Intervertebral disc herniation effects on multifidus muscle composition and resident stem cell populations

Absract Background Paraspinal muscles are crucial for vertebral stabilization and movement. These muscles are prone to develop fatty infiltration (FI), fibrosis, and atrophy in many spine conditions. Fibro‐adipogenic progenitors (FAPs), a resident muscle stem cell population, are the main contributors of muscle fibrosis and FI. FAPs are involved in a complex interplay with satellite cells (SCs), the primary myogenic progenitor cells within muscle. Little is known about the stem cell composition of the multifidus. The aim of this study is to examine FAPs and SCs in the multifidus in disc herniation patients. Multifidus muscle samples were collected from 10 patients undergoing decompressive spine surgery for lumbar disc herniation. Hamstring muscle was collected from four patients undergoing hamstring autograft ACL reconstruction as an appendicular control. Multifidus tissue was analyzed for FI and fibrosis using Oil‐Red‐O and Masson's trichrome staining. FAPs and SCs were visualized using immunostaining and quantified with fluorescence‐activated cell sorting (FACS) sorting. Gene expression of these cells from the multifidus were analyzed with reverse transcription‐polymerase chain reaction and compared to those from hamstring muscle. FI and fibrosis accounted for 14.2%± 7.4% and 14.8%±4.2% of multifidus muscle, respectively. The multifidus contained more FAPs (11.7%±1.9% vs 1.4%±0.2%; P<.001) and more SCs (3.4%±1.6% vs 0.08%±0.02%; P=.002) than the hamstring. FAPs had greater α Smooth Muscle Actin (αSMA) and adipogenic gene expression than FAPs from the hamstring. SCs from the multifidus displayed upregulated expression of stem, proliferation, and differentiation genes. Conclusion The multifidus in patients with disc herniation contains large percentages of FAPs and SCs with different gene expression profiles compared to those in the hamstring. These results may help explain the tendency for the multifidus to atrophy and form FI and fibrosis as well as elucidate potential approaches for mitigating these degenerative changes by leveraging these muscle stem cell populations.


| INTRODUCTION
Chronic low back pain (CLBP) is one of the world's leading debilitating conditions, and is the most common, noncancer reason for opioid prescription in the United States. 1 The paraspinal muscles, particularly the multifidus, have an important biomechanical role by serving to stabilize posture and limit excessive intervertebral movement. 2,3 Clinically, paraspinal muscles have been shown to develop fatty infiltration (FI), fibrosis, and muscle atrophy in a number of spine conditions, such as scoliosis, spinal stenosis, and disc herniation. [4][5][6][7][8] These degenerative changes are not without consequence as studies have found correlations between FI, muscle atrophy, back pain, and compensatory spinal biomechanics. 6,[9][10][11][12] The often multifaceted etiology of back pain creates ambiguity in understanding the pathophysiology underlying multifidus degeneration and symptoms across different spinal disorders, leaving room for further investigation. 4,13,14 A better understood source of back pain is intervertebral disc herniation which often results in spinal nerve root compression, leading to denervation and associated multifidus FI and atrophy. [15][16][17][18] Changes in paraspinal histologic fibrosis profiles in patients with disc herniation have also been reported. 19 In addition, avoidance of activities that worsen pain stemming from the herniation may lead to disuse related alterations that exacerbate these degenerative features. 20,21 As of now, strategies to mitigate FI and fibrosis in disc herniation patients are lacking, and attempts to reverse their progression in paraspinal muscle have proven to be challenging. For example, studies examining the effects of high intensity exercise regimens on muscle morphology in patients with CLBP have shown little or no efficacy in reducing paraspinal FI. 22,23 These results suggest the need to explore additional avenues to more effectively treat these patients.
Fibro-adipogenic progenitors (FAPs), a subset of resident muscle stem cells characterized by platelet-derived growth factor receptor alpha (PDGFRα) expression, have recently been found to serve an important role in muscle regeneration, FI, and fibrosis. Upon injury, FAPs rapidly proliferate and contribute to prodifferentiation signaling for satellite cells (SCs), the main myogenic progenitors in muscle.
