the influence of tissue geometry on compaction and ... · pdf filethe influence of tissue...
TRANSCRIPT
1
The influence of tissue geometry on compaction and contractile properties of ovine myofibroblasts
BMTE 09.21
Chiara Tamiello
January 2009 – May 2009
Coaches:
Anita Driessen-Mol
Marijke Van Vlimmeren
2
ABSTRACT
Background: Currently, tissue engineered heart valves are cultured with the leaflets attached to each other as
this has shown to provide good tissue formation because of the generated stress within the scaffold. However,
when detaching the leaflets and exposing them to physiologic systemic flow conditions the stress generated
within the tissue is released resulting in shrinkage of the tissue, referred as tissue compaction, which, on its turn,
results in loss of functionality of the heart valve. It is hypothesized that the geometry of the tissue itself
influences the degree of stress generated in the tissue and, thereby, the resulting compaction after release of the
constraints.
In this study effect of the geometry of the scaffold on tissue compaction, cell orientation and contractile
properties of the cells are investigated.
Methods: Strips, rectangular, triangular and circular scaffolds of rapid-degrading polyglycolic acid (PGA)
meshes coated with poly-4-hydroxybutyrate were seeded with saphenous vein derived myofibroblasts. These
tissues were cultured under static constraint for four weeks (n = 4 constrained constructs and n = 1 unconstrained
construct per group). Subsequently, constraints were released partially for strips, rectangular and triangular
constructs and completely for the circular constructs. Compaction was assessed from photographs of the released
constructs obtained over 48 hours. Finally, cell orientation and contractile properties of the cells were
qualitatively investigated by immunostaining of tissues sections along different directions while tissue formation
was analyzed by means of histology.
Results: Quantification of the compaction during the culture demonstrated that the rectangular constructs
compacted more than the other geometries. Culturing the constructs unconstrained resulted in clumped tissues.
After release of the constraints, all geometries continued to compact over 48 hours. All geometries, except the
circular construct, compacted in an unpredictable way. In contrast, the circular construct compacted in the
direction of its radius for the first 24 hours, starting to curl after that time. Immunostaining demonstrated that in
the circular scaffold cells are randomly oriented before and after the release of the constraints. Results did not
allow for a qualitative assessment of the contractile properties of the cells. Histology showed a sufficient tissue
formation in the constructs, except in the rectangular construct.
Conclusion: The results indicate that compaction is a continuous process. The circular tissues compact in a more
predictable way than the other geometries. This is most likely due to being either constrained in all directions, or
only partially. This study may provide insights towards the design of a heart valve in which geometry is
adjusted to the expected compaction in order to compensate for the loss in the functionality that usually occurs.
3
TABLE OF CONTENTS
INTRODUCTION ................................................................................................................................................................... 4
2 MATERIALS AND METHODS ......................................................................................................................................... 8
2.1 CULTURING SHEEP VENA SAPHENA CELLS ......................................................................................................... 8 2.2 PREPARATION OF THE CONSTRUCTS ..................................................................................................................... 8 2.3 SEEDING ...................................................................................................................................................................... 10 2.4 EXPERIMENTAL DESIGN ......................................................................................................................................... 11 2.5 RELEASE OF CONSTRAINTS OF THE CONSTRUCTS ........................................................................................... 11 2.6. ASSESSMENT OF DEGREE OF COMPACTION ..................................................................................................... 12
2.6.1 IMAGE PROCESSING .......................................................................................................................................... 12 2.7 TISSUE PROCESSOR AND EMBEDDING ................................................................................................................ 13 2.8 IMMUNOSTAINING AND HYSTOLOGY ................................................................................................................. 14
3. RESULTS ........................................................................................................................................................................... 15
3.1 COMPACTION OF THE CONSTRUCTS .................................................................................................................... 15 3.1.1 COMPACTION OF THE CONSTRAINED CONSTRUCTS DURING THE CULTURING PERIOD.................... 15 3.1.2 COMPACTION OF THE UNCONSTRAINED CONSTRUCTS DURING THE CULTURING PERIOD .............. 16 3.1.3 QUALITATIVE EVALUATION OF COMPACTION DURING 48 HOURS AFTER RELEASE OF CONSTRAINTS
........................................................................................................................................................................................ 17 3.2 CELL ORIENTATION AND CONTRACTILE PROPERTIES ................................................................................... 19
3.2.1 Strip ........................................................................................................................................................................ 19 3.2.2 Rectangle................................................................................................................................................................ 19 3.2.3 Triangle .................................................................................................................................................................. 20 3.2.4 Circle ..................................................................................................................................................................... 20
3.3 TISSUE FORMATION ................................................................................................................................................. 22
4. DISCUSSION ..................................................................................................................................................................... 24
5. CONCLUSION .................................................................................................................................................................. 26
REFERENCES ...................................................................................................................................................................... 28
APPENDIX 1: CULTURING SHEEP VENA SAPHENA CELLS ................................................................................... 29
APPENDIX 2: PREPARATION OF SCAFFOLD FOR TISSUE ENGINEERING CONSTRUCTS ........................... 32
APPENDIX 3: AREA MEASURAMENT BY MEANS OF IMAGEJ SOFTWARE ...................................................... 37
APPENDIX 4: SECTIONING PARAFFIN BLOCKS CONTAINING SCAFFOLD MATERIAL ............................... 39
APPENDIX 5: IMMUNO-FLUORESCENT STAINING VIMENTIN, DESMIN AND Α-SMA ................................... 41
APPENDIX 6: HEMATOXYLIN & EOSIN (H&E) STAINING ..................................................................................... 43
APPENDIX 7: RESULTS – COMPACTION DURING CULTURE ................................................................................ 46
4
INTRODUCTION
Surgical replacement of diseased heart valves with biomechanical and bioprosthetic valve substitutes is
the most common treatment for end-stage valvular heart diseases, with approximately 85,000 valve
replacements performed each year in the US and 275,000 worldwide [1]. Replacement of diseased
valves reduces the morbidity and mortality associated with valvular disease, but come at the expense of
risking complications unique to the implanted prosthetic device. These complications include primary
valve failure (valve durability), prosthetic valve endocarditis (PV), prosthetic valve thrombosis (PVT),
thromboembolism, anticoagulant-related hemorrhage, and mechanical hemolytic anemia [2]. These
problems have been attributed to the design as well as the durability and biological incompatibility of
materials used in mechanical valves.
Recent advances in tissue engineering have demonstrated the potential of in the development of
customized living and autologous replacement body parts. The advantages of a tissue-engineered
implant include the ability to regenerate over prosthetic and mechanical implants and grow. Further, the
risks related to immunological complication and pathogen transfer are diminished. [3]. In the case of
tissue engineered heart valves, the feasibility of the autologous tissue engineering concept for heart
valve application has been investigated, demonstrated and improved for the last decade[4]. The
approach involves the seeding of autologous cells onto a porous synthetic polymer or biological
material configured in the shape of the heart valve. The cells are subsequently stimulated to develop a
complete autologous, living heart valve replacement [2].
In the rapidly growing field of tissue engineering, the functional properties of tissue substitutes are
recognized as being of utmost importance. The optimal functionality of a tissue is strictly correlated to
its appropriate histological organization. It is known indeed that abnormal tissue structures resulting
from various pathologies are responsible for functional defects [5].
In previous studies, good tissue formation in tissue engineered heart valve leaflets was provided when
culturing them constrained and attached to each other. Due to contractile properties of the seeded
myofibroblasts, contractile forces develop within the tissue, which has been demonstrated to be
beneficial for the tissue architecture and functionality. However, before implantation the leaflets have
to be separated and due to the stress release within the tissue coupled to the removal of the constraints,
5
the leaflets compacted resulting in leakage of the valve, thus in its loss of functionality [6].This is
clarified in Fig. 1.
Fig. 1. The effects of stress generation. Schematic image representing the positive (left) and negative (right) effects of stress
generation within the tissue in a tissue engineered heart valve cultured with the three leaflets attached to each other: tissue
architecture is optimized while the release of the stress after separation of the leaflets results in compaction and, therefore,
in leakage of the heart valve.
