Key Engineering Materials Vols. 342-343

Abstract: The reimplantation method of cultured chondrocytes broadly has been offered as an alternative for articular cartilage repair. A variety of biologically derived and synthetic polymeric and hydrogel materials also have been investigated for good cell delivery efficiency. Preciously, we examined the feasibility of fibrin gel, mixed with hyaluronic acid(HA) as a cell delivery carrier. In order to reinforce the material, hybrid biomaterials of fibrin/HA composite gels with fibrinolysis inhibition factors(FIFs: aprotinin, DI101, EACA) have been investigated in the present work because we did not satisfy a little progress. These fibrin/HA composite gels added FIFs maintained their structural integrity in long-term culture over 4th weeks. Contrary to our expectation the mass of the fibrin/HA composite with DI 101 was significantly superior to the ones of other combinations. In histological evidence, all of them are showed good positive result of stain of Safranin-O and alcian blue during the culture period. In gross examination, samples of all groups grossly resembled cartilage in color and were resistant to external compression. Our study demonstrates that most favorable polymer can be used good quality tissue engineered cartilage and in this culture systems have been useful for studying the basic biology of chondrocyte biosynthesis of ECM and new cartilage matrix formation without a loss of volume. After all, we proved the safety of inhibitors of the fibrinolytic system without hazardous effect on cell behavior and found out that DI 101 would be the most effective agent.

Abstract: We have developed a direct conjugation method of RGD (Arg-Gly-Asp) into polylactide (PLA) using co-solvent system, which showed high reaction yield compared to one solvent system presumably due to good solubility of reactants. First, hydroxyl groups at the end of PLA were changed into carboxyl acid by ring opening reaction of succinic anhydride. For a coupling reaction with high solubility, RGD was dissolved in water and then PLA in dioxane was added into RGD solution in the presence of 1-(3-dimenthylaminopropyl)3-ethylcarbodiimide hydrochloride (EDC). The conjugation of RGD was confirmed by 1H-NMR, GPC and elementary analysis. The RGD conjugated PLA (RGD-PLA) on poly(lactide-co-glycolide) (PLGA) film significantly increased the number of attached cells compared to PLGA without RGD-PLA, while it did not change static contact angle of PLGA. This result demonstrated this conjugation method would be a useful tool to increase cell affinity of biomaterials without change in hydrophilic/hydrophobic property for tissue engineering applications.

Abstract: This study was designed to investigate the influence of demineralized bone particles (DBP)/PLGA hybrid scaffold on angiogenesis and osteogenesis in a calvarial defect model. DBP/PLGA scaffolds were manufactured by solvent casting/salt leaching method, and each scaffold contained 0, 10, 20, 40, and 80 wt% DBP of PLGA, respectively. A total of 34 rats were operated and bicortical holes were placed on their calvaria. The defects were filled with different ratio DBP/PLGA scaffolds. After 3, 7, 14, and 28 days, specimens were taken and, histologic, immunohistologic and RT-PCR analyses were carried out concerning number of vessels and density of regenerated bone, and angiogenic activation. On days 7, in all experimental groups, bone formation occurred in a direction from defected margin of calvarium to center of implanted scaffold and new vessel formation took place in front of the osteogenic regeneration front. We found that the 20 and 40 wt% DBP/PLGA scaffold was superior in its ability to regenerate new bone, induced more intensive formation of microvasculature and expressed in a higher level of osteocalcin mRNA than other groups.

Abstract: To ascertain the properties of human dental stem cells, various postnatal human stem cells were isolated from extracted human teeth and jawbones. Isolated dental stem cells were plated until losing their ‘stemness’ and were evaluated for their proliferation rate, colony forming efficiency, and expression of a specific stem cell marker. These dental stem cells have the potential to proliferate for more than 10 passages, except for the maxillary bone marrow stem cells (MXBMSCs). In particular, stem cells obtained from the periapical follicle (PAFSCs) were definitely superior to the other dental stem cells in proliferation, colony forming efficiency and expression of specific stem cells marker.

Abstract: Gelatin nanofibers were obtained by the use of TFE/deionized water co-solvent system without adding any other fiber forming material. The diameter of nanofibers was in the rage from 200 to 360 nm. The water-resistant ability and mechanical properties of electrospun gelatin nanofibers were improved by crosslinking in GTA or FA vapor, which increased with crosslinking time. Cytotoxicity evaluation indicates that the crosslinked gelatin membranes support the proliferation of human dermal fibroblast.

