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Geometry as a Factor for Tissue Growth: Towards Shape

Optimization of Tissue Engineering Scaffolds

Cécile M. Bidan, Krishna P. Kommareddy, Monika Rumpler, Philip Kollmannsberger,

Peter Fratzl,* and John W. C. Dunlop

of cell and tissue responses and to design

Scaffolds for tissue engineering are usually designed to support cell viability optimal scaffolds for in vivo experiments
with large adhesion surfaces and high permeability to nutrients and oxygen. and applications.
Recent experiments support the idea that, in addition to surface roughness, Cells are known to adapt to the phys-
ical properties of their surroundings by
elasticity and chemistry, the macroscopic geometry of the substrate also con- integrating the mechanical equilibrium
tributes to control the kinetics of tissue deposition. In this study, a previously established at their adhesion sites.[5] The
proposed model for the behavior of osteoblasts on curved surfaces is used to resulting mechanical cue is translated
predict the growth of bone matrix tissue in pores of different shapes. These into a biochemical signal that triggers bio-
predictions are compared to in vitro experiments with MC3T3-E1 pre-osteob- logical decisions of the cells.[6] As cells are
mechanically attached to each other, either
last cells cultivated in two-millimeter thick hydroxyapatite plates containing
directly or via their extracellular matrix,
prismatic pores with square- or cross-shaped sections. The amount and they are also able to synchronize their
shape of the tissue formed in the pores measured by phase contrast micro- response on a larger scale. For example,
scopy confirms the predictions of the model. In cross-shaped pores, the ini- patterning in cell differentiation arises as
tial overall tissue deposition is twice as fast as in square-shaped pores. These a response to stiffness[7] or strain[8] pat-
results suggest that the optimization of pore shapes may improve the speed terns, and the distribution of proliferation
activity also correlates with the stress dis-
of ingrowth of bone tissue into porous scaffolds. tribution in a layer of cells.[9]
Cell fate has also been investigated
in three-dimensional artificial scaffolds.
1. Introduction Adhesion, proliferation, differentiation and mineralization of
cells and tissues have been compared in several scaffolds with
Three-dimensional scaffolds are needed for tissue engineering varying structures.[10,11] Recently, Kumar et al.[12] showed that
applications and may also help to study the effect of the environ- gene expression, and thus cell differentiation, is more affected
ment on tissue growth in vitro. The material used,[1] the fabrica- by the structural properties of the substrate than by its com-
tion process,[2] and the architecture of the scaffold[3,4] are known position. Furthermore, pore size and porosity need to satisfy
to influence the biological interactions with the host organism. the compromise between a high permeability that enables
Although all these parameters are difficult to decouple, quanti- cell migration and nutrient diffusion within the scaffold, and
fying their effects in vitro is necessary to understand the nature a large surface area for cell adhesion and extracellular matrix
production.[3] Many fabrication processes produce structures
C. M. Bidan, Dr. K. P. Kommareddy,
with random pores in a large range of sizes and interconnec-
Dr. P. Kollmannsberger, Prof. P. Fratzl, tivities difficult to control. Rapid prototyping techniques are
Dr. J. W. C. Dunlop much more accurate in that respect.[13] The direct printing of
Department of Biomaterials the scaffold enables to control the architecture and thus many
Max Planck Institute of Colloids and Interfaces mechanical properties of the structure.
14424 Potsdam, Germany Rumpler et al.[14] used rapid prototyping to build artificial
E-mail: [email protected]
macro-pores of different controlled geometries and showed that
Dr. M. Rumpler
Ludwig Boltzmann Institute of Osteology at the Hanusch Hospital cells locally respond to high curvature by producing tissue. Their
of WGKK and AUVA Trauma Centre Meidling hypothesis of local tissue growth proportional to curvature has
1th Medical Department been confirmed experimentally, not only in pores but also on
Hanusch Hospital, Vienna, Austria open surfaces,[15] however with the additional observation that
Dr. P. Kollmannsberger tissue does not grow on convexities. The interfacial evolution
Department of Health Sciences and Technology (D-HEST)
ETH Zurich, 8093 Zurich, Switzerland
derived from a curvature-driven tissue growth model matched
the experimental observations as well as the in vivo expecta-
DOI: 10.1002/adhm.201200159 tions when comparing with the typical geometries involved in

