6 research outputs found
Effects of Three-Dimensional Culture of Mouse Calvaria-Derived Osteoblastic Cells in a Collagen Gel with a Multichannel Structure on the Morphogenesis Behaviors of Engineered Bone Tissues
Bone
has a complex hierarchical structure that contributes to its
superior mechanical properties. Therefore, reproducing the complex
hierarchical structure of bone tissue is a promising strategy to construct
functional engineered bone tissues. In this study, we aimed to reproduce
this complex hierarchical structure by developing a method for the
three-dimensional culture of MC3T3-E1 osteoblastic cells in a collagen
gel with a multichannel structure (MCCG), which mimics the parallel
arrangement of Haversian canals in bone tissue. MCCG was homogeneously
calcified via the biomineralization properties of MC3T3-E1s. Confocal
laser scanning microscopy revealed that MCCG could support the growth
and proliferation of MC3T3-E1 cells in the deeper parts of the engineered
bone tissue and that the cells formed a toroidal structure on the
channel surface and a network-like structure in the gel matrix region.
Furthermore, quasi-quantitative measurement of osteocalcin and dentin
matrix protein 1 expression indicated the coexistence of two types
of cells with different morphologies and differentiation phenotypes.
Thus, three-dimensional culture of MC3T3-E1 cells in MCCG yielded
engineered tissues mimicking the hierarchical structures of bone tissues.
Engineered bone tissues with a biomimetic hierarchical structure could
be used as a model system for investigating bone metabolism and evaluating
the efficacy of novel drugs
Application of Multichannel Collagen Gels in Construction of Epithelial Lumen-like Engineered Tissues
Introduction
of epithelial lumen-like structures such as blood
and lymphatic vessels, as well as renal tubules, is a prerequisite
for successful construction and function of artificially engineered
giant tissues. Here, we demonstrate a methodology for construction
of various epithelial lumen-like structures by using multichannel
collagen gels (MCCGs). MCCGs were prepared and used as template scaffolds
for constructing epithelial lumen structures in a controlled fashion.
The effect of NaCl concentration on the multichannel structure of
MCCGs was investigated by using confocal laser scanning microscopy
along with fluorescent staining. The channel diameter increased with
increasing NaCl concentrations in the collagen solution and the phosphate
buffer solution. In contrast, the channel number decreased with increasing
NaCl concentrations. Engineered tissues with various lumen-like structures
were constructed by seeding and culturing Madin–Darby canine
kidney cells on MCCGs. The diameter of the lumen and the number of
lumens per unit area were controllable by regulating the multichannel
structure of cylindrical MCCG. We believe that our methodology for
the construction of engineered tissues possessing epithelial lumen-like
structures will prove helpful in regeneration of giant tissues with
various hierarchical structures
Application of Multichannel Collagen Gels in Construction of Epithelial Lumen-like Engineered Tissues
Introduction
of epithelial lumen-like structures such as blood
and lymphatic vessels, as well as renal tubules, is a prerequisite
for successful construction and function of artificially engineered
giant tissues. Here, we demonstrate a methodology for construction
of various epithelial lumen-like structures by using multichannel
collagen gels (MCCGs). MCCGs were prepared and used as template scaffolds
for constructing epithelial lumen structures in a controlled fashion.
The effect of NaCl concentration on the multichannel structure of
MCCGs was investigated by using confocal laser scanning microscopy
along with fluorescent staining. The channel diameter increased with
increasing NaCl concentrations in the collagen solution and the phosphate
buffer solution. In contrast, the channel number decreased with increasing
NaCl concentrations. Engineered tissues with various lumen-like structures
were constructed by seeding and culturing Madin–Darby canine
kidney cells on MCCGs. The diameter of the lumen and the number of
lumens per unit area were controllable by regulating the multichannel
structure of cylindrical MCCG. We believe that our methodology for
the construction of engineered tissues possessing epithelial lumen-like
structures will prove helpful in regeneration of giant tissues with
various hierarchical structures
Multiscale Analysis of Changes in an Anisotropic Collagen Gel Structure by Culturing Osteoblasts
Mimicking the complicated anisotropic
structures of a native tissue is extremely important in tissue engineering.
