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3-D integrated organ printing for ear reconstruction
PLASTIC/MAXILLOFACIAL SURGERY Microfluidic single cell transcriptional analysis reveals subpopulations of adipose derived stromal cells with enhanced angiogenic potential Elizabeth R Zielins, MD, Michael Januszyk, MD, PhD, Anna Luan, Elizabeth A Brett, Kevin Paik, Graham G Walmsley, Geoffrey C Gurtner, MD, FACS, Michael T Longaker, MD, MBA, FACS, Derrick C Wan, MD, FACS Stanford University, Stanford, CA
3-D integrated organ printing for ear reconstruction Carlos Kengla, Hyun Wook Kang, PhD, John D Jackson, PHD, Sang Jin Lee, James Yoo, MD, PhD, Anthony Atala, MD, FACS Wake Forest School of Medicine, Winston-Salem, NC INTRODUCTION: The auricular cartilage of the outer ear is composed of chondrocytes embedded in an extracellular matrix (ECM) predominated by collagen (type II), elastin, and various glycosaminoglycan macromolecules. Tissue engineered cartilage for outer ear reconstruction has several disadvantages including fibrocartilage instead of elastic cartilage and weak structural integrity. This study seeks to demonstrate the feasibility of using an Integrated Organ Printing (IOP) platform to fabricate a construct able to develop cartilage tissue in a stable 3-D shape.
INTRODUCTION: Cell-assisted lipotransfer (CAL), addition of autologous adipose-derived stromal cells (ASCs) from the stromal vascular fraction (SVF) of adipose tissue, has shown promise for improvement of fat graft retention, likely via promotion of early revascularization. As ASCs are extremely heterogeneous, identification of subpopulations with enhanced secretion of pro-angiogenic growth factors for use in CAL may improve fat grafting outcomes.
METHODS: Primary rabbit chondrocytes were isolated from New Zealand White rabbits. Cells were plated and expanded up to passage 2. Constructs were printed with the IOP system. Constructs were cultured for one month in chondrogenic differentiation media or implanted subcutaneously after culture overnight. The in vitro cultured and in vivo implanted constructs were evaluated at 1 month by histology, immunohistochemistry, biochemical assay, and biomechanical testing.
METHODS: Human lipoaspirate was processed for SVF and individual cells were isolated in a 96-well plate containing RT-PCR reagents by flow cytometry. Single cell lysates were loaded onto a 96.96 IFC Chip, with primers for angiogenic genes and multiple cell surface markers. A Fuzzy C-Means algorithm was employed to determine functionally distinct clusters based on angiogenic gene expression, and linear discriminant analysis used to identify surface markers capable of distinguishing clusters. Isolation of cells based on identified markers and qRT-PCR for angiogenic gene expression was performed to validate findings.
RESULTS: 3-D bioprinted ear constructs, analyzed after 1 month of culture in chondrogenic media, demonstrated positive staining for cartilage formation throughout the growing tissue. Retrieved constructs after one month of implantation showed positive staining for cartilage.
RESULTS: Eight cell-surface markers delineating functional subgroups of ASCs based on angiogenic gene expression were identified. Analysis of freshly harvested SVF by flow cytometry using CD248, the strongest correlating surface marker, showed 16% of cells were CD248+ and 84% were CD248-. qRTPCR demonstrated CD248+ cells had significantly higher VEGFa expression than both unsorted and CD248- cell populations.
CONCLUSIONS: Based on histological findings, this study shows that the IOP platform is able to print living chondrocytes, resulting in the production of cartilaginous tissue. The constructs can be printed in 3D shapes including that of the outer ear. Important aspects for successful clinical translation may include the optimization of construct architecture, polymeric biomaterial for appropriate degradation time/rate, and reliable cell sources and combinations for cartilage and perichondrium regeneration.
ª 2015 by the American College of Surgeons Published by Elsevier Inc.