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bone remodeling (osteon and hemi-osteon).[16] An interesting simple shapes used up to now.[14,15] Figure 1 shows the growth

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consequence of curvature-driven growth was also observed by behavior expected in square-, star- and cross-shaped pores nor-
Rumpler et al.[14] Despite seeing local differences in growth malized with respect to the perimeter of their section (Pmedium =
rates in prismatic pores with different convex sections (circle, 4.71 mm). Although the interface between tissue and medium
square and triangle) but identical surface areas, the total tissue tends to adopt a circular shape in all cases (Figure 1(a)), the
growth was found to be independent of the shape. This could initial kinetics of growth is expected to be significantly affected
be understood using Fenchel’s law,[17] which states that the by the geometry of the straight sided pore (Figure 1(b)). For
average curvature in a convex shape, is inversely proportional to example, tissue is predicted to grow twice as fast in a cross-
the perimeter. This would imply that the average growth rate, if shaped pore as in a square-shaped pore or any other convex
curvature-controlled, would also be the same. shape (Figure 1(c)).
This paper aims at understanding how tissue production
can be enhanced simply by controlling the geometry of the sur-
face by exploring non-convex pore geometries. The model of 2.2. Evolution of the Shape in the in vitro System
curvature-driven growth as implemented in a previous work[15]
was first used to predict growth in pores with cross-, star- (non- In order to verify the predictions obtained with the model of
convex) and square- (convex) shaped sections. The simulations curvature-driven growth, tissue was cultured in 2 mm thick
predict higher initial growth rates in non-convex shapes and hydroxyapatite (HA) plates containing straight sided pores of
even a two-fold increase by growing cells in a cross-shaped pore square- (convex) and cross- (non-convex) shaped sections normal-
compared with a square-shaped pore. To verify these predic- ized with respect to their perimeter. Once seeded on the scaffolds,
tions, straight sided pores with cross- and square-shaped sec- the MC3T3-E1 pre-osteoblasts proliferate and start to produce
tions are designed in hydroxyapatite scaffolds and incubated collagenous extracellular matrix (ECM). Tissue deposition was
with MC3T3-E1 pre-osteoblast cells for in vitro tissue culture. followed in each pore by phase contrast microscopy over 28 days
Not only the motion of the tissue-medium interfaces and the and quantified in terms of projected tissue area (PTA).
evolution of their curvature profiles compare well to the model, Three sets of 5 pores of each size (Pmedium = 4.71 mm, Plarge =
but the quantitative analysis of tissue production also matches 6.28 mm) and each shape (Sq stands for square and Cr for cross)
the outcomes from the curvature-controlled growth model. have been independently seeded and showed similar results.
Understanding the mechanisms involved in such a phenom- The paper presents the data from one representative set.
enon is of high interest for developing tools to design scaffolds Figure 2(a) shows phase contrast images taken at 4 different
with the optimal geometry and meeting the numerous criteria time points during culture (D2, D7, D14 and D21). As already
for tissue engineering and clinical applications. observed by Rumpler et al. in convex shapes,[14] tissue depo-
sition starts in the corners whereas no growth occurs on flat
surfaces until the surrounding tissue deposition modifies the
2. Results local geometry. In cross-shaped pores, the concave regions of
the branches are also quickly filled with tissue. However, the
2.1. Predictions of the Theoretical Model 4 convex points (indicated by red circles on the figure) seem to
act as ‘flat surfaces’ since no growth occurs until local curva-
The model of curvature-controlled growth proposed in pre- ture becomes positive through the global interfacial evolution.
vious studies was applied to non-convex geometries to predict This effect of the sign of curvature has already been pointed
potential changes of growth behavior in comparison to the out in previous studies.[15] In both cases, the tissue-medium

