Cell therapy
for denervated tissue
Roine El-Habta
Department of Integrative Medical Biology, Anatomy Umeå 2020
This work is protected by the Swedish Copyright Legislation (Act 1960:729) The original articles were reproduced with permission from the publishers.
ISBN: 978-91-7855-228-3 ISSN: 0346-6612 New Series No 2077
Cover illustration by Yerai Ibarria Figures illustrated by the author
Electronic version available at: http://umu.diva-portal.org/
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“Science knows no country, because knowledge belongs to humanity, and is the torch which
illuminates the world.”
– Louis Pasteur
Table of Contents
Abstract ... iv
Populärvetenskaplig sammanfattning ... vi
Abbreviations ...viii
List of original papers ... x
Introduction ... 1
The musculoskeletal system ... 3
Tendon development ... 3
Development of skeletal muscle ... 3
Gross anatomy of tendons ... 4
Tendon pathology ... 5
Gross anatomy of skeletal muscle ... 6
Skeletal muscle contraction... 6
The musculotendinous junction ... 7
Acetylcholine: synthesis, origin, and receptors ... 8
Denervation and its consequences ... 8
Short-term degenerative changes ... 9
Long-term degenerative changes ... 10
Apoptotic response to muscle denervation ... 11
Treatment strategies ... 12
Experimental cell therapy ... 13
Adipose tissue-derived stem cells ... 14
Differentiated adipose tissue-derived stem cells ... 14
The stromal vascular fraction ...15
Potential mechanism of cell therapy in vivo ... 16
Hypothesis and Aims ... 19
Materials and Methods ... 23
Animal model... 25
Ethical considerations ... 25
Denervation model ... 25
Intramuscular stem cell injections ... 25
Muscle and tendon samples... 26
Cell culture model ... 26
Ethical considerations ... 26
Myoblast cell lines ... 26
Primary myoblasts... 27
Cells isolated from adipose tissue ... 28
Experimental setup ... 32
Methods of analysis ... 33
General morphology ... 33
Immunohistochemistry ... 35
Measurement of muscle fiber area and diameter ... 35
Immunocytochemistry ... 36
Analysis of proliferation and metabolic activity ... 36
Analysis of apoptosis ... 37
Quantitative reverse transcription PCR (RT-qPCR)... 38
Enzyme-linked immunoassay (ELISA) ... 40
Western blotting ... 40
Statistics ... 43
How data is presented ... 43
Unpaired t-test ... 43
One-way ANOVA with post hoc test (Bonferroni correction) ... 43
Results ... 45
Consequences of denervation for tendon ... 47
Hypercellularity ... 47
Changes in cell shape, collagen production and organization ... 47
Expression of signaling molecules... 47
Consequences of denervation for skeletal muscle ... 47
Muscle fiber area and diameter ... 47
Expression of muscarinic ACh receptors ... 48
Activation of apoptotic signaling ... 48
Effects of ASC treatment on the proliferation of myoblasts ... 48
Differences between uASC and dASC co-cultures ... 48
Expression of ACh-related proteins in ASCs ... 49
Exogenous ACh and its effects on proliferation ... 49
Effects of atropine on dASC-induced proliferation ... 50
Effects of SVF treatment on the proliferation of myoblasts ... 50
Proliferation of myoblasts in SVF co-cultures ... 50
The SVF cell secretome ...51
Exogenous HGF and its effects on proliferation ...51
Effects of Norleual on SVF-induced proliferation ...51
Possible sources of HGF ...51
Effects of SVF treatment on the differentiation of myoblasts ... 52
Effects of SVF on myotube formation ... 52
HGF and its role in myoblast differentiation ... 53
SVF cell differentiation ... 53
Effects of SVF treatment on the survival of myoblasts ... 55
TNF-a-induced apoptosis in myoblasts ... 55
Effects of SVF on TNF-a-induced apoptosis ... 55
Effects of intramuscular injections of SVF on denervated muscle ... 55
Discussion ... 57
Methodological aspects ... 59
Indirect co-culture as a model to study the SVF secretome ... 59
The use of primary cells vs. cell lines ... 59
The duration of denervation ... 60
Use of contralateral muscles and tendons as controls ... 60
Sample preparation ... 61
Tendinosis-like changes in denervated tendons ... 62
Hypercellularity ... 62
Collagen turnover ... 64
The regenerative potential of SVF cells ... 64
Proliferation... 64
Myogenic potential of SVF cells... 66
Anti-apoptotic effects ... 67
Clinical implications ... 68
The role of the tendon in the recovery after denervation ... 68
Cell therapy for denervated tissue ... 69
Major findings ... 71
Overall conclusions of this thesis ... 74
Acknowledgements ... 75
References ... 77
Abstract
Background
Peripheral nerve injury results in denervation of tendons and muscles. The bi- ology of denervated muscle has been well studied but little is known about the associated tendons. Denervation of muscle leads to atrophy which includes muscle fiber shrinkage and cell death, a process that is influenced by the lack of acetylcholine (ACh) signaling to the muscle cells. Recovery of long-term de- nervated muscle function is often poor. This thesis describes how a cell therapy approach using adipose tissue-derived stromal vascular fraction (SVF) may be used to protect and regenerate denervated muscle. Previous studies have shown how adipose tissue-dervied stem cells (ASCs), commonly expanded from the SVF, have pro-regenerative effects on the injured peripheral nervous system, and how ASCs differentiated towards a “Schwann cell-like phenotype” (dASCs) reduce muscle atrophy. In this thesis work, we studied the possible mechanisms underlying the regenerative potential of both SVF and culture expanded dASCs.
Hypotheses
We hypothesized that: 1) denervated tendon displays morphological and bio- chemical properties that resemble the chronic degenerative tendon condition known as tendinosis; 2) denervated muscle up-regulates expression of musca- rinic acetylcholine (ACh) receptors and apoptosis-associated signaling mecha- nisms; 3) dASCs enhance the proliferation of myoblasts in vitro through secre- tion of ACh; 4) SVF influences the proliferation, differentiation, and survival of myoblasts in vitro via secretion of growth factors; and 5) SVF can preserve de- nervated muscle tissue. To test our hypotheses, two model systems were used:
an in vitro model based on indirect co-culture, and an in vivo rat sciatic nerve transection model.
Results
Denervated tendon displayed morphological changes similar to tendinosis, in- cluding hypercellularity, disfigurement of cells, and disorganized collagen ar- chitecture, along with an increased expression of type I and type III collagen.
