Ronald P. Silverman

Injectable tissue-engineered cartilage using a fibrin glue polymer

The purpose of this study was to demonstrate the feasibility of using a fibrin glue polymer to produce injectable tissue-engineered cartilage and to determine the optimal fibrinogen and chondrocyte concentrations required to produce solid, homogeneous cartilage. The most favorable fibrinogen concentration was determined by measuring the rate of degradation of fibrin glue using varying concentrations of purified porcine fibrinogen. The fibrinogen was mixed with thrombin (50 U/cc in 40 mM calcium chloride) to produce fibrin glue. Swine chondrocytes were then suspended in the fibrinogen before the addition of thrombin. The chondrocyte/polymer constructs were injected into the subcutaneous tissue of nude mice using chondrocyte concentrations of 10, 25, and 40 million chondrocytes/cc of polymer (0.4-cc injections). At 6 and 12 weeks, the neocartilage was harvested and analyzed by histology, mass, glycosaminoglycan content, DNA content, and collagen type II content. Control groups consisted of nude mice injected with fibrin glue alone (without chondrocytes) and a separate group injected with chondrocytes suspended in saline only (40 million cells/cc in saline; 0.4-cc injections). The fibrinogen concentration with the most favorable rate of degradation was 80 mg/cc. Histologic analysis of the neocartilage showed solid, homogeneous cartilage when using 40 million chondrocytes/cc, both at 6 and 12 weeks. The 10 and 25 million chondrocytes/cc samples showed areas of cartilage separated by areas of remnant fibrin glue. The mass of the samples ranged from 0.07 to 0.12 g at 6 weeks and decreased only slightly by week 12. The glycosaminoglycan content ranged from 2.3 to 9.4 percent for all samples; normal cartilage controls had a content of 7.0 percent. DNA content ranged from 0.63 to 1.4 percent for all samples, with normal pig cartilage having a mean DNA content of 0.285 percent. The samples of fibrin glue alone produced no cartilage, and the chondrocytes alone produced neocartilage samples with a significantly smaller mass (0.47 g at 6 weeks and 0.46 g at 12 weeks) when compared with all samples produced from chondrocytes suspended in fibrin glue (p < 0.03). Gel electrophoreses demonstrated the presence of type II collagen in all sample groups. This study demonstrates that fibrin glue is a suitable polymer for the formation of injectable tissue-engineered cartilage in the nude mouse model. Forty million chondrocytes per cc yielded the best quality cartilage at 6 and 12 weeks when analyzed by histology and content of DNA, glycosaminoglycan, and type II collagen.

The use of acellular dermal matrix to prevent capsule formation around implants in a primate model.

Background: Implant-based breast reconstruction is a popular option after mastectomy, but capsular contracture may detract from long-term outcomes. The authors have observed that breast implants covered with acellular dermal matrix (AlloDerm) are less likely to develop a capsule in the area where the implant is in direct contact with the acellular matrix. The authors tested this observation experimentally by comparing capsular formation around implants in the presence and absence of AlloDerm in primates. Methods: Eight smooth-surfaced tissue expanders were implanted into eight African green monkeys. In four experimental animals, a sheet of AlloDerm was draped over the tissue expander so as to cover the implant. Four control animals underwent placement of a tissue expander only. Animals were killed after 10 weeks and specimens underwent histologic and immunohistochemical analysis. Results: Hematoxylin and eosin staining of control specimens revealed the presence of a distinct layer of wavy, parallel arrays of collagen fibers consistent with capsule formation. Immunostaining identified abundant myofibroblasts, a profibrotic cell found in breast capsules. In the AlloDerm-covered specimens, no capsule layer was visible, and specimens stained weakly for myofibroblasts. The difference in myofibroblast staining intensity was statistically significant. Conclusions: The use of AlloDerm to partially enclose implants effectively prevented formation of a capsule in areas where AlloDerm contacted the implant at 10 weeks. Long-term studies will be required to determine whether this is a durable result that can be reproduced in humans.

