Additional benefits of combined ceftriaxone and adipose-derived mesenchymal stem cells on revamping the outcomes in rodent after acute spinal infection

doi:10.21203/rs.3.rs-5310715/v1

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Additional benefits of combined ceftriaxone and adipose-derived mesenchymal stem cells on revamping the outcomes in rodent after acute spinal infection

https://doi.org/10.21203/rs.3.rs-5310715/v1

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This study tested whether combined ceftriaxone and adipose-derived mesenchymal stem cells (ADMSCs) would defend the spinal cord against acute spinal infection (ASI) in rodent. Adult-Male-SD rats were grouped into groups 1 (SC)/2 (ASI)/3 (ASI + ceftriaxone from days 2 to 28 after ASI induction)/4 (ASI + allogenic ADMSCs from day 2 for a total of 3 doses/3 consecutive intervals by intravenous injection)/5 (ASS + combined ceftriaxone and ADMSC) and spinal cord tissues were harvested by day 28. Circulatory levels of TNF-α/IL-6 at days 7 and 28, and these two parameters in spinal fluid at day 28 were lowest in group 1, highest in group 2, significantly lower in group 5 than in groups 3/4, and significantly lower in group 3 than in group 4 (all p < 0.0001). The day-28 bacterial colony formation unit (CFU) in vertebral bone and circulatory WBC counts at the time points of days 7/14/28, and the protein expressions of upstream (TRL-2/TLR-4/MYD88/TRAF6/IKKα/IKKβ /IKBβ/p-NF-κB) and downstream (IL-1β/IL-6/TNF-α/IFN-γ/iNOS) inflammatory signalings displayed a similar pattern of inflammatory biomarkers in spinal fluid among the groups (all p < 0.0001). By day 28, the bone injury score/bone marrow density/ratio of bone volume (BV) to the bone tissue volume (TV)/ratio of bone surface (BS) to BV/ratio of BS to bone TV/trabecular number exhibited an opposite, whereas the trabecular space exhibited an alike pattern of inflammatory biomarkers among the groups (all p < 0.0001). Combined ceftriaxone and ADMSCs therapy offered an additional benefit on protecting the vertebral bone/spinal cord against ASI damage.

acute spinal cord infection

antibiotics

mesenchymal stem cells

inflammatory reaction

Spinal infection (SI) is an infectious disease involving the intervertebral disc, vertebral body, and/or adjacent paraspinal tissue (Lener et al. 2018). The SI contributes to 2–7% of all musculoskeletal infections in the clinical observation, usually resulting in seriously destructive spinal cord tissues if not treated promptly and properly (Lener et al. 2018). The histopathological findings have identified that the sources of infection can be spread to the spine by hematogenous dissemination from distant site, by diffusion from contiguous tissues, or/and by external inoculation (trauma, surgery) (Lener et al. 2018; Gregori et al. 2019).

The mortality rate of SI has been reported to be 2% and 20%, respectively (Lener et al. 2018; Aagaard et al. 2016; Artenstein et al. 2016; Fantoni et al. 2012). Patients who suffer from SI generally express non-specific symptoms, including back pain (85%), fever (48%), and paresis (32%) (Fantoni et al. 2012; Gregori et al. 2019; Lener et al. 2018). Although treatment strategies of SI remain controversial and sometimes ineffective, use of concomitant antibiotics treatment under non-surgical (i.e., immobilization with rigid orthosis) or surgical managements (i.e., debridement, decompression, etc.) is still the common choice of treatment for SI, depending on different clinical manifestation and disease course as well as the general conditions (Gregori et al. 2019; Lener et al. 2018). Conclusively, the management of SI is still an unmet need that raises the urgency to conduct a comprehensive investigation to establish an effective and innocuous treatment for SI patients.

