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

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

Engineered vascularized bone grafts

Clinical protocols utilize bone marrow to seed synthetic and decellularized allogeneic bone grafts for enhancement of scaffold remodeling and fusion. Marrow-derived cytokines induce host neovascularization at the graft surface, but hypoxic conditions cause cell death at the core. Addition of cellular components that generate an extensive primitive plexus-like vascular network that would perfuse the entire scaffold upon anastomosis could potentially yield significantly higher-quality grafts. We used a mouse model to develop a two-stage protocol for generating vascularized bone grafts using mesenchymal stem cells (hMSCs) from human bone marrow and umbilical cord-derived endothelial cells. The endothelial cells formed tube-like structures and subsequently networks throughout the bone scaffold 4–7 days after implantation. hMSCs were essential for stable vasculature both in vitro and in vivo; however, contrary to expectations, vasculature derived from hMSCs briefly cultured in medium designed to maintain a proliferative, nondifferentiated state was more extensive and stable than that with hMSCs with a TGF-β-induced smooth muscle cell phenotype. Anastomosis occurred by day 11, with most hMSCs associating closely with the network. Although initially immature and highly permeable, at 4 weeks the network was mature. Initiation of scaffold mineralization had also occurred by this period. Some human-derived vessels were still present at 5 months, but the majority of the graft vasculature had been functionally remodeled with host cells. In conclusion, clinically relevant progenitor sources for pericytes and endothelial cells can serve to generate highly functional microvascular networks for tissue engineered bone grafts

A Highly Tunable Biocompatible and Multifunctional Biodegradable Elastomer

Biodegradable elastomers synthesized under mild conditions with highly tunable mechanical properties are described. These elastomeric biomaterials are biocompatible, exhibit minimal deformation following cyclical tensile loading, and permit tight control over the release kinetics of encapsulated bioactive molecules. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim