Retinoic Acid-induced Differentiation of Rat Mesenchymal Stem Cells into beta-Cell Lineage / 대한이식학회지

BACKGROUNDS: Type I diabetes mellitus (T1DM), an autoimmune disease, is associated with insulin deficiency due to the death of beta-cells. Bone marrow-derived mesenchymal stem cells (BM-MSCs) are capable of tissue repair and thus are a promising source of beta-cell surrogates. METHODS: In this study, the therapeutic potential of BM-MSCs as beta-cell replacements was analyzed both in vitro and in vivo. First, we used retinoic acid (RA) to induce rat BM-MSCs to differentiate into cells of endodermal/pancreatic lineage. Then, differentiated rat BM-MSCs were syngeneically injected under the renal capsule of rats. RESULTS: Analysis of gene expression revealed that rat BM-MSCs showed signs of early pancreatic development, and differentiated cells were qualitatively and quantitatively confirmed to produce insulin in vitro. In vivo study was performed for short-term (3 weeks) and long-term (8 weeks) period of time. Rats that were injected with differentiated MSCs exhibited a reduction in blood glucose levels throughout 8 weeks, and grafted cells survived in vivo for at least 3 weeks. CONCLUSIONS: These findings show that RA can induce differentiation of MSCs into the beta-cell lineage and demonstrate the potential of BM-MSCs to serve as therapeutic tools for T1DM.

Changes in Serum Cytokine Profile after AEB071 (Sotrastaurin) or Tacrolimus versus Their Combinations in Rat Heterotopic Cardiac Allografts / 대한이식학회지

BACKGROUND: AEB071, an orally available PKC inhibitor, prevents organ rejection after transplantation in rodents and man. Furthermore, pro-inflammatory cytokines and inflammatory processes are important mediators of transplanted organ rejection. We therefore examined whether single or combination therapies of AEB071 and/or tacrolimus affect cytokine profiles in a rat cardiac allograft model. METHODS: AEB071 (60 mg/kg twice a day) and tacrolimus (0.6 or 1.2 mg/kg once a day) were orally administered daily after cardiac transplantation. Interferon (IFN)-gamma, interleukin (IL)-1beta, IL-2, IL-4, IL-6, IL-10, and tumor necrosis factor (TNF)-alpha levels in serum were subsequently measured 5 days after cardiac transplantation using a multiplex protein assay system. RESULTS: All cytokine levels were significantly depressed in cardiac transplanted rats treated with AEB071, whereas tacrolimus only reduced IFN-gamma, IL-2, IL-4, IL-6, and IL-10 levels. When administered in combination, AEB071 and low- or high-dose tacrolimus had additive effects on IFN-gamma, IL-4, IL-6, and TNF-alpha. CONCLUSIONS: These results suggest that AEB071 inhibits T cell activation by blocking the production of proinflammatory cytokines, and that tacrolimus combined with AEB071 can effectively regulate inflammatory cytokines in the transplantation setting.

A Case of a Giant Cell Tumor of Tendon Sheath in a Child / 대한피부과학회지

A giant cell tumor of tendon sheath is a slow growing benign soft tissue tumor that is known by a variety of names including fibrous histiocytoma of tendon sheath and fibrous xanthoma of the synovium. Clinically, it presents as a 1~3 cm firm, non-mobile, painless, nontender mass, and mostly occurs at interphalangeal joints of fingers. It shows female predominance and can occur at any age, but it is most common between the third and fifth decades and is rare in children. We now report the case of a 10-year-old girl with a giant cell tumor of tendon sheath on the toe.

Effects of Sinusoidal Electromagnetic Field on Structure and Function of Different Kinds of Cell Lines

