Electrospun biomimetic microfibers for pelvic floor repair ...€¦ · options for recurrence of POP. Therefore, new principles for effective repair or replacement of deficient connective

Electrospun biomimetic microfibers for pelvic

floor repair tested in a rat abdominal wall

defect model

Cecilie Lærke Glindtvad [email protected]

Tel: +45 20978986

Cecilie L. Glindtvad Speciale Opgave 29.04.2015

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Resumé

Introduktion: Fra 50 års alderen vil halvdelen af den kvindelig befolkning opleve nedsynkning i

underlivet pga. udtynding og bristning i støttende bindevæv og fascier i bækkenet. Der er ingen

fuldstændig effektiv behandling for nedsynkning i underlivet, som giver et varigt resultat og er uden

komplikationer. Vi forslår en ny behandlingsmetode med rekonstruktion af de tre dimensionale

anatomiske strukturer i bækkenbunden.

Formål: At undersøge en nedbrydelig mesh med fibroblast vækstfaktor (bFGF) i forhold til mekaniske

egenskaber og effekt på collagen og elastin dannelse i nyt bindevæv.

Materiale og metode: 40 rotter fik indopereret en elektrospundet polycaprolactone(PCL)/

polyethylene oxid (PEO) mesh af mikrofibre. Test gruppen havde bFGF i kernen af mesh fibrene, og

hos kontrol gruppen var bFGF ikke tilsat den indopererede mesh. Meshen blev testet i en abdominal

defekt model i rotterne. Testområdet blev udtaget efter 4, 8 og 24 uger og testet for styrke,

forlængelse, stivhed, energi absorption, total collagen mængde, elastin samt mRNA expression og

protein mængde af collagen-I, collagen-III og fibronectin. Den samlede effekt af meshen blev vurderet

ud fra størrelsen af det hernie, som en stor del af rotterne udviklede.

Resultater: Studiet viste en forøgelse af styrken, stivheden og energien over tid. Desuden sås en

signifikant forøgelse efter 4 uger i gruppen med bFGF for styrke, stivhed og energi absorption. Der var

ingen forskel mellem grupperne efter 8 og 24 uger. Der var en signifikant forøgelse af mRNA

expression for collagen-I og collagen-III efter 4 uger. Der blev for grupper med og uden bFGF ikke

fundet en forskel for protein mængden, elastin eller total collagen. Histologien viste en hæmmet celle

indvækst ved 4 uger med bFGF, men ved 8 og 24 uger var der ingen forskel. Desuden sås tegn på

elastin fibre ved 24 uger. Analyse af hernie størrelse i forhold til variablerne viste, at øget hernie

størrelse medførte øget styrke og stivhed.

Konklusion: Analyserne viste at en mesh af PCL/PEO fremmer dannelse af nyt væv med øget styrke,

stivhed og energi absorption over tid. Meshen med bFGF havde kun en effekt efter 4 uger på styrke,

stivhed, energi absorption og mRNA expression for collagen-I og collagen-III. Der var øget styrke og

stivhed med stigende hernie størrelse.


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Introduction

Half of the female population experiences disorders of the pelvic floor caused by weakening and

rupture of supportive connective tissue sheet or fascia [1]. This disorder can lead to pelvic organ

prolapse (POP) seen as a herniation of the anatomical structures (bladder, uterus and intestines) into

the vagina [2]. The frequency of POP generally increases with age and affects quality of life in terms of

social, psychological, physical, sexual, body image, and overall wellbeing [3]. Consequently, these

problems occupy a significant proportion of the healthcare budget now and expenses will continue in

the future [4].

POP affects up to 50% of all women over the age of 50 [5]. The mean prevalence is 19.7%, ranging

from 3.4-56.4% in developed countries [6]. The lifetime risk for undergoing POP surgery is estimated to

11.1-19% [5,7]. Furthermore, the risk of reoperation because of recurrence is up to 30% [8].

