Kevin C. Olbrich

Long- and short-term effects of biological hydrogels on capsule microvascular density around implants in rats.

Fibrous capsule formation around implants can inhibit solute exchange between implantable devices and the circulation. Parylene-n coated polycarbonate disks surrounded with growth factor reduced Matrigel (MG) or several gelatin-based matrices were implanted intramuscularly into rats for 21 or 50 days. MG supplemented with vascular endothelial growth factor (VEGF) or basic fibroblast growth factor (bFGF) increased capsule microvascular density at 21 days (p < 0.05) when compared to bare parylene-coated polycarbonate disks (control). The increased microvascular density around VEGF- and bFGF-treated implants regressed by 50 days and was no longer significantly different from controls. The microvascular density induced by the gelatin-based matrices was not significantly different from controls at 21 days, but was increased at 50 days (p < 0.05), suggesting a slower, long-term effect. Disks treated with MG and gelatin-based matrices had thinner capsules at 21 days (p < 0.05). By 50 days, the capsule thicknesses around these implants were no longer statistically thinner than controls. The capsule thickness around implants treated with VEGF, bFGF, and essential gelatin-based matrix was thinner than controls at 50 days (p < 0.05). These results indicate that it is possible to increase functional microvascular density within fibrous capsules using angiogenic growth factors and gelatin-based matrices. However, this effect may be short-lived, requiring chronic administration of growth factors.

Metabolic and Functional Characterization of Human Adipose-Derived Stem Cells in Tissue Engineering

Background: The use of adipose-derived stem cells for tissue engineering involves exposing them to metabolically adverse conditions. This study examines the metabolism, proliferation, and differentiation of adipose-derived stem cells under various conditions. Methods: Adipose-derived stem cells were cultured in 16 media conditions containing 0.6, 2.4, 4.3, or 6.1 mM glucose; 0.1, 2.5, 4.1, or 6.1 mM glutamine; and then grown in either 0.1% or 20% oxygen. Conditioned media were collected and assayed for glucose, lactate, and pyruvate. Cell proliferation and cell death were measured at several time points. Osteogenic differentiation was analyzed by alizarin red staining/quantification and alkaline phosphatase activity, measured weekly over 4 weeks. Results: Adipose-derived stem cells remained metabolically active in all nutrient and oxygen conditions tested. Glucose consumption and lactate production increased under hypoxic conditions, but pyruvate consumption was jointly dependent on oxygen and glucose concentration. The 20% oxygen environment produced greater proliferation and cell death compared with the hypoxic environment. Osteogenic differentiation of adipose-derived stem cells was observed only when glucose and/or oxygen concentrations were physiologically normal to high. Conclusions: Adipose-derived stem cells are an excellent source of multipotent cells and are capable of advancing current tissue engineering methodologies. These data show that adipose-derived stem cells remain viable under adverse conditions of low glucose, glutamine, and oxygen concentrations. However, there are variable levels of differentiation in the various culture conditions, which could lead to challenges in de novo osteogenesis and other forms of tissue engineering. Therefore, these results should be used in developing specific strategies to ensure successful application of adipose-derived stem cells in bone engineering and similar applications.

Combined bone allograft and adipose-derived stem cell autograft in a rabbit model.

Currently available options for the repair of bony defects have substantial limitations. Much work has looked to the possibility of engineering bone using stem cells. These tissue-engineering efforts have focused on calvarial defect models, which have the advantages of minimal load-bearing and a large surface area. This study aims to solve the somewhat more challenging problem of repairing segmental bony defects such as those of the mandible and long bones. Four groups of decellularized bone tubes with cortical perforations were implanted subcutaneously in a rabbit model: empty bone tubes, bone tubes containing fibrin glue alone, bone tubes containing fibrin glue and freshly isolated autologous adipose-derived stem cells (ASCs), and bone tubes containing fibrin glue and predifferentiated autologous ASCs. Results showed a foreign body response characterized by fibrous capsule formation with minimal angiogenesis and no evidence of osteoblastic activity. Substantial changes are needed if this model is to become viable.