In a previous study, we developed an anisotropic collagen gel scaffold
(ACGS) having a hierarchical structure and a properties gradient.
In this study, our objective was to see how cells remodel the scaffolds
through the cells–ACGS interaction. For this purpose, we cultured
osteoblastic cells on ACGS, which we regarded as a model system for
the cells–extracellular matrix (cell-ECM) interaction. Changes
in the ACGS–cell composites structure by cell-ECM interactions
was investigated from a macroscopic level to a microscopic level.
Osteoblastic cells were also cultured on an isotropic collagen gel
(ICGS) as a control. During the cultivation, mechanical stimuli were
applied to collagen-cell composites for adequate matrix remodeling.
Confocal laser scanning microscope (CLSM) was used to observe macroscopic
changes in the ACGS–cell composite structure by osteoblastic
cells. Small-angle X-ray scattering (SAXS) measurements were performed
to characterize microscopic structural changes in the composites.
Macroscopic observations using CLSM revealed that osteoblastic cells
remained only in the diluted phase in ACGS and they collected collagen
fibrils or formed a toroidal structure, depending on the depth from
the ACGS surface in the tubular diluted phase. The cells were uniformly
distributed in ICGS. SAXS analysis suggests that collagen fibrils
were remodeled by osteoblastic cells, and this remodeling process
would be affected by the structure difference between ACGS and ICGS.
These results suggest that we directly regulate cell-ECM interaction
by the unique anisotropic and hierarchical structure of ACGS. The
cell–gel composite presented in this study would promise an
efficient scaffold material in tissue engineering
Anisotropic Growth of Hydroxyapatite in Stretched Double Network Hydrogel
Bone
tissues possess excellent mechanical properties such as compatibility
between strength and flexibility and load bearing owing to the hybridization
of organic/inorganic matters with anisotropic structure. To synthetically
mimic such an anisotropic structure of natural organic/inorganic hybrid
materials, we carried out hydroxyapatite (HAp) mineralization in stretched
tough double network (DN) hydrogels. Anisotropic mineralization of
HAp took place in stretched hydrogels, as revealed by high brightness
synchrotron X-ray scattering and transmission electron microscopic
observation. The <i>c</i>-axis of mineralized HAp aligned
along the stretching direction, and the orientation degree <i>S</i> calculated from scattering profiles increased with increasing
in the elongation ratio λ of the DN gel, and <i>S</i> at λ = 4 became comparable to that of rabbit tibial bones.
The morphology of HAp polycrystal gradually changed from spherical
to unidirectional rod-like shape with increased elongation ratio.
A possible mechanism for the anisotropic mineralization is proposed,
which would be one of the keys to develop mechanically anisotropic
organic/inorganic hybrid materials
Studies on the Formation Mechanism and the Structure of the Anisotropic Collagen Gel Prepared by Dialysis-Induced Anisotropic Gelation
We have found that dialysis of 5 mg/mL collagen solution
into the
phosphate solution with a pH of 7.1 and an ionic strength of 256 mM
at 25 °C results in a collagen gel with a birefringence and tubular
pores aligned parallel to the growth direction of the gel. The time
course of averaged diameter of tubular pores during the anisotropic
gelation was expressed by a power law with an exponent of 1/3, suggesting
that the formation of tubular pores is attributed to a spinodal decomposition-like
phase separation. Small angle light scattering patterns and high resolution
confocal laser scanning microscope images of the anisotropic collagen
gel suggested that the collagen fibrils are aligned perpendicular
to the growth direction of the gel. The positional dependence of the
order parameter of the collagen fibrils showed that the anisotropic
collagen gel has an orientation gradient