Role of Muscle Stem Cells During SkeletalRegenerationSTEM CELLS 2015;33:1501–1511 www.StemCells.com

Impact factor: 7.532

報告學生： 郭啟甲 指導老師： 鄭伯智老師 , 林宏榮老師

Introduction Skeletal muscle and bone (common mesodermal origin) ar

e closely linked across development, growth, and aging Muscle provides a source of mechanical stimuli for bone Muscle mass, bone mass, and strength are highly correlate

d. The clinical importance of muscle in fracture repair (esp op

en fractures)proper vascularization, release of osteogenic factors, and/or recruitment of stem cells

Roles of muscle to support bone repair have not been elucidated at the cellular and molecular levels

Introduction Skeletal muscle and bone regenerative capacities supported by e

ndogenous muscle stem cells, also called satellite cells, and skeletal stem cells

Satellite cells marker: Pax3 and Pax7 Skeletal muscle regulates inflammation during bone regeneratio

n Tumor necrosis factor-abone morphogenetic proteins (BMPs) insulin-like growth factor 1 (IGF1)fibroblast growth factor 2 (FGF2)myostatin

Skeletal stem cells: local periosteum and muscle-derived Satellite cells can differentiate into osteoblasts and chondrocytes

and bone

Introduction

We investigate the role of satellite cells as a source of cells and molecular signals, and show that satellite cells are activated in the muscle adjacent to the fractured bone leading to increased production of growth factors that are essential for bone regeneration.

Materials and methods

Animals C57BL/6, Pax7CreERT2 beta-actin-GFP, Pax7-/-, Pax3Cre, D

TAf/f (DTA5diphtheria toxin fragment A), R26RLacZ, and R26ReYFP mice

Pax7CreERT2 were mated with DTAf/f, R26RLacZ, and R26ReYFP mice.

Pax3Cre mice were bred with R26RLacZ. Three-month-old male mice and age-matched wildtype ma

le To induce Cre recombinase activity, mice received intraper

itoneal injections of Tamoxifen (Tmx) In our hands, cre recombination and satellite cell ablation

efficiency was 80%

Closed non-stabilized fractures

Under anesthesia in the mid-diaphysis of the right tibia via three-point bending

The tibia was placed on the fracture jig, and a 500 g weight was dropped from 3.5 cm

Mice were revived and monitored closely until sacrifice.

rhBMP2 treatment

At the time of the fracture, Tmx-induced Pax7CreERT2/1;DTAf/f mice received a single injection of 10 mg of recombinant human BMP2 (rhBMP2) in phosphate buffered saline (0.7 mg/ml) between the fractured tibia ends using a syringe and 30-gauge needle

Open non-stabilized fractures

Osteotomy Following osteotomy, the muscles were s

utured on the anterior part of the tibia Soft filter In control samples, the muscle was separ

ated from the bone without disrupting the periosteum.

Histological and Histomorphometric Analyses Briefly, mice were sacrificed at days 5, 7, 14, and 21

postfracture Serial 10 mm longitudinal paraffin sections were st

ained with Safranin-O/ Fast Green to detect cartilage and modified Milligan’s Trichrome to detect bone.

Images were analyzed with Adobe Photoshop (Adobe, Inc., San Jose, CA) and Visiopharm (Visiopharm, Horsholm, Denmark) to determine the component volumes of bone and cartilage formation and reference volumes of callus tissues

Transplantation of Bone Grafts Bone grafts, with or without muscle, were isolat

ed from the tibia of Rosa26 donor mice that expressed LacZ

To follow cells derived from Rosa26 muscle combined with the periosteum, muscle was left attached to the periosteum.

Bone grafts containing intact periosteum, with or without muscle, were placed in a tibial cortical bone defect adjacent to the fracture site of 3-month-old recipient C57BL/6 male mice

Muscle Transplantation EDL (extensor digitorum longus) from beta-acti

n-GFP donor mice, expressing green fluorescent protein (GFP) ubiquitously, and transplanted into 3- month-old recipient C57BL/6 host male mice

Prior to open nonstabilized fracture injury, GFP-EDL muscle was transplanted adjacent to the tibia.