In most countries, Staphylococcus aureus is still one of the most common pathogens that causes SI. According to the previous reports, the incidence of methicillin-resistant S. aureus (MRSA) accounts from 6.8–30% of pathogens for SI (Gregori et al. 2019; Lener et al. 2018). Such pathogens might further generate complicated process of localized or systemic inflammatory reactions, such as overwhelming immune response, induction of oxidative stress and reactive oxygen species (ROS) as well as over-compensatory inflammatory reaction (Gregori et al. 2019; Lener et al. 2018; Sung et al. 2016). Although use of antimicrobial agents is the gold standard treatment of bacterial-induced infection, the morbidity, mortality, hospital length of stay (LOS), and health costs are still relatively high (Camino Willhuber et al. 2019). This could be mainly due to the reason that antimicrobial agents cannot effectively control the exaggerated immune response and overwhelming inflammatory reaction effectively (Camino Willhuber et al. 2019; Sung et al. 2016). Therefore, in addition to the advisable choice of antimicrobial agents, control of the exaggerated immune response and overwhelming inflammatory reaction may be critical to reduce SI-induced morbidity and mortality (Artenstein et al. 2016; Camino Willhuber et al. 2019).

It is well known that ceftriaxone (Sileshi et al. 2016; Hedlund 2011; Raja et al. 2020) is an antibiotic that pertains to a category of medicine recounted to as cephalosporin antibiotics, and it effectively treats a variety of bacterial infections by ceasing the growth of bacteria in variety of bacterial infections in different organs, including bone and spinal cord bacterial infection. However, growing data have shown that ceftriaxone-resistant Gram-negative bacilli, especially those caused by extended-spectrum β-lactamases (Paterson et al. 2020; Harris et al. 2018) becomes more and more popular in our daily clinical practice.

Not only has abundant evidence showed that stem cell therapy attenuates inflammation and innate/adaptive immune responses (Sung et al. 2016; Le Blanc et al. 2003; Sun et al. 2011), but adipose-derived mesenchymal stem cells (ADMSCs) have also been shown to possess the intrinsic capacity of immunomodulation and tissue regeneration (Sun et al. 2011; Camino Willhuber et al. 2019; Sung et al. 2016). Interestingly, limited investigations suggest that, in the setting of ischemia-reperfusion injury and sepsis, ADMSCs treatment could attenuate morbidity and mortality through regulation of ischemia-reperfusion injury and sepsis-induced exaggerated immune response and overwhelming inflammatory reaction as well as control of ROS and oxidative stress generation (Sung et al. 2016; Krasnodembskaya et al. 2012; Mei et al. 2010). Additionally, our previously experimental studies have demonstrated that ADMSCs therapy effectively controlled sepsis syndrome (SS) (Sung et al. 2016; Chen et al. 2014), whereas combined therapy with antibiotics and ADMSCs even more effectively controlled the SS and reduced the rodent mortality (Sung et al. 2016).

On the other hand, there has been no experimental study that focuses on the therapeutic effect of stem cell therapy compared with that of ceftriaxone antimicrobial agents as well as that when the two strategies are combined in the setting of experimental model of SI. Therefore, we conducted this study to test the hypothesis that ceftriaxone-ADMSC combined therapy was superior to ceftriaxone monotherapy in animal model of microbial-induced SI to attenuate inflammation/oxidative stress as well as morbidity and mortality.

Ethical statement

All animal procedures were approved by the Institute of Animal Care and Use Committee at Kaohsiung Chang Gung Memorial Hospital (Affidavit of Approval of Animal Use Protocol No. 2020121605) and performed in accordance with the Guide for the Care and Use of Laboratory Animals.

Animals were housed in an Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC; Frederick, MD, USA)-accredited animal facility in Kaohsiung Chang Gung Memorial Hospital under temperature (22 ± 1 ◦C), humidity (55 ± 5%) and light (12: 12/light: dark photo-cycle, light on at 08:00) control. Standard laboratory rat chow and tap water were available ad libitum. All animals were allowed to acclimatize to the animal facility for 14 days prior to experimental manipulations.

