery (p<0.0001). High dose MSC-S had a more potent analgesic effect than low dose MSC-S. A statistically higher analgesic effect was observed in the high dose MSC-S group compared to morphine at the 3 hours post-surgery hot plate assessment (p<0.002). Morphine (10mg/kg) administration resulted in a signi cant increase in hot plate withdrawal threshold compared to vehicle at 1.5 hours (p<0.0001) and 3hours (p<0.008) post-surgery. Animals that received morphine displayed signi cantly less allodynia than vehicle control animals at 1.5 hours post-surgery (p<0.0001).

Conclusions
The present study demonstrated that the MSC-S topically administered in a rat post-operative pain model signi cantly improved thermal nociceptive thresholds. The MSC-S has potential as a non-opioid based therapy for the treatment of acute post-operative pain.

Background
The prevalence of post-operative pain varies from 10-80% in patients following surgery (1)(2)(3)(4). Acute post-operative pain is caused by post-surgical trauma resulting in an in ammatory reaction and hyperalgesia (5,6). Hyperalgesia at the region of the surgical trauma is mediated by sensitisation of the peripheral afferents and hyperexcitability of A-Delta and C-bre nociceptors (5).
Although post-operative pain is a phenomenon normally described in acute settings, recent studies have demonstrated that poorly managed post-operative pain can lead to chronic pain (1). In this scenario, chronic pain can develop via central sensitisation, where higher brain centre systems play a role in the development and persistence of post-operative pain long after the trauma has healed (5).
Numerous biopsychosocial factors can contribute to acute post-operative pain (7). Various interventions are currently utilised to mitigate the factors that exacerbate exaggerated pain states and achieve effective post-operative pain management, including access to acute management teams, patient education, balanced use of analgesic medication, and regular pain assessments to enable personalised care (7).
Currently, acute post-operative pain management involves initial opioid analgesic administration.
However, physicians aim to minimise the duration of opioid use to avoid common adverse events associated with opioid use (8). These strategies have resulted in decreased patient morbidity and an increase in patients' physical and mental state post-surgery, thereby reducing the risk of chronic pain development (5,9). Nevertheless, there is a need for more effective post-operative pain treatments. This is because despite the improvements in post operative pain management 50% percent of patients remain sub-optimally treated (10). Ineffective post-operative pain management leads to increased morbidity, decreased quality of life, delayed recovery, prolonged opioid use, and higher healthcare costs (4).
Effective treatment of acute post-operative pain can reduce psychological stress contributing to central sensitisation seen in chronic post-operative pain patients (5). Therefore, an alternative, safe, preferably non-opioid analgesic treatment is urgently needed to address this signi cant unmet need.
Adipose-derived mesenchymal stem cells (AdMSCs) are multipotent adult stem cells with signi cant therapeutic potential in various indications. It is now accepted that MSCs do not exert their therapeutic effect via their differentiation potential but rather via direct cell-cell contact and through paracrine effects of their bioactive secretome (11). These MSC-S contain a myriad of bioactive substances, many of which have been examined for their antinociceptive and antiallodynic qualities in isolation and are hypothesised to contribute to the MSCs reversal and prevention of the induction of neuropathic pain mechanical allodynia and thermal hyperalgesia . However, further investigation is warranted to understand the MSCs mechanism of action via their MSC-S factors. Therefore, we aimed to conduct the rst study to investigate the bene ts of the adipose-derived MSC-S in a rat incisional acute post-operative pain model, with a benchmarking of the analgesic effect to a robust dose of subcutaneous morphine. We hypothesised that topical application of MSC-S in the post-operative model of pain in rats would create signi cant non-opioid dependent analgesia.

ADMSC growth and Secretome Production
Adipose tissue was acquired from an HREC approved procurement process under ethics approval number 2014-01-006. Adipose tissue was digested with collagenase as per (41), washed, and frozen as a stromal vascular fraction (SVF) in liquid nitrogen.
AdMSCs were thawed and isolated via plastic adherence in tissue culture asks. AdMSC identi cation was performed as per ISCT guidelines (42)  was collected, centrifuged at 4000g to remove cell debris, and a portion frozen at -80 C to produce the low dose MSC-S group. The remaining portion was concentrated 5x using a 3kDa Tangential Flow Filtration (TFF) (Sartorius, Germany) to produce the high dose group before freezing at -80 C.

Study Design
The rat incision induced post-operative pain study was conducted by MD Biosciences Ltd (Weizmann Science Park Ness Ziona, Israel).