To overcome the leaking of tissue engineered valves cultured with the leaflets attached to each other, it
is relevant to understand the different aspects that influence the compaction of the leaflets. It is
hypothesized that the geometry of the tissue itself influences the degree of stress generation and
thereby, the resulting compaction after release of the constraints [5]. The degree of stress generated in
the tissue cannot be measure directly, while analysis of the compaction and of the contractile properties
of the cells can provide an indirect measure of it. When the influence of geometry on the compaction is
known, the design of a heart valve could perhaps be adjusted to take this compaction into account.
6
The aim of this study is to investigate the effects of the geometry of the scaffold on the degree of
compaction of the tissue after release of the constraints applied during culture. The correlation of the
contractile cell properties and cell orientation within the tissue to the compaction of the tissue are also
studied together with an evaluation of the tissue formation in the different constructs.
For this purpose, four different scaffold geometries were studied: strip, rectangle (shorter and wider
scaffolds compared to strips), triangle and circle.
The scaffolds cut off of rapid-degrading polyglycolic acid (PGA) meshes were coated with poly-4-
hydroxybutyrate and seeded with saphenous vein derived myofibroblasts. These tissues were cultured
under static constraint for four weeks (n = 4 constrained constructs and n = 1 unconstrained construct
per group). Subsequently, constraints were released partially for strips, rectangular and triangular
tissues and completely the circular tissues and the compaction was assessed from photographs obtained
over 48 hours. Further, orientation and contractile properties of the cells were qualitatively investigated
by means of immunostaining of tissue sections along different directions: staining of the cytoskeleton
with phalloidin was used to visualize cell orientation while α-Smooth Muscle Actin (α-SMA)
expression was used to assess the contractility of the cells [7]. Finally, the density of the tissue in the
different scaffolds was assessed by means of the Hematoxylin and Eosin (H&E) staining.
Differences in the compaction and in the cell orientation were expected especially between
strips/rectangles and circles/triangles because of the differences in the stress generation [5]. In the
circular scaffold, compactions after release of the constraints was expected in all directions, while in
the strips and rectangular constructs shrinkage was expected mostly along the longitudinal direction.
Further, in the case of the triangular constructs compaction was expected along the base and height of
the scaffold, if the base was released from the constraining ring.
Cell orientation was expected random in the constrained and released circular scaffold. In the
rectangles and in the strips constrained to the ring, cells were expected to align mostly along the
longitudinal direction, namely the direction along which stress developed (axis of tension).On the other
hand, in the released sample a random orientation was expected because of stress release along several
directions. The triangular scaffold was expected to have a random orientation when being constrained
as well as after release of the constraints.
7
α-SMA expression was supposed to be higher in the constrained samples than in the released ones.
During constrained culture, myofibroblasts tend to develop contractile forces and pull on the
constraining frame, while contractility is supposed to decrease when the stress is released because of
releasing of the constraints.
8
2 MATERIALS AND METHODS
2.1 CULTURING SHEEP VENA SAPHENA CELLS
Ovine vena saphena cells (isolation I, passage 3) were cultured using regular cell culture methods
[Appendix 1]. The medium used for cell culture consisted of DMEM Adanced (Gibco/Invitrogen Corp)
supplemented with 10% Lamb serum, 1% GlutaMAX (Gibco, UK), 1% PenStrep. Cell cultures were
maintained in a humidified incubator at 37° C and 5% CO2 and medium was replaced every 3 days.
Cells were cultured until passage 6.
2.2 PREPARATION OF THE CONSTRUCTS
For each geometry, 5 scaffolds (Table 1), were cut out of rapid-degrading nonwoven polyglycolic acid
meshes (PGA; thickness 1.0 mm, specific gravity 70 mg/cm3, lot# MD00645 and MD00283) and
coated with a thin layer of poly-4-hydroxybutyrate (P4HB (1.75 % w/v)) dissolved in tetrahydrofuran
(THF; Fluka; Germany).[Appendix 2]
Geometry Constrained Not constrained
strip 25* 5 mm 18*5 mm
rectangle 22*11 mm 18*11 mm
triangle b = 20 mm, h = 20 mm b = 20 mm, h = 20 mm
circle r = 12.5 mm r = 9 mm
Table 1. Scaffold measures. b, base of the triangle; h, height of the triangle; r, radius of the circle
9
Note that the unconstrained constructs are smaller than the constrained ones because the seeding area
only is taken into consideration. For the triangle the same measures were kept in order to maintain the
geometry of the construct; however, the actual area seeded was the same.
The constructs were glued with polyurethane (PU) glue (20% w/v ; DSM, Netherlands) to a stainless
steel ring (inner diameter 16 mm, outer 25mm), (Fig. 2a). The strips and the rectangular constructs
were glued at both ends, the triangles at the three vertices and the circles along their perimeter. A
custom-built set up was required for the circular scaffold in order position the tissue near the surface of
the medium to provide good oxygen perfusion (Fig. 2b). This set up consisted of two white tips
(Greiner Bio, Austria) where glued onto each other and attached to the ring at opposite positions with
PU. The white tips are free from DNase, RNase and human DNA, free from endotoxin (pyrogen) and
are non-cytoxic.
(a)
(b)
Fig. 2. Set up used in the experiment. PGA-Scaffold glued to a stainless steel ring (a): this set up was used for strips,
rectangular and triangular constructs. Set-up for the circular scaffold (b): the white tips were glued onto each other and
attached to the ring with PU. The circular scaffold was already glued to the stainless steel ring.
The samples were dried over night in the vacuum oven to allow for vaporization of the leftover THF.
The white tips in the circular set-up enlarged in the contact points with the ring. The cause is not
known, but this was probably caused by a reaction between PU, plastic and stainless steel.
Thereafter, the rings with strips, rectangular and triangular scaffolds were placed in 6-wells plates
while the circular constructs were put in medium tubes as a different amount of medium was required
10
because a density of 1.5x106 cells per cm
3 was used to seed the scaffolds and 0.25x10
6 cells are usually
cultured in 1 ml of medium. After sterilization with 70% ethanol and washing with PBS, the wells were
filled with TE medium and placed in the incubator overnight.
2.3 SEEDING
The seeding with ovine vena saphena myofibroblasts was performed the day after sterilization of the
scaffold constructs, as described previously [Appendix 2]. Briefly, the cells (passage 6) were
enzymatically detached with trypsin and after a centrifuging step they were counted and resuspended in
a thrombin solution. Subsequently, fibrinogen was added and the cells were evenly distributed over the
scaffolds at a density of 1.5x106 cells per cm
3.
The seeding area (Table 2) was equal in the constrained and not constrained constructs. Therefore the
number of cell used was also the same in each group. The constructs were placed in the incubator for
30 minutes to let the fibrin gel further polymerize. After that, Tissue Engineering medium (TE
medium) made with DMEM Advanced and supplemented with additional 1% lamb serum, 1%
Pen/Strep, 1% Glutamax and 130 mg L-ascorbic acid 2-phosphate (0.26 mg/mL, Sigma, Germany) was
added in order to promote extracellular matrix production. The amounts of TE medium used are
summarized in Table 2.The constructs were cultured for four weeks and the medium was changed
every 3 to 4 days.
Table 2. Seeding area, amount of cells seeded and amount of medium for each geometry.
geometry Seeding area
(cm2)
Cells (x106) Medium (mL)
strip 0.9 1.35 5
rectangle 2.0 3.0 12
triangle 1.8 2.7 10
circle 2.5 3.8 15
11
2.4 EXPERIMENTAL DESIGN
Four experimental groups were studied. Each group (n = 5 samples) was representative for a different
geometry: strips, rectangular, triangular and circular geometry. For each group, n = 4 samples were
cultured constrained to a stainless steel ring (constrained constructs, Table 3), while one of them was
cultured not constrained (unconstrained constructs, Table 3). In order to study the compaction of
different geometries, three out of four constrained constructs were released partially or completely from
the constraining ring (released constructs, Table 3). -SMA expression and cell orientation were
analyzed in slices from a released construct and a constrained construct used as a control.