To create an animal model of acute bacterial spinal vertebral bone infection (ASI) in SD rat

The procedure and protocol were based on the previous study with some modification (Ofluoglu et al. 2007; Dworsky et al. 2017) with some modification. Pathogen-free, adult male Sprague-Dawley (SD) rats, weighing 320-350 g (Charles River Technology, BioLASCO Taiwan Co. Ltd., Taiwan), were used in the present study. In detail, the animals were anesthetized by inhalation of isoflurane, i.e., isoflurane was generally used at concentrations of 5% for induction, and 2 % for maintenance of general anesthesia and 1.0% for recovery stage and placed prone on a warming pad. The hair on the skin on the lower back around the spine was shaved. The skin was incised at the midline of the spine at the T11 - L2 level, and the paraspinal muscles were carefully dissected subperiosteally to expose the lamina. A sterile self-tapping screw (diameter 1 mm, length 4 mm) was screwed into the vertebral bone between T13 - L1, following by unscrewing the above screw, the Staphylococcus aureus subsp. aureus (S. aureus, ATCC® 19636 ™) suspension (5×105 cells in 50μL) was inject into the hole that was drilled by the screw, and then reset the screw. Finally, the incision was sutured, and the animals were left awake on a warm pad at 37°C.

Animal grouping and animals were euthanized

Adult male SD rats (n = 42) were categorized into group 1 [sham-operated control (SC), i.e., merely opening the skin and muscle layer of spinal cord, followed by closure of these two layers over the spinal cord, n = 8], group 2 [acute spine infection (ASI), n = 10], group 3 [ASI + ceftriaxone (5 mg/kg/day by subcutaneous administration over the spinal area from days 2 to 28 after ASI induction, n = 8), group 4 [ASI + allogenic ADMSCs (1.0 x 10⁶ cells/rat from day 2 for a total of 3 doses/for 3 consecutive intervals with 9 days after ASS by intravenous injection), n = 8] and group 5 (ASS + combined ceftriaxone and ADMSC, n = 8), respectively.

The dosage of antibiotics to be utilized in the present study was based on the previous report with some modification (Sung et al. 2016). On the other hand, the dosage of ADMSCs to be utilized in the present study was based on our previous reports with some modification (Chang et al. 2018; Chang et al. 2019).

Euthanasia of animals was performed under anesthesia with overdose of isoflurane inhalation (i.e., more than 5.0%) and collected the blood by more than 10 ml by day 28 after ASI induction. The vertebral bone, cartilage and spinal cord tissues were harvested after the respiratory arrest for individual study.

Methodology for preparing and culturing allogenic ADMSCs

Additional 20 rats were utilized for allogenic ADMSCs isolation. The procedure and protocol for allogenic ADMSC isolation and culture have been described in our previous reports (Fantoni et al. 2012; Sung et al. 2016). Briefly, adipose tissue surrounding the epididymis was dissected, excised and prepared by day 14 prior to acute kidney IR induction. For purification, the harvested cells were cultured in Dulbecco’s modiﬁed Eagle's medium (DMEM)-low glucose medium containing 10% FBS for 14 days. By this time, plentiful ADMSCs (i.e., approximately 2.5 - 3.0 x 10⁶cells) were obtained in the culture plate and were collected to treat ASI animals.

ELISA examination

Collect the circulatory blood samples at days 14 and 28 after ASI induction in each group of the animals for determining the circulating levels of IL-6 and TNF-α levels. Additionally, these parameters of spinal fluid were also collected and measured at the end of study period, i.e., by day 28 after ASI induction. These biomarkers were measured by ELISA standard method was according to the manufacturer’s instruction.

Histopathological examination

By day 28 after ASI induction, the histopathological examination was performed after routine fixation, decalcification, and paraffin embedding for assessment of vertebral bone/cartilage destruction, the bacterial counts in the bone and adjacent region and inflammatory cell infiltration (i.e., polymorphonuclear cells).

