Ozone therapy with local cellular immune modulation and disc progenitor cell implant is safe, effective and efficient

Low Back Pain (LBP) is a major cause of morbidity with signifi cant economic impact through loss of work (15% in the United Kingdom). Additionally, there is the cost of health care and social support for the affected individual and their family. It is estimated that more than half the population (49%70%) will experience signifi cant LBP during their lives. Point prevalence from 12% to 30% is reported in western countries [1,2].


Introduction
Low Back Pain (LBP) is a major cause of morbidity with signifi cant economic impact through loss of work (15% in the United Kingdom). Additionally, there is the cost of health care and social support for the affected individual and their family.
It is estimated that more than half the population (49%-70%) will experience signifi cant LBP during their lives. Point prevalence from 12% to 30% is reported in western countries [1,2].
LBP is usually defi ned as pain, muscle tension, or stiffness localized below the costal margin and above the inferior gluteal folds, with or without leg pain (sciatic pain). These symptoms cause signifi cant disability. LBP is defi ned as acute when it persists for less than six weeks, subacute between six weeks and three months, and chronic when it lasts longer than three months [2,3].
The anatomical basis of this pathology is associated with degeneration of the intervertebral disc (IVD), disc endplates and subchondral bones [4][5][6][7][8]. Etiology has been attributed to, weight overload of the affected joins (through excessive physical labor and/or extreme sport activity), aging, obesity and genetic predisposition [3].
The pathobiology of this condition assumes that an initial acute trauma or degeneration induces a strong infl ammatory response. This infl ammatory response causes the attraction and activation of macrophages in the IVD structures and adjacent bones [9][10][11][12][13]. The concept of this initial infl ammation is to promote healing of damaged tissues [14,15]. While infl ammation removes the damaged tissues [16][17][18][19][20], additionally through cytokines secretion and the developing of Th1 specifi c response the differentiation of local progenitor cells replace the damaged tissues and rebuild the Extracellular Matrix (ECM) of each affected tissue [16][17][18][19][20][21][22][23][24][25]. When the pathogenic factors persist, the infl ammatory pro regenerative reaction become profi brotic and the activated macrophages lead to a chronic infl ammation developing a Th2 [25][26][27][28] immune reaction, increasing their phagocytic and lysosomal activity [29]. These events are responsible for the damage of the Annulus Fibrosus (AF) of the IVD. Then, the AF matrix loses the normal organization of the collagen fi brils [26][27][28]. The Nucleus Pulposus (NP) dehydrates and decreases in overall height with a large loss of cell numbers, whereas the collagen type 2 fi bers became type 1 with increasing concentration and thickness. End-plate changes are associated with subchondral sclerosis. These late fi ndings are clear signs of the loss of normal IVD function. (Figure 1). In late stages of degeneration, the IVD has less elasticity and becomes thin; the NP and AF regions become indistinguishable from each other [10,11,[26][27][28].
Initially the treatment for LBP is limited to the use of short-term pain relief with Nonsteroidal Anti-Infl ammatory Citation: Grangeat  Drugs (NSAIDs) which are helpful. In addition to the NSAID's a second treatment approach is physiotherapy with the aim to improve movements of the spine and correct posture. When conservative treatments fail, more invasive therapies are considered including epidural injection of corticosteroids and anesthetics or trigger point injections. When less invasive approaches fail, surgical interventions are considered to decompress the spine and or do a spinal fusion. Unfortunately, surgical treatments do not reverse the Degenerative Disease (DDD) or restore the IVD tissues [2,3].
Oxygen-ozone therapy, used to treat this problem, mainly as local but also as systemic administration, has been shown to induce an up regulation of antioxidant enzymes resulting in improved blood circulation delivering more oxygen to ischemic tissues. The improved oxygen delivery up regulates the activity of the immune system by macrophage activation [30][31][32][33][34][35][36][37][38].
These multifactorial effects of the Oxygen-Ozone therapy are the rational explanation for the high clinical effi cacy on chronic DDD. It is highly effective controlling, and even abolishing, the LBP [39][40][41][42][43][44][45][46][47][48][49]. The limitation of this therapy is that it does not affect fi brosis of the affected area, which is mainly composed of collagen type 1 which inhibits the re growing of new cartilage.
Wick G and co-workers [25], has established that the fi brosis process is induced under a Th2 immune environment and the Th1 atmosphere may induce fi broblast and other mesenchymal cells to produce a more pro-regenerative Extra Cellular Matrix (ECM). Th1 and Th2 immune polarization are reciprocally controlled by Macrophage Type 1 (Mo1) and Mo2. These 2 types of cells are different phenotype expressions of the same cell according the microenvironment characteristics [18].
The single use of IVD injections of chondrocytes or different kind of stem cells has been studied in the past decade in various animal models of induced DDD. The preclinical success of these treatments has failed to produce a functional and prolonged effects in the clinical setting. Detailed basic research on IVD cells and their niche indicates that transplanted cells are unable to survive and adapt in the avascular, collagen 1 fi brotic and highly pro-apoptotic in the niche of the DDD. As confi rmation of these assumptions, animal and human data on the regenerative potential of injected chondrocytes or disc cells are promising for regeneration of early IVD degeneration [50][51][52][53][54][55][56][57][58]. It is felt that the microenvironment of the IVD in acute patients remains healthy and allows the engraftment, growth and differentiation of the implanted cells. [56][57][58][59][60] Based on these concepts, combining local cellular immune modulation with specifi c cellular progenitor cells [60][61][62][63][64][65][66][67][68], for chronic fi brotic pathologies, have induced the simultaneous recovery of parenchymal cells and ECM as well as their mutual integration in different clinical settings [69][70][71][72][73].
Moreover, pre-clinical [24] data suggested that the triple approach of oxygen-ozone therapy, followed by intradiscal injection of Effector Immune cells and Disc Progenitor Cells has a synergistic effect, recovering the damaged vertebral body and IVD structure.