Table 3. Overview of the experimental design.
2.5 RELEASE OF CONSTRAINTS OF THE CONSTRUCTS
After four weeks, three out of four constrained constructs were released from the ring (released
constructs). Following the inner diameter of the rings, the strips and the rectangles were cut from one
side, by means of a scalpel. For the triangular constructs 2 vertices were cut loose while the circles
geometries Tot.
Unconstrained
constructs
Constrained
constructs
Released
constructs
Cell orientation (slices
from)
α-SMA expression(
slices from)
circle
n = 5 n =1 n = 4 completely cut
loose
n=3
1 released construct
+
1 constrained construct
(control)
1 released construct +
1 constrained construct
(control)
rectangle n = 5 n=1 n = 4 one side cut
loose
n=3
1 released construct
+
1 constrained construct
(control)
1 released construct +
1 constrained construct
(control)
triangle n = 5 n=1 n = 4 2 vertices cut
loose
n=3
1 released construct
+
1 constrained construct
(control)
1 released construct +
1 constrained construct
(control)
strip n = 5 n=1 n = 4 one side cut
loose
n=3
1 released construct
+
1 constrained construct
(control)
1 released construct +
1 constrained construct
(control)
12
were completely cut loose from the ring. One construct for each group was left constrained to the ring
in order to be used as a control for further analysis. After assessing the degree of compaction over 48
hours (2.6) in the released constructs, the samples were stored overnight at 4°C in 3.7% formalin. After
24 hours they were put in PBS and stored in the 4°C fridge until use for embedding and sectioning as
described in 2.7.
2.6. ASSESSMENT OF DEGREE OF COMPACTION
During culture, due to the contractile properties of the seeded myofibroblasts, contractile forces
develop within the tissue resulting in reorganization of the tissue and compaction of the construct. In
order to quantify the degree of compaction during the culturing period, at the end of every culture
week, digital photographs of the TE constructs were taken with a digital camera (Canon, Japan). The
amount of compaction was measured with the help of ImageJ software and results were represented as
the relative compaction of the constrained constructs by comparing the surface area at the end culturing
week (t = 4 week) with the seeding area (t = 0).
After releasing the constraints, photographs of the TE constructs were taken at each time point (i.e. t=0,
25, 45 min. and t =1, 1.5, 2, 2.5, 3, 6, 8, 24, 48 hours from release of the constraints) for macroscopic
appearance. The camera was placed on a stative to keep magnification and position the same in all
photographs. The background used was a black paper to avoid the effects of shadows in the
photographs. A scale (1 mm) for calibration was also present in the image.
2.6.1 IMAGE PROCESSING
ImageJ software was used in order to measure the surface area of the constrained constructs at the end
of culturing (t = 4 weeks).The inner perimeter of the ring was selected and the image was then
transformed in 8-bit level. Then, a threshold operation was performed and the Region of Interest (ROI)
was assigned to a red mask. After entry of the Known Distance, which was “1”, and the Unit of Length,
which was “mm”, the area was calculated by the software and the results displayed the area in mm2.
13
Finally, the results were represented as the relative compaction of the TE construct by comparing the
calculated surface area relative at week 4 to the seeding area at t = 0 (Table3, seeding area).The
procedure is described in Appendix 3.
2.7 TISSUE PROCESSOR AND EMBEDDING
After fixation, one released construct representative for each geometry and the relative constrained
construct used as a control were completely detached from the rings and put in the tissue processor
(Adamas) overnight. Tissue processing dehydratated the constructs and then removed the dehydrant
with a substance that was miscible with the embedding medium (paraffin). Thus, the constructs were
embedded in paraffin. The analysis of cell orientation needed to be studied along at least two different
directions. For this reason the samples were cut before embedding following the plan that is described
in Fig.3.
(a)The rectangular scaffolds and the strips were cut along
directions y and x. Thus along NO and MO segments. The
embedding was carried out in order to obtain sections
parallel y and x directions.
(b)The triangular scaffold was analyzed along two
directions: y direction and z direction. Thus, the scaffold was
cut along AO and the BO segments and the embedding was
carried out in order to obtain sections parallel to y and z
directions.
(c)The circular scaffold was cut along AO and BC segments.
Thus, the two quarters of the circle were embedded in order
to obtain a section parallel to y and x directions while the
remnant half of the circle was embedded so that the scaffolds
could be sectioned through the plane parallel to its surface.
Thus, three different sections were available for this
construct.
Fig.3. Directions considered for the embedding and the sectioning. Strips and rectangular constructs (a) were cut and
embedded along y and x directions, triangular constructs (b) were cut and embedded along y and z directions and circular
constructs (c) were cut and embedded along y and x directions and on the plane parallel to the surface.
14
2.8 IMMUNOSTAINING AND HYSTOLOGY
10µm-thick sections were cut from the embedded samples and air-dried (Appendix 4). In order to
visualize the cytoskeleton for cell orientation and the expression of α-SMA, sections were
deparaffinized, blocked with PBS containing 1% bovine serum albumin (BSA) and incubated with
rhodamine-conjugated phalloidin (1:200, Sigma), with a mouse monoclonal antibody (DAKO, Canada)
directed against α-SMA (1:500), and with a secondary antibody coupled to Alexa 488(goat anti-mouse,
1:300) as described in Appendix 5.As a control, sections were additionally stained, the primary
antibody was omitted. Nuclei were counterstained with DAPI. The sections were viewed with a
fluorescence microscope (Zeiss, Germany); particularly, the intensity of the green signal (α-SMA
expression) was used as measure of α-SMA expression. Further, sections of the control constructs
were histologically examined by Hematoxylin and Eosin stain (H&E stain) in order to have a visible
look at the nucleus of the cells and their state of activity (tissue formation) (Appendix 6). The sections
were viewed with an optical microscope (Zeiss, Germany).
15
3. RESULTS
3.1 COMPACTION OF THE CONSTRUCTS
3.1.1 COMPACTION OF THE CONSTRAINED CONSTRUCTS DURING THE
CULTURING PERIOD
By means of pictures taken during the 4 culturing weeks it was possible to assess the compaction of the
different geometry constructs. Obviously, circular construct could not compact because they were
constrained, thus they were not taken into account for this analysis.
Compaction during the culturing period was investigated in order to get some insight into changes in
compaction over time and to elucidate differences between the groups. It was observed that compaction
for two out of three rectangles, started at the end of week 2. One of the rectangles was not properly
fixated, so one end detached from the constraining ring during the culture. For strips and triangles,
compaction started during the third week of culturing. Compaction was not observed in one of the
triangles, which therefore was excluded for further analysis. Overall, the tendency towards compaction
was visible for all the geometries over the four weeks of culturing, starting after week 2.
Quantification of compaction performed with the help of ImageJ software is provided in Table 4 and in
Appendix 5. It was pointed out that the strips and rectangular constructs compacted to a greater extent
(37 % and 41%), compared to the triangular constructs (7%). Overall, rectangles compacted more than
the other constructs, with the lowest SD as can be seen in Table 4.
geometry Average relative compaction SD
Strip 37 % 5.0
Rectangle 41% 0.5
Triangle 7 % 7.5
Table4. Compaction during culturing
16
3.1.2 COMPACTION OF THE UNCONSTRAINED CONSTRUCTS DURING THE
CULTURING PERIOD
For the unconstrained constructs, it was macroscopically visible that at the end of week 2 strips and
rectangular unconstrained constructs (n = 1) started to bend while the triangle and the circles had not
changed their shape. One week later, the circular tissue bended and tended to form a cylindrical
construct while for the triangle, only one vertex started to curl. For the strip and the rectangle
compaction continued until the end of the fourth week of culturing when they reduced to a clump; the
triangle was also completely curled into a clump and the circle formed a structure similar to a tube, as
can be seen in Fig.3. Results are summarized in Table 5.