In addition, levels of neurokinin 1 receptor (NK-1R) were upregulated in the tendon cells. In denervated muscle, there was an increased expression of mus- carinic ACh receptors, as well as of genes associated with apoptosis, such as
caspases, cytokines (e.g., tumor necrosis factor-alpha; TNF-a), and death do- main receptors. We subsequently used TNF-a as an inducer of apoptosis in an in vitro rat primary myoblast culture model. TNF-a activated/cleaved caspase 7 and increased poly ADP-ribose polymerase (PARP) levels. Moreover, Annexin V and TUNEL were increased after TNF-a treatment. Indirect co-culture with SVF significantly reduced all these measures of apoptosis. Proliferation studies showed that both dASCs and SVF enhanced growth of myoblasts in vitro. With dASCs, the effect was partially explained by secretion of ACh, and for SVF by released growth factors, such as hepatocyte growth factor (HGF). In both cases, the signal was mediated via phosphorylation of ERK1/2 (MAPK). HGF also had an inhibitory effect on the differentiation of myoblasts into myotubes. Finally, the protective effects of SVF were confirmed in vivo: injections of SVF into de- nervated muscle significantly increased the mean fiber area and diameter, as well as reduced the expression of apoptotic genes and TUNEL reactivity.
Conclusions
Denervated tendons undergo severe degenerative changes similar to tendinosis.
Furthermore, SVF has the ability to reduce muscle atrophy in vivo. Using in vitro systems, we showed that this might occur through secretion of growth factors which activate MAPK signaling and anti-apoptotic pathways. In conclusion, SVF offers a promising approach for future clinical application in the treatment of denervated muscle.
Populärvetenskaplig sammanfattning
Vårt nervsystem kan delas in i det centrala nervsystemet, bestående av hjärnan och ryggmärgen, och det perifera nervsystemet, bestående av alla de nervtrådar som förbinder det centrala nervsystemet med kroppens övriga vävnader. Det finns två olika sorters nervtrådar i vår kropp: så kallade sensoriska nervtrådar, som skickar information från kroppen till det centrala nervsystemet, och moto- riska nervtrådar, som skickar information från det centrala nervsystemet till kroppens alla muskler och körtlar. I kroppen ligger dessa nervtrådar ofta pack- ade tillsammans. Skador på nerver kan därför leda till både känselbortfall och oförmåga att kunna röra sig.
Nerver växer med en hastighet av cirka 1 millimeter per dag, vilket innebär att om avståndet är långt kan det ta flera år innan målorganet (muskeln) återfår kontakten med nervsystemet efter en nervskada. Efter så lång tid har muskeln förändrats så till den grad att den inte längre går att reparera; muskelmassa har successivt bytts ut mot fett och ärrvävnad. Då spelar det inte längre någon roll om nerven växer tillbaka eftersom muskeln ändå inte går att använda.
I den här avhandlingen har vi undersökt cellterapi som en potentiell behand- lingsmetod för bevarandet av muskelvävnad. Tanken är att celler från buk- och underhudsfett, även kallat stromal vascular fraction (SVF), ska kunna skydda nervskadade muskler under den tid det tar för nerven att växa tillbaka. För att undersöka detta har vi använt oss av en metod som heter indirekt co-kultur som går ut på att odla muskelceller och SVF i samma vätska men utan att de kommer i direkt kontakt med varandra. På så sätt har vi kunnat studera betydelsen av de signalsubstanser som cellerna frisätter. Vi har även använt oss av en djurmodell där ischiasnerven (som löper ned till benet) hos råttor har skurits av varpå SVF har injicerats i vadmuskeln. Vi har därefter samlat in muskel- och senvävnad och analyserat dels vad som händer i respektive vävnad efter en nervskada, dels om injektioner med SVF verkligen kan motverka nedbrytning av muskelvävnad.
Våra resultat visar att det i senvävnad sker signifikanta förändringar efter två veckor av denervering (artikel I). Bland annat ökar antalet celler i senan och även produktionen av kollagen I och kollagen III (det ”sjuka” kollagenet). Vi kunde även konstatera att kollagenet låg ”huller om buller” istället för i paral- lella stråk, vilket skulle kunna förklara varför cellerna var rundare till formen istället för avlånga, vilket är det normala. I dessa senor fann vi också en ökad
mängd av smärtproteinet substans P och dess receptor NK-1R – fynd som ofta förknippas med sjukdomstillståndet tendinos.
Vidare har vi kunnat visa att en en mindre population av celler som återfinns i SVF, så kallade adipose tissue-derived stem cells (ASC), kan öka celldelningen hos muskelceller genom frisättning av acetylkolin (en signalsubstans som nervsyste- met själv använder för att kommunicera) och att denerverad muskelvävnad in- tressant nog ökar sitt uttryck av acetylkolinreceptorer (artikel II). Vi har också visat att SVF kan åstadkomma samma sak fast via en annan molekylär mekan- ism, nämligen frisättning av tillväxtproteinet HGF (artikel III). Slutligen har vi data som visar att injektioner med SVF kan förhindra celldöd (”apoptos”) och motverka nedbrytning av muskelvävnad genom en minskning av antalet ”döds- receptorer” hos muskelfibrer som det inflammatoriska proteinet TNF-a binder till (artikel IV).
Sammantaget har studierna i denna avhandling bidragit till den kunskapspool som kan komma att ligga till grund för framtida behandling av denerverad väv- nad.
Abbreviations
ACh Acetylcholine
ASC Adipose tissue-derived stem cell
BrdU Bromodeoxyuridine (5-bromo-2’-deoxyuridine) ChAT Choline acetyltransferase
Chrm Choline receptor muscarinic
c-Met Tyrosine-protein kinase Met (HGF receptor) dASC Differentiated adipose tissue-derived stem cell DMEM Dulbecco’s modified eagle medium
ERK1/2 Extracellular signal-regulated kinase 1 and 2 GFP Green fluorescent protein
HGF Hepatocyte growth factor MAPK Mitogen-activated protein kinase MEK MAP kinase kinase
MEM Minimum Essential Medium Mrf4 Myogenic regulatory factor 4
mRNA Messenger RNA
MSC Mesenchymal stem cell MTJ Musculotendinous junction Myf5 Myogenic factor 5
MyHC Myosin heavy chain
MyoD Myoblast determination protein
Myog Myogenin
NMJ Neuromuscular junction Pax3 Paired box gene 3
PARP Poly ADP-ribose polymerase PCR Polymerase chain reaction
RT-qPCR Quantitative reverse transcription PCR Scx Scleraxis transcription factor
SVF Stromal vascular fraction (adipose tissue-derived) Tac1 Tachykinin precursor 1 (Substance P)
Tacr1 Tachykinin receptor 1 (NK-1R) TGF-b1 Transforming growth factor b 1 TNF-a Tumor necrosis factor alpha
Tnfrsf Tumor necrosis factor receptor superfamily
TUNEL Terminal deoxynucleotidyl transferase dUTP nick end labeling uASC Undifferentiated adipose tissue-derived stem cells
VAChT Vesicular acetylcholine transporter
List of original papers
I. Tendinosis-like changes in denervated rat Achilles tendon
El-Habta R, Chen J, Pingel J, Backman LJ.
