A novel flow-perfusion bioreactor supports 3D dynamic cell culture.

BACKGROUND: Bone engineering requires thicker three-dimensional constructs than the maximum thickness supported by standard cell-culture techniques (2 mm). A flow-perfusion bioreactor was developed to provide chemotransportation to thick (6 mm) scaffolds. METHODS: Polyurethane scaffolds, seeded with murine preosteoblasts, were loaded into a novel bioreactor. Control scaffolds remained in static culture. Samples were harvested at days 2, 4, 6, and 8 and analyzed for cellular distribution, viability, metabolic activity, and density at the periphery and core. RESULTS: By day 8, static scaffolds had a periphery cell density of 67% +/- 5.0%, while in the core it was 0.3% +/- 0.3%. Flow-perfused scaffolds demonstrated peripheral cell density of 94% +/- 8.3% and core density of 76% +/- 3.1% at day 8. CONCLUSIONS: Flow perfusion provides chemotransportation to thick scaffolds. This system may permit high throughput study of 3D tissues in vitro and enable prefabrication of biological constructs large enough to solve clinical problems.

The proximally based peroneal vascular bundle: an insulated extension cord for free flap reconstruction.

Large, traumatic wounds around the proximal third of the lower extremity may have disrupted local vasculature, potentially obviating local pedicled options. However, free-tissue transfer to this area is technically challenging given the resulting paucity of recipient options and the depth of principal blood vessels. We present an anatomic and radiographic study of the proximally based peroneal vascular bundle as a recipient option in the proximal leg. Optimal approach was prone, through an incision over the fibula with dissection between lateral and posterior compartments. Magnetic resonance angiography demonstrated consistent vascular anatomy between patients. A proximally based peroneal vascular bundle protected by a cuff of flexor hallucis longus was used as a recipient vessel in free flap reconstruction of an open knee wound. The bundle itself does not require coverage by virtue of its own local muscle cuff. Caveats for its use include the need for adequate leg inflow and foot outflow.

How many people work in your operating room? An assessment of factors associated with instrument recounts within plastic surgery.

BACKGROUND: Intraoperative instrument recounts are performed to avoid retained foreign surgical items. These additional counts, however, beget risks of their own, including prolonged operative times, exposure to radiation, and increased cost. Our study aimed to identify factors that increase the likelihood of instrument recounts during plastic surgery procedures, and use our findings to guide potential solutions for preventing unnecessary recounts across all surgical fields. STUDY DESIGN: This is a retrospective review of all plastic surgical cases in the main operating setting at New York University Langone Medical Center (NYULMC) between March 2014 and February 2015. RESULTS: Of 1285 plastic surgery cases, 35 (2.7%) reported a missing instrument necessitating a recount. Of all subspecialties within plastic surgery, only microsurgery conferred an increased risk of a recount event. We identified multiple factors that increased the odds of a recount event, including increased operative time, number of surgical sites, and intraoperative instrument handoffs. CONCLUSION: Instrument recounts, although designed to prevent inadvertently retained surgical items, present inherent risks of their own. In a large retrospective review of plastic surgery cases at our medical center, we identified many factors that increased the likelihood of an instrument recount. On the basis of our findings and prior literature, we recommend limiting the number of staff handling instrument, the number of handoffs, and a heightened awareness by surgeons and perioperative staff of specific procedures and factors that increase the risk of a miscount event.

Design and validation of a dynamic cell-culture system for bone biology research and exogenous tissue-engineering applications.

Bone lacunocanalicular fluid flow ensures chemotransportation and provides a mechanical stimulus to cells. Traditional static cell-culture methods are ill-suited to study the intricacies of bone biology because they ignore the three-dimensionality of meaningful cellular networks and the lacunocanalicular system; furthermore, reliance on diffusion alone for nutrient supply and waste product removal effectively limits scaffolds to 2-3 mm thickness. In this project, a flow-perfusion system was custom-designed to overcome these limitations: eight adaptable chambers housed cylindrical cell-seeded scaffolds measuring 12 or 24 mm in diameter and 1-10 mm in thickness. The porous scaffolds were manufactured using a three-dimensional (3D) periodic microprinting process and were composed of hydroxyapatite/tricalcium phosphate with variable thicknesses, strut sizes, pore sizes and structural configurations. A multi-channel peristaltic pump drew medium from parallel reservoirs and perfused it through each scaffold at a programmable rate. Hermetically sealed valves permitted sampling or replacement of medium. A gas-permeable membrane allowed for gas exchange. Tubing was selected to withstand continuous perfusion for > 2 months without leakage. Computational modelling was performed to assess the adequacy of oxygen supply and the range of fluid shear stress in the bioreactor-scaffold system, using 12 × 6 mm scaffolds, and these models suggested scaffold design modifications that improved oxygen delivery while enhancing physiological shear stress. This system may prove useful in studying complex 3D bone biology and in developing strategies for engineering thick 3D bone constructs. Copyright © 2013 John Wiley & Sons, Ltd.

Characterization of adipose-derived mesenchymal stem cell combinations for vascularized bone engineering.

