Key Engineering Materials Vols. 342-343

Abstract: The aim of this study was to estimate the mechanical properties and evaluate the biocompatibility of silk and PGA scaffolds as an artificial ligament to an ACL reconstruction. The scaffold for the artificial ligament was braided / knitted silk or PGA thread. The mechanical properties, cell growth, and subcutaneous tissue reactions were determined for both types of scaffolds. The breaking load of the PGA scaffold was double that of the sericin removed silk scaffold (SRSS). However, the initial attachment and growth of human ACL cells on the SRSS was superior to the PGA scaffold. In addition, the immune response was significantly higher on the PGA scaffold after 72 h (p<0.05) compared with the sericin removed silk scaffold by T lymphocyte and mononuclear cells (MNCs) in vitro cultures. In vivo, the ACL scaffold made from silk or PGA were implanted in the subcutaneous layer in rats and harvested 1 week later. A histological evaluation of the scaffolds explants revealed the presence of monocytes in the SRSS, and an absence of giant cells in all cases. An inflammatory tissue reaction was more conspicuous around the silk scaffold containing sericin and even more around the PGA scaffold compared with SRSS. These results support the conclusion that a properly prepared SRSS, aside from providing benefits in terms of biocompatibility both in vitro and in vivo, can provide suitable scaffolds for the support of ACL cell growth. These results suggest that a SRSS for ACL repair can overcome the current limitations with the PGA scaffold. And SRSS is biocompatible, and the in vitro T cell and MNCs culture model showed inflammatory responses that were comparable to those observed in vivo.

Abstract: Poly ε-caprolactone(PCL)/hydroxyapatite(HA) composite scaffolds were fabricated by particulate leaching and freeze drying routes with different HA content. Porosity was decreased with HA addition, while mean pore size was maintained at around porogen size regardless of HA content. Compressive modulus was increased with increasing HA content. In this study, the optimum content of HA was around 40% in weight against PCL to obtain the highest compressive modulus with keeping porosity above 85%. HA apparently enhanced proliferation of osteoblast-like MG63 cells in PCL/HA composite scaffolds. Typical adhesion, migration and aggregation procedure of MG63 cells were found on PCL, while spreading morphology only was found on HA even at the early stage of adhesion without migration or aggregation.

Abstract: Adhesions are abnormal attachments between tissues, caused by an inflammatory stimulus or trauma. It was generally used physical barriers and various agents to prevent from adhesions formation. In this study, we made an experiment on animals with wound covering material of substance to prevent tissue adhesion. It was performed in sub-acute toxicity, and tested local effects after implantation.

Abstract: Attachment and viability of different cell types(fibrioblast, chondrocyte and osteoblast ) was observed on two forms of silk (mat & Three-dimensional scaffolds). The osteoblasts behaviors cultured on silk mat were significantly higher than that found on 3-D silk fibroin scaffold (3-D SF scaffold). In the MTT assay, the cell viability of fibroblasts, chondrocyte and osteoblasts seeded on 2-D nanofiber mat was (2-D mat) significantly higher than that found on 3-D SF scaffold. Similar result could be seen from SEM observation and cell attachment study. However, alkaline phosphatase activity was significantly increased on 3-D SF scaffold than on2-D nanofiber

Abstract: Despite many outstanding research works on cartilage tissue engineering, actual clinical application is not quite successful because of the absorption and progressive distortion of tissue engineered cartilage. We have developed a new method of cartilage tissue engineering comprising chondrocyte mixed Pluronic F-127 and cultured chondrocyte cell sheet which entirely cover the cell-Pluronic complex. We believe the addition of cultured chondrocyte cell sheet enhances the efficacy of chondrogenesis in vivo. Human ear cartilage piece was enzymatically dissociated and chondrocyte suspension was acquired. Chondrocytes were cultured and expanded as the routine manner. Cultured chondrocytes were plated in high-density monolayer and cultured with Chondrogenic media in 5% CO2 incubator. After 3 weeks of culture, chondrocyte cell sheet was formed and complete single sheet of chondrocyte could be harvested by gentle manipulation of culture plate with a cell scraper. Chondrocyte-Pluronic complex was established by mixing 1x 106 cells with 0.5 of Pluronic F- 127. Chondrocyte-Pluronic complex was completely covered with a sheet of cultured chondrocyte. The completed tissue engineered constructs were implanted into the subcutaneous tissue pocket of nude mice on the back. Tissue engineered constructs without cultured cell sheet were used as control. Samples were harvested at 8 weeks postoperatively and they were subjected to histological analysis and assayed for glycosaminoglycan (GAG), and type II collagen. Grossly, the size of cartilage specimen of cultured chondrocyte cell sheet covered group was larger than that of the control. On histologic examination, the specimen of cultured chondrocyte cell sheet covered group showed lacunae-containing cells embedded in a basophilic matrix. The chondrocyte cell sheet covered group specimen resembled mature or immature cartilage. The result of measurement of GAG and type II collagen of cartilage specimen of cultured chondrocyte sheet covered group was higher than that of the control. In conclusion, the new method of cartilage tissue engineering using chondrocyte cell sheet seems to be an effective method providing higher cartilage tissue gain and reliable success rate for cartilage tissue engineering.