BMC Musculoskelet Disord 2018; 19; 426
II. Adipose stem cells enhance myoblast proliferation via acetylcho- line and extracellular signal-regulated kinase 1/2 signaling
El-Habta R, Kingham PJ, Backman LJ.
Muscle Nerve 2018; 57; 305-311
III. The adipose tissue stromal vascular fraction secretome enhances the proliferation but inhibits the differentiation of myoblasts
El-Habta R, Sloniecka M, Kingham PJ, Backman LJ.
Stem Cell Res Ther 2018; 9; 352
IV. Anti-apoptotic effect of adipose tissue-derived stromal vascular fraction in denervated rat muscle
El-Habta R, Andersson G, Kingham PJ, Backman LJ.
Manuscript
The original papers in the thesis will be referred to by their Roman numerals. Figures from the papers will be referred to by the Roman numeral of the paper followed by the figure number in the corresponding paper (e.g., Fig III: 4 = Figure 4 in paper III).
Introduction
Introduction
The musculoskeletal system
Tendon development
Tendon is a vital component of the musculoskeletal system as it transmits the mechanical force of muscle contraction to the bone and thereby the joints. Ten- dons of different parts of the body have distinct embryological origins, but most of them originate from mesoderm (Gaut and Duprez, 2016). Tendons are com- posed of highly aligned collagen fibrils organized as fibers, which are laid out in a parallell fashion with tendon cells (tenocytes) inbetween. Studies on pri- marily chick embryos have demonstrated the progressive assembly of collagen fibrils during fetal development, which involves collagen molecule assembly, linear fibril growth, and lateral fibril growth to generate large diameter fibrils (Zhang et al., 2005). Unlike skeletal muscle, which has “master genes” driving the formation of muscular tissue, no such genes have been identified in tendon (Gaut and Duprez, 2016). Furthermore, none of the extracellular matrix (ECM) components involved in tendon formation, mainly type I collagen, are specific to tendon, but are involved in collagen fibrillogenesis in other tissues as well.
To date, the only early tendon marker in vertebrates is the transcription factor Scleraxis (Scx) (Gaut and Duprez, 2016), which is expressed in tendon progeni- tor cells in somites and limbs (Schweitzer et al., 2001). In Scx mutant mice, ten- don still develops and the mice remain viable and mobile, but force transmis- sion is severely disrupted (Murchison et al., 2007). Thus, it is possible that Scx needs one or several partners to fulfill the function of “master gene” for teno- genesis.
Development of skeletal muscle
Skeletal muscle development can be divided into three stages: first, there is primary myogenesis, which refers to the formation of muscular tissue during embryonic development; then, there is secondary myogenesis, which occurs in the fetus and gives rise to the largest part of fetal muscle; and finally, postnatal muscle growth, which involves the stem cells (satellite cells) of skeletal muscle (Buckingham et al., 2003). Primary myogenesis relies on mesodermal cells. Be- fore skeletal muscle forms, the mesoderm at either side of the neural tube is segmented into well-defined blocks of tissue called somites (Christ and Ordahl, 1995). Somites will form the voluntary musculature of the neck, body wall, and
limbs. As the somite matures, its various regions become committed to forming specific cell types, such as myogenic precursor cells (myoblasts). Myoblasts then start to migrate from the somite to the limb bud, the area in the embryo that will develop into the limb (Buckingham et al., 2003). This process depends on the paracrine secretion of hepatocyte growth factor (HGF) and the presence of its receptor c-Met, which in turn depends on the transcription factor Pax3 (Epstein et al., 1996). Studies have shown that Pax3-deficient mice fail to de- velop limb muscles, because the myoblasts cannot reach their final destination (Tremblay et al., 1998). Additionally, knockout of Hgf and c-Met in mice are em- bryonically lethal (Kato, 2017). Another crucial step in myogenesis is the com- mitment to the myogenic lineage, which is dependent on expression of myo- genic factor 5 (Myf5) and myoblast determination protein 1 (MyoD). When either Myf5 or MyoD are individually inactivated in mice, there are no muscle defects, but loss of both Myf5 and MyoD results in complete abscence of muscle devel- opment (Rudnicki et al., 1993). Once the myoblasts have reached the limb bud, they start to express the myogenic regulatory factors (MRFs) Myogenin and Mrf4, which terminally differentiate them into myocytes. Myocytes can then start to fuse to form elongated, multinucleated, cylindrical structures called myotubes.
During this process, contractile proteins such as actin and myosin heavy chain (MyHC) accumulate in the cytoplasm and start to form myofilaments. Thereaf- ter, entire muscles are formed and the myotubes continue to grow in diameter because of the formation of more myofilaments. Muscles increase in length and width to accompany the growth of the skeleton, and their ultimate size depends for example on the amount of exercise that is performed in the adult.
Gross anatomy of tendons
Tendons are tough bands of fibrous connective tissue connecting muscle to bone. Tendon tissue is mainly composed of type I collagen, but also small amounts of type III and V, along with sparse tenocytes (Gaut and Duprez, 2016).
Type I collagen accounts for 90 % of the collagen subtypes and contributes to the mechanical strength of the tendon tissue. The tenocytes are located between the collagen fibrils, which are oriented parallell to the tendon axis. The tenocytes synthesize the collagen molecules and secrete them to the extracellu- lar space where they assemble into collagen fibrils. Collagen type III and V are important for the fibril formation. Multiple collagen fibrils form collagen fibers, and bundles of fibers are wrapped into fascicles by a fine layer of loose connec- tive tissue also containing the blood vessels and nerves (Gaut and Duprez, 2016).
