Search

Mammalian skin impacts metabolic efficiency system-wide, controlling the rate of heat loss and consequent heat production. Here we compare the unique fat depots associated with mouse and human skin, to determine whether they have corresponding function and regulation. For human, we assay a skin-associated fat (SAF) body-wide depot to distinguish it from the subcutaneous fat pads characteristic of abdomen and upper limbs. We show that the thickness of SAF is not related to general adiposity; it is much thicker (1.6-fold) in women than men, and highly subject-specific. We used molecular and cellular assays of β-adrenergic induced lipolysis and found that dermal white adipose tissue (dWAT) in mice is resistant to lipolysis; in contrast, the body-wide human SAF depot becomes lipolytic, generating heat in response to β-adrenergic stimulation. In mice challenged to make more heat to maintain body temperature (either environmentally or genetically), there is a compensatory increase in thickness of dWAT: A corresponding β-adrenergic stimulation of human skin adipose (in vivo or in explant) depletes adipocyte lipid content. We summarize the regulation of skin-associated adipocytes by age, sex, and adiposity, for both species. We conclude that the body-wide dWAT depot of mice shows unique regulation that enables it to be deployed for heat preservation; combined with the actively lipolytic subcutaneous mammary fat pads they enable thermal defense. The adipose tissue that covers human subjects produces heat directly, providing an alternative to the brown adipose tissues.KEY POINTS SUMMARYSeveral distinct strategies produce and conserve heat to maintain body temperature of mammals, each associated with unique physiologies, with consequence for wellness and disease susceptibilityHighly regulated properties of skin offset the total requirement for heat productionWe hypothesize that the adipose component of skin is primarily responsible for modulating heat flux; here we evaluate the relative regulation of adipose depots in mouse and human, to test their recruitment to heat production and conservationWe found that insulating mouse dermal white adipose tissue accumulates in response to environmentally- and genetically-induced cool stress; this layer is one of two adipose depots closely apposed to mouse skin, where the subcutaneous mammary gland fat pads are actively recruited to heat productionIn contrast, the body-wide adipose depot associated with human skin produces heat directly, potentially creating an alternative to the centrally regulated brown adipose tissue

Extracellular functions of the endoplasmic reticulum chaperone protein calreticulin (CRT) are emerging. Here we show novel roles for exogenous CRT in both cutaneous wound healing and diverse processes associated with repair. Compared with platelet-derived growth factor-BB-treated controls, topical application of CRT to porcine excisional wounds enhanced the rate of wound re-epithelialization. In both normal and steroid-impaired pigs, CRT increased granulation tissue formation. Immunohistochemical analyses of the wounds 5 and 10 days after injury revealed marked up-regulation of transforming growth factor-beta3 (a key regulator of wound healing), a threefold increase in macrophage influx, and an increase in the cellular proliferation of basal keratinocytes of the new epidermis and of cells of the neodermis. In vitro studies confirmed that CRT induced a greater than twofold increase in the cellular proliferation of primary human keratinocytes, fibroblasts, and microvascular endothelial cells (with 100 pg/ml, 100 ng/ml, and 1.0 pg/ml, respectively). Moreover, using a scratch plate assay, CRT maximally induced the cellular migration of keratinocytes and fibroblasts (with 10 pg/ml and 1 ng/ml, respectively). In addition, CRT induced concentration-dependent migration of keratinocytes, fibroblasts macrophages, and monocytes in chamber assays. These in vitro bioactivities provide mechanistic support for the positive biological effects of CRT observed on both the epidermis and dermis of wounds in vivo, underscoring a significant role for CRT in the repair of cutaneous wounds.

To properly evaluate the potential benefits of in utero repair for cleft lip and palate, an animal model of cleft lip and palate formation is needed that simulates human cleft morphology. We have developed a fetal lamb model in which incisional or excisional unilateral cleft lips were created early in gestation and later repaired. Through a maternal celiotomy and hysterotomy, six excisional and six incisional clefts were created in early first trimester fetal lambs. Clefts were created by a novel space helmet technique and made completely through the lip and the alveolus. Two weeks later, the wound edges were freshened and repaired in all but one lamb in each group. In the early third trimester, the fetuses were harvested and studied. We found that the incisional clefts healed spontaneously with replacement of the native reticular collagen pattern and with regeneration of the skin appendages. However, the excisional clefts did not autorepair and, when repaired surgically, healed without a collagen scar but showed no regeneration of the skin appendages. This preliminary study is the first successful large animal model where cleft lips and palates were created, allowed to develop with the cleft, and later repaired in utero. Prior to repair, the edges of the excisional clefts epithelialize. When repaired early in the third trimester, excisional clefts heal without a dermal scar but exhibit incomplete regeneration of the skin appendages.

