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ORIGINAL ARTICLE
Year : 2021  |  Volume : 69  |  Issue : 6  |  Page : 1565-1570

Bone Marrow-Derived Mesenchymal Stem Cells Augment Regeneration of Intervertebral Disc in a Reproducible and Validated Mouse Intervertebral Disc Degeneration Model


1 Department of Neurological Sciences, Christian Medical College, Bagayam, Tamil Nadu, India
2 Department of Radiodiagnosis, Christian Medical College, Bagayam, Tamil Nadu, India
3 Department of Pathology, Christian Medical College, Bagayam, Tamil Nadu, India
4 Centre for Stem Cell Research, Christian Medical College, Bagayam, Tamil Nadu, India

Date of Submission28-Jan-2020
Date of Decision23-May-2020
Date of Acceptance15-May-2021
Date of Web Publication23-Dec-2021

Correspondence Address:
Dr. Krishna Prabhu
Professor of Neurosurgery, Department of Neurological Sciences, Christian Medical College, Vellore - 632 004, Tamil Nadu
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0028-3886.333531

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 » Abstract 


Background: Back pain and radicular pain due to disc degeneration are probably the most common problems encountered in neurosurgical practice. The experience and results of stem cell therapy in animal disc degeneration model will help us while doing clinical trials.
Objective: To study the effect of bone marrow-derived mesenchymal stem cells in an established mouse disc degeneration model.
Methods: An easily reproducible mouse coccygeal (Co) 4-5 disc degenerated model by CT-guided percutaneous needle injury was established. The mesenchymal stem cells (MSCs) were cultured from mouse bone marrow and validated. By an established technique, 24 mice disc degenerative models were generated and divided equally into 3 groups (test, placebo, and control). The test group received MSCs with fibrin glue scaffold and placebo group received only scaffold after 6 weeks of degeneration. The control group did not receive any injection. The effects of MSCs were analyzed 8 weeks post injection.
Results: The test group showed a significant change in disc height index (%) in micro CT, whereas in the placebo and control groups, there was no change. The Safranin O staining showed an increase in glycosaminoglycan content and the polarized imaging of picrosirius red staining showed restoration of the collagen fibers in annulus fibrosus, which was statistically significant.
Conclusion: Intradiscal MSC injection restored disc height and promoted regeneration in the discs at the end of 8 weeks. MSC's niche depends on the microenvironment of the host tissue. These findings will be helpful for clinical trials.


Keywords: Disc height index (%), intervertebral disc, mesenchymal stem cells, mouse disc degeneration model, regeneration
Key Message: Mesenchymal stem cells derived from mouse bone marrow have good potential to regenerate mouse intervertebral disc if placed in the disc space.


How to cite this article:
Baldia M, Mani S, Walter N, Kumar S, Srivastava A, Prabhu K. Bone Marrow-Derived Mesenchymal Stem Cells Augment Regeneration of Intervertebral Disc in a Reproducible and Validated Mouse Intervertebral Disc Degeneration Model. Neurol India 2021;69:1565-70

How to cite this URL:
Baldia M, Mani S, Walter N, Kumar S, Srivastava A, Prabhu K. Bone Marrow-Derived Mesenchymal Stem Cells Augment Regeneration of Intervertebral Disc in a Reproducible and Validated Mouse Intervertebral Disc Degeneration Model. Neurol India [serial online] 2021 [cited 2022 Jan 18];69:1565-70. Available from: https://www.neurologyindia.com/text.asp?2021/69/6/1565/333531




Back pain and radicular pain are probably the most common problems encountered in neurosurgery and orthopedic practice. Globally, they create a significant socioeconomic burden and cause 83 million disability-adjusted life years (DALY) annually.[1] Stem cells, with their property of pluripotency, are believed to cure or halt many disease processes. Due to ethical concerns, no standard treatments or human trials using embryonic stem cells are done. However, adult stem cell therapy has been used for many years, with excellent outcomes, to treat leukemia and related neoplasms through bone marrow transplants.[2],[3] With this background, we decided to study the effect of bone marrow-derived mesenchymal stem cells (BM-MSCs) in a mouse disc degeneration model.