Recently, no efficient treatment without side effects or risk of recurrence has been invented. Instead,

each country and sometimes each hospital in a country use different operations and treatment-

techniques. So far, surgeons all over the world have tried to treat POP operatively with native tissue

repair, biological graft, and synthetic mesh in different shapes and sizes.

Synthetic, non-degradable mesh made of polypropylene (PP), has been used for operative correction

of complicated POP cases and recurrence occurs in up to 9.7% of cases [9-11]. Moreover, serious

complications of non-degradable meshes are seen with poor tissue integration, inflammation,

exposition of the mesh through the vaginal wall, and erosion into the bladder or bowel [12].

Consequently, usage of non-degradable mesh is reduced, even though there are few other treatment

options for recurrence of POP. Therefore, new principles for effective repair or replacement of

deficient connective tissue are needed. Collagen I, III, and V are the principal components of the

connective tissue in the pelvic floor providing strength to the soft tissue [13]. Furthermore, the pelvic

organs rely on the tensile strength of the pelvic floor for support; in this matter elastin is a part of

extension, resilience and recoil of tissue.

In recent years, approaches based on tissue engineering and regenerative medicine principles have

emerged as strategies for pelvic tissue reconstruction [14,15]. In this matter, we suggest that

treatment of pelvic floor disorders could be brought forward by functional reconstruction of the three

dimensional, anatomical structures by deploying a degradable, electrospun mesh. Our approach


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includes release of basic fibroblast growth factor (bFGF) with the purpose to enhance the formation of

new connective tissue, and in particular collagen deposition within the degrading mesh [16]. An

engineering challenge is to balance mesh degradation and implant weakening with the increase of

structural stability accompanying collagen deposition. In vitro studies show that a polycaprolactone

(PCL) /poly ethylene oxide (PEO) mesh with bFGF increases fibroblast cell proliferation with 331% after

9 days of culture, and that 72% of the bFGF is released. Studies show that the PCL/PEO fibers without

bFGF have no toxic effect and that added bFGF increases cell viability [17]. The results of the former in

vitro study indicated that a PCL mesh with bFGF could be a potential treatment for pelvic organ

prolapse and was chosen to test in vivo.

The purpose of this study is to investigate the in vivo effects of a degradable, bFGF releasing,

electrospun mesh on collagen and elastin production and study the influence on the temporal

mechanical properties of the neotissue in a rat abdominal wall defect model.

Materials and Methods

The mesh was made of biodegradable, biocompatible PCL/PEO microfibers (97% PCL and 3% PEO) and

consisted of randomly placed hollow fibers with bFGF in the core. The microfibers were continuous

with high porosity and high spatial interconnectivity, which mimicked the intricate interweaving

structure of extracellular matrix composed of fibrillar collagen and elastin promoting cell adhesion. The

microfibers were produced by coaxial electrospinning, fabricating two-component fibers by forcing

solutions through a coaxial spinneret in the presence of a high electric field. The fibers consisted of PCL

in the outer wall, polyethylene oxide (PEO) and bovine serum albumin (BSA) in the inner wall, and

bFGF in the core. The fibers in the present mesh measured 1.6 µm in shell thickness and had a fiber

diameter of 7.489 ± 0.445 µm. The amount of bFGF in the mesh was 60 ng bFGF per mg mesh

corresponding to approximately 0.34851 µg bFGF per implanted mesh.


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Figure 1 – Scanning electronic picture of the mesh. Cross-section (a) and surface (b) for mesh without bFGF. Cross-section (c) and surface (d) for mesh with bFGF [17].

Mesh with bFGF was not visibly morphologically different from mesh without bFGF as shown in Figure

1. Three-dimensional structure of the mesh and thereby its ability to mimic the extracellular matrix

composition was not changed with bFGF in the core [17].