Microvascular techniques are important in making routine anastomoses quicker and difficult anastomoses easier. The authors have developed three types of sleeve anastomoses for different size vessels: a three-stitch sleeve anastomosis, four-stitch sleeve anastomosis, and sidecut sleeve anastomosis. Arteriography, in vivo observation, pulsed ultrasound Doppler hemodynamic study, histology, and ultrastructural examination with scanning electron microscopy at 2 to 90 days demonstrated that all three groups of sleeve anastomoses do not stenose, do not show hemodynamically significant vasospasm, and are not complicated by aneurysm formation by 90 days. The hemodynamics were comparable to control sutured anastomoses at 2 to 4 hr postoperatively. The sleeve anastomoses left no suture material within the vessel lumen. Reendothelialization was comparable with controls at 14 days, and the long-term patency rates were similar (p > 0.05). The operative times for all three types of sleeve anastomoses were significantly shorter than sutured control anastomoses (p < 0.001). Although limited by available vessel length and diameter, the sleeve anastomosis is a useful technique for the microsurgeon.

Using a newly developed Doppler probe, we have found that a standard suture anastomosis in a rat femoral artery is accompanied by significant (30-60%) cross-sectional area stenosis, which dissipates to baseline levels within 24 hours. We hypothesized that spasm, deposition of coagulation products, or the suture technique itself was responsible. Topical vasodilators (papaverine, sodium nitroprusside, lidocaine) and intravenous thromboxane A2 synthetase inhibitor and receptor blocking agent (Ridogrel, 4 mg/ml), anticoagulants heparin and SC4992 (an experimental platelet inhibitor/arginine-glycine-aspartic acid analogue), were administered. No drug had any significant effect on preventing postoperative stenosis. Varied suture bites affected stenosis measurements. Scanning electron microscopy and light microscopy displayed "bunching" of vessel wall in the suture ties. This was confirmed with methyl methacrylate corrosion casts and microangiography. "Sham" anastomoses also produced stenosis, which was relieved when sutures were removed. We conclude that suture anastomosis of small vessels is accompanied by significant cross-sectional stenosis caused by the physical action of tensioned sutures. This effect dissipates over a 24-hour postoperative period. The mechanism behind these changes and the clinical importance of this effect are still under investigation.

Wound contraction is an important component of healing but, in the extreme, may lead to excessive scar formation and pathological wound contracture. Fetal rabbit wounds heal without contraction or scarring, whereas excisional fetal sheep wounds have been shown to contract, but no scarring or pathological wound contracture is noted. We used an in vitro model, the fibroblast-populated collagen lattice, to study the ability of fetal fibroblasts to coordinate contraction of a collagen matrix and the modulating effects of epidermal growth factor and transforming growth factor-beta 1 on this contraction. With increasing gestational age, fibroblasts increased the degree of collagen lattice contraction. Epidermal growth factor inhibited contraction by fetal fibroblasts, whereas transforming growth factor-beta 1 stimulated it. These findings suggest that while intrinsic differences between fetal and adult fibroblasts exist, polypeptide growth factors may operate at the site of tissue repair to alter cell phenotype. Further work is underway to delineate the role of soluble protein factors responsible for the absence of scarring and contracture seen in the fetal wound.

The ability of fetal skin wounds to heal without scar formation is remarkable. The mechanisms that endow the fetus with this unique healing ability remain unknown. We have developed an in vitro explant model using fetal sheep skin to investigate fetal wound healing. This model eliminates the complex systemic mechanisms that modulate in vivo wound healing. We demonstrated that using an enriched medium, midgestation fetal sheep skin explants following wounding reepithelialized within 4 days. By 7 days after wounding the confluent epidermis was thicker, but the dermal wound remained open. This model demonstrates that it is possible to achieve conditions in culture that maintain tissue viability and support reepithelialization. This model may allow us to resolve some of the individual components that participate in the process of scarless fetal skin healing.

