Intradiscal injection of allogeneic bone marrow derived clonal mesenchymal stromal cells in discogenic low back pain: a phase I study on safety and feasibility (RELIEF: phase I)

Abstract

Aims

This is a phase I trial, assessing the feasibility, safety, and potential efficacy of intradiscal injection of allogeneic bone marrow-derived clonal mesenchymal stromal cells (BM-cMSCs) in patients suffering from discogenic low back pain (DLBP).

Methods

Five patients underwent single intradiscal injection of 9 × 10⁶ allogeneic BM-cMSCs and were followed at predefined intervals for 24 weeks. Safety outcomes included monitoring adverse events (AEs), serious adverse events (SAEs), and laboratory assessments. Efficacy endpoints were evaluated over 24 weeks and included Visual Analog Scale (VAS), Oswestry Disability Index (ODI), and SF-36 quality-of-life scores. MRI was performed to assess disc height, apparent diffusion coefficient (ADC), and modified Pfirrmann grading.

Results

No SAE was observed. Transient injection site pain was the most common AE which resolved within 4–8 days after injection. VAS decreased significantly in three patients, with pain relief sustained through 24 weeks. ODI indicated functional improvement in three patients, with two achieving minimal disability. SF-36 quality of life questionnaire outcomes demonstrated improvements in physical functioning and pain domains in three patients. MRI findings showed modest increases in disc height and apparent diffusion coefficient (ADC) improvements in 2 patients.

Conclusions

Intradiscal injection of allogeneic BM-cMSCs appears to be safe and feasible as a potential therapy for DLBP, with promising efficacy in reducing pain and improving function and quality of life. Further studies with larger cohorts and extended follow-ups are needed to confirm these findings and explore long-term structural benefits.

Trial registration

IRCT, IRCT20080728001031N30. Registered 21 October 2021, https://irct.behdasht.gov.ir/trial/58723.

Supplementary Information

The online version contains supplementary material available at 10.1186/s13287-025-04606-w.

Introduction

Low back pain (LBP) is a compound term describing a wide spectrum of clinical conditions with complex pathogenesis and different etiologies. LBP is among the most common disabilities affecting 619 million people worldwide (almost 10% of the world’s population) in 2020. It is estimated that by 2050, 843 million will suffer from LBP [1]. Discogenic low back pain (DLBP) is a term frequently used to describe back pain associated with intervertebral disc degeneration (IVD), occurring without herniation, structural deformities, or other identifiable causes of pain and functional impairment. The annual costs for society due to chronic DLBP are substantial, with estimates ranging from €7,911.95 to €18,940.58 per patient, depending on the cost calculation method [2]. Patients with DLBP experience severe pain, significant physical limitations, and a serious loss of quality of life. Although DLBP is primarily caused by IVD, this condition involves complex biomechanical and biochemical processes that lead to pain. Degeneration of the intervertebral disc initiates inflammatory responses that recruit immune cells, establishing a chronic inflammation. This persistent inflammation promotes neovascularization and nerve ingrowth into the inner layers of the intervertebral disc, resulting in increased sensory innervation, nociceptive sensitization, and pain. Key cytokines, including tumor necrosis factor-α (TNF-𝛼) and pro-inflammatory interleukins, alongside nerve growth factor, drive this inflammatory process and contribute to the pain of degenerated discs [3]. Progressive disc degeneration increases collagenase production, causing mechanical instability and lumbar hypermobility, which worsen pain. While growth factors (bFGF, TGF-β), macrophages, and mast cells aid in annulus fibrosus repair, they may also contribute to further degeneration [4]. A critical factor underlying disc degeneration and pain is the imbalance in extracellular matrix (ECM) components, including a significant loss of proteoglycans and collagen, which accelerates degeneration and perpetuates pain [5].

Management of DLBP includes lifestyle modifications, pharmacologic treatments, and non-pharmacologic interventions. While anti-inflammatory medications are commonly used, they are often less effective. Current treatments focus on pain management and biomechanical adjustments, but many patients fail to show significant improvement, highlighting the limited efficacy of existing options [6]. In addition, restoring normal physiology and inducing regeneration of the affected disk is largely neglected in these therapeutic methods.

