Christopher Blewett

Studies in cranial suture biology: in vitro cranial suture fusion.

The biology underlying craniosynostosis remains unknown. Previous studies have shown that the underlying dura mater, not the suture itself, signals a suture to fuse. The purpose of this study was to develop an in vitro model for cranial-suture fusion that would still allow for suture-dura interaction, but without the influence of tensional forces transmitted from the cranial base. This was accomplished by demonstrating that the posterior frontal mouse cranial suture, known to be the only cranial suture that fuses in vivo, fuses when plated with its dura in an organ-culture system. In such an organ-culture system, the sutures are free from both the influence of dural forces transmitted from the cranial base and from hormonal influences only available in a perfused system. For the cranial-suture fusion in vitro model study, the sagittal sutures (controls that remain patent in vivo) and posterior frontal sutures (that fuse in vivo) with the underlying dura were excised from 24-day-old euthanized mice, cut into 5 x 4 x 2-mm specimens, and cultured in a chemically defined, serum-free media. One hundred sutures were harvested at the day of sacrifice, then every 2 days thereafter until 30 days in culture, stained with H & E, and analyzed. A subsequent cranial-suture without dura in vitro study was performed in a similar fashion to the first study, but only the calvariae with the posterior frontal or sagittal sutures (without the underlying dura) were cultured. Results from the cranial-suture fusion in vitro model study showed that all sagittal sutures placed in organ culture with the underlying dura remained patent. More importantly, the posterior frontal sutures with the underlying dura, which were plated-down as patent at 24 days of age, demonstrated fusion after various growth periods in organ culture. In vitro posterior frontal mouse-suture fusion occurred in an anterior-to-posterior direction but in a delayed fashion, 4 to 7 days later than in vivo posterior frontal mouse-suture fusion. In contrast, the subsequent cranial-suture without dura in vitro study showed patency of all sutures, including the posterior frontal suture. These data from in vitro experiments indicate that: (1) mouse calvariae, sutures, and the underlying dura survive and grow in organ-culture systems for 30 days; (2) the local dura, free from external influences transmitted from the cranial base and hormones from distant sites, influences the cells of its overlying suture to cause fusion; and (3) without dura influence, all in vitro cranial sutures remained patent. By first identifying the factors involved in dural-suture signaling and then regulating these factors and their receptors, the biologic basis of suture fusion and craniosynostosis may be unraveled and used in the future to manipulate pathologic (premature) suture fusion.

Influence of epidermal growth factor and transforming growth factor beta-1 on patterns of fetal mouse lung branching morphogenesis in organ culture

Transforming growth factor‐β (TGF‐β), a potent inhibitor of epithelial cell proliferation, and epidermal growth factor (EGF), a mitogenic polypeptide that binds to cell surface receptors, are important regulators of cell differentiation; however, their distinct role(s) in lung development and their mechanisms of action are not well understood. We evaluated the effects of these factors on lung morphogenesis in murine fetal lungs at gestational day 14 (time:zero) and again after 7 days in culture. Baseline controls were cultured after tracheal transection in supplemented BGJb medium, and other tracheally transected lungs were cultured following addition of EGF (10 ng/ml BGJb), TGF‐β1 (2 ng/ml BFJb), or with both in combination added to the medium.

Reduced hospitalization cost for patients with pectus excavatum treated using minimally invasive surgery

Background: Currently, few data exist regarding the relative costs associated with open and minimally invasive pectus excavatum repair. The aim of this study was to compare the surgical and hospitalization costs for these two surgical techniques and to identify factors responsible for cost differences. Methods: A retrospective review of hospital charts, patient and parent questionnaires, and hospital accounting records was performed for 68 patients who underwent surgical correction of pectus excavatum between June 1996 and December 1999. Results: In this series, 25 patients underwent open repair, whereas 43 patients underwent minimally invasive repair of pectus excavatum (MIRPE). The patient ages ranged from 4 to 19 years. The average ages for open repair (12 years) and MIRPE (11 years) did not differ significantly. As compared with open repair, MIRPE was associated with a 27% lower overall cost of hospitalization (p < 0.05). The operating room costs were 12% higher for the patients who underwent MIRPE (p < 0.05). The mean operative time for open repair was 3 h 15 min, whereas MIRPE required 1 h 10 min (p < 0.001). The hospital stay for open repair averaged 4.4 days, as compared with 2.4 days for MIRPE (p < 0.001). In contrast to other published series, the postoperative analgesia after MIRPE in this series consisted of narcotics, ketorolac, and methocarbamol. No patient received epidural analgesia, regardless of the repair technique selected. The postoperative complication rate was 4% in the open group and 14% in the MIRPE group. Most of the patients treated with either open or MIRPE reported postoperative oral narcotic usage for 2 weeks or less and returned to routine activities within 3 weeks. The patients and parents alike reported good to excellent overall outcomes in 85% or more of the open repair cases and 90% or more of the MIRPE cases. Conclusions: These data demonstrate for the first time that the use of an alternate pain management strategy including, narcotics, NSAIDs, and methocarbamol, but without epidural catheters, results in reduced hospital length of stay and decreased overall hospitalization costs for MIRPE, as compared with open pectus repair. This cost benefit was achieved without compromising pain management or patient satisfaction with surgical care.

