Local injection of autologous bone marrow cells to regenerate muscle in patients with traumatic brachial plexus injury

Article focus

Is autologous bone marrow-derived mononuclear cell injection in partly denervated muscle of traumatic brachial plexus patients safe?

Can mononuclear cell injection cause reinnervation and regeneration of the denervated muscle?

Key messages

Following autologous bone marrow-derived mononuclear cell injection no adverse effects were seen

After cell transplantation quantitative needle electromyography improved, suggestive for muscle reinnervation

Computerised muscle tomography and histological assessment showed muscle improvement, suggestive for myogenic regeneration

Strengths and limitations

Strength: a profound pilot study using blinded histology, quantitative needle electromyography and computerised muscle tomography as objective effect parameters

Limitations: the small number of patients and the lack of a control patient group. The promising results of the study require confirmation in a larger controlled clinical study

Introduction

Brachial plexus (BP) injury is often a result of a high-energy road traffic accident in young adults.^1,2 The upper BP (C5-C6) is commonly affected, resulting in paresis of shoulder function and elbow flexion.^1-4 Nerve surgery is aimed at restoring innervation of the bicepsmuscle, but often a deficit in the functionality of the arm remains. In that case, secondary surgery can improve arm function by transfer of the flexor-pronator group of the forearm (Steindler procedure) for active elbow flexion.^5-10 However, neither nerve surgery nor tendon transfers come close to restoring normal elbow flexion.⁵

Any procedure that improves elbow flexion without sacrificing the function of transferred donor muscles will have advantages for the patient. One approach is to improve the regenerative capacity of the partially denervated muscles. Denervation causes loss of contractile force and muscle atrophy. If innervation is restored quickly, good restorative ability exists. Conversely, prolonged interruption of innnervation results in irreversible muscle atrophy, interstitial fibrosis and muscle fattening.^11-14 Since regeneration of denervated muscles not only depends on nerve supply, but also on its regenerative capability, the muscle can be a target organ for cell therapy.

Muscle satellite cells are responsible for repair and maintenance of skeletal muscles; they are the main source of new myofibres. Myogenic precursor cells proliferate, fuse and form new myofibres in response to muscle damage.¹⁵ Exhaustion of the pool of available satellite cells may contribute to poor functional recovery of long-term denervated muscle.^16,17 A progressive and rapid decrease in the capillary-to-muscle fibre ratio occurs, further limiting regeneration.¹⁸

Transplantation of primary satellite cells has been shown to improve the properties of reinnervated skeletal muscles in rabbits.¹⁹ However, poor cellular survival and limited cell dissemination hampers successful satellite cell transplantation. Furthermore, only few cells fuse with host fibres. This suggests that a subpopulation of myogenic cells (i.e. stem cells) may be optimally suited for transplantation.^20,21 Bone marrow (BM)-derived cells migrate to the site of muscle injury and contribute to the satellite cell pool.^22-24

The injection of autologous BM-derived mononuclear cells (MNCs) has been applied in clinical studies focusing on the muscles of the heart and leg.^25-28

The primary objective was to assess the safety of autologous BM-derived MNC injection in partially denervated muscles of BP patients. The secondary objective was to obtain a first estimate on regenerative potential of injected BM-derived MNCs in partially denervated muscles.

Patients and Methods

Results

Baseline patient and injected MNC characteristics are shown in Table I. During hospital stay and follow-up, no adverse events in vital signs (blood pressure, pulse rate, temperature), BM aspiration site, injection sites or surgical wound (haematoma, infection) were observed. Post-operatively, the mean decrease in haemoglobin (Hb) concentration was 0.2 mmol/l (group A), 1.0 mmol/l (group B), and 1.8 mmol/l (group C). Hb concentration was normalised in all patients six months after BM aspiration.

In order to assess the safety of MNC injection at a local muscular level, quantitative CT analysis and histology were used. With quantitative CT analysis, the MMD of the injured biceps muscle was compared with injured and non-injured control muscles. A significant decrease in the MMD of the injured biceps muscles in the total patient group was observed (p = 0.03). The decrease was 32% at three months (p = 0.04) and 48% at six months follow-up (p = 0.009). No dose effect was observed. The non-injured contralateral biceps and the injured (non-injected) triceps muscle showed no significant change in MMD (Fig. 1).

Figs. 1a - 1b

Graphs showing the mean muscle density (MMD) of the injured and non-injured biceps a) and triceps b) muscle pre-transplantation and at three and six months follow-up for the total patient group. Error bars denote 95% confidence interval (HU, Hounsfield unit).

A Masson’s Trichrome staining was performed to analyse the possible formation of muscle fibrosis after cell injection. Compared with the pre-injection biopsy, a decrease of 52% in mean areas of fibrosis of the injured biceps muscle was measured at three months after cell injection in the total patient group (p= 0.01). No dose effect was observed (Fig. 2).

Figs. 2a - 2b

Histological images showing Masson’s trichrome staining of a representative muscle biopsy a) pre- and b) three months post-transplantation (magnification 5×).