These signals increase the rate of new SC myotube formation and initially aid in overall muscle regeneration. [24][25][26] However, after FAPs rapid expansion and transient promyogenic function, these cells continue toward adipocyte and fibroblast differentiation, becoming the main contributors of muscle FI and fibrosis. 27,28 Currently, little is known about the composition of paraspinal muscle in regard to FAPs and SCs and their association with paraspinal degeneration. A deeper understanding of such may provide opportunities for novel therapeutics that leverage these resident stem cell populations. In this study, we analyze multifidus muscle collected from disc herniation patients, examine the degree of degenerative pathology present, and investigate FAP and SC quantity and gene expression profiles. We hypothesized that there would be increased numbers of FAPs with greater adipogenic and fibrogenic expression profiles in the multifidus compared to control hamstring muscle.

| Muscle tissue collection
The need for institutional review board (IRB) approval for this study was considered prior to commencement. To access the vertebra for decompression surgery, muscle tissue, including the multifidus is dissected away from the spine for proper surgical exposure. Much of the muscle tissue adjacent to the facet joint is discarded during this process as gaining proper exposure results in significant disruption of the muscle-bone interface which is not amenable to repair. Given that the collected muscle specimens were taken from tissue routed to surgical waste as part of the surgical approach, IRB approval was not required by our institution. Although IRB approval was not required from our institution, all muscle samples were properly deidentified to protect patient health information. Ten patients undergoing decompressive spine surgery for lumbar disc herniation were selected for multifidus waste collection. During the operation, small pieces of multifidus muscle removed from the lumbar region as surgical waste were collected.
The samples were obtained from the muscle directly adjacent to and overlying the facet joint capsule that was removed as part of the surgical approach. The muscle removed from this location is reliably multifidus based on the anatomy and consistent surgical approach used by the surgeons. The tissue was then immediately transferred to the lab on ice for same day processing. Each specimen weighed approximately 1 g. The muscle samples were then partitioned for histology and fluorescence-activated cell sorting (FACS) cell sorting. To serve as a control from an appendicular anatomic location, hamstring muscle was harvested from patients undergoing ACL reconstruction with hamstring tendon autograft.

| Cell isolation
Muscle specimens underwent digestion separately at 37 C in 0.2% collagenase for 90 minutes followed by 0.4% dispase for 30 minutes. to quantify the expression of genes. A total of 5n g of cDNA was used per reaction. Primer sequences of the genes tested are summarized in Supplemental Table S1. The expression level of each gene was normalized to that of the housekeeping gene of S26. Fold difference relative to hamstring controls was calculated using double delta cycle threshold.

| Demographics
The mean age of patients from which multifidus muscle was collected was 62.2 ± 17.6 years. Patient age range was 37 to 83 years. There were eight males and two female patients. The anterior cruciate ligament (ACL) reconstruction with hamstring autograft cohort consisted of three females and one male. The mean patient age was 60.5 ± 11.6 with a range of 47 to 74 years. 3.3 | Multifidus muscle contains a higher number of FAPs and SCs FACS sorting for FAPs and SCs from the muscle specimens were performed ( Figure 2). There were significantly more FAPs in the multifidus than in the hamstring (11.7% ± 1.9% vs 1.4% ± 0.2%; P < .001) ( Figure 3). Similarly, the multifidus contained significantly more SCs than the hamstring muscle (3.4% ± 1.6% vs 0.08% ± 0.02%; P = .002). In addition, immunostaining using antibodies against PDGFRα and Pax7 revealed large quantities of FAPs and SCs within the multifidus (Figure 4).

