Safety of autologous nucleus pulposus cell transplantation with fibrin glue following endoscopic discectomy in chronic low back pain: a preliminary phase I trial

Abstract

Background

Intradiscal cell transplantation has been proposed as a minimally invasive therapeutic modality in chronic low back pain (CLBP) and may prevent future recurrence after surgical procedures. Since nucleus pulposus (NP) cells with the capability to synthesize appropriate extracellular matrix and withstand the pathological microenvironment of degenerative discs could be more practical in CLBP management.

Methods

five patients with CLBP were enrolled and underwent the intradiscal injection of autologous NP cells after endoscopic therapeutic discectomy. They were followed up for 24 months by the evaluation of clinical measurements and magnetic resonance imaging (MRI).

Results

The primary outcome, safety assessment, was evaluated by recording any complications of surgery or NP cell transplantation during 24 months follow-up. Neither adverse events (AEs) nor serious adverse events (SAEs) were reported. Visual analogue scale (VAS), Oswestry disability index (ODI), and MRI were assessed as secondary outcomes. Reduction of ODI and VAS scores in all five patients indicated improvement in CLBP. Moreover, a comparison of MRI findings between the baseline and endpoint demonstrated improvement in disc protrusion status, less bulging, and more hydration.

Conclusions

According to these promising results and MRI findings after two years follow-up, intradiscal injection of NP cells after endoscopic discectomy seems safe and could be considered as an effective therapeutic method in CLBP with no recurrence.

Trial registration

Iranian registry of clinical trials (IRCT): IRCT20080728001031N35; Registration Date: 08/10/2022.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12891-025-09186-7.

Introduction

Chronic low back pain (CLBP), is one of the most common causes of disability in societies with high global burden and growing prevalence [1]. CLBP typically is common after the second decade of Life, and disc herniation and intervertebral disc disease often appear during the third and fourth decades of Life. This chronic health condition affects approximately 20% of the global population with the higher female incidence [2]. CLBP is characterized with the persistent pain for a period of more than three months [3]. Within the spectrum of CLBP, intervertebral disc degeneration emerges as the most prevalent cause, giving rise to disc degeneration and lumbar disc herniation (LDH). In general, intervertebral disc degeneration stands out as the most common underlying etiology of CLBP [4, 5]. Lumbar disc degeneration is a multifactorial degenerative disorder which is attributed to the numerous risk factors. Substantial physical loading, obesity, trauma, genetic predisposition, and life style are among the factors leading to discopathy [6, 7]. Although the consideration of symptoms, medical history and physical examination are rudimental diagnosis factors, disc degeneration mainly confirmed by magnetic resonance imaging (MRI) [8]. MRI findings including disc dehydration, bulging, protrusion and herniation are usually interpreted as the hallmarks of disc degeneration [9]. Conservative approaches (rehabilitation, non-steroidal anti-inflammatory drugs (NSAIDs), steroid injections, and etc.) as the first line of the treatment lead to improvement in most patients. However, in advanced discopathy and chronic pain surgical interventions (most commonly therapeutic discectomy) are implemented to alleviate symptoms and pain [10, 11]. Nevertheless, the possibility of recurrent symptoms has remained a challenge even after discectomy. Therefore, the necessity for revision interventions arises as a consequential aspect of post-surgery care. Recurrent disc herniation post-surgery implies that patients endure increased pain, necessitate more intricate revision procedures, experience more complications and incur heightened cost [12]. Regenerative medicine approaches particularly cell-based therapies have been proposed in order to induce disc regeneration and prevent the disease recurrence [13].

In recent decade, an increasing number of clinical trials have investigated the potential of cell-based therapies, including autologous or allogeneic mesenchymal stromal cells (MSCs) discogenic cells, and nucleus pulposus (NP) cells for the treatment of CLBP [14–16]. In this regard, Schol et al. in a recent systematic review highlighted the overall safety and potential efficacy of these interventions in improving pain and functional outcomes, as well as early signs of disc repair based on imaging studies. Despite these promising findings, the cell-based therapy in CLBP still faces several challenges, such as variability in cell sources, delivery methods, adjunctive carriers, optimized cell dose, and clinical endpoints [17].