Animals
This study was performed following approval of an application form submitted to the Committee for Ethical Conduct in the Care and Use of Laboratory Animals by MD Biosciences Ltd stating that the study complies with the rules and regulations of the ethics committee from MD Biosciences.
A total of 40 male Sprague Dawley (SD) rats weighing 180 to 200g (Envigo RMS (Israel), Ltd) were used for this study. Animals were acclimatised for at least ve days and provided ad libitum with a commercial, sterile rodent diet and free access to drinking water. Environmental conditions were maintained at 17-23 C with a relative humidity of 30-70%, a 12:12 hour light: dark cycle and 15-30 air changes/h in the study room. Animals were randomly assigned to experimental groups. Each dosing group was kept in a separate cage to avoid cross-contamination, which can occur through the consumption of faecal matter. At the end of the study, animals were euthanised by sodium pentobarbital.

Induction of Post-Operative Pain
All animals were anesthetised by ketamine/xylazine solution. Under anaesthesia, a 1 cm longitudinal incision over the plantar surface of the right hind paw was performed, and the plantaris muscle was incised longitudinally. Following the surgery, the incision was closed with two stitches.

Treatment Administration
At 60 min post-surgery, 1ml of either vehicle, low dose MSC-S or high dose MSC-S were applied topically on a soaked gauze pad placed on top of the incision for 15 min. Morphine (10 mg/kg; Group 2) was administered intraperitoneally 30 min post-surgery.

Behavioural Testing
Von Frey lament test for mechanical induced hyperalgesia and allodynia: Von Frey bres were used to measure mechanical hyperalgesia and allodynia. Each rat was placed in an enclosure and positioned on a metal mesh surface but allowed to move freely. The test began after the cessation of exploratory behaviour. Fibres used ranged from 1.65 to 6.65 in size and 0.008 to 300 g force applied.
Hotplate test for assessing thermal hyperalgesia: Thermal hyperalgesia was tested using a hotplate apparatus. Animals were placed on a hotplate apparatus (57±1ºC), and the time until a rst response was recorded for both legs. The maximal time of testing was set at 12 s.

Statistical Analysis
Statistical analysis of thermal and mechanical withdrawal thresholds was undertaken using a repeated

MSC-S creates dose-dependent alleviation of thermal postoperative pain in the rodent model
The POP model caused signi cant thermal hypernociception at 1.5 hours, as demonstrated by the vehicle control group where baseline measurements are signi cantly higher than 1.5 hours (p<0.0001) and 3 hours (p<0.05), which resolved by 5 hours (Figure 2). Treatment with all interventions caused signi cant attenuation of the hypernociception at 1.5 hours with high dose MSC S and morphine sustaining analgesia until 3 hours post-surgery ( Figure 2). Hotplate measurements indicate a highly signi cant effect from the high dose MSC-S on thermal nociceptive thresholds at 1.5 hours (p<0.0001) and 3 hours post-surgery (p<0.001) when compared to Vehicle at the corresponding timepoint. A similar result was observed following morphine administration at 10 mg/kg (p-values of <0.001 and p<0.04 at 1.5 and 3 hours, respectively). However, treatment with high dose MSC-S resulted in signi cantly higher thermal nociceptive thresholds than morphine at 3 hours post-surgery (p<0.04). Signi cantly higher withdrawal thresholds were recorded in animals at 1.5 hours post-surgery in the low dose MSC-S group when compared to Vehicle (p<0.001).

MSC-S failed to alleviate post-operative mechanical allodynia in the rodent model
The POP model induced signi cant (p<0.0001) mechanical allodynia from 1.5 hours that was maintained throughout the observation window. Only morphine signi cantly attenuated this mechanical allodynia at 1.5 hours post-surgery (p<0.03).
Whilst rats that received morphine displayed signi cantly less allodynia than vehicle control mice at 1.5 hours post-surgery, this effect was not long-lasting ( Figure 3). There were no other statistically signi cant differences in withdrawal threshold at any timepoint. No recovery to uninjured baseline withdrawal thresholds was observed at the 5-hour timepoint.

The post-operative rodent model does not impact animal body weight
Bodyweight measurements (Figure 4) were taken 1 hour prior to surgery. Body weights were not signi cantly different between groups as analysed using ordinary 2-way ANOVA using uncorrected Fishers LSD test. Bodyweight measurements indicate no variation between test groups indicating maintenance of animal welfare prior to the model being conducted.

Multiplex protein analysis of MSC-S reveal the complex bioactives present
The complex bioactives secreted by MSC that hypothesised to produce the physiological actions of MSC have been explored previously (43) and (38,44). This underlies the complex nature of the physiological activities of MSCs. A multiplex protein analysis was performed to provide information on the nature of the complex mixture of MSC-S utilised here. Our MSC-S contained detectable amounts of bioactive proteins, including IL-6, IL-8, MCP-1, VEGF-A, HGF, SDF-1a, TNF-R1 and TIMP-1 ( gure 5).