Objectives
In patients with symptomatic DDD Ozone therapy, and Disc Progenitor Cells will be used to control LBP syndrome and recover the damaged vertebral and IVD structures. We hypothesized that ozone therapy, associated to local immune modulation, and local implant of Disc Progenitor Cells, has a synergic effect that will improve the results of the treatment of severe chronic degenerative disc disease.  [74,75]. The pain associated to the MRI characteristics should mark greater than or equal to 5 in the Visual Analogue Scale (VAS) [77] and/or deconditioning due to pain greater than 80% in the ODI scale (Oswestry Disability Index). Patients must sign the Informed Consent Form [78].  Patients with jaundice or liver failure. Pregnant patients.

Patients population
Dependence on alcohol or any other type of addiction.

Ethical considerations
This is a report of therapeutic results of clinical cases treated with an innovative approach. Therefore, this is not

Ozone therapy
Selected Patients received 10 sessions of ozone therapy.
It is an outpatient bases using a portable generator of ozone from medical liquid oxygen (Ozomed®, Kastber Praxisbedarf GMBH-Medizintechnik), 10 ml of Oxygen-Ozone gas at 10 μg/ ml, twice a week. It was administered through an Intramuscular injection into the muscles adjacent to the involved IVD [39][40][41].
The day of the cell implant, (under image guidance) the patient received a dose oxygen-ozone into the most affected discs. This procedure was done to mark the site of cellular implants and preconditioning the local microenvironment [42][43][44][45][46][47][48][49].

Preparation of immune effector cells (EC)
Autologous Mononuclear Cells (MNC) were obtained from the patient by removing 250 ml of blood. The MNCs were purifi ed by a Ficoll Hypaque gradient. Cells were washed and cultivated in DMEM enriched with 1% enzymatic hydrolysate (Laboratorio Villar, Argentina) of complete calf spine cervical tissues (cartilage, muscles and bones), ranitidine (5 ng/ml), and indomethacin (5 ng/ml). After 4 days of culture, as described previously [23,34,[69][70][71][72][73], only lymphocytes pre-sensitized in vivo against the antigen present in the medium were activated and amplifi ed by the clonal selection principle of Burnet [78].
The rest entered apoptosis. The addition of ranitidine and indomethacin made it diffi cult to enrich the culture in Mo2 and regulatory lymphocytes (Tregs) [79]. Consequently, the suspension was characterized by Fluorescence-Activated Cell Sorting (FACS) as CD3 + CD20 + CD68 + CD183 + CD163 -CD25 - [79][80][81]. As evidence of the immune-specifi city of the ECs, different EC aliquots were cultured separately in the presence of muscle, brain, or kidney hydrolysate. After 24 hours, the in vitro reaction of tetrazolium blue [81] had a selective increase for calf spine cervical tissues, with values greater than 25% of those for the other antigens.