Strip rectangle triangle Circle
Week1 NO NO NO NO
Week2 + + NO NO
Week3 ++ ++ + +
Week4 +++ +++ +++ ++
Table 5. Compaction of the unconstrained constructs during the culturing period. +, starting of the compaction; ++, the
degree of compaction increases, +++, the construct reduces to a clump.
Strip (a) Rectangle (b) Triangle (c) Circle (d)
Fig.3. Unconstrained constructs at the end of the 4th
week of culturing. The strip (a), the rectangle (b) and the triangle (c)
compacted and reduced to a clump; the circle formed a structure similar to a tube (d).
17
3.1.3 QUALITATIVE EVALUATION OF COMPACTION DURING 48 HOURS AFTER RELEASE OF CONSTRAINTS
Compaction was investigated macroscopically from photographs of the released constructs during 48
hours after release of the constraints.
Strips
At t = 25 min. after sacrifice the strips started to curl and to compact, as can be seen in Fig. 4a. The
compaction along the axis of tension, as expected, was common to all the constructs and continued
over 48 hours, as depicted in Fig. 7b and Fig. 7c.
Rectangles
Compaction started soon after the release of the constraints, as can be seen in Fig. 5d (t = 25 min.) and
continued until the time point t = 48 hours (Fig. 4e -4f). The TE constructs compacted along the axis of
tension as expected and the longer edges curled towards the centre of the construct.
Triangles
Compaction was observed already at t = 25 min. (Fig. 4g).At time points t = 24 and t = 48 hours, two
out of 3 triangles completely bend on themselves while continuing to compact. An example is shown if
Fig. 4h-4i. The triangle that did not compacted during culturing did compact during the 48 hours along
the height and base directions, while maintaining its own shape.
Circles
All the circular constructs showed a similar behaviour during 48 hours: they started to compact in the
radial direction soon after release from the constraining ring (Fig. 4l, t = 25 min.) maintaining their
circular shape until t = 24h when the edges visibly started to curl (Fig. 4m). Shrinkage and curling
continued until t = 48h (Fig. 4n)
18
t=25min.(a) t =24 (b) t=48h (c)
t = 25min. (d) t=24 h (e) t = 48h (f)
t = 25min. (g) t = 24h (h) t = 48 h (i)
t =25min. (l) t = 24h (m) t = 48h (n)
Fig. 4. Photographs of the released construct an overview of the compaction during 48 hours after release of the
constraints. Strips (a, b, c): at t = 25 min. after sacrifice the strip starts to curl and to compact (a); comparison of (b)and
(c) points out that compaction continues until t = 48h. Rectangles (d, e, f): compaction starts soon after the release of the
constraints (d, t = 25min.).The shrinkages continues until the time point t = 48 hours (e, f).Triangles (g, h, i ) : compaction
is observed already at t = 25 min. (g).At t = 24h the triangle depicted in (h) bends on itself and continues to fold until t =
48h (i).Circles (l, m, n): the circle after release of the constraints, maintains its shape while compaction in the radial
direction, as depicted at t = 25min (l). At t= 24h (m) its edges start to curl. Shrinkage and curling are more pronounced at
time point t = 48h (n).
19
3.2 CELL ORIENTATION AND CONTRACTILE PROPERTIES
Sections were double immunostained for cell orientation analysis and with α-SMA as a measure for the
contractile properties of the cells. Controls confirmed the suitability of the stainings.
3.2.1 Strip
When studying in the x direction, both surface layers were aligned in the released construct (Fig. 5a),
while only one of them was aligned in the control (constrained construct). This could be due to the fact
that the cut along that direction was not orthogonal to the width of the sample. However, the alignment
was more pronounced (Fig. 5b). Along the y direction, the released construct showed a slight alignment
of cells along the surface layers (Fig. 5c), while in the control the cells were more randomly oriented
(Fig. 5d). When comparing the constrained construct along y (Fig. 5d) and x (Fig. 5b) directions, it was
visible that the cell nuclei were more rounded along the y direction while they were more stretched
along the x direction. The cells were indeed expected to align mostly along the axis of tension, namely
the x direction in the constrained construct, while in the released one any cellular alignment was not
predictable.
-SMA expression was observed but differences were not noticed between control and the released
construct, even if the same settings for exposure time were used.
3.2.2 Rectangle
Results about rectangle were not obtained because the control section along y direction detached from
the poly-L-lysine coated slides during deparaffinization. This could be due to the size of the section or
the fair tissue formation. The analysis along x direction did not provide any insight in the orientation
and in the α-SMA expression
20
3.2.3 Triangle
Cell alignment was visible in both samples along the y direction: in the released construct a thick layer
of aligned cells was present (Fig. 5f), while in the control the cells clearly had a different orientation
within some micrometers as can be seen in Fig. 5g.
Along the z direction, the cells had no organization in the released construct (Fig. 5h).In contrast, the
control showed a strong orientation of the surface layer (Fig. 5i).
A lot of scaffold (red autofluorescence) was still visible in the middle of the control (Fig. 5i); while
fewer scaffold was observed in the released construct (Fig. 5h).
Results did not allow for view of differences in the expression of α-SMA between the two constructs.
3.2.4 Circle
The comparison between the directions x and y pointed out that, in both directions, the cells are much
more aligned in the control while, in the released construct, they had a more random orientation, as can
be seen in Fig. 5m and Fig. 5n.The scaffold was really compacted in the middle of the released
construct (Fig. 5m), while in the constrained construct there was more empty space in the middle of the
construct (Fig. 5n). The slices cut in the plane parallel to the surface showed very little scaffold
remaining in the control and cells randomly oriented, as expected (Fig. 5q). The released construct had
much more scaffold and randomly oriented cells as well (Fig. 5r). The cells showed some alignment
along the edge but this result is not reliable because the circle curled after 24 hours making it
impossible to have a useful section containing all the surface of the scaffold.
α-SMA expression, based on the intensity of the green signal seen through the microscope, appeared to
be higher in the control
21
Released construct Constrained construct (control)
Dir.x
(a)
(b)
(e)
Dir.y
(c)
(d)
Dir. y
(f)
(g)
(l)
Dir. z
(h)
(i)
22
Dir.y
(m)
(n)
(q)
Dir. p
ara
llel to su
rface
(o)
(p)
Fig. 5. Cross sections of the released and constrained (control) constructs stained with phalloidin and immunolabeled with
antibodies directed against α-SMA (magnification x200).Strip: along the x direction, (a) cells are organized predominantly
in the surface layer in the released construct,(b) the same happens in the constrained construct (control )but the alignment
is more pronounced. Along the y direction cells are randomly oriented in both constructs (c, released construct) and (d,
constrained construct). (e), directions considered for sectioning the strips. Cross sections of triangular construct
demonstrates that, along the y direction, (f) cells organize themselves in a thick layer of aligned cells in the released
construct while, in the control, one of the surface layers shows that cells have a different orientation within some
micrometers from the surface (g). Along the z direction, (h) cells are not organized in the released construct, while a strong
alignment is visible on the surface layers of the constrained construct (i). (l), directions considered for sectioning the
triangular constructs. Circular construct: along the y direction, (m) cells of the released construct demonstrate that they
have a slight organization; (n) in the control, there is a much stronger alignment. (o) and (p) show sections cut in the plane
parallel to the surface: a lot of scaffold (red autofluorescence) and random cell orientation can be seen in the released
construct (o), very little scaffold and random cell orientation in the constrained construct (p). (q), directions considered for
sectioning the circular constructs.
3.3 TISSUE FORMATION
Histology (hematoxylin and eosin staining) confirmed that cells were successfully seeded in the
constructs but tissue formation resulted in some slight differences among the constructs. Strip tissue
appeared to be dense and almost uniform over the cross section (Fig. 6a).There was organization of the
23
tissue predominantly in outer layer. The rectangular construct showed holes that were probably just a
cutting artifact due to the fair density of the tissue. The black line is probably caused by an air bubble
(Fig. 5 b). In the triangular construct the cells were clearly visible and the tissue was almost uniform
across the section (Fig. 5c). Finally, cross section of the circular construct showed denser and more
organized tissue formation near the surface layers than in the middle (Fig. 5d).Overall, sufficient tissue
formation was shown, except for the rectangular construct.
