Enhanced achilles tendon healing by fibromodulin gene transfer Anthony Delalande Ph.D, Marie-Pierre Gosselin MSc, Arnaud Suwalski MSc, William Guilmain Ph.D, Chlo´e Leduc MSc, Mathieu Berchel Ph.D, Paul-Alain Jaffr`es, Patrick Baril Ph.D, Patrick Midoux Ph.D, Chantal Pichon PII: DOI: Reference:
S1549-9634(15)00119-7 doi: 10.1016/j.nano.2015.05.004 NANO 1131
To appear in:
Nanomedicine: Nanotechnology, Biology, and Medicine
Received date: Revised date: Accepted date:
27 January 2015 30 April 2015 18 May 2015
Please cite this article as: Delalande Anthony, Gosselin Marie-Pierre, Suwalski Arnaud, Guilmain William, Leduc Chlo´e, Berchel Mathieu, Jaffr`es Paul-Alain, Baril Patrick, Midoux Patrick, Pichon Chantal, Enhanced achilles tendon healing by fibromodulin gene transfer, Nanomedicine: Nanotechnology, Biology, and Medicine (2015), doi: 10.1016/j.nano.2015.05.004
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Enhanced Achilles tendon healing by fibromodulin gene transfer. Anthony Delalande1*, Marie-Pierre Gosselin1*, Arnaud Suwalski1, William Guilmain1,
T
Chloé Leduc1, Mathieu Berchel2, Paul-Alain Jaffrès2, Patrick Baril1, Patrick Midoux1
RI P
and Chantal Pichon1. 1
Centre de Biophysique Moléculaire, rue Charles Sadron, Orléans 45071 CEDEX 2,
SC
France. 2
Université de Brest, Brest, France
MA NU
CEMCA, CNRS UMR 6521, IFR148 ScInBioS, Université Européenne de Bretagne,
*: These authors contributed equally to this work.
Abstract: 147 words
PT
Body text: 4962 words
AC
3 tables
CE
37 references 6 figures
ED
Corresponding author: [email protected]
1 supplementary material The authors disclose no conflict of interest. This work was supported by the Association Française contre les Myopathies (A.F.M).
1
ACCEPTED MANUSCRIPT Abstract Tendon injury is a major musculoskeletal disorder with a high public health impact.
T
We propose a non-viral based strategy of gene therapy for the treatment of tendon
RI P
injuries using histidylated vectors. Gene delivery of fibromodulin, a proteoglycan involved in collagen assembly was found to promote rat Achilles tendon repair in vivo
SC
and in vitro. In vivo liposome-based transfection of fibromodulin led to a better healing after surgical injury, biomechanical properties were better restored compared
MA NU
to untransfected control. These measures were confirmed by histological observations and scoring. To get better understandings of the mechanisms underlying fibromodulin transfection, an in vitro tendon healing model was developed.
ED
In vitro, polymer-based transfection of fibromodulin led to the best wound enclosure speed and a pronounced migration of tenocytes primary cultures was observed.
PT
These results suggest that fibromodulin non-viral gene therapy could be proposed as
Key words
CE
a new therapeutic strategy to accelerate tendon healing.
AC
Tendon healing ; Gene therapy ; Lipoplex ; Polyplex
2
ACCEPTED MANUSCRIPT 1. Background The incidence of work-related musculoskeletal disorders (WMSD) is increasing every
T
year due to modern life. Tendon injuries represent the main pathology of WMSD and
RI P
healing requires a long rest period 1. It has been proven that growth factors like PDGF, TGF-β or VEGF can considerably accelerate this wound-healing process by
proliferation and migration
SC
regulating the inflammation phase, the production of extracellular matrix and the cell 2-5
. The delivery of these growth factors has some
MA NU
limitations due to their short half-life time requiring repetitive injections and the extensive cost of recombinant protein production and purification. Gene therapy is the best approach to produce growth factors in situ and can be exploited in tendon disorders 6.
ED
Transient gene expression after gene transfer by non-viral vectors would be useful
PT
for wound-healing because molecules involved in tissue repair have to be expressed only in a short-time period 7. Synthetic vectors are chemically controlled compounds
CE
easy to handle, and show a low immunogenicity with a weak risk of transgene
AC
integration.
