Contact Cooling of Random-Pattern Cutaneous Flaps: Does it Increase Necrosis?

BACKGROUND: Cooling after surgery reduces pain, swelling and ecchymosis. However, the fear of adverse effects of vasoconstriction caused by cooling may prevent its use when the skin is undermined extensively, for example, after rhytidectomy. The purpose of this study is to determine whether the contact cooling of random-pattern skin flaps increases the area of necrosis observed. METHODS: Twenty-eight random-pattern skin flaps (4 × 10 cm) were raised on four pigs. Flaps were divided into three groups: control, intermittently cooled and continuously cooled. Pads connected to a ThermaZone cooling device delivered local hypothermia in the range of 4-6 °C for 24 h postoperatively. ImageJ software was used to calculate the area of necrosis on each flap on postoperative day 7, confirmed with histological analysis. RESULTS: The average areas of necrosis observed were as follows: control (17.61 cm2; SD 5.23), intermittent cooling (15.65 cm2; SD 3.76) and continuous cooling (14.16 cm2; SD 3.91). An ANOVA revealed no statistically significant differences between the three interventions (p = 0.35). CONCLUSIONS: Postoperative continuous or intermittent cooling does not increase the area of necrosis in random-pattern flaps. In fact, a trend was observed, demonstrating decreasing area of necrosis with increased periods of hypothermia. NO LEVEL ASSIGNED: This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors. www.springer.com/00266 .

The Impact of Age Upon Healing: Absolute Quantification of Osteogenic Genes in Calvarial Critical-Sized Defects.

BACKGROUND: The current study was performed to elucidate changes in growth factor expression over time in critical-sized calvarial defects in rats from infancy to skeletal maturity. MATERIALS AND METHODS: Critical-sized parietal defects of 5, 6, and 8 mm were created in postnatal day 6 (P6), postnatal day (P20), and postnatal day (P84) adult rats, respectively. Dura was harvested at 3, 7, or 14 days after surgery, and serial micro-computed tomography imaging was performed through 12 weeks postoperatively. Absolute quantitative polymerase chain reaction was performed for Bone Morphogenic Protein-2 (BMP-2), Fibroblast Growth Factor-2 (FGF-2), Insulin-like Growth Factor-1 (IGF-1), and Transforming Growth Factor-ß1 (TGF-ß). RESULTS: The P6 (6-d-old) rats showed the greatest difference in gene expression between the dura derived from the defect side and the dura derived from the control side, demonstrating significant differences in TGF-ß1, BMP-2, IGF-1, and FGF-2 at various time intervals. Absolute gene expression in the defect dura was highest in the P6 rats and declined with age. Significant differences were noted at limited time points in the P20 rats for TGF-ß1 and BMP-2 as well as in the P84 rats for TGF-ß1. TGF-ß1 was the only gene studied that showed significant differences at postoperative days 3, 7, and 14 in varying age groups. CONCLUSIONS: The P6 rats have a higher osteogenic potential accompanied by a more vigorous alteration in growth factor expression compared with the P20 or P84 rats. Decrease in BMP-2 and FGF-2 as well as relative increase in TGFß-1 messenger RNA were observed in healing defects. These data provide valuable insight into the mechanism of healing of critical-sized defects and may be of use to engineer factor-releasing implants to correct skull defects.

Mechanical analysis of resorbable plates for long-term soft-tissue molding.

BACKGROUND: Resorbable plates are used with increasing frequency in pediatric craniofacial surgery. A recent innovation has been the use of these plates in long-term soft-tissue molding. However, these plates were not designed for these applications, and an unanswered question remains the mechanical rigidity and stability of the plate when placed in a prolonged out-of-plane bend. METHODS: Ten varieties of resorbable plates were folded to a right angle and subjected to a standardized 80-g load at body temperature over a duration of 2 weeks. Angle height was measured as an index of the ability of the plate to maintain its out-of-plane bend against an axial force. Each plate was subjected to 3 successive tests, after bending back to its original configuration. RESULTS: All plates showed a progressive decrease in angle height over 2 weeks. Thickness of the plate appeared to correlate with mechanical rigidity and ability to maintain out-of-plane bend. The plates that performed the best were the Synthes PLGA (85/15) 1.2-mm and BioGeneral PLA (poly-D,L-lactic acid) 1.0-mm plates. Decrease in angle height occurred over shorter intervals in successive tests. CONCLUSIONS: Of all the plates tested, none were able to maintain initial out-of-plane bend over 2 weeks of testing. Further research and development by industry are required to optimize mechanical properties of resorbable plates for applications in soft-tissue molding.

RNA interfering molecule delivery from in situ forming biodegradable hydrogels for enhancement of bone formation in rat calvarial bone defects.