This study investigated that whether a 2 mT, 60 Hz, sinusoidal electromagnetic field (EMF) alters the structure and function of cells. This research compared the effects of EMF on four kinds of cell lines: hFOB 1.19 (fetal osteoblast), T/G HA-VSMC (aortic vascular smooth muscle cell), RPMI 7666 (B lymphoblast), and HCN-2 (cortical neuronal cell). Over 14 days, cells were exposed to EMF for 1, 3, or 6 hours per day (hrs/d). The results pointed to a cell type-specific reaction to EMF exposure. In addition, the cellular responses were dependent on duration of EMF exposure. In the present study, cell proliferation was the trait most sensitive to EMF. EMF treatment promoted growth of hFOB 1.19 and HCN-2 compared with control cells at 7 and 14 days of incubation. When the exposure time was 3 hrs/d, EMF enhanced the proliferation of RPMI 7666 but inhibited that of T/G HA- VSMC. On the other hand, the effects of EMF on cell cycle distribution, cell differentiation, and actin distribution were unclear. Furthermore, we hardly found any correlation between EMF exposure and gap junctional intercellular communication in hFOB 1.19. This study revealed that EMF might serve as a potential tool for manipulating cell proliferation.

Effects of Sinusoidal Electromagnetic Field on Structure and Function of Different Kinds of Cell Lines

This study investigated that whether a 2 mT, 60 Hz, sinusoidal electromagnetic field (EMF) alters the structure and function of cells. This research compared the effects of EMF on four kinds of cell lines: hFOB 1.19 (fetal osteoblast), T/G HA-VSMC (aortic vascular smooth muscle cell), RPMI 7666 (B lymphoblast), and HCN-2 (cortical neuronal cell). Over 14 days, cells were exposed to EMF for 1, 3, or 6 hours per day (hrs/d). The results pointed to a cell type-specific reaction to EMF exposure. In addition, the cellular responses were dependent on duration of EMF exposure. In the present study, cell proliferation was the trait most sensitive to EMF. EMF treatment promoted growth of hFOB 1.19 and HCN-2 compared with control cells at 7 and 14 days of incubation. When the exposure time was 3 hrs/d, EMF enhanced the proliferation of RPMI 7666 but inhibited that of T/G HA- VSMC. On the other hand, the effects of EMF on cell cycle distribution, cell differentiation, and actin distribution were unclear. Furthermore, we hardly found any correlation between EMF exposure and gap junctional intercellular communication in hFOB 1.19. This study revealed that EMF might serve as a potential tool for manipulating cell proliferation.

Tissue Engineered Intervertebral Disc by Atelocollagen Scaffolds and Growth Factors / 대한척추외과학회지

STUDY DESIGN: In vitro experimental study. OBJECTIVES: To examine the cellular proliferation, synthetic activity and phenotypical expression of intervertebral disc (IVD) cells seeded on types I and II atelocollagen scaffolds, with the stimulation of TGF-beta1 and BMP-2. SUMMARY OF LITERATURE REVIEW: Recently, tissue engineering is regarded as a new experimental technique for the biological treatment of degenerative IVD diseases, and has been highlighted as a promising technique for the regeneration of tissues and organs in the human body. Research on cell transplantation in artificial scaffolds has provided that the conditions for tissue engineering have to be equilibrated, including the cell viability and proliferation, maintenance of characteristic phenotype, suitable scaffolds in organisms and biologically stimulated growth factor. MATERIAL AND METHOD: Lumbar IVD cells were harvested from 10 New Zealand white rabbits, with the nucleus pulposus cells isolated by sequential enzymatic digestion. Each of 1% types I and II atelocollagen dispersions were poured into a 96-well plate (diameter 5 mm), frozen at -70 degrees C, and then lyophilized at -50 degrees C. Fabricated porous collagen matrices were made using the cross-linking method. Cell suspensions were imbibed by surface tension into a scaffold consisting of atelocollagen. The cell cultured scaffolds were then treated with TGF-beta1 (10 ng/ml) or BMP-2 (100 ng/ml) or both. After 1 and 2 week culture periods, the DNA synthesis was measured by [3H] thymidine incorporation, and newly synthesized proteoglycan by incorporation of [35S] sulphate. Reverse transcription-polymerase chain reactions for the mRNA expressions of type I and II collagen, aggrecan and osteocalcin were performed. The inner morphology of the scaffolds was determined by scanning electron microscopy (SEM). RESULTS: The IVD cultures in collagen type II with TGF-beta1 demonstrated an increase in proteoglycan synthesis and up regulation of aggrecan and types I and II collagen mRNA expressions compared to the control. IVD cultures in the type I atelocollagen scaffold with growth factors exhibited an increase in DNA synthesis and up regulation of the type II atelocollagen mRNA expression. With all combinations of growth factor, the IVD cultures in types I and II atelocollagen scaffolds showed no up regulation of the osteocalcin mRNA expression. Furthermore, there was no synergistic effect of TGF-beta1 and BMP-2 in the matrix synthesis or for the mRNA expression of the matrix components. CONCLUSIONS: Nucleus pulposus cells from rabbit were viable in atelocollagen types I and II atelocollagen scaffolds. The type I atelocollagen scaffold was suitable for cell proliferation, but the type II atelocollagen scaffold was more suitable for extracellular matrix synthesis. The IVD cells in both scaffolds were biologically responsive to growth factors. Taken together, nucleus pulposus cells in atelocollagen scaffolds, with anabolic growth factors, provide a mechanism for tissue engineering of IVD cells.