These last years, few reports have shown the potentiality of in vivo gene delivery for tendon regeneration. They mainly concern the delivery of genes encoding growth factors
by
using
electroporation
liposomes,
adenoviral
vectors,
silica
nanoparticles
and
8-11
. The main effect of the expression of growth factors is the
induction of tenocytes proliferation and collagen I production. But, the tendon strength depends also on a good matrix assembly which relies on the collagen fibrillogenesis that depends on the activity of proteoglycans like fibromodulin (Fmod) or lumican (Lum)
12, 13
. Moreover, proteoglycans have been shown to enhance the
3
ACCEPTED MANUSCRIPT repair and remodelling of injured cornea
14
, skin15, and ligament
16
. They have a
specific expression profile in injured tendons compared to normal tissue
17
.
T
In this study, we investigated as nanomedicine approach whether Fmod or Lum gene
RI P
transfer could improve rat Achilles tendon healing. Gene transfer was performed either with imidazole cationic lipids (Lip 100 liposomes) or histidylated linear
SC
polyethyleneimine (PTG1) in Achilles tendons in vivo and in primary cultures of tenocytes. Wound-healing effect was assessed by histological analyses and stiffness
MA NU
measurements after transfection of fibromodulin. Finally, we performed in vitro experiments including wound-healing, cell proliferation and cell migration assays to get a better understanding of the benefic effect brought by fibromodulin gene
AC
CE
PT
ED
transfer.
4
ACCEPTED MANUSCRIPT 2. Methods 2.1. Plasmids
T
pNFCMV-Luc (pLuc), a 7.5 kb homemade plasmid DNA (pDNA) encoding the
RI P
firefly luciferase under control of the strong cytomegalovirus promoter was used as a reporter gene. This plasmid has five consecutive NFκB motifs (termed NF that
SC
recognize the NFB transcription factor) inserted upstream of the promoter. The second reporter pDNA was pMaxGFPTM, a 3.5 kb plasmid encoding the eGFP gene
MA NU
(Lonza, Basel, Switzerland). pPDGF plasmid (Invitrogen, Cergy-Pontoise, France) was encoding the human PDGFB gene. Plasmids pCEP4-FBM (pFmod) and pCEP4LUM (pLum) plasmids (generous gift from Dr. Peter Roughley) were encoding human 18
. pQE30, a mock plasmid (pMock)
ED
fibromodulin and lumican genes, respectively
that did not encode any gene was used as control in wound-healing experiments.
PT
2.2. Liposomes and polymers.
Histidylated liposomes (Lip100) were prepared by mixing O,O-dioleyl-N-[3Niodide)propylene]
CE
(N-methylimidazolium
phosphoramidate
and
O,O-dioleyl-N-
19, 20
AC
histamine phosphoramidate. These lipids were synthetized as previously described . Histidylated linear polyethylenimine (PTG1) was produced by Polytheragene
(Evry, France)
21
. Lipoplexes and polyplexes were formed at vector/DNA weight ratio
of 3:1 and 6:1, respectively. The detailed procedure for DNA complexes preparation is presented in supplementary methods. 2.3. Animal studies 2.3.1. In vivo transfection Adult Wistar rats were bred in CBM animal facility at 22°C for at least one week before experiments and groups were randomly made. Experiences were conducted according to the guidelines of the French Ministry of Agriculture for 5
ACCEPTED MANUSCRIPT experiments with laboratory (law 87848, C. Pichon accreditation). Rats were anesthetized by intraperitoneal injection of ketamine (50 mg/kg) and xylazine (10
T
mg/kg) in 0.9% NaCl. Skin was incised on few millimeters in Achilles tendon region.
RI P
For tendon healing study, Achilles tendon was incised through the tendon sheath along its total length three times using a carbon steel surgical blade n°12 (Swann-
SC
Morton, Sheffield, UK) (figure 1A). Fifty microliters of lipoplexes or polyplexes containing 20 µg of plasmid DNA were slowly injected in the middle section of
MA NU
Achilles tendon using a 31G Hamilton PBS600 (Hamilton, Bonaduz, Switzerland) repeater delivery system. Skin was sutured and rats were closely observed until their wake-up. Painkiller treatments were managed by subcutaneous injection of Finadyne at 250 µl/kg (Schering-Plough, Kenilworth, NJ, USA).
