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Reconstruction of Multiple Facial Nerve Branches Using Skeletal Muscle-Derived Multipotent Stem Cell Sheet-Pellet Transplantation.

ABSTRACT: Head and neck cancer is often diagnosed at advanced stages, and surgical resection with wide margins is generally indicated, despite this treatment being associated with poor postoperative quality of life (QOL). We have previously reported on the therapeutic effects of skeletal muscle-derived multipotent stem cells (Sk-MSCs), which exert reconstitution capacity for muscle-nerve-blood vessel units. Recently, we further developed a 3D patch-transplantation system using Sk-MSC sheet-pellets. The aim of this study is the application of the 3D Sk-MSC transplantation system to the reconstitution of facial complex nerve-vascular networks after severe damage. Mouse experiments were performed for histological analysis and rats were used for functional examinations. The Sk-MSC sheet-pellets were prepared from GFP-Tg mice and SD rats, and were transplanted into the facial resection model (ST). Culture medium was transplanted as a control (NT). In the mouse experiment, facial-nerve-palsy (FNP) scoring was performed weekly during the recovery period, and immunohistochemistry was used for the evaluation of histological recovery after 8 weeks. In rats, contractility of facial muscles was measured via electrical stimulation of facial nerves root, as the marker of total functional recovery at 8 weeks after transplantation. The ST-group showed significantly higher FNP (about three fold) scores when compared to the NT-group after 2-8 weeks. Similarly, significant functional recovery of whisker movement muscles was confirmed in the ST-group at 8 weeks after transplantation. In addition, engrafted GFP+ cells formed complex branches of nerve-vascular networks, with differentiation into Schwann cells and perineurial/endoneurial cells, as well as vascular endothelial and smooth muscle cells. Thus, Sk-MSC sheet-pellet transplantation is potentially useful for functional reconstitution therapy of large defects in facial nerve-vascular networks.

SUBMITTER: Saito K

PROVIDER: S-EPMC4570662 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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Reconstruction of Multiple Facial Nerve Branches Using Skeletal Muscle-Derived Multipotent Stem Cell Sheet-Pellet Transplantation.

Saito Kosuke K Tamaki Tetsuro T Hirata Maki M Hashimoto Hiroyuki H Nakazato Kenei K Nakajima Nobuyuki N Kazuno Akihito A Sakai Akihiro A Iida Masahiro M Okami Kenji K

PloS one 20150915 9

Head and neck cancer is often diagnosed at advanced stages, and surgical resection with wide margins is generally indicated, despite this treatment being associated with poor postoperative quality of life (QOL). We have previously reported on the therapeutic effects of skeletal muscle-derived multipotent stem cells (Sk-MSCs), which exert reconstitution capacity for muscle-nerve-blood vessel units. Recently, we further developed a 3D patch-transplantation system using Sk-MSC sheet-pellets. The ai ...[more]

PMID: 26372044

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Project description:Vibrissal whisking is often employed to track facial nerve regeneration in rats; however, we have observed similar degrees of whisking recovery after facial nerve transection with or without repair. We hypothesized that the source of non-facial nerve-mediated whisker movement after chronic denervation was from autonomic, cholinergic axons traveling within the infraorbital branch of the trigeminal nerve (ION). Rats underwent unilateral facial nerve transection with repair (N=7) or resection without repair (N=11). Post-operative whisking amplitude was measured weekly across 10weeks, and during intraoperative stimulation of the ION and facial nerves at ⩾18weeks. Whisking was also measured after subsequent ION transection (N=6) or pharmacologic blocking of the autonomic ganglia using hexamethonium (N=3), and after snout cooling intended to elicit a vasodilation reflex (N=3). Whisking recovered more quickly and with greater amplitude in rats that underwent facial nerve repair compared to resection (P<0.05), but individual rats overlapped in whisking amplitude across both groups. In the resected rats, non-facial-nerve-mediated whisking was elicited by electrical stimulation of the ION, temporarily diminished following hexamethonium injection, abolished by transection of the ION, and rapidly and significantly (P<0.05) increased by snout cooling. Moreover, fibrillation-related whisker movements decreased in all rats during the initial recovery period (indicative of reinnervation), but re-appeared in the resected rats after undergoing ION transection (indicative of motor denervation). Cholinergic, parasympathetic axons traveling within the ION innervate whisker pad vasculature, and immunohistochemistry for vasoactive intestinal peptide revealed these axons branching extensively over whisker pad muscles and contacting neuromuscular junctions after facial nerve resection. This study provides the first behavioral and anatomical evidence of spontaneous autonomic innervation of skeletal muscle after motor nerve lesion, which not only has implications for interpreting facial nerve reinnervation results, but also calls into question whether autonomic-mediated innervation of striated muscle occurs naturally in other forms of neuropathy.