Transition from fetal to adult repair occurring in mouse forelimbs maintained in organ culture

In previous wound healing experiments with the use of midgestation murine fetal forelimb explants, wounds were made before or immediately after amputation from the fetus. This experimental technique allows one to ask the question: Do circulatory elements initiate or sustain the repair process in vitro? The hypotheses tested in the current study were that repair occurs in organ culture in the absence of systemic influences and that the in vivo transition from fetal‐like to adult‐type repair persists in an unperfused in vitro system. Gestational day‐14 mouse forelimbs were harvested and placed in serum‐free culture medium. Before amputation, control forelimbs received linear full‐incision microscalpel wounds that were closed primarily. The animals in the other group were not immediately wounded but cultured for 4 days and then wounded with primary wound closure. All limbs were cultured for 7 days after wounding and then processed for histologic analysis. In the immediately wounded limbs, scarless healing occurred with collagen fibers deposited in a reticular form. In contrast, the delay‐wounded limbs had collagen organized in parallel arrays (disordered), constituting repair by scarring. Wound repair proceeded as a local phenomenon in the absence of systemic mediators. We conclude that day‐14 gestation forelimbs undergo maturation in culture, causing a transition from scarless to adult scar repair.

Inhibition of wound closure by transforming growth factor-β and dexamethasone in a fetal mouse limb organ culture model

Tissue repair comprises several physiologic processes including the deposition of a newly synthesized connective tissue matrix and the regeneration of the epidermis by reepithelialization. A fetal mouse limb organ culture system facilitates the investigation of both reepithelialization and connective tissue deposition in repair within a controlled environment. A sutured closed wound in an intact 18.5‐day old fetal mouse limb completely heals by 7 days. Including dexamethasone in the media inhibited both reepithelialization and connective tissue deposition. Concurrent administration of transforming growth factor‐β with dexamethasone restored the deposition of connective tissue but did not restore reepithelialization. When transforming growth factor‐β was given alone, connective tissue deposition was enhanced at both the wound site and in contiguous dermis, but reepithelialization did not proceed. Transforming growth factor‐β inhibited wound closure by blocking the migration of epidermal cells. The organ‐cultured, wounded fetal mouse limb system is sufficiently sensitive to show both mesenchymal cell‐enhancing activity as well as epithelial migration inhibiting activity by transforming growth factor‐β. The in vitro repair of fetal mouse limbs may serve as an in vitro system to test the influence of soluble agents on the repair process.

Regenerative healing of incisional wounds in murine fetal lungs maintained in organ culture

Although fetal dermal repair is known to be fundamentally different from adult healing, the response to wounding in other organs is less well characterized. Scarless repair in mid-gestation dermis with a transition to adult-type healing at term has been shown in fetal organ culture. A lung explant culture system was used to investigate whether wound repair in the fetal lung shows characteristics similar to those found in fetal dermis. Lungs from 14-day and 18-day Cd-1 murine fetuses and 2-day-old newborns, (term = 20 days, n = 24) were wounded by linear incision and incubated at 37 degrees C, in a 21% O2, 5% CO2 environment, in BGJb supplemented with vitamin C and antibiotics. Medium was changed daily. Samples were fixed at 7 days and embedded in paraffin. Sections were stained with hematoxalyn-eosin and Masson Trichrome. Additional 14-day and 18-day samples were frozen in freon and immunohistochemical staining for TGF-beta performed. Other frozen tissues from each time point were homogenized and used to assay for endogenous TGF-beta levels by Western blot analysis. Histology showed reconstitution of tissue architecture across the wound in 14-day and 18-day specimens. In representative histological sections, intact bronchial architecture developed across the previous wound site. No cellular inflammatory response was observed, and collagen deposition was undetectable at the site of the wound by Trichrome staining. By 22 days the lung explants showed a much less ordered repair, including disorganized collagen deposition.(ABSTRACT TRUNCATED AT 250 WORDS)

Endoluminal retrieval of a dislodged umbilical vein catheter--a case report.

Endoluminal retrieval of foreign bodies in the pediatric and infant population is an uncommon and challenging procedure for the endovascular specialist. The alternative is an open exploration of these often-fragile patients. The availability of smaller catheter systems allows retrieval with minimally invasive techniques. We report retrieval of a catheter fragment using an Amplatz loop snare through the umbilical vein and review the literature.