Abstract: Poly (D,L-latic-co-glycolic acid) (PLGA) has been used as the artificial scaffold for blood vessel formation. In order to hinder smooth muscle cell (SMC) angiogenesis, new scaffold design method of loading Epigallocatechin 3-O-gallate (EGCG) on PLGA film was introduced. PLGA and EGCG were dissolved in acetone and film-shape scaffold was manufactured. Antiangiogenetic effect of EGCG released on scaffold was analyzed for SMC and human umbilical vein endothelial cell (HUVEC) and method for selective inhibition from the difference of growth of SMC and HUVEC was suggested.

Abstract: This paper compares the characteristics of chondrocyte adhesion on two types of threedimensional (3-D) scaffold: types A and B. These 3-D scaffolds can be repeatedly constructed with the same dimensions using microstereolithography, a system that allows the fabrication of predesigned internal structures, such as pore size and porosity, by stacking the photopolymerized material. In tissue engineering, chondrocyte adhesion to a scaffold should have a major effect on the regeneration of cartilage. In this regard, we evaluated chondrocyte adhesion to two types of scaffold and found that chondrocyte adhesion was better on the type B scaffold than on the type A, demonstrating the importance of scaffold geometry in chondrocyte adhesion.

Abstract: The purpose of this study is to confirm the possibility of regenerating actual fat tissue using human adipose tissue-derived stem cells (ASCs) and hyaluronic acid-collagen sponge in animal model. Human ASCs of young female adults were isolated and culture expanded in basal media. At the second passage, cultured ASCs suspension containing 106 cells was applied on prewetted scaffolds the hyaluronic acid-collagen sponge and the sponges was exposed to adipogenic media for the 1week. Then the tissue engineered constructs were implanted into the subcutaneous pocket on the back of immunodeficient athymic nude mice for 3 weeks. Hyaluronic acid-collagen sponges without human ASCs were used as the control. After 3 weeks, specimens were harvested and adipogenic potentials were assessed with histological examination, RT-PCR for PPAR-γ2 expression and G-3-PDH activity. Tissue engineered fat tissue from ASCs and hyaluronic acid-collagen sponges demonstrated PPAR-γ2 positive expression and positive Oil red O staining. The histologic study showed definitive adipose tissue and rich vascular tissue within the engineered fat. Two-fold higher activities of G-3-PDH were identified in experimental group after 3 weeks as compared to control. By contrast, the specimen from control group did not show active vessel ingrowth and contained only few cellular elements within the scaffold. The control specimens failed to demonstrate adipogenic gene markers and were negative in oil red O staining. In conclusion, human ASCs can be differentiated into adipocytes and actual fat tissue engineering was possible with combination of adequate scaffold materials, such as hyaluronic acid-collagen sponges. These data demonstrate that fat tissue engineered from human ASCs can retain predefined shape and dimension for soft tissue augmentation and reconstruction of defects.

Abstract: To improve ostegenic healing efficiency by demineralized bone matrix, we evaluated the ectopic bone formation induced by variously demineralized allogenic cortical bone matrices at subcutaneous and muscular sites in rats. The rat tubular cortical bone matrices were demineralized in heated 0.6N HCl at 60 °C for 5 and 20 mins, respectively, using a controlledheat ultrasonic cleaner and implanted in rat dorsal subcutaneous pouches and thigh muscles for 1-3 weeks. The influence of the demineralized condition of bone matrix on cellular proliferation and osteogenic differentiation was also evaluated in vitro by MTT assay and ALP staining. The cortical matrices were completely demineralized within 20 mins by sonication and heating of diluted 0.6 N HCl. The sonicated bone matrices in heated acidic solution at 60 °C revealed no adverse immunogenic and inflammatory response in vivo regardless of demineralized condition. Cellular proliferation and osteoblastic differentiation was facilitated by more fully demineralized. Ectopic bone formation was induced only by demineralized bone matrices and were more favorable in fully demineralized matrices. The ectopic bone induction was more favorably in subcutaneous pouches than in muscular tissue. These findings suggest that a fully demineralized cortical bone matrix maximizes osteogenic repair by exposing more bioactive molecules which in turn induce chondro- and osteognic differentiation of mesenchymal cells around the implanted matrices, and that the sonication of diluted 0.6 N HCl heated at 60 ° C is a rapid and effective method for sterile demineralized graft preparation.

Abstract: Porous and bioactive composite scaffolds based on poly ε-caprolactone(PCL) and hydroxyapatite(HA) were successfully fabricated by solvent casting and salt leaching method. The scaffolds have interconnected pore structure with pore size ranging from 10μm to 500μm. The pore size of PCL scaffold and PCL/HA scaffold were similar to that of the salt particles. The pore walls became thick and the small pores on the surface of macropores were formed as the HA increased. MTT assay showed that HA content did not affect initial cell attachment in both PCL scaffolds and PCL/HA scaffolds. The osteoblasts proliferated in both scaffolds, but the cell number was higher in the PCL/HA composite scaffolds. It was found that the incorporation of hydroxyapatite enhances bone cell proliferation rather than initial cell attachment in PCL/HA composite scaffolds. The results suggest that the PCL/HA composite scaffolds have a potential for the bone tissue engineering applications.