Hidetomi Terai

A biodegradable nanofiber scaffold by electrospinning and its potential for bone tissue engineering

Microporous, non-woven poly( epsilon -caprolactone) (PCL) scaffolds were made by electrostatic fiber spinning. In this process, polymer fibers with diameters down to the nanometer range, or nanofibers, are formed by subjecting a fluid jet to a high electric field. Mesenchymal stem cells (MSCs) derived from the bone marrow of neonatal rats were cultured, expanded and seeded on electrospun PCL scaffolds. The cell-polymer constructs were cultured with osteogenic supplements under dynamic culture conditions for up to 4 weeks. The cell-polymer constructs maintained the size and shape of the original scaffolds. Scanning electron microscopy (SEM), histological and immunohistochemical examinations were performed. Penetration of cells and abundant extracellular matrix were observed in the cell-polymer constructs after 1 week. SEM showed that the surfaces of the cell-polymer constructs were covered with cell multilayers at 4 weeks. In addition, mineralization and type I collagen were observed at 4 weeks. This suggests that electrospun PCL is a potential candidate scaffold for bone tissue engineering.

Microfabrication Technology for Vascularized Tissue Engineering

This work describes the application of advanced microfabrication technologies including silicon micromachining and polymer replica molding towards the field of tissue engineering of complex tissues and organs. As a general approach, tissue engineering of skin, bone and cartilage using cell transplantation on biodegradable matrices has achieved great success. However, such techniques encounter difficulties when applied to complex tissues and vital organs. The principal limitation for such applications is the lack of an intrinsic blood supply for the tissue engineered organ, which experiences significant cell death when the tissue thickness is increased above the 1–2 mm range. In this work, the concept of microfabricated scaffolds is introduced, with the goal of producing organ templates with feature resolution of 1 micron, well in excess of that necessary to fashion the capillaries which comprise the microcirculation of the organ. Initial efforts have resulted in high resolution biocompatible polymer scaffolds produced by replica molding from silicon micromachined template wafers. These scaffolds have been successfully seeded with endothelial cells in channels with dimensions as small as the capillaries.

Endothelialized networks with a vascular geometry in microfabricated poly(dimethyl siloxane).

One key challenge in regenerating vital organs is the survival of transplanted cells. To meet their metabolic requirements, transport by diffusion is insufficient, and a convective pathway, i.e., a vasculature, is required. Our laboratory pioneered the concept of engineering a vasculature using microfabrication in silicon and Pyrex. Here we report the extension of this concept and the development of a methodology to create an endothelialized network with a vascular geometry in a biocompatible polymer, poly(dimethyl siloxane) (PDMS). High-resolution PDMS templates were produced by replica-molding from micromachined silicon wafers. Closed channels were formed by bonding the patterned PDMS templates to flat PDMS sheets using an oxygen plasma. Human microvascular endothelial cells (HMEC-1) were cultured for 2 weeks in PDMS networks under dynamic flow. The HMEC-1 cells proliferated well in these confined geometries (channel widths ranging from 35 μm to 5 mm) and became confluent after four days. The HMEC-1 cells lined the channels as a monolayer and expressed markers for CD31 and von Willebrand factor (vWF). These results demonstrate that endothelial cells can be cultured in confined geometries, which is an important step towards developing an in vitro vasculature for tissue-engineered organs.

In vitro engineering of bone using a rotational oxygen-permeable bioreactor system