Proximal: sutured to the patella tendon Distal: sutured to peroneus muscle

Myoblast Culture Hind limb muscles were dissected from 2-m

onth-old betaactin- GFP male mice and digested with 400 UI/ml collagenase type II

Dissociated into single myofibers and digested with 0.5 U/ml Dispase and 0.2% collagenase type II

Satellite cells were liberated from myofibers and plated into culture dish in growth media (Hams F-10, 20% fetal bovine serum (FBS), 5 ng/ml bFGF). Primary myoblast cultures

Fluorescent-Activated Cell Sorting of Satellite Cells Satellite cells were freshly sorted from Pax7Cre

ERT2/1;R26ReYFP/1 male mice myogenic mononucleated cells

Satellite cells were freshly sorted based on the expression of YFP (yellow fluorescent protein).

Live cells were identified by negative staining for Sytox Blue (1 mg/ml)

Only pure sorted satellite cells (99%) were used for cell transplantation

Cell Transplantation An open tibial fracture was performed as described above

on 3- month-old recipient C57BL/6 host male mice. To transplant the cells to the fracture site, Tissucol kit (Bax

ter, France) was used Myoblasts and freshly sorted muscle stem cells (105 cells)

were embedded in highly and fast resorbable Tissucol fibrin scaffold obtained by adding 15 ml of fibrinogen (30 mg/ml) followed by 15 ml of thrombin (18 mg/ml).

After gentle mixing, cells embedded into resorbable fibrin scaffold were transplanted into the fracture site of the C57BL/6 host mice (n55).

Mice were revived and monitored closely until sacrifice.

Antibodies

Affinity-purified rat anti-mouse CD31/PECAM antibody to detect endothelial cells.

Affinity-purified Chicken anti-GFP antibody to detect GFP or YFP proteins.

Immunofluorescence Following transplantation of GFP-EDL, GFP myoblasts, and freshl

y sorted Pax7 YFP1 satellite cells into the fracture site, fracture calluses were harvested.

Samples were fixed and embedded for cryostat sectioning. Immunofluorescence was performed on slides prepared from sec

tions located 300 mm apart throughout the callus. Tissue sections for immunohistochemistry were fixed in 4% PFA f

or 10 minutes, washed, permeabilized in 0.2% PBS, Triton X-100, and incubated with blocking solution (10% goat serum) for 30 minutes.

Sections were stained in GFP antibody overnight at 4C and subsequently revealed with Alexa fluorophore conjugated chicken anti-IgG antibodies with DAPI for 1 hour at room temperature.

PECAM Immunohistochemistry and StereologicalAnalyses Anti-PECAM immunohistochemistry was performed on callus tissue After deparaffinization, sections were treated with 0.1% trypsin in PBS 13

for retrieval of antigenicity Endogenous peroxidase activity and nonspecific binding sites were block

ed by incubating sections in 0.3% H2O2 in phosphate buffer saline (PBS) 13 and 5% goat serum in PBS 13

Sections were then incubated with diluted primary antibody in 5% goat serum (1:100)

Sections were next incubated with diluted secondary antibody in 5% goat serum (1:250).

Subsequently, sections were incubated with avidin/ biotin enzyme complex

Staining was detected using diaminobenzidine, and the tissue was counterstained with 0.02% Fast Green.

Stereological analyses of endothelial cell surface density were performed as previously described

X-gal Staining and Quantification Beta-galactosidase activity was detected by X-gal (5-bro

mo-4-chloro-3-indolyl-D-b-galactoside) staining Callu stissues were fixed in 0.2% glutaraldehyde solution, washed

three times for 15 minutes in a solution containing 2 mM MgCl2, 0.01% sodium deoxycholate, 0.02% Nonidet P40 in PBS, and stained overnight in wash solution containing 1 mg/ml X-gal, 2.1 mg/ml potassium ferrocyanide, 1.64 mg/ ml potassium ferricyanide, and 20 mM Tris-HCl, pH 7.3, and lightly counterstained with eosin.

To exclude X-Gal staining due to endogenous b-galactosidase activity in osteoclasts, we performed double staining for b-galactosidase followed by tartrate resistant acid phosphatase (TRAP) staining with a leukocyte acid phosphatase kit

Quantification of LacZ donor contribution was performed to count X-gal-positive cells excluding the bone marrow compartment (n=5 or 6 per group).