Fig. 6. Histology of the constrained constructs by H&E stain (magnification x100). (a), strip tissue is organized near the
outer layer and the density is almost uniform throughout the section. (b), cross section of the rectangular construct
demonstrates fair tissue formation that resulted in cutting artifacts (holes in the tissue). (c), cross section of the triangular
construct shows dense tissue throughout the section. (d), tissue of the circular construct is not composed of dense tissue in
the middle of the construct, while more organized and dense tissue is shown on the surface layers.
a b
c d
24
4. DISCUSSION
The described analysis conducted on the degree of compaction of the constructs gave some insight into
the trend of compaction during culturing time for different geometries of constructs cultured either
constrained to a ring or not. Differences between the compaction of different geometries were also
studied after release of the constraints.
The overall results indicate that the shrinkage of the tissue during culturing started during the third
week of culturing. Taken together, the results suggest that the PGA scaffold starts to degrade after 2
weeks allowing the cells to pull on the extracellular matrix (ECM). During culturing, compaction
occurred mainly in the constrained rectangular constructs. This can be caused by the fair density of the
tissue formation that has been found by means of histology. The even distribution of the generated
stress in the triangular constructs could explain a less pronounced compaction in the constrained
triangular construct.
In this study it was also proved that it is not possible to obtain functional tissue when culturing
unconstrained constructs. Differences in compaction over time suggest also that the orientation of the
fibers in the PGA scaffold might influence the tendency to compaction of geometrically different
constructs. The constructs that were cut parallel to the PGA fibres started to curl one week earlier than
the ones that were not cut along the fiber direction. This could be further investigated in future studies.
One of the most interesting finding of this study is that compaction does not stop but continues until 48
hours from the release of the constraints for all the geometries taken into consideration. Further studies
are needed in order to establish whether compaction continues beyond that point.
It was also relevant to note that circular constructs started to bend after 24 hours from sacrifice had
passed. All the released circular constructs maintained their flattened shape while compacting in the
radial direction and compacted to the same extent during the first day after release of the constraints, as
expected. Therefore, there is a good possibility that the shrinkage of this kind of scaffold is predictable
and can be taken into account before culturing a heart valve with the three leaflets attached to each
other.
25
The results from immunofluorescence staining were restricted to strips, triangular and circular
constructs. It is clear that orientation of the cells can be predicted in the case of the circular scaffold:
the cells of the control appeared to be locally stretched along the directions taken into account in this
study, but the overall orientation is random because it is likely that the same results could be found in
all the every radial directions. In contrast, in the experimental sample, the cells are not stretched
because they released the stress generated within the tissue during culturing. Thus, they have a local
and overall random orientation. In the strip and in the triangular construct the unpredictable bending
and compacting, thus the remodelling of the tissue is influencing the orientation of the cells within the
tissue. However, based on this experiment it was important to note that the results for strips were in
agreement with the expectations only in the case of the constrained construct: alignment was more
pronounced in the longitudinal direction and cells were not randomly oriented like in the circle not only
in the constrained construct but also in the released sample. This is likely to be due to stress release
along the axis of tension, which is the direction along which contractile forces developed during
constrained culture. Further, no other major findings can be derived.
One of the limitations of this study is clear: the assessment of -SMA expression is not reliable.
Nevertheless, the exposure time used was the same for the experimental sample and the control, it was
impossible to determine from the fluorescent green signal clear differences in the expression of -SMA
expression. This limitation could be overcome using ELISA analysis, which allows a more reliable
quantification of -SMA expression. In this way, the trend of -SMA expression after release of the
constraints could be elucidated. Further, it could be relevant to achieve a quantitative measurement of
the compaction in the different scaffold in order to provide a more accurate indirect measure of the
stress generated in the tissue engineered heart valve leaflet.
Finally, if further studies will be conducted on the contractile properties of cells within tissue from
different scaffolds it should be taken into consideration that in this study it was not possible to achieve
a proper sectioning in the plane of the surface because the released samples tended to curl and bend.
Therefore, it was not possible to have the scaffold completely parallel to the blade in the microtome.
Further, when performing the cuts along different directions, the cuts must be performed perpendicular
to each other in order to have useful sections to stain for cell orientation.
26
5. CONCLUSION
The aim of this study was to investigate the effects of the geometry of the scaffold on the compaction
of the tissue and the correlation of the contractile cell properties and cell orientation within the tissue to
the compaction. This was done by culturing four different PGA-scaffold geometries seeded with ovine
myofibroblasts constrained to a stainless steel ring. Compaction of the tissue during the culturing
period and during 48 hours after partial or total release of the constraints was assessed by means of
photographs. The contractile properties of the cells and their orientation were investigated by means of
immunostaining, while histology (H&E stain) gave some insight about tissue formation in the
constructs.
In this study it was shown that geometry indeed influences the degree and the way of compaction of the
constructs. The circular construct appeared to have the most predictable way of compacting during 48
hours after detachment from the constraining ring. This might be connected to the random organization
of the cells in the tissue that appeared to be denser on the surface layers. Immunostaining pointed out
that the randomness in the orientation was maintained during compaction after release of the
constraints but the cells were much less stretched in the released construct due to stress release. The
behaviour of the circular construct suggests that culturing the scaffold completely attached to a frame
lead to a predictable compaction after detachment. It is likely that this kind of scaffolds are more
attractive for the optimization of heart valve cultured with the three leaflets attached to each other in
order to avoid leakage after their detachment [6].
A remarkable finding of this research is that compaction is continuing at least until 48 hours after
sacrifice of the constructs. More research is needed in order to quantify the amount of compaction over
a longer period in order to elucidate when it eventually stops.
-SMA expression is also an important parameter that should be objectively quantified by ELISA in
order to elucidate the correlation with the differences in the geometry and the stress generated within
the tissue.
In summary, this study describes the influences of the tissue geometry on the compaction of PGA
scaffolds seeded with ovine myofibroblasts. Results demonstrated that circular constructs do not curl
27
and compact in a predictable way during the first 24 after release of the constraints. This finding can
give some useful insights to help the design of a scaffold for a tissue engineered heart valve cultured
with the leaflets attached to each other in order to avoid any loss of functionality due to compaction of
the tissue.
28
REFERENCES [1] E. Rabkin and F.J. Schoen. Cardiovascular tissue engineering. Cardiovasc Pathol, 11:305-317, 2002
[2] Eric Kardon, MD, FACEP, Associate Staff, Division of Emergency Medicine, Athens Prosthetic
Heart Valves, Feb 26, 2007; http://emedicine.medscape.com/article/780702-overview
[3] Tissue engineered tri-leaflet heart valve-preliminary fabrication of biodegradable polymer
scaffolds. Teoh, S.H.; Lan, C.Y.; Ranawake, M.; Hutmacher, D.W.; Chew, Y.T.; Sim,
K.W.[Engineering in Medicine and Biology, 1999. 21st Annual Conf. and the 1999 Annual Fall
Meeting of the Biomedical Engineering Soc.] BMES/EMBS Conference, 1999. Proceedings of the
First Joint Volume 2, Issue, Oct 1999 Page(s): 748 vol.2
[4] Driessen - Mol, Functional Tissue Engineering of Human Heart Valve Leaflets, PhD. Thesis, 2005,
Eindhoven University of Technology
[5] Grenier Guillaume; Rémy-Zolghadri Murielle; Larouche Danielle; Gauvin Robert; Baker Kathleen;
Bergeron François; Dupuis Daniel; Langelier Eve; Rancourt Denis; Auger François A; Germain Lucie
Tissue reorganization in response to mechanical load increases functionality. Tissue engineering 2005;
11(1-2):90-100
[6] J. Kortsmit , N. J. B. Driessen, M. C. M. Rutten and F. P. T. Baaijens. Real Time, Non-Invasive
Assessment of Leaflet Deformation in Heart Valve Tissue Engineering. Annals of biomedical
engineering (Ann Biomed Eng); 2009-Mar; vol. 37 (issue 3): pp 532-41
[7] Guillaume Grenier, Murielle Rémy-Zolghadri, François Bergeron, Rina Guignard, Kathleen Baker,
Raymond Labbé, François A. Auger, Lucie Germain. Mechanical Loading Modulates the
Differentiation State of Vascular Smooth Muscle Cells. Tissue Engineering. November 2006, 12(11):
3159-3170.
