41.16 Three Dimensional Confocal Imaging Analysis of Adipose Tissue

Posted on January 25, 2017

N. N. Chung1, C. P. Blackshear1, C. Ransom1, D. Irizarry1, D. Nguyen1, M. Longaker1,2, D. C. Wan1  1Hagey Laboratory For Pediatric Regenerative Medicine,Stanford University Medical Center,Stanford, CA, USA 2Institute For Stem Cell Research And Regenerative Medicine,Stanford University Medical Center,Stanford, CA, USA

Introduction:  Autologous fat grafting is frequently used to correct soft tissue defects, but outcomes remain unpredictable. Studies have shown stromal cell enrichment of fat grafts to improve volume retention, but the mechanism of this effect has been primarily explained through inferential gene expression analysis and two-dimensional histologic staining. In this study, we employed novel three-dimensional confocal imaging to determine how graft architecture changes with stromal cell enrichment.

Methods:  Confocal microscopy with optical sectioning was employed to reconstruct three-dimensional data of whole mounted tissue using Imaris. Unprocessed human abdominoplasty fat, as well as unenriched human fat grafts and stromal cell enriched fat grafts after two, four, six, and eight weeks of implantation in immunocompromised mice were evaluated. Supplemental stromal cells were fluorescently tagged to determine three-dimensional distribution throughout the fat graft. Isosurface rendering was employed to determine individual adipocyte size and variability, vascular density, and proximity of stromal cells to new vasculature.

Results: Preliminary data has shown that confocal imaging of adipose tissue results in superior images that elucidate tissue architecture and more closely approximates the graft in situ. Images allowed for clear visualization of supplemented ASCs within the graft environment, showing an intimate relationship with revascularization patterns in the fat graft. Heterogeneity in adipocyte size was also appreciated with fat grafts supplemented with stromal cells appearing more similar to unprocessed abdominoplasty fat.

Conclusion: Confocal imaging allows for direct three-dimensional visualization of the fat graft environment that approximates in situ, which holds promise for future study of CAL grafts and retention.

 

41.04 A mouse model of mandibular distraction osteogenesis

Posted on January 25, 2017

R. C. Ransom1,2, T. Leavitt1, C. D. Marshall1, L. Barnes1, D. C. Wan1, M. T. Longaker1,2  1Hagey Laboratory For Pediatric Regenerative Medicine,Department Of Surgery, Division Of Plastic Surgery, Stanford University School Of Medicine,Palo Alto, CA, USA 2Institute For Stem Cell Biology And Regenerative Medicine,Stanford University,Stanford, CA, USA

Introduction: Distraction osteogenesis (DO) refers to the gradual mechanical lengthening of a bone segment, resulting in the growth of new bone. DO is a powerful method of endogenous bone tissue engineering that has been applied to the craniofacial skeleton in patients with critical-sized bone defects. However, the cellular and molecular signaling that regulates this process of de novo bone formation is not well understood. We aimed to develop a rigorous and genetically dissectable mouse model of mandibular DO.

Methods: Mandibular DO devices were manufactured using computer-aided design (CAD) software and 3D printing. Animals were divided into four groups: sham-operated (n = 6 per time point), fracture (n = 6 per time point), acutely lengthened (n = 8 per time point) and gradually distracted (n = 8 per time point) right hemimandibles. Gradual DO was performed at a rate of 0.15 mm every 12 h over the course of 10 days to a total length of 3 mm.  Acute lengthening was performed at a single time point to a total length of 3 mm. Therefore, the total amount of distraction lengthening (3 mm) was maintained across both groups. Specimens were harvested at mid-consolidation (POD 29) and end consolidation (POD 43) time points for micro-computed tomography (CT) and histological analysis.

Results: Histologic and CT analyses confirmed that fractured mandibles healed normally through a cartilaginous callous with complete bone bridging by POD 43. However, gradually distracted mandibles revealed a remarkable pattern of new bone formation. Two weeks into consolidation (POD 29), greater than half of the distraction gap was filled with trabecular bone. No cartilage was seen within the distraction gap, indicating that the regenerate was produced through direct intramembranous bone formation. At the end of consolidation (POD 43), trabecular bone completely bridged the osteotomy site of all gradually distracted specimens. Trabecular bone along the edges of the osteotomies was remodelled to lamellar, cortical bone. Three-dimensional reconstruction of the CT images of gradually distracted specimens clearly showed complete osseous bridging of the osteotomy gap. In marked contrast to the above data, the distraction gap of all mandibles undergoing acute lengthening was filled with fibrous tissue at end-consolidation and there was minimal histological or radiographic evidence of new bone formation at the osteotomized bone.

Conclusions: We have developed and a model of mandibular DO in mice, and begun to characterize the processes that lead to the formation of new bone. This model will afford tremendous opportunities to utilize transgenic mouse systems that are not available in larger animals. Experiments utilizing this unique model will offer valuable insight into the biology of de novo bone formation and mechanical forces guiding distraction osteogenesis, speeding the development of clinical therapies for patients suffering from critical-sized bone defects.