Western blot analysis from isolated spinal-cord tissues

The protocol and procedure were described by our previous reports (Chang et al. 2019; Chang et al. 2018; Sung et al. 2016; An et al. 2006). Briefly, equal amounts (50 µg) of protein extracts were loaded and separated by SDS-PAGE using 8-12% acrylamide gradients. After electrophoresis, the separated proteins were transferred electrophoretically to a polyvinylidene difluoride (PVDF) membrane (Amersham Biosciences). Nonspecific sites were blocked by incubation of the membrane in blocking buffer (1X TBS, 0.1% Tween-20 with 5% w/v nonfat dry milk) overnight. The membrane was incubated with indicated primary antibodies Toll-like receptor 2 (TLR2) (1:1000, Abcam), TLR4 (1:1000, Novusbio), tumor necrosis factor receptor associated factor 6 (TRAF6)1:1000, Abcam), phosphorylated nuclear factor (p-NF)-κB (1:1000, Abcam), interleukin (IL)-1ß (1:1000, Cell Signaling), IL-6 (1:1000, Abcam), IKKα(1:5000, Abcam), IKKβ (1:1000, Cell Signaling), IKBβ (1:1000, Abcam), interferon (IFN)-γ (1:4000, Abcam), tumor necrosis (TNF)-α (1:1000, Cell Signaling), inducible nitric oxide synthase (iNOS) (1:1000, Abcam), MYD88 (1:1000, Abcam) and Actin (1:6000, Millipore)] for 1 hour at room temperature. Horseradish peroxidase-conjugated anti-rabbit immunoglobulin IgG (1:5000, Sigma) was used as a secondary antibody for one-hour incubation at room temperature. The washing procedure was repeated eight times within one hour. Immunoreactive bands were visualized by enhanced chemiluminescence (ECL; Amersham Biosciences, Amersham, UK) and exposed to Biomax L film (Kodak, Rochester, NY, US). For the purpose of quantification, ECL signals were digitized using Labwork software (UVP, Waltham, MA, US).

Micro-CT examination of decalcified vertebral/spinal-cord bone

By day 28, i.e., at the end of study period, the animals were euthanized, and the bone-articular joints were harvested from each animal. After well preparation, these specimens were assessed by the Bruker Micro-CT (SkyScan 1176, Bruker BioSpin, Germany, with Bruker CTAn Micro-CT Software for analysis) for verifying the destructive features of the vertebral/spinal-cord bone, respectively.

Definition for BMD, ratios of BV/TV, BS/BV and BS/TV, TN and TS

Bone mineral density (BMD) measures the density of minerals in the bone. It can be used to detect osteoporosis, determine the risk of future fractures, and assess the outcome of osteoporosis treatment.

Ratio of bone volume (BV) to bone tissue volume (TV) (i.e., percentage of bone volume) refers to the proportion of deducted dense bone to bone TV in all tissues. This index is a common index used for evaluating the bone mass of compact bone and spongy bone. An increase in this value indicates that bone metabolism is greater than catabolism and bone mass increases correspondingly, and vice versa, which indirectly reflects the state of bone metabolism. Ratio of bone surface to bone volume (BS/BV) indirectly reflects the amount of bone mass. Ratio of bone surface to bone tissue volume (BS/TV) also indirectly reflects the amount of bone mass.

Trabecular number (TN) refers to the number of intersection points produced by the intersection of the plate-shaped structure and the round rod-shaped structure, and the structure woven by these intersection points is the trabecular bone. When osteoporosis occurs, the TN value will decrease relatively.

Trabecular separation (TS) refers to the average width of the medullary cavity between the trabecular bone. The increase of TS corresponds the increase of bone loss leading to the increase of cavity space between bone trabeculae, so osteoporosis may occur.

Definitions of Classification of bone destructive core and infectious bone deposition

The definitions were based on the previous report (Ofluoglu et al. 2007) with minimal modifications:

(1) Classification of vertebral bone destruction: on a scoring of 1 to 5, indicating that the bone was damaged (i.e., by infection) and could not be repaired by itself, which included the bone marrow destructive space.