Disc degeneration commonly appears with some irreversible pathologic changes in disc morphology and extracellular matrix (ECM) composition. Meanwhile, the damaged disc losses resident cells, including those of the NP and annulus fibrosus (AF). This pathological condition is the consequence of the interruption in ECM anabolic and catabolic pathways [18]. Therefore, a potential therapeutic strategy would be the augmentation of the disc cells to restore normal biologic functions, repopulate and produce matrix. Among the different cell sources such as NP cells, chondrocytes, mesenchymal stromal cells (MSCs), induced pluripotent stem cells (iPSC), etc., NP cells are uniquely capable of producing a proteoglycan- and collagen type II-rich ECM suitable for regeneration within the IVD harsh microenvironment [19–21]. NP cells can endure the degenerative disc microenvironment, while the enclosed acidic microenvironment makes it difficult for the other cells to be effective in disc regeneration [22, 23]. While numerous preclinical studies have demonstrated the regenerative potential of autologous NP cells in animal models of disc degeneration [24, 25], clinical investigations remain limited. Notably, Mochida et al. [26] investigated the safety and efficacy of activated NP cells in patients with disc degeneration and Xuan et al. [16]conducted a comparative clinical trial evaluating autologous discogenic cell transplantation after discectomy versus only-discectomy.

In spite of the numerous advantages, cell leakage has remained as a momentous concern in cell-based therapies in intervertebral disc (IVD) diseases. Cell leakage may lead to osteophyte formation in the spine or around the joints [27, 28]. Biocompatible scaffolds, especially hydrogel-based scaffolds have been proposed for not only preventing cell leakage but also providing biomechanical support for cell growth and tissue reconstruction [29]. Hyaluronic acid (HA), fibrin glue, and platelet rich plasma (PRP) are known as the common cell carriers in intradiscal cell transplantation which prevent cell leakage and reduce osteophyte formation [30, 31]. Among the different scaffolds, fibrin glue which is known as fibrin sealant, is a degradable, bio-absorbable, non-immunogenic and, easily prepared scaffold that has been well studied in pre-clinical and clinical studies [28].

This phase I clinical study aimed to evaluate the safety of autologous NP cells loaded in fibrin glue implantation in patients who underwent therapeutic endoscopic discectomy. In this regard, the patients were monitored by the use of laboratory tests, clinical examination and recording any side effect during the follow up period. Furthermore, pain and disability-related questionnaires and magnetic resonance imaging (MRI) were used for the safety and probable efficacy assessment.

Methods

Study design and patients enrollment

The current study is a phase I open-label non-randomized clinical trial which was approved by the Ethics committee of Royan institute (IR.ACER.ROYAN.REC.1394.36) and registered in the Iranian registry of clinical trials (IRCT) with ID code number: IRCT20080728001031N35. This research was conducted with the collaboration of Royan institute and Noor medical center, Tehran, Iran, between May 2021 and July 2023 and was in accordance with the good clinical practice (GCP) guidelines and the latest version of Helsinki Declaration. Signed informed consent was obtained from all participants after comprehensive explanation of the procedure by the expert physicians.

This study included patients who were diagnosed with CLBP and were considered candidates for endoscopic discectomy. The recruitment process for identifying eligible patients was conducted at Noor Medical Center, Tehran, Iran. The patients had CLBP, that was refractory to conservative conventional treatments for at least 6 months, and were candidates for endoscopic discectomy. The inclusion and exclusion criteria of the patients are listed in Table 1.

Table 1.