Tissue Engineered Intervertebral Disc by Atelocollagen Scaffolds and Growth Factors / 대한척추외과학회지

STUDY DESIGN: In vitro experimental study. OBJECTIVES: To examine the cellular proliferation, synthetic activity and phenotypical expression of intervertebral disc (IVD) cells seeded on types I and II atelocollagen scaffolds, with the stimulation of TGF-beta1 and BMP-2. SUMMARY OF LITERATURE REVIEW: Recently, tissue engineering is regarded as a new experimental technique for the biological treatment of degenerative IVD diseases, and has been highlighted as a promising technique for the regeneration of tissues and organs in the human body. Research on cell transplantation in artificial scaffolds has provided that the conditions for tissue engineering have to be equilibrated, including the cell viability and proliferation, maintenance of characteristic phenotype, suitable scaffolds in organisms and biologically stimulated growth factor. MATERIAL AND METHOD: Lumbar IVD cells were harvested from 10 New Zealand white rabbits, with the nucleus pulposus cells isolated by sequential enzymatic digestion. Each of 1% types I and II atelocollagen dispersions were poured into a 96-well plate (diameter 5 mm), frozen at -70 degrees C, and then lyophilized at -50 degrees C. Fabricated porous collagen matrices were made using the cross-linking method. Cell suspensions were imbibed by surface tension into a scaffold consisting of atelocollagen. The cell cultured scaffolds were then treated with TGF-beta1 (10 ng/ml) or BMP-2 (100 ng/ml) or both. After 1 and 2 week culture periods, the DNA synthesis was measured by [3H] thymidine incorporation, and newly synthesized proteoglycan by incorporation of [35S] sulphate. Reverse transcription-polymerase chain reactions for the mRNA expressions of type I and II collagen, aggrecan and osteocalcin were performed. The inner morphology of the scaffolds was determined by scanning electron microscopy (SEM). RESULTS: The IVD cultures in collagen type II with TGF-beta1 demonstrated an increase in proteoglycan synthesis and up regulation of aggrecan and types I and II collagen mRNA expressions compared to the control. IVD cultures in the type I atelocollagen scaffold with growth factors exhibited an increase in DNA synthesis and up regulation of the type II atelocollagen mRNA expression. With all combinations of growth factor, the IVD cultures in types I and II atelocollagen scaffolds showed no up regulation of the osteocalcin mRNA expression. Furthermore, there was no synergistic effect of TGF-beta1 and BMP-2 in the matrix synthesis or for the mRNA expression of the matrix components. CONCLUSIONS: Nucleus pulposus cells from rabbit were viable in atelocollagen types I and II atelocollagen scaffolds. The type I atelocollagen scaffold was suitable for cell proliferation, but the type II atelocollagen scaffold was more suitable for extracellular matrix synthesis. The IVD cells in both scaffolds were biologically responsive to growth factors. Taken together, nucleus pulposus cells in atelocollagen scaffolds, with anabolic growth factors, provide a mechanism for tissue engineering of IVD cells.