Figure 1. The computational simulation of curvature-driven growth was run on artificial images representing square-, star-, and cross- shaped pores of
medium size (Pmedium = 4.71 mm). a) The tissue-medium interface evolves toward a circular shape. b) Initial kinetics of growth are significantly affected
by the geometry of the pore section before reaching a circular interface. c) Initial growth rates calculated on the 40 first steps of the simulation suggest
that a two-fold increase in tissue deposition can be expected in cross-shaped pores compared to square-shaped pores. Adapted from Bidan et al.[23]

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Figure 2. Tissue growth in square- and cross-shaped pores. a) Phase contrast images of the pore taken 2, 7, 14, and 21 days after seeding the MC3T3-
E1 on the scaffolds. b) The superposition of the interfaces obtained experimentally is compared with the predictions of the curvature-driven growth
simulation applied to the actual geometry of the experimental pore at D2. 7, 14 and 21 days of experiments are obtained with 36, 120, and 204 steps
of simulation with r = 8.5pxl, α = 12steps, t0 = 4 d. c) Curvature profiles of the tissue–medium interface measured at D2, D7, D14, and D21 in a square-
(i) and a cross-shaped pore (ii). d) Curvature profiles are measured on the interfaces predicted by the curvature-driven growth model after 7, 14, and
21 days of culture in ideal square- (iii) and cross-shaped pores (iv). The curvature measurements were smoothed using a mask size of r = 14.5pxl.

interface evolves toward a circle, as predicted by the model of immunofluorescence methods. On Figure 3(a), nuclei staining
curvature-driven growth applied to the actual geometry of the (red) reveals the homogeneous distribution of cells within the
pore, derived from the experimental images (Figure 2(b)). tissue whereas actin fibers (green) are mostly concentrated and
The evolution of the geometry was quantified in terms of highly oriented along the tissue-medium interface.
curvature. Figure 2(c) presents the curvature profiles measured In larger pores, polarized microscopy enables to image
on experimental images taken at different time points. The the preferential orientation of the fibrous extracellular matrix
behavior compares well with the interfacial curvature profiles deposited by the cells. As shown on Figure 3(b), collagen fibers
measured on images obtained with the curvature-driven growth deep in the tissue are oriented parallel to the substrate, whereas
model applied to ideal square and cross shapes (Figure 2(d)). those at the interface have a direction similar to the cells.
The 4 peaks of curvature characterizing the corners of a square-
shaped pore vanish as the tissue grows; the curvature profile of
the interface flattens and becomes characteristic of a circle. In a
cross-shaped pore, 8 peaks correspond to the 8 concave corners 2.4. Kinetics of Bone-Like Tissue Growth
and 4 regions of highly negative curvature are related to the
4 convex corners. As in the square, all the peaks–whatever their Kinetics of new tissue formation within the pores of the scaf-
sign–tend to vanish and curvature profiles become smooth as folds was followed in the in vitro system by measuring the pro-
tissue is deposited. Note that differences in curvature values are jected tissue area (PTA) on phase contrast images taken twice a
due to the imperfections of the HA scaffolds, and that the final week. As predicted by the model of curvature-driven growth in
profiles are not totally smooth due to the discrete character of ideal shapes (Figure 1), Figure 4(a) and 4(b) reveal that after any
the binarized images. time of culture, more tissue has been produced in the cross-
than in the square-shaped pores, and this for two different pore
sizes (Plarge = 6.28 mm and Pmedium = 4.71 mm respectively).
2.3. Structure Reporting tissue growth rates calculated between D4 and D14
also confirms that initial growth rates are almost two times
The organization of the cells and collagen fibers within the faster in cross- than in square-shaped pores, independent of the
newly formed tissue was investigated qualitatively using size (Figure 4(c) and 4(d)).