Abstract Tissue engineering of bone may supersede the need in the future of autograft procedures to treat bone defects resulting from trauma or developmental diseases. A Rotational Oxygen-Permeable Bioreactor System (ROBS) has recently been developed in our laboratory to reproduce dynamic and gas-permeable culture conditions that would supply optimal oxygen and continuous loading to cell/polymer constructs in culture. The cell culture media in ROBS were examined at 1, 24 and 48 h to evaluate the kinetics of p O 2 , p CO 2 and pH without culturing cells. The results were compared to the kinetics in 100 mm diameter cell culture dishes (Control I: static, gas permeable) and 50 ml centrifuge tubes (Control II: dynamic, non-gas permeable). The results showed the same kinetics in ROBS and Control I, whereas Control II failed to maintain the gas conditions of the media. Next, osteoblasts derived from mesenchymal stromal cells (MSCs) of neonatal rats were cultured in three-dimensional poly( dl -lactide-co-glycolide) (PLGA) foams using ROBS to study the effectiveness of this bioreactor system to support cell growth and differentiation. Mineralization was observed within 2 weeks of culture and was shown throughout the polymer at 7 weeks with embedded osteocytic cells. This study demonstrates the usefulness of ROBS for in vitro bone tissue engineering.

Osteoclastogenesis on tissue-engineered bone.

Bone remodeling plays an important role in bone function. To date, bone tissue-engineering research has focused primarily on bone formation from osteoblasts. This study demonstrates that osteoclastogenesis can occur on a mineralized polymer scaffold. Porcine bone marrow-derived mesenchymal stem cells (pMSCs) and hematopoietic cells were isolated from the bone marrow of Yucatan minipigs (n = 3) and cultured separately. pMSCs were differentiated into osteoblasts, seeded on porous poly(D,L-lactic-co-glycolic acid) foams, and cultured in a rotating oxygen-permeable bioreactor system. Once the cell-polymer constructs had started to mineralize, the hematopoietic cells were added and cocultured to include osteoclastogenesis. The cultured constructs were evaluated by histochemical and microscopic examination. Our results show that osteoblasts and osteoclasts were successfully differentiated from bone marrow on the scaffolds. This is the first demonstration of osteoclast formation on mineralized polymer surfaces.

Microfluidics for Tissue Engineering Microvasculature: Endothelial Cell Culture

Development of an integrated blood supply is a critical step towards tissue engineering vital organs. We are attempting to engineer microvasculature using microfluidic networks for the guidance of endothelial cell growth. In this work, we have focused on fabrication of the microfluidic scaffold, in vitro seeding, and extended cell culture in the device. Capillary networks were fabricated in biocompatible PDMS, sterilized, coated with cell adhesion molecules, and seeded with cells. Cell-containing devices were then connected to a closed-loop bioreactor for long term culture. We have used the device to demonstrate continuous-flow culture of endothelial cells for up to 4 weeks without occlusion or contamination.

Capillary Formation In Microfabricated Polymer Scaffolds

One of the primary challenges for engineering thick, complex tissues such as vital organs is the requirement for a vascular supply for nutrient and metabolite transfer. Earlier work has shown that Solid Freeform Fabrication techniques such as Three-Dimensional Printing (3DP) are capable of producing biodegradable scaffolds for the subsequent formation of a wide range of tissues and organs. While this approach shows great promise as a method for constructing complex tissues and organs in vitro, the resolution of the process is currently limited to length scales larger than the narrowest capillaries in the microcirculation. In this work, microfabrication technology is demonstrated as an approach for organizing endothelial cells in vitro at the size scale of the microcirculation. Standard process techniques utilized to build MEMS (MicroElectroMechanical Systems) devices include photolithography, silicon and glass micromachining, and polymer replica molding. Photolithography is used to print a model network of blood vessels on silicon wafers; the network is designed to replicate the fluid dynamics of the vasculature of a particular tissue or organ. A reverse image of the channel network is formed either by Deep Reactive Ion Etching (DRIE) of silicon or through the use of a thick negative-polarity photoresist (SU-8). Polymeric scaffolds are formed by replica molding, using the silicon wafer as a master mold. Microfluidic chambers have been constructed from PDMS and other biocompatible polymers. Initial cell seeding experiments demonstrate that rat lung endothelial cells attach in a single layer to the walls of these structures without occluding them, providing early evidence that MEMS process technology can serve as a method for organizing capillary networks.