Reconstruction after Resection of Undifferentiated Pleomorphic Sarcoma and Invasive Ductal Carcinoma in a Patient with Li-Fraumeni Syndrome.

This case exhibits a presentation of multiple primary malignancies in a patient with Li-Fraumeni syndrome, necessitating surgical excision and multistaged reconstruction. Due to Li-Fraumeni syndrome patients' predisposition to developing malignancies, management includes lifelong surveillance and aggressive treatment of cancers. Plastic surgeons can minimize damage to patient's quality of life by carrying out reconstruction in a thoughtful manner that maximizes function and considers a potential lifetime of future reconstructive needs.

Evaluation of modified frailty index for predicting post-operative outcomes after lower-extremity free-flap reconstruction.

BACKGROUND: Risk stratification for complex procedures such as microsurgical reconstruction of the lower extremities is an important part of preoperative planning and counseling. The aim of this study was to determine the effectiveness of the modified five-item frailty index (5-mFI) score, a validated tool for assessing risk in surgical patients, in predicting postoperative complications after lower extremity (LE) free flap reconstruction. METHODS: A retrospective review of the American College of Surgeons National Surgical Quality Improvement Program (ACS-NSQIP) database was conducted from 2010 to 2020 on patients who underwent LE free-flap reconstruction. 5-mFI scores were calculated and patients were categorized as 5-mFI ≥2 or <2. The primary endpoint was the presence of 30-day overall complications. The secondary endpoints included 30-day readmission, need for reoperation, and need to discharge to a care facility. Comparisons were made using one-way analysis of variances, Pearson's chi-squared test, or Fisher's extract test. Multivariable logistic regression models were performed for sex, age, BMI, smoker status, operative time, and ASA classification. RESULTS: Total of 294 (61.6% males) patients were identified. Univariate analysis showed 5-mFI ≥2 had higher rates of overall complications (p = .043) and hematologic complications (p = .033). In this population, there were also higher rates of reoperation (p = .003) and discharge to care facility (p < .001). Multivariable regression models further substantiated that 5-mFI ≥2 was independently associated with increased overall complications [2.46, CI: 1.10-5.59, p = .031], hematologic complications [2.55, 1.02-6.35, p = .046], reoperation [4.55, 1.54-13.3, p = .006], and discharge to facility [2.86, 1.27-6.45, p = .011]. CONCLUSIONS: There is a strong association of 5-mFI ≥2 with adverse post-operative outcomes in male patients undergoing LE free-flap reconstruction. This can be a valuable adjunct in the counsel of patients for whom lower extremity salvage is feasible.

Literature Review: Robotic-Assisted Harvest of Deep Inferior Epigastric Flap for Breast Reconstruction.

INTRODUCTION: Robotic-assisted surgery is gaining popularity because of reported improvement in aesthetic outcomes while reducing the occurrence of complications compared with conventional surgical methods. Deep inferior epigastric perforator (DIEP) flap harvesting has a long track record as a viable procedure for autologous reconstruction of the breast. In this literature review, we describe the feasibility of using the robotic platform in DIEP flap harvest. METHODS: The Preferred Reporting Items for Systemic Reviews and Meta-Analysis methodology was to guide the literature review. PubMed and Scopus databases were searched from inception to June 6, 2022. The Medical Subject Heading terms and keywords used to conduct this search are as described: "Robotic AND deep inferior epigastric perforator AND Breast reconstruction." RESULTS: Seven publications, detailing a total of 56 robotic-assisted DIEP flap harvest procedures, were selected for review. Four publications used the transabdominal preperitoneal approach, whereas 2 exclusively used a totally extraperitoneal approach, and 1 compared the 2 approaches. The measured outcomes included technical feasibility of flap harvest in cadavers, viable flap harvest in live patients, harvest time and pedicle dissection time, pedicle length, fascial incision length, donor site pain, need for postoperative narcotic, donor site morbidity, and hernia formation. Overall, the reviewed articles demonstrated successful DIEP flap harvesting without the need for conversion to the conventional open procedure. Postoperative complications were minimal. Robotic DIEP flap harvest was shown to be safe and there were no reports of donor-site morbidity in the studies reviewed. The main advantages of the robotic approach include decreased postoperative pain and length of hospital stay, along with improved aesthetic outcomes. The main disadvantages are increased operative time and cost. CONCLUSIONS: Although at its current iteration, the robotic-assisted DIEP flap is feasible, it may not be practical in all settings. Furthermore, the true benefit of the robotic platform is yet to be determined, as more long-term studies are necessary.