 » Materials and Methods Top


Creation of the model

A novel tool was designed to create disc injury in the mouse tail (described in a separate article of ours).[4] A total of 24 C57 mice were equally divided into three groups: test, placebo, and control (n = 8). Each animal was anesthetized with an intraperitoneal injection of ketamine (180 μl/100 g, 1 ml = 50 mg) and Xylazine (50 μl/100 g, 1 ml = 20 mg). High-resolution CT of the coccyx (Co) was done to measure disc height index (DHI) and to mark the disc level by inserting a subcutaneous needle from one side tailored at Co 4-5. After confirmation, the level was externally marked on the dorsum of the tail with ink [Figure 1]. Preinjury DHI was measured as shown in [Figure 2]. Following marking the level, the mouse was placed on the extended glass platform of our tool and the tail was clamped in the groove [Figure 1]b. A 3-directional control of the syringe holder was used to position a 32 G needle over the tail marking. The needle syringe fixed in the syringe holder was brought down using the coarse adjustment knob of the microscope stage and a controlled 2 mm penetration was made into the disc. Post penetration, the needle hub was detached from the syringe; the syringe holder was moved up leaving behind the needle in the disc space. For confirming the needle position, the mouse with needle in-situ was imaged with CT [Figure 3]. The needle was removed post confirmation of location and the mouse was transferred to a cage. All three groups (test, placebo, and control) were kept for 6 weeks after the injury.
Figure 1: (a) A mouse placed on the glass slab attached to an old microscope body. There is a syringe holder containing 32G needle attached to the microscope stage. On the glass slab, there is a tail groove for the tail to fit in and a clamp to immobilize. (b) The dorsum of the tail with the marked entry point (circled) of the needle and arrow showing the tip of the needle

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Figure 2: Describes how to measure disc height index on mid sagittal section of CT coccygeal spine (Co) and the formula to calculate the change in disc height index (%) (DHI %)

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Figure 3: CT images of the mouse with the needle in the disc. (a) 3D image of mouse skeleton. (b and c) Sagittal and axial sections of the Coccygeal vertebra with needle in-situ

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Culture of mouse bone marrow-derived mesenchymal stem cells (BM-MSCs)

Marrow from the femur and tibia was extracted from a single donor mouse [Figure 4] and partial digestion with 100 U/ml collagenase and 16 U/ml hyaluronidase for 3 to 5 h in separate spinner flasks was done [Figure 4]. The tissue was cultured in Dulbecco Modified Eagles Medium (DMEM), and 10% fetal bovine serum (FBS) supplemented with antibiotics (5% CO2 at 37°C). Plastic adherent cells migrated out after culturing for 1 week. The cell count and the viability were measured using Trypan Blue. Mononuclear cells obtained were cryopreserved in 90%FBS/10% DMSO (vol/vol) until further use.
Figure 4: Harvested mouse femur and tibia for bone marrow cells

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Injection of BM-MSCs into mouse models

Six weeks post creation of the disc degeneration models, isolated BM-MSCs [Figure 5] (at the end of 2 passages and in a dose of 30,000 cells/3 μl) in fibrin glue scaffold were injected into the Co 4-5-disc space of test group (8) with the help of a 33G-μl syringe. In the placebo group (8), only fibrin glue was injected, and in the control group (8), no injection was given. Post-intervention, all mice were observed for 8 weeks.
Figure 5: The mouse bone marrow-derived MSCs at 3 weeks (10×)

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Post injection of BM-MSC's CT imaging and histology

After 8 weeks of BM-MSC injection, all mice underwent CT for DHI measurement, and the change in DHI % was calculated as shown in [Figure 2]. Post CT, the animals were sacrificed, and sections of their tails were taken for histological staining.

Harvested mouse spine segments were fixed with 10% neutral buffered formalin for 1 week and then decalcified in 22.5% formic acid and 10% sodium citrate for 3 days. The material was processed for paraffin embedding, and sagittal sections of 4-μm thickness obtained using a microtome. The sections were stained with Safranin O and Sirius Red stains. Disc degeneration in all the groups was graded using the Masuda disc degeneration score[5] [Table 1]. Polarized imaging with Sirius Red stain was done to check the orientation of collagen fibers in the annulus fibrosus (AF) of the discs.
Table 1: Masuda disc degeneration score