Esophageal/pyloric ligation enhances development of the murine fetal stomach in organ culture

PURPOSE The authors hypothesized that increased intraluminal pressure in the fetal stomach would enhance development in a murine organ culture model. METHODS Gestation day 14 (Gd14) fetal stomachs from time-dated pregnant CD-1 mice (term, 20 days) were maintained in organ culture for 7 days. Some stomachs were ligated at the gastroesophageal (GE) and pyloroduodenal (PD) junctions. Others were left unligated. Gd14, Gd16, and Gd18 stomachs were taken as well to compare organogenesis in vivo. Tissues were processed for histological, morphometric, and immunohistochemical analysis, as well as total protein and DNA determination. RESULTS The ligated stomachs were visibly distended compared with unligated stomachs in organ culture after 7 days. The length and width of the 7-day in vitro ligated stomachs were significantly increased compared with unligated (2.97+/-0.04 mm v 2.48+/-0.05 mm and 2.14+/-0.04 mm v 1.57+/-0.08 mm, respectively, P < .05). Mucosal epithelial cells showed nuclear polarization, and there was a distinct outer muscle layer in the ligated stomachs, but not in the unligated stomachs, which demonstrated pseudostratified epithelial cells in the mucosa. The ligated stomachs had increased in mucosal thickness compared with unligated (31.4+/-1.3 microm vs 24.9+/-0.9 microm, p < 0.05). The ligated stomachs also had significantly increased protein and DNA content when compared with unligated stomachs (65.8+/-3.1 microg and 23.3+/-1.2 microg v 55.0+/-2.7 microg and 19.0+/-1.2 microg, respectively, P < .05). However, there were no significant differences noted between the protein to DNA ratios. Immunohistochemical staining for proliferating cell nuclear antigen (PCNA), a marker for cell proliferation, demonstrated increased proliferative activity of the mucosal epithelial cells in the ligated stomachs. CONCLUSIONS Esophageal and pyloric ligation enhanced the development of the fetal stomach in vitro in comparison with unligated stomachs cultured under similar conditions. Developmental characteristics of the ligated stomachs paralleled that of Gd16 stomachs in vivo.

Wound complications after chemo-port placement in children: Does closure technique matter?

PURPOSE Wound dehiscence after chemo-port placement is a rare but potentially significant complication. We hypothesize that by using a simple running skin closure technique during chemo-port placement the rate of wound dehiscence and overall wound complications can be significantly decreased. METHODS IRB approval was obtained and patients <18years that received a tunneled central line with port from June 2012 to April 2016 were analyzed. Data collected on patients included patient demographics, skin closure type, and wound complications within 30days. Chi-square was performed to examine the univariate association with skin closure technique and wound dehiscence. Logistic regression was performed to examine the multivariable association between skin closure type and wound dehiscence and to compute odds ratios. RESULTS There were 259 ports placed in this cohort: 125 used simple running skin closure technique, and 134 used the subcuticular skin closure. Patients were found to not have any difference in rate of dehiscence or overall wound complications based on gender, age, location of port, or use of steroids or chemotherapy within 1week of port placement. When compared, only 1 case (0.80%) in the simple running group vs 10 cases (7.46%) in the subcuticular group experienced a wound dehiscence [unadjusted OR=14.07 (1.69, 116.99) p=0.0144]. When comparing overall wound complications the simple running group had 3 (2.4%) versus 12 (8.96%) in the subcuticular group [unadjusted OR=4.78 (1.27, 17.94) p=0.0203]. When adjusting for port-number both dehiscence and overall wound complications remained statistically significant. CONCLUSION We conclude that the simple running skin closure for chemo-port placement in children has superior outcomes in regards to prevention of dehiscence and overall wound related complications when compared to the subcuticular technique.

Stapled intestinal anastomoses with endoscopic staplers in premature infants

PURPOSE The safety and effectiveness of a stapled intestinal anastomosis in adults, children, and infants is well documented. However, in neonates it is not well validated. We hypothesized that premature infants who received a stapled bowel anastomosis utilizing endoscopic staplers had similar outcomes compared to patients with a handsewn anastomosis. METHODS A retrospective study was performed reviewing premature infants who underwent an intestinal anastomosis over a 4-year period. Patients greater than 36weeks gestational age at birth or a weight greater than 5kg at surgery were excluded. Patient demographics, type of intestinal anastomosis, and anastomotic related complications within 3months were collected and analyzed. RESULTS Sixty-five patients underwent 71 operations involving an intestinal anastomosis: 33 cases were handsewn, and 38 cases were stapled. Groups were noted to have differences in age, weight, and diagnosis. Complications including leak and anastomotic stricture did not differ between groups. Reports of blood per rectum after surgery were more common in the stapled group (24% versus 6%, p=0.0522), but this did not reach statistical significance. CONCLUSION There were no significant differences in anastomotic complications when comparing the handsewn and stapled intestinal anastomosis techniques in premature infants weighting less than 5kg. TYPE OF STUDY Treatment Study. LEVEL OF EVIDENCE III.