In order to assess any regenerative potential of the injected MNCs, quantitative needle EMG and histology were performed. The mean amplitude, duration, and number of phases of the MUPs of the injured biceps muscle were compared with injured and non-injured control muscles. After three months, the increase was 36% in mean amplitude (p = 0.045), 22% in duration (p = 0.005) and 29% in number of phases (p = 0.002). The increase in number of phases were sustained at six months follow-up (p = 0.001). A dose-effect relation between group A, B and C was observed only in the mean amplitude (p = 0.03). The non-injured contralateral biceps muscle, the injured (non-injected) brachialis muscle and non-injured brachialis muscle showed no significant changes in amplitude, duration, or number of MUP phases. The mean number of analysed MUPs was 5.3 (sd 2.2). This low number represents the severity of the nerve lesions in which patients were unable to recruit more motor units (Fig. 3).

Figs. 3a - 3f

Graphs showing the mean amplitude (a and b), duration (c and d) and number of phases (e and f) of motor unit potentials of the injured and non-injured biceps (left column) and brachialis muscle (right column) pre-transplantation and at three and six months follow-up for the total patient group. Error bars denote 95% confidence interval.

The myogenic repair potential of the injected MNC cells was also evaluated using the histology of the injured biceps muscle. Analysis of all patients showed a mean increase of 80% in myofibre diameter three months after the cell injection compared with the pre-injection biopsy (p = 0.007). Pax7 staining demonstrated a 50% increase in the number of satellite cells (p = 0.045). Von Willebrand Factor staining showed an increase of 83% in the number of capillaries per myofibre (p< 0.001). No significant increase was demonstrated in the number of centronucleated myofibres or CD56 positive myofibres after cell therapy (Table II). Histological muscle regeneration was most apparent in group B, which observed a 126% increase in myofibre diameter, a 186% increase in percentage of centronucleated myofibres, a 117% increase in number of satellite cells, a 100% increase in capillaries per myofibre and a 70% decrease in the area of fibrosis. No dose-effect relation was observed in any of the histological measurements. Figure 4 shows a representative example of H& E, Pax7, and vWF and CD56 staining of a muscle biopsy before and after cell therapy in group B. After cell therapy, the muscle biopsy of one patient in group A did not contain 200 muscle fibres and was therefore excluded from analysis. In group A, no analysis of the CD56 staining could be performed because one patient’s muscle biopsy after cell therapy contained only 120 myofibres and was therefore excluded.

Figs. 4a - 4h

Histological images for specimens taken pre-transplantation (left column) and at three months follow-up (right column), showing staining of a representative muscle biopsy with haematoxylin and eosin (a and b; ×20), Pax7 (c and d, ×40), vWF (e and f, ×40) and CD56 (g and h, ×40).

In order to observe function improvement after MNC injection, we measured the active flexion ROM and strength of the injured elbow before and after cell therapy compared with the injured and non-injured control muscles. In addition, quality of life questionnaires (SF-36 and DASH) and the VAS score for pain were used. Overall, all patients had a significant increase in active flexion ROM and flexion strength. The flexion ROM increase was 152% at three months (p < 0.001) and 147% at six months follow-up (p< 0.001). The flexion strength increase was 38% at three months (p< 0.001) and 46% at six months follow-up (p< 0.001). As expected, the increase in active elbow flexion ROM and flexion strength was most prominently significant in patient groups where cell therapy was combined with a modified Steindler procedure (groups B and C). No significant changes in SF-36, DASH or VAS score were observed comparing pre- and post-injection at three months (Table III).

Discussion

Autologous BM-derived MNC injection in a partially denervated biceps muscleis safe and shows no adverse events with respect to vital signs, BM aspiration sites, injection sites or surgical wounds. Only a mild anaemia was observed, which recovered fully. The interstitial fibrosis present at long-term denervation decreased by 52% after MNC injection. These results run parallel to the results of CT scan analysis, which demonstrated a decrease of 48% in MMD.

Our secondary aim was to estimate myogenic repair and reinnervation of partial denervated muscles after MNC injection. Histological analysis, quantitative needle EMG and CT scan analysis demonstrated muscle improvement after MNC injection, which was not present in the control muscles of the sound arm or in the non-injected injured muscles of the affected arm.

Our study has some limitations. While useful to quantify fatty degeneration of rotator cuff muscles, CT analysis only provides an indirect measure of the amount of muscle lipid or fibrosis.³³ We observed a significant decrease in MMD of the injured biceps muscles after cell injection, which could reflect decrease in muscle fibrosis and increase in muscle vascularisation, muscle fattening or oedema from the procedure.³⁶ Histology of the muscle biopsies demonstrated a 52% decrease in muscle fibrosis. In addition, an 83% increase in capillaries per myofibre was observed. Using routine H& E staining, no increase in muscle fattening was seen, thus the decrease in MMD reflects either a decrease in muscle fibrosis or an increase in muscle vascularisation. The presence of muscle denervation was assessed using quantitative needle EMG. Analysing a minimum of 20 MUPs per analysed muscle is generally accepted in the literature.^34,37,38 Unfortunately, we could not obtain a minimum of 20 MUPs per analysed muscle (mean 5.3 MUPs [sd 2.2]), due to the partial denervation. Nevertheless a significant difference in mean amplitude, duration and the number of phases was found.

