Bone loss due to trauma or disease is prevalent in the U.S. Over 3 million orthopaedic procedures are performed every year; approximately 500,000 of these are bone grafting procedures making bone second only to blood as the most transplanted material. Bone loss is usually treated using autografts or allografts. Although they do have their benefits, each of these materials has a set of drawbacks which limit the extent of their use. With this in mind, the objective of this project is to use tissue engineering to create new scaffolds as practical and functional alternatives for bone replacement and regeneration. The new scaffolds will have a nanofibrous structure and be structurally similar to natural bone, containing both cortical and trabecular areas and microvascularization. To accomplish this task we will complete the following objectives: 1) Perform a finite element analysis to discover the nanofiber orientation necessary for the scaffold to bear the appropriate load. 2) Construct fully mineralized, porous, nanofibrous scaffolds for trabecular bone by combining techniques for nanofiber mineralization, pore creation, and sintering. 3) Creating cortical bone like structures with vascular channels using electrospinning techniques with microfibers and nanofiber mineralization. 4) Creating a full thickness, porous load bearing scaffold by combining the trabecular and cortical scaffolds with sintering techniques. The resulting structure will be engineered from mineralized poly (L-lactic acid) nanofibers and will be designed to withstand the forces experienced in load bearing bones while having enough porosity to allow for full thickness cellular and tissue infiltration.

Intellectual Merit: Although many studies have created scaffolds to replace bone, most of these seek to only replace trabecular bone. No currently available scaffold or technique seeks to mimic the structure and properties of both trabecular and cortical bone, including correctly placed vasculature. The creation of this scaffold would also lead to a solution to the problem of cell movement in nanofiber scaffolds. Typically the pores in nanofibrous scaffolds are too small to allow significant cellular infiltration. The method of micro-porous nanofibrous scaffold fabrication described in this project would solve this problem and could be used for the other tissue engineering applications.

Broader Impacts: The proposed research will advance the field of tissue engineering through the creation of nanofibrous scaffolds that do not hinder cell motility and the production of a full thickness bone graft. This research will provide an opportunity for many students to gain experience in experimental design, data analysis, engineering, and the ability to work in a group environment. Dr. Freeman is devoted to establishing outreach programs to recruit students from underrepresented groups into engineering and science. Currently, he mentors graduate students from all segments of underrepresentation. Since arriving at Virginia Tech over 2 years ago he has also mentioned 3 undergraduates of underrepresented groups. Dr. Freeman is involved in the Multicultural Academic Opportunities Program (MAOP) and the Center for the Enhancement of Engineering Diversity (CEED) at Virginia Tech. Students will be chosen from these programs to conduct research based on this project. He will use aspects from this project in his presentations to encourage student interest in math, science, and engineering.

Project Report

RESEARCH PERFORMED Mineralization: We have treated scaffolds with NaOH in order to create carboxylic acid groups for calcium phosphate binding and incubated them in concentrated simulated body fluid (SBF) to induce rapid mineralization. While it increased the degree of mineralization on PLLA, the treatment weakened the scaffolds. Therefore, we investigated PLLA-gelatin nanofibers. The nucleation sites present in gelatin (a derivative of collagen, which causes mineral nucleation in bone) allow scaffold mineralization without damage due to NaOH treatment. Trabecular Bone Scaffold: To increase scaffold porosity, as seen in trabecular bone, we developed a technique to add salt to electrospun mats. Salt particles are deposited while polymer is being electrospun, resulting in particles embedded into the electrospun mat. The salt crystals can be removed by leaching, leaving behind pores, Fig. 1. This technique allows us to create large pores (greater than 100 μm) and mesoporous pore walls (nanoscale) to match tissue repair requirements, and the cells’ ability to infiltrate the scaffold, Fig. 2. Cortical Bone Scaffold: Cortical bone is composed of mineralized collagenous tubes, we have produced similar tubes by electrospinning onto rotating polyethylene oxide (PEO) microfibers, Fig 3. We have also delivered cells and microbeads into the scaffolds and shown that these they stimulate osteoblast proliferation, Fig 4. 3-D Scaffolds: We have refined our technique for sintering nanofiber matrices together while maintaining their morphology. We can bind multiple electrospun mats together without damaging nanofiber structure; the process increases the number of bonds between nanofibers causing an increase in strength. This allows us to build complex 3-D scaffolds using 2-D mats. We have tested unmineralized structures; the properties are similar to those of trabecular bone, Fig. 5. Vascularization: We have shown that the mineralized PLLA-gelatin nanofibers support the proliferation of human vascular endothelial cells (HVECs), Figure 6. OUTREACH PERFORMED Elementary School Interactions: In order to change students’ perspectives on careers in science and engineering we have used a series of programs designed to give elementary school children an idea about what biomedical engineers do. One method has been the use of webchats. We talk to the students using web cameras and programs such as Skype. Dr. Freeman also exposed students to research through a series of DVDs. In these DVDs he describes his research and one of his graduate students carries out the research while explaining their work. The videos from the DVDs have also been placed online on Dr. Freeman’s laboratory website (in an outreach section) and posted on YouTube. Middle School Interactions: Dr. Freeman has worked with the Center for the Enhancement of Engineering Diversity on their middle school focused "Imagination" summer camp. The camp is designed to excite middle school students about math, science, and engineering through hands on laboratory experiences and talks. High School Interactions : Dr. Freeman provides laboratory experiences for C-Tech2 (Computers and Technology at Virginia Tech) which brings female high school students to Virginia Tech to help develop and sustain their interests in engineering and the sciences. Dr. Freeman played a similar role with the NASA INSPIRE (Interdisciplinary National Science Program Incorporating Research and Education Experience) program, a two-week summer residential program involving high school students from around the country. As part of a program with the College of Agriculture and Life Sciences, Dr. Freeman mentored a student from Uniondale High School in Long Island, NY. At the end of the program she gave a presentation at a student symposium at Virginia Tech. Dr. Freeman also began a 5 week paid summer research program in conjunction with University High School in Newark, NJ. Dr. Freeman has integrated undergraduate research into all of his research projects. The students are actively involved in the research. The lab has been included in many summer undergraduate research programs such as the Bioengineering and Bioinformatics Summer Institute (BBSI) and the summer diversity research program sponsored by the Virginia Tech School of Engineering.

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