Implantation

Forty inbred Wistar female rats (adult, weight 250–300 grams) were purchased from Taconic,

Denmark. A pilot study included eight rats and the primary study included 32 rats. Half of the rats

served as a control group with mesh without bFGF and the other half as test group with bFGF. After an

abdominal midline incision, the skin was loosened from the muscle layer and a full thickness fascia-

muscle defect (1.5 cm x 3.0 cm) was made. Then, the defect was repaired by the mesh with a 0.5 cm

overlap to the surrounding tissue in all directions. The mesh was independently fixated with single

sutures (Prolene 5-0, Ethicon®) in the corners and with continuously absorbable sutures (Vicryl 4-0

Ethicon®) at the peripheral edges (Figure 2). The skin was closed by staples.


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Figure 2 – Overview of the mesh and explantation schedule. The grey square is the mesh, the red square is the muscle defect, the dashed squares is the pieces for mechanical testing, and the blue lines are the permanent stitches.

From implantation to explantation of the mesh the operation area was observed for herniation, fluid

collection and complications in general. During the study, one rat died because of peritonitis (24 weeks

+ bFGF) and two rats were sacrificed: one because of infection in the operation wound (24 weeks –

bFGF) and one because of suspicion of incarcerated hernia (8 weeks + bFGF).

Explantation

After four, eight, and 24 weeks rats were sacrificed by Pentobarbital overdose (1 ml of Pentobarbital®

200 mg/ml with 10% Lidocaine® intraperitoneally) (Table 1).

4 weeks 8 weeks 24 weeks In total

Mesh without bFGF 4 rats 6 rats 9 rats 19 rats

Mesh with bFGF 4 rats 5 rats 9 rats 18 rats

Table 1 - Time schedule for explantation

The implantation area with one cm surrounding tissue was excised and divided into four sections. One

section for total collagen, elastin, and mechanical testing which was wrapped air-tight and stored at

minus 20 degrees. Two sections were snap frozen in liquid nitrogen and used for evaluating mRNA

expression and protein content. The fourth section was formalin fixed and used for histology (Figure

2).


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Mechanical testing

After thawing, the material was kept moistened with a 50 mM Tris/HCl (pH 7.4) buffer. Four mm wide

strips were cut with a multicutter (parallel mounted razor blades) and adjusted to 18 mm in length.

The thickness of the central part of the strip was measured by an electronic length gauge (MT25/ND

281B, Heidenhain, Traunreut, Germany) under standardized stress (25 kPa) for 10 seconds. For

mechanical testing, the ends of the strip were gripped in clamps mounted with 10 mm gage distance in

a material testing machine (Alwetron TCT5, Kista, Sweden) equipped with a 20 N load cell. To avoid

slippage the clamp jaws were coated with emery cloth (grain size 320) and screwed together to an

adjusted and standardized moment with a torque wrench (22 cNm). During testing at 30 mm/min the

strip was soaked in the Tris/HCL buffer. The strip was subjected to loading until failure. The ruptured

pieces were collected for total collagen analysis.

mRNA expression by real-time PCR

mRNA was extracted from tissue flaps using Trizol reagent (Invitrogen). For each sample 0.5 µg RNA

was reverse-transcribed using RT-Superscript III (Invitrogen) and polymerase chain reaction (PCR) was

carried out by using iCycler™. All the reactions were performed with SYBR® Green under the same

conditions as follows: 95°C for five minutes followed by 45 cycles 30 seconds at 95°C , one minute at

60°C , 45 seconds at 72°C , and five minutes at 72°C. A standard curve was mixed from all the copy

DNA samples and included in each PCR dilution (1:5, 1:25, 1:125, 1:625). To quantify the expression of

the genes, they were compared to the housekeeping gene Glyceraldehyde 3-phosphate

Dehydrogenase (GAPDH), and the results were expressed as a ratio. The primer pairs used in the study

were: forward collagen-I 5’-3’ ATG TTC AGC TTT GTG GAC CT, reverse collagen-I 5’-3’ CAG CTG ACT

TCA GGG ATG T, forward collagen-III 5’-3’ CGG AAT TGC AGA GAC CTG AA, reverse collagen-III 5’-3’

ACA GTC ATG GGA CTG GCA TTT AT ,forward fibronectin 5’-3’ GTG GCT GCC TTC AAC TTC TC and

reverse fibronectin 5’-3’GTG GGT TGC AAA CCT TCA AT.