Regenerative medicine and advanced therapy medicinal products (ATMPs) offer new hope for treating previously challenging diseases. Mesenchymal stromal cells (MSCs), in particular, have been widely utilized for treatment of inflammatory conditions due to their well-documented immunomodulatory and anti-inflammatory properties. MSCs exert strong immunomodulatory effects by reducing the phagocytic and antigen-presenting functions of monocytes/macrophages [7]. In addition, these cells release immunosuppressive molecules, such as IL-10 and programmed cell death ligand. They inhibit dendritic cell maturation, regulating proinflammatory cytokine production, and T-cell stimulation [8]. MSCs also modulate the development and activity of proinflammatory Th1, Th17, and CD8 + T cells while promoting T regulatory cells and IL-10-producing B cells. By impeding B-cell differentiation and antibody secretion, MSCs modulate immune responses into anti-inflammatory state through various soluble molecules [9]. In addition to their strong immunomodulatory properties, MSCs are ideal candidate for treating inflammatory diseases due to their low immunogenicity and the relative ease of culturing and cell banking [10]. Among the various pathologic conditions in which MSC therapy efficacy has been explored, musculoskeletal disorders have benefited significantly from cell therapy. Several studies have investigated the safety and feasibility of using MSCs for treating chronic LBP [11–14]. Intradiscal injection of MSCs have proven to be a safe intervention [15]. It is of note that previous studies have incorporated MSCs obtained from different sources [11–13, 16]. However, MSCs are highly heterogeneous, which poses significant challenges for their clinical application and therapeutic efficacy which affects their therapeutic outcomes. This heterogeneity arises from various factors, including differences in subpopulations, tissue sources, donor variability, and in vitro expansion processes [17]. Inconsistent sourcing, phenotypic characterization, and variable potency of MSCs have led to unpredictable clinical results and inconclusive therapeutic benefits [18].

Various methods have been proposed to produce a homogeneous MSC population, including surface marker-based purification (e.g., STRO-1) and sub-fractionation culturing method (SCM) [19]. While surface marker selection faces technical challenges due to the lack of consensus, SCM is a promising approach that generates clonal MSCs (cMSCs) by isolating single colony-forming units (CFUs). In our previous study, we showed that BM-cMSCs at passage 15 (P15) are superior to conventional heterogenous MSCs (at P3) derived from the same sample, especially in regard to higher proliferation and less doubling time, as well as non-senescence cells in P15 [20]. Furthermore, we believe that the high efficacy of BM-cMSCs, as demonstrated in several of our previous studies, is attributed to their greater homogeneity compared to the MSCs obtained through conventional methods [21–24].

When evaluating basic MSCs characteristics, including proliferation rate, senescence, surface marker expression, immunomodulation, and more lineage differentiation capacity, BM-cMSC exhibited significantly more youthful properties compared to lower-passage. An allogeneic BM-cMSC bank addresses the main challenges posed by the inherent heterogeneity of BM-MSCs, such as accelerated cellular aging, limited large-scale expansion, and variability in inflammatory responses [20]. In this study, we aim to assess the safety, feasibility of the intradiscal injection of BM-cMSCs in DLBP patients. Assessment of the efficacy of the intervention will be the secondary outcome of this study.

Materials and methods

Study design

This is a prospective open label phase I clinical study that received ethical clearance from the ethics committee of the Royan institute under the reference number of IR.ACECR.REC.1400.040. This trial is registered in the Iranian registry of clinical trials with ID code number: IRCT20080728001031N30. The data and safety monitoring board (DSMB; Royan institute) at the Royan institute was in charge of monitoring the trial and independent experts advised researchers and clinicians. The study adhered to the principles of good clinical practice (GCP) and was carried out in accordance with the declaration of Helsinki. Five patients were included in the study, after obtaining informed consent in a stepwise order. The first patient was observed for three months, and if no AE occurred, the next patient was enrolled in the study and was observed for 2 months. Moreover, if the treatment was safe, the next patient was included in the study. Subsequent patients were included in the study if the third patient showed no AEs for one month after treatment. All patients followed for 24 weeks after first injection.

Patients

Patient recruitment took place in the LBP clinic of the Shariati and Yas hospitals of Tehran university of medical sciences. Patients with LBP were assessed by principal investigator of the study and were recruited if the inclusion and exclusion criteria were met. Patients enrolled as subjects in the study presented with symptomatic moderate to severe DLBP as defined according to the following criteria: chronic low back pain that lasted at least six months; undergone non-operative management for at least three months without resolution; shown a pathologic change in normal disc morphology as defined by MRI (magnetic resonance imaging) evaluation; have a modified Pfirrmann (MRI) score of 3–6; have a Modic grade II change or less; disc height loss of < 30% compared to an adjacent non-pathologic disc; pre- treatment baseline Oswestry Disability Index (ODI) score of at least 30 on the 100-point scale; and pre-treatment baseline low back pain of at least 30 mm on the 100 mm visual analogue scale (VAS). Detailed inclusion and exclusion criteria are reported in Table-1. Written informed consent was obtained from all study subjects after thorough explanation of the study and possible outcomes.

Table 1.