ED
2.3.2. Ex vivo luciferase assay
PT
The transfection efficiency obtained in tendons of pLuc-injected rats was determined as follows. Rats were euthanized by lethal CO 2 inhalation at indicated
CE
day post-surgery. At day 1, 3, 6 and 9 post-treatment, tendons were harvested and
AC
crushed in liquid nitrogen with a mortar and a pestle. The powder was added in an ice-cold lysis buffer (Luciferase Cell Culture Lysis, Promega, Madison, USA) and the mixture was kept for 3 hours on ice before luminescence measurement. The level of luciferase activity was measured with the Lumat LB9507 luminometer (Berthold, Wildbarch, Germany) after adding 100 µl of luciferin (Luciferase Assay System, Promega) to 20 µl of tendon lysates and expressed as relative luminescence unit (RLU) per mg of proteins. Proteins content of tendons were quantified by bicinchoninic acid assay. 2.3.3. In vivo bioluminescence imaging and quantification
6
ACCEPTED MANUSCRIPT Bioluminescence imaging was conducted in vivo using a Hamamatsu Photonics cooled CCD camera mounted on a dark box chamber (Hamamatsu,
T
Japon). Signal intensity was recorded 2 minutes after luciferin injection (100 µg,
RI P
Promega) in the tendon area and quantified as the mean of photons per second (p/s) of time exposure within a region of interest prescribed over the tendon.
SC
2.3.4. Biomechanical tests
Rats were euthanized by lethal CO2 inhalation at indicated times. Achilles
MA NU
tendons were harvested and stored in PBS at -20°C before use. They were dissected and cleaned to remove muscular and adherent surrounding tissues. Then the tendon was mounted vertically on a mechanical test machine (MTS synergie 400, Eden Prairie, MN, USA) and preloaded with a velocity of 0.6 mm/min by a triple cyclic
ED
loading, stretching up to 110% of its initial length (L0). After pre-conditioning,
PT
specimens were stretched to failure at the same stretching speed and the force was recorded. The stiffness of the tendons was calculated by the linear regression of the
CE
curve when plotting the displacement (mm) versus the load (N). During all the
AC
experiments the tendons were kept wet by spraying distilled water. Each group was composed of 5 tendons (healthy group was composed of 20 tendons). Values are expressed as means ± SEM. 2.3.5. Histological analysis Tendons were harvested 14 days post-injury and were fixed in 10% pformaldehyde solution, paraffin embedded, sliced in serial sections (thickness: 4 μm), mounted on glass slides and counterstained with HES (5 tendons per group were used for histological analyses). Histological process and blind analyses were carried out by In-Cell-Art (Nantes, France). The wound healing status of the tendons was evaluated by 3 criteria: the number of nuclei (hypercellularity), the matrix continuity
7
ACCEPTED MANUSCRIPT and the presence of inflammation. For each criteria a score from 1 to 3 was given, 1: poor, 2: average, 3: good 22.
T
2.4. In vitro studies
RI P
2.4.1. Primary culture of tenocytes
Tenocytes were obtained from adult Wistar rat Achilles tendon explants.
SC
Tendons were harvested, cut in small pieces in PBS and placed in tenocyte culture medium composed of MEM alpha, 10% Fetal Calf Serum, ascorbic acid (50 µg/ml),
MA NU
primocin (100 µg/ml, InvivoGen, Toulouse, France). Tenocytes were cultured at 37°C under humidified atmosphere containing 5% CO2. Half of the medium volume was replaced every two to three days. 2.4.2. In vitro transfection
ED
Two days before experiment, 105 tenocytes were seeded on 24-well plates.
PT
Then, tenocytes were transfected with indicated DNA formulations as described in supplementary material. After 4 hours, transfection medium was removed and cells
CE
were cultured for two days in tenocytes medium. The transfection efficiency was
23
.