Protein content by Western-blots

Total protein was extracted from the tissue samples by homogenization using a homogenization buffer

containing 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), protease and phosphatase

inhibitors. Each sample was homogenized with 20 µl buffer/mg wet tissue and ceramics beads on a

Precellys. The protein concentration was determined by the Micro BCA Protein Assay kit (Pierce).


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Running samples were mixed in Laemmli Buffer and 20µg sample was loaded on a 4-15% gel Criterion

TGX Stain-Free BioRad. Rainbow marker (2.5µl) was used. The gel was run on a vertical electrophorese

(SDS-Page). After 2.5min UV activation of the gel the protein was transferred to a membrane using

BioRad TurboTrans HIGH MW. After transfer, a stainfree picture of the membrane was made and used

for detection of reference protein as total protein on the membrane instead of using beta-actin

concentration as a reference [18]. After blocking of the membrane it was blotted with primary

antibodies for Collagen-I (Abcam, cat.no: ab90395, 1:1000), Collagen-III (GenWay Biotech cat.no:

292343, 1:2500) or Fibronectin (DAKO cat.no: A0245, 1:5000) over night. Afterwards, the membranes

were washed inTTBS buffer before placing in secondary antibody; Collagen-I (anti-mouse SC-2005,

1:1000), Collagen-III (anti-rabbit SC-2054, 1:5000) and fibronectin (anti-rabbit SC-2054, 1:10.000).

Again, the membranes were washed before the induction with SuperSignal West Dura Extended

Duration Substrate (Pierce) on Imagelab (BioRad). The procedure for collagen-I was not successful and

ELISA was used instead.

ELISA

100µl of serial dilutions of Collagen l standard (from 12.5 to 0.39 ng/ml) diluted in PBS, (cat:1200-01

Southern Biotechnology, USA) and 100µl of serial dilution of sample (1:4000 to 1:160000 ) diluted in

PBS was coated in an ELISA microplate and incubated overnight at RT. After blocking for 30 minutes

with 5% Milk in PBST the plate was washed and incubated for 90 minutes at RT with 100µl 1:1000

dilutions of goat-anti-collagen l biot. (cat: 1310-08, Southern Biotechnology, USA) Wells were washed

and incubated with 100µl 1:1000 Streptavidin-HRP (cat:7100-05, Southern Biotechnology, USA). The

wells were washed before the induction with TMB substrate (Cat: TMBW-100, SMS, Denmark). The

reaction was stopped with 0.2M H2SO4. The Optical Density was determined at 450nm microplate

reader. Standard absorbance versus its concentration was plotted on graph to construct a standard

curve. The result is shown as a ratio between Collagen l and total protein. Results for four weeks

samples were not achieved because of lack of test material.

Total collagen content

The collected strips from mechanical testing were defatted in acetone 2 × 1 hour at room temperature,

air-dried and weighed. To remove the remaining PCL in the strips, they were treated with chloroform:

methanol 2:1, for 21 hours at 4°C, air-dried, and weighed. Before hydrolysis in 6 N HCl at 100°C for 16

hours, the strips were freeze-dried and weighed. Hydroxyproline (Hyp) was determined colorimetric


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[19]. Collagen content was calculated as Hyp × 7.46 [20]. The total collagen amount was correlated to

the cross-sectional area of the samples [mg/mm2] to neutralize differences in sample size.

Histology

The samples for histology were fixated in four percent neutral formaldehyde buffer for 24-48 hours.