Interaction of Mesenchymal Stem Cells and Osteoblasts for in vitro Osteogenesis

It has recently been reported that bone marrow-derived mesenchymal stem cells (MSCs), which are systemically administrated to different species, undergo site-specific differentiation. This suggests that the tissue specific cells may cause or promote the differentiation of the MSCs toward their cell type via a cell-to-cell interaction that is mediated not only by hormones and cytokines, but also by direct cell-to-cell contact. In this study, in order to assess the possible synergistic interactions for osteogenesis between the two types of cells, the MSCs derived from rabbit bone marrow were co-cultured with rat calvarial osteoblasts in direct cell-to-cell contact in a control medium (CM) and in an osteogenic medium (OM). The cell number, alkaline phosphatase activity, and amount of calcium deposition were assayed in the cultures of MSCs, osteoblasts, and co-cultures of them in either OM or CM for up to 40 days. The cell numbers and the alkaline phosphatase activities in the co-culture were somewhere in between those of the osteoblast cultures and the MSC cultures. The amounts of deposited calcium were lower in the co-culture compared to those of the other cultures. This suggests that there are little synergistic interactions during osteogenesis in vitro between the rat osteoblasts and rabbit MSCs.

The Effects of Recombinant Human BMP-7, Prepared from a COS-7 Expression System, on the Proliferation and Differentiation of Rat Newborn Calvarial Osteoblasts

A family of proteins, the bone morphogenetic proteins (BMPs), which promote osteoblast differentiation and bone mineralization, have recently been identified. One, BMP-7, has shown the ability to induce cartilage and bone formation processes. In this report, the possibility that other cell lines, to CHO cells, may also be available as host cells for the expression of hBMP-7 was validated. Recombinant human BMP (rhBMP) -7 was produced in COS-7 cells, as a processed mature disulfide-linked homodimer, with an apparent molecular weight of 36, 000. Examination of the expressions of the markers characteristic of osteoblast phenotypes showed that the rhBMP-7 specifically stimulated the inductions of alkaline phosphatase (ALP) (5-fold increase at 100 ng of rhBMP-7/ml), parathyroid hormone (PTH) -mediated intracellular cAMP production (4-fold increase at 100 ng of rhBMP-7/ml) and osteocalcin synthesis (5-fold increase at 100 ng of rhBMP-7/ml). In summary, the in vitro mineralization assay results provide evidence that the rhBMP-7 peptide, produced by COS-7 expression system, possesses intact biological activity. A similar pattern of biological activity was observed for the BMP-7 in COS-7 cells compared to the corresponding CHO cell expression system. Thus, these findings can be experimentally utilized for the production of rhBMPs for in vitro or in vivo studies.

Behavior of Fibroblasts on a Porous Hyaluronic Acid Incorporated Collagen Matrix

A hyaluronic acid (HA) incorporated porous collagen matrix was fabricated at -70 degree C by lyophilization. The HA incorporated collagen matrix showed increased pore size in comparison with collagen matrix. Biodegradability and mechanical properties of matrices were controllable by varying the ultraviolet (UV) irradiation time for cross-linking collagen molecules. Addition of HA to collagen matrix did not effect ultimate tensile stress after UV irradiation. HA incorporated collagen matrices demonstrated a higher resistance against the collagenase degradation than collagen matrix. In an in vitro investigation of cellular behavior using dermal fibroblasts on the porous matrix, HA incorporated collagen matrix induced increased dermal fibroblast migration and proliferation in comparison with collagen matrix. These results suggest that the HA incorporated collagen porous matrix assumes to enhance dermal fibroblast adaptation and regenerative potential.

Evaluation of the Cytotoxicity of Polyetherurethane (PU) Film Containing Zinc Diethyldithiocarbamate (ZDEC) on Various Cell Lines