Inclusion and exclusion criteria of the study. DDD: degenerative disc disease; CLBP: chronic low back pain; MRI: magnetic resonance imaging; VAS: visual analogue scale; ODI: Oswestry disability index;

Inclusion Criteria	Exclusion Criteria
> 18 years	BMI > 35 kg/m2
Both gender	Knee osteoarthritis ≥ Kellgren Lawrence 3
Early to moderate DDD of 1–3 lumbar discs	Congenital or acquired diseases leading to spine deformity
Modified Pfirrmann Grade 3–6	Spinal segmental instability, spinal canal stenosis, spondylitis, spondyloarthropathy
Modic Grade II or less on MRI (± disc protrusion)	Previous surgery at the involved or adjacent level
CLBP for at least 3 months in the last 6 months, unresponsive to conservative treatment (medical and physical therapy)	Any infection or lesion at the site of injection, History of any intradiscal injection, History of epidural injection within 3 months
VAS: 3–9	Uncontrolled comorbidities (Diabetes, organ failure)
ODI: 30–90	Movement disorders
LBP > radiculopathy	History of any neoplasm
Decrease of disc height < 30%	Immunosuppression or taking systemic immunosuppressant
Central disc bulging	Positive viral tests, or abnormal hematologic or biochemical lab tests
Informed consent	Any coagulopathies or taking anticoagulants
	Pregnancy or breast-feeding
	Alcohol or drug abuse
	History or diagnosis of any psychologic disorder
	Allergy to protein (gentamicin, bovine or horse serum)

Eligible patients underwent pretreatment evaluation which included baseline lumbosacral MRI and lab test including: complete blood count (CBC), fasting blood gugar (FBG), blood urea nitrogen (BUN), serum creatinine (Cr), hepatitis viral tests, liver function tests, thyroid function tests, erythrocyte sedimentation rate (ESR), C-reactive protein (CRP) and urine analysis (U/A).

Allogeneic BM-cMSCs Preparation and characterization

In this study, allogeneic BM-cMSCs were supplied by the Royan Advanced Therapy Medicinal Products Technology Development Center (Royan ATMP-TDC). These cells were isolated using the SCM approach, and BM-cMSC cell banks was established at the GMP-certified facility of Royan ATMP-TDC, as previously documented in our earlier report [20]. Bone marrow (BM) was collected from the iliac crest of a healthy donor after obtaining informed consent and cultured in a tissue culture dish with Minimum Essential Medium Eagle-Alpha Modification (Alpha MEM; Thermo Fisher Scientific, 11,900,073, USA), supplemented with 15% fetal bovine serum (FBS; HyClone, SH30070-03, USA). The culture was incubated at 37 °C and 5% CO₂, with the suspended cells transferred daily to fresh dishes for nine days. Selected colonies were enzymatically isolated with TrypLE (Thermo Fisher, A1277, USA) and further cultured in Alpha MEM with FBS until passage 3. Following characterization, the most suitable clone was selected from the seed stock and expanded in Alpha MEM with 5% human platelet lysate (hPL; Bioscience, PLS-500.03. FDi, USA) for biobanking.

For clonal isolation of MSCs, SCM [19] was used to isolate the individual clones of MSCs from BM. Of the 27 isolated clones, 21 could be cryopreserved as seed stock in 3 vials at passage 3 (P3). During a process of clone selection, three clones were selected based on their ability to maintain long-term passage. In the next step, one vial of each clone from the seed stock was thawed and used to establish the following serial cell banks at different passages: an initial cell bank (ICB; P6), a master cell bank (MCB; P10), a working cell bank (WCB; P13), and end‐of‐product cell bank (EoPCB; P15). The culture medium used for clonal isolation contained FBS until seed stock, but for the establishment of all four cell banks, (ICB, MCB, WCB, and EoPCB) the culture medium contained hPL. The hPL used in these studies was from Bioscience Co. that is fibrinogen-depleted (Bioscience, Fibrinogen-depleted Human Platelet Lysate, no anticoagulant required – GMP Grade – US origin).

Characterization of clonal mesenchymal stromal cells (cMSCs) adhered to the international society for cell and gene therapy (ISCT) criteria, assessing safety, efficacy, quality, and identity. Sterility testing was conducted using BD BACTEC™ to detect aerobic and anaerobic bacteria and fungi. Mycoplasma contamination was evaluated using a Genesig^® Standard Kit and quantitative real-time PCR. The endotoxin level was assessed with the Gel Clot LAL ENDOSAFE^® method, and viral contamination was screened by real-time PCR. Genetic stability was confirmed through karyotyping (G-banding) and comparative genomic hybridization (CGH) array. Tumorigenicity was evaluated by injecting cMSCs into ten nude male mice (six to seven weeks old) to assess oncogenic potential.

Detailed characterization protocols and further functional and immunological assays are discussed in supplementary materials and are shown in Table S1.

