Rapid Prototyping of Flexible Structures for Tissue Engineered Ear Reconstruction

The tissue engineered ear has been an iconic symbol of the field since 1991, when the report of an engineered ear in a mouse model was first published A tissue engineered ear has an inherent advantage over conventional approaches because the structure is derived from the patient's own cartilage. In this approach, autologous auricular chondrocytes are harvested from the patient and grown within an ear-shaped scaffold. However, as the scaffold degrades or remodels, the ear-shaped structure undergoes significant distortion, resulting in a skewed ear shape that is smaller and often unrecognizable In order to maintain the desired ear geometry, a composite scaffold concept was developed Methods Several functional requirements for the manufacturing process were identified. First, the wire framework must be created with arbitrary three dimensional (3D) control, and with a diameter significantly smaller than the thickness of normal ear cartilage, which is about 2 mm. The bending stiffness must be sufficiently high so that shape is maintained during neocartilage maturation and sufficiently low such that flexibility of the overall structure is not impaired. The material must be approved for clinical use, and must not cause an inflammatory reaction. Finally, the manufacturing process must be capable of producing single, custom parts without significant cost burden. Plastic surgeons identified titanium and stainless steel as preferred materials due to their long history of success in medical implants Three manufacturing processes were identified that are capable of producing arbitrary shapes with the listed metals: wire bending, direct metal laser sintering (DMLS) Results Ear frameworks produced using DMLS and EBM technology are shown in Interpretation Ear frameworks produced using DMLS and EBM technology are shown i

Dynamic Seeding and in Vitro Culture of Hepatocytes in a Flow Perfusion System

Our laboratory has investigated hepatocyte transplantation using biodegradable polymer matrices as an alternative treatment to end-stage liver disease. One of the major limitations has been the insufficient survival of an adequate mass of transplanted cells. This study investigates a novel method of dynamic seeding and culture of hepatocytes in a flow perfusion system. In experiment I, hepatocytes were flow-seeded onto PGA scaffolds and cultured in a flow perfusion system for 24 h. Overall metabolic activity and distribution of cells were assessed by their ability to reduce MTT. DNA quantification was used to determine the number of cells attached. Culture medium was analyzed for albumin content. In Experiment II, hepatocyte/polymer constructs were cultured in a perfusion system for 2 and 7 days. The constructs were examined by SEM and histology. Culture medium was analyzed for albumin. In experiment I, an average of 4.4 X 106 cells attached to the scaffolds by DNA quantification. Cells maintained a high metabolic activity and secreted albumin at a rate of 13 pg/cell/day. In experiment II, SEM demonstrated successful attachment of hepatocytes on the scaffolds after 2 and 7 days. Cells appeared healthy on histology and maintained a high rate of albumin secretion through day 7. Hepatocytes can be dynamically seeded onto biodegradable polymers and survive with a high rate of albumin synthesis in the flow perfusion culture system.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/63347/1/107632700320874.pd

Extensively Expanded Auricular Chondrocytes Form Neocartilage In Vivo

Objective: Our goal was to engineer cartilage in vivo using auricular chondrocytes that underwent clinically relevant expansion and using methodologies that could be easily translated into health care practice. Design: Sheep and human chondrocytes were isolated from auricular cartilage biopsies and expanded in vitro. To reverse dedifferentiation, expanded cells were either mixed with cryopreserved P0 chondrocytes at the time of seeding onto porous collagen scaffolds or proliferated with basic fibroblast growth factor (bFGF). After 2-week in vitro incubation, seeded scaffolds were implanted subcutaneously in nude mice for 6 weeks. The neocartilage quality was evaluated histologically; DNA and glycosaminoglycans were quantified. Cell proliferation rates and collagen gene expression profiles were assessed. Results: Clinically sufficient over 500-fold chondrocyte expansion was achieved at passage 3 (P3); cell dedifferentiation was confirmed by the simultaneous COL1A1/3A1 gene upregulation and COL2A1 downregulation. The chondrogenic phenotype of sheep but not human P3 cells was rescued by addition of cryopreserved P0 chondrocytes. With bFGF supplementation, chondrocytes achieved clinically sufficient expansion at P2; COL2A1 expression was not rescued but COL1A1/3A1genes were downregulated. Although bFGF failed to rescue COL2A1 expression during chondrocyte expansion in vitro, elastic neocartilage with obvious collagen II expression was observed on porous collagen scaffolds after implantation in mice for 6 weeks. Conclusions: Both animal and human auricular chondrocytes expanded with low-concentration bFGF supplementation formed high-quality elastic neocartilage on porous collagen scaffolds in vivo

Bone marrow Derived Pluripotent Cells are Pericytes which Contribute to Vascularization

Pericytes are essential to vascularization, but the purification and characterization of pericytes remain unclear. Smooth muscle actin alpha (α-SMA) is one maker of pericytes. The aim of this study is to purify the α-SMA positive cells from bone marrow and study the characteristics of these cells and the interaction between α-SMA positive cells and endothelial cells. The bone marrow stromal cells were harvested from α-SMA-GFP transgenic mice, and the α-SMA-GFP positive cells were sorted by FACS. The proliferative characteristics and multilineage differentiation ability of the α-SMA-GFP positive cells were tested. A 3-D culture model was then applied to test their vascularization by loading α-SMA-GFP positive cells and endothelial cells on collagen-fibronectin gel. Results demonstrated that bone marrow stromal cells are mostly α-SMA-GFP positive cells which are pluripotent, and these cells expressed α-SMA during differentiation. The α-SMA-GFP positive cells could stimulate the endothelial cells to form tube-like structures and subsequently robust vascular networks in 3-D culture. In conclusion, the bone marrow derived pluripotent cells are pericytes and can contribute to vascularization