Necrotizing Soft Tissue Infection of Abdominal Wall after Lipoabdominoplasty: Complication following Medical Tourism.

Necrotizing soft tissue infection (NSTI) is an acute life-threatening complication of cosmetic surgery. We present a case study of a 48-year-old woman diagnosed with NSTI of the abdominal wall following liposuction, abdominoplasty, and ventral hernia repair performed in Latin America. In the operating room, bowel perforation at the hepatic flexure was observed requiring emergent fascial debridement, bowel resection, and broad-spectrum antibiotics. She required multiple washouts and split-thickness skin grafts. Complications such as NSTI are possible after lipoabdominoplasty and are prevalent in cosmetic medical tourism, due, in part, to varying institutional standards and accreditations, and in some cases lack of consistent adherence to set standards. Complications after medical tourism contribute a significant burden to medical staff and the healthcare system in patients' home countries. Patient education by their surgeons and plastic surgery societies should be prioritized. It is important for domestic emergency physicians, plastic surgeons, and other care providers to understand complications postcosmetic surgery to promote timely management.

Palmar Venous Malformation: A Case Report of a Rare Occurrence and a Treatment Algorithm.

Palmar venous malformations are not well described in the surgical literature and are a therapeutic challenge due to the intricate anatomy of the hand. We present the case of a 19-year-old right-handed female patient from Mexico who was referred to us with a right palmar lesion present since birth, previously diagnosed as a venous malformation. The patient reported that she had undergone sclerotherapy 10 years before in addition to a prior aborted attempt at excision. Venous palmar malformations are rare occurrences. Although an individualized approach is important, the development of an algorithm may assist in the standardization of the treatment of this pathology to preserve the hand's functional status.

Not all flaps are created equal: Assessing the impact of active smoking in muscle-only versus perforator flaps for patients undergoing nonelective extremity-free tissue transfer-A case control study.

INTRODUCTION: Active smoking is known to impair wound healing following free tissue transfer for reconstruction due to its vasoconstrictive effect on the microcirculation. The aim of this study was to evaluate the impact of flap selection on flap loss, in nonelective, traumatic extremity-free soft tissue transfer in active smokers. METHODS: All patients undergoing a free tissue transfer for acute trauma of the extremity at a level I trauma center from 2011 to 2017 were identified. Breast reconstruction and osseous/osseocutaneous flaps were excluded. The study population was divided in two groups based on the type of flap used (muscle versus perforator flap). Factors known to be associated with impaired wound healing were extracted from the database. Primary outcome was major smoking related complications (complete/partial flap loss). Secondary outcomes included minor flap-related complications (infection, dehiscence) and donor site complications. The impact of smoking was assessed for the different type of flaps using multivariate analyses. RESULTS: A total of 118 flaps were identified during the study period. Out of those, 52 were perforator-based fasciocutaneous flaps, while 66 were muscle flaps. Active smoking status resulted in a statistically significant increase in the incidence of major and minor complications in the perforator flap group (36% vs. 4%, adjusted odds ratio, AOR [95%CI]: 2.31[1.48,19.30], adj-p = 0.021 and 32% vs. 17%, AOR [95% CI]: 1.23[1.11,14.31], adj-p = 0.034) but had no impact in the muscle group. CONCLUSIONS: The present study suggests a higher incidence of flap related complications in smokers when a perforator flap was selected but no impact when a muscle flap was utilized.

Impact of Blood Transfusion in Free Flap Breast Reconstruction Using Propensity Score Matching.