Inclusion and exclusion criteria

Inclusion Criteria	Exclusion Criteria
1. 18 < Age < 65	1. BMI >30.5 kg/m²
2. Lumbar disc herniation involving one or two discs associated with predominant back pain after conservative treatment for over 6 months.	2. Spinal segmental instability, spinal canal stenosis, isthmus pathology, and other conditions that may compromise the study.
3. VAS Index Score ≥ 4 cm	3. Immune deficiency
4. ODI of 30% or higher	4. Modic type III changes on MRI images.
5. Confirmation of fibrous ring integrity for cell implantation, by MRI	5. Congenital or acquired diseases leading to spine deformations that may upset cell application.
6. Decrease of disc height of more than 50% (By comparing with adjacent normal discs).	6. History of allergy to gentamicin, or to bovine, cattle or horse serum.
7. Absence of spinal infection.	7. Hepatitis B, C, HIV, syphilis infections.
8. Hematological and biochemical analysis with no significant alterations that contraindicates intervention.	8. History of Renal failure (Cr > 2 mg/dL), liver malfunction (ALT or AST > 100 IU/L), heart diseases (EF < 45%), and diabetes.
9. Informed written consent of the patient.	9. Pregnancy or lactation
	10. Neoplasia
	11. Participating in another clinical trial.
	12. Unwillingness to cooperate in the study and follow-ups.

VAS visual analogue scale, ODI oswestry disability index, BMI body mass index, MRI magnetic resonance imaging, HIV human immunodeficiency virus

Patients underwent endoscopic discectomy surgery after signing informed consent. MRI was performed prior to the intervention to assess the integrity of the fibrous ring and confirm its capability to maintain the cell implantation. During the operation, the protruded part of the nucleus pulposus, was removed and the sample biopsies of nucleus pulposus were sent to the IR-FDA approved good manufacturing practice (GMP)-certified clean room facility, Royan Institute, for in vitro culture of NP cells. One month later, when the number of cultured cells reached total number of 20 × 10⁶ cells/disc, these cells were re-implanted to the same degenerated discs using intradiscal injection. Patients were evaluated for side effects according to Common Terminology Criteria for Adverse Events (CTCAE) version 5.0. Allergic reaction, anaphylaxis, fever, chills, pain, infection, and sudden death NOS, paresthesia, peripheral motor neuropathy, peripheral sensory neuropathy, right sided muscle weakness, left sided muscle weakness, spinal cord compression, and any other adverse reaction have been monitored for two years. Figure 1 illustrates an overview of the study design.

Fig. 1.

Study Flowchart

Endoscopic lumbar discectomy and autologous nucleus pulposus-derived cells preparation

All five patients underwent transforaminal endoscopic lumbar discectomy under general anesthesia and sterile conditions. In cases with two affected discs, the procedure was performed at each affected level. The patients were positioned prone on the radiolucent operating table. Based on preoperative imaging, a 20-gauge needle was inserted into the foramen at the level of the herniated disc. A guidewire was placed over the needle, followed by a 1-cm incision made with a no. 11 blade. A dilator was then placed through the guidewire and advanced into the foramen. An operative cannula was inserted into the foramen. Then, the foramen was opened, the herniation was identified, and the disc material was removed. Degenerated lumbar discs were removed and the NP tissue were carefully harvested (approximately 1 cm³). Manual separation was employed to isolate the NP tissue from the AF and any fibrotic or degenerated tissue. Only the healthy NP tissue with a viable, chondrocyte-like phenotype was used for the cell culture process. The tissue was then carefully examined under a microscope to ensure that only non-fibrotic and healthy NP tissue was included, while any fibrotic or necrotic material was discarded. The harvested tissue was washed with phosphate-buffered saline (PBS) containing Penicillin/Streptomycin (Pen/Strep) solution. The tissue samples were sliced into 1–2 mm sections and incubated with collagenase type II (64 U/ml) for 24 h at 37 °C in a humidified incubator with 5% CO₂. After digestion, the isolated NP cells were passed through a 70 μm mesh filter to remove any residual tissue debris and were cultured in DMEM/F-12 media supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. The cells were seeded at a density of 5 × 10⁴ cells/cm² in culture flasks and maintained under standard culture conditions with an oxygen tension of 21% at 37 °C. The cells were passaged when they reached approximately 80% confluence, and only passage 3 cells were used for the subsequent transplantation procedure [32]. Autologous NP-derived cells were harvested, isolated and cultured in the IR-FDA approved GMP-certified clean room and all procedures in this study conducted under GCP conditions.

