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Paper Title Page
Abstract: Organ engineering remains a challenge to researchers. We tried to reconstruct kidney-like tissue
using tissue engineering technique. Kidney fragments were isolated from rat E16 embryonic
kidneys and seeded on either ECM gel or on polyglycolic acid (PGA) fibers, then implanted in vivo
into the subcutaneous tissue of nude mice for 2 and 3 weeks, respectively. As a negative control,
ECM alone was implanted. Results showed that kidney like tissue could be formed in ECMfragment
constructs after 2 or 3 weeks of implantation, including the formation of glomeruli,
tubules and collecting ducts. In addition, these structures seemed more mature in 3 weeks
specimens than that of 2 weeks. Further development of this model may lay a solid foundation for
kidney tissue engineering.
1
Abstract: The purpose of the present study was to observe the response and changes of cranial
suture to the distraction forces in growing goats and to examine the expression patterns of TGF-β
and BMP during suture distraction.Twenty growing goats were divided into three groups: control
(n=4), experimental (n=12), and sham (n=4). A pure titanium distractor was placed in the coronal
suture in both the sham and experimental groups. After healing, the distractor was activated for
distraction of the coronal suture at a rate of 0.5 mm/day for 8 days in the experimental group. Three
animals were killed respectively, at 0,2,4 and 8 weeks after completion of suture distraction. No
force was applied in the sham group. X- Ray examination was taken and the coronal suture samples
were harvested and processed for histological analysis and scanning electron microscopic analysis
and immunohistochemistry of TGF-β and BMP. The coronal sutures of experimental group were
separated successfully. Signs of intramembranous bone formation and remodeling were found in the
distracted suture,and the sutural structure almost return to its normal state at 8 weeks after end of
distraction. At 0 and 2 weeks after completion of suture distraction, the collagen fiber bundles were
strengthened and aligned in the direction of the distracted forces. Strong expression of BMP and
TGF-β were detected in the fibroblast-like cells and the active osteoblasts. At 4 weeks after suture
distraction, signs of intramembranous ossification were found in the edge areas of the distracted
suture, and the positive staining of BMP and TGF-β was still noted in the osteoblasts around the
newly formed bone trabeculae. This study suggests that cranial suture expansion can be achieved in
growing animal by distraction osteogenesis. Mechanical strain resulted from distractor can induce
the adaptive remodeling in the cranial suture of growing goats. It also suggests BMP and TGF-β
may play very important roles in the process of bone formation and remodeling during suture
distraction osteogenesis.
5
Abstract: Tendon is an important supportive tissue of human body responsible for normal physical
activity. However, tendon damage and defect remain an important factor for causing disability. The
rise of tissue engineering technology provides an effective means of tendon reconstruction and
repair, which will bring promise for functional recovery. In our center, tendon engineering is one of
major research areas. We have performed the in vivo study by using tenocytes and polyglycolic acid
fibers to reconstruct and repair tendon defect in hen and porcine models. The results demonstrated
the successful regeneration and repair of tendon defects created in different models. In addition,
tendon function was also well recovered by generated autologous tendon tissue that possesses strong
biomechanical property. Recently, we have also successfully generated tendon tissue in vitro by
using static strain device and bioreactors, which could be potentially transplanted as the tendon graft
for tendon defect repair.
9
Abstract: Multi-directional mechanical properties of human cancellous bone tissue were never
measured using a compressive test with microscopic cubic specimens. In this study, a small scale
compressive testing machine with nano meter resolution and a measurement system for Poisson’s
ratio with sub-nano meter resolution were developed to measure accurate microscopic mechanical
properties of human CBT. The measured mean longitudinal (E1), postero-anterior (E2), and lateromedial
(E3) elastic moduli were 3.47 GPa (S.D. ±0.41), 2.57 GPa (S.D. ±0.28), and 2.54 GPa (S.D.
±0.22), respectively. ANOVA showed that the longitudinal elastic modulus (E1) was significantly
(p < 0.01) greater than the postero-anterior (E2) and latero-medial (E3) elastic moduli. For Poisson’s
ratios, ν12 was significantly (p <0.01) higher than ν23 and ν31.