BACKGROUND: Microvascular thrombosis has been associated with cytokine release and inflammatory syndromes which can occur as a result of blood transfusions. This phenomenon could potentially lead to complications in breast free flap reconstruction. The aim of this study was to evaluate the impact of perioperative blood transfusion in free flap breast reconstruction using large population analysis. METHODS: The American College of Surgeons National Quality Improvement Program database was queried for delayed free flap breast reconstructions performed in 2016. The study population was divided based on perioperative blood transfusion within 24 hours of the start of the operation. Propensity score matching analysis was used to ensure homogeneity between the two study groups. Primary outcome was unplanned return to the operating room (OR) within 30 days. Secondary outcomes were readmission and complications. RESULTS: A total of 1,256 patients were identified. Out of those, 91 patients received a perioperative blood transfusion. All the patients received only one unit of PRBC within the first 24 hours. Those patients were matched with similar patients who did not receive a transfusion on a ratio of 1:3 (273 patients). Patients who received a transfusion had a significantly higher incidence of reoperation (42 vs. 10%, p < 0.001). Patients who received a transfusion were more likely to return to the OR after 48 hours from the initial operation (13 vs. 5%, p = 0.001). All returns to the OR were due to flap-related complications. Perioperative blood transfusion increased the incidence of wound dehiscence (9 vs. 2%, p = 0.041) but had no protective effect on the development of other postoperative complications. CONCLUSION: Perioperative blood transfusion in free flap breast reconstruction is associated with an increased probability of flap-related complications and subsequent return to the OR without decreasing the probability of developing other systemic postoperative complications.

Perioperative Fluid Resuscitation in Free Flap Breast Reconstruction: When Is Enough Enough?

Perioperative liberal fluid resuscitation (LFR) can result in interstitial edema and venous congestion and may be associated with compromised perfusion of free flaps and higher incidence of wound complications. We hypothesized that restrictive intraoperative fluid resuscitation improves flap perfusion and lowers the wound complication rate in free flap breast reconstruction. METHODS: Patients undergoing free flap reconstruction of the breast from 2010 to 2018 were retrospectively reviewed. The study population was divided into 2 groups, LFR (≥7 ml/kg/h) and restrictive fluid resuscitation (RFR) (<7 ml/kg/h). Mean percutaneous oximetry readings of the flap over the first 24 hours were recorded. Primary outcome was development of any wound complication. Secondary outcomes were mean percutaneous oximetry readings within the first 24 hours, length of stay, and development of acute kidney injury. RESULTS: One hundred twenty-six patients were identified, with 41 patients undergoing LFR. The mean fluid received for the RFR group was 5.5 versus 10.2 ml/kg/h for the LFR group (P < 0.001). LFR resulted in a significantly higher incidence of wound complications (76% versus 15%, P < 0.001). The mean oximetry readings within 24 hours were significantly lower for the LFR group (41% versus 56%, P < 0.001). Urine output intraoperatively and within the first 24 hours was similar between the 2 groups. No patients developed acute kidney injury. CONCLUSION: RFR in free flap breast reconstruction is associated with increased flap perfusion and lower incidence of wound-related complications and should be considered whenever possible.

BRCA1-associated R-loop affects transcription and differentiation in breast luminal epithelial cells.

BRCA1-associated basal-like breast cancer originates from luminal progenitor cells. Breast epithelial cells from cancer-free BRCA1 mutation carriers are defective in luminal differentiation. However, how BRCA1 deficiency leads to lineage-specific differentiation defect is not clear. BRCA1 is implicated in resolving R-loops, DNA-RNA hybrid structures associated with genome instability and transcriptional regulation. We recently showed that R-loops are preferentially accumulated in breast luminal epithelial cells of BRCA1 mutation carriers. Here, we interrogate the impact of a BRCA1 mutation-associated R-loop located in a putative transcriptional enhancer upstream of the ERα-encoding ESR1 gene. Genetic ablation confirms the relevance of this R-loop-containing region to enhancer-promoter interactions and transcriptional activation of the corresponding neighboring genes, including ESR1, CCDC170 and RMND1. BRCA1 knockdown in ERα+ luminal breast cancer cells increases intensity of this R-loop and reduces transcription of its neighboring genes. The deleterious effect of BRCA1 depletion on transcription is mitigated by ectopic expression of R-loop-removing RNase H1. Furthermore, RNase H1 overexpression in primary breast cells from BRCA1 mutation carriers results in a shift from luminal progenitor cells to mature luminal cells. Our findings suggest that BRCA1-dependent R-loop mitigation contributes to luminal cell-specific transcription and differentiation, which could in turn suppress BRCA1-associated tumorigenesis.