RNA Isolation and RTqPCR Mice were sacrificed as described above at days 3 a

nd 5 postfracture. Callus tissues and all adjacent tissues located 0.5 c

m distal and proximal to the callus boundaries were collected at day 5 postfracture to analyze osteogenic and chondrogenic markers.

Only the adjacent muscles surrounding callus were collected after 3 days of bone regeneration.

Total RNA was extracted from muscles Real-time PCR

Statistical Analyses A minimum of five samples was used for

each group. Statistical significance was calculated wit

h GraphPad Prism v6.0a. Student’s t test, one-way, and two-way

ANOVA were used for statistical analyses. In all experiments, p values <.05 were co

nsidered significant.

Results

Figure 1

Figure 2

Figure 3

Figure 4

Fogure 5

Figure 5E

Figure 6

Discussion 10% of all skeletal injuries bone regeneration is del

ayed or impaired, and there is even greater risk of delayed union or nonunion in patients with soft tissue damage.

Muscle may be essential at several stages of the bone repair process.

Muscle supports the normal process of bone healing by a direct interaction with the periosteum and by providing osteogenic/chondrogenic factors through activation of satellite cells in the muscle adjacent to the fracture callus.

Our results provide a mechanism by which muscle grafts covering soft tissue injuries stimulate healing.

The periosteum plays an indispensible role in bone regeneration and is a major source of skeletal stem cells for cartilage and bone formation.

Skeletal muscle adjacent to bone interacts with the periosteum and is essential for its activation in response to bone injury.

Muscle obstruction, using a porous filter, impaired bone regeneration by inhibiting periosteal activation in areas where direct muscle-periosteum interactions were blocked, delaying chondrogenesis and osteogenesis, and most importantly preventing bone bridging at later time points.

Using a periosteal graft model, we also showed that muscle enhanced the periosteal contribution to bone regeneration confirming the importance of skeletal muscle during bone regeneration as a source of growth factors and/or stem cells.

Satellite cells play a crucial role in bone regeneration as satellite cell loss in Pax7-/- mice and satellite cell ablation in Pax7CreERT2/1;DTAf/f mice severely impaired bone regeneration.

Interestingly, in both the Pax7-/- and Pax7CreERT2/1;DTAf/f mice, angiogenesis was increased indicating that skeletal progenitors within blood vessels did not compensate for the defect in bone regeneration.

Muscle-lineage analyses in Pax3Cre/1;R26RLacZ/1 mice revealed a contribution of satellite cells as an endogenous source of chondrocytes during bone regeneration.

Local muscle injury surrounding the callus activates satellites cells in regenerating fibers. These activated satellite cells may be released to be integrated in the callus and become exposed contribution to cartilage within the callus compared to sorted satellite cells, suggesting that the GFP-myoblast cell population was maybe a heterogeneous cell population allowing a better contribution to cartilage within the callus.

BMPs may be produced by many cell types at the fracture site including inflammatory cells, bone matrix, and bone cells.

Satellite cells provide a source of growth factors during bone regeneration. (BMPs, FGF, IGF etc)

When treated with rhBMP2, the delayed bone regeneration in Pax7CreERT2/1;DTAf/f mice was improved.

The role of muscle in periosteal activation via a BMP-dependent mechanism may be particularly relevant in the context of endochondral ossification, as we previously showed that cell fate within the periosteum was regulated by BMP2

Conclusion1. Altogether, our results elucidate the functional role of muscle du

ring bone regeneration, via the cellular and molecular contribution of satellite cells, the muscle stem cells, in the process of endochondral ossification.

2. Muscle-derived growth factors are primary actors in this context. 3. Our findings may lead to direct clinical applications for the treat

ment of nonunion and for better understanding the bases of musculoskeletal repair defects associated with musculoskeletal diseases and with musculoskeletal trauma.

4. Indeed, muscle may provide a more efficient source of stem cells for cell therapies, as muscle stem cells may reveal superior in vivo regenerative capacities compared to bone marrow-derived mesenchymal stem cells used widely in tissue engineering approaches.