MRI protocol

To assess the efficacy and safety of the cell therapy, disc height, disc fluid content (Apparent Diffusion Coefficient (ADC)), and Pfirrmann grading was measured by quantitative T2 MRI with the Siemens MAGNETOM Prisma 3T scanner between baseline and 24 weeks follow up. For calculating the ADC, diffusion-weighted imaging (DWI) was performed. By placing region of interests (ROIs) in the nucleus pulposus, the ADC can be measured, providing a quantitative estimate of water content. An experienced radiologist evaluated the intravertebral disc degeneration using Pfirrmann classification. Sagittal T-2 weighted images were used to analyze the height and signal of the disc.

Treatment protocol

Intradiscal injections took place at Yas hospital and Mehregan pain clinic and experienced neurosurgeon carried out the procedure. Before cell implantation, pretreatment MRI studies were evaluated and symptomatic discs were chosen for stem cell injection. Before the cell injection, injection site was treated with local anesthetic. While in prone position, a 22G spinal needle was placed in the center of the nucleus of the affected disc. The placement of the needle was confirmed by fluoroscopy in 2 planes (Figure-1). After placement of the spinal needle, 3 ml cell suspension including: 1 ml of hyaluronic acid (HA), 1 ml of normal saline and 1 ml of cell product containing 9 × 10⁶ cells were implanted in the disc. After injection, patients were observed for their vital signs and any AEs in the facility for 4 h. At the time of discharge, in addition to receiving the follow-up schedule, patients also received the necessary recommendations regarding possible side effects.

Fig. 1.

Fluoroscopic view of needle placement in L4-L5 disc of the patient. After local anesthesia, 22G needle is placed in the desired space and will be verified by taking fluoroscopic scans of the disc in anteroposterior (AP) and lateral views

Outcomes

To evaluate the safety and feasibility of intradiscal injection of BM-cMSC, patients were in direct contact with the study coordinator. Moreover, adverse events (AEs) and serious adverse events (SAEs) were assessed during the scheduled follow-up visits at baseline (prior to the injection), week 1, 4, 12 and, 24 after the cell injections. AEs included any short-term and long-term complications, systemic or local and severe or mild complications related and unrelated to the cell injection procedure. SAEs were defined as adverse events that result in death, require either inpatient hospitalization or prolongation of hospitalization, life-threatening events, resulting in a persistent or significant disability/incapacity. AEs and SAEs were assessed through common terminology criteria for adverse events (CTCAE) v5. AEs and SAEs were recorded in the patients’ case report form (CRF). Lab tests were carried out on the baseline and 24 weeks after injection for safety assessment which included: CBC, FBG, BUN, Cr and U/A.

Secondary outcome of the study was the efficacy of the intervention. Efficacy was evaluated by patient reported outcome measures (PROMs) and MRI of the lumbar discs. PROMs included: pain evaluation using VAS, ODI, and short form-36 (SF-36) life quality questionnaire. VAS and ODI were recorded on baseline and weeks 4, 12 and, 24 after injections. SF-36 was collected on the baseline and the last follow up visits. MRI data included measurements of disc height and disc water content using ADC mapping, along with Pfirrmann grading. MRI was conducted at the baseline and the final visit.

Results

Demographic data

From July 2022 to September 2023, five DLBP patients (Three males and 2 females) were enrolled in the study and received intradiscal injection of the allogeneic BM-cMSCs. Their age ranged between 20 and 49 years. Except one patient (P2) which had normal body weight, four patients were obese (BMI > 30). The flow diagram of the study is presented in Figure-2. Table-2 represents baseline characteristics of the study subjects. For 3 patients, injections were carried out on L4-L5 disc and two patients received cell injection on L5-S1 discs.

Fig. 2.

Flow diagram of the study

Table 2.

Demographic characteristics of the patients. VAS: visual analogue scale; ODI: Oswestry disability index; LBP: low back pain; BMI: body mass index; HTN: hypertension;

Patients	P1	P2	P3	P4	P5
Age (y)	38	20	49	47	37
Gender	Male	Male	Female	Female	Male
Affected Disc	L4-L5	L5-S1	L4-L5	L4-L5	L5-S1
History of LBP (y)	4	2	5	4	3
Comorbidities	None	None	HTN	HTN	None
VAS score at baseline	7	8	7	5	6
ODI score at baseline	38	30	34	60	32
BMI	32.3	24.5	33.6	34.1	31.8
Pfirmann grade at baseline	5	6	5	5	6

Adverse events (AEs) and serious adverse events (SAEs)

All the patients discharged from the surgical facility without any SAEs. The most common AEs was injection site pain which all patients reported in the course of study. Pain started a few hours after the procedure and lasted for 4–8 days. Injection site pain was treated by oral administration of Acetaminophen. No radiculopathy, numbness, motor dysfunction and muscle weakness were observed during the study. Apart from the pain, one patient (P3) reported a generalized pruritis 6 h after injection which responded well to 10 mg of cetirizine orally. The same patient suffered from headache 6 h after injection which resolved the next day. Fortunately, no SAE occurred during the course of the study. In addition, lab tests of the patients revealed no significant changes. Safety findings of the study are summarized in Table-3.