Fibrin glue preparation

Umbilical cord blood-derived Fibrin glue (ROCOSEAL, lot.02134, Royan cell therapy center, Tehran, Iran) including two separate solutions; solution A containing fibrinogen (80 mg/ml) and factor VIII (150 IU/ml) and solution B containing thrombin (500 IU/ml) and CaCl₂ (40 µmol/ml), has been used as a carrier for NP cells transplantation. Suspended NP cells in normal saline (20 × 10⁶ cells/1 ml normal saline) were mixed with solution A (1 ml), then solution B (1 ml) were infused using dual syringe injection system. The fibrin glue clotting time was within 10–15 s.

Nucleus pulposus cells transplantation

Nucleus pulposus (NP) cells (20 × 10⁶ cells/1 ml normal saline) in combination with 2 ml fibrin glue (1 ml solution A and 1 ml solution B) were transplanted intradiscally into the degenerated discs via dual syringe injection system using 22-gauge spinal needle. In sterile condition, the patients underwent the local anesthesia followed by intradiscal injection. In the absence of prior investigation involving dose escalation, the choice of cell dose of 20 × 10⁶ was determined for the current study. This decision was informed after a comprehensive review of the previous studies that have explored intradiscal cell transplantation in disc degeneration, thus serving as a refer point for our dosage selection [30, 33, 34]. The findings of these investigations declared the middle dose of cells (around 20 × 10⁶ cells) is more effective than low dose (below the 10 × 10⁶ cells) and any meaningful differences have not been reported between middle and high cell dosage (40 × 10⁶ cells).

Outcome measures

Adverse events (AEs) and serious adverse events (SAEs) were used to assess the safety profile as the main study outcomes at baseline (prior to the injection), 1 week, 1, 3, 6, 12, and 24 months after cell transplantation. AEs and SAEs were recorded in the patients’ history. In addition, general and specific laboratory tests including complete blood count (CBC), erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), hemoglobin (Hb), hematocrit (Hct), sodium (Na), potassium (K), calcium (Ca), serum glutamic-oxaloacetic transaminase (SGOT), serum glutamic-pyruvic transaminase (SGPT), total and direct bilirubin (Bil (T, D)), alkaline phosphatase (ALP), vitamin D3 (Vit D3), fasting blood sugar (FBS), triglycerides (TG), cholesterol (Chol), low density lipoprotein (LDL), high-density lipoprotein (HDL), blood urea nitrogen (BUN), creatinine (Cr), prothrombin time (PT), partial thromboplastin time (PTT), international normalized ratio (INR), bleeding time (BT), clotting time (CT), hepatitis B surface antibody (HBS Ab), hepatitis B surface antigen (HBS Ag), human immunodeficiency virus (HIV), hepatitis B core antibody (HBc Ab), hepatitis C antibody (HCV Ab), thyroid stimulating hormone (TSH), thyroxine (T4), and urinalysis (U/A) were carried out at the baseline and 6 months after treatment.

Pain and disability-related questionnaires including visual analogue scale (VAS) [35] and Oswestry disability index (ODI) [36] were evaluated at the baseline and in 3, 6, 12 and 24 months after treatment. The VAS is a general measurement tool that is commonly used to evaluate perceived pain in clinical research [37]. While, ODI is a validated and reliable tool for evaluating the level of disability and functional impairment in the individuals with low back pain (LBP). VAS and ODI questionnaires were assessed by the expert and good clinical practice (GCP)-certified general practitioner during the follow-up period.

MRI evaluations were performed via 1.5 Tesla system (Sigma, GE Medical Systems, USA) with standard lumbar MRI imaging protocol (T1-weighted and T2-weighted sequences). Transvers and sagittal planes were reviewed and the stage of disc herniation as well as Pfirrmann grading were assessed by an experienced radiologist at Royan institute, who was blinded to patient identifiers, clinical data, and time points to minimize interpretation bias.

Statistical analysis

Descriptive data, including mean ± SD, were calculated using GraphPad Prism version 8.4.3. The software was also used to generate visual representations of outcomes during the study timeline. In this phase I study with 5 patients, the results are presented descriptively to illustrate trends. However, Wilcoxon signed-rank test was used to compare VAS, ODI, and Pfirmann at 24 months post-intervention with baseline. The corresponding p-values are reported in the “Supplementary Material”.