A polyetherurethane (PU) film containing 0.1% zinc diethyldithiocarbamate (ZDEC) is the international standard reference material for testing the in vitro cytotoxicity of polymer based biomaterials. Nowadays, culturing L929 or BALB/3T3 cells in direct contact or in an extract dilution condition is the most frequently using method for evaluating the cytotoxicity from biomaterials and medical devices. However, the results often vary, because it is directly related to the cellular functions and the mechanism of the toxicity of the contacting cells. In this study, 13 cell lines originating from various tissues were used to detect the cytotoxic activities of a PU film containing 0.1% ZDEC (PU-ZDEC). The correlation between the reactivity zone size and the relative cytotoxicity by quantifying the released total protein from each cell in the direct contact testing method was investigated. Hepa-1c1c7 cells demonstrated the highest sensitivity in the reactivity zone size, while CHO/dhFr(-) cells were the most sensitive in terms of the relative cytotoxicity. A correlation between the two processes in each cell line was not found (r=-0.478). In the extract dilution method, which involved cultivating the cells in the medium with various ZDEC concentrations prepared by diluting the PU incubation, the cytotoxicity increased with increasing ZDEC concentration in all cell lines. The BALB/ 3T3 cells demonstrated the highest sensitivity in the extract dilution method. No correlation in a comparison of the relative cytotoxicity from the direct contact method with the extract dilution method in each cell line, was found (r=-0.445). In this experiment, Hepa-1c1c7, BALB/3T3, CHO/dhFr(-) and L-929 cells among the 13 types of cell lines were the sensitive cell lines according to the two methods. The preliminary results suggest that a comparison of at least one or more cytotoxicity testing methods and many cell lines is necessary for an in vitro cytotoxicity test of biomaterials.

Effect of Centrifugal Force on Cellular Activity of Osteoblastic MC3T3-E1 Cells in vitro

The effects of centrifugal force on growth and differentiation of osteoblastic cells cultured in alpha-MEM containing 1% Fetal bovine serum were investigated by assays of DNA synthesis, alkaline phosphatase activity and osteocalcin- production in osteoblastic MC3T3-E1 cells. Centrifugation of the cells in low concentrations (1%) of fetal bovine serum caused a 1.9-fold increase of [3H] thymidine incorporation on day 3 from the start of centrifugation, and gradually decreased with culture up to day 9. Alkaline phosphatase activity was not affected by centrifugal force until day 5, and increased rapidly after day 7. Stimulation of DNA synthesis by centrifugation was abolished in the presence of H-7, an inhibitor of protein kinase C. These results suggest that centrifugal force stimulates the proliferation of osteoblastic cells through an autocrine secretion of some diffusable growth- promoting activity. Additional centrifugation of the cells also slightly stimulated alkaline phosphatase activity, although this did not directly influence the cell's osteocalcin-production activity.

Calcification Comparison of Polymers for Vascular Graft

Polytetrafluoroethylene (PTFE), polyurethane (PU) and silicone are widely known biocompatible polymers which are commonly used for vascular grafts. However, in vitro and in vivo calcifications of these polymers have been found to seriously compromise their quality as biomaterials. In consideration of this problem, the present study compared the calcification rate and extent of PTFE, PU and silicone. Using the in vitro flow-type method, PTFE, PU and silicone films were tested for 1, 4, 7, 10, 14 and 21 days. After 21 days of in vitro calcification test, the calcium levels on PTFE, PU and silicone were 35.89 5.01 microgram /cm2, 23.73 0.68 microgram/cm2 and 19.86 5.28 microgram/cm2, respectively. The higher observed calcium level for PTFE may be due to the effect of the rough surface of PTFE in accumulating calcium ions on the polymer surface. From the 7th day of test, the [Ca]/[P] molar ratio started to decrease over time, and PTFE showed a faster calcification process. This decreasing [Ca]/[P] molar ratio demonstrated the typical calcification mechanism consisting of phosphorus ion accumulation following calcium ion accumulation. This study concluded that PU and silicone are less calcified than PTFE film, a finding in good agreement with previously published studies.

Preparation of Collagen Modified Hyaluronan Microparticles as Antibiotics Carrier

Hyaluronan (HA), a natural glycoaminoglycan featuring an extracellular matrix, has been suggested as an effective biocompatible material. In this study, the effectiveness of HA microparticles as a carrier system for antibiotics was evaluated, and their physicochemical characteristics were determined. Microparticles were fabricated by the gelation of sulfadiazine (SD) loaded HA solution with calcium chloride through either a granulation (GR-microparticles) or encapsulation (EN-microparticles) process, and atelocollagen was incorporated into the microparticles as an additive in order to improve their physical properties. The characteristics of the microparticles were examined by scanning electron microscopy (SEM), differential scanning calorimetry (DSC), and swelling test. In vitro release experiments were performed for 7 days and the released amount of SD was determined using high-performance liquid chromatography (HPLC). Microscopic observations revealed that the collagen incorporated HA particles had a more compact surface than the HA particles. DSC analysis determined a loss of SD crystallinity in the particles. Calcium chloride retarded the swelling of particles, whereas the loaded drug contents did not affect this property. Both GR-and EN-microparticles sustained SD release with initial bursting effect. SD release from EN-microparticles was faster than from GR- microparticles. In addition, the release rate was dependent on the SD content in the microparticles. These results suggest that collagen modified HA microparticles have a potential as a release rate controlling material for crystalline drugs such as SD.