Table 3.

Adverse events and serious adverse events of the study.sae: serious adverse event; AE: adverse event;

Patients	SAEs		AEs		Description		Date of Onset		Duration		Action taken
P1		-		+		Injection site pain		Hours after injection		7 days		Acetaminophen 500 mg
P2		-		+		Injection site pain		Hours after injection		5 days		Acetaminophen 500 mg
P3		-		+		Injection site pain Pruritis Headache		1 day after injection Hours after injection Hours after injection		7 days 1 day 1 day		Acetaminophen 500 mg Cetirizine 10 mg Acetaminophen 500 mg
P4		-		+		Injection site pain		Hours after injection		8 days		Acetaminophen 500 mg
P5		-		+		Injection site pain		Hours after injection		4 days		Acetaminophen 500 mg

Efficacy outcomes

Patient reported outcomes

Secondary endpoints of the study were assessed by quantifying changes in VAS, ODI and SF-36. Throughout the 24-week study period, three patients (P1, P2, and P5) showed a decreasing trend in pain intensity based on the VAS. Among the patients who reported pain reduction, improvement began around 10 days post-injection and remained stable through the study’s conclusion. One patient (P3), initially not indicating pain relief, experienced significant pain reduction within the first month. However, this improvement was not consistent, with symptoms increasing notably by the third month. Another patient’s (P4) pain level remained unchanged, even peaking in the third month. Patients P1, P2 and P5 show substantial and sustained pain relief by week 24 (Figure-3 A).

Fig. 3.

VAS (A) and ODI (B) score of the patents during the course of study. A: Most patients showed a reduction in pain over time, although some fluctuations were observed. B: While some patients exhibited a steady decline in disability, others showed variability in their response

Each line represents an individual patient, with different symbols denoting distinct patients. VAS: Visual analogue scale; ODI: Oswestry Disability Index

The impact of back pain on daily activities was assessed using the ODI (Figure-3 B). During the study, four patients reported improvements in daily activities, while one reported no change (P3). Six months after intervention, three patients described their level of back pain–related disability as minimal, while one patient (P4) experienced a reduction yet continued to report moderate disability. One patient (P3) reported no significant change in the ODI score. Patients P1, P2, and P5 showed sustained reductions in ODI scores over the six-month period, indicating improved functionality and quality of life. P2 appears to benefit the most, showing the lowest ODI score at the 3-month mark, with sustained low disability through the 6-month follow-up. P3 showed a stable but modest reduction in ODI scores, indicating moderate functional improvement without marked long-term progression. P4 initially showed some improvements but eventually returned close to baseline disability by the 6-month visit, suggesting either a temporary benefit or potential factors limiting the effectiveness of the BM-cMSC therapy in this patient.

The SF-36 questionnaire was used to evaluate the impact of intradiscal MSCs injections on patients’ quality of life. The findings showed effectiveness in 3 out of 5 patients (P1, P2 and, P5) (Figure-4). P1 and P2 generally showed improvements across multiple domains, especially in physical functioning (PF), bodily pain (BP), and mental health (MH), P4 and P5 demonstrated relatively stable scores across domains. However, P3 demonstrated declines in RP and RE values, reflecting potential challenges in physical and emotional role functioning, with minimal improvements in other areas. Among the five subscales assessing physical health in the SF-36, improvements were noted in three parameters, i.e., PF, BP, and RP. However, the results for mental health aspects related to quality of life were mixed, with no clear trend emerging across the patients.

Fig. 4.

SF-36 score of the patients at baseline and 24 weeks after the injection. Eight domains of the Short Form-36 (SF-36) health survey were assessed at baseline (week 0) and after 24 weeks in five patients (P1–P5). The SF-36 scores vary across individuals, with some showing improvements in quality-of-life measures, particularly in bodily pain and physical functioning, while others exhibit variable trends in other domains. SF-36: Short form 36; BP: Bodily pain; RE: Role limitations due to emotional problems; RP: Role limitations due to physical health; GH: General health; VT: Vitality; SF: Social functioning; MH: Mental health; PF: Physical functioning

MRI analysis

The results of MRI analysis are summarized in Table-4; figure-5. In the current, changes in disc height, ADC, and modified Pfirrmann grade were analyzed based on MRI findings before and 6 months after the intervention in five patients. Minimal increases in disc height were observed in all patients and the range of improvement was modest, varying from 0.03 mm to 0.49 mm. P5 showed the largest increase, from 8 mm to 8.49 mm (+ 0.49 mm) followed by P4 with 0.28 mm increase in disc height. P2 showed the least amount of changes in disc height.