(2) Classification of infectious bone deposition: including the severity of scale from 1 to 5, indicating abnormal bone hyperplasia/deposition. Thus, bone injury score was the comprehensive score of cartilage destruction (1) and infectious bone deposition (2) in the current study.

Statistical analysis

Quantitative data were expressed as mean ± SD. Statistical analysis was adequately performed by ANOVA, followed by Bonferroni multiple comparison post hoc test. SAS statistical software for Windows version 8.2 (SAS Institute, Cary, NC, USA) was utilized. A probability value <0.05 was considered statistically significant.

Body weight, mortality rate, and time courses of circulatory level of white blood cell counts (WBC) (Fig. 1)

The ratio of the initial (i.e., by day 0) to final (i.e., by day 28) body weight significantly lower in group 4 (ASI + ADMSC) than in groups 1 (SC), 2 (ASI), 3 (ASI + ceftriaxone), and 5 (ASS + combined ceftriaxone and ADMSC)] and significantly lower in groups 2 and 5 than in groups 1 and 3, but it showed no difference between groups 2 and 5 or between 1 and 3 (n = 8 for each group).

The total mortality rate within 48h prior to grouping was 9.1% (5/55). Animals were groups at day 2 prior to the treatment and the mortality rate after grouping prior to being euthanized by day 28 were 0% (0/8) in group 1, 30.0% (3/10) in group 2 and 0% (0/8) in groups 3, 4 and 5, respectively. Regarding the statistical analysis of the mortality rate, the variables were compared with one-way analysis of variance (ANOVA), followed by Fisher’s least significant difference (LSD) for comparison of the difference between two groups. Difference of alphabetic characters (^{a or b}) in superscript indicates statistical significance of p-value < 0.05 after comparison, p = 0.012 (n = 10 for each group).

The circulating level of WBCs at baseline did not differ among the five groups (n = 8 for each group). However, by day 3 after the ASI induction, the WBC count was significantly lower in group 1 than in other groups, significantly lower in group 5 than in groups 2, 3 and 4. However, this parameter did not differ among the latter three groups. Additionally, by days 7 and 14 after ASI, this parameter was significantly lower in 1 than in other groups, and significantly higher in group 2 than in groups 3, 4, and 5, but it was similar among the groups 3 to 5. However, by days 28 after ASI induction, the WBC count was lowest in group 1, highest in group 2, significantly lower in group 5 than in groups 3 and 4, but it showed no difference between the latter two groups (n = 8 for each group).

Circulatory and spinal cord fluid levels of proinflammatory cytokines and bacterial CFU in vertebral bone and circulation (Fig. 2 and Table 1)

To elucidate whether the ceftriaxone or ADMSCs treatment would suppress the circulating and spinal cord fluid levels of IL-6 and TNF-α, the ELISA methodology was utilized in the present study. The result showed that by day 7 after ASI induction, the circulating levels of IL-6 and TNF-α, were lowest in group 1, highest in group 2, significantly lower in group 5 than in groups 3 and 4 and significantly lower in group 3 than in group 4 (n = 8 for each group). Additionally, by day 28 after ASI induction, the circulating and spinal cord fluid levels of these two parameters displayed an alike pattern as those of day 7 (n = 6–8 for each group).

Additionally, Table 1 illustrated that the bacterial CFU in vertebral bone (n = 6 for each group) was significantly higher in group 2 than in groups 3, 4, and 5, and significantly higher in groups 3 and 4 than in group 5, but it showed no difference between groups 3 and 4. Furthermore, bacterial CFU in circulation (n = 8 for each group) was significantly higher in group 2 than in groups 3 to 5, but this parameter did not differ within these latter three groups.