Results

Demographic data

Five patients aged 23–57 years were enrolled for the safety evaluation of the intradiscal implantation of autologous NP cells following the therapeutic endoscopic discectomy for regeneration of degenerated lumbar disc. The characteristics of the patients are listed in Table 2. Five patients are identified as P1, P2, P3, P4 and P5. Patients with CLBP referred to Noor medical center, Tehran, Iran, 2–3 months before the endoscopic surgery. The affected discs and different stages of disc herniation were detected in the patients by lumbar MRI. The MRI findings of each patient were included in Table 3. All participants were followed up for 2 years after treatment.

Table 2.

Demographic characteristics of the patients

Patients	P1	P2	P3	P4	P5	Mean ± SD
Age (year)	29	57	43	33	23	37.00 ± 13.34
Gender (M, F)	M	M	F	M	M	-
BMI (Kg/m 2 )	18.21	25.71	28	22.69	25.25	23.97 ± 3.73
Disease duration (month)	6	30	24	8	6	14.80 ± 11.37
Pfirrmann grade	IV	IV, V	IV	IV, IV	IV	-

M male, F female, BMI body mass index 

Table 3.

Magnetic resonance imaging (MRI) findings

Patient ID No.	Degenerated disc Location	Pre implantation status	Pre implantation Pfirmann	Post implantation status		Post implantation Pfirmann
P1	L5-S1	Right paracentral disc protrusion	IV	Improvement in disc hydration, Mild bulging, no osteophyte formation	III
P2	L4-L5	Disc dehydration and moderate bulging	IV	Disc dehydration, moderate bulging, no osteophyte formation	IV
	L5-S1		V		IV
P3	L5-S1	Right paracentral disc protrusion	IV	Disc height preserved, mild bulging, no osteophyte formation	III
P4	L3-L4	Right paracentral disc protrusion and central protrusion in L5-S1	IV	Improvement in disc hydration, Bulging, no osteophyte formation	III
	L4-L5		IV		IV
P5	L5-S1	Disc dehydration and severe bulging	IV	Disc height preserved, mild bulging, no osteophyte formation	III

Autologous nucleus pulposus cell’s characterization

The sterility of the cultured cells was verified through multiple stages including microbial contamination, endotoxins, and mycoplasma detection tests. Additionally, to determine the properties of the NP cells, their chondrocyte like phenotype, and morphological characteristics were investigated. Inverted light microscopic images demonstrates polygonal to elongated morphology of NP cells (Figure S1). In addition, immunohistochemistry findings indicates remarkable expressions of type II collagen and SOX9; which are common markers of healthy NP cells (Figure S2).

Safety outcomes

All patients underwent endoscopic discectomy with the approximate average operation time of 1 h with minimal blood loss in all cases. No complications were observed during the endoscopic discectomy. Post-operation, patients were monitored for vital signs, neurological status, and local wound condition. Recovery period was uneventful in all cases. These findings support the minimally invasive nature of the procedure and contribute to the favorable safety profile of the combined discectomy and NP cell implantation approach.

During the 24 months follow-up, neither AEs nor SAEs were reported in any of the patients based on CTCAE version 5.0. Moreover, the results of laboratory data such as ESR, CRP, etc. during the follow-up period indicated no clinical abnormalities in none of the patients. In addition, MRI findings confirmed no signs of ectopic tissue formation, infection, or osteophyte formation following the NP cells implantation.

Clinical outcomes

The clinical results of this study were analyzed using VAS and ODI questionnaires. Figure 2A illustrates a remarkable reduction in the VAS during the 24 months follow-up. The level of VAS score, which is known as an indicator of pain level, decreased from 7, 8, 9, 7, and 7 to 0, 1, 0, 1, and 1 for P1, P2, P3, P4, and P5 respectively, after 24 months. Moreover, Fig. 2B represents ODI scores. This candlestick chart illustrates a significant reduction in average ODI at the end of 24 months. The decrease in ODI score, which is an indicator of improvement of patients’ conditions, initially showed a rapid decline after 3 months, and the decrease in average ODI score continued until 24 months. Details of VAS and ODI assessments have been presented in the supplementary data (Table S1 and Table S2).