Basic fibroblast growth factor expression following surgical delay of rat transverse rectus abdominis myocutaneous flaps.

Partial transverse rectus abdominis myocutaneous (TRAM) flap loss in breast reconstruction can be a devastating complication for both patient and surgeon. Surgical delay of the TRAM flap has been shown to improve flap viability and has been advocated in “high-risk” patients seeking autogenous breast reconstruction. Despite extensive clinical evidence of the effectiveness of surgical delay of TRAM flaps, the mechanisms by which the delay phenomenon occurs remain poorly understood. To examine whether angiogenic growth factors such as basic fibroblast growth factor (bFGF) may play a role in the delay phenomenon, the authors studied the expression of bFGF in rat TRAM flaps subjected to surgical delay. Thirty-five female Sprague-Dawley rats were randomly assigned to one of four TRAM flap groups: no delay (n = 6), 7-day delay (n = 12), 14-day delay (n = 10), or 21-day delay (n = 7). Surgical delay consisted of incising skin around the perimeter of the planned 2.5 × 5.0-cm TRAM flap followed by ablation of both superior epigastric arteries and the left inferior epigastric artery, thus preserving the right inferior epigastric artery (the nondominant blood supply to the rectus abdominis muscle of the rat). TRAM flaps were then elevated after 7, 14, and 21 days of delay by raising zones II, III, and IV off the abdominal wall fascia. Once hemostasis was assured, the flaps were sutured back in place. All flaps were designed with the upper border of the flap 1 cm below the xiphoid tip. Three days after the TRAM procedure, postfluorescein planimetry was used to determine percent area viability of both superficial and deep portions of TRAM flaps. All rats were euthanized and full-thickness TRAM specimens were taken from zones I, II, III, and IV for enzyme-linked immunoabsorbent assay analysis of bFGF levels. Statistical testing was done by t test (percent viability) and two-way analysis of variance (bFGF levels). All delayed flaps had significantly higher bFGF levels when compared with all nondelayed control flaps (p < 0.05). The bFGF levels were not different in the rats that received TRAM flaps 7, 14, or 21 days after delay surgery. There was also no significant difference in bFGF levels among zones I through IV. Control rats had more peripheral zone necrosis compared with all delayed TRAM rats. All delayed flaps had a significantly higher area of flap viability superficially than nondelayed control flaps (p < 0.05). There was no difference in deep flap viability. Surgical delay of rat TRAM flaps is associated with improved flap viability and significantly elevated levels of bFGF over nondelayed TRAM flaps at postoperative day 3 after TRAM surgery. The increases in bFGF noted at this time point suggests that bFGF may play a role in the improved TRAM flap viability observed after delay surgery. Further investigation is needed to evaluate the role bFGF may play in the delay phenomenon.

Expanded Polytetrafluoroethylene Membrane Alters Tissue Response to Implanted Ahmed Glaucoma Valve

Purpose: Long-term intraocular pressure control by glaucoma drainage implants is compromised by the formation of an avascular fibrous capsule that surrounds the glaucoma implant and increases aqueous outflow resistance. It is possible to alter this fibrotic tissue reaction and produce a more vascularized and potentially more permeable capsule around implanted devices by enclosing them in a porous membrane. Methods: Ahmed glaucoma implants modified with an outer 5-μ m pore size membrane (termed porous retrofitted implant with modified enclosure or PRIME-Ahmed) and unmodified glaucoma implants were implanted into paired rabbit eyes. After 6 weeks, the devices were explanted and subject to histological analysis. Results: A tissue response containing minimal vascularization, negligible immune response, and a thick fibrous capsule surrounded the unmodified Ahmed glaucoma implant. In comparison, the tissue response around the PRIME-Ahmed demonstrated a thinner fibrous capsule (46.4 ± 10.8 μ m for PRIME-Ahmed versus 94.9 ± 21.2 μ m for control, p < 0.001) and was highly vascularized near the tissue-material interface. A prominent chronic inflammatory response was noted as well. Conclusions: Encapsulating the aqueous outflow pathway with a porous membrane produces a more vascular tissue response and thinner fibrous capsule compared with a standard glaucoma implant plate. Enhanced vascularity and a thinner fibrous capsule may reduce aqueous outflow resistance and improve long-term glaucoma implant performance.