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Additionally, Figure 4 shows that the model of curvature-
driven growth applied to the actual geometry of the pores, also
predicts quantitatively the growth behavior for the first two
weeks of cell culture. The model is fitted with a single param-
eter which sets the time scale of the simulation and is calculated
with the experimental tissue growth rate in a square-shaped
pore. For this set of experiments, 12 steps of simulation repre-
sent 1 day of culture (α = 12steps). A lag time t0 = 4d accounting
for the time that cells need to settle was incorporated to overlap
the curves.

3. Discussion
In this study, a curvature-driven growth model[14,15] was applied
to different (non-convex) geometries. The growth behaviors
obtained by computational simulations were verified experi-
mentally using an in vitro tissue culture system that offers the
possibility to vary the geometry of a substrate in a controlled
way, independently of the chemistry. Not only the qualitative
and quantitative geometrical evolution of the tissue-medium
interface, but also the faster tissue generation by MC3T3-E1
cells in non-convex-shaped pores (cross) could be derived from
the simple hypothesis that the local growth rate is locally pro-
portional to the curvature (if it is positive).
Despite a well-defined experimental protocol, some limita-
tions remain. The hydroxyapatite scaffolds produced by casting
and sintering present the expected geometry on the millimeter
scale, but the roughness of the surfaces is difficult to control,
especially in non-convex shapes. This drawback also justifies the
necessity of a computational tool able to quantify the geometry
in terms of curvature profile and apply the curvature-controlled
growth model directly on experimental images, and therefore
take into account the interfacial defects.

3.1. Convex Versus Partially Non-Convex Pores

To get a simple analytical estimate of the growth rate based on

local curvature, we use the following considerations. In non-
convex-shaped pores, the growth law can be written as:


ds λκ + , κ > 0
= (1)
dt 0 ,κ ≤ 0

Or in terms of projected area:

d P T A por e ds +
= P = Pλ κave (P)
dt dt
Figure 3. a) Whatever the geometry of the pore, cells are homogene-  + 
Pκave (P) (2)
ously distributed in the tissue (nuclei in red) but actin concentration = 2πλ = 2π λκ +∗
is much higher at the interface which tends to be circular. b) Polarized 2π
microscopy reveals collagen fibers having the same orientation as the
cells, i.e., parallel to the tissue–medium interface. c) The geometrical con- +
with κave (P) being the positive curvature averaged over the
struction that considers tissue as an assembly of tensile elements repre-
perimeter P of the section.
senting contractile cells[15] applies to convex and non-convex geometries. +
d) Tissue stained for actin fibers reveals stretched cells organized along The calculation of κave (P) is based on the demonstration of
dPTA por e
the interface as predicted by the chord model. Fenchel’s law (see Supporting Information (SI)). As
dt

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Figure 4. Growth kinetics measured in square- and cross-shaped pores of large (a) and medium (b) size. Experimental and simulated growths are
reported in terms of projected tissue area (PTA) (α = 12steps, t0 = 4d). Growth rates are calculated between D4 and D14 with experimental and simulated
data in large (c) and medium (d) pores. ANOVA analysis shows no significant differences between the methods used (Exp, Sim) but a significant dif-
ference in the tissue growth rates achieved in square and cross (p < 0.05). Dots and error bars represent mean values and standard errors respectively
(n = 5).