Fig. 2.

Timeline of changes in VAS and ODI. Changes in the A: VAS and B: ODI during the 24-months follow-up after implantation of nucleus pulposus-derived cells in five patients. VAS, visual analog scale; ODI, Oswestry low back pain questionnaire. Each bar represents an individual patient. The line graphs indicate mean ± SD for VAS and ODI

MRI findings

Identification of changes in disc herniation was achieved by comparing the MRI findings before and 24 months after the intradiscal implantation of NP cells in the degenerated discs. Table 3 listed the changes before and two years after intervention in detail. This table indicates prevention of osteophyte formation in all patients and improvement in disc protrusion, disc hydration, and disc height preservation in four patients (P1, P3, P4, P5), and no changes in P2. Figure 3 illustrates the transverse and sagittal views for disc herniation of P3 before and 2 years after the injection. Figure 3A and C indicate the sagittal and transverse views of the affected disc (indicated with white arrows) before the endoscopic surgery, respectively. Figure 3B and D illustrate the sagittal and transverse views for this patient after 24 months follow up, with white arrows highlighting the observed improvements in the affected disc. Implantation of the NP cells led to a remarkable improvement in the L5-S1 herniation of this patient. Comparison of MRI findings demonstrated that the stage of disc degeneration in P3 changed from right paracentral disc protrusion to mild bulging. The MRI images before and after treatment for the other patients have been presented in the supplementary data (Figure S3-S6).

Fig. 3.

MRI analysis of P3. Figure A and C indicate the sagittal and transverse views of the affected disc before the intervention, respectively. Figure B and D illustrate the sagittal and transverse views for this patient after 24 months follow up. White arrows highlight the amelioration of disc herniation in L5-S1 after the intervention

Discussion

The current study investigated the safety and efficacy of NP cells transplantation in patients with LDH who underwent discectomy as a conventional treatment. In this regard, clinical outcomes and MRI findings evaluated during 24 months. Clinical outcome measures including ODI and VAS showed improvement during the 24 months follow-up. In addition, 24 months post transplantation MRI findings compared to the baseline indicated improvement in degenerated discs.

Intradiscal cell transplantation has been proposed as an innovative and promising minimally invasive therapeutic option for discogenic CLBP by induction of disc regeneration. In this regard, transplantation of different cells such as mesenchymal stromal cells (MSCs) has been assessed in numerous clinical trials. For instance, Henriksson et al. [38] and Kumar et al. [30], evaluated the intradiscal transplantation of autologous bone marrow-derived MSCs (BMSCs) and adipose-derived MSCs (AD-MSCs), respectively. In the other clinical trial conducted by Xuan et al.., safety and feasibility of intradiscal injection of autologous discogenic cells following the discectomy evaluated in 40 patients (19 patients received autologous discogenic cells after discectomy and 21 patients only underwent discectomy in control group) with IVD. They administrated the average cell dose of 5.57 × 10⁶ cells/disc. They reported based on radiological analysis, transplantation of discogenic cells may slow the progression of degeneration in the index discs and reduce post-discectomy complications [16]. The other similar study, Meisel et al. investigated the feasibility of autologous cultured disc-derived chondrocytes (ADCT) transplantation following microdiscectomy. Their findings supported the practicality of combining discectomy with cell-based strategies [39]. In this regard, Meisel et al. conducted a systematic review on cell therapy for IVD and evaluated 8 clinical studies. They revealed that cell therapy in IVD was associated without any SAEs and showed promising early efficacy in reducing discogenic pain and improving function, although heterogeneity across trials limits direct comparison [40]. Moreover, the other systematic review conducted in 2024 suggested that intradiscal injection of MSCs may be effective for managing CLBP [41].