13
Abstract: Fibrin is a natural substrate for growth, adhesion, and migration of mature endothelial
cells (ECs) and a candidate coating material in approaches to graft endothelialization. Adipose
tissue represents an abundant, practical source of donor tissue for stem cells which may be a useful
source for engineering of vascular grafts. However, the optimal substrates that promote
differentiation of adipose tissue-derived stem cells (ASCs) into ECs remain to be elucidated. In the
present study, we investigated whether fibrin can be used as a substratum to support in vitro ECs
differentiation of ASCs and whether fibrinogen concentration can be affect on ECs differentiation of
ASCs. For determination of phenotypic characteristics of ASCs used in this experiment, we
performed flow cytometry analysis. ASCs were plated on fibrin composed of varying
concentrations of fibrinogen and induced into ECs differentiation in presence of VEGF. Before
inducing into ECs, ASCs did not express any markers of hematopoietic cells (CD34, CD45), ECs
(CD31, CD34), and endothelial progenitor cells (CD34, CD133, Flk-1). The degree of ECs
differentiation was determined by capillary network formation, ECs-specific gene expression, and
F-actin assembly. During the first 12 h after seeding, cells spread randomly, moved and formed
small interconnected clusters. These clusters decreased in size and formed a capillary tube at 48 h.
During the further incubation in presence of VEGF for 7 days, ASCs expressed mRNA and protein
of von Willebrand factor (vWF). The degree of ECs differentiation of ASCs was consistently
decreased as fibrinogen concentration increase. Fibrin may be used as biomatrix to promote
differentiation of ASCs into ECs for tissue engineering.
17
Abstract: New strategies to make cultured fibroblasts grafts more appealing are aimed at reducing
the time spent in culture and improving the handling and biologic properties. In the present study,
we developed a simple and effective method to fabricate dermal fibroblasts-populated membrane
based on (1) the use of fibrin as a 3-dimensional matrix and (2) the use of cell- mediate contraction
to make a self assembled, detachable cells-populated membrane. Human dermal fibroblasts were
cultured by explants method. The fibroblasts encapsulated in fibrin were transferred into 6-well
culture plates which pretreated with Sigmacoat® to prevent cell binding on surface of culture dish.
Fibroblasts populated fibrin matrix (FPFM) was cultured in attached condition for 7 days and in
free floating condition for 1 day. The FPFM were contracted, spontaneously released from culture
plate, compacted, and formed tissue-like membrane. The fabricated FPFM revealed uniformly
distributed cells and newly synthesized extracellular matrix was deposited in matrix. FPFM could
successfully graft into full-thickness cutaneous defect of nude mice, and showed significantly
increased wound closure rate. Our results demonstrate that the FPFM membrane delivery system
allows for restoration of both the epidermal and dermal compartments.
21
Abstract: To obtain an enhanced population of cardiomyocytes from differentiating mouse
embryonic stem (ES) cells, we confirmed the role of noggin treatment during the cardiac
differentiation of mouse ES cells. ES cells were cultured in ES medium containing both noggin and
LIF for 3 days on the mouse embryonic fibroblast feeder layer, followed by dissociated and
suspension culture without LIF to form the embryoid body (EB). The next day, noggin was
eliminated and EBs were cultured continuously. Noggin treated ES cells showed a relatively rapid
increase of cardiac marker genes, while the vehicle (PBS) treated group showed no significant
cardiac marker expression at 4 days after the EB formation. Furthermore, Noggin treated ES cells
showed 68.00±9.16% spontaneous beating EBs at 12 days after the EB formation. To develop a
more efficient cardiomyocyte differentiation method, we tested several known cardiogenic reagents
(ascorbic acid, 5’-Azacytidine, LiCl, oxytocin, FGF2 and PDGF-BB) after noggin induction or we
cultured noggin treated ES cells on various extracellular matrixes (collagen, fibronectin and
Matrigel). Quantitative RT-PCR and immunocytochemistry results showed a significantly increased
cardiac differentiation rate in the FGF2 treated group. Differentiation on the collagen extracellular
matrix (ECM) could slightly increase the cardiac differentiation efficiency. These results show the
possibilities for the establishment of selective differentiation conditions for the cardiac
differentiation of mouse ES cells.
25
Abstract: I tested the effectiveness of a particulate dentin and plaster of Paris mixture as a bone
substitute. Histologic analysis indicated that all of the bone defects surrounding the implants treated
with particulate dentin/plaster of Paris were filled with new bone 6 and 12 weeks after surgery. No
significant differences were observed in the new bone forming activity in any species (human,
bovine, pig, rabbit, and dog). No cytotoxicity was detected in cell cultures with added particulate
dentin extract and no specific allergic reactions were seen in the hypersensitivity test. These results
suggested that the combination of particulate dentin and plaster is suitable as an alternative bone
substitute.
29