Calreticulin (CRT), an intracellular chaperone protein crucial for the proper folding and transport of proteins through the endoplasmic reticulum, has more recent acclaim as a critical regulator of extracellular functions, particularly in mediating cellular migration and as a requirement for phagocytosis of apoptotic cells. Consistent with these functions, we show that the topical application of CRT has profound effects on the process of wound healing by causing a dose-dependent increase in epithelial migration and granulation tissue formation in both murine and porcine normal and impaired animal models of skin injury. These effects of CRT are substantiated, in vitro, as we show that CRT strongly induces cell migration/wound closure of human keratinocytes and fibroblasts, using a wound/scratch plate assay, and stimulates cellular proliferation of human keratinocytes, fibroblasts, and vascular endothelial cells, providing mechanistic insight into how CRT functions in repair. Similarly, in both animal models, the histology of the wounds show marked proliferation of basal keratinocytes and dermal fibroblasts, dense cellularity of the dermis with notably increased numbers of macrophages and well-organized collagen fibril deposition. Thus, CRT profoundly affects the wound healing process by recruiting cells essential for repair into the wound, stimulating cell growth, and increasing extracellular matrix production.

The postoperative outcome of hand flexor tendon repair can be complicated by adhesions between the repair site and surrounding tissue. To date, the biology of hand flexor tendon wound healing remains controversial--both intrinsic (resident tenocyte) and extrinsic (tendon sheath fibroblast and inflammatory cell) processes may contribute to repair. Transforming growth factor beta-1 is a cytokine that plays multiple roles in wound healing but is also implicated in the pathogenesis of excessive scar formation. This study examines the activation of transforming growth factor beta-1 mRNA in a rabbit zone II flexor tendon wound-healing model. Forty New Zealand White rabbit forepaws underwent complete transection and repair of the middle digit flexor digitorum profundus tendon in zone II. Tendons were harvested at increasing time intervals (1, 3, 7, 14, 28, and 56 days) and analyzed by in situ hybridization and immunohistochemistry to determine the expression patterns of transforming growth factor beta-1. A small number of tenocytes exhibited expression of transforming growth factor beta-1 mRNA at baseline in nonwounded control tendon specimens. The surrounding tendon sheath in these control specimens also revealed low numbers of fibroblasts and inflammatory cells expressing transforming growth factor beta-1 mRNA. In contrast, flexor tendons subjected to transection and repair exhibited increased signal for transforming growth factor beta-1 mRNA in both resident tenocytes and infiltrating fibroblasts and inflammatory cells from the tendon sheath. These data demonstrate that (1) normal unwounded tenocytes and tendon sheath cells are capable of transforming growth factor beta-1 production, (2) this cytokine is activated in the tendon wound environment, as evidenced by mRNA upregulation, and (3) the upregulation of this cytokine in both "intrinsic" tenocytes and "extrinsic" tendon sheath fibroblasts and inflammatory cells supports dual mechanisms for tendon repair. Because transforming growth factor beta-1 is thought to contribute to the pathogenesis of excessive scar formation, the findings presented here suggest that perioperative biochemical modulation of transforming growth factor beta-1 levels may help limit flexor tendon adhesion formation.

Multiple microvascular anastomotic techniques have been described with varying rates of success. This experimental study presents the results of a comparison of three types of venous microanastomotic techniques: the Unilink system, the sleeve technique, and the suture technique. Twenty male Sprague-Dawley rats, 40 femoral veins, were used for this study. In vivo observation and microvasculography demonstrated that patency rates between the Unilink system and suture techniques were comparable (p > 0.05) and were significantly superior to the sleeve anastomosis (p < 0.05). The anastomotic time for the sleeve technique was significantly shorter than for the suture technique (p < 0.001). Compared with suture and sleeve anastomoses, the anastomotic time employing the Unilink system was significantly the shortest (p < 0.001). The Unilink system proved to be the fastest method with the highest patency rate. These results suggest that the use of the Unilink system is superior with regard to anastomotic time and patency rate, when compared to suture and sleeve techniques for venous microanastomosis.