is proportional to P.κ +ave(P), which turns to be constant and growth and the higher tissue organization in non-convex shapes
characteristic of the shape but not the size neither the propor- (Figure 3(a)). This approach is further supported by actin stained
+
tions, κ +∗ = P.κave
2π
(P)
is defined as a constant dimensionless tissues showing cells locally oriented parallel to the tissue-
value characteristic of the “non-convexity” of the shape. κ +∗ for medium interface (Figure 3(a) and 3(d)). Considering that this
convex shapes and κ +∗ for non-convex shapes. preferential organization is also followed by the collagen fibers
In cross-shaped pores, the curvature is negative in four synthesized by the cells during tissue growth (Figure 3(b)), one
points, positive in the eight right angle corners and null else- could transfer the geometrical construction to the tissue level in
where. In squares, the curvature is positive in the 4 right angle a similar way to the cable model of Bischof et al.[24]
corners and null elsewhere. If the negative curvature plays no The curvature-driven growth model predicts a decrease
role in the growth rate, then the positive curvature averaged in tissue growth rate in cross-shaped pores as soon as the
on the perimeter in the cross is twice the one of a square and tissue-medium interface becomes convex. However, as already
growth should be twice faster. Both simulation and experiments observed in similar experiments, the tissue growth slows down
meet this prediction. after 18 days of culture, independently of the shape of the
pore. Three main hypotheses have been proposed to explain
this phenomenon.[15] i) Ageing and differentiation affect the
3.2. Gradual Slowdown of Growth Rates proliferative activity of the cells and thus their ability to pro-
duce tissue. ii) Not only cells mature, but also the ECM they
The patterns obtained with the curvature-controlled growth produce. With maturing collagen cross-links the matrix could
model and the ones observed in the experiments can also be become locally denser, which may be implemented into the
derived from the simple geometrical construction using ten- model as a gradual reduction of the growth rate. iii) Consid-
sile elements.[15] In essence, this model represents a cell by its ering the projected tissue area to quantify growth supposes
internal actin filaments (stress fibers) connecting adhesion sites that tissue grows homogeneously all along the height of the
of the cell. This “chord model” explains intrinsically not only three-dimensional pore, which is unlikely. Therefore, one
the absence of growth on convexities but also the faster tissue needs two principal curvatures to describe the geometry of

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Figure 5. The effect of the curvature in the third dimension may be partly responsible for the slowdown of growth observed experimentally on the pro-
jected plane. a) Schematic representation of tissue repartition in the pore with the associated geometrical descriptors in the middle plane. b) A numer-
ical derivation was done with λ = 0.01 mm.timeunit − 1, L = 2 mm in pores of medium and large sizes. The dashed line indicates a linear growth.

the interface along the vertical axis, and they are likely to be of 2 in the case of cross-like pores) giving a new opportunity to opti-
opposite sign. mize the architecture of scaffolds for tissue repair.
To discuss this last point further, one can analytically esti-
mate the impact of the convexity appearing in the third dimen-
sion in a cylinder of radius R0. As depicted on Figure 5(a), if 3.3. Towards Optimizing Pore Geometry in a Scaffold
the inward curvature is approximated by a circle (red), the two
principal curvatures at the point M are: Integrating a scaffold in a host organism often implies to pro-
duce as much tissue as possible in a short time. In that respect,

κ1 = R1 > 0 a lot of highly non-convex pores would be useful. However,
(3) having small pores filling fast and completely with agglomer-
κ2 = − 2(R0 −R) <0
2 L2
(R0 −R) + 4 ates of cells is also not desired. Indeed, diffusion of nutrients
would be impaired and cell viability affected. Moreover, cells
where R is the radius of the pore at time t and L is the depth of also need space to migrate and lay down extracellular matrix.
tissue deposition. Mean curvatures H = κ1 +2 κ2 in each point of Pores should then be large enough to guarantee a good per-
meability and leave room for the formation of new tissue and
the interface in the middle plane are lower than the one meas- angiogenesis.[26]
ured on the projection (κ1) and, therefore, As shown in this study, the geometry of individual pores not
only influences density, permeability and the amount of tissue
d P T A por e d R2 produced in the scaffold, but also the speed and repartition of
= −π = 2π R λ H(R) tissue deposition. In the cross-shaped pore, for example, tissue
dt dt
is generated with a high rate in the branches in a first stage,