| DISCUSSION
Our study demonstrates that multifidus tissue in lumbar disc herniation patients contains high levels of FI and fibrosis. We also demon- Our data regarding multifidus fat and fibrosis percentages is similar to that of other studies with histologic methods. One study that used histologic assessment of fat and fibrosis reported 11.7% adipose tissue and 26.1% fibrotic tissue in multifidus biopsies from patients with degenerative disc disease. 8 Another recent study which evaluated fat and fibrosis percentages in multifidus biopsies for a cohort of surgical spine cases, including patients undergoing lumbar disc herniation decompressive surgery, reported a multifidus fat percentage of 14.3% ± 12.3% and a fibrosis percentage of 21.2% ± 12.7%. 37 Most studies, however, quantify disc herniation paraspinal FI with magnetic resonance imaging (MRI) often using semiquantitative methods of which tend to have a wider range of values and greater averages. 4,17,38 These differences in disease and quantification methods make direct comparisons challenging as MRI FI assessment often includes fat consolidations surrounding muscle fascicles and compartments not normally captured in small multifidus histology sections.
Histologic evaluation employed in our study likely provides a more accurate assessment of intrafiber and interfiber FI of the multifidus than macroscopic imaging techniques.
The mechanism underlying the high quantity and elevated αSMA and adipogenic gene expression of FAPs in the multifidus, as found in our study, may be multifactorial. Baseline quantitative and qualitative differences in FAPs based on anatomic location are one possibility.
Previous work by our lab demonstrated that uninjured mouse F I G U R E 5 Comparative gene expression profile of FAPs from multifidus normalized to FAPs from hamstring. Gray bar designates fibrogenic gene expression. Black bars designate adipogenic gene expression. Y-axis scale represents logarithmic fold difference. * denotes P < .05. FAP, fibro-adipogenic progenitor F I G U R E 6 Comparative gene expression profile of SCs from multifidus normalized to SCs from hamstring. Gray bars designate stem/proliferation gene expression. Black bars designate differentiation gene expression. Y-axis scale represents logarithmic fold difference. * denotes P < .05. SC, satellite cell paraspinal muscles had higher concentrations of FAPs that exhibited increased proliferation and adipogenic potency compared to FAPs taken from hind limb muscles. 39 Interestingly, greater adipogenic gene expression was also noted in the human FAPs from the multifidus in this current study. Other studies have also detailed baseline differences in FAP populations. [40][41][42][43][44] Davies et al observed upregulated adipogenic genes and increased FI in the rotator cuff of mice compared to the gastrocnemius after analogous tendon-nerve transection injury at each site. 45 These results taken to together may provide some explanation for paraspinal muscle's proclivity to develop FI. FAPs residing is specific regions may be more primed for adipogenic differentiation than those in other muscle groups.
Beyond possible inherent differences between FAP populations within the multifidus and hamstring, the disuse-denervation injury specific features of disc herniation may also play a role. Pagano et al demonstrated upregulated expression of PDGFRα and adipogenic markers as well as increased intramuscular FI in a short-term disuse model. 46 In an amyotrophic lateral sclerosis mouse model that resulted in diffuse denervation, increases in muscle atrophy, fibrosis, and FAP number were observed. 47  Our study demonstrated markedly elevated percentages of SCs within the multifidus compared to the hamstring. Shahidi et al examined SCs in human multifidus muscle biopsies from patients with degenerative lumbar disc disease and found SCs at a rate of 13 ± 9 per 100 muscle fibers. 8 Due to differences in disease type and quantification methods, a direct comparison to our study is not possible.
However, discovering a large quantity of SCs in our study residing in muscle plagued by atrophy is seemingly paradoxical and not fully understood at this time. It is possible that the large amounts of SCs present in the multifidus are simply reflective of higher baseline concentrations as has been reported in literature regarding other muscle groups. 53,54 Alternatively, active degeneration of the muscle may trigger SC expansion; however, their inability to reliably regenerate the multifidus may point to overwhelming perturbations in the SC niche that hinders their effectiveness.
