OBJECTIVETo investigate a method that constructing a tissue-engineered tendon with a continuous and heterogeneous transition region.

METHODSFibroblasts derived from rabbit epithelial tissue were cultured in vitro and collagen gel was prepared. The experimental groups were scaffold only group, fibroblasts+ chondrocytes group (Fb+ CC group), fibroblasts+ osteoblasts group (Fb+ OB group), fibroblasts+ chondrocytes+ osteoblasts group (Fb+ CC+ OB group). Heterogeneous cell populations(fibroblasts, chondrocytes and osteoblasts) with collagen gel were seeded within three predesigned specific regions (fibrogenesis, chondrogenesis, and osteogenesis) of decellularized rabbit achilles tendons to fabricate a stratified scaffold containing three biofunctional regions supporting fibrogenesis, chondrogenesis, and osteogenesis. The tests of morphology, architecture and cytocompatibility of the scaffolds were performed. Gradient tissue-specific matrix formation was analysed within the predesignated regions via histological staining and immunofluorescence assays.

RESULTSThe HE staining and scanning electron microscopy analysis demonstrated that no major cell fragments or nuclear material was evident, and increased intra-fascicular and inter-fascicular spaces were found, the cytocompatibility of the scaffolds showed that the numbers of viable cells on the scaffold surfaces increase steadily, no significant differences were found between the scaffold only containing ordinary culture medium and scaffold containing gel groups. Histological staining and immunofluorescence assays demonstrated that the cartilage-related markers (GAG, COL2A1) were found only in the chondrogenesis region, but bone-related proteins only in the osteogenesis region of bone tunnel, and fibrosis was remarkable for the fibrogenesis region in the joint cavity. The transitional architecture with ligament-fibrocartilage-bone was constructed in the ligament-bone tunnel interface.

CONCLUSIONSA transitional interface (fiber-fiberocartilage-bone) could be replicated in a decellularized tendon through stratified tissue integration in vitro. The cell-tendon complex offers the advantages of a multi-tissue transition involving controlled cellular interactions and matrix heterogeneity.

Animals ; Bone and Bones ; Cells, Cultured ; Chondrocytes ; cytology ; Collagen ; Fibroblasts ; cytology ; Ligaments ; Osteoblasts ; cytology ; Rabbits ; Tendons ; Tissue Engineering ; methods

*Bone Marrow Transplantation ; Cell Differentiation/physiology ; Cell Division/physiology ; Ligaments/*cytology ; Ligaments/surgery ; *Mesenchymal Stem Cell Transplantation ; Tendon Injuries/*surgery ; Tendons/*cytology ; *Tissue Engineering ; Wound Healing/physiology

Ligament injury always has an unsatisfied outcome because of the poor blood supply and scar tissue formation. This may result in severe joint dysfunction. Tissue engineering, as a most prospective field, may provide an effective approach for the treatment of ligament injury. This paper has reviewed some recently published articles focusing on the sources of seed cells in ligament tissue engineering, application of growth factors, screening of scaffold materials with specific mechanical and biodegradable properties, and interaction between cells and scaffold materials. At present, what should be extensively studied are scaffolds with specific mechanical and biodegradable properties, and bioreactors providing three-dimensional culture microenvironment mimic in vivo.
Animals ; Biocompatible Materials ; Cell Differentiation ; Cells, Cultured ; Humans ; Ligaments ; injuries ; surgery ; Ligaments, Articular ; injuries ; surgery ; Mesenchymal Stromal Cells ; cytology ; Tissue Engineering

OBJECTIVETo culture fibroblast cells from the knee ligaments and to study the biological characteristics of these cells.

METHODSCells of the anterior cruciate ligament (ACL) and the medial collateral ligament (MCL) from New Zealand white rabbit were cultured in vitro. Cellular growth and expression of the collagen were analyzed. Moreover, an in vitro wound closure model was established and the healing of the ACL and the MCL cells was compared.

RESULTSMaximal growth for all these cells were obtained with Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, but RPMI 1640 and Ham's F12 media were not suitable to maintain these cells. Morphology of both ACL and MCL cells from New Zealand white rabbit was alike in vitro, but the MCL cells grew faster than the ACL cells. Both cell types produced similar amount of collagen in culture, but the ratio of collage type I to type III produced by ACL cells was higher than that produced by MCL cells. Wound closure assay showed that at 36 hours after injury, cell-free zones created in the ACL cultures were occupied partially by the ACL cells; in contrast, the wounded zone in the MCL cultures was almost completely covered by the cells.

CONCLUSIONSAlthough the ACL cells and the MCL cells from New Zealand white rabbit show similar appearance in morphology in culture, the cellular growth and the biochemical synthesis of collagen as well as the healing in vitro were significantly different. These differences in intrinsic properties of the two types of cells in vitro might contribute to the differential healing potentials of these ligaments in vivo.

Animals ; Anterior Cruciate Ligament ; cytology ; Cell Division ; physiology ; Cells, Cultured ; Collagen ; metabolism ; Collateral Ligaments ; cytology ; Culture Media ; Female ; Fibroblasts ; physiology ; Male ; Rabbits ; Sensitivity and Specificity

Bone marrow mesenchymal stem cells (BMSCs) can be directed to differentiate into a variety of cell types depending on their micro-environment. In this study, rat BMSCs were co-cultured with rat ligament fibroblasts during different time courses. The mRNA expressions of type I, type III collagens and tenascin-C were measured by real time RT-PCR, and the corresponding protein levels of type I and type III collagens by radioimmunoassay. Results show that the mRNA expressions of type I and type III collagens in the BMSCs were 2 times up-regulated after a 6-day co-culture, and the relative mRNA expressions of type I and type III collagens were 3.9 +/- 0.2 and 1.9 +/- 0.2, while they were 1.9 +/- 0.3 and 0.8 +/- 0.1 in the control groups, respectively. The protein syntheses of these two collagens were also increased after a 12-day co-culture; the type I and type III collagens synthesis were 13.6 +/- 1.3 ng/microg and 5.9 +/- 0.5 ng/microg in co-culture groups and 12.4 +/- 0.8 ng/microg and 5.0 +/- 0.4 ng/microg in their control groups, respectively. Likewise, there was a 2 times enhancement in tenascin-C mRNA expression after the 12-day co-culture (0.07 +/- 0.02 by control group and 0.14 +/- 0.02 by co-culture group, P < 0. 05). These data suggest that the presence of the ligament fibroblast promotes the syntheses of type I and the III collagens and tenascin-C in the rat BMSC.
Animals ; Bone Marrow Cells ; cytology ; Cells, Cultured ; Coculture Techniques ; Collagen Type I ; metabolism ; Collagen Type III ; metabolism ; Fibroblasts ; cytology ; Ligaments, Articular ; cytology ; Mesenchymal Stromal Cells ; cytology ; RNA, Messenger ; metabolism ; Rats ; Tenascin ; metabolism