[8] Visual Guide IV: Measuring cells with ImageJ, http://naranja.umh.es/~atg/tutorials/VGIV-
MeasuringCellsImageJ.pdf
29
APPENDIX 1: CULTURING SHEEP VENA SAPHENA CELLS
MEDIUM:
DMEM Advanced (500 ml)
10% Lamb Serum (50 ml)
1% Glutamax (5 ml)
1% Penicillin/Streptomycin (5 ml)
THAWING THE CELLS
Normally the cells are frozen in an amount of 3x106
cells per vial. This is a good amount to start with in
a T150 flask. The passage that is on the vial is the passage at which they were frozen, so add a passage
when you set up the cells.
Add 25 ml of medium to a T150 flask
Take a vial out of the liquid nitrogen
Warm up between your hands
Carefully open the vial to let the N2 escape
Transfer the content of the vial to the flask and rinse the vial once with medium from the flask.
Mix the cells with the medium gently and place the flask in the incubator
Change the medium one day after thawing to remove dead cells.
MEDIUM CHANGE
The medium should be changed every three or four days. Most convenient is to do this every
Thursday and Friday or every Monday and Tuesday.
Discard the medium
30
Rinse the cells once with PBS
Add new medium up to 25 ml per flask.
SUBCULTURING THE CELLS
Sheep cells grow rather fast in comparison to human cells and they are much smaller so more cells fit
in one flask. After about 5 days the flask is confluent and the cells are ready to be subcultures. When
confluent there are about 20x106 cells in one flask .You can transfer these cells 1:6 (approximately
3x106 per flask). Do never let the cells grow too confluent as this will influence their growth.
Discard the medium
Rinse the cells twice with PBS
Add trypsin (2.5 ml per flask) and distribute evenly over the bottom of the flask.
Place the flask in the incubator for 7 minutes
Tap the flask several times and check whether all the cells are rounded up and de-attached. If
not placed the flask into the incubator for a few more minutes.
Add 5ml of medium to the flask and mix the cells and trypsin
Transfer the medium to a centrifuge tube
Add 10 ml of PBS to the flask and pipette up and down to the walls of the flask to get all the
leftover cells transferred into the centrifuge tube
Check the flask microscopically for leftover cells. If there are still many cells in there rinse the
flask again with PBS and add this to the centrifuge tube.
Centrifuge 7 minutes at 1000rpm(or 5 min at 1500 rpm)
Discard the supernatant and resuspend the cells in 4 ml medium in order to count them.
After counting, divide them the cells over 6 new flasks.
Fill each flask up to 25 ml with medium and place the flasks in the incubator.
31
NB. Ovine sheep vena cava cells can be subculture up to passage 8-9, after that passage they will not
be the same cells anymore as it can be noticed in a changed morphology and a different growth speed.
COUNTING THE CELLS
Transfer 1 to a eppendorf tube
Add 50 of buffer A
Spin the vial for 5 seconds
Add 50 of buffer B
Spin the vial for 5 seconds
Use the nucleon counter to count the cells
32
APPENDIX 2: PREPARATION OF SCAFFOLD FOR TISSUE
ENGINEERING CONSTRUCTS
Introduction:
This is a standardized protocol for the preparation of the scaffold for Tissue Engineering control strips,
prior to the seeding of the cells onto the scaffold. Polyglycolic acid (PGA) is used for the scaffold
material and poly-4-hydroxybutyrate (P4HB) is used for the coating of the scaffold. The rectangular
scaffold strips are attached to RVS rings using polyurethane (PU). Two sizes of rings may be used but
the use of small rings is advised. This protocol is based on the original protocol (Tissue Engineering of
tissue strips in 6-wells plate) written by M. Stekelenburg, which is based on the tissue engineering
protocol, written by A.Mol.
Precautions
Perform operations and materials handling according to safe microbiological procedures.
Several hazardous chemicals are being used in this protocol. Use the appropriate personal protection
(gloves, fume-hood, etc) and read the concomitant MSDS’s.
Requirements
1. PGA
2. P4HB solution dissolved in THF
3. Polyurethane (PU) dissolved in THF
4. RVS rings (OD 25 mm, ID 16 mm)
5. 6 wells-plate
6. 70% ethanol
7. PBS(sterile)
8. TE medium
33
Preparation of reagents
The medium used for tissue engineering differs from the medium used for cell culture
TE medium sheep
DMEM Advanced(500 ml)
1% Lamb serum (5 ml)
1% Glutamax (5 ml)
1% Penicillin/Streptomycin(5 ml)
L-ascorbic acid 2-phosphate, Sigma(130 mg)
N.B: L-ascorbic acid 2-phosphate has to be solved in DMEM Advanced and sterile filtered: therefore,
add 130 mg of L-ascorbic acid 2-phosphate to a 50 ml tube and add 10 ml of DMEM Advanced, put
this for several minutes in the warm water bath (in warm medium L-ascorbic acid 2-phosphate
dissolves quicker). After about 10 minutes it will be dissolved, sterile filter it into the rest of DMEM.
Then add the rest of the components to the medium.
PH4B solution
Dissolve 1.75 gram of P4HB in 100 ml THF to obtain a 1.75 w/v solution
Procedures
Day1: COATING AND GLUEING OF THE PGA
Cut the scaffolds out of the PGA with the desired measured (Remember to cut the strips parallel
to the fibres in the PGA sheet).
Coat the PGA scaffolds with P4HB by dipping the scaffold into the solution and discard the
P4HB solution afterwards (in the fume -hood).
Leave the scaffold to dry on a glass plate in the safety cabinet until P4HB is white.
34
The scaffolds can then be mounted onto the RVS rings. Put some PU solution in a glass beaker.
Put a droplet of the viscous PU solution on either sides of the PGA scaffold edges or vertices
that you intend to glue. Make sure that the PU runs from the scaffolds ends in one streak over
the side and bottom of the RVS ring. This will ensure attachment of the scaffold to the ring.
This procedure is necessary as the PU does not adhere to RVS.
Let the scaffolds dry in the safety cabinet for approximately 24 hours.
Day2: DRYING IN THE VACUUM OVEN
Let the scaffolds further dry in the vacuum stove overnight.
Day 3: STERILIZING AND PREPARING THE SCAFFOL FOR SEEDING
Fill the wells with 70% ethanol and leave it for 30 minutes(work in the LAF cabinet)
Remove the alcohol and rinse thoroughly with PBS.
Fill the wells with TE medium and place in an incubator overnight.
Day 4: SEEDING OF THE SCAFFOLD
Prepare the thrombin solution: weight about 1-1.5 mg of thrombin and transfer to a centrifuge
tube.
Add an amount of TE medium to obtain a concentration of 10 IU/ml
Shake the solution and put on ice for about 10 minutes
Sterile filter the solution with the syringe and the sterile filter
Store the sterile solution on ice until use
Prepare the fibrinogen solution : weight about 50 mg of fibrinogen and transfer to a centrifuge
tube
Add an amount of TE medium to obtain a concentration of 10 mg actual protein per lm medium
35
Mix (gently shake) the solution for a couple of minutes until (almost all) the fibrinogen is
dissolved and sterile filter this.
Store the sterile solution on ice until use.
Before you start , calculate the amount of cells you need .The seeding density should be
Rinse the desired amount of flasks with the cells twice with PBS
Add 2.5 ml of trypsin and distribute evenly over the bottom.
Place the flask for 7 minutes in the incubator
Check whether all the cells are rounded up and de-attached. If not place the flask back in the
incubator for a few more minutes
Add 5ml of medium to the flasks and mix the medium with the cells and trypsin
Transfer the medium with cells to a centrifuge tube
Rinse the flask with PBS and add to the tube
Centrifuge 7 minutes at 1000 rpm
Discard the supernatant and resuspend the cells in 5 ml of medium for counting.