The differences in gene expression of stem, proliferation, and differentiation markers between SCs from multifidus and the hamstring detailed in our study further raise the possibility that changes in overall SC activation could play a role in the mismatch of increased SC quantity and atrophy. Upon injury, SCs proliferate and differentiate asymmetrically of which the majority of daughter cells continue toward myoblasts that form new myofibers. A small fraction of these daughter cells replenish the SC pool and retain their stemness as designated by continued Pax7 positivity. Studies have found that after denervation injury, SC populations within muscle increase, although this increase may be somewhat dependent on the duration of denervation. [55][56][57][58] Our study reveals upregulation of genes that preserve stemness and promote proliferation. 59 We also happened to observe elevated levels of UCP1 expression in FAPs from the multifidus compared to those from the hamstring.
FAPs have been shown to adopt a brown/beige fat (BAT) phenotype characterized by elevated expression of UCP1 that represents a more metabolically active tissue than its white fat (WAT) counterpart that comprises the majority of muscle FI. 24,65,66 BAT has been shown to have positive effects on myogenesis likely through paracrine signaling and "batokine" factor secretion. [67][68][69][70] Transplantation of BAT in rotator cuff muscles after cardiotoxin injury increased muscle mass, contractile force, and fiber cross-sectional area. 71 Wang et al demonstrated that reversal of rotator cuff FI in mice after suprascapular nerve compression and release likely involved the "browning" of white adipocytes. 66 Our findings of high multifidus FAP percentage and UCP1 expression may represent an opportunity for novel therapeutic approaches to reduce the amount of FI and curtail muscle atrophy by driving FAPs into the more beneficial BAT phenotype. Future studies are warranted to evaluate pharmacologic and transplantation strategies to increase adoption of the BAT phenotype by FAPs in paraspinal muscle and assess effects on FI, muscle quality, and function.
There are limitations to our study. We compare multifidus muscle after disc herniation to hamstring muscle. It is possible that ACL injury results local inflammation, disuse injury, and biomechanical changes that could impact hamstring morphology and stem cell populations, as has been demonstrated within the vastus lateralis by Fry et al 72 Ideally, uninjured multifidus would be used as a control for such a study.
However, due to the nature of human clinical research, procuring uninjured multifidus muscle from patients not undergoing a concurrent spine surgery was not feasible at this time. Future studies will attempt to obtain samples from other sources to allow for more robust direct comparisons. Additionally, procuring samples from patients undergoing surgery for different spinal conditions could further elucidate disc herniation specific features of FAPs and SCs related to paraspinal muscle degeneration Studies have shown that αSMA expression and collagen gene expression is significantly regulated by the TGFβ-SMAD signaling pathway. 31,73,74 Other studies have evaluated FAP αSMA expression in the setting of fibrogenesis and have detailed αSMA expression in myofibroblasts, which bear close similarity to FAPs. 74,75 However, there are likely additional genes involved in multifidus FAP-derived fibrosis other than αSMA examined in our study. Future studies will aim to expand the number of genes tested to further characterize FAP-fibrosis pathways in paraspinal muscle. Finally, the age range of the patients in our study is relatively wide. Age may impact baseline levels of stem cell populations as well as their responses to a variety of injury types, although we observed relatively narrow SC and FAP percentage standard deviations in this study. Future work will aim to expand the sample size and overall scope of the present study to better characterize the relationships between age, gene expression, muscle stem cell activity, and muscle pathology in disc herniation patients. These types of studies can identify potential therapeutic targets and help distinguish for which patient subsets they would be most effective.
In conclusion, this study demonstrates that multifidus muscle in patients undergoing surgery for disc herniation contain large quantities of FAPs and SCs which display different gene expression profiles compared to their counterparts within hamstring tissue. These results may help explain the multifidus' propensity to form FI and fibrosis and can shed light on novel strategies to target this cell population to improve muscle quality. In addition, the discovery of a large SC population within the multifidus in these patients who often suffer from muscle atrophy may prove useful in developing cell-based approaches that harness the SC pool to mitigate or reverse degeneration.