Discussion
This study demonstrates that the MSC-S, when administered topically to rats following a hind paw incision, signi cantly affects thermal withdrawal latencies and, hence, hyperalgesia. Importantly, these antinociceptive actions were equivalent to 10 mg/kg morphine. To our knowledge, this is the rst report of the pain-relieving effects of the MSC-S on post-operative pain.
Post-operative pain arises as a result of an in ammatory reaction caused by post-surgical trauma. During the normal injury process, an in ammatory response involving increased proin ammatory cytokine release at injury sites to promote immunoregulation and immune cell recruitment occurs (42). Although proin ammatory cytokine release can be bene cial for wound healing (42), it can contribute to the pathogenesis of pain as TNF-α and IL-1 cause rapid activity in C bre and A-Delta nociceptors reducing mechanical activation thresholds (45). It has been demonstrated that IL-1 exposure to dorsal root ganglion induces spontaneous acute hyperexcitability of peripheral nociceptors and heat-induced hyperalgesia through sensitisation of the capsaicin and heat-sensitive cation channel TRPV1 receptors (45). The behavioural effects of this process resulting from the post-operative pain model were observed in this study, with heat-induced hyperalgesia being evident in Vehicle control animals. Our results illustrate a signi cant effect of topical administration of the high dose MSC-S in prolonging thermal latencies. However, no effect was observed in mechanical allodynia assessed by Von Frey testing. This is an intriguingly speci c effect of the MSC-S and suggests the complex bioactives in the MSC-S either directly preferentially block the hyperexcitability in discrete thermal sensitive afferents, or reduces the generation of the endogenous factors that create the thermal rather than the mechanical sensitising agents. The complexity of this redundancy and parallel signalling pathways is exempli ed in the recent pain interactome published by Science Signalling (46). Future studies examining these speci c receptor and ligand effectors are warranted to identify which and how.
The MSC-S contains a milieu of chemokines, cytokines, growth factors and extracellular vesicles that promote angiogenesis and have anti-in ammatory properties ( Figure 5) through T-cell suppression and immunoregulation (17,21,25,28,38). The potential therapeutic bene ts of the MSC-S are well reported.
However, given the duration of this study and the rapid effect of the MSC-S on thermal latencies, T cell suppression may not be the main mechanism of action due to the immediate response with a topical application. We postulate that molecules such as IL-1Ra, HGF, and TIMP-1 contained within the MSC-S may be responsible for the effects seen. IL-1Ra has also been shown to combat IL-1β induced hyperexcitability of peripheral nociceptors responsible for thermal hyperalgesia development (47)(48)(49).
HGF and TIMP-1 were present in high MSC-S ( Figure 5) levels and have been demonstrated to exert analgesic and immunoregulatory effects (50,51). HGF may inhibit pain mediating genes in sensory neurons via interaction with c-MET receptors causing acute anti-hyperalgesic responses driven by the MSC-S (50). TIMP-1 has been demonstrated to attenuate in ammatory pain through matrix metalloproteinase inhibition and receptor-mediated cell signalling (51). IL-1Ra levels in the MSC-S were not measured in this study. Future studies will include an analysis of IL-1Ra levels in the MSC-S and blocking experiments to further investigate the role of key cytokines in pain. The upregulation of IL-1Ra secretion from MSCs under in ammatory stimulation has implications in pain-speci c pathways and plays an important role in pain reduction and regulation (47)(48)(49).

Conclusions
The present study demonstrated that the MSC-S signi cantly improved thermal nociceptive thresholds when administered topically in a rat post-operative pain model.
Further investigation is required to elucidate the mechanism of action of the MSC-S. Speci cally studies using cytokine depleted MSC-S and further immunological analysis through mRNA analysis at the site of injury would be bene cial to elucidate the effects of the MSC-S on hypernociception. A dynamic plantar aesthesiometer could be used in future studies due to increased sensitivity and repeatability compared to manual Von Frey bres for the assessment of mechanical allodynia. The MSC-S has potential as a nonopioid based therapy for the treatment of acute post-operative pain. Ethics approval and consent to participate Adipose tissue was obtained with informed consent under ethics approved program, approval number, 2014-01-006. The animal study was performed following approval of an application form submitted to the Committee for Ethical Conduct in the Care and Use of Laboratory Animals by MD Biosciences Ltd and stated that the study complies with the rules and regulations of the AEC.

Consent for publication Not Applicable
Availability of data and materials The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Competing interests
Graham Vesey is a shareholder and executive director of Regeneus Ltd.

Funding
Funding was provided from Regeneus Ltd. MRH is the recipient of an ARC Future Fellowship (FT180100565).
Authors' contributions CM interpreted data and wrote the manuscript. GV, SB and FM revised the work and manuscript. MH contributed to the manuscript and data analysis. signi cantly different from Vehicle at the same timepoint *** p<0.001 signi cantly different from Vehicle at the same timepoint **** p<0.0001 signi cantly different from Vehicle at the same timepoint # p<0.04 Signi cantly different from Morphine at the same timepoint