Bone is a complex, highly structured, mechanically active, three-dimensional (3-D) tissue composed of cellular and matrix elements. We previously published a report on in situ collagen gelation using a rotary 3-D culture system (CG–RC system) for the construction of large tissue specimens. The objective of the current study was to evaluate the feasibility of bone tissue engineering using our CG–RC system. Osteoblasts from the calvaria of newborn Wistar rats were cultured in the CG–RC system for up to 3 wk. The engineered 3-D tissues were implanted into the backs of nude mice and calvarial round bone defects in Wistar rats. Cell metabolic activity, mineralization, and bone-related proteins were measured in vitro in the engineered 3-D tissues. Also, the in vivo histological features of the transplanted, engineered 3-D tissues were evaluated in the animal models. We found that metabolic activity increased in the engineered 3-D tissues during cultivation, and that sufficient mineralization occurred during the 3 wk in the CG–RC system in vitro. One mo posttransplantation, the transplants to nude mice remained mineralized and were well invaded by host vasculature. Of particular interest, 2 mo posttransplantation, the transplants into the calvarial bone defects of rats were replaced by new mature bone. Thus, this study shows that large 3-D osseous tissue could be produced in vitro and that the engineered 3-D tissue had in vivo osteoinductive potential when transplanted into ectopic locations and into bone defects. Therefore, this system should be a useful model for bone tissue engineering.

Tools and methods for analyzing differences in embryos resulting from somatic cell nuclear transfer (NT) in comparison to those derived from normal fertilization are needed to define better the nature of the nuclear reprogramming that occurs after NT. To this end, a collection of bovine blastocyst-derived cell lines was created. In vitro expanded or hatched blastocysts, used as primary culture tissue, were from NT; in vitro maturation, fertilization, and culture (IVF); or parthenogenetic (P) activation. Also, five in vivo-fertilized and developed blastocysts were collected by uterine flushing on the eighth d postfertilization. Whole blastocysts were physically attached to STO feeder layers to initiate all of the cell lines generated. The majority of the cell lines in the collection are trophectoderm, 38 NT-derived, 6 in vivo-derived, 20 IVF-derived, and 13 P-derived. Trophectoderm identity was ascertained by morphology and, in many cases, interferon-tau production. Several visceral endoderm cell lines and putative parietal endoderm cell lines were also established. At approximately 5% efficiency, epiblast masses from NT and IVF blastocysts survived and were isolated in culture. Two epiblast masses were also isolated from P blastocysts. Spontaneous differentiation from the epiblast outgrowths resulted in the establishment of fibroblast cell lines. The use of the trophectoderm cell lines as a comparative in vitro model of bovine trophectoderm and placental function is discussed in relation to NT reprogramming.

Two porcine cell lines of yolk-sac visceral endoderm, designated as PE-1 and PE-2, were derived from in vivo 11-d porcine blastocysts that were either ovoid (PE-1) or at the early tubular stage of elongation (PE-2). Primary and secondary culture of the cell lines was done on STO feeder cells. The PE-1 and PE-2 cells morphologically resembled visceral endoderm previously cultured from in vivo-derived ovine and equine blastocysts and from in vitro-derived bovine blastocysts. Analysis of the PE-1- and PE-2-conditioned medium by 2D-gel electrophoresis and matrix-assisted laser desorption/ionization–time-of-flight–mass spectrometry demonstrated that they produced serum proteins. Reverse transcriptase polymerase chain reaction analysis showed that the cells expressed several genes typical for yolk-sac endoderm differentiation and function including GATA-6, DAB-2, REX-1, HNF-1, transthyretin, alpha-fetoprotein, and albumin. Unlike a porcine liver cell line, the PE-1 and PE-2 cell lines had relatively low inducible P-450 content and EROD activity, and, while they cleared ammonia from the cell culture medium, they did not produce urea. Transmission electron microscopy revealed that the cells were a polarized epithelium connected by complex junctions resembling tight junctions and by lateral desmosomes. Rough endoplasmic reticulum was prominent within the cells. Immunocytochemistry indicated that the PE-1 cells expressed cytokeratin 18 and had robust microtubule networks similar to those observed in in vivo porcine yolk-sac endoderm. Metaphase spreads prepared at passage 26 of the PE-1 cell line indicated a diploid porcine karyotype of 38 chromosomes. The cells have been grown for over 1 yr for multiple passages at 1:10 or 1:20 split ratios on STO feeder cells. The cell lines will be of interest as an in vitro model of the porcine preimplantation yolk-sac tissue.

During the process of angiogenesis, the normally quiescent endothelial cells that line the vasculature are induced to proliferate, migrate and align to form new blood vessels by angiogenic stimuli. Assays for angiogenic factors mostly involve in vivo approaches. The two most commonly used in vivo assays—the chick chorioallantoic membrane (CAM) assay and the rabbit corneal assay are tedious to perform and are technically demanding. Several in vitro assays have also been developed, based on the ability of endothelial cells to form tubes in 3-D matrices. Here, we describe the modification of a microcarrier bead-based assay. This assay combines cells grown on Cytodex-3 microcarrier beads with Matrigel to provide an easy, rapid, and reliable method for evaluating and measuring angio-genic activity. We also describe the differential behavior of normal and transformed endothelial cells cultured in Matrigel.

Human, rat, and mouse studies have demonstrated the existence of a population of adipose mesenchymal stem cells (AMSCs) that can undergo multilineage differentiation in vitro. Understanding the clinical potential of AMSCs may require their use in preclinical large-animal models such as pigs. Thus, the objectives of this study were to establish a protocol for the isolation of porcine AMSCs from adipose tissue and to examine their ex vivo differentiation potential to adipocytes and osteoblast. The porcine AMSCs from passage 4 were selected for differentiation analysis. The adipocytes were identified morphologically by staining with Oil Red O, and the adipogenic marker genes were examined by RT-PCR technique. Osteogenic lineage was documented by deposition of calcium stained with Alzarin Red S, visualization of alkaline phosphatase activity, and expression of marker gene. Our result indicates that porcine AMSCs have been successfully isolated and induced differentiation into adipocytes and osteoblasts. This study suggested that porcine AMSCs are also a valuable model system for the study on the mesenchymal lineages for basic research and tissue engineering.

Two cell lines, i.e., BmE-SWU1 and BmE-SWU2, were established from silkworm embryonic tissues of the reversion phase through primary culture in Grace's medium supplemented with 20% fetal bovine serum. The BmE-SWU1 cell line mainly included diploid spindle cells and round cells, which were large and had severe heteroploidy karyotypes. The population doubling time of the 30th passage of the cell line was 58.7 hr. BmE-SWU2 cells were oblong or round, and small. The population doubling time for the 30th passage of the cell line was 46.6 hr. Of BmE-SWU2 cells 89.9% were diploid (2n=56). Both strains were attached to epithelial-like cell lines and were susceptible to Bombyx mori nucleopolyhedroviruse (BmNPV). Inter simple sequence repeat (ISSR) fingerprinting of silkworm embryonic cell line was obtained.