Vascular endothelial growth factor expression in pig latissimus dorsi myocutaneous flaps after ischemia reperfusion injury

Exogenous administration of vascular endothelial growth factor (VEGF) improves long-term viability of myocutaneous flaps. However, endogenous expression of this substance in flaps following ischemia-reperfusion injury has not been reported previously. Endogenous production of VEGF was measured in myocutaneous pig latissimus dorsi flaps after ischemia-reperfusion injury. Latissimus dorsi myocutaneous flaps (15 x 10 cm) were simultaneously elevated bilaterally in six Yorkshire-type male pigs (25 kg). Before elevation, three flap zones (5 x 10 cm) were marked according to their distance from the vascular pedicle. After isolation of the vascular pedicle, ischemia-reperfusion injury was induced in one flap by occlusion of the thoracodorsal artery and vein for 4 hours, followed by 2 hours of reperfusion. The contralateral flap served as a control. Perfusion in each zone was monitored by laser Doppler flowmetry at baseline, during ischemia, and during reperfusion. At the end of the protocol, skin and muscle biopsies of each flap zone and adjacent tissues were obtained for later determination of VEGF protein levels. VEGF concentrations were quantified using the Quantikine human VEGF immunoassay. Skin perfusion was similar among all flap zones before surgery. Flow fell in all flaps immediately after flap elevation. After 4 hours of ischemia, blood flow in the ischemic flaps was significantly decreased (p < 0.05) compared with nonischemic control flaps. After 2 hours of reperfusion, flow in ischemic flap skin recovered to levels similar to those in control flaps. VEGF protein concentrations in muscle tissue exceeded concentrations in skin and decreased from zones 2 to 3 in control and ischemic flaps. No significant differences in VEGF concentrations between ischemic and control muscle zones were observed. However, the concentration of VEGF in all muscle zones was significantly higher (p < 0.05) than muscle adjacent to the flap. Concentrations in skin zones 1 and 2 were significantly higher (p < 0.05) in ischemic flaps than in control flaps, but levels in zone 3 (most ischemic flaps) showed no significant difference.

Immunohistochemical identification of Vascular Endothelial Growth Factor in pig Latissimus dorsi musculocutaneous flaps following ischemia-reperfusion injury

Vascular Endothelial Growth Factor (VEGF), a potent angiogenic, mitogenic and vascular permeability enhancing protein, appears to improve survival of ischemic flaps independent of its route of administration. The purpose of this study was to examine VEGF protein expression in biopsies of surgical flaps with immunohistochemical techniques. In 6 male Yorkshire-type pigs, 10 cm × 15 cm Latissimus dorsi musculocutaneous flaps were elevated bilaterally. Flap zones I, II, and III were established according to their distance from the vascular pedicle. After isolation of the thoracodorsal artery and vein, one flap was randomly assigned to ischemia by temporary occlusion of the vascular pedicle. Ischemia (4 hours) was followed by 2 hours of reperfusion (ischemia group, n = 6). The contralateral (nonischemic) flap served as a control (control group, n =6). Skin and muscle biopsies of flaps were taken at the end of the protocol for immunohistochemical staining using a VEGF antihuman monoclonal antibody. Epidermis of flap skin did not demonstrate VEGF-positive staining, but the dermis and subcutaneous tissue did. Muscle components of biopsies demonstrated staining of interfascicular septa and staining of myocytes. A semi-quantitative scoring system with a scale of 0 to 3 was used for grading of immunohistochemical staining. In skin, areas adjacent to the flap showed an overall mean VEGF staining score of 0.7. All zones of ischemic flaps showed increased mean immunohistochemical staining for VEGF (scores = 1.2, 1.6, and 1.4 in zones I, II, and III, respectively). In muscle, however, only zone I showed increased VEGF immunohistochemical staining from 0.7 in adjacent areas to 1.7 in ischemic flaps. The results indicate only moderate endogenous up-regulation of VEGF in flaps, supporting the utilization of exogenous VEGF as an adjunct in microsurgical therapy.