Postoperative monitoring of free flap reconstruction: A comparison of external Doppler ultrasonography and the implantable Doppler probe.

HYPOTHESIS: The time to detection of vascular compromise and the postoperative time to re-exploration are shorter using the implantable Doppler (ID) probe, thereby resulting in earlier surgical re-exploration and a higher flap salvage rate. METHODS: A single-centre experience with 176 consecutive free flap reconstructions in 167 patients from 2000 to 2008 in a university-based teaching hospital by retrospective chart review is presented. RESULTS: There was a significant difference in overall flap survival (ID 98.0%, external Doppler [ED] 89.3%) and total flap loss (ID 2.0%, ED 10.7%) between the two groups (P=0.03). The difference in flap salvage rate was not significant (ID 90.9%, ED 63.6%; P=0.068). The false-positive (ID 0%, ED 3%; P=0.18) and false-negative rates (ID 0.0%, ED 4.5%; P=1.0) were not significantly different. There was also a lower median postoperative time to re-exploration for the ID group, from 48 h to one week after initial surgery (ID 74.5 h, ED 136.8 h; P=0.05). CONCLUSION: The present analysis revealed a potential benefit for the ID probe in the postoperative monitoring of free tissue transfers.

HYPOTHÈSE: Le délai entre la détection d'une atteinte vasculaire et la réexploration postopératoire est plus court à l'aide de la sonde Doppler implantable (DI), ce qui favorise une réexploration chirurgicale plus rapide et un taux plus élevé de sauvegarde des lambeaux. MÉTHODOLOGIE: Les auteurs présentent l'expérience monocentrique de 176 reconstructions consécutives par lambeaux libres chez 167 patients entre 2000 à 2008 dans un hôpital universitaire au moyen d'une analyse de leurs dossiers. RÉSULTATS: Il y avait une différence importante dans la survie globale des lambeaux (DI 98,0 %, Doppler externe [DE] 89,3 %) et perte totale des lambeaux (DI 2,0 %, DE 10,7 %) entre les deux groupes (P=0,03). La différence en matière de taux de sauvegarde des lambeaux n'était pas significative (DI 90,9 %, DE 63,6 %; P=0,068). Les taux de résultats faussement positifs (DI 0 %, DE 3 %; P=0,18) et de résultats faussement négatifs (DI 0,0 %, DE 4,5 %; P=1,0) n'étaient pas sensiblement différents. Le délai postopératoire médian avant la réexploration était également plus court dans le groupe DI, soit de 48 heures à une semaine après l'opération initiale (DI 74,5 h, DE 136,8 h; P=0,05). CONCLUSION: La présente analyse a révélé l'avantage potentiel de la sonde DI lors de la surveillance postopératoire des transferts de tissus libres.

Deep Inferior Epigastric Artery Perforator Flap Breast Reconstruction without Microsurgery Fellowship Training.