Count the cells
Mix gently the cells and the medium
Divide the cells in vials according to the number of cells needed by each scaffold.
Remove the medium from the well and aspirate the remaining medium from the strip. The
scaffold must be nearly dry before seeding.
Decide the amount of thrombin and fibrinogen you want to use to seed the scaffold. Add the
desired amount of thrombin to the cells Take the fibrinogen in the pipette tip and put the pipe to
the final volume estimated for each scaffold. When fibrinogen is added to the thrombin, the
solution starts to gel very quickly. Therefore you need to adjust the volume of the pipette before
you start mixing. The total volume of fibrinogen and thrombin is not enough because the cells
have a volume as well. Not to loose the cells, take a larger volume.
36
Mix the cell/thrombin solution gently with the fibrinogen for several times and the immediately
pipette the mixture at several spots of the scaffold.
After seeding all the scaffolds, place them with the cells in the incubator for about 30 minutes
to let the fibrin gel further polymerize
Carefully add medium to the constructs and place them back into the incubator on a shaking
table to allow mixing of the medium.
37
APPENDIX 3: AREA MEASURAMENT BY MEANS OF IMAGEJ
SOFTWARE
The method that will be described in this section was used to measure the surface area of the
constrained constructs at the end of culturing (t = 4 weeks) by means of ImageJ software. In order to
improve the representation in the display and enhance the contrast the colours can be redistributed over
the full colour range. Further, the equalization of an image allows a much better visual discrimination
of image details and features. ImageJ provides such functionalities via the Enhance Contrast-function
from the Process-menu (equalization option selected, 0.5% saturation of pixel) [8]. This was performed
on the photographs. Therefore, the selection of the region of interest for all further image analysis/
measurement was facilitated (Fig.7a).
Subsequently, the inner perimeter of the ring was selected via the elliptical or brush selection and the
outside was cleared (Edit-menu / Clear outside-function) (Fig.7b). The image was cropped to allow a
better visualization (Fig.7c) and then inverted (Edit-menu/Invert-function). (Fig7d).
Contrast enhanced picture (a) Inner diameter selected and
outside cleared (b)
Cropped image (c) Inverted image(d)
Fig.7. First steps of the image processing
The image was then transformed in 8-bit level via the Type-function from the Image-menu(Fig. 8a)
This resulted in an image showing white colour (corresponding to 255) out of the region of interest
(ROI), while the ROI was represented by high levels of grey (close to 0). If some regions out of the
ROI were still incorporated in the image because of the non-uniform luminance texture of the image,
38
they were discarded carefully selecting the areas with the freehand selection tool after zooming on the
image (Fig.7b).
In order to measure the area a threshold operation was needed. Selecting the Red entry in the pop up
menu inside the Threshold dialogue from Image/Adjust menu it was possible to assign the ROI to a red
mask. As a result of that, the ROI was the only masked area (Fig.7c).
Once the ROI was selected with the Wand-tool from the ImageJ toolbar, the contour of the selection
was displayed in the image with a yellow line.
Before calculating the area the scale must be set. For this purpose the Set Scale-function from the
Analyze menu of ImageJ had to be selected and the reference scale in the picture (1cm) was used to
draw a correspondent straight line with the tool in the toolbar.
After entry of the Known Distance, which was “1”, and the Unit of Length, which was “mm”, the
Analyze Particles–function was chosen from the Analyze menu. Previous window settings were not
changed and the Outline entry in the pop up menu was selected in order to double check which areas
were measured (Fig.7d). The results displayed the area in mm2. Finally, the results were represented as
the relative compaction of the TE construct by comparing the calculated surface area relative at week 4
to the seeding area at t = 0 .
8 bit image (a) Regions out of ROI have been
cleared (b)
Red masked ROI (c) Outline of the surface area (d)
Fig.8. Final steps of the image processing
39
APPENDIX 4: SECTIONING PARAFFIN BLOCKS CONTAINING
SCAFFOLD MATERIAL
Sectioning paraffin blocks containing scaffold material, such as in tissue engineered tissues is quite
difficult due to differences in stiffness between the paraffin, tissue, and the scaffold material.
Successful sectioning depends on many factors:
- Sectioning technique: this protocol describes the optimal settings for the microtome to be used
when scaffold material is present. With this protocol and a lot of patience you should be able to
obtain some nice sections.
- Type and amount of scaffold: blocks with tissues based on PGA and P4HB can be sectioned.
When the coating percentage increases, sectioning will be more difficult, but still possible. PCL
is too stiff to be sectioned using paraffin and this still can be better embedded in plastic. Do not
section pure scaffolds as these will not adhere to the slides. You need tissue to prevent the
sections from being lost during staining.
- Amount of tissue: blocks with scaffolds with good tissue formation are always easier to cut
compared to scaffolds with less tissue. Keep that in mind when sectioning static controls!
- Temperature of the block and sectioning speed: the block needs to be very cold to diminish the
differences in stiffness between the various components. Therefore, always store the blocks at -
20C and cool them frequently during sectioning. Furthermore, the slower you perform the
sectioning, the better the sections.
Preparation:
- Turn on the cold plate, the microtome and the hot plate.
- Take the blocks out of the -20C one for one for sectioning and not all at the same time. They have
to be really cold when you start. Place the block on the cold plate.
- Set the thickness for trimming at 30 µm and for sectioning at 10 µm.
- Place a new blade in the microtome. Always start sectioning at the most left side of the blade. Make
sure the angle of the blade is set at 10.
Trimming the block:
- Make sure the handle is locked and place the block in the microtome holder. Try to use the
orientation in which the construct will be divided equally over the blade.
- Make sure the block is positioned properly with respect to the blade and adjust the holder if
necessary. Make sure the blade holder is fixed before starting sectioning!
To give you an idea about the time required for sectioning:
If you would like to have 6 slides with 2 good quality sections per slide from one paraffin
block it will take between 30 and 60 minutes, depending on the amount of times you cool
the block in between.
40
- Set the sectioning window
- Set the microtome for trimming and use continuous mode. Start trimming at speed 2-5. Let the
microtome trim until you have reached the block. Then slow down the speed to about 1 and check
the sections if you are already in your sample (use the pins to prevent the sample from curling). If
this is the case stop trimming and place the block back on the cold plate.
Sectioning the block:
- Prepare some poly-L-lysine coated slides by writing down the sample name (use a crayon as other
pens will be washed off in stainings) and putting on water.
- Place the block back into the microtome holder in the same position as used for trimming.
- Set the microtome for sectioning and use continuous mode. Start sectioning at speed 1-2 until you
are in the sample again (this normally takes 3 to 4 sections). When you are in the sample again stop
sectioning.
- Now use single mode and set the speed at near to zero (really near to zero). Start sectioning and use
the pins to prevent the section from curling. When finished, transfer the section to the glass slide on
top of the water. You can in this way cut some sections (varying from 2-6) which are useful. You
will notice it immediately when the block is not cold enough anymore and you will need to place it
on the cold plate again and repeat the sectioning part for more sections (see comments in textbox).
- Transfer the slides containing the water and the sections to the hot plate and see the sections being
stretched. As soon as stretching is complete remove the water using a tissue. Do not touch the
coupes.
- Leave the slides drying in the special slide holder overnight.
41
APPENDIX 5: IMMUNO-FLUORESCENT STAINING VIMENTIN, DESMIN
AND α-SMA
Precautions
- Perform operations and materials handling according to safe microbiological procedures.
- Several hazardous chemicals are being used in this protocol. Use the appropriate personal
protection (gloves, fume-hood, etc) and read the concomitant MSDS’s.