It has been shown that the collagen-producing cells, whether isolated from the
cornea, tendon, or the skin, autonomously arrange themselves according to their in vivo origins (Doane and Birk, 1991). This suggests that the cells contain intrinsic tissue-specific information that drives the parallell organization of type I collagen fibrils. This organization is also dependent on the direction of force transmission. Disruption of the highly organized collagen architecture is a com- mon finding in tendon pathology.
The innervation of the Achilles tendon, the tendon studied in this thesis, orig- inates from the nerves that innervated the triceps surae muscle, as well as from cutaneous branches of the sural nerve. The main part of the innervation is found in the connective tissue surrounding the tendon, paratenon (Józsa and Kannus, 1997). Many of the nerve fibers in the paratenon terminate as sensory nerve endings involved in transmission of pain (Ackermann et al., 2002). Some nerves fibers are also shown to enter the tendon tissue proper following the blood vessels in the connective tissue in-between the fiber bundles, in the so called endotenon (Józsa and Kannus, 1997). These nerve fibers anastomose obliquely and transversely inside the tendon tissue proper, ultimately forming the nerve endings.
Tendon pathology
Tendon pathology can be of different ethiology, and range from chronic to acute. Chronic overuse injuries, such as tendinosis, are characterized by pain and swelling around the tendon, with degenerative processes commonly visu- alized using ultrasound, magnetic resonance imaging, or histopathological ex- amination. Although tendinosis was originally believed to be a degenerative le- sion without an inflammatory component (Khan et al., 1999), lately inflamma- tion has become accepted as a model of tendinosis (Cook et al., 2016). It remains unclear, however, what role inflammation plays in the development of tendi- nosis.
Tendinosis is characterised by collagen disruption and disorganization, in- creased vascularity, hypercellularity, and the rounded appearance of tenocytes instead of their normal spindle-shape (Chuen et al., 2004; Kannus, 2000). Alter- ations in the collagen content is also seen with elevated collagen type III levels, and an increased ratio of type III collagen to type I collagen. Additionally, there is an ingrowth of vessels into the tendon, which is accompanied by nerve fibers.
The tenocytes start to produce signaling substances traditionally believed to be confined to neurons, such as glutamate, catecholamines, acetylcholine (ACh),
and substance P (SP) (Danielson, 2009). These non-neuronally produced sub- stances have a diversity of known effects, e.g., pain signaling, but are also known to cause the histopathological changes seen in tendinosis (Ackermann et al., 2003; Andersson et al., 2011). Interestingly, tenocytes of tendinosis tendons also express the specific receptors for these signaling substances (Andersson et al., 2008), which has raised the suggestion of a biochemical theory, i.e., that these substances exert effects in an autocrine/paracrine manner possibly driving the development of tendinosis and/or maintaining the state of the disease. There- fore, future studies on the role of the tenocytes in tendinosis are needed.
Gross anatomy of skeletal muscle
Skeletal muscle is striated and under voluntary control of the somatic nervous system (Levy et al., 2009). Skeletal muscle is composed of a multitude of muscle fibers, with each fiber representing a single, multinucleated cell. Surrounding the muscle is a sheet of connective tissue, the epimysium, and inside the mus- cle, septa of connective tissue surround groups of muscle fibers, forming fasci- cles (Fig 1). Skeletal muscle fibers are broadly classified as “slow-twitch” or “fast twitch” (Talbot and Maves, 2016). Slow-twitch fibers are called type 1 fibers and express myosin heavy chain 1 protein, whereas fast-twitch fibers, referred to as type 2 fibers, express myosin heavy chain 2 protein with the isoforms 2A, 2B and 2X found in mammalian skeletal muscles. In a single muscle, the different fiber types are intermingled.
Skeletal muscle contraction
Skeletal muscle contraction is initiated when impulses from the motor cortex of the brain are passed down the spinal cord to activate alpha lower motor neu- rons in the ventral horns (Levy et al., 2009). From there, the axon exits the spinal cord via the ventral roots and synapses on the skeletal muscle fibers. Once the signal has reached the axon terminal, acetylcholine (ACh) is released at the neu- romuscular junction (NMJ). The NMJ comprises three components: the nerve terminal, the muscle fiber, and perisynaptic terminal Schwann cells (Wu et al., 2010). The nerve terminals contain ACh-containing vesicles; the muscle mem- branes express ACh receptors (AChRs); and the Schwann cells insulate the syn- aptic cleft. The binding of ACh to its receptors in the muscle fiber membrane initiates an action potential that ultimately leads to muscle contraction.
Figure 1. Schematic illustration of muscle in cross-section. Muscle fibers are packed with contractile filaments such as actin and myosin which form the basic functional unit of the muscle. Muscle fibers are bundled into fascicles containing vessels, lymphatics and nerves. The fascicles are grouped together and form the gross structure of the mus- cle, which is enclosed by the epimysium, a sheet of fibrous connective tissue.
The musculotendinous junction
The site where tendons attach to muscle is called the musculotendinous junc- tion (MTJ). At the MTJ, the sarcolemma of muscle fibers is folded into finger- like extensions that connect the myofibrils to the tendinous extracellular matrix (Charvet et al., 2012). The function of these finger-like processes is to increase the interface area and to allow the sarcolemma to better resist to muscle con- traction forces. Molecules on both sides of the junction are involved in the for- mation of the linkage. On the muscle side, laminins and collagen IV are the major constituents on the surface of the sarcolemma, whereas collagen I and the glycoprotein tenascin-C are the major molecules enriched at the tendon side of the MTJ (Charvet et al., 2012). Paracrine signaling between muscle fibers and tenocytes is important for the establishment of a stable MTJ (Liu and Geisbrecht, 2011).
Acetylcholine: synthesis, origin, and receptors
Acetylcholine (ACh) is synthesized from acetyl-CoA and choline in a reaction catalyzed by the enzyme choline acetyltransferase (ChAT). Once synthesized, a vesicular acetylcholine transporter (VAChT) loads ACh into secretory vesicles in exchange for protons. ACh is synthesized by practically all living cells, which has changed the traditional view of ACh acting solely as a neurotransmitter (Wessler and Kirkpatrick, 2008).
There are two types of ACh receptors (AChRs): muscarinic and nicotinic. These receptors are functionally different. Nicotinic ACh receptors (nAChRs) are the primary receptors mediating muscle contraction (Levy et al., 2009). Nicotinic receptors consist of five subunits: two a subunits, and a combination of the b, d, and either g/e subunits, which together form an ion channel pore. The a sub- units each have a binding site for ACh, and both binding sites must be occupied for the receptor to be activated (Levy et al., 2009), thus only relatively high con- centrations of ACh can activate nAChRs.