RNA interference (RNAi) may be an effective and valuable tool for promoting the growth of functional tissue, as short interfering RNA (siRNA) and microRNA (miRNA) can block the expression of genes that have negative effects on tissue regeneration. Our group has recently reported that the localized and sustained presentation of siRNA against noggin (siNoggin) and miRNA-20a from in situ forming poly(ethylene glycol) (PEG) hydrogels enhanced osteogenic differentiation of encapsulated human bone marrow-derived mesenchymal stem cells (hMSCs). Here, the capacity of the hydrogel system to accelerate bone formation in a rat calvarial bone defect model is presented. After 12 weeks post-implantation, the hydrogels containing encapsulated hMSCs and miRNA-20a resulted in more bone formation in the defects than the hydrogels containing hMSCs without siRNA or with negative control siRNA. This localized and sustained RNA interfering molecule delivery system may provide an excellent platform for healing bony defects and other tissues. STATEMENT OF SIGNIFICANCE: Delivery of RNAi molecules may be a valuable strategy to guide cell behavior for tissue engineering applications, but to date there have been no reports of a biomaterial system capable of both encapsulation of cells and controlled delivery of incorporated RNA. Here, we present PEG hydrogels that form in situ via Michael type reaction, and that permit encapsulation of hMSCs and the concomitant controlled delivery of siNoggin and/or miRNA-20a. These RNAs were chosen to suppress noggin, a BMP-2 antagonist, and/or PPAR-γ, a negative regulator of BMP-2-mediated osteogenesis, and therefore promote osteogenic differentiation of hMSCs and subsequent bone repair in critical-sized rat calvarial defects. Simultaneous delivery of hMSCs and miRNA-20a enhanced repair of these defects compared to hydrogels containing hMSCs without siRNA or with negative control siRNA. This in situ forming PEG hydrogel system offers an exciting platform for healing critical-sized bone defects by localized, controlled delivery of RNAi molecules to encapsulated hMSCs and surrounding cells.

Endochondral Ossification in Critical-Sized Bone Defects via Readily Implantable Scaffold-Free Stem Cell Constructs.

The growing socioeconomic burden of musculoskeletal injuries and limitations of current therapies have motivated tissue engineering approaches to generate functional tissues to aid in defect healing. A readily implantable scaffold-free system comprised of human bone marrow-derived mesenchymal stem cells embedded with bioactive microparticles capable of controlled delivery of transforming growth factor-beta 1 (TGF-ß1) and bone morphogenetic protein-2 (BMP-2) was engineered to guide endochondral bone formation. The microparticles were formulated to release TGF-ß1 early to induce cartilage formation and BMP-2 in a more sustained manner to promote remodeling into bone. Cell constructs containing microparticles, empty or loaded with one or both growth factors, were implanted into rat critical-sized calvarial defects. Micro-computed tomography and histological analyses after 4 weeks showed that microparticle-incorporated constructs with or without growth factor promoted greater bone formation compared to sham controls, with the greatest degree of healing with bony bridging resulting from constructs loaded with BMP-2 and TGF-ß1. Importantly, bone volume fraction increased significantly from 4 to 8 weeks in defects treated with both growth factors. Immunohistochemistry revealed the presence of types I, II, and X collagen, suggesting defect healing via endochondral ossification in all experimental groups. The presence of vascularized red bone marrow provided strong evidence for the ability of these constructs to stimulate angiogenesis. This system has great translational potential as a readily implantable combination therapy that can initiate and accelerate endochondral ossification in vivo. Importantly, construct implantation does not require prior lengthy in vitro culture for chondrogenic cell priming with growth factors that is necessary for current scaffold-free combination therapies. Stem Cells Translational Medicine 2017;6:1644-1659.

Augmentation of intraorbital volume with fat injection.

BACKGROUND: Enophthalmos is a challenging surgical problem to correct. Standard techniques to adjust orbital volume require invasive maneuvers such as osteotomies. Fat injection may provide a simple and less-invasive way of augmenting orbital volume to correct enophthalmos. METHODS: The right eye orbital volume of 10 New Zealand White rabbits was augmented with fat. Autologous fat was diced and injected into the retrobulbar space. Computed tomographic scans were evaluated for changes in globe position and retrobulbar volume. Visually evoked potentials were conducted to test the integrity of the optic tract. Rabbits were killed at 12 weeks after surgery. Orbital exenterations were performed to allow for gross and histologic evaluation. RESULTS: Right globe position showed a mean increase in eye proptosis of 3.4 mm at postoperative day 1 and 0.9 mm at 11 weeks postoperatively in comparison with the left globe position. No significant change was noted in the left globe position. Retrobulbar volume demonstrated an initial mean increase of 31 percent and a final mean increase of 9.8 percent at 11 weeks in the right eye compared with the left eye. Visually evoked potentials revealed intact optic pathways in all animals. Gross anatomical evaluation showed deposition of fat grafts. Histologic analysis showed both revascularized and necrotic areas of fat. No retinal or optic nerve damage was identified. CONCLUSIONS: Fat injection can augment orbital volume in an animal model and preserve visual function. Further investigation is necessary to document the clinical safety and value of this technique in humans.

Pc 4 photodynamic therapy of U87-derived human glioma in the nude rat.

BACKGROUND AND OBJECTIVES: As a potential therapy for malignant glioma, we tested the phthalocyanine photosensitizer Pc 4 for: (1) rapid clearance from the vasculature, (2) specificity for glioma, and (3) tumoricidal photosensitizing capability. STUDY DESIGN/MATERIALS AND METHODS: Parenchymal injection of U87 cells into athymic rat brains (N = 100) was followed after 12 days by tail vein injection of 0.5 mg/kg Pc 4. After 1 day, the tumor was illuminated with either 5 (N = 11) or 30 (N = 16) J/cm(2) red light at 672 nm. Sacrifice was 1 day later. The brains from these 27 animals underwent H&E (necrosis) and TUNEL assay (apoptosis) histology. Pc 4 concentration of explanted brains and tumors (N = 16), and all blood samples (N = 52) were determined by HPLC-MS 1 day post Pc 4 administration. RESULTS: Tumor-specific apoptosis was almost uniformly seen; however, necrosis was found mostly in the high-light-dose group. Pc 4 concentration in bulk tumor averaged 3.8 times greater than in normal brain. CONCLUSIONS: These results warrant expanding this pre-clinical study to seek effective baseline Pc 4 drug- and light-doses and infusion-to-photoirradiation timing that would be necessary for a Pc 4-mediated PDT clinical trial for glioma patients.