BACKGROUND: Deep inferior epigastric artery perforator (DIEP) flap breast reconstruction requires complex microsurgical skills. Herein, we examine whether DIEP flap breast reconstruction can be performed safely without microsurgical fellowship training. METHODS: A total of 28 patients and 34 DIEP flaps were included in the study. We reviewed the medical records of patients for donor site and flap-related complications and analyzed the correlation between the complications and preoperative risk factors. We also performed a literature review to compare complication rates in our series with the literature. RESULTS: We observed total flap necrosis in 1 patient (2.9%), partial flap necrosis in 5 patients (14.7%), infection in 1 patient (2.9%), hematoma/seroma in 3 patients (8.8%), donor site complications in 5 patients (18.5%), venous occlusion in 4 patients (11.7%), and arterial occlusion in 1 patient (2.9%). We did not observe any correlation between complications and preoperative risk factors. Literature review yielded 18 papers that met our inclusion criteria. Partial flap necrosis rate was significantly higher in our series compared with literature (14.7% vs 1.6%, P = 0.003). Venous complication rate was marginally higher in our series compared with literature (11.7% vs 3.3%, P = 0.057). However, total flap loss rate in our series was comparable with the literature (2.9% vs 2.2%, P = 0.759). CONCLUSION: With proper training during plastic surgery residency, DIEP flap can be performed with acceptable morbidity.

Differentiated adipose-derived stem cell cocultures for bone regeneration in polymer scaffolds in vivo.

Critical-sized bone defects can lead to significant morbidity, and interventions are limited by the availability and donor-site morbidity of bone grafts. Polymer scaffolds seeded with cells have been explored to replace bone grafts. Adipose-derived stem cells have shown great promise for vascularization and osteogenesis of these constructs, and cocultures of differentiated stem cells are being explored to augment vessel and bone formation. Adipose-derived stem cells were differentiated into endothelial cells and osteoblasts, and in vitro studies showed increased proliferation of cocultured cells compared with undifferentiated adipose-derived stem cells and monocultures of endothelial cells and osteoblasts. The cells were seeded into polylactic acid gas-plasma-treated scaffolds as cocultures and monocultures and then implanted into critical-sized rat calvarial defects. The cocultures were in a 1:1 osteoblast to endothelial cell ratio. The increase in proliferation seen by the cocultured cells in vitro did not translate to increased vascularization and osteogenesis in vivo. In vivo, there were trends of increased vascularization in the endothelial cell group and increased osteogenesis in the osteoblast and endothelial monoculture groups, but no increase was seen in the coculture group compared with the undifferentiated adipose-derived stem cells. Endothelial cells enhance vascularization and osteoblast and endothelial cell monocultures enhance bone formation in the polymer scaffold. Predifferentiation of adipose-derived stem cells is promising for improving vascularization and osteogenesis in polymer scaffolds but requires future evaluation of coculture ratios to fully characterize this response.

Analysis of free flap complications and utilization of intensive care unit monitoring.

We aimed to determine the optimal time for intensive care unit (ICU) monitoring after free flap reconstruction based on the timing of surgical complications. We reviewed retrospectively 179 free flaps in 170 subjects during an 8-year period at University Hospital. Thirty-seven flaps were reoperated due to vascular (n = 16, 8.9%) and nonvascular complications (n = 21, 11.7%). Vascular complications presented earlier relative to nonvascular complications (10.8 versus 99.3 hours). The flap survival rate was 93.2% with a mean ICU length of stay of 6.2 days. The lack of standardized monitoring protocols can lead to overutilization of ICU. Sometimes, flap monitoring is not the limiting factor, as patients with other comorbidities necessitate longer ICU stays. However, our study suggests that close monitoring of flaps seems most critical during the first 24 to 48 hours, when most thrombotic complications occur and prompt identification and re-exploration is critical. Some thrombosis and most hematomas present within 72 hours, and thus close monitoring is still warranted. We suggest close monitoring of free flaps in the ICU or dedicated flap monitoring unit where nursing can check the flap on an every-1-to-2-hour basis for the first 72 hours postoperatively to assure optimal surveillance of any potential problems.

Effect of endothelial differentiated adipose-derived stem cells on vascularity and osteogenesis in poly(D,L-lactide) scaffolds in vivo.