Cultured Chondrocytes Produce Injectable Tissue-Engineered Cartilage in Hydrogel Polymer

The purpose of this study was to determine if chondrocytes cultured through several subcultures at very low plating density would produce new cartilage matrix after being reimplanted in vivo with or without a hydrogel polymer scaffold. Chondrocytes were initially plated in low-density monolayer culture, grown to confluence, and passaged four times. After each passage cells were suspended in purified porcine fibrinogen and injected into the subcutaneous space of nude mice while simultaneously polymerizing with thrombin to reach a final concentration of 40 million cells/cc. Controls were made by injecting fresh, uncultured cells with fibrin polymer and by injecting the cultured cells in saline (without polymer). All samples were harvested at 6 weeks. When injected in polymer, both fresh cells and cells that had undergone only one passage in culture produced cartilaginous nodules. Cultured cells did not produce cartilage, regardless of length of time spent in culture, when injected without polymer. Cartilage was also not recovered from samples with cells kept in culture for longer than one passage, even when provided with a polymer matrix. All samples harvested were subjected to histological analysis and assayed for total DNA, glycosaminoglycan (GAG), and type II collagen. There was histological evidence of cartilage in the groups that used fresh cells and cultured cells suspended in fibrin polymer that only underwent one passage. No other group contained areas that would be consistent with cartilage histologically. All experimental samples had a higher percent of DNA than native swine cartilage, and there was no statistical difference between the DNA content of the groups containing cultured or fresh cells in fibrin polymer. Whereas the GAG content of native cartilage was 8.39% of dry weight and fresh cells in fibrin polymer was 12.85%, cultured cells in fibrin polymer never exceded the 2.48% noted from first passage cells. In conclusion, this study demonstrates that porcine chondrocytes that have been cultured in monolayer for one passage will produce cartilage in vivo when suspended in fibrin polymer.

What is the best surgical margin for a Basal cell carcinoma: a meta-analysis of the literature.

Background: Current management of basal cell carcinoma is surgical excision. Most resections use predetermined surgical margins. The basis of ideal resection margins is almost completely from retrospective data and mainly from small case series. This article presents a systematic analysis from a large pool of data to provide a better basis of determining ideal surgical margin. Methods: A systematic analysis was performed on data from 89 articles from a larger group of 973 articles selected from the PubMed database. Relevant inclusion and exclusion criteria were applied to all articles reviewed and the data were entered into a database for statistical analysis. Results: The total number of lesions analyzed was 16,066; size ranged from 3 to 30 mm (mean, 11.7 ± 5.9 mm). Surgical margins ranged from 1 to 10 mm (mean, 3.9 ± 1.4 mm). Negative surgical margins ranged 45 to 100 percent (mean, 86 ± 12 percent). Recurrence rates for 5-, 4-, 3-, and 2-mm surgical margins were 0.39, 1.62, 2.56, and 3.96 percent, respectively. Pooled data for incompletely excised margins have an average recurrence rate of 27 percent. Conclusions: A 3-mm surgical margin can be safely used for nonmorpheaform basal cell carcinoma to attain 95 percent cure rates for lesions 2 cm or smaller. A positive pathologic margin has an average recurrence rate of 27 percent.