CONCLUSIONS: Significant heterogeneity exists among ASCs, but using a single cell transcriptional profiling strategy, we identified a functionally distinct subpopulation with enhanced angiogenic capacity. The cell-surface marker CD248 may be employed to isolate this subgroup, and use of these cells may enhance early revascularization and fat graft volume retention.
e26
http://dx.doi.org/10.1016/j.jamcollsurg.2015.08.365 ISSN 1072-7515/15
3-D integrated organ printing for ear reconstruction Carlos Kengla, Hyun Wook Kang, PhD, John D Jackson, PHD, Sang Jin Lee, James Yoo, MD, PhD, Anthony Atala, MD, FACS Wake Forest School of Medicine, Winston-Salem, NC INTRODUCTION: The auricular cartilage of the outer ear is composed of chondrocytes embedded in an extracellular matrix (ECM) predominated by collagen (type II), elastin, and various glycosaminoglycan macromolecules. Tissue engineered cartilage for outer ear reconstruction has several disadvantages including fibrocartilage instead of elastic cartilage and weak structural integrity. This study seeks to demonstrate the feasibility of using an Integrated Organ Printing (IOP) platform to fabricate a construct able to develop cartilage tissue in a stable 3-D shape.
INTRODUCTION: Cell-assisted lipotransfer (CAL), addition of autologous adipose-derived stromal cells (ASCs) from the stromal vascular fraction (SVF) of adipose tissue, has shown promise for improvement of fat graft retention, likely via promotion of early revascularization. As ASCs are extremely heterogeneous, identification of subpopulations with enhanced secretion of pro-angiogenic growth factors for use in CAL may improve fat grafting outcomes.
METHODS: Primary rabbit chondrocytes were isolated from New Zealand White rabbits. Cells were plated and expanded up to passage 2. Constructs were printed with the IOP system. Constructs were cultured for one month in chondrogenic differentiation media or implanted subcutaneously after culture overnight. The in vitro cultured and in vivo implanted constructs were evaluated at 1 month by histology, immunohistochemistry, biochemical assay, and biomechanical testing.
METHODS: Human lipoaspirate was processed for SVF and individual cells were isolated in a 96-well plate containing RT-PCR reagents by flow cytometry. Single cell lysates were loaded onto a 96.96 IFC Chip, with primers for angiogenic genes and multiple cell surface markers. A Fuzzy C-Means algorithm was employed to determine functionally distinct clusters based on angiogenic gene expression, and linear discriminant analysis used to identify surface markers capable of distinguishing clusters. Isolation of cells based on identified markers and qRT-PCR for angiogenic gene expression was performed to validate findings.
RESULTS: 3-D bioprinted ear constructs, analyzed after 1 month of culture in chondrogenic media, demonstrated positive staining for cartilage formation throughout the growing tissue. Retrieved constructs after one month of implantation showed positive staining for cartilage.
RESULTS: Eight cell-surface markers delineating functional subgroups of ASCs based on angiogenic gene expression were identified. Analysis of freshly harvested SVF by flow cytometry using CD248, the strongest correlating surface marker, showed 16% of cells were CD248+ and 84% were CD248-. qRTPCR demonstrated CD248+ cells had significantly higher VEGFa expression than both unsorted and CD248- cell populations.
CONCLUSIONS: Based on histological findings, this study shows that the IOP platform is able to print living chondrocytes, resulting in the production of cartilaginous tissue. The constructs can be printed in 3D shapes including that of the outer ear. Important aspects for successful clinical translation may include the optimization of construct architecture, polymeric biomaterial for appropriate degradation time/rate, and reliable cell sources and combinations for cartilage and perichondrium regeneration.
ª 2015 by the American College of Surgeons Published by Elsevier Inc.
CONCLUSIONS: Significant heterogeneity exists among ASCs, but using a single cell transcriptional profiling strategy, we identified a functionally distinct subpopulation with enhanced angiogenic capacity. The cell-surface marker CD248 may be employed to isolate this subgroup, and use of these cells may enhance early revascularization and fat graft volume retention.
e26
http://dx.doi.org/10.1016/j.jamcollsurg.2015.08.365 ISSN 1072-7515/15