Steven M. Sultan

Fat grafting accelerates revascularisation and decreases fibrosis following thermal injury

BACKGROUND Fat grafting has been shown clinically to improve the quality of burn scars. To date, no study has explored the mechanism of this effect. We aimed to do so by combining our murine model of fat grafting with a previously described murine model of thermal injury. METHODS Wild-type FVB mice (n=20) were anaesthetised, shaved and depilitated. Brass rods were heated to 100°C in a hot water bath before being applied to the dorsum of the mice for 10s, yielding a full-thickness injury. Following a 2-week recovery period, the mice underwent Doppler scanning before being fat/sham grafted with 1.5cc of human fat/saline. Half were sacrificed 4 weeks following grafting, and half were sacrificed 8 weeks following grafting. Both groups underwent repeat Doppler scanning immediately prior to sacrifice. Burn scar samples were taken following sacrifice at both time points for protein quantification, CD31 staining and Picrosirius red staining. RESULTS Doppler scanning demonstrated significantly greater flux in fat-grafted animals than saline-grafted animals at 4 weeks (fat=305±15.77mV, saline=242±15.83mV; p=0.026). Enzyme-linked immunosorbent assay (ELISA) analysis in fat-grafted animals demonstrated significant increase in vasculogenic proteins at 4 weeks (vascular endothelial growth factor (VEGF): fat=74.3±4.39ngml(-1), saline=34.3±5.23ngml(-1); p=0.004) (stromal cell-derived factor-1 (SDF-1): fat=51.8±1.23ngml(-1), saline grafted=10.2±3.22ngml(-1); p<0.001) and significant decreases in fibrotic markers at 8 weeks (transforming growth factor-ß1(TGF-ß): saline=9.30±0.93, fat=4.63±0.38ngml(-1); p=0.002) (matrix metallopeptidase 9 (MMP9): saline=13.05±1.21ngml(-1), fat=6.83±1.39ngml(-1); p=0.010). CD31 staining demonstrated significantly up-regulated vascularity at 4 weeks in fat-grafted animals (fat=30.8±3.39 vessels per high power field (hpf), saline=20.0±0.91 vessels per high power field (hpf); p=0.029). Sirius red staining demonstrated significantly reduced scar index in fat-grafted animals at 8 weeks (fat=0.69±0.10, saline=2.03±0.53; p=0.046). CONCLUSIONS Fat grafting resulted in more rapid revascularisation at the burn site as measured by laser Doppler flow, CD31 staining and chemical markers of angiogenesis. In turn, this resulted in decreased fibrosis as measured by Sirius red staining and chemical markers.

Human fat grafting alleviates radiation skin damage in a murine model.

Background: Autogenous fat grafting has been observed to alleviate the sequelae of chronic radiodermatitis. To date, no study has replicated this finding in an animal model. Methods: The dorsa of adult wild-type FVB mice were shaved and depilated. The dorsal skin was then distracted away from the body and irradiated (45 Gy). Four weeks after irradiation, 1.5-cc fat or sham grafts were placed in the dorsal subcutaneous space. Gross results were analyzed photometrically. The animals were euthanized at 4 and 8 weeks after fat or sham grafting and their dorsal skin was processed for histologic analysis. Results: Hyperpigmentation and ulceration were grossly improved in fat-grafted mice compared with sham-grafted controls. This improvement manifested histologically in a number of ways. For example, epidermal thickness measurements demonstrated decreased thickness in fat-grafted animals at both time points (20.6 ± 1.5 &mgr;m versus 55.2 ± 5.6 &mgr;m, p = 0.004; 17.6 ± 1.1 &mgr;m versus 36.3 ± 6.1 &mgr;m, p = 0.039). Picrosirius red staining demonstrated a diminished scar index in fat-treated animals at both time points as well (0.54 ± 0.05 versus 0.74 ± 0.07, p = 0.034; and 0.55 ± 0.06 versus 0.93 ± 0.07, p = 0.001). Conclusion: Fat grafting attenuates inflammation in acute radiodermatitis and slows the progression of fibrosis in chronic radiodermatitis.

Endogenous stem cell therapy enhances fat graft survival.