Protein levels of upstream and downstream inflammatory signalings (Fig. 3)

To verify the impact of ceftriaxone and ADMSCs treatments on alleviating the inflammatory reaction, the Western blot analysis (n = 6 for each group) for the protein levels of parameters were conducted in the present study. The result demonstrated that protein expressions of TRL-2, TLR-4, MYD88, TRAF6, IKK-α, IKK-β, IKB-β and p-NFκ-B, eight upstream inflammatory signalings, were lowest in group 1, highest in group 2, significantly lower in group 5 than in groups 3 and 4, and significantly lower in group 3 than in group 4. Additionally, the protein expressions of IL-1β, IL-6, TNF-α, IFN-γ and iNOS, five downstream inflammatory signalings, displayed an identical pattern among the five groups, implicating that combined Cef and ADMSCs offered additional benefit than merely one on suppressing ASI induced inflammatory reaction.

To clarify the impact of ceftriaxone and ADMSCs treatments on attenuating the bone injury score (Fig. 4)

To verify whether ceftriaxone-ADMSCs would offer benefit on protecting the bone and cartilage against ASI damage, the H&E staining (n = 5–10 for each group) was conducted in the present study. The result showed that vertebral bone destructive score and infectious bone deposition score, i.e., defined as the bone injury score, were significantly higher in group 2 than in other groups, significantly higher in groups 3 and 4 than in groups 1 and 5 and significantly higher in group 5 than in group 1, but it showed no difference between groups 3 and 4.

Micro-CT examination of vertebral/spinal-cord bone for identification of bone/bone marrow damage (Fig. 5 and Table 2)

To verify the ceftriaxone and ADMSCs on protecting the vertebral bone against ASI induced damage, the micro-CT specific for small animals was utilized in the present study (n = 6 for each group). The result showed that the bone marrow density (BMD) was significantly higher in group 5 than in other groups, significantly higher in group 1 than in groups 2 to 4 and significantly higher in group 3 and 4 than in group 2, but it showed no difference between the groups 3 and 4. Our finding implied that combined ceftriaxone and ADMSCs treatment increasing the BMD could be mainly due to the tissue regeneration effect of ADMSCS.

Additionally, the BV/TV bone volume/trabecular volume (i.e., BV/TV), a ratio of bone volume to the trabecular volume, was significantly higher in groups 1 and 5 than in the groups 2 to 4 and significantly higher in groups 3 and 4 than in group 2, but it showed no difference between groups 1 and 5 or between 3 and 4 (n = 6 for each group), implicating that the combined ceftriaxone and ADMSCs was superior to merely one treatment on protecting the bone materials again infection-induced loss.

Furthermore, the bone surface/trabecular volume (i.e., BS/TV), i.e., a ratio of bone surface to trabecular volume reflecting the bone density, and the numbers of trabeculae, an indicator of trabecular bone density, were significantly higher in groups 1 and 3 to 5 than in those of the group 2, but these parameters were similar among the groups 1 and 3 to 5 (n = 6 for each group), implicating that ceftriaxone-ADMSCs therapy significantly protected bone materials against the bacterial infection damage.

Schematically illustrated the underlying mechanism of bacterial spinal cord infection in rodent and the therapeutic impact of ceftriaxone-ADMSCs on ASI (Fig. 6)

Finally, through the results from Figs. 1 to 6, we could graphically depict the underlying mechanistic basis of overwhelming inflammatory reactions in circulatory levels and bone tissue, resulting in vertebral bone and cartilage destruction that were remarkably reversed by ceftriaxone-ADMSCs therapy.

Our present study which investigated the therapeutic effect of ceftriaxone-DMSCs on ASI delivered some striking connotations. First, we successfully created a reproducible animal model of ASI for testing the aftermath of ceftriaxone-ADMSCs on this disease entity and could be extended to the other strategic management of ASI. Second, the result of the current study demonstrated that ADMSCs were comparable with ceftriaxone for securing the spinal cord against the ASI damage. Third, combined ceftriaxone and ADMSCs therapy was superior to either one on sheltering the spinal cord against the ASI injury. Finally, we found that upstream and downstream inflammatory signaling acted as a foremost role on spinal cord damage in setting of ASI.