The formalin fixed paraffin embedded tissue was cut in 3 µm thick slices, collected on slides, treated in

tissueclear and stored in water until staining. Sections were stained with Hematoxylin Eosin performed

on a Combitec slide stainer 4009 from HISTOLAB Products, Göteborg Sweden and Masson’s trichrome

staining was performed with the three colors; Harris Hemotoxylin, Ponceau Red and Anilin Bleu. After

staining, the sections were dehydrated and fixated with the mounting medium Pertex. After staining,

the samples were blinded within the groups of four, eight, and 24 weeks and investigated for visual

differences in relation to amount of mesh left, collagen amount and inflammatory response.

Ethical

The study was approved by the Danish Inspectorate for Animal Experimentation under the Danish

Ministry of Justice with authorization number 2014-15-2934-01021. The animals were anesthetized

and treated with analgesics according to ethical guidelines and looked for by professional animal

keepers under guidance from a veterinarian.

Statistics

The number of animals was chosen from former short term studies with an identical animal model

[21,22]. Data were tested for normality. Differences between groups were assessed by Student’s t-test.

The mechanical testing was expressed in mean results calculated from one to five test samples for

each rat. The cross-sectional area and volume used, was the one priori to testing. Furthermore, the

removed abdominal wall from the implantation of the mesh was used as a reference and a goal for the

mechanical properties for the newly formed tissue. The mesh values were reference for the starting

point for the mechanical properties. Data were analyzed for association between hernia size and the

various outcomes by two-way ANOVA. Spearmen’s Rho was performed when a trend seemed possible.

The statistic program StataIc version 13 was used. Significance level: P ≤ 0.05.


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Results

The mechanical tensile tests are presented as a mean stress-strain curve for each group (Figure 3).

Strain is fractional increase ((l-l0)/l0) in sample length (l) where l0 is the length of the sample at a

recorded minimal load value (0.003 N). The vertex of the curve is the maximum stress representing the

tensile strength of the sample, given as the force measured by the load cell divided by the initial cross

sectional area of the sample. The maximum slope of the stress-strain curve represents the maximum

stiffness of the specimen. The area below the stress-strain curve until failure represents the energy

absorption needed for the tissue deformation.

Figure 3 – Stress-strain curve of mean values for each group, mesh, and explanted muscle. The work curve for the mesh continues to break at mean strain 8.70 (870%) and stress at 2.1 MPa. Four animals in the two four weeks groups, Five and six rats in the eight weeks groups and nine rats in each of the 24 weeks groups.

Figure 4 shows tensile strength (P=0.0021), stiffness (P=0.0002), and energy absorption(P=0.0097) in

the newly formed tissue with a significant difference between four weeks samples with and without

bFGF, but no difference at eight and 24 weeks. For strain at tensile strength, no differences at any

point between the groups with and without bFGF were seen. For all parameters except strain at tensile

strength, there was a significant difference over time from four to 24 weeks (P<0.0002) (Figure 4).


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Figure 4– Bar graph of tensile strength, stiffness, energy absorption, and strain at tensile strength in the newly formed tissue.

A significant increase in mRNA expression for collagen-I (P= 0.0060) and collagen-III (P= 0.0086) was

seen in the group with bFGF at four weeks. This difference was equalized at eight and 24 weeks (Figure

5). For fibronectin mRNA expression there was only a significant increase at 24 weeks in the group

without bFGF (P= 0.0104) (Figure 5).


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Figure 5–Bar graphs for collagen-I, collagen-III, and fibronectin mRNA expression, comparing groups with and without bFGF at four, eight and 24 weeks.

No difference between groups with and without bFGF for protein amount for collagen-I, collagen-III

and fibronectin was seen at any time.

For total collagen amount in relation to cross-sectional area no significant difference between the

mesh with and without bFGF was seen. But over time, there was a significant increase in the collagen

amount (P=0.0000) (Figure 6).