Abstract: Recently, it has been studied tissue engineered technique as novel approaches for treatment of the degenerative intervertebral disc (IVD). We designed the hybrid type of IVD mimicked scaffolds with poly(lactide-co-glycolide) (PLGA) and methoxypoly(ethyleneglycol)- poly(
-caprolactone) (MPEG-PCL) diblock copolymers in order to application for the tissue engineered IVD. The MPEG-PCL solutions formed a gel-to-sol phase transitions as the temperature was increasesd. MPEG-PCL diblock copolymers were prepared by ring opening polymerization, and then nucleus pulposus (NP) cell was impregnated. Also, in order to restore annulus fibrosus (AF), we fabricated PLGA scaffold by solvent casting/salt leaching method. We confirmed disc cell function in manufactured scaffold through MTT assay in vitro and gross morphology and special staining in vivo for the possibility of the application of tissue engineering techniques.

Abstract: In this study, we developed an injectable carrier for demineralized bone matrix (DBM) which consists of chitosan and glycerol phosphate (GP). The phase transition behaviors of chitosan and GP solution characterized as a function of temperature. The chitosan and GP solutions were shown to form a sol at room temperature, and the chitosan solution with 10-20 wt% concentrations were found to undergo sol-to-gel phase transitions as the temperature was increased. In situ gel forming implant in vivo was successfully fabricated by simple subcutaneous injection of chitosan and GP solutions with demineralized bone matrix (DBM). von Kossa images of the gel implant formed from solution with DBM revealed the presence of mineral deposits.

Abstract: We have developed tensile and porous neutralized chitosan scaffold (NCS) whose pore size was controlled by freezing temperatures. At -70 oC, mean pore size of 112 μm was obtained. At -196 oC, mean pore size was approximately 70 μm at surface. The scaffold processed at -196 oC showed homogeneous small pores with structural integrity, which may be more useful as a guidance dermal scaffold for inward cell migration. The scaffold processed at –70 oC may be more useful for cell loading scaffold requiring wider pore. Since biodegradability and biocompatibility are crucial parameters for the development of dermal scaffold, we evaluated the rate of NCS degradation in the culture medium containing lysozyme by measuring weight loss as well as mean molecular weight of the scaffold. Approximately 40% weight loss at one week and 70% weight loss at 30 days was observed, which means that 70% of the scaffold will be degraded and releasable if the wound microenvironment is similar to the test condition. Again, mean molecular weight of the scaffold based on gel permeation chromatography was less than 1x105 after 10 day incubation. This result suggests that degradation of the NCS begins earlier than the observation of gross weight loss. We also evaluated whether degradation product of NCS are toxic to the human dermal fibroblast or not. Chitosan oligomer up to 1.0 mg/ml, which corresponds to 10% of the total degradation derivatives of the NCS, did not affect the viability of the dermal fibroblast based on MTT assay. This result along with the biodegradation data suggests that the NCS can be developed as suitable dermal scaffold.

Abstract: The porous neutralized chitosan scaffold (NCS) was prepared by freeze-dry method. Its poor cell binding capacity was improved approximately five folds by mixing or coating of atelomeric type I collagen. In order to recreate wound-healing microenvironment within the NCS for the better wound healing effect, various concentrations of bFGF and fibronectin (FN) were supplied in the secondary freeze-dry process of the scaffold. NCS+ bFGF and NCS+FN improved the cell binding capacity by four folds and three folds respectively. Therefore supplementation of collagen, b-FGF and/or fibronectin in the NCS can improve the biocompatibility of the chitosanbased scaffold which itself revealed poor cell binding capacity.

Abstract: In this study, such behaviors of vascular smooth muscle cells (VSMCs), as proliferation and migration, with serum stimulation were investigated onto (−)-epigallocatechin-3-O-gallate (EGCG)-blended poly(L-lactide-co-ε-caprolactone, PLCL) copolymers (EGCG-b PLCL). VSMCs were primarily cultured from rat aorta, and EGCG-b PLCL films were fabricated by mixing PLCL with EGCG. The proliferation of VSMCs cultured onto EGCG-b PLCL film was significantly suppressed in spite of serum induction. Moreover, recovery of denuded area by VSMCs receiving conditioned media obtained from EGCG-b films was completely inhibited, whereas VSMCs onto intact films migrated into denuded area in response to serum showing essentially complete recovery. These results suggest that inhibition in the behaviors of serum-stimulated VSMCs may be mediated through the anti-proliferative effects of EGCG released from polymer films, and EGCG-b polymers can be applied for fabricating an EGCG-eluting vascular stent.