AC
determined by measuring the luciferase activity in cell lysates as described previously
2.4.3. Monolayer wound-healing assay The tenocyte wound-healing model was created by plating 9×104 tenocytes two days before experiments on a CytoSelect™ 24-Well Wound-Healing Assay (Cell Biolabs Inc., San Diego, CA, USA) allowing creation of a 0.9 mm-wide standardized gap between confluent tenocytes monolayers. Inserts were removed and cells were transfected with indicated polyplexes formulations for 4 hours at 37°C. Transfection medium was replaced by fresh medium and the wound closure was monitored by videomicroscopy using a Zeiss Axiovert 200M (Carl Zeiss Inc., Thornwood, NY, USA)
8
ACCEPTED MANUSCRIPT fully motorized microscope during 72 hours (scan speed: 2 images/hour). Cells were incubated in an atmosphere and temperature controlled chamber at 37°C and 5%
T
CO2. Quantitative analysis of the cell free area was performed using the Axiovision
RI P
Rel. 4.7 (Carl Zeiss Inc.). The level of wound-healing for a time x was evaluated by calculating the percentage of the cell free area at tx divided by the cell free area at
SC
the initial state. Experiments were at least repeated 6 times. In a set of experiments wound closure was followed when the tenocyte wound-healing model was incubated
2.4.4. Quantitative real-time PCR
MA NU
with medium from transfected tenocytes, collected 24 hours after transfection.
Tenocytes were harvested 18 and 43 hours post-transfection and total RNA
ED
was extracted using an RNA II NucleoSpin® mini kit (Macherey-Nagel EURL, Hoerdt, France). For all experiments, 50 ng of RNA was used for reverse transcription using
PT
200 units of M-MLV Reverse Transcriptase (New England Biolabs, MA, USA). The gene expression was evaluated from 100 ng of cDNA by qPCR using Quantifast®
CE
PCR Master Mix (Qiagen, Venlo, Netherlands) with 0.1 µM of Fmod, Lum, PDGF or
AC
RNA6S specific primers (Table 1). The qPCR was performed on a Light Cycler II 480 (Roche Applied Science, Meylan, France), the cycles consisted of 10 seconds at 95°C for DNA denaturation followed by 30 seconds at 60°C for primer annealing and polymerization repeated 40 times. Fmod, Lum and PDGF primers were specific to the human transcripts of cDNA inserted into the pFmod, pLum and pPDGF plasmids. The relative quantification was made using the ΔΔCt method using the RNA6S housekeeping gene expression.
2.4.5. Tenocytes proliferation
9
ACCEPTED MANUSCRIPT The evaluation of tenocytes proliferation was monitored by MTT colorimetric assay 18, 43 and 72 hours after transfection. Cells were incubated during 4 hours at
T
37°C, 5% CO2 in presence of 500 µg/ml of 3-(4,5-dimethylthiazol-2-yl)-2,5-
RI P
diphenyltetrazolium bromide (Sigma Aldrich, Saint Louis, MO, USA). Cells were washed and lysed with acidified isopropanol containing 3% of SDS (Sigma Aldrich)
SC
allowing the solubilization of tetrazolium salts. Quantification was made measuring optical density at 560 nm; the control was made using cells having received the same
MA NU
manipulations without transfection. Experiments were done in triplicate and repeated two times.
2.4.6. Migration analysis of tenocytes
Migration analysis was defined as the capacity of tenocytes to migrate through
ED
8 µm cell culture insert membranes (BD Falcon, Franklin Lakes, NJ, USA). Bottom
PT
wells were filled with complete medium and inserts seeded with 10 5 transfected tenocytes. Cells were incubated at 37°C in a 5% CO2 atmosphere during 18 hours.