2 ( R0 − R) R (4)
= 2π λ 1 − 2 which could help anchoring the scaffold faster in the host
( R0 − R)2 + L4
organism. As the interface smoothens and becomes circular
PTA(t) can be derived numerically from Equation (4) and (convex), the growth rate slows down, leaving time and space
some results presented in Figure 5(b) show, indeed, a slowing for exchanges through the pore. Experimentally, the slowdown
down of the growth that is not predicted by the two dimensional occurs a bit earlier for the reasons discussed above.
model but appears in experiments. These principles apply to single pores and, when up-scaling
Although the observations and calculations proposed in this them to scaffolds with multiple pores, one has to consider
paper are simple, they have important consequences for the design that geometry also determines the number of pores npores
of tissue engineering scaffolds. For example, the results suggest which geometrically fit in a scaffold with defined size Ascaff and
that for purely convex channels, the rate of new tissue growth in a porosity ϕ:
single pore is independent of size and geometry. This implies that A por e
pore shape can be modified to satisfy other criteria (e.g. strength, φ = n por es (5)
As ca f f
fatigue resistance, permeability, etc)[25] without changing the rate
of tissue ingress. Moreover, introducing non-convexities into the Therefore, the global tissue growth rate in a scaffold can be
pore shapes can greatly increase the growth rate (by a factor of estimated as (based on Equation (2)):

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d P T Atot d P T A por e As ca f f of the implant in the host body. The total initial tissue growth
= n por es = φ 2π λκ +∗ (6) rate obtained in such a scaffold can be estimated by adapting
dt dt A por es
Equation (8):
This can be rewritten in terms of the circularity C, which is
a dimensionless shape factor that can be used to describe the d P T Atot   1   κ +∗ 
i
= 8π 2 λAs ca f f φi (9)
pores. This value depends on the geometry but not on the size: dt Pi2 Ci
i
A
C = 4π (7) with ϕi the contribution of the shape i to the global density.
P2
where P is the pore perimeter. Figure 6(a) classifies pore shapes
with respect to their “non-convexity” and their “circularity”, 4. Conclusion
two geometrical parameters that influence respectively tissue
growth rate in an individual pore and the global porosity of a This work lays stress on the determining role of the geometry
scaffold made of those pores. The global tissue growth rate can of a substrate on the kinetics of tissue deposition. We show that
then be expressed as a function of the scaffold and the pores
characteristics:
   +∗ 
d P T Atot 1 κ
= 8π 2 λ φ As ca f f (8)
dt P2 C
Equation (8) shows that the global tissue growth rate in
the scaffold is a product of independent terms characterizing
i) cell activity, ii) scaffold properties, iii) pore size and iv) pore
geometry.
Figure 6(b) shows how the total tissue growth rate depends
on the size and the geometry of the pores. The initial tissue
growth rate is considered, i.e. the growth rate achieved until the
interface becomes convex. In a plate-like scaffold of a given area
(20 mm2) and given porosity (0.9), small and non-convex pores
give rise to higher growth rates (white areas on the bottom
right).
However, a common concern in tissue engineering is the
permeability of the scaffold to guarantee cell migration as well
as nutrient and waste diffusion necessary for cells to survive.
Tissue engineering literature suggests that pores should be at
least 300 µm large to ensure a good permeability of the scaf-
fold.[27] For each shape, the size of the inner circle is taken as
a limitation for pore size. An inner radius of 150 µm leads
to the minimum relevant perimeter. Maximum realistic ini-
tial growth rates are estimated for each shape (small shapes
on Figure 6(b)). Considering this aspect, the fastest initial
growth rate can be obtained using regular crosses with thick
branches. The circular interface being quickly reached, the
amount of tissue produced at that high rate is however low.
The remaining space and the slower growth from this time
point could be profitable for angiogenesis and facilitate diffu-
sion as the pore is closing.
All these calculations assume that the totality of the scaffold
area can be covered by assembling pores of the same shape.
This statement is true for squares, triangles and regular crosses
with k = 0.33. However, it is known that the maximum density
achieved by packing circles in a hexagonal arrangement on a
surface is 0.90 and star shapes are also not likely to be packed Figure 6. a) Pore shapes can be classified using the “non-convexity”
in an optimal arrangement. It is therefore relevant to envisage which determines tissue growth rate in the pore and the “circularity”
scaffolds containing pores of different geometries and sizes. which influences the number of pores fitting in a scaffold. b) Contour plot
representing the influence of pore geometry and pore size on the total
Few large highly non-convex shapes can promote the anchoring
tissue growth rate in a 20 mm2 scaffold with a porosity of 0.9. Grey level
of the scaffold at the early time points and facilitate diffusion as decreases with the growth rate. For each shape, the dot shows the perim-
growth progresses, whereas smaller (mainly convex) pores fitted eter corresponding to an inner radius of 150 µm (considered as the limit
in between can provide additional surfaces for cells to deposit for good permeability properties). In that respect, smaller perimeters are
tissue with a slower rate, but that will also support integration not relevant for tissue engineering purposes.