Prevascularization of engineered bony constructs can potentially improve in vivo viability. However, the effect of endothelial cells on osteogenesis is unknown when placed in poly(D,L-lactide) (PLA) scaffolds alone. Adipose-derived stem cells (ASCs) have the ability to differentiate into both osteoblasts and endothelial cells by culture in specific media. We hypothesized that ASC-derived endothelial cells would improve vascularity with minimal contribution to bone formation when placed in scaffold alone. ASCs were successfully differentiated into endothelial cells (ASC-Endo) and osteoblasts (ASC-Osteo) using media supplemented with vascular endothelial growth factor and bone morphogenic protein 2, respectively. Tissue-engineered constructs were created with PLA matrices containing no cells (control), undifferentiated ASCs (ASCs), osteogenic-differentiated ASCs (ASC-Osteo), or endothelial differentiated ASCs (ASC-Endo), and these constructs were evaluated in critical-size Lewis rat calvarial defect model (n = 34). Eight weeks after implantation, the bone volume and microvessel population of bony constructs were evaluated by micro-computed tomography analysis and histologic staining. Bone volumes for ASCs and ASC-Osteo constructs, 0.7 and 0.91 mm(3), respectively, were statistically greater than that for ASC-Endo, 0.28 mm(3) (P < 0.05). There was no statistical difference between the PLA control (0.5 mm(3)) and ASC-Endo (0.28 mm(3)) constructs in bone formation. The percent area of microvessels within constructs was highest in the ASC-Endo group, although it did not reach statistical significance (0.065). Prevascularization of PLA scaffold with ASC-Endo cells will not increase bone formation by itself but may be used as a cell source for improving vascularization and potentially improving existing osteoblast function.

Effect of adipose tissue-derived osteogenic and endothelial cells on bone allograft osteogenesis and vascularization in critical-sized calvarial defects.

The use of processed bone allograft to repair large osseous defects of the skull has been limited, given that it lacks the osteogenic cellularity and intrinsic vascular supply which are essential elements for successful graft healing and, at the same time, the areas to be targeted through tissue-engineering applications. In this study, we investigated the effect of predifferentiated rat adipose tissue-derived osteoblastic cells (OBs) and endothelial cells (ECs) on calvarial bone allograft healing and vascularization using an orthotopic critical-sized calvarial defect model. For this purpose, thirty-seven 8 mm critical calvarial defects in Lewis rats were treated with bone allografts seeded with no cells, undifferentiated adipose tissue-derived stem cells (ASC), OBs, ECs, and OBs and ECs simultaneously. After 8 weeks, the bone volume and mineral density were calculated using microcomputed tomography and the microvessel formation using immunohistochemical staining and imaging software. The amount of bone within the 8 mm defect was significantly higher for the allografts treated with ECs compared with the allografts treated with OBs (p=0.05) and simultaneously with the two cell lineages (p=0.02). There were no significant differences in bone formation between the latter two groups and the control groups (allografts treated with no cells and undifferentiated ASC). There were no significant differences in bone mineral density among the groups. The amount of microvessels was significantly higher in the group treated with ECs relative to all groups (p=< 0.05). Our results show that the implantation of ASC-derived ECs improves the vascularization of calvarial bone allografts at 8 weeks after treatment. This cell-based vascularization strategy can be used to improve the paucity of perfusion in allogenic bone implants. However, in this study, the treatment of allografts with OBs alone or in combination with ECs did not support bone formation or vascularization.

The use of visible light spectroscopy to measure tissue oxygenation in free flap reconstruction.

The loss of a free flap is a feared complication for both the surgeon and the patient. Early recognition of vascular compromise has been shown to provide the best chance for flap salvage. The ideal monitoring technique for perioperative free flap ischemia would be noninvasive, continuous, and reliable. Visible light spectroscopy (VLS) was evaluated as a new method for predicting ischemia in microvascular cutaneous soft tissue free flaps. In an Institutional Review Board-approved prospective trial, 12 patients were monitored after free flap reconstructions. The tissue hemoglobin oxygen saturation (StO (2)) and total hemoglobin concentration (THB) of 12 flaps were continuously monitored using VLS for 72 hours postoperatively. Out of these 12 flaps 11 were transplanted successfully and 1 flap loss occurred. The StO (2 )was 48.99% and the THB was 46.74% for the 12 flaps. There was no significant difference in these values among the flaps. For the single flap loss, the device accurately reflected the ischemic drop in StO (2) indicating drastic tissue ischemia at 6 hours postoperatively before the disappearance of implantable Doppler signals or clinical signs of flap compromise. VLS, a continuous, noninvasive, and localized method to monitor oxygenation, appeared to predict early ischemic complications after free flap reconstruction.