Adhesion of tissue-engineered cartilage to native cartilage

Reconstruction of cartilaginous defects to correct both craniofacial deformities and joint surface irregularities remains a challenging and controversial clinical problem. It has been shown that tissue-engineered cartilage can be produced in a nude mouse model. Before tissue-engineered cartilage is used clinically to fill in joint defects or to reconstruct auricular or nasal cartilaginous defects, it is important to determine whether it will integrate with or adhere to the adjacent native cartilage at the recipient site. The purpose of this study was to determine whether tissue-engineered cartilage would adhere to adjacent cartilage in vivo. Tissue-engineered cartilage was produced using a fibrin glue polymer (80 mg/cc purified porcine fibrinogen polymerized with 50 U/cc bovine thrombin) mixed with fresh swine articular chondrocytes. The polymer/chondrocyte mixture was sandwiched between two 6-mm-diameter discs of fresh articular cartilage. These constructs were surgically inserted into a subcutaneous pocket on the backs of nude mice (n = 15). The constructs were harvested 6 weeks later and assessed histologically, biomechanically, and by electron microscopy. Control samples consisted of cartilage discs held together by fibrin glue alone (no chondrocytes) (n = 10). Histologic evaluation of the experimental constructs revealed a layer of neocartilage between the two native cartilage discs. The neocartilage appeared to fill all irregularities along the surface of the cartilage discs. Safranin-O and toluidine blue staining indicated the presence of glycosaminoglycans and collagen, respectively. Control samples showed no evidence of neocartilage formation. Electron microscopy of the neocartilage revealed the formation of collagen fibers similar in appearance to the normal cartilage matrix in the adjacent native cartilage discs. The interface between the neocartilage and the native cartilage demonstrated neocartilage matrix directly adjacent to the normal cartilage matrix without any gaps or intervening capsule. The mechanical properties of the experimental constructs, as calculated from stress-strain curves, differed significantly from those of the control samples. The mean modulus for the experimental group was 0.74 ± 0.22 MPa, which was 3.5 times greater than that of the control group (p < 0.0002). The mean tensile strength of the experimental group was 0.064 ± 0.024 MPa, which was 62.6 times greater than that of the control group (p < 0.0002). The mean failure strain of the experimental group was 0.16 ± 0.061 percent, which was 4.3 times greater than that of the control group (p < 0.0002). Finally, the mean fracture energy of the experimental group was 0.00049 ± 0.00032 J, which was 15.6 times greater than that of the control group. Failure occurred in all cases at the interface between neocartilage and native cartilage. This study demonstrated that tissue-engineered cartilage produced using a fibrin-based polymer does adhere to adjacent native cartilage and can be used to join two separate pieces of cartilage in the nude mouse model. Cartilage pieces joined in this way can withstand forces significantly greater than those tolerated by cartilage samples joined only by fibrin glue.

Abdominal wall reconstruction following severe loss of domain: the R Adams Cowley Shock Trauma Center algorithm.

Background: Large, complex, posttraumatic and recurrent abdominal hernias present a reconstructive challenge. Multiple techniques have been described to restore the integrity of the abdominal wall, although the indications and applications can be difficult to navigate. The authors propose an algorithm that facilitates the assessment and treatment of secondary large ventral defects. Methods: The algorithm described involves a systematic approach to abdominal wall reconstruction and was applied to 23 consecutive patients at the R Adams Cowley Shock Trauma Center. Data collected from the chart review included age, body mass index, mechanism of injury, placement of skin graft and use of resorbable mesh before definitive reconstruction, size of defect, number of tissue expanders placed, length of follow-up, and complications. Results: There were six female patients and 17 male patients, with an average age of 36 years. The average follow-up was 7 months. Seventeen patients had posttraumatic laparotomies, five patients had aggressive abdominal wall debridement following necrotizing fasciitis, and one patient developed a large abdominal wall hernia following complications from gastric bypass surgery. All patients underwent delayed abdominal wall reconstruction, with an average time to initial reconstruction of 19.5 months. Sixteen patients had no postoperative complications. Seven patients had complications, including one with an enterocutaneous fistula, one with a partial small bowel obstruction, two with seromas, one with a superficial wound infection, and two with recurrent abdominal wall laxity. Conclusions: The reconstructive ladder for large, complex abdominal hernias is poorly defined. The proposed algorithm provides a systematic staged approach that incorporates available techniques used for delayed reconstruction of the abdominal wall.