Table 4.

Changes of disc in MR imaging. ADC: apparent diffusion coefficient

Patient Number	Disc level	Disc height (mm)		ADC (×10⁻³ mm²/s)		Pfirrmann Grade
		Before	After 24 weeks	Before	After 24 weeks	Before	After 24 weeks
P1	L4-L5	11.32	11.45	--	--	5	5
P2	L5-S1	8	8.06	1123	611	6	6
P3	L4-L5	9.83	9.86	1185	1430	5	5
P4	L4-L5	11.72	12	1730	1234	5	5
P5	L5-S1	8	8.49	771	1973	6	6

Fig. 5.

Changes of disc height (A) and apparent diffusion coefficient (ADC) score (B) of patients observed in MRI. A: Minimal changes in disc height were observed across patients. B: ADC increased in most patients, suggesting potential changes in disc hydration and microstructure. ADC: Apparent diffusion coefficient

Water content of the disc were evaluated with ADC changes of the disc in DWI. In one patient (P3) although the disc height changes were minimal, ADC increased significantly from 1185 to 1430, suggesting potential improvement in hydration of the disc. The most significant change occurred in P5 (771 to 1973), indicating significant hydration improvement. On the other hand, P4 MRI showed a deterioration of disc hydration (ADC changes from 1730 to 1234). In regard to modified Pfirrmann grading, no changes were observed after the intervention in all the patients. Among the patients, P5 showed the most favorable imaging outcomes which can be observed in figure-6.

Fig. 6.

Assessment of disc degeneration using diffusion weighted imaging (DWI) (A and B) and T2-weighted MRI images (C and D). DWI imaging of the P5 shows the improvement of disc hydration 24 weeks after cell injection (A and B). T2- weighted imaging of the same patient shows the improvement of disc height 24 weeks after injection

Discussion

Due to high prevalence of DLBP and complexity of the pathogenesis of this disease and limited therapeutic options, MSCs therapy for DLBP has attracted remarkable attention. In this open label pilot study, it was observed that intradiscal injection of BM-cMSCs is safe and feasible. Apart from temporary injection site pain which resolved within 10 days after procedure, no other AEs was observed among all study subjects. Administration of allogeneic BM-cMSCs suspended in HA carrier was deemed feasible in DLBP patients. Among the secondary outcomes, the VAS data suggested that intradiscal injection of BM-cMSCs might provide substantial and sustained pain relief for some patients with DLBP. These results also suggested that BM-cMSC therapy could provide functional benefits for some patients with DLBP, as evidenced by reduced ODI scores. However, analysis of the SF-36 domains underscored the heterogeneity in patient responses to BM-cMSC therapy for discogenic pain. While some patients experience enhanced physical functioning, reduced pain, and improved mental health, others showed limited or no improvement. MRI analysis of discs of these patients showed modest improvements in disc height and water content (as indicated by ADC), although these changes do not seem to significantly alter the overall degenerative grading (Pfirrmann scale) within the timeframe of this study.

Our safety results align with previous studies on intradiscal MSC injections in patients with DLBP, which consistently demonstrated a favorable safety profile. Previous clinical trials and cohort studies indicated that intradiscal MSC injections are safe, irrespective of MSC origin, and do not provoke immunogenic responses or other adverse reactions. In a large-scale randomized controlled trial, Pers et al.. confirmed the safety and efficacy of MSC injections in the disc space, showing no significant safety concerns [11]. Additionally, the SafeCell study—a comprehensive systematic review and meta-analysis—assessed the safety of MSC therapy across various clinical applications. This analysis reported few serious adverse events across different delivery routes, including intradiscal injections, reinforcing the favorable safety profile of MSC-based treatments [25]. A meta-analysis specifically focused on MSC therapy for lumbar discogenic pain in patients with intervertebral disc degeneration further supported the safety of intradiscal MSC injections [15].