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Figure 6–Total collagen amount in relation to

cross-sectional area.

Histologically, the Weigert’s elastin stain showed no elastin fibers in the newly formed tissue at four

and eight weeks. At 24 weeks there were areas of elastin in eight of 18 samples independent of bFGF

status but five of the eight samples were from rats with bFGF (Figure 7).

Maisson trichrome staining showed that the four weeks samples with bFGF had a clear blue area of

collagen at the edge of the sections (Figure 8A). The mesh was seen as an area of light pink with

collagen ingrowth at the edge (Figure 8a). In the four weeks samples without bFGF no clear area of

mesh was seen, instead small stripes of mesh surrounded by cells were seen. Figure 8B shows

ingrowth of collagen throughout almost the entire depth of the mesh. Equally, all the four weeks

samples presented giant cells with multiple nuclei (Figure 8b). At eight weeks, there was no clear

difference between the sections with and without bFGF (Figure 8 C and D), but clear fibers of collagen

and multiple giant cells (Figure 8 c and d). After 24 weeks still no difference between with and without

Figure 7 – Representative cross section of 24 samples with and without bFGF. Weigert’s elastin stain - Magnification x40 with scale bar indicating 100 µm. Blue-black fibers in the left of the picture are elastin fibers.


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bFGF was seen (Figure 8 E and F), instead there was a decrease in the number of giant cells and an

increase in the amount of collagen fibers compared to eight weeks samples (Figure 8 e and f).

Figure 8 – Representative cross section of four (A and B), eight (C and D) and 24 weeks (E and F) samples with and without bFGF. Masson’s Trichrome staining; A, B, C, D, E, and F are magnification x10 with scale bar indicating 200 µm. a, b, c, d, e, and f are magnification x40 with scale bar indicating 100 µm.


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The results of 24 weeks samples were categorized according to hernia size no matter if they were with

or without bFGF (Table 2). There was a significant increase of tensile strength and stiffness with

increasing hernia size, and for fibronectin mRNA expression a decrease with increasing hernia size was

seen (Figure 9). For the remaning variables no difference was oberserved (Table 3).

Category Hernia size

Group 1 None Group 2 Up to 3x3 cm Group 3 Over 3x3 cm

Table 2 – Categorization of hernia size.

P-value

Tensile strength [MPa] 0.0118 Strain at tensile strength 0.0867 Stiffness [MPa] 0.0214

Energy absorption [mJ/mm3] 0.1730

Total collagen amount [mg/mm2] 0.4078 mRNA – Collagen-I 0.2667 mRNA – Collagen-III 0.0902 mRNA - Fibronectin 0.0126 Protein – Collagen-I 0.6722 Protein – Collagen-III 0.1548

Table 3 – P-values for correlation between tensile strength, strain, stiffness, energy absorption, mRNA expression, protein amount, and total collagen amount in relation to hernia size.

Figure 9– Tensile strength, stiffness, and fibronectin mRNA expression in relation to hernia size. Continued. .


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Figure 9 continued – Tensile strength, stiffness, and fibronectin mRNA expression in relation to hernia size.

Discussion

The present study investigated a degradable PCL/PEO mesh from a mechanical point of view but also

regarding collagen and elastin formation in the newly formed tissue. The idea was to test a new mesh

treatment for POP in an animal model with the future aspect to be able to treat POP in women. The

optimal outcome of an in vivo mesh study would be strong, elastic neotissue with growth of collagen

suitable for treatment of POP. By adding bFGF to the mesh, an in vitro study showed enhanced

fibroblast proliferation with a promising rate of formation and sufficient supportive mechanical

properties of the new connective tissue [17]. The effect of the mesh in the present study was explored

by mechanical testing, histology, total collagen amount and analysis of mRNA expression and protein

amount of collagen-I, collagen-III and fibronectin. These components were considered the most

important in strong connective tissue. Despite the hypothesis based on promising in vitro studies, we

did not succeed to show significant effect of adding bFGF to the mesh in our in vivo study. But the

mesh itself showed an effect on tissue formation.