CE
Inserts were cut and the number of cells present on the other side of the insert was
AC
evaluated by microscopy. The migration factor was calculated as the number of cells present at the bottom side of the insert after transfection divided by the number of cells present in non-transfected condition. Each condition was repeated two times in triplicate. 2.5. Statistical analysis Data were expressed by means ± SD (in vitro experiments) or SEM (in vivo experiments). All statistical differences were analyzed by a bilateral Mann–Whitney U-test when statistical difference was found by Kruskal-Wallis statistical test. XLStat 2014 software was used (Addinsoft, Paris, France), significance was defined as pvalue
S1549-9634(15)00119-7 doi: 10.1016/j.nano.2015.05.004 NANO 1131
To appear in:
Nanomedicine: Nanotechnology, Biology, and Medicine
Received date: Revised date: Accepted date:
27 January 2015 30 April 2015 18 May 2015
Please cite this article as: Delalande Anthony, Gosselin Marie-Pierre, Suwalski Arnaud, Guilmain William, Leduc Chlo´e, Berchel Mathieu, Jaffr`es Paul-Alain, Baril Patrick, Midoux Patrick, Pichon Chantal, Enhanced achilles tendon healing by fibromodulin gene transfer, Nanomedicine: Nanotechnology, Biology, and Medicine (2015), doi: 10.1016/j.nano.2015.05.004
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Enhanced Achilles tendon healing by fibromodulin gene transfer. Anthony Delalande1*, Marie-Pierre Gosselin1*, Arnaud Suwalski1, William Guilmain1,
T
Chloé Leduc1, Mathieu Berchel2, Paul-Alain Jaffrès2, Patrick Baril1, Patrick Midoux1
RI P
and Chantal Pichon1. 1
Centre de Biophysique Moléculaire, rue Charles Sadron, Orléans 45071 CEDEX 2,
SC
France. 2
Université de Brest, Brest, France
MA NU
CEMCA, CNRS UMR 6521, IFR148 ScInBioS, Université Européenne de Bretagne,
*: These authors contributed equally to this work.
Abstract: 147 words
PT
Body text: 4962 words
AC
3 tables
CE
37 references 6 figures
ED
Corresponding author: [email protected]
1 supplementary material The authors disclose no conflict of interest. This work was supported by the Association Française contre les Myopathies (A.F.M).
1
ACCEPTED MANUSCRIPT Abstract Tendon injury is a major musculoskeletal disorder with a high public health impact.
T
We propose a non-viral based strategy of gene therapy for the treatment of tendon
RI P
injuries using histidylated vectors. Gene delivery of fibromodulin, a proteoglycan involved in collagen assembly was found to promote rat Achilles tendon repair in vivo
SC
and in vitro. In vivo liposome-based transfection of fibromodulin led to a better healing after surgical injury, biomechanical properties were better restored compared
MA NU
to untransfected control. These measures were confirmed by histological observations and scoring. To get better understandings of the mechanisms underlying fibromodulin transfection, an in vitro tendon healing model was developed.
ED
In vitro, polymer-based transfection of fibromodulin led to the best wound enclosure speed and a pronounced migration of tenocytes primary cultures was observed.
PT
These results suggest that fibromodulin non-viral gene therapy could be proposed as
Key words
CE
a new therapeutic strategy to accelerate tendon healing.
AC
Tendon healing ; Gene therapy ; Lipoplex ; Polyplex
2
ACCEPTED MANUSCRIPT 1. Background The incidence of work-related musculoskeletal disorders (WMSD) is increasing every
T
year due to modern life. Tendon injuries represent the main pathology of WMSD and
RI P
healing requires a long rest period 1. It has been proven that growth factors like PDGF, TGF-β or VEGF can considerably accelerate this wound-healing process by
proliferation and migration
SC
regulating the inflammation phase, the production of extracellular matrix and the cell 2-5
. The delivery of these growth factors has some
MA NU
limitations due to their short half-life time requiring repetitive injections and the extensive cost of recombinant protein production and purification. Gene therapy is the best approach to produce growth factors in situ and can be exploited in tendon disorders 6.
ED
Transient gene expression after gene transfer by non-viral vectors would be useful
PT
for wound-healing because molecules involved in tissue repair have to be expressed only in a short-time period 7. Synthetic vectors are chemically controlled compounds
CE
easy to handle, and show a low immunogenicity with a weak risk of transgene
AC
integration.