Although clinical improvement was reported after MSCs injection in patients with discogenic LBP in several clinical trials, disc regeneration has not been asserted by MRI in most of them [38, 42], MRI-based evidence of disc regeneration, such as improved Pfirrmann grades, has been reported only occasionally, as highlighted in the systematic review by Schol et al. [17]. Therefore, symptoms alleviation may be related to the immunomodulatory effects of MSCs [43, 44]. NP cells are naturally resident within the IVD and are responsible for ECM synthesis that provides mechanical properties of the discs. In degenerated discs, the NP cells undergo senescence or apoptosis, leading to irreversible changes in physical characteristics including reduction in disc height. This alteration causes compression on the nerves that results in CLBP. Therefore, transplantation of healthy NP cells has been proposed as a potential therapeutic modality for CLBP [45, 46]. In the current study, NP cells were isolated from relatively well-preserved regions within degenerated discs. The expressions of SOX9 and type II collagen, alongside with polygonal to elongated chondrocyte-like morphology in cultured NP cells, firmly supports the preservation of anabolic function and capacity for ECM synthesis. The sustained expressions of these markers suggests that the NP cells retained sufficient capacity for ECM production, alleviating concerns about senescence or apoptosis [47, 48].

NP cells are uniquely adapted to the harsh microenvironment of the intervertebral disc, characterized by low oxygen tension, limited nutrient supply, and high osmotic pressure, which supports their specialized role in maintaining ECM homeostasis and disc function. In this regard, in vivo models highlight that the ability of NP cells to survive and function in this challenging environment is critical for successful regenerative therapies aimed at restoring disc structure and alleviating CLBP [49]. Considering the therapeutic potential of NP cells, Mochida et al. [50] in 2015 transplanted NP cells into the degenerated discs of nine patients with disc degeneration after reactivated them via co-culture with autologous BMSCs. They reported no AEs in none of the patients during the 3 years follow-up. Their MRI findings indicated a mild improvement in only one case while any remarkable changes have not been reported in the other patients. Furthermore, recent clinical studies evaluated the safety and clinical effectiveness of intradiscal injection of 100 mg of NP allograft (VIA disc NP allograft) in patients with symptomatic degenerated discs. Their results indicated that viable disc tissue allograft is safe and associated with improvements in pain and function in patients with discogenic CLBP [51, 52]. Nevertheless, the capability of NP cells in disc regeneration has been studied in numerous animal studies. In this regard, the potential of NP cells and MSCs in disc regeneration was investigated in a preclinical study using rabbit, rat and canine animal models [24, 53, 54].

ODI and VAS are the most common outcome-measure questionnaires for evaluating the efficacy of the treatment modalities in CLBP and also tracking the patients [30, 55, 56]. These questionnaires were used in the present study to investigate the related changes in the patients’ health status. Assessment of these questionnaires together revealed the improvement of the patients’ health condition after the transplantation of NP cells. The reduction of ODI and VAS after NP cells transplantation in our study was in accordance with results of other studies which investigated cell-based therapeutic methods in CLBP. For instance, Gornet et al. [34] in a food and drug administration (FDA)-approved clinical trial investigated the efficacy of different doses of allogeneic disc progenitor cells in disc degeneration by the assessment of VAS, ODI, and EQ-5D. The results of all outcome measures indicated significant improvement in the high dose group (9 × 10⁶ cells/ml) 104 weeks after treatment. While, changes of ODI and EQ-5D in the low dose group (3 × 10⁶ cells/ml) revealed no remarkable difference from baseline. Moreover, in a study conducted in 2022, ODI and VAS score decreased significantly after 1 year follow-up in patients with discogenic LBP who underwent the bone marrow aspirate concentrate (BMAC) transplantation. The reduction of VAS and ODI values indicated the improvement of patients’ status after BMAC intradiscal injection [57].

In the current study, the observed reductions in VAS and ODI scores exceeded thresholds commonly considered clinically meaningful [58]. This suggests that the intervention had an impact on patients’ pain and function. Although a formal cost-effectiveness analysis was not performed, it is reasonable to speculate that reducing recurrence and avoiding revision surgeries may help justify the additional cost of cell therapy. Future trials with larger sample size, control groups and economic evaluations are warranted to explore this potential benefit.