Experimental strengths and weaknesses

This study is a rethinking of POP treatment with a three dimensional repair of the pelvic floor with

formation of new connective tissue. Hereby, long-term complications from foreign bodies as seen with

non-degradable meshes were reduced. Another strength was the long follow-up time with results at


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eight and 24 weeks preceded by a four weeks pilot study. The pilot study showed no indications for

changing the design of the animal model. Without the long follow-up time we might not have seen the

effect of bFGF wear off.

The animal model itself had both strength and weakness. The mesh was tested as a patch over a full

wall muscle defect on the abdomen of the rat which gave the opportunity to explant only newly

formed tissue for analysis. The weakness was that the mesh was implanted on the abdomen of a

rodent walking on four legs. In the “real world” a mesh would be used for women walking in an upright

position with a heavy load on mesh and pelvic floor. Furthermore, a mesh on the abdomen was

surrounded by muscle instead of connective tissue, which can influence the results negatively.

The small number of animals in each study group was a weakness, that could have affected and

underestimated the results. A larger number of rats in each group might have shown more significant

results than we did. Nevertheless, the total number of rats was large compared to similar studies

[21,22]. In relation to the use of bFGF, the release of approximately 75% of the bFGF in 9 days as seen

in in vitro studies could be a problem not knowing when the last 25% was released in our in vivo study

[17]. A steady state release over a longer period of time would be preferable. However, the release

profile in vitro was hard to mimic in the in vivo environment.

The usage of bFGF

Despite in vitro studies where bFGF enhanced fibroblast cell proliferation, our in vivo study did not

show the same effect, at least judged from accumulation of cells. The only effect of bFGF was seen

after four weeks in collagen-I and collagen-III mRNA expression, tensile strength, strain, stiffness, and

energy absorption.

The detectable effect on mRNA level but not on protein level could be explained by the short time of

bFGF release with a peak release of 10.6% of the bFGF in the first hour and 52.9% during the following

day one to six [17]. In addition, there was a histologically difference between the two four weeks

groups. It seemed as if bFGF inhibited cell accumulation and collagen deposition within the mesh. This

effect was difficult to explain because in vitro results suggested that the mesh was non toxic and that

bFGF increased fibroblast cell proliferation [17].

Heybeli et al used an identical animal model and tested the effect of bFGF loaded polyprolene mesh

compared to polyprolene mesh without bFGF. After four weeks they showed no difference between


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the groups. After eight weeks there was an increase in tensile strength in the group with bFGF but also

increased vascularization, a higher amount of fibroblasts, and a greater amount of collagen fibers in

the bFGF group than the group without bFGF [22].

The difference between the study by Heibeli et al. and the present study could be due to different

dosages of bFGF and a difference in the release profile. We used 0.34851 µg bFGF per implanted mesh

and Heybeli et al. used 4-12 times more in the dosage of bFGF. [22] Furthermore, 75% of the bFGF

dosage used in this study was released in 9 days in vitro [17]. From our in vitro study, we know that

our mesh had a burst release of bFGF, and the release profile in the study by Heybeli et al. is not

described.

In our study, the histology showed a clear difference at four weeks, where bFGF seemed to inhibit cell

popularization and collagen deposition within the mesh. The inhabitation was reflected on the

mechanical results where the four weeks samples with bFGF resembled the results of the mesh.

Thereby, the four weeks samples with bFGF were significant stronger and stiffer than the samples

without bFGF. The higher stiffness results from less scaffold degradation in this case and thus, the

presence of bFGF seamed to alter not only collagen deposition but also scaffold degradation by giant

cells.