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 » Results Top


Histology findings

Eight weeks after injection, Safranin O stained sections of the test group showed cartilage regeneration with vacuolation in the center, and restoration of collagen fibers in the AF in 4 out of 8 animals. In the other four mice, disc degeneration persisted due to leakage of stem cells into the subcutaneous plane [Figure 6] with partial cartilaginous metaplasia at the leak site. In the placebo and control groups, there were no features of cartilage regeneration.
Figure 6: Safranin O stained histology sections of test group showed the formation of nucleus pulposus in the center and restoration of AF collagen fibers in 4 of the 8 animals. The other 4 mice (on the right-side images) had shown severe disc degeneration. The leaked cells which had differentiated into cartilage (circled) as evident in Safranin O stain are seen subcutaneously

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The comparison of placebo and control groups with the test group showed a clear demarcation between degenerated and regenerated discs [Figure 7]. The Masuda scores of the test, placebo, and control groups were 10.0, 11.7, and 12, respectively, which were statistically significant (P < 0.001) [Table 2].
Figure 7: The histology of test, placebo, and control groups demonstrating clear demarcation between the regenerated and degenerated discs after 8 weeks of MSC injection (upper row: Safranin O stain; lower row: plain polarized light microscopy with Sirius Red stain)

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Table 2: Masuda disc degeneration grade of three groups post intervention

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Polarized imaging with Sirius Red stain of AF at 400× magnification in the test group showed restoration of the orientation of collagen fibers, suggestive of healing [Figure 8]. In the control and placebo groups, the disorientation persisted, and no signs of healing were seen.
Figure 8: shows histology slide of test group (a) 400X magnification of Sirius red stain without polarized light microscopy. (b) Plain polarized light microscopy following Sirius red stain of annulus fibrosus with 400X magnification showed restoration of orientation of collagen fibers, suggestive of healing along the track of the needle used for injuring the disc, in the animals of MSC transplanted cohort

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CT findings

Six weeks after needle injury, there was a significant increase in DHI (%) but, after treating the test models with BM-MSCs, the change in DHI (%) had reduced significantly at the end of 8 weeks, whereas in the placebo and control groups, it remained same [Graph 1].




 » Discussion Top


This is the second study to describe the effect of mesenchymal stem cells in a percutaneous needle injury mouse disc degeneration model. Other needle puncture studies have been done in rabbits, rats, and porcine models.[5],[6],[7],[8],[9] Our model is unique, as the disc injury was created using a sophisticated novel tool leading to accurate placement of the needle, confirmed by CT (We discussed the outcomes and comparison of our model in a separate article).[4] We chose a mouse tail model for ease of handling and less operative morbidity.

Injection of MSCs and fibrin glue scaffold

MSCs are the preferred choice for disc repair because of their capacity to differentiate into the chondrocyte lineage and produce collagens and proteoglycans.[10],[11] Feng et al.[6] recommended MSCs as ideal for the regeneration of the nucleus pulposus (NP) than NP cells. Yang et al.[9] demonstrated that BM-MSCs arrest mouse intervertebral disc degeneration through chondrocyte differentiation and stimulation of endogenous cells. They also showed disc regeneration by upregulated proteoglycan genes and accumulation of extracellular matrix in NP after BM-MSC injection.

Different scaffolds are available for injecting stem cells,[12],[13],[14],[15] but we found fibrin glue (a conventional sealant) to be superlative. One of its unique features is that it provides chemotaxis for inflammatory cells in the repair of damaged tissues.[16],[17] These fibrin sealants are three-dimensional scaffolding agents, capable of maintaining cell survival with no interference with differentiation and useful in drug delivery. Fibrin glue has properties of an appropriate scaffold for stem cells as it is suitable for every need, cost-effective, and does not transmit infectious diseases from the blood.[17] Fibrin glue (gel scaffold) is easily injectable into the disc space but may also leak into the subcutaneous space as happened in four of our animals.

Evidence of disc regeneration

In the test group, four mice (50%) had restoration of DHI% with a decrease in Masuda degeneration score, whereas, in the remaining 4, the disc degeneration persisted. The change in DHI% showed a statistically significant reduction in the test group in comparison to the placebo and control groups, indicating restoration of disc height following MSC injection [Graph 1]. It may be noted that the Masuda degeneration score was 10 in the test group, and this was significantly lower than the scores of placebo and control groups [Table 2]. There was no significant difference in the Masuda grade between the placebo and control groups, indicating that it was the MSC injection and differentiation that resulted in regeneration of discs in treated animals and not the natural healing process.