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tissue growth can be promoted simply by tuning the curvature and tissue (represented in black in the binarized images) to be
of the surfaces where cells deposit their extracellular matrix. A distinguished from the medium (represented in white).
simple geometrical model based on the tensile behavior of the Measurement of Tissue Growth: Tissue growth in the pores is
quantified by determining the projected tissue area (PTA) formed in the
cells, which leads to curvature-controlled growth, can predict pores. As this measurement is two-dimensional, it is only a proxy for
both the kinetics achieved and the distribution of tissue depo- quantifying the volume of growth into the depth of the pore. The free
sition. As such simple principles could be of high interest for section of a pore, corresponding to the white regions in the binarized
tissue engineering, we propose some methods to optimize pore images, decreases with time. The PTA is then calculated by subtracting
design when considering a porous scaffold intended for tissue the binarized image at an initial time point from the image at the time of
repair. interest, and then calculating the remaining area. As cells need time to
settle on the scaffold and start tissue deposition, the initial pore section
is taken on the second day after seeding (D2).
Curvature Measurement: The curvature profile of the interface between
5. Experimental Section the tissue and the medium on each binarized image is calculated using
Frette’s algorithm[18,22] implemented in a custom made Matlab code
Curvature-Driven Growth Simulation: A model for curvature-driven (Matlab 7.8.0 R2009a, MathWorks, Natick, MA) as described in.[15]
tissue growth was proposed by Rumpler et al.[14] and implemented by Briefly, the algorithm first locates the pixels on the tissue-medium
Bidan et al.[15] in a Matlab (Matlab 7.8.0 R2009a, MathWorks, Natick, MA) interface in the binarized image and the local curvature κ associated
code based on a method for measuring curvature on digital images.[18] with an interface pixel is then estimated from the ratio of the number of
This computational simulation is run on binarized images of the black to white pixels lying within a given radius from the interface with
pores in which the scaffold is black and the medium is white. Each the formula:
iteration consists in a) attributing a value of effective curvature to each  
pixel representing what cells sense from the geometry of the surface, 3π A 1
κ= − (10)
b) transforming the white pixels having a positive effective curvature to r A tot 2
black and represent tissue deposition in concave regions. The process
is then repeated to simulate curvature-controlled growth. To make a where A is the number of pixels in the mask and on the outer side of
quantitative comparison with the experimental results, this simple the interface, Atot is the number of pixels in the mask and r is the mask
model only requires the input of a single parameter accounting for the radius. The calculation is made for all pixels on the interface on each
number of iterations needed to simulate one day of culture. This value side of the border. The local curvature in one position of the interface is
is calculated using the experimental growth rate measured in a convex taken as the mean value of the curvatures measured on the outer pixel
shape, the square in this study. and the inner pixel. In the limit of a perfectly smooth interface and an
In order to be equivalent to the geometrical interpretation described infinitely small radius, this ratio corresponds to the local curvature. In
previously,[15] when the computational tool is used for modeling the context of this paper, concave surfaces have a positive curvature.
√
purposes, the mask radius is set to r = 8.5pxl, i.e. 3 2 times of the size To quantify interfacial geometry at different time points, the local