These last years, few reports have shown the potentiality of in vivo gene delivery for tendon regeneration. They mainly concern the delivery of genes encoding growth factors
by
using
electroporation
liposomes,
adenoviral
vectors,
silica
nanoparticles
and
8-11
. The main effect of the expression of growth factors is the
induction of tenocytes proliferation and collagen I production. But, the tendon strength depends also on a good matrix assembly which relies on the collagen fibrillogenesis that depends on the activity of proteoglycans like fibromodulin (Fmod) or lumican (Lum)
12, 13
. Moreover, proteoglycans have been shown to enhance the
3
ACCEPTED MANUSCRIPT repair and remodelling of injured cornea
14
, skin15, and ligament
16
. They have a
specific expression profile in injured tendons compared to normal tissue
17
.
T
In this study, we investigated as nanomedicine approach whether Fmod or Lum gene
RI P
transfer could improve rat Achilles tendon healing. Gene transfer was performed either with imidazole cationic lipids (Lip 100 liposomes) or histidylated linear
SC
polyethyleneimine (PTG1) in Achilles tendons in vivo and in primary cultures of tenocytes. Wound-healing effect was assessed by histological analyses and stiffness
MA NU
measurements after transfection of fibromodulin. Finally, we performed in vitro experiments including wound-healing, cell proliferation and cell migration assays to get a better understanding of the benefic effect brought by fibromodulin gene
AC
CE
PT
ED
transfer.
4
ACCEPTED MANUSCRIPT 2. Methods 2.1. Plasmids
T
pNFCMV-Luc (pLuc), a 7.5 kb homemade plasmid DNA (pDNA) encoding the
RI P
firefly luciferase under control of the strong cytomegalovirus promoter was used as a reporter gene. This plasmid has five consecutive NFκB motifs (termed NF that
SC
recognize the NFB transcription factor) inserted upstream of the promoter. The second reporter pDNA was pMaxGFPTM, a 3.5 kb plasmid encoding the eGFP gene
MA NU
(Lonza, Basel, Switzerland). pPDGF plasmid (Invitrogen, Cergy-Pontoise, France) was encoding the human PDGFB gene. Plasmids pCEP4-FBM (pFmod) and pCEP4LUM (pLum) plasmids (generous gift from Dr. Peter Roughley) were encoding human 18
. pQE30, a mock plasmid (pMock)
ED
fibromodulin and lumican genes, respectively
that did not encode any gene was used as control in wound-healing experiments.
PT
2.2. Liposomes and polymers.
Histidylated liposomes (Lip100) were prepared by mixing O,O-dioleyl-N-[3Niodide)propylene]
CE
(N-methylimidazolium
phosphoramidate
and
O,O-dioleyl-N-
19, 20
AC
histamine phosphoramidate. These lipids were synthetized as previously described . Histidylated linear polyethylenimine (PTG1) was produced by Polytheragene
(Evry, France)
21
. Lipoplexes and polyplexes were formed at vector/DNA weight ratio
of 3:1 and 6:1, respectively. The detailed procedure for DNA complexes preparation is presented in supplementary methods. 2.3. Animal studies 2.3.1. In vivo transfection Adult Wistar rats were bred in CBM animal facility at 22°C for at least one week before experiments and groups were randomly made. Experiences were conducted according to the guidelines of the French Ministry of Agriculture for 5
ACCEPTED MANUSCRIPT experiments with laboratory (law 87848, C. Pichon accreditation). Rats were anesthetized by intraperitoneal injection of ketamine (50 mg/kg) and xylazine (10
T
mg/kg) in 0.9% NaCl. Skin was incised on few millimeters in Achilles tendon region.
RI P
For tendon healing study, Achilles tendon was incised through the tendon sheath along its total length three times using a carbon steel surgical blade n°12 (Swann-
SC
Morton, Sheffield, UK) (figure 1A). Fifty microliters of lipoplexes or polyplexes containing 20 µg of plasmid DNA were slowly injected in the middle section of
MA NU
Achilles tendon using a 31G Hamilton PBS600 (Hamilton, Bonaduz, Switzerland) repeater delivery system. Skin was sutured and rats were closely observed until their wake-up. Painkiller treatments were managed by subcutaneous injection of Finadyne at 250 µl/kg (Schering-Plough, Kenilworth, NJ, USA).