There are five major subtypes of muscarinic ACh receptors (M1-M5) and most of them exert their effects in the brain, but also in organs such as heart, and smooth muscle, and exocrine glands (Wessler and Kirkpatrick, 2008). The mAChRs are coupled to different G proteins and can cause a variety of re- sponses in target cells. For example, activation of mAChRs enhances prolifera- tion of keratocytes via MAPK-dependent signaling (Sloniecka et al., 2015). The M3 receptor subtype has been shown to be central in mediating these prolifera- tive effects. Activation of mAChRs also reduces apoptosis via modulation of apoptotic factors such as Bcl-2, Bad, and Bid (Sloniecka et al., 2016). Addition- ally, an upregulation of the ACh-synthesizing machinery, including enhanced anti-ChAT and anti-M2 immunoreactivity, was shown in chronic patellar tendi- nosis (Danielson et al., 2007).
Denervation and its consequences
Denervation refers to loss of nerve supply and may be caused by an injury or a disease. Each year, several hundreds of thousands of people in Europe and the United States suffer from peripheral nerve injuries (Wiberg and Terenghi, 2003). Denervation results in muscle atrophy and the biology of denervated muscle is well-documented (Carlson, 2014). In contrast, the biology of dener- vated tendon is very limited. The only study that exist describe how healing of the rat Achilles tendon is impaired after sciatic neurectomy, as evidenced by
irregular collagen alignment, hypercellularity, and reduced tensile stress and stiffness (Scott and Bahr, 2009). The local expression of neuromodulators, how- ever, such as SP, has not been studied in denervated tendon. This is a major concern since a pathological tendon may result in pain and restrict movement even after a denervated muscle has re-established its neural connection.
The consequences of muscle denervation can be divided into short-term (<2 months), and long-term (>2 months) changes. Some of these changes are illus- trated in Figure 2. It should also be noted that the biology of denervated mus- cles is in many ways similar in experimental animal models as in humans. In humans however, the different stages of denervation usually last for longer pe- riods of time, whereas similar changes in laboratory animals are observed at earlier time-points (Carlson, 2014).
Short-term degenerative changes
Immediately after a peripheral nerve injury, the voluntary control over the mus- cle is lost. Spontaneous fibrillation activity, however, can still be observed, and is in fact one of the earliest indications of denervation (Hník and Škorpil, 1962).
These contractions are believed to reflect the spread of nicotinic ACh receptors across the muscle fiber membrane, thus rendering the muscle “supersensitive”
to ACh (Merlie et al., 1984). In fact, in one of the earliest studies of contractile response of denervated muscles, it was reported that the threshold to elict a muscle contraction was about 1 000 times lower for denervated muscle com- pared to normal muscle in the cat (Brown, 1937). In contrast to the nicotinic ACh receptors, the response of muscarinic ACh receptors to denervation has not been as extensively studied. The only reports that exist describe a compen- satory sprouting mechanism of mAChRs at neuromuscular junctions (Wright et al., 2009). Interestingly, in the same study, inhibition of all mAChR subtypes with atropine enhanced muscle fiber atrophy, thus suggesting a potential role of mAChR in preserving muscle tissue during denervation.
Within a week after nerve injury, bone-marrow-derived cells start to infiltrate the tissue. These cells, mainly macrophages, secrete growth factors such as transforming growth factor beta 1 (TGF-b1) that stimulate local fibroblasts to produce type I collagen (Mochizuki et al., 2005). As time goes by, more and more collagen accumulates around muscle fibers, which, in combination with the loss of capillaries (Borisov et al., 2000), creates a highly hypoxic environment for the
cells. During the progressive atrophy and deterioration of muscle fibers, satel- lite cells (the muscle stem cells) exit their normal quiescent state and begin to proliferate, at which state they are often referred to as myogenic precursor cells or myoblasts (Charge and Rudnicki, 2004). This phase is followed by differenti- ation and fusion of myoblasts with damaged muscle fibers in a process that is remniscent of embryonic muscle development. If the muscle is transplanted into an innvervated site during the first two months in rats, the muscle is capa- ble of restoration equal to control muscle (Carlson et al., 1996; Gulati, 1990).
Figure 2. Schematic illustration of some of the consequences of denervation on skeletal muscle. Immediately after a nerve injury, the voluntary control over the muscle is lost, which is followed by muscle fiber atrophy and weight loss. With prolonged denerva- tion, sarcomeric organization is lost, as evidenced by loss of individual actin and myosin filaments. In parallell, satellite cells are activated which enables some endogenous re- generation of the muscle. As for the tendon, little is known of the consequences of de- nervation.
Long-term degenerative changes
After two months of denervation, the muscle drops its restorative capacity dra- matically (Carlson et al., 1996). The decrease in muscle fiber area depends on the muscle fiber type: fast muscle fibers (type 2) undergo rapid atrophy, whereas slow muscle fibers (type 1) retain their mean fiber area for at least two months
(Borisov et al., 2001; Lu et al., 1997). Long-term denervated muscle is character- ized by massive fibrosis and the presence of adipocytes within the interstitium (Lu et al., 1997). With increasing amounts of collagen fibers, nerve channels become obstructed, making it difficult for Schwann cells to aid reinnervation (Bradley et al., 1998). In this harsh environment, satellite cells continue to pro- liferate and differentiate into newly forming muscle fibers. By two months, the number of satellite cells per muscle fiber is approximately three times higher (9.1 % compared to 2.8 %) compared to control muscle (Viguie et al., 1997). From there, the cell population slowly starts to decline until they fall well below con- trol levels. Parallell to these changes is the dramatic loss of capillaries, with a 90 % reduction by 18 months in rats (Borisov et al., 2000). Loss of myonuclei is also common and is believed to be the result of apoptotic mechanisms, includ- ing DNA fragmentation (Siu, 2009). After seven months of denervation, virtually everything measured (mass, contractile force, satellite cell numbers, and capil- lary density) has reached a stable baseline state (Lu et al., 1997).
Apoptotic response to muscle denervation
Apoptosis is a form of programmed cell death that leads to destruction of intact single-nucleated cells. The situation in skeletal muscle is different because of the multinucleated structure of muscle cells, which means that there can be loss of one or more nuclei without having to sacrifice the entire cell. Since cell death in denervated muscle is distinct from classical apoptosis (Borisov and Carlson, 2000), the interrelations of muscle fiber atrophy, apoptosis, and activation of myogenesis is complex and poorly understood. The available literature suggests two major mechanisms for atrophy: proteolysis through activation of the ubiq- uitin-proteosome pathway, achieved by factors such as tumor necrosis factor alpha (TNF-a) (Glass, 2003), and reduction of protein synthesis due to the loss of myonuclei (referred to as “nuclear apoptosis”) (Alway et al., 2003; Siu, 2009).