Characterization of UV-irradiated dense/porous collagen membranes: morphology, enzymatic degradation, and mechanical properties

Collagen-based membranous materials of various shapes (gel, film, sponge) are known to be the most promising materials in terms of facilitating the regeneration of dermal defects. In this study, dense and porous collagen membranes were fabricated using air-drying and freeze-drying processes, respectively, and the effect of ultraviolet (UV) radiation on the degree of membrane crosslinking was evaluated by in vitro biodegradation and mechanical testing. A non-irradiated membrane group was used as the negative control and a glutaraldehyde (GA) treated group as the positive control. Scanning electron microscopy showed that, as the freezing temperature decreased to -196 degrees C, the resultant mean pore sizes also decreased; optimal pore size was obtained at a freezing temperature of -70 degrees C. In vitro biodegradation and mechanical testing demonstrated that GA treatment or 4 hours of exposure to UV radiation significantly increased both resistance to collagenase and mechanical strength versus the untreated controls, regardless of the collagen membrane type (dense or porous). Our results suggest that UV treatment is a useful tool for the fabrication of collagen membranes designed to be used as dermal dressings.

A bone replaceable artificial bone substitute: morphological and physiochemical characterizations

A composite material consisting of carbonate apatite (CAp) and type I atelocollagen (AtCol) (88/12 in wt/wt%) was designed for use as an artificial bone substitute. CAp was synthesized at 58 degrees C by a solution-precipitation method and then heated at either 980 degrees C or 1,200 degrees C. In this study, type I AtCol was purified from bovine tail skins. A CAp-AtCol mixture was prepared by centirfugation and condensed into composite rods or disks. The scanning electron-microscopic (SEM) characterization indicated that the CAp synthesized at 58 degrees C displayed a crystallinity similar to that of natural bone and had a high porosity (mean pore size: about 3-10 microns in diameter). SEM also revealed that the CAp heated at 980 degrees C was more porous than that sintered at 1,200 degrees C, and the 1,200 degrees C-heated particles were more uniformly encapsulated by the AtCol fibers than the 980 degrees C-heated ones. A Fourier transformed-infrared spectroscopic analysis showed that the bands characteristic of carbonate ions were clearly observed in the 58 degrees C-synthesized CAp. To enhance the intramolecular cross-linking between the collagen molecules, CAp-AtCol composites were irradiated by ultraviolet (UV) ray (wave length 254 nm) for 4 hours or vacuum-dried at 150 degrees C for 2 hours. Compared to the non cross-linked composites, the UV-irradiated or dehydrothermally cross-linked composites showed significantly (p < 0.05) low collagen degradation and swelling ratio. Preliminary mechanical data demonstrated that the compressive strengths of the CAp-AtCol composites were higher than the values reported for bone.

Viability evaluation of engineered tissues

Biohybrid artificial organs encompass all devices capable of substituting for an organ or tissue function and are fabricated from both synthetic materials and living cells. The viability of engineered tissue could be related to the viability of implanted cells. The system of viability assay for mammalian cell culture can be applied to the determination of cell viability for engineered tissue. This review explores various methods of cell viability assay which can be applied to the viability evaluation of engineered tissue. The major criteria employed in viability assays include survival and growth in tissue culture, functional assay, metabolite incorporation, structural altercation, and membrane integrity. Each viability assay method is based on different definitions of cell viability, and has inherent advantages and disadvantages. In order to be able to assess the viability of cells with one assay method, it is desirable to compare the viability measurements from various assays derived from different criteria.