Since bone repair and regeneration depend on vasculogenesis and osteogenesis, both of these processes are essential for successful vascularized bone engineering. Using adipose-derived stem cells (ASCs), we investigated temporal gene expression profiles, as well as bone nodule and endothelial tubule formation capacities, during osteogenic and vasculogenic ASC lineage commitment. Osteoprogenitor-enriched cell populations were found to express RUNX2, MSX2, SP7 (osterix), BGLAP (osteocalcin), SPARC (osteonectin), and SPP1 (osteopontin) in a temporally specific sequence. Irreversible commitment of ASCs to the osteogenic lineage occurred between days 6 and 9 of differentiation. Endothelioprogenitor-enriched cell populations expressed CD34, PECAM1 (CD31), ENG (CD105), FLT1 (Vascular endothelial growth factor [VEGFR1]), and KDR (VEGFR2). Capacity for microtubule formation was evident in as early as 3 days. Functional capacity was assessed in eight coculture combinations for both bone nodule and endothelial tubule formation, and the greatest expression of these end-differentiation phenotypes was observed in the combination of well-differentiated endothelial cells with less-differentiated osteoblastic cells. Taken together, our results demonstrate vascularized bone engineering utilizing ASCs is a promising enterprise, and that coculture strategies should focus on developing a more mature vascular network in combination with a less mature osteoblastic stromal cell.

Cutis aplasia: perioperative management and case report.

Aplasia cutis congenita, a rare congenital disorder involving defects of some or all of the layers of the cranium, is associated with potential life-threatening complications. Although treatment involves both nonsurgical and surgical techniques, the importance of perioperative management cannot be overstressed. A multidisciplinary team, including personnel from nursing, neonatology, pediatrics, radiology, neurosurgery, and plastic surgery services, diagnosed aplasia cutis congenita and planned local wound care, surgical correction, and prevention of potentially life-threatening complications in a 1-day-old boy with a 6×5-cm full-thickness scalp defect.

Flow perfusion maintains ex vivo bone viability: a novel model for bone biology research.

Encased in lacunae, osteocytes receive nutrition and biomechanical signals through the lacunocanalicular system. We have developed a novel flow-perfusion bioreactor designed to support lacunocanalicular fluid flow. We hypothesize that ex vivo fluid flow can maintain endochondral bone viability and, ultimately, serve as a novel model to study bone biology in vitro. Sprague-Dawley rat femurs were harvested, stripped of soft tissue, loaded into a custom-designed bioreactor and perfused with osteogenic culture medium. After 14 days of flow-perfusion or static culture, the bones were harvested, fixed, decalcified, embedded, sectioned and stained with haematoxylin and eosin. Fresh long bone samples were similarly processed for comparison. Osteocyte viability and function were also evaluated, using thiazolyl blue tetrazolium bromide (MTT), fluorospectrophotometric DNA quantification, alkaline phosphatase (ALP) colorimetric assay and fluorochrome labelling of mineralizing surfaces. All samples remained free of infection throughout the study period. After 14 days of flow perfusion, histological analysis showed normal-appearing bony architecture, with 72% of lacunae being osteocyte-filled compared with 93% in freshly harvested samples and only 36% in static samples. MTT staining and assay confirmed osteocyte viability in the flow-perfusion samples as well as in fresh samples. DNA quantification demonstrated DNA to be preserved in flow-perfused samples when compared with freshly harvested samples. ALP activity in flow-perfusion explants was upregulated compared with fresh and static samples. Fluorochrome-labelled mineralizing surfaces were seen throughout the explanted flow-perfused samples. This is the first demonstration that flow perfusion provides adequate chemotransportation to explanted murine endochondal bones.

Lower extremity arterial injury patterns and reconstructive outcomes in patients with severe lower extremity trauma: a 26-year review.

BACKGROUND: Management of severe traumatic lower extremity injuries remains a considerable challenge. Free tissue transfer is now a standard part of reconstruction for Gustilo IIIB and IIIC injuries. There is limited information on arterial injury patterns in this population. We undertook a review of our experience to gain insight on vascular injury patterns and surgical outcomes. STUDY DESIGN: A 26-year retrospective analysis was performed of all lower extremity Gustilo IIIB and IIIC injuries requiring microvascular reconstruction at New York University Medical Center. Patient demographics, Gustilo classification, angiographic findings (conventional/computed tomographic angiography/magnetic resonance angiography), recipient vessels, elapsed time from injury, flap choices, and outcomes were examined. RESULTS: Two hundred twenty-two free flaps on 191 patients were performed from September 1982 until March 2008. There were 151 males and 40 females ranging in age from 4 to 83 years (median age 33 years). Patients sustained either Gustilo IIIB (170 patients) or IIIC (21 patients) open fractures. One hundred fifty-four patients had angiograms (78.2% IIIB, 100% IIIC). Sixty-six (42.9%) had normal 3-vessel runoff and 88 (57.1%) were abnormal. Sixty-one patients (31.9%) had anterior tibial injuries, 17 patients (8.9%) had posterior tibial injuries, and 30 (15.7%) had peroneal injuries. Sixty-three complications occurred (11 early thrombosis, 33 requiring secondary procedures, and 10 requiring amputation). CONCLUSIONS: Angiography of severe lower extremity injuries requiring free flap reconstruction usually revealed arterial injury and is generally indicated. In our experience, the anterior tibial artery is most commonly injured and the posterior tibial artery is most likely to be spared and used as a recipient.