ED
2.3.2. Ex vivo luciferase assay
PT
The transfection efficiency obtained in tendons of pLuc-injected rats was determined as follows. Rats were euthanized by lethal CO 2 inhalation at indicated
CE
day post-surgery. At day 1, 3, 6 and 9 post-treatment, tendons were harvested and
AC
crushed in liquid nitrogen with a mortar and a pestle. The powder was added in an ice-cold lysis buffer (Luciferase Cell Culture Lysis, Promega, Madison, USA) and the mixture was kept for 3 hours on ice before luminescence measurement. The level of luciferase activity was measured with the Lumat LB9507 luminometer (Berthold, Wildbarch, Germany) after adding 100 µl of luciferin (Luciferase Assay System, Promega) to 20 µl of tendon lysates and expressed as relative luminescence unit (RLU) per mg of proteins. Proteins content of tendons were quantified by bicinchoninic acid assay. 2.3.3. In vivo bioluminescence imaging and quantification
6
ACCEPTED MANUSCRIPT Bioluminescence imaging was conducted in vivo using a Hamamatsu Photonics cooled CCD camera mounted on a dark box chamber (Hamamatsu,
T
Japon). Signal intensity was recorded 2 minutes after luciferin injection (100 µg,
RI P
Promega) in the tendon area and quantified as the mean of photons per second (p/s) of time exposure within a region of interest prescribed over the tendon.
SC
2.3.4. Biomechanical tests
Rats were euthanized by lethal CO2 inhalation at indicated times. Achilles
MA NU
tendons were harvested and stored in PBS at -20°C before use. They were dissected and cleaned to remove muscular and adherent surrounding tissues. Then the tendon was mounted vertically on a mechanical test machine (MTS synergie 400, Eden Prairie, MN, USA) and preloaded with a velocity of 0.6 mm/min by a triple cyclic
ED
loading, stretching up to 110% of its initial length (L0). After pre-conditioning,
PT
specimens were stretched to failure at the same stretching speed and the force was recorded. The stiffness of the tendons was calculated by the linear regression of the
CE
curve when plotting the displacement (mm) versus the load (N). During all the
AC
experiments the tendons were kept wet by spraying distilled water. Each group was composed of 5 tendons (healthy group was composed of 20 tendons). Values are expressed as means ± SEM. 2.3.5. Histological analysis Tendons were harvested 14 days post-injury and were fixed in 10% pformaldehyde solution, paraffin embedded, sliced in serial sections (thickness: 4 μm), mounted on glass slides and counterstained with HES (5 tendons per group were used for histological analyses). Histological process and blind analyses were carried out by In-Cell-Art (Nantes, France). The wound healing status of the tendons was evaluated by 3 criteria: the number of nuclei (hypercellularity), the matrix continuity
7
ACCEPTED MANUSCRIPT and the presence of inflammation. For each criteria a score from 1 to 3 was given, 1: poor, 2: average, 3: good 22.
T
2.4. In vitro studies
RI P
2.4.1. Primary culture of tenocytes
Tenocytes were obtained from adult Wistar rat Achilles tendon explants.
SC
Tendons were harvested, cut in small pieces in PBS and placed in tenocyte culture medium composed of MEM alpha, 10% Fetal Calf Serum, ascorbic acid (50 µg/ml),
MA NU
primocin (100 µg/ml, InvivoGen, Toulouse, France). Tenocytes were cultured at 37°C under humidified atmosphere containing 5% CO2. Half of the medium volume was replaced every two to three days. 2.4.2. In vitro transfection
ED
Two days before experiment, 105 tenocytes were seeded on 24-well plates.
PT
Then, tenocytes were transfected with indicated DNA formulations as described in supplementary material. After 4 hours, transfection medium was removed and cells
CE
were cultured for two days in tenocytes medium. The transfection efficiency was
23
.