The idea of nuclear apoptosis is complementary to the proposed “domain hy- pothesis” (Allen et al., 1999; Hikida et al., 1997) which states that each nucleus in a muscle fiber controls a defined cytoplasmic area (domain) and that the ad- dition of new nuclei is required to support muscle hypertrophy, and removal of nuclei is needed for muscle atrophy. This theory is supported by the fact that activation of satellite cells contributes to myogenesis by adding new nuclei to the muscle cells (Charge and Rudnicki, 2004). Although the exact mechanism behind the loss of nuclei during muscle atrophy is largely unknown, both caspase-dependent and caspase-independent signaling have been proposed (Siu, 2009). There are, however, findings that challenge the idea that muscle
loss is coupled to apoptosis. In a study by Bruusgaard and Gundersen, imaging of single muscle fibers for up to 28 days following denervation atrophy showed no loss of myonuclei in mice, despite a greater than 50 % reduction in mean fiber area (Bruusgaard and Gundersen, 2008). Labeling of fragmented DNA us- ing TUNEL revealed high levels of apoptotic nuclei, but they were all confined to stromal and satellite cells. Thus, DNA breaks may have some unidentified physiologic function in coordinating the remodeling of myofibers in response to denervation.
To date, there are no widely accepted methods for delaying or preventing mus- cle atrophy following denervation. Hence, more research is needed to unravel the mechanisms that would allow denervated muscle to retain its potential for functional recovery. If muscle atrophy is a consequence of the loss of myonu- clei, then recovery of strength would require replenishment of nuclei from sat- ellite cells. Potential intervention therapy may therefore include mitogenic and anti-apoptotic signaling.
Treatment strategies
The main method for treatment of peripheral nerve injuries is surgery. The ex- tent of the nerve injury determines the type of repair that is performed. Short gaps can be repaired by joining the proximal and distal nerve stumps together and suturing, whereas long nerve gaps require some sort of bridge, such as a section of a nerve taken from elsewhere in the body (Wiberg and Terenghi, 2003). The autologous nerve grafts provide the regenerating axons with a natu- ral guidance structure to cross the gap but have some drawbacks, including lim- ited availability of autologous nerves, size mismatches, loss of sensation, and donor site tenderness. Therefore, as an alternative, the use of conduit structures has become increasingly popular in tissue engineering and regenerative medi- cine applications. Following nerve injury, when the axon is disconnected from the cell body, its distal segment progressively degenerates and eventually dis- appears, a feature known as Wallerian degeneration (Waller, 1850). Schwann cells which are devoid of contact with axons adopt a phenotype specialized to promote repair (Jessen and Mirsky, 2016). These cells then form a cell strand called the bands of Büngner which provide a trophic and physical support for axons to regrow and reinnervate correctly their targets (Boerboom et al., 2017).
Due to their pivotal role in peripheral nerve regeneration, many attempts to boost regeneration have been made using conduits filled with Schwann cells
(Levi et al., 2016; Madduri and Gander, 2010). These studies have generally re- ported positive results, but their therapeutic effect has remained significantly below that achieved with autologous nerve grafts. Also, the use of Schwann cells is not ideal because of donor site morbidity, and the time it takes to obtain clin- ically useful cell numbers. As such, adipose tissue with all of its regenerative cell types (Zuk et al., 2001) has emerged as a promising candidate for the treat- ment of peripheral nerve injuries. These cells have been shown to promote pe- ripheral nerve regeneration in their undifferentiated state (Suganuma et al., 2013), as well as after induction into a Schwann cell-like phenotype (Schaakxs et al., 2017). However, because injured neurons regenerate at a rate of 1 mm/day, nerve injury with a long distance to the target organ often results in irreversible muscle atrophy. There is, in other words, a need for muscular protective ther- apy during the time required for axon regeneration and subsequent reinnerva- tion to occur. To date, no such direct therapy exists, although attempts have been made using electrical stimulation (Nakagawa et al., 2017) and experimental stem cell injections (Schaakxs et al., 2013).
Experimental cell therapy
Stem cells are unspecialized cells of the human body. They are capable of re- newing themselves through cell division and can differentiate into other cell types depending on the stimulatory signals they encounter. Stem cells can be divided into different catogeories depending on their potency (Zakrzewski et al., 2019). Pluripotent stem cells can give rise to all of the cell types that make up the human body. Embryonic stem cells are an example of these. Multipotent stem cells can develop into more than one cell type, but are more limited than pluripotent cells, i.e., they have a narrower spectrum of differentiation. Adult stem cells, such as mesenchymal stem cells (MSCs), are considered to be mul- tipotent. MSCs can be isolated from almost all tissues, with bone marrow being the most frequently utilized (Ullah et al., 2015). MSCs have been shown to be effective modulators of regeneration and used in the treatment of many dis- eases, including myocardial infarction, diabetes, and liver cirrhosis (Wei et al., 2013). They also exert strong therapeutic effects in the musculoskeletal system (De Bari et al., 2003; Wang et al., 2015). Recent research has suggested that the regenerative effects of MSCs are closely related to their secretome, i.e., soluble factors produced and released by the cells in a paracine fashion (Bruno and Camussi, 2013; Drago et al., 2013). These factors include cytokines, chemokines, and growth factors, and have been shown to promote neurite outgrowth, in- crease angiogenesis, and inhibit apoptosis and scarring.
Adipose tissue-derived stem cells
MSC-like cells can be obtained from adipose tissue using minimally invasive methods, such as liposuction (Zuk et al., 2001). All the cells from the lipoaspirate are collectively named the stromal vascular fraction (SVF) and include endothe- lial cells, smooth muscle cells, immune cells, and cells with a stem cell pheno- type, the so-called adipose tissue-derived stem cells (ASCs) (Ramakrishnan and Boyd, 2018). ASCs are defined as plastic-adherent cells expressing a multitude of surface antigens (Bourin et al., 2013), including CD73 and CD90, which are also surface antigens of MSCs, but not CD31 and CD45. ASCs are also positive for CD34 and CD36, and negative for CD106. To isolate and expand the ASC population, the SVF is plated in culture which allows the ASCs to adhere to the tissue culture plastic so that the non-adherent cells can be removed.