Background: Lipoaspirate centrifugation creates graded density of adipose tissue. High-density fat contains more vasculogenic cytokines and progenitor cells and has greater graft survival than low-density fat. The authors hypothesize that accelerating the bone marrow–derived progenitor cell response to injected low-density fat will improve its graft survival. Methods: Male 8-week-old FVB mice (n = 60) were grafted with either high-density (n = 20) or low-density (n = 40) human lipoaspirate. Half of the mice receiving low-density fat (n = 20) were treated with a stem cell mobilizer for 14 days. Grafted fat was harvested at 2 and 10 weeks for analysis. Results: Low-density fat, low-density fat plus daily AMD3100, and high-density fat had 26 ± 3.0, 61.2 ± 7.5, and 49.6 ± 3.5 percent graft survival, respectively, at 2 weeks (low-density fat versus low-density fat plus daily AMD3100 and low-density fat versus high-density fat, both p < 0.01). Similar results were observed 10 weeks after grafting. Mice receiving low-density fat plus daily AMD3100 had significantly more vasculogenic progenitor cells per cubic centimeter of peripheral blood (p < 0.01) and more new blood vessels (p < 0.01). Both low-density fat plus daily AMD3100 and high-density fat contained more stromal-derived factor-1&agr; and vascular endothelial growth factor mRNA/protein. Conclusion: Endogenous progenitor cell mobilization enhances low-density fat neovascularization, increases vasculogenic cytokine expression, and improves graft survival to a level equal to that of high-density fat grafts.

Interval Cranioplasty: Comparison of Current Standards

Background: Although different cranioplasty storage methods are currently in use, no study has prospectively compared these methods. The authors compare freezing and subcutaneous storage methods in a rat model. Methods: Trephine defects (10 mm) were created in 45 Sprague-Dawley rats. The cranial bone grafts were stored in an autologous subcutaneous pocket (n = 15), frozen at –80°C (n = 15), immediately analyzed (n = 12), or immediately replanted into the defect (n = 3). After 10 days of storage, the subcutaneous or frozen grafts were either replanted (subcutaneous, n = 3; frozen, n = 3) or analyzed (subcutaneous, n = 12; frozen, n = 12). Grafts underwent histologic analysis, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, alkaline phosphatase assay, mechanical testing, and micro–computed tomographic imaging. Results: After 10 days of storage, physiologic assays demonstrated a significant decrease in cellular functionality (e.g., alkaline phosphatase assay concentration: fresh, 18.8 ± 0.77 mM/mg; subcutaneous, 12.2 ± 0.63 mM/mg; frozen, 8.07 ± 1.1 mM/mg; p < 0.012 for all comparisons). Mechanical integrity (maximal load) of fresh grafts was greatest (fresh, 9.26 ± 0.29 N; subcutaneous, 6.27 ± 0.64 N; frozen, 4.65 ± 0.29 N; fresh compared with frozen, p < 0.001; fresh compared with subcutaneous, p = 0.006). Replantation of subcutaneously stored and frozen grafts resulted in limited bony union and considerable resorption after 12 weeks; in contrast, replanted fresh grafts demonstrated bony union and little resorption. Conclusions: Current preservation methods for interval cranioplasty do not maintain bone graft viability. Subcutaneous storage appears to provide a small advantage compared with freezing.

Augmenting neovascularization accelerates distraction osteogenesis.

Background: Distraction osteogenesis has revolutionized the treatment of craniofacial deformities, but it is limited by lengthy consolidation periods and tenuous healing in certain clinical settings, such as irradiated tissue. In this study, the authors aim to investigate whether increasing neovascularization by progenitor cell mobilization accelerates bone formation during distraction. Methods: Sprague-Dawley rats aged 8 weeks (n = 36) were subjected to unilateral mandibular distraction with 3-day latency, 7-day activation (0.25 mm twice daily), and 21-day consolidation periods. From the beginning of the consolidation period, animals received daily injections of either AMD3100 (bone marrow progenitor cell mobilizing agent) or sterile saline. Animals were euthanized on postoperative day 31; mandibles were harvested; and bone regeneration was assessed using micro–computed tomography, immunohistochemistry, bone morphogenetic protein-2 enzyme-linked immunosorbent assay, and mechanical testing. Results: Immunohistochemistry demonstrated that AMD3100 treatment increased vascular density and bone formation. Micro–computed tomography and dual-emission x-ray absorptiometry demonstrated that AMD3100-treated animals had improved bone generation compared with sham-treated controls. Greater force was required on three-point testing to break AMD3100-treated bone. Bone morphogenetic protein-2 expression was up-regulated with AMD3100. Interestingly, the nondistracted contralateral hemimandibles treated with AMD3100 were also stronger than sham-treated counterparts. Conclusions: Progenitor cell mobilization improves bone regeneration in a rat distraction model. Furthermore, because this effect is seen in healthy bone and in ischemic bone healing during distraction, the mechanism is not merely related to oxygenation, but could be a phenomenon of fluid flow.