Roman et al. investigated the use of adipose-derived stem cells (ADSC) and oral fibroblasts on poly-L-

lactic acid (PLA) scaffolds for tissue engineered POP repair material.[23]. They found that ADSC

resulted in more total collagen than oral fibroblasts. Under restrained conditions, the two cell types

enhanced the same collagen amount and an increased elastin production indicating that both cell

sources were proper candidates for tissue engineering. We tested mesh with bFGF. Comparing our

minimal effect on collagen formation and the promising results of Roman et al., stem cells in general

seem to be a better choice for the future research in the field.

Rate of new tissue formation and mesh degradation

The mesh alone was the stiffest material of the examined samples, followed by the four weeks

samples with bFGF, 24 weeks samples, muscle from the implantation site, and last 8 and 4 weeks

samples without bFGF. This might reflect that the mesh was too stiff compared to the muscle. The

mesh was also stronger than the muscle and any of the test groups. From comparing work curves in

figure 3 it was seen that in the low strain domain ( < 0.1 strain) a toe region existed in the degraded


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meshes and muscle tissue, which was not present in the initial mesh. The consequence was, that cells

situated within muscle tissue or degraded meshes were thus subjected to higher strains at the same

load, compared to cells in the initial mesh. It is known that mechano transduction influences cell

maturation and growth [24], and that dynamic stretching alter adhesion, proliferation, locomotion,

morphology, and the synthetic profile of cells [25]. Additionally, the work curve of the mesh did not

show a vertex at approximately 0.5 strain as all other samples did. The later was not expected to

influence cell dynamics as in vivo loads never implied such high strain levels. But these factors could

indicate that the mesh had the wrong mechanical properties in comparison with the target tissue and

therefore led to herniation.

Finally, the work curve shows that the newly formed tissue was the most fragile at eight weeks, in

consistence with the start of herniation at nine weeks. This could indicate that the degradation of the

mesh was too fast in comparison with the formation of the new connective tissue leading to a period

where herniation could appear. At 24 weeks, the strength of the tissue increased to the level of the

four weeks samples without bFGF indicating that a formation of new tissue could have strengthened

the area of the mesh.

Herniation

In the animal study, it was possible to investigate the factors important for developing or preventing

hernia when dealing with newly formed connective tissue. The categorization of the degree of

herniation into three groups made it possible to test the impact of the measured variables on hernia

formation. The results showed that only tensile strength and stiffness were increased with increasing

hernia size. Former studies investigating samples from the pelvic floor from women with and without

POP showed that women with POP have weaker and stiffer tissue than women without POP [26,27].

These results were supported by the findings of the present study were we expected that dynamic

loading at low strain levels would lead to increased collagen deposition. Meaning, development of a

mesh which enhances formation of new strong and elastic tissue seems to be a theoretical possible

treatment of POP. The design of a toe-region in the stress-strain curve of the mesh together with an

improved and sustained release of growth factor is a strategy to be tested.


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Conclusion

A tissue-engineered treatment for POP with a degradable PCL/PEO mesh could be possible judged

from the results of the present animal model. The degradable mesh promoted tissue formation with

increasing collagen amount and biomechanical mesh properties being temporal adjusted toward the

behavior of muscle. However, the addition of bFGF (60 ng bFGF per mg mesh) did not add any long-

term positive effects. The occurrence of hernias accompanied with an increase in total collagen,

strength and stiffness over time, leads to an assumption of a mismatch between the degradation time

of the mesh and the formation of the new tissue. The results suggest that the degradation of the mesh

is too fast compared to the formation of new tissue and thereby leading to herniation. Regarding the

hernia analysis, the mesh should not be too stiff since such an environment seems to limit collagen

formation and consequently increase the strength and stiffness of the new tissue. The outcome can be

improved through mesh redesign incorporating tailored mechanical properties and bFGF release to

match mesh degradation time with a tissue formation that will prevent herniation.


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Documents

Electrospun biomimetic microfibers for pelvic floor repair ...€¦ · options for recurrence of POP. Therefore, new principles for effective repair or replacement of deficient connective