Like other types of MSCs, ASCs can be differentiated in vitro into cells of mes- oderm origin such as osteogenic, chondrogenic, adipogenic lineages but possi- bly also myogenic lineage, by culturing the cells in culture media supplemented with specific growth factors (Zuk et al., 2001). Moreover, the potential of ASCs to differentiate into skeletal muscle in vivo has been demonstrated (Forcales, 2015). GFP-labeled ASCs were shown to be incorporated into muscle fibers fol- lowing ischemia where 20 % of all fibers were GFP-positive after one week (Di Rocco et al., 2006), and intramuscular injections of ASCs into the tibialis ante- rior muscle of mdx mice, an animal model of Duchenne muscular dystrophy, resulted in 50 % of muscle fibers expressing dystrophin after ten days, compared to no dytrophin-positive fibers in the non-injected group (Rodriguez et al., 2005). However, despite their high regenerative capacity in vivo, few studies have explored the effects of ASCs on muscle atrophy following denervation. In a re- cent study, ASCs were injected into denervated rat gastrocnemius muscles. At six weeks after nerve injury, the muscle mass and fiber area of ASC-treated muscles were evaluated, and both of these parameters were significantly im- proved compared to control (Schilling et al., 2019). In addition, muscles having received ASCs showed increased expression of the proliferation marker Ki-67.
However, in another study, injections of ASCs had no significant effect unless the cells were first differentiated into a Schwann cell-like phenotype (dASC) (Schaakxs et al., 2013).
Differentiated adipose tissue-derived stem cells
As first described by Kingham et al., ASCs were induced into a Schwann cell- like phenotype (dASC) using a mixture of mitogenic and differentiating factors
(Kingham et al., 2007). These cells exhibited a spindle-like morphology similar to Schwann cells and up-regulated expression of p75NTR (p75 neurotrophin re- ceptor), S-100 protein and GFAP (glial fibrillary acidic protein), all markers co- expressed by Schwann cells. As described previously, Schwann cells play a piv- otal role in peripheral nerve regeneration, and are thus an attractive therapeutic agent. However, due to donor site morbidity and the time required to culture and expand the cells, Schwann cells have limited clinical application. As such, dASCs offer a promising alternative as they are more easily accessible and evoke a similar response to Schwann cells in a model of peripheral nerve repair (Kingham et al., 2007). A significant number of reports have described the ben- efits of using dASCs in vivo (Kingham et al., 2014b), with effects on neurite out- growth and inhibition of apoptosis, most likely through secretion of neu- rotrophic factors such as nerve growth factor (NGF), brain-derived neu- rotrophic factor (BDNF), and glial cell-dervied neurotrophic factor (GDNF) (Kolar and Kingham, 2014). In addition to these neuroprotective functions, dASCs also possess musculoprotective functions such as the ability to attenuate muscle atrophy (Schaakxs et al., 2013). However, despite the benefits of dASCs in regenerative medicine, the manipulation of cells creates regulatory and man- ufacturing challenges. By using SVF instead of culture expanded dASCs, the therapeutic cellular product can be instantly obtained, making it safer to use and associated with lesser regulatory criteria.
The stromal vascular fraction
Recent advances in the field of regenerative medicine have demonstrated that SVF cells are in many aspects better than ASCs. For example, intravenous in- jections of freshly isolated SVF cells improved the outcome after acute myocar- dial infarction by reducing the infarction size in rats, and was proven safer than cultured ASCs (van Dijk et al., 2011). SVF cells also performed better than ASCs in forming new cartilage matrix when co-cultured with primary human chon- drocytes (Wu et al., 2016), making it a promising alternative for cartilage regen- eration. SVF cells also possess potent immunomodulatory functions. In a recent study, the effectiveness of SVF cell therapy in a model of multiple sclerosis (MS) was demonstrated and compared to ASCs (Bowles et al., 2017). In this study, mice with MS-like symptoms (EAE mice) were subjected to intraperitoneal in- jections of SVF cells. After ten days of treatment, there was a significant im- provement in the clinical scoring, behavior, motor function, and histopatho- logic analyses in these mice. Although ASCs had a similar effect, treatment with SVF resulted in significantly higher frequencies of T cells, helper T cells, B
cells, and macrophages within the spleen, lymph nodes, and peripheral blood.
This was accompanied by high levels of interleukin-10 in the CNS and periph- eral tissues. Based on these findings, the authors concluded that SVF treatment promoted an anti-inflammatory milieu that may reduce autoimmunity and pro- mote immune tolerance. A similar study in the same animal model was per- formed by Semon et al., who showed that SVF treatment was statistically more effective than ASCs (Semon et al., 2013). Some of the benefits of using SVF over ASCs are listed in Table 1.
Table 1. A comparison of the SVF and dASC characteristics. The table was originally published by Bora et al. (Bora and Majumdar, 2017).
Factors SVF ASCs / dASCs
Cell population Heterogenous Homogenous
Cell type ASCs, endothelial cells,
smooth muscle cells,
fibroblasts, immune cells ASCs / dASCs only
Application range Autologous Autologous and allogenic
Immune rejection Not anticipated Immune monitoring
required
Properties Angiogenic, immuno-
modulatory, and differen- tiative
Immunomodulatory and differentiative
Ex vivo exposure Low (hours) High (weeks)
Potential mechanism of cell therapy in vivo
There are two major hypotheses on how exogenously administered cells such as ASCs or SVF might establish tissue regeneration. The first proposes that the cells differentiate in vivo into the cells of the damaged tissue, and that this pro- cess is supported by factors produced by the surrounding cells. Over the last two decades, the body of literature supporting a cell secretome-based therapy has grown exponentially. The second hypothesis postulates that the administered cells themselves have trophic functions through their secretome that are im- portant for extracellular matrix remodeling and tissue regeneration (Kingham et al., 2014a). There is also the possibility of a dual function, in which a small fraction of cells has a function in replacing damaged tissue, while the remaining part of cells maximizes the intrinsic regenerative capacity by producing growth factors and cytokines. Such mechanisms speak in favor of using the SVF, since this comprises such different cell types.