Despite various modalities have been reported for the treatment (Artenstein et al. 2016; Camino Willhuber et al. 2019; Gregori et al. 2019; Lener et al. 2018), there is still lacking an unanimously acknowledged effective management that could rationally explained why the morbidity, mortality, hospital length of stay and health costs are still unacceptably high in ASI and chronic SI setting (Camino Willhuber et al. 2019). One important finding in this preclinical study demonstrated that one kind of antibiotic (i.e., ceftriaxone) was identified to be comparable with ADMSCs for the efficacy on safeguarding the spinal cord damage and bettering the outcomes in ASI rodent. The most important finding in this preclinical study proclaimed that combined antibiotic and ADMSCs therapy was superior to just one therapy for boosting the outcomes in ASI rodent. In this way, our findings, in addition to extending the findings from previous studies (Artenstein et al. 2016; Camino Willhuber et al. 2019; Gregori et al. 2019; Lener et al. 2018), highlight that this combined regimen may be a therapeutic novelty for ASI patients, especially for those patients who are refractory to the conventional therapy (Camino Willhuber et al. 2019).

Two essential findings from this current study were that the bacteria in the circulation and derived from vertebral bone were remarkably higher in ASI animals than those of ASI animals after receiving ceftriaxone-ADMSCs treatment. Interestingly, when looked at the findings from H&E staining, we identified that the bone injury score was remarkably increased in ASI animals without than with the ceftriaxone-ADMSCs treatment. It is well known that the bacteria in chronic osteomyelitis patients are always too difficult to be eradicated through antibiotics (i.e., by bactericidal effect) mainly due to the antibiotics are rather difficult to penetrate in the bone parenchyma and could be also due to the bone distinctively complicated structure always serving as a good harbor for bacteria concealed there. Perhaps, our findings (i.e., by H.E. staining) of destructive vertebral bone (i.e., including bone marrow, trabeculae, and cartilage) would serve as a good sanctuary for bacterial occultation.

We were very concerned the underlying mechanism for how ceftriaxone-ADMSCs treatment ensured the spinal cord/vertebral bone in setting of ASI. It is well recognized that MYD88 played the role as an essential innate immune signal transduction adaptor (Deguine et al. 2014). Additionally, plentiful studies have established that this inflammatory-immune signaling takes part in damage of the tissues/organs in organ ischemic settings (Chen et al. 2020; Yang et al. 2021). Intriguingly, when looked at MYD88 innate immune signal, we found that the protein expressions of upstream and downstream inflammation/innate immune signalings were much increased in ASI animals than in SC animals. Our finding was consistent with the findings of previous studies (Chen et al. 2020; Deguine et al. 2014; Yang et al. 2021). Of paramount important finding in the present study was that these molecular-cellular perturbations were noteworthily repressed by ceftriaxone or ADMSCs therapy and further noteworthily repressed by combined ceftriaxone + ADMSCs treatment, highlighting that our finding in addition to extending the findings of previous studies (Chen et al. 2020; Deguine et al. 2014; Yang et al. 2021), encourages the use of these two regimens for those acute or chronic SI or even extrapolates to the treatment of osteomyelitis patients, especially when they are refractory to conventional therapy. We suggest this combination therapy offering great benefits to those of ASI animals and improving their outcomes could be due to the synergic effect of anti-inflammation (i.e., the essential effect of ceftriaxone) and anti-inflammation/immunomodulation (i.e., the principal therapeutic effect of ADMSCs) along with possible tissue regeneration (another therapeutic impact of MSCs).

An especially interesting finding in the present study was that as compared with the ASI animals without treatment, the mortality rate was significantly lower in those of ASI animals treated by ADMSCs. Undoubtedly, MSCs treatment could not directly killed the bacterial. However, when we looked at the circulatory derived CFU, we found that the CUF was markedly reduced in ASI animals with than in without ADMSCs treatment. Additionally, it is well known that systemic inflammatory response syndrome (SIRS) is an exaggerated defense response of the body to a noxious stressor, i.e., such as bacterial infection, trauma, surgery, or acute inflammation…etc. Based on the aforementioned issues, we suggested that perhaps, the mortality rate was notably lower in ASI animals with than in without ADMSCs treatment could be, at least in part, due to the fact that HUCDMSCs has the property of anti-inflammation and immunomodulation (Le Blanc et al. 2003; Sun et al. 2011; Sung et al. 2016).