In polarized light microscopy with the Sirius Red stain, we could see the restoration of fiber orientation in disrupted regions of the AF in the test group. The margin between the AF and NP in the test group was distinctly seen, whereas, in control and degenerated groups, it was not. This was explained by the degenerated NP region being filled with newly formed thin green collagen fibers. The Safranin O stain revealed an increase in GAG content of the NP, as well as marked microvacuole formation and an increase in the number of cartilage cells in the test group. Overall, there was restoration of the NP, which was well demonstrated by the histology findings, degeneration score, and change in DHI%.

Comparison with other studies

In various studies, it has been shown that MSCs maintain the annular structure, reconstitute glycosaminoglycan and keratin sulfate in the disc nucleus, and also partially restore disc height and hydration.[9],[18],[19] Our results show similar evidence of recovery as seen in the previous animal studies done by Yang et al., Sakai et al., and Omlor et al.[7],[9],[18]

Effect of intradiscal environment on MSC differentiation

In the test group, 50% (4) of animals did not show regeneration due to leakage of injected stem cells into the subcutaneous tissue. MSCs which migrated into subcutaneous tissue differentiated incompletely into cartilage, as revealed by Safranin O stained histological sections. This finding was not seen in the placebo or control groups, which confirms that it was due to the migration of MSCs with fibrin glue outside the disc space. This incomplete cartilage metaplasia in our study has proven the in vivo importance of MSC implantation and microenvironment for its differentiation. Similar findings were also seen in Vadala et al.[20] study. They had migration of injected MSCs in a rabbit disc degeneration model and led to anterolateral osteophyte formation without any regeneration effects in the disc. The osteophytes had enhanced green fluorescent protein (eGFP) labelled MSCs which confirmed the migration.

Every tissue has its matrix of intracellular and extracellular components. The fate of MSCs between growth, differentiation, and apoptosis is highly dependent on the available environmental cues, cell-cell contacts, cell-extracellular matrix contacts, and physical forces.[21] The presence of cell-cell contact is probably essential for the differentiation of MSCs into NP cartilage. The identification of differences between the extracellular matrix of the nucleus pulposus and cartilage for MSC differentiation was stressed by Mwale.[22] Lech et al.[23] demonstrated substantial divergence of MSC differentiation with the regulation of microenvironment. In their study, they highlighted the need to implement more sensitive and selective methods to predict and control the fate of stem cells in-vitro before their use in vivo.

Intervertebral discs have a very different microenvironment (low glucose and low oxygen) when compared to other cartilage tissue in the body. The microenvironment of the disc following the onset of degeneration has a significant effect on the survival of transplanted mesenchymal stem cells. Post degeneration, the release of different proteases and cytokines like matrix metalloproteinases (MMPs), interleukins (IL- 6, IL-8), and tumor necrosis factor (TNF-alpha) has an effect on BM-MSCs.[24] IL-6 and TNF cause paracrine effects on BM-MSC, whereas an increase in intradiscal pressure andMMPs, lead to migration of BM-MSCs. The factors favoring chondrocytic differentiation of BM-MScs are NP cells, appropriate hypoxia, MMPs, acidic PH, and mechanical loading.[24] The complexities of these molecular mechanisms lead to a challenging task for these MSCs; however, recent studies on homing MSCs to Tie-2 positive progenitor cells prevents apoptosis and provides a proliferative response.[25] As shown by various studies in the literature, the maximum survival of MSCs in a scaffold is approximately 6 months.[8],[26] We assume our cells survived until 8 weeks, as was the case in the previous similar studies.[6],[8],[9],[26]

Limitations

We could not track these MSCs following their transplantation into the mice because of the lack of a stable reporter gene. Quantification tests like gene expression analysis, GAG quantification, and NP cell quantification could have added further information on the regeneration process. CT is suboptimal for evaluation of soft tissue structure and, ideally, MRI is required for the best display of structural detail.


 » Conclusion Top


Intradiscal MSC injection restored disc height and promoted regeneration in injured discs by 8 weeks. A unique finding due to leakage of cells demonstrated the significance of macro and microenvironment for MSC differentiation. These findings will be helpful while using MSCs for clinical trials.

Acknowledgements

Mr. Parameshwaran and Dr. Suresh R. Devasahayam (Bio-engineering).

The manuscript submitted does not contain any patient data/medical device/drug(s) information.

This research was approved by IRB (IRB no- 9263) and animal ethics committee (IAEC).

Prior publication - Nil.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
 » References Top

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25.
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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8]
 
 
    Tables

  [Table 1], [Table 2]



 

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