MSC preparation
Adipose Mesenchymal Stem Cells (aMSC) were obtained from abdominal subcutaneous fat tissue by minimal lipectomy. The tissue was mechanically and enzymatically dissociated (Collagenase IV, Thermo Fisher). The obtained cell suspension was cultivated in DMEM enriched with recombinant human insulin (INULIN® and 2% of human platelet lysate supernatant (homemade). After 3 days of culture, the cells were reseeded. They were cultured until a total amount of 5x10 7 per disc to be treated was obtained [71][72][73]. According to International Society for Cellular Therapy (ISCT) criteria, their identity and purity were monitored by fl ow cytometry, which showed CD73 + CD90 + CD105 + CD34 -CD45 - [82]. As a test of multipotency, their ability to differentiate into cartilage, bone, and nerve tissue was demonstrated [69][70][71][72][73].

Obtaining disc progenitor cells
According to the results in other biological scenarios, we hypothesized that the co-incubation of anti-muscle EC with MSC was suffi cient for the latter to differentiate into DPC [23,24,[69][70][71][72][73]. To test this hypothesis, EC and aMSC were incubated at a 1:1 ratio. No other substance was added to the culture medium. For controls, immunohistochemical staining was performed to detect the phenotypic expression of CD 271 (Nerve Growth Factor Receptor) [83], Collagen Type 2, and Aggrecan [84,85]. As controls of this co-culture, MSC were cultured in IVD tissues-enriched medium with inactivated MNC and medium enriched with the supernatant of the EC.

Safety and tumorigenicity tests effect of treatment with human EC and DPC in animals
After the animal experiment protocol was approved by the ethics committee, a group of six nude mice was injected through the tail vein with 1x10 6 MSC per animal. A second batch was injected with 1×10 6 EC + 1×10 6 DPC (both humans). A third batch was injected with 1x10 4 cells of the 4T1 syngeneic tumor line. The animals were cared for and kept under observation for 120 days in the Argentinean National Commission of Atomic Energy animal facility. Necropsies were performed on animals that developed and died from tumors as well as those that survived the 13-week observation period. The latter were euthanized with carbon monoxide at the end of this period. During the necropsy, the brain, lungs, heart, liver, both kidneys with adrenal glands, and spleen were collected for further study, as were the small intestine and large intestine after visual inspection. These organs were fi xed in 10% buffered formaldehyde and processed for pathological study.

Patients safety and clinical follow-up
During the 6 weeks of the schedule therapy the patients were Citation: Grangeat  for adverse events (version 3.0) [86]. VAS and ODI [76,77] were assessed at 3 months and MRI of the spine treated areas at 6 months after the end of the treatment [74,75].

Statistical analysis
All the Statistic Analysis were done with the MedCalc Software from MedCalc Inc. The test used for media, Standard error calculation and graphic plotting was ANOVA for repeat measures. The Signifi cance difference Level established was P< 0.01

Preclinical safety results
After 120 days of observation, all nude mice that were treated with 5×10 5 4T1/kg of body weight intravenously developed tumors and died from these tumors before day 45. None of the animals injected with 5×10 7 MSCs or DPCs/kg of body weight, both of human origin, died during the observation period. While the autopsy of the organs injected with 4T1 showed metastases and parenchymal alteration fi gures, no histological alterations were observed in the post-euthanasia organs at 120 days after the animals were injected with human MSCs and human MPCs.
All the organ anatomopathological results and laboratory study related to the adverse events associated with the treatment of the animals showed no abnormalities. The only minor event detected was local infl ammation at the site of injection but not any sign of animal suffering, controlled under the Grimace scale [87,88], was detected. No other variation in the recorded parameters was found in any of the treated animals.