Transdermal photopolymerized adhesive for seroma prevention

The purpose of this study was to determine whether or not a synthetic photopolymerized tissue adhesive (polyethylene oxide hydrogel) is useful in seroma prevention using a well established rat mastectomy seroma model. Twenty-three Sprague-Dawley rats received mastectomies. The rats were randomly assigned to either the control group (n = 13) or the experimental group (n = 10). The control animals received 0.2 cc of saline into the wound before closure. The experimental group received either 0.2 cc (n = 5) or 0.4 cc (n = 5) of the polyethylene oxide polymer into their wounds before closure. The experimental animals were placed under an ultraviolet A lamp for 3 minutes to polymerize the adhesive. On postoperative day seven, the resultant seromas were quantified, and wound tissues were harvested for histologic evaluation. The rats in the control group had a mean seroma volume of 3.25 cc (SD = 2.41), whereas the rats treated with polymer had a mean seroma volume of 0.37 cc (SD = 0.51). A Students t test was performed showing a statistically significant difference between the control and experimental groups (p < 0.005). The volume of polymer used (0.2 cc versus 0.4 cc) did not significantly impact the volume of the resultant seromas. This study demonstrates that photopolymerizable polyethylene oxide hydrogels can be used as a tissue adhesive and that such an adhesive significantly reduces seroma formation in the rat mastectomy model.

The Modified Reverse Sural Artery Flap Lower Extremity Reconstruction

INTRODUCTIONnThe reverse sural artery flap eliminates the need for long and technically demanding free tissue transfers, which have become the gold standard for significant tissue defects in the distal third of the leg and ankle. Unfortunately, the originally described reverse sural artery flap technique has a risk of partial or total flap necrosis as high as 25%. We hypothesized that delaying the flap (the delay time ranged from 48 hours to 2 weeks) and using a 4-cm wide pedicle would decrease the amount of partial flap necrosis that commonly occurs with this flap.nnnPATIENTSnFive patients (3 women, 2 men) with open wounds in the distal lower extremity were treated with delayed fasciocutaneous reverse sural artery flaps elevated on a 4-cm wide pedicle.nnnRESULTSnThe patients ranged from 22 to 75 years of age and had sustained defects in the ankle region resulting from trauma. All five wounds healed with favorable functional and asthetic results without any evidence of flap necrosis.nnnCONCLUSIONSnIn patients with known vasculopathy, a surgical delay of 1 week and increasing the pedicle size to 4 cm may increase the likelihood of graft survival and decrease the amount of partial flap necrosis by dilating the arterial network.

Omental transposition flap for sternal wound reconstruction in diabetic patients

In 2004, we published our 12-year experience with tissue transfer for deep sternal wound infection after median sternotomy, finding increased rates of reoperation for diabetic patients. Therefore, we decided to alter our treatment approach to diabetic patients to include sternal debridement followed by omental transposition. Eleven diabetic patients underwent omental transposition by our division during the study period. Hospital records were retrospectively reviewed to determine outcomes and complications. We found that diabetic patients treated after implementation of the new treatment approach were 5.4 times less likely to require reoperation for sternal wound management than were patients in the previous series, most of whom had been treated with pectoralis muscle flaps (95% confidence interval, 0.5–50.5). By altering our treatment approach to use omental transposition as the initial surgical therapy, we were able to demonstrate a trend toward decreased need for flap revision in diabetic patients.

Flap perfusion mapping: TRAM flap after abdominal suction-assisted lipectomy.

This technique or its modification (using other dyes) may play a beneficial role in other clinical scenarios where the reconstructive plastic surgeon preoperatively needs to know the integrity of vessels that are too small to image using standard angiographic techniques. In addition, flap perfusion mapping can demonstrate the pattern of skin that is physiologically perfused by the intact vessels. Knowledge of the perfusion characteristics of the tissues to be transferred before surgery may, at the least, alter the design of the tissues to be transferred and, in the extreme case, could affect the nature of the operative choice altogether.