Another limitation was that in groups B and C, the BM-derived MNC injection was combined with a flexor-pronator transfer, which improves elbow flexion. Since the flexor-pronator group has to be fixated to the distal humerus with a bicortical screw, it is possible that BM may leak underneath the screw, stimulating the muscle in its vicinity (biceps triceps). However, CT changes were not seen in the control triceps muscle, nor were EMG improvements in the injured but non-injected brachialis muscle. A final limitation was low number of patients and lack of a control patient group (i.e., modified Steindler in a partially denervated biceps muscle with no MNC injection).

Quantitative needle EMG was used to determine muscle innervation. After injection of BM-derived MNC cells, an increase of 36% in amplitude, 22% in duration, and 29% in number of phases of MUPs was observed, suggestive for muscle reinnervation. Both human and animal studies have shown that BM stromal cells can differentiate into neural cells and improve nerve regeneration.^39-42

Histology was used to evaluate the myogenic repair on a microscopic level. Upon muscle denervation, animal studies have shown a decrease from 2104 µm to 50 µm in myofibre diameter, a decrease from 1.6% to 0.2% in capillary-to-myofibre ratio and an increase from 11% to 35% in fibrosis.^14,17,18 Our data are consistent with these studies, demonstrating a mean myofibre diameter of 193 µm, a mean capillary-to-myofibre ratio of 0.6 and a mean fibrosis of 25% before cell injection.

The number of cells in the satellite pool within a muscle appears to be correlated to postnatal growth characteristics of the muscle (Pax7). In adult muscle, up to 10% Pax7 positive myofibres have been reported.^16,17,43 Before cell injection, we observed mean Pax7 positive myofibres of 11.9% (sd 6.5). This higher percentage of Pax7 fibres is probably due to the partially denervated biceps muscle in the current study.

After MNC injection, we observed an increase of 50% in Pax7 positive myofibres and an increase of 83% in vWF positive capillaries per myofibre. This may demonstrate that MNC injection can result in restoration of the satellite cell pool and vascular bed, which can play an important role in functional recovery following long-term denervation. Indeed, the increase of 80% in myofibre diameter suggested myofibre regeneration and thus restoration of muscle atrophy. Muscle regeneration was most prominently observed in patient group B, with an increase of 117% in Pax7 positive myofibres, 100% in capillary-to-myofibre ratio, 126% in myofibre diameter, 186% in centronuclear myofibres and a decrease of 70% in fibrosis. These results are suggestive of an optimal dose in group B, although a dose-effect response was not significant.

Skeletal muscle is a regenerative tissue in which mononuclear precursor cells respond to injury by dividing and fusing with damaged fibres.¹⁵ As such, it is an attractive target for cell-based therapy. Muscle fibre regeneration levels of up to 12% have been achieved by using appropriate transplant variables, including cell dose and mode of cell delivery.²⁴ The present study is the first in which BM transplantation in a human muscle denervation model was assessed with objective measurements (histology, CT muscle and EMG). In humans, transplantation of the MNC fraction of the adult BM enhanced cardiac- and skeletal muscle function in post-ischaemic heart failure and peripheral artery disease.^25-28 These effects could be attributed to the incorporation of stem cells and building muscle or by supplying the logistics required for efficient proliferation of innate cells via paracrine effects.^44,45 Heterochronic parabiosis was shown to restore the regenerative potential of satellite cells, which suggests that satellite cell activity can be modulated by their microenvironment.⁴⁶ Chronic inflammation does not affect the in vitro regenerative potential of human satellite cells, which underscores the in vivo influence of the microenvironment on muscle regeneration.⁴⁷ Unravelling the molecular mechanism behind the observed regeneration in BP injury patients is a challenge that still has to be met. In the future, this can be studied in newly developed animal models representative for BP injury.^48,49

In conclusion, BM-derived MNC injection is safe in partially denervated muscle of traumatic BP patients. Significant muscle improvement has been observed in muscle biopsies, quantitative needle EMG and CT scan analysis. Although promising, the preliminary results of the present study require confirmation in a larger controlled clinical study.

Acknowledgements: We thank A. Kawakami for the Pax7 antibody, obtained from the Developmental Studies Hybridoma Bank, developed under the auspices of the NICHD and maintained by the University of Iowa, Department of Biological Sciences, Iowa City, Iowa. The assistance on the research protocol provided by J. H.N. Lindeman was greatly appreciated. We thank F. A. Prins, I. M. Hegeman and C. Welling of the Department of Pathology, Leiden University Medical Center, Leiden, The Netherlands for their technical assistance.