Differentiation of stem cells into cells of the damaged tissue
As descibred in the previous sections, due to their inherent multipotency, ASCs are capable of directly integrating into diseased organs and tissues. In summary, transplantation of ASCs has been reported to be beneficial in peripheral nerve repair, regeneration of cartilage and bone, urinary tract reconstruction and more (Zuk, 2010). In addition, ASCs have been shown to be able to differentiate into all kinds of muscle tissue, both in vitro and in vivo (Bajek et al., 2016). How- ever, although many studies have reported positive results using ASCs, the level of ASC engraftment into the diseased organ is often minimal, suggesting that factors produced by the ASCs promote tissue regeneration via complex para- crine actions rather than the cells themselves. The differentiation of ASCs into target cells may instead become important in late-stage organ repair.
Trophic functions of the secretome from stem cells; cell secretome-based therapy It is now commonly recognized that in addition to differentiation, many of the positive effects of adipose tissue-derived cells are related to the secretome and the soluble factors found within it (Salgado et al., 2010). Great effort is currently being made to map the molecules that compose the secretome. So far, a pleth- ora of molecules have been identified with effects on oxidative stress, apoptosis, neurite outgrowth, angiogenesis and revascularization, immunomodulation, wound healing and tissue regeneration (Kapur and Katz, 2013; Pires et al., 2016).
In a screening of the ASC secretome, the expression of physiologically relevant levels of hepatocyte growth factor (HGF) and vascular endothelial growth factor (VEGF) was detected, which significantly increased the metabolic viability and cell density of neuronal cells (Ribeiro et al., 2012). In another study, the contri- bution of HGF to ASCs potency was evaluated by silencing HGF expression (Cai et al., 2007). When HGF production was restricted, ASCs could no longer contribute to inducing endothelial cell proliferation, migration, and survival in a mouse hindlimb ischemia model. Moreover, treating endothelial cells with conditioned medium taken from ASCs promoted the formation of capillary-like tube structures via VEGF and insulin-like growth factor-1 (IGF-1) (Hsiao et al., 2012). In addition to these pro-angiogenic effects, which constitute a large por- tion of the ASC secretome (Kapur and Katz, 2013), ASCs also express a range of neurotrophic factors including NGF, GDNF and BDNF that promote neurite outgrowth (Kalbermatten et al., 2011). The expression of these factors is upreg- ulated in ASCs induced into a Schwann cell-like phenotype (dASC) (Kingham et al., 2014a). Other factors of interest include granulocyte and macrophage col- ony stimulating factors, interleukins (ILs) 6, 7, 8, and 11, and tumor necrosis
factor-a (TNF-a) (Kilroy et al., 2007), factors co-expressed by SVF cells (Blaber et al., 2012).
In addition to individual paracrine factors, extracellular vesicles (EVs) produced by ASCs may deliver critical information to target cells for tissue regeneration.
EVs released from ASCs have been found to exert effects on angiogenesis, cell survival and apoptosis, inflammation, and tissue regeneration (Wong et al., 2019). It is believed that EVs released from stem cells may, at least in part, ac- count for many of the therapeutic effects of secreted factors (Bruno and Camussi, 2013). To mention a few examples: microvesicles from ASCs were shown to mimic the effects of the living cells by improving function, electro- physical recordings and muscle mass measurements after sciatic nerve injury in rats (Raisi et al., 2014); and ASCs treated with platelet-derived growth factor (PDGF) stimulated the secretion of EVs, which enhanced the angiogenic po- tential of ASCs (Lopatina et al., 2014).
There are benefits of using secreted molecules in replacement of cells: 1) it would minimize the stem cell-related ethical issues; 2) it would provide a faster and safer route to clinical translation of the cell therapy as there is no need to expand the cells in culture; and 3) it would allow for precise dosing and localized delivery, with minimally invasive techniques.
Hypothesis and Aims
Hypothesis and Aims
Hypothesis
The overall hypothesis of the studies presented in this thesis is that denervated tendons undergo degenerative changes, and that cells from adipose tissue have the potential to protect and regenerate denervated muscle through secretion of soluble factors.
Aims
The specific aims of the studies of this thesis were:
1. To determine whether denervated tendon exhibits morphological changes that resemble tendinosis, such as hypercellularity, disor- ganized collagen architecture, and production of substance P (SP) and its preferred receptor NK-1R (Paper I).
2. To determine whether denervated muscle exhibits altered expression of muscarinic ACh receptors, as well as changes in apoptosis-associ- ated signaling mechanisms involving caspases, cytokines, and death domain receptors (Paper II and IV).
3. To determine whether the ASC secretome enhances the proliferation of myoblasts in vitro via secretion of ACh (Paper II).
4. To determine whether the SVF cell secretome influences the prolifer- ation, differentiation, and survival of myoblasts in vitro via secretion of growth factors (Paper III, IV).
5. To determine whether intramuscular injections of SVF cells can pre- serve denervated muscle tissue in vivo and reduce apoptosis (Paper IV).
Materials and Methods
Materials and Methods
Animal model
Ethical considerations
Ten to 12-week-old female Sprague-Dawley rats (Taconic Europe A/S) were used for in vivo experiments. Animal care and experimental procedures de- scribed in this thesis were carried out in accordance with the Directive 2010/63/EU of the European Parliament and of the Council on the protection of animals used for scientic purposes, with the approval of the Northern Swedish Committee for Ehics in Animal Experiments (No. A186-12 and A50-13). All sur- gical procedures were performed under general anesthesia using isoflurane in- halation (Attane vet, 1000 mg/g, Oiramal Healthcare, UK). Animals were given the analgesic Finadyne perioperatively (Schering-Plough, Denmark, 2.5 mg/kg, s.c.). Each animal was housed alone after surgery and exposed to 12-hour light/dark cycles, with free access to water and food. Animals were euthanized by exsanguination by cardiac puncture under deep anaesthesia (4 % isoflurane inhalation).
Denervation model
Surgery was performed using aseptic conditions under general anesthesia with isoflurane gas. The left sciatic nerve and its branches were exposed using a dor- sal gluteal muscle-splitting incision. The cut in the sciatic nerve was made ap- proximately 5 mm proximal to the bifurcation of the tibial and common pero- neal nerves. The proximal nerve stumps were capped with polyethylene tubes to prevent muscle reinnervation before the wound was closed in layers.
Intramuscular stem cell injections
Following the sciatic nerve transection, some animals received intramuscular injections of SVF cells before closure of the wound. Cells (1 x 106) in a total volume of 150 µl were aspirated into a syringe and injected slowly into the de- nervated gastrocnemius muscle. The injection was made around the point of entry of the tibial nerve. The control group received sham injections of 150 µl of normal growth medium alone in a similar manner. The animals received one injection during the experimental period.