of a cell (about 50 µm for an elongated osteoblast). curvature is given as a function of the position along the interface
Production of the Hydroxyapatite (HA) Scaffolds: 2 mm thick HA normalized with respect to its perimeter. In order to reduce the
scaffolds containing straight sided pores are produced by slurry noise induced on the curvature profiles by both the roughness of
casting as mentioned in previous studies.[14,15] Pore sections the experimental interfaces and the digitalization, the mask radius r
represent squares or crosses and are normalized with respect to their of the computational tool is set to r = 14.5pxl and the resulting profile
perimeter (Pmedium = 4.71 mm, Plarge = 6.28 mm). Molds are designed is then smoothed using a running average algorithm with a sampling
using the computer-aided design (CAD) software Alibre Design proportion of 5% of the total length of the perimeter.
(Alibre Inc., Richardson, TX) and produced with a three-dimensional Immunofluorescence Staining: Scaffolds are washed with phosphate
wax printer, Model Maker II (Solidscape Inc., Merrimack, NH) as buffered saline (PBS), fixed with 4% paraformaldehyde for 5 min and
described by Manjubala et al.[19] The molds are then filled with a HA permeabilized overnight with 1% Triton-X100 (Sigma-Aldrich, Steinheim,
slurry made of methacrylamide (MAM) monomers (15 g), N-N´- Germany) at room temperature. Once washed in PBS, the tissue is stained
Methylenebisacrylamide (BMAM) (5 g), water (75 g), Dextran (12.5 g) for actin stress fibers by incubating with Alexa-Fluor 488–phalloidin
and HA powder (300 g), and cross-linked with ammonium persulfate (Invitrogen, Molecular Probes) (3 × 10−7 M) for 90 min. Nuclei are then
and N,N,N´,N´-Tetramethylethylenediamine (TEMED). The structures stained with TO-PRO 3 692–661 (Invitrogen, Molecular Probes) (3 ×
are slowly air dried by heating the samples to 50 °C at a rate of 5 °C 10−6 M) for 5 min. Fluorescent images of the stress fibers are obtained
per day and then are held at this temperature for one day. The dried using a confocal laser scanning microscope (Leica TCS SP5).
samples are then pre-sintered at 600 °C for 48 h to remove the wax
molds and are finally sintered at 1100 °C for 24 h.[20]
Cell Culture: Murine pre-osteoblastic cells MC3T3-E1 (provided by the Supporting Information
Ludwig Boltzmann Institute of Osteology, Vienna, Austria) are seeded
with a density of 105 cells.cm−2 on the surface of the HA scaffolds Supporting Information is available from the Wiley Online Library or
and cultured for 28 days in α-MEM (Sigma-Aldrich, St. Louis, MO) from the author.
supplemented with fetal calf serum (PAA laboratories, Linz, Austria)
(10%), ascorbic acid (Sigma-Aldrich, St. Louis, MO) (0.1%) and
gentamicin (Sigma-Aldrich, Steinheim, Germany) (0.1%) in a humidified Acknowledgements
atmosphere with CO2 (5%) at 37 °C.
Imaging: Each pore is imaged every 3 to 4 days using a phase contrast We acknowledge funding from the Leibniz prize of PF running under
microscope (Nikon Eclipse TS100, Japan) equipped with a digital camera DFG contract number FR2190/4-1. C.B. is a member of the Berlin-
(Nikon Digital sight DS 2Mv). All pictures are taken with a 4× objective, Brandenburg School for Regenerative Therapies (GSC 203).
yielding a final image resolution of 205 pixels per mm.
Image Analysis: The digital phase contrast images are semi- Received: May 11, 2012
automatically binarized using ImageJ (National Institutes of Health, Revised: August 7, 2012
Bethesda).[21] The contrast in the images is sufficient to enable scaffold Published online: November 19, 2012

Adv. Healthcare Mater. 2013, 2, 186–194 © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim wileyonlinelibrary.com 193
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