In vitro fluid dynamics of the Ahmed glaucoma valve modified with expanded polytetrafluoroethylene.

Purpose: Long-term intraocular pressure reduction by glaucoma drainage devices (GDDs) is often limited by the fibrotic capsule that forms around them. Prior work demonstrates that modifying a GDD with a porous membrane promotes a vascularized and more permeable capsule. This work examines the in vitro fluid dynamics of the Ahmed valve after enclosing the outflow tract with a porous membrane of expanded polytetrafluoroethylene (ePTFE). Materials and Methods: The control and modified Ahmed implants (termed porous retrofitted implant with modified enclosure or PRIME-Ahmed) were submerged in saline and gelatin and perfused in a system that monitored flow (Q) and pressure (P). Flow rates of 1–50 μl/min were applied and steady state pressure recorded. Resistance was calculated by dividing pressure by flow. Results: Modifying the Ahmed valve implant outflow with expanded ePTFE increased pressure and resistance. Pressure at a flow of 2 μl/min was increased in the PRIME-Ahmed (11.6 ± 1.5 mm Hg) relative to the control implant (6.5 ± 1.2 mm Hg). Resistance at a flow of 2 μl/min was increased in the PRIME-Ahmed (5.8 ± 0.8 mm Hg/μl/min) when compared to the control implant (3.2 ± 0.6 mm Hg/μl/min). Conclusions: Modifying the outflow tract of the Ahmed valve with a porous membrane adds resistance that decreases with increasing flow. The Ahmed valve implant behaves as a variable resistor. It is partially open at low pressures and provides reduced resistance at physiologic flow rates.

Orthogonal Polarization Spectral Imaging und derzeitige Perspektiven in der Plastischen Chirurgie

ZusammenfassungDie Technologie des seit 1999 in den USA patentierten Orthogonal Polarization Spectral Imaging (OPSI) wird aufgrund ihrer einfachen Handhabung und der Möglichkeit der kontinuierlichen Untersuchung des Mikrogefäßsystems als Alternative zur konventionellen Intravitalmikroskopie (IVM) propagiert. In den vorgestellten Versuchsreihen wurde das OPS Imaging auf Bereiche der plastisch-rekonstruktiven Chirurgie übertragen. Zwei tierexperimentelle Modelle und ein klinisches Beispiel werden nachfolgend dargestellt. Die Ergebnisse zeigen, dass die bisherige Bildqualität eine differenzierte Beurteilung des Mikrogefäßsystems nicht erlaubt. Die Datenanalyse des Bildmaterials ist derzeit nur mit Einschränkungen möglich. Nach Abschluss technischer Verbesserungen, vor allem im Bereich der Bildauflösung und Software, ist der Einsatz des Systems in vielen experimentellen und klinischen Bereichen der Plastischen Chirurgie vorstellbar.AbstractThe technology of Orthogonal Polarization Spectral Imaging (OPSI), patented in the United States in 1999, has been heralded as an alternative to conventional intravital microscopy for several reasons, including ease of application and ability to perform continued observations. In the following examples, OPSI was applied to reconstructive plastic surgery. Two experimental and one clinical model are presented. Currently, the image quality does not allow a qualitative analysis of the microvasculature. The data analysis of images remains inconclusive. However, a technically modified version with improved image resolution and software could become a valuable experimental as well as clinical tool in the field of reconstructive plastic surgery in the future.