Advanced Biomaterials VII

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.

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.

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.

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.

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.

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.

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.

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.