Although the current study was not powered to definitively assess treatment efficacy, some patients demonstrated enhanced physical functioning, reduced pain, and improved mental health. Conversely, others experienced limited or no improvement, reflecting variability in therapeutic response. These findings align with previous studies on MSC administration for chronic LBP (CLBP), which, while generally reported favorable outcomes, have shown inconsistent results across different investigations. In a feasibility study of intradiscal injection of iron-labeled autologous MSCs in CLBP patients, the PROMs demonstrated modest numerical improvements. However, these changes were not statistically significant over time. Additionally, no correlations were identified between the clinical outcomes and variables such as the cell yield from bone marrow aspiration, duration of ex vivo expansion, number of cell culture passages, or the ability of the MSCs to form pellets [26]. Notably, this study recruited patients who were on the waiting list for transforaminal lumbar interbody fusion (TLIF) or total disc replacement (TDR) due to degenerative disc disease. These patients exhibited more severe disability compared to those typically included in similar trials, reflecting a more advanced stage of pathology and a higher baseline level of impairment. Additionally, in a randomized, double-blind, controlled study of intradiscal injection of 20 million allogeneic BM-MSC in CLBP, despite the progressive improvement in functional and pain indices (including improvement if at least 20% in VAS and ODI), no significant difference between placebo and treatment group was observed [11]. Interestingly, in the mentioned study, strong placebo effect was reported which highlights the strong placebo responses in trials involving pain and mobility assessments. Such effects could overshadow modest but clinically meaningful benefits of new treatments [27]. In contrast to the previous study, another randomized controlled trial investigating the use of 25 million autologous BM-MSCs in 24 patients with degenerative disc disease reported significant long-term pain relief and disability improvement in the MSC-treated group compared to the placebo group [13, 28]. In addition, the MSC-treated patients in this study exhibited two distinct response patterns: the subpopulation of “responders” who showed outcomes near the “perfect treatment” line, and “non-responders” whose results were indistinguishable from those of the control group. In our study, patients who responded well to the treatment demonstrated a sustained therapeutic effect, comparable to the “responders” group observed in the previously mentioned study [28]. It can be postulated that patients with more advanced stages of DLBP may demonstrate a reduced response to cell therapy. Noteworthy, in our study, patients with the highest BMI reported the poorest outcome. A robust meta-analysis indicated that both overweight and obesity are the main risk factors for low back pain in both men and women, with obesity showing a stronger association. The odds ratios for overweight and obesity compared to normal weight are 1.15 and 1.36, respectively, indicating an increased risk for LBP in obese population [29]. It is important to note that variations in the study design, including differences in cell dosage, frequency of injection (s), intervals between the injections, preparation methods, and post-injection follow-up duration, may limit the comparative analysis between this study and existing literature.

MRI analysis of our study, revealed a modest amelioration of disease in the patients. Disc height increased minimally, and ADC values improved significantly in most patients, indicating enhanced hydration and stabilization. However, one patient showed decreased ADC. It is of note that no changes in Pfirrmann grading were observed, suggesting limited structural reversal within the study period. Despite the promising clinical outcomes, imaging outcomes of the studies using MSC in DLBP treatment is less favorable. In a recent study, Lee et al. in spite of positive clinical outcomes, radiologic improvement of patients was limited and modified Pfirrmann grade remained consistent after 6 months of follow up [16]. MRI analysis from the most recent randomized clinical trials (RCTs) revealed no differences in disc water content or modified Pfirrmann staging between the two arms. However, a two-year follow-up reported a non-significant increase in the water content signal in the BM-MSC group compared to the placebo group [11]. Interestingly, improved water content was observed at 24 months but not at 12 months. Additionally, the most favorable imaging outcomes were observed in the studies with longer follow up periods. These findings suggest that structural changes in discs following MSC injection may require a longer timeframe to become apparent. One notable finding in studies on LBP is the lack of a strong correlation between imaging changes in the disc and clinical outcomes or PROMs. In a multicenter, randomized, controlled study investigating the safety and efficacy of a single injection of STRO-3⁺ adult allogeneic mesenchymal precursor cells (MPCs) in CLBP, regardless of the promising clinical outcomes, no significant MRI changes was evident in the study groups [12]. Similarly, a population-based cohort evaluating the associations between common lumbar degenerative changes observed on MRI and present or future LBP, revealed that MRI degenerative findings do not have clinically important associations with LBP [30]. As a result, MRI assessments in LBP studies should be interpreted considering that radiologic changes do not necessarily reflect clinical symptoms.

Two clinical studies have evaluated the safety and efficacy of co-administering HA with MSCs [12, 14]. These studies have shown that the combination of MSCs and HA is generally safe and well-tolerated in patients with chronic LBP. Furthermore, the combination has been effective in improving clinical outcomes, including reductions in pain and disability scores. Notably, significant improvements in VAS and ODI scores were observed, indicating that this treatment can effectively alleviate low back pain symptoms. Adding HA to the injected MSCs is believed to reduce cell leakage and osteophyte formation in the disc [31]. Studies have shown that HA can enhance the efficacy of intradiscal MSC injections (mesoblast unpublished) [32].