In vitro osteogenic differentiation of adipose stem cells after lentiviral transduction with green fluorescent protein.

BACKGROUND: Adipose-derived stem cells (ASCs) have the potential to differentiate into osteogenic cells that can be seeded into scaffolds for tissue engineering for use in craniofacial bone defects. Green fluorescent protein (GFP) has been widely used as a lineage marker for mammalian cells. The use of fluorescent proteins enables cells to be tracked during manipulation such as osteogenic differentiation within three-dimensional scaffolds. The purpose of this study was to examine whether ASCs introduced with GFP-encoding lentivirus vector exhibit adequate GFP fluorescence and whether the expression of GFP interfered with osteogenic differentiation of ASCs in both monolayer and three-dimensional scaffolds in vitro. METHODS: Primary ASCs were harvested from the inguinal fat pad of Sprague Dawley rats. Isolated ASCs were cultured and infected with a lentiviral vector encoding GFP and plated into both monolayers and three-dimensional scaffolds in vitro. The cells were then placed in osteogenic medium. Osteogenic differentiation of the GFP-ASCs was assessed using alizarin red S, alkaline phosphate staining, and immunohistochemistry staining of osteocalcin with quantification of alizarin red S and osteocalcin staining. RESULTS: The efficacy of infection of ASCs with a lentiviral vector encoding GFP was high. Cell-cultured GFP-ASCs remained fluorescent over the 8 weeks of the study period. The GFP-ASCs were successfully induced into osteogenic cells both in monolayers and three-dimensional scaffolds. Whereas the quanitification of alizarin red S revealed no difference between osteoinduced ASCs with or without GFP, the quantification of osteocalcin revealed increased staining in the GFP group. CONCLUSIONS: Transduction of isolated ASCs using a lentiviral vector encoding GFP is an effective method for tracing osteoinduced ASCs in vitro. Quantification data showed no decrease in staining of the osteoinduced ASCs.

An experimental design to study adipocyte stem cells for reconstruction of calvarial defects.

Autogenous osteogenic differentiated rat adipose stromal cells (ASCs) were used to repopulate cadaveric processed bone in a critical calvarial defect model to improve bone healing as compared with the standard treatment of cadaveric bone implantation alone. Forty-two skeletally mature Sprague-Dawley rats were randomized to 5 groups including bone graft only, bone/osteogenic differentiated ASCs, fibrin glue/osteogenic differentiated ASCs, and bone/fibrin glue/osteogenic differentiated ASCs; 2 animal calvarias were left empty. Adipose stromal cells were isolated from the inguinal fat pad of rats and differentiated into osteogenic cells, verified using von Kossa and alkaline phosphatase staining, and osteocalcin immunohistochemistry. These cells were added to sterilized, 8-mm cadaveric bone graft disks and placed back into calvarial defect for 6 weeks. The rat calvarias then underwent bone density analysis and histology. Intact cells were observed in the bone graft of the bone/osteogenic differentiated ASC group only. Islands of bone were seen in the bone-graft-only group, the bone/osteogenic differentiated ASC group, and the bone/fibrin/osteogenic differentiated ASC group. The bone-graft-only group and bone/osteogenic differentiated ASC group were similar in bone mineral density (1397 +/- 184.5 vs 1365 +/- 160.4). The bone/fibrin/osteogenic differentiated ASC group density was less than the bone and bone/osteogenic differentiated ASC groups at 835.2 +/- 319.5 (P < 0.001). Allograft bone scaffolds with autogenous osteogenic differentiated ASCs showed cellularity within the bone grafts and had larger bone islands. The presence of osteogenic differentiated ASCs did not increase overall bone density compared with bone graft only.