Lacunocanalicular Fluid Flow Transduces Mechanical Tension Stress During Distraction Osteogenesis

Abstract The mechanotransduction mechanisms linking distraction device activation to new bone formation remain unknown. We hypothesize that the tension stress of activation during distraction osteogenesis is transmitted through lacunocanalicular fluid flow to initiate the osteogenic signaling cascade. Adult Sprague-Dawley rats (N = 24) were subjected to mandibular osteotomy and application of an external distraction device. After a 3-day latency period, half the animals (n = 12) underwent device activation at 0.25 mm twice daily for 6 days (total activation, 3 mm), and the other half (n = 12) had no activation. On day 10, the animals were injected with fluorescent reactive red lacunocanalicular tracer before killing. Mandibles were harvested, embedded, and sectioned, and reactive red epifluorescence lacunocanalicular flow was measured. Protein was harvested for focal adhesion kinase 1 (FAK1), NESPRIN1, SUN1, LAMIN A/C, and SMAD1 Western blotting as well as for bone morphogenetic protein (BMP)-2 enzyme-linked immunosorbent assay and alkaline phosphatase assay. Lacunocanalicular fluid flow was significantly greater in the distracted samples (60.5 ± 14 vs 10.3 ± 4 molecules of equivalent soluble fluorochrome per megapixel, P = 0.01). Flow distribution demonstrated the highest lacunocanalicular flow near the center of the distraction gap. Increased lacunocanalicular flow resulted in increased FAK1 (P = 0.009), NESPRIN1 (P = 0.01), SUN1 (P = 0.01), and LAMIN A/C (P = 0.008) expression. Focal adhesion kinase 1 activation in the presence of BMP-2 protein expression (P = 0.001) resulted in increased intranuclear SMAD1 phosphorylation (P = 0.04) and alkaline phosphatase activity (P < 0.0001). These findings suggest that activation of the distraction osteogenesis device affects cellular response through changes in lacunocanalicular fluid flow.

1: LACUNOCANALICULAR FLOW AND DISTRACTION OSTEOGENESIS: THEORY AND THERAPEUTICS

Introduction: Our hypothesis is that the tension stress of activation increases lacunocanalicular flow and upregulates the mechanotransductive osteogenic pathway. Furthermore, we hypothesize improving vascularization by endothelial progenitor cell (EPC) mobilization enhances lacunocanalicular flow in the consolidation period, maintaining upregulation of the mechanotransductive pathway to improve and accelerate osteogenesis.

Human Fat Grafting Alleviates Radiation Skin Damage in a Murine Model by Neovascularization and Decreased Fibrosis

METHODS: This study was carried out using a previously developed murine model of radiation skin damage. 4 weeks following radiation exposure (45 Gy) animals received either 1.5 cc of subcutaneous fat graft, or 1.5 cc of saline in the subcutaneous space. Tissue oximetry and gross photometric analysis were carried out biweekly thereafter. Animals were sacrificed at 4 and 8 weeks following fat/saline grafting and their dorsal skin was processed for histological analysis. This analysis Smad-3 staining, Picrosirius Red staining and epidermal thickness measurements.

Lacunocanalicular Flow in Osseous Repair and Regeneration

INTRODUCTION: Our hypothesis is that the tension stress of activation increases lacunocanalicular flow and upregulates the mechanotransductive-osteogenic pathway. Furthermore, we hypothesize improving vascularization by endothelial progenitor cell (EPC) mobilization enhances lacunocanalicular flow in the consolidation period, maintaining upregulation of the mechanotransductive pathway to accelerate osteogenesis. We develop strategies to manipulate this mechanism to recapitulate lacunocanalicular flow and optimize the biomimicry of tissueengineered implants.