Clinical results
Clinical individual patient conditions, as well as VAS and ODI evaluation results, are summarized in Table 1.    Previous his treatment the patient was unable to play golf. At 3 months after treatment, he could recover his full capacity to play the game. test demonstrate that these differences were highly signifi cant.
In order to shed light on the mode of action of this combined therapy is necessary to analyze the individual mode of action for each therapy, as well as the reported results when they are applied as individual therapies.
O 3 was used for more than a century as a disinfectant for its oxidative power. However, about 30 years ago, it was reported that when it is locally applied in the tissues at low concertation, O 3 up regulate the production of antioxidant enzymes becoming a powerful stimulating of oxygen metabolism and activating the immune system [30,33,37]. Moreover, O 3 may reach many other tissues for its ability to dissolve in the aqueous component of plasma [37,38] and phase II enzymes of drug metabolism. Many of these enzymes act as free radical scavengers clinically relevant to a wide variety of diseases [33,37,38].
Several clinical reports and clinical trials have proved that O 3 therapy is effective to control LBP associated to NP Herniated and/or DDD [34,[39][40][41][42][43][44][45][46][47][48][49]. The two main route of administration used were the intramuscular injection in the paravertebral muscles of the muscles innervated for the nerves of affected segments [39][40][41] and the percutaneous Intra disc injection [42][43][44][45][46][47][48][49]. Both administration route showed effi ciency to control the LBP in around 60 to 80% of treated patients with a diminishing of 60 to 75% in the VAS index. Both routes seem to control the local pathology stress but only diminishing of the IVD volume (measured on CT scan or MRI images) was observed when the percutaneous intradiscal route was used [42,43]. No other morphological change was detected on the pre and post image analysis. The major cause of medical failure was and advanced degree of lumbar-sacrum osteo arthrosis related to a continuous and severe pain that lasted more than one year. Because these morphologic and clinical limitations were described by the fi rst reported treated patients [39][40][41][42][43], Supporting the fi ndings of Apuzzo, et al [45], Rahimzadeh, et al [46], compared percutaneous intradiscal ozone injection with laser disc decompression in patients that suffer of discogenic LBP. VAS reduction in both groups was similar, but with a slight superiority after a year of patient follow up for O 3 threated patients [46].
As a result of the analysis of these data, only 60% of patients with LBP qualify to be treated with O 3 therapy focusing in the long-lasting resolution of the problem.
Between the new approaches intended to solve this medical necessity cellular therapy emerge as one that may have large application [50][51][52][53][54][55][56][57][58]. The DDD is a problematic scenario for tissue regeneration due to the harsh microenvironment that resident cells are immersed [54,56]. It is very important to control the interactions between implanted cells and the disc microenvironment (oxygen tension, nutrients, pH, osmolarity, cytokine levels related with the kind of infl ammatory cells present, ECM scar and mechanical load). It may be the reason for the poor and transitory result obtained for different cellular treatments [50][51][52][53][54][55][56][57][58].
Proposed cell-based strategies include the implantation of autologous cells into the degenerate NP [50][51][52][53][54][55][56]. Autologous NP cells have demonstrated improvements in terms of pain relief and disc hydration upon injection into degenerate human IVDs [51,56]. However, harvesting of NP cells yields a limited number of them and requires invasive procedures, which have, themselves, been shown to initiate degenerative changes.
Allogeneic juvenile chondrocytes [53][54][55][56] have been explored as an alternative cell source. However, caution must be taken as the matrix produced by articular chondrocytes may not be the most appropriate for IVD tissue engineering/repair [56,59,61].
A more immediate autologous progenitor cell source is adult mesenchymal stem cells (MSCs), which can be isolated easily from multiple sources, mainly bone marrow (BM-MSCs) [50][51][52][53][54] or adipose tissue (AD-MSCs) [51,[54][55][56], divide rapidly, and are able of differentiating into cells of different lineage. They are also capable of differentiating into NP-like cells. Adult MSCs appear to offer a promising cell type for IVD regeneration, due to the relative ease of acquisition and their ability to undergo discogenic differentiation. Autologous MSCbased therapies, in a pilot study, have been shown to reduce the pain induced by IVD [53]. The only opportunity to see a Long-lasting effective result of a single kind of cell therapy was when they were used to treat early stages of disc degeneration [56,58]. There was also reported that tissue specifi c progenitor cells seem to be more effective than MSC [56][57][58].
A possible explanation for these discordant effects (effi cacy in early stages and poor response in late stages) is to consider, that the DDD is an active process [11], that progressively affect different essential structures of the vertebral to vertebral joint [4][5][6][7]. The deterioration of these structures is double: The fi brotic ECM do not allow an appropriate differentiation of the stem cell [59][60][61]. Early stages have partially preserved the physiologic structure of ECM allowing cell implant engraftment and differentiation to the appropriated tissues [59][60][61]. The fi brotic transformation of the ECM is associated with chronic infl ammation in DDD pathogenesis [8][9][10][11]. Moreover, the destruction of structures that allow the nourish of the tissues [4][5][6][7] produce a shift of a healthy microenvironment to a more anoxic and catabolic one with ROS which leads to very low extracellular pH [25][26][27][28][29] which in turn adds more stress to any kind of tissue regeneration.
In order to overcome this adverse effect, we have tested in the animal setting [24] and now in this case report the use of O 3 therapy. It is justifi ed because through their secondary Citation: Grangeat

Conclusion
These pilot cases support the hypothesis that ozone therapy, associated to local immune modulation, and local implant of Disc Progenitor Cells, has a synergic effect that improve the functional and structural results of the treatment of severe chronic degenerative disc disease.