Our study has some limitations that should be addressed in the future research studies. Due to the invasive nature of intradiscal injection, the absence of prior clinical experience with BM-cMSCs for this indication, and the exploratory safety focus of the study, we had a limited sample size, which limits the generalizability of the findings. In addition, the absence of a control group prevents definitive conclusions regarding the efficacy of BM-cMSC therapy which could be addressed in the advanced phases of the study. Furthermore, the lack of subgroup analyses by age, gender, BMI, or hormonal factors, limit the interpretation of potential patient-specific outcome after intervention. Additionally, the 24-week follow-up period may not be sufficient to capture the full extent of regenerative changes, as structural improvements in intervertebral discs are likely to occur over a longer timeframe. Future studies should include larger population and randomized controlled trials with extended follow-up periods to validate these preliminary findings and to account for potential confounding factors such as age, gender, body mass index (BMI), and hormonal status. Advanced imaging techniques, along with molecular and biochemical analyses, could provide deeper insights into the mechanisms underlying BM-cMSC therapy and help identify biomarkers predictive of treatment response.

Conclusion

The intradiscal injection of allogeneic BM-cMSCs demonstrated a favorable safety profile and potential therapeutic benefits in patients with DLBP. While preliminary results are promising, further research is needed to optimize patient selection, dosing strategies, and treatment protocols.

Supplementary Information

Below is the link to the electronic supplementary material.

Abbreviations

LBP

Low back pain

DLBP

Discogenic low back pain

IVD

Intervertebral disc degeneration

TNF-α

Tumor necrosis factor-α

ECM

Extracellular matrix

ATMP

Advanced therapy medicinal products

MSC

Mesenchymal stromal cell

SCM

Sub-fractionation culturing method

cMSC

Clonal MSC

CFU

Colony forming unit

DSMB

Data and safety monitoring board

GCP

Good clinical practice

MRI

Magnetic resonance imaging

ODI

Oswestry disability index

VAS

Visual analogue scale

CBC

Complete blood count

FBG

Fasting blood glucose

BUN

Blood urea nitrogen

Cr

Creatinine

ESR

Erythrocyte sedimentation rate

CRP

C-reactive protein

U/A

Urine analysis

BM

Bone marrow

MEM

Minimum Essential Medium

FBS

Fetal bovine serum

hPL

Human Platelet lysate

ICB

Initial cell bank

MCB

Master cell bank

WCB

Working cell bank

ISCT

International Society for Cell & Gene Therapy

CGH

Comparative genomic hybridization

ADC

Apparent diffusion coefficient

DWI

Diffusion weighted imaging

ROI

Region of interest

HA

Hyaluronic acid

SF-36

Short form 36

AE

Adverse event

SAE

Serious adverse event

CTCAE

Common Terminology Criteria for Adverse Events

CRF

Case report form

PROM

Patient reported outcome measures

PF

Physical functioning

BP

Bodily pain

MH

Mental health

TLIF

Transforaminal lumbar interbody fusion

TDR

Total disc replacement

RCT

Randomized controlled trial

MPC

Mesenchymal precursor cells

Author contributions

A.B.M. drafting, data analysis, visits and patient’s follow-up. M.K. and A.A. interventional procedure. H.M, N. M., M.N., H.H., B.S., S.K., E.H., N.H., M.B.E and Sh.F. data analysis, review the manuscript and edit. H.B, R.K., and M.V, conceptualization, study design, review and final edit, supervising and approval.

Funding

This work was supported by grant from the Royesh Venture Capital Fund and Royan Atitech Pharmed fund to Hossein Baharvand. The funding source had no responsibilities in the study design; data collection, analysis, and interpretation, manuscript writing, and the decision to submit this paper for publication.

Data availability

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Declarations

Ethics approval and consent to participate

This study received ethical clearance from the ethics committee of the Royan institute under the title of “Safety evaluation of intra-discal transplantation of allogeneic bone marrow derived clonal mesenchymal stromal cells (human BM-cMSCs) in patients with low back pain induced by degenerative disc disease: Interventional clinical trial, phase I” with the reference number of IR.ACECR.REC.1400.040 on the July 13th of 2021. This trial is registered in the Iranian registry of clinical trials with ID code number: IRCT20080728001031N30. The data and safety monitoring board (DSMB; Royan institute) at the Royan institute was in charge of monitoring the trial and independent experts advised researchers and clinicians. The study adhered to the principles of good clinical practice (GCP) and was carried out in accordance with the declaration of Helsinki.

Consent for publication

The patients included in this study provided written informed consent for the publication of anonymized data in a scientific journal. No identifiable personal information is disclosed in this manuscript.

Competing interests

The authors declare that they have no conflict of interest.

Artificial intelligence

The authors declare that they have not use AI-generated work in this manuscript.