In addition to the enhancement of the clinical conditions, MRI assessment indicated a remarkable improvement in the degenerated discs. MRI findings revealed that NP cells transplantation prevented the progression of disc degeneration and also led to the amelioration of the damaged discs. Evaluation of the degenerated disc by MRI has been considered in similar studies. In this regard, Bates et al. [59] assessed MRI changes in patients with disc degeneration after the administration of 10 × 10⁶ autologous adipose-derived mesenchymal stem cells (ADMSCs). According to the results, slight improvement in disc protrusion and annular fissure was reported in patients after 12 months and ADMSCs injection prevented progression of disc degeneration. However, their findings showed no enhancement in disc height in the patients. In another clinical trial, Atluri et al. [42] in 2022 evaluated the efficacy of autologous bone marrow mesenchymal stem cells (BMSCs) in CLBP. The results of this study showed no correlation between MRI findings and perceived pain by patients after the treatment. Comparison between the baseline and 6 months post injection MRI findings in BMSCs group indicated slight improvement in only 40% of patients. While remarkable improvement was reported in clinical outcomes including ODI and VAS. According to the lack of any meaningful MRI changes in spite of the pain relief, the authors suggested that CLBP occurred due to the inflammation rather than mechanical or structural changes.

Improved outcome measures in the current study attributed to the combination of NP cell transplantation and the endoscopic discectomy. Although pain reduction is expected following endoscopic discectomy, the absence of symptom recurrence over 24 months and MRI evidence of improved disc hydration and preserved height suggest potential additive effects from NP cell transplantation. According to the previous investigations, the rate of recurrent disc herniation after discectomy surgery (micro-discectomy and endoscopic discectomy) was reported up to 25% [60]. Nonetheless, since there was no control group, it is not possible to determine with certainty how much of the improvement was specifically due to the cell therapy. Further research with more arms can address this question.

In the context of this safety assessment trial, it is crucial to acknowledge the study’s inherent limitation concerning its small sample size involving only five patients. Due to the limited sample size, drawing definitive conclusions about the treatment’s efficacy may be challenging and a larger number of patients should be included in future studies. Furthermore, design of future studies for dose escalation is suggested for providing valuable insights into safety, feasibility and efficacy of NP cells transplantation after endoscopic discectomy. In addition future randomized-controlled trials are suggested to incorporate to enhance the validity of NP cells application after discectomy in discogenic LBP.

Conclusion

This phase I study provides preliminary evidence that autologous intradiscal transplantation of 20 × 10⁶ NP cells was well-tolerated and not associated with any AEs and SAEs in five patients with CLBP. The observed improvements in clinical outcomes and MRI findings after two years suggest that NP cell transplantation following endoscopic discectomy may be beneficial. However, further investigations on a larger population and controlled studies is needed for more evaluation of the efficacies.

Authors’ contributions

B.S. and N.L. collected data. B.S. reviewed literature and drafted the manuscript. M.N. and M.H. analyzed MRI findings. M.E., B.S., H.M., A.B.M., S.F., A.B., M.H. and N.L. participated in the interventions and study procedures. H.M., ABM, N.H.K., S.K., A.B., M.H. and S.F. reviewed the manuscript. M.V. conceived the design of the study, reviewed and confirmed the final manuscript.

Funding

This work has received grants from Royan Institute for Stem Cell Biology and Technology (ID: RI.94000027). The funding sources had no responsibilities in the study design; data collection, analysis, and interpretation; manuscript writing; and in the decision to submit this paper for publication.

Data availability

No datasets were generated or analysed during the current study.

Declarations

Ethics approval and consent to participate

This study was approved by the Ethics committee of Royan institute (IR.ACER.ROYAN.REC.1394.36) and registered in the Iranian registry of clinical trials (IRCT, https://www.irct.ir/trial/34) with identifier IRCT20080728001031N35 (Registration date: 08/10/2022). The study included only adults and written informed consent have been obtained from the patients. All procedures were conducted in accordance with the relevant approved regulations, guidelines, and the Declaration of Helsinki.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.