(Hazardous) Chemicals
- Triton-X-100 is harmful; R22,41/S26,36,39
- Ethanol is highly flammable and harmful; R11,20,21,22,36,37,38,40/ S7,16,24,25,36,37,39,45
- Acetone is highly flammable; R11,36,41,66,67/S9,16,26
- Hydrochloric acid (HCl) is toxic, corrosive and dangerous to the environment;
R23,24,25,34,36,37,38/S26,36,37,39,45
- DAPI is mutagenic, reprotoxic, eye irritant; R36/S26
- Paraclear (Xylene) is harmful and highly flammable; R10,20,22,36,37,38
Requirements
- PBS
- 1% BSA in PBS
- TwPBS (0.1%Tween 20 in PBS)
- 1% Triton-X-100 in PBS
- Antigen retrieval solution
- Your primary and secondary antibodies
- Monoclonal Mouse anti-αSMA (IgG2a,DAKO, M0851, 1:500)
- Rhodamin-conjugated phalloidin (1:200, P1951, Sigma)
- Anti-mouse goat IgG2a alexa 488 (1:300).
- DAPI, to stain the nuclei as well
- Mowiol embedding agent (ready made)
- Cover glasses
Procedures
1. Deparaffinize slides (25min):
42
1. 2 x 5 min Paraclear (Xylene)
2. 2 x 2 min Ethanol 100%
3. 2 min Ethanol 96%
4. 2 min Ethanol 80%
5. 2 min Ethanol 70%
6. 2 min Ethanol 50%
2. 5 min PBS
3. Incubate 20 min. with epitope retrieval (Tris-EDTA Buffer cook it till 100˚C than incubate 20min.
at RT)
4. Incubate 30 min. with 1% BSA in PBS
5. Wash 5 minutes in 0.1% Tween 20 PBS agitate gently
6. Wash 5 minutes in 1% Triton X-100 agitate gently
7. Wash 5 minutes in PBS agitate gently
8. Dry the glasses carefully and draw limiting circles with the wax-pen.
9. Incubate 2 hours at room temperature with primary antibody solution in 1% BSA in PBS
- 1:500 aSMA …… l aSMA + …… l 1% BSA in PBS
10. Wash 5 minutes in 0.1% Tween-20 PBS agitate gently
11. Wash 3 x 5 minutes in PBS agitate gently
12. Incubate 30 minutes with secondary antibody with Rhodamin-conjugated phalloidin
- 1:200 Rhodamin-conjugated phalloidin
- 1:300 alexa 488 IgG2a
- …… l phalloidin + …… l Alexa 488 + ……. l PBS
13. Wash 5 minutes in 0.1% Tween 20 PBS agitate gently
14. Wash 3 x 5 minutes in PBS agitate gently
15. Incubate 5 minutes with DAPI in PBS (1:1000)
16. Wash 5 minutes in 0.1% Tween-20 PBS
17. Wash 3 x 5 minutes in PBS
18. Embed in Mowiol
19. Store slices in the dark at 4°C.
43
APPENDIX 6: HEMATOXYLIN & EOSIN (H&E) STAINING
Introduction
In routine histology the hematoxylin and eosin stain (better known as the ‘H&E’ stain) gives a visible
look at the nucleus of the cells and their present state of activity.
The H&E stain uses two separate dyes: hematoxylin is a dark purplish dye that will stain the
chromatin (nuclear material) within the nucleus, leaving it a deep purplish-blue color and eosin is an
orange-pink to red dye that stains the cytoplasmic material including connective tissue and collagen,
and leaves an orange-pink counterstain. This counterstain acts as a sharp contrast to the purplish-blue
nuclear stain of the nucleus, and helps identify other entities in the tissues such as cell membrane
(border), red blood cells, and fluid.
Performing the H&E stain: After the tissue has been paraffin embedded, sectioned, placed on a slide
and the slide dried in an oven, the slide is taken through brief changes of Xylene, alcohol and water to
‘hydrate’ the tissue. This process is called ‘running the slides down to water’ and must be done to
give the cells an affinity for the dyes. The slides are then stained with the nuclear dye (hematoxylin)
and rinsed, then stained in the counterstain (eosin). They are then rinsed, run in the reverse manner
from the run down (taken back through water, alcohol, and Xylene), then coverslipped.
Precautions
- Perform operations and materials handling according to safe microbiological procedures.
- Several hazardous chemicals are being used in this protocol. Use the appropriate personal
protection (gloves, fume-hood, etc) and read the concomitant MSDS’s.
(Hazardous) Chemicals
- Xylol/Xylene is harmful and highly flammable; R10,20,22,36,37,38
- Hematoxylin is harmful; R22,36,37,38
- Eosin is harmful and irritant; R36,37,38/S26,36
- Ethanol is highly flammable and harmful; R11,20,21,22,36,37,38,40/S7,16,24,25,36,37,39,45
- Acetic Acid is harmful and corrosive; R10,35/S23,26,45
- Entallan contains Toluene which is highly flammable, toxic and teratogenic;
R11,23,24,25/S16,25,29,33
- You are working with (very) toxic, harmful and flammable solutions; so wear gloves and
labcoat and work as much as possible in the fumehood!
44
Requirements
* Reagents and solutions:
- Xylol (Xylene, see chemical list)
- 100% Ethanol (EtOH, see chemical list)
- Diluted EtOH, 96% and 70%
- Hematoxylin solution (Mayers’, Sigma, cat# MHS16), can be re-used several times; don’t throw
back in original solution!
- Eosine Y solution (aqueous, Sigma, cat# HT110-2-16), before use slowly add 1 ml glacial acetic
acid per 200 ml Eosine Y solution to acidify the solution. Can be re-used several times; don’t
throw back in original solution!
- Tap water, MilliQ water
- Entallan (contains Toluene, see chemical list)
* Equipment and disposables:
- Dewaxing/ hydration/ dehydration series
- cover slips
Procedures
Before starting procedure fill in the administration list: date, name, which sequence, staining, number
of slides! After about 1000 slides or when staining become faint the hematoxylin and eosin should be
changed.
Check the “Dewaxing/ hydration/ dehydration series” if there are still enough solutions in the buckets
(EtOH and Xylol evaporate during time) and whether the solutions are clean (e.g. redness in the
dehydrating range caused by rests of eosin). If not: refill and/or change solutions.
1) Dewax and rehydrate the sections:
a) 2x 5 minutes Xylol
b) 3x 2 minutes 100% EtOH
c) 1x 2 minutes 96% EtOH
d) 1x 2 minutes 70% EtOH
e) 1x 2 minutes MilliQ water
2) Stain 10 minutes in Mayer’s hematoxylin solution
3) Wash in slow running tap water for 5 minutes
4) Stain in acidified aqueous eosin Y solution for 30 sec-3 minutes (depends on tissue and thickness of
the section, 30 sec 20 dips)
5) Wash in slow running tap water for 1 minute
6) Dehydrate, mount and cover the sections:
a) 10 dips in 70% EtOH
b) 10 dips in 96% EtOH
c) 3x 10 dips in 100% EtOH
45
d) 2x 3 minutes Xylol
e) Entallan and coverslip
f) Let dry O/N in fumehood
Expected results
Nuclei: Purple, Purple/ Black
Cytoplasm, extracellular matrix: different shades of pink till orange
References
- Sigma-Aldrich, H&H Informational Primer, “Hematoxylin & Eosin” (The Routine
Stain) by H. Skip Brown, BA, HT (ASCP)
- Administratieruimte: Book “Theory and practice of histological techniques”
- Internet: www.Histosearch.com/histonet
46
APPENDIX 7: RESULTS – COMPACTION DURING CULTURE
Construct Area seeded
(cm2)
Surface area at week 4
(cm2)
Compaction % Average
compaction
SD
Strip1 0.9 0.534 40
37
(+)
5.0
Strip2 0.9 0.510 43
Strip3 0.9 0.600 33
Strip4 0.9 0.605 33
Rectangle1 1.98 1.17 41
41
(++)(excluding R2)
0.5
Rectangle2 1.98 0.756 62
Rectangle3 1.98 1.15 42
Rectangle4 1.98 1.144 42
Triangle1 1.8 1.8 0
6.75
7.5
Triangle2 1.8 1.764 2
Triangle3 1.8 1.607 11
Triangle4 1.8 1.501 16