AC
determined by measuring the luciferase activity in cell lysates as described previously
2.4.3. Monolayer wound-healing assay The tenocyte wound-healing model was created by plating 9×104 tenocytes two days before experiments on a CytoSelect™ 24-Well Wound-Healing Assay (Cell Biolabs Inc., San Diego, CA, USA) allowing creation of a 0.9 mm-wide standardized gap between confluent tenocytes monolayers. Inserts were removed and cells were transfected with indicated polyplexes formulations for 4 hours at 37°C. Transfection medium was replaced by fresh medium and the wound closure was monitored by videomicroscopy using a Zeiss Axiovert 200M (Carl Zeiss Inc., Thornwood, NY, USA)
8
ACCEPTED MANUSCRIPT fully motorized microscope during 72 hours (scan speed: 2 images/hour). Cells were incubated in an atmosphere and temperature controlled chamber at 37°C and 5%
T
CO2. Quantitative analysis of the cell free area was performed using the Axiovision
RI P
Rel. 4.7 (Carl Zeiss Inc.). The level of wound-healing for a time x was evaluated by calculating the percentage of the cell free area at tx divided by the cell free area at
SC
the initial state. Experiments were at least repeated 6 times. In a set of experiments wound closure was followed when the tenocyte wound-healing model was incubated
2.4.4. Quantitative real-time PCR
MA NU
with medium from transfected tenocytes, collected 24 hours after transfection.
Tenocytes were harvested 18 and 43 hours post-transfection and total RNA
ED
was extracted using an RNA II NucleoSpin® mini kit (Macherey-Nagel EURL, Hoerdt, France). For all experiments, 50 ng of RNA was used for reverse transcription using
PT
200 units of M-MLV Reverse Transcriptase (New England Biolabs, MA, USA). The gene expression was evaluated from 100 ng of cDNA by qPCR using Quantifast®
CE
PCR Master Mix (Qiagen, Venlo, Netherlands) with 0.1 µM of Fmod, Lum, PDGF or
AC
RNA6S specific primers (Table 1). The qPCR was performed on a Light Cycler II 480 (Roche Applied Science, Meylan, France), the cycles consisted of 10 seconds at 95°C for DNA denaturation followed by 30 seconds at 60°C for primer annealing and polymerization repeated 40 times. Fmod, Lum and PDGF primers were specific to the human transcripts of cDNA inserted into the pFmod, pLum and pPDGF plasmids. The relative quantification was made using the ΔΔCt method using the RNA6S housekeeping gene expression.
2.4.5. Tenocytes proliferation
9
ACCEPTED MANUSCRIPT The evaluation of tenocytes proliferation was monitored by MTT colorimetric assay 18, 43 and 72 hours after transfection. Cells were incubated during 4 hours at
T
37°C, 5% CO2 in presence of 500 µg/ml of 3-(4,5-dimethylthiazol-2-yl)-2,5-
RI P
diphenyltetrazolium bromide (Sigma Aldrich, Saint Louis, MO, USA). Cells were washed and lysed with acidified isopropanol containing 3% of SDS (Sigma Aldrich)
SC
allowing the solubilization of tetrazolium salts. Quantification was made measuring optical density at 560 nm; the control was made using cells having received the same
MA NU
manipulations without transfection. Experiments were done in triplicate and repeated two times.
2.4.6. Migration analysis of tenocytes
Migration analysis was defined as the capacity of tenocytes to migrate through
ED
8 µm cell culture insert membranes (BD Falcon, Franklin Lakes, NJ, USA). Bottom
PT
wells were filled with complete medium and inserts seeded with 10 5 transfected tenocytes. Cells were incubated at 37°C in a 5% CO2 atmosphere during 18 hours.
CE
Inserts were cut and the number of cells present on the other side of the insert was
AC
evaluated by microscopy. The migration factor was calculated as the number of cells present at the bottom side of the insert after transfection divided by the number of cells present in non-transfected condition. Each condition was repeated two times in triplicate. 2.5. Statistical analysis Data were expressed by means ± SD (in vitro experiments) or SEM (in vivo experiments). All statistical differences were analyzed by a bilateral Mann–Whitney U-test when statistical difference was found by Kruskal-Wallis statistical test. XLStat 2014 software was used (Addinsoft, Paris, France), significance was defined as pvalue