Project description:Loss of vital functions in the somatic motor and sensory nervous systems can be induced by severe peripheral nerve transection with a long gap following trauma. In such cases, autologous nerve grafts have been used as the gold standard, with the expectation of activation and proliferation of graft-concomitant Schwann cells associated with their paracrine effects. However, there are a limited number of suitable sites available for harvesting of nerve autografts due to the unavoidable sacrifice of other healthy functions. To overcome this problem, the potential of skeletal muscle-derived multipotent stem cells (Sk-MSCs) was examined as a novel alternative cell source for peripheral nerve regeneration. Cultured/expanded Sk-MSCs were injected into severely crushed sciatic nerve corresponding to serious neurotmesis. After 4 weeks, engrafted Sk-MSCs preferentially differentiated into not only Schwann cells, but also perineurial/endoneurial cells, and formed myelin sheath and perineurium/endoneurium, encircling the regenerated axons. Increased vascular formation was also observed, leading to a favorable blood supply and waste product excretion. In addition, engrafted cells expressed key neurotrophic and nerve/vascular growth factor mRNAs; thus, endocrine/paracrine effects for the donor/recipient cells were also expected. Interestingly, skeletal myogenic capacity of expanded Sk-MSCs was clearly diminished in peripheral nerve niche. The same differentiation and tissue reconstitution capacity of Sk-MSCs was sufficiently exerted in the long nerve gap bridging the acellular conduit, which facilitated nerve regeneration/reconnection. These effects represent favorable functional recovery in Sk-MSC-treated mice, as demonstrated by good corduroy walking. We also demonstrated that these differentiation characteristics of the Sk-MSCs were comparable to native peripheral nerve-derived cells, whereas the therapeutic capacities were largely superior in Sk-MSCs. Therefore, Sk-MSCs can be a novel/suitable alternative cell source for healthy nerve autografts.

Project description:Background. Significant and/or complete rupture in the musculotendinous junction (MTJ) is a challenging lesion to treat because of the lack of reliable suture methods. Skeletal muscle-derived multipotent stem cell (Sk-MSC) sheet-pellets, which are able to reconstitute peripheral nerve and muscular/vascular tissues with robust connective tissue networks, have been applied as a "bio-bond". Methods. Sk-MSC sheet-pellets, derived from GFP transgenic-mice after 7 days of expansion culture, were detached with EDTA to maintain cell-cell connections. A completely ruptured MTJ model was prepared in the right tibialis anterior (TA) of the recipient mice, and was covered with sheet-pellets. The left side was preserved as a contralateral control. The control group received the same amount of the cell-free medium. The sheet-pellet transplantation (SP) group was further divided into two groups; as the short term (4-8 weeks) and long term (14-18 weeks) recovery group. At each time point after transplantation, tetanic tension output was measured through the electrical stimulation of the sciatic nerve. The behavior of engrafted GFP(+) tissues and cells was analyzed by fluorescence immunohistochemistry. Results. The SP short term recovery group showed average 64% recovery of muscle mass, and 36% recovery of tetanic tension output relative to the contralateral side. Then, the SP long term recovery group showed increased recovery of average muscle mass (77%) and tetanic tension output (49%). However, the control group showed no recovery of continuity between muscle and tendon, and demonstrated increased muscle atrophy, with coalescence to the tibia during 4-8 weeks after operation. Histological evidence also supported the above functional recovery of SP group. Engrafted Sk-MSCs primarily formed the connective tissues and muscle fibers, including nerve-vascular networks, and bridged the ruptured tendon-muscle fiber units, with differentiation into skeletal muscle cells, Schwann cells, vascular smooth muscle, and endothelial cells. Discussion. This bridging capacity between tendon and muscle fibers of the Sk-MSC sheet-pellet, as a "bio-bond," represents a possible treatment for various MTJ ruptures following surgery.

Project description:BackgroundThe occurrence of hyperplasia, through myofibre splitting, remains a widely debated phenomenon. Structural alterations and fibre typing of skeletal muscle fibres, as seen during regeneration and in certain muscle diseases, can be challenging to interpret. Neuromuscular electrical stimulation can induce myofibre necrosis followed by changes in spatial and temporal cellular processes. Thirty days following electrical stimulation, remnants of regeneration can be seen in the myofibre and its basement membrane as the presence of small myofibres and encroachment of sarcolemma and basement membrane (suggestive of myofibre branching/splitting). The purpose of this study was to investigate myofibre branching and fibre type in a systematic manner in human skeletal muscle undergoing adult regenerative myogenesis.MethodsElectrical stimulation was used to induce myofibre necrosis to the vastus lateralis muscle of one leg in 5 young healthy males. Muscle tissue samples were collected from the stimulated leg 30 days later and from the control leg for comparison. Biopsies were sectioned and stained for dystrophin and laminin to label the sarcolemma and basement membrane, respectively, as well as ATPase, and antibodies against types I and II myosin, and embryonic and neonatal myosin. Myofibre branches were followed through 22 serial Sects. (264 μm). Single fibres and tissue blocks were examined by confocal and electron microscopy, respectively.ResultsRegular branching of small myofibre segments was observed (median length 144 μm), most of which were observed to fuse further along the parent fibre. Central nuclei were frequently observed at the point of branching/fusion. The branch commonly presented with a more immature profile (nestin + , neonatal myosin + , disorganised myofilaments) than the parent myofibre, together suggesting fusion of the branch, rather than splitting. Of the 210 regenerating muscle fibres evaluated, 99.5% were type II fibres, indicating preferential damage to type II fibres with our protocol. Furthermore, these fibres demonstrated 7 different stages of "fibre-type" profiles.ConclusionsBy studying the regenerating tissue 30 days later with a range of microscopy techniques, we find that so-called myofibre branching or splitting is more likely to be fusion of myotubes and is therefore explained by incomplete regeneration after a necrosis-inducing event.

Dataset Information

Reconstruction of Multiple Facial Nerve Branches Using Skeletal Muscle-Derived Multipotent Stem Cell Sheet-Pellet Transplantation.

Publications

Reconstruction of Multiple Facial Nerve Branches Using Skeletal Muscle-Derived Multipotent Stem Cell Sheet-Pellet Transplantation.

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