Viability of cells in cryopreserved canine cardiovascular organs for transplantation

To determine applicability of the cryopreservation procedure for vessel grafts, the viability of endothelial cells (ECs) among the whole cells in three kinds of organs artery, vein, trachea in mongrel dogs was evaluated on the basis of histological analysis. The Griffonia simplicifolia agglutins-fluorescein isothiocyanate (GSA-FITC) and propidium iodide (PI) double staining methods were combined with flow cytometry (FCM), which was able to simultaneously determine the viability of whole cells and ECs from the same tissue, were performed after harvesting, after antibiotic solution treatment, and after cryopreservation and thawing. In most cases, the viability of ECs is lower than that of whole cells from veins and arteries. The viability of whole cells in veins was maintained until the antibiotic solution treatment and then decreased significantly after cryopreservation and thawing, while the ECs began to decrease significantly after the antibiotic solution treatment and more markedly decreased after thawing. The viability of ECs and whole cells from arteries was similar to that of the veins' conditions. The viability of whole cells from the trachea decreased with a similar pattern to that of the ECs from vessels. In consideration of maintaining cell viability among the three kinds of organs, the viability of arteries was better than that of the others. The cells in the trachea demonstrated a lower viability than the vessels. The effect of antibiotic solution treatment on the reduction of cell viability depends on the treatment time and temperature.

Tissue restoration, tissue engineering and regenerative medicine

Recently, thanks to the rapid progress of new technologies in cell modulation, extracellular matrix fabrication and synthetic polymers mimicking bodily structures, the self-regeneration of bodily defects by host tissue has been considered by many researchers. The conventional science of art in biomaterials has been concerned with restoring damaged tissue using non-biological materials such as metals, ceramics and synthetic polymers. To overcome the limitations of using such non-viable materials, several attempts to construct artificial organs mimicking natural tissue by combining modulated cells with extracellular matrix-hybridized synthetic polymers have produced many worthy results with biologically functioning artificial tissues. The process involved in manufacturing biomaterials mimicking living tissue is generally called tissue engineering. However recently, the extension of knowledge about cell biology and embryology has naturally moved the focus from tissue restoration to tissue regeneration. Especially, embryonic and mesenchymal stem cells are attractive resources due to their potential for the differentiation of various tissue cells in response to signal transduction mediated by cytokines. Although no one knows yet what is the exact factor responsible for a stem cell's ability to differentiate between specific cells to generate specific tissue, what has been agreed is that delivering stem cells into the body provides a strong potential for the regeneration of tissue. In this review, the historical issues and future possibilities involved in medical tissue restoration and tissue regeneration are discussed.

Viability and enzymatic activity of cryopreserved porcine heart valve

Fibroblast viability of a natural tissue valve for replacing a defective heart valve through allograft or xenograft has been suggested to affect its clinical durability. In this study, the cell viability and enzymatic activity of porcine heart valve leaflets were examined in regard to concerning to the preservation process [variable warm ischemic time (WIT), cold ischemic time (CIT), and cryopreservation]. Porcine heart enblocs were obtained and valve dissection was performed after 2, 12, 24, or 36 hours, in respective groups A, B, C, and D, as WIT. Each group was stored for 24 hours as CIT and cryopreserved. Leaflets were dissected from a valved conduit after each process, and cell viability and enzymatic activity in the leaflet were investigated using trypan blue staining and API ZYM kits. WIT extension significantly decreased fibroblast viability (p < 0.05, 92.25 +/- 2.7% at 2 hours, 84.9 +/- 6.7% at 12 hours, 57.0 +/- 10.2% at 24 hours, 55.9 +/- 7.9% at 36 hours), while CIT for 24 hours was also influenced significantly (p < 0.05), whereas cryopreservation demonstrated no effect on cellular viability. In enzyme activity observation, several enzymes related to lipid or nucleotide degradation (esterase, esterase lipase, particularly phosphatase, phosphohydrolase) were remarkably changed following the valve-fabrication process. After 24 hours CIT, these enzymatic activities in groups B, C and D significantly increased, but the activities decreased after cryopreservation. Particularly, both the viability and enzymatic activity showed remarkable changes after CIT in group B (WIT = 12 hours). These results suggest that WIT is more important than CIT in maintaining viability of the valve, and that completing all the cryopreservation process within 12 hours after acquisition is recommended.