Scientists would be interesting in understanding the underlying mechanism for how the combined ceftriaxone and ADMSCs could offer synergic effect on protecting the vertebral bone and cartilage against ASI damage. In fact, abundant data have shown that ceftriaxone kills many types of bacteria by suppressing the mucopeptide synthesis of bacterial cell wall. The β-lactam core of ceftriaxone irreversibly binds to carboxypeptidases, endopeptidases, and transpeptidases in the bacterial cytoplasmic membrane (Lamb et al. 2002). Thus, ceftriaxone is characterized as one kind of bactericidal antibiotics. During bacterial infection, especially those of gram-negative bacteria always release endotoxin that would elicit overwhelming inflammatory reaction, enhance innate immune reaction and inflammatory cell infiltration in infectious organs, resulting in activate the inflammatory signalings and generation of proinflammatory cytokines, subsequently destructs the tissues, organs and bone as well as the cartilage. On the other hand, the ADMSCs have been shown to have intrinsic ability of anti-inflammation, immunomodulation and tissue regeneration (Sun et al. 2011; Camino Willhuber et al. 2019; Sung et al. 2016). Accordingly, the above-mentioned issues (Sun et al. 2011; Camino Willhuber et al. 2019; Sung et al. 2016; Lamb et al. 2002) and the findings of the present study could, at least in part, explain why combined ceftriaxone and ADMSCs therapy was superior to merely one on protecting the vertebral bone and cartilage against ASI damage (referred to Fig. 7).

Study limitations

Our study has some constraints. Frist, the study period was only 28 days, implicating that it was relatively short, i.e., just reflected the ASI outcomes. Accordingly, the chronic outcome of SI after receiving these therapeutic management is currently unclear. Second, the underlying mechanism of bacterial spinal cord injury in rodent and therapeutic impact of ceftriaxone-ADMSCs on ASI was merely dependent on the results of our study that may not really delineate the complicated mechanistic basis of vertebral bone/spinal-cord damage in the setting of ASI.

The complications of spinal cord infection are commonly observed in gravely destructive spinal cord tissues and uncontrolled infection, consequently, leave grievous sequalae and unacceptable high morbidity and mortality. On the other hand, how to effective treatment for ASI, especially in those patients who already develop destructive vertebral bone and cartilage damage, remains a formidable challenge. Our study demonstrated that combined ceftriaxone-ADMSCs treatment provided synergic effect on safeguarding the vertebral bone/spinal cord in opposition to ASI damage.

Acknowledgments

This study was supported by a program grant from Chang Gung Memorial Hospital, Chang Gung University (CMRPG8M0581).

Availability of data and materials

The datasets of present study can be available from the corresponding author upon request.

Authors’ contributions

T. -C. Y., P.-H. S., H.-K. Y. designed the study. T. -C. Y., H. -S. L., P.-H. S., C.-H. Y., investigated experiments. T. -C. Y., P.-H. S., C.-H. Y., H.-K. Y curated data. H. -S. L., P.-H. S., K.-H. C., C.-H. Y. and H.-K. Y. did formal analysis. K.-H. C. was responsible for funding acquisition. H.-K. Y. and K.-H. C. administered and supervised the project. K.-H. C., J.-Y. C., H.-K. Y. wrote the first draft of the manuscript and all named authors contributed in revising the manuscript.

Ethics approval and consent to participate

Patient consent for publication

Not applicable.

Conflict of interest

The authors declare that they have no competing interests.

Funding

This study was supported by a program grant from Chang Gung Memorial Hospital, Chang Gung University (CMRPG8M0581).

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Table 1 and 2 are available in the Supplementary Files section.

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Additional benefits of combined ceftriaxone and adipose-derived mesenchymal stem cells on revamping the outcomes in rodent after acute spinal infection

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