A new technique for microvascular four-stitch sleeve anastomosis of arteries is described. Modifications from previously described techniques include absence of suture material within the arterial lumen, and a decreased proximal to distal artery overlap. In 40 rat arterial anastomoses, 100% patency without evidence of stenosis or early aneurysm formation was seen up to 6 weeks postoperatively by in vivo observation, arteriography and Doppler flow study. This new anastomosis was also successful in repairing two clinical cases of complete radial artery transection as evident from physical examination and doppler flow study. We believe that this new four-stitch sleeve anastomosis is faster and easier to perform and has patency and flow characteristics similar to those of the conventional end-to-end anastomosis.

This study assessed the postoperative microvascular hemodynamics in end-to-side arterial anastomoses of varied angles in order to determine whether the angle of the anastomosis affects arterial flow. Thirty male rats were divided into three groups of ten. Carotid to carotid end-to-side anastomoses were performed with anastomotic angles of 45, 90, and 135 degrees. Postoperative flow was assessed using high frequency pulsed Doppler ultrasound (HFPDU) at 30 and 120 min postoperatively. Measurements at 30 min demonstrated significant differences in flow, with the 45 degrees grafted vessels utilizing 56.5% of total carotid flow, and the 90 degrees and 135 degrees anastomoses appropriating 46.5% and 43.2% of flow, respectively (comparing 45 degrees to both 90 degrees, P < 0.05, and 135 degrees, P < 0.002). The change from baseline in 45 degrees and 90 degrees groups dissipated over a 2 hr postoperative period, but flow in the grafted vessels in the 135 degrees group continued well below 50% at 39.9% (comparing to both 45 degrees and 90 degrees, P < 0.001), thereby displaying a significant difference in the postoperative arterial flow of varied angled microanastomoses.

Evolving microsurgical technique has allowed for the anastomosis of smaller diameter vessels. Standard suture anastomoses cause a measure of stenosis and intimal damage during application and therefore cannot be dependably used in the anastomosis of very small vessels. We developed and tested a fibrin glue-based anastomosis applied over a meltable stent made of mono- di- and tri-glycerides. In vivo rodent studies using the 0.35 mm diameter inferior epigastric artery have shown immediate and short-term patency rates better than those associated with suture technique. The stent technique is significantly faster and easier than the suture technique. The glyceride stent method suffers from decreased late patency due to aneurysm formation. In addition, we developed a glyceride-coated polyethylene glycol-based stent for use in lasered anastomoses. Work on both projects is ongoing.

Several researchers and clinicians have taken advantage of the omentum's rich vascular arcades to support skin grafts. We have previously described an experimental model using the omentum as a vascular carrier for prefabricated free flaps in the rat. In this study, we used this model to compare three different sizes of free flaps using the same size omental carrier. Twenty-four male Sprague-Dawley rats were used for this study. A 2.5 × 4 cm patch of omentum with gastroepiploic vessels and its rich vascular arcades was transferred under a bipedicled 2.5 × 6 cm (group I), a 2.5 × 8 cm (group II), and a 4 × 10 cm (group III) right abdominal panniculocutaneous flap. On the seventh postoperative day, the skin pedicles were divided and the skin flap raised as a composite island flap vascularized only by the underlying omental patch. The composite flap was then sutured back in place. Prefabricated flaps examined 7 days postoperatively demonstrated a dye florescence index percent (DFI) of 38.19 ± 7.52 and 98.13 ± 3.72% flap survival (FS) in the 6 ± 2.5 cm skin flap group; a DFI of 39.96 ± 6.81% and FS 94.88 ± 7.08% in the 8 ± 2.5 skin flap group (P > 0.05) and a DFI 29.71 ± 2.85% and FS 57.06 ± 9.52% in the 10 × 4 cm skin flap group (P < 0.05). India ink injection study and histologic examination confirmed revascularization of the overlying skin at 7 days. This study confirms that omentum can be used as a vascular carrier for prefabricated flaps. However, there is a limit to the size of the flap. A 10 cm2 carrier can support 57% of a 40 cm2 (10 × 4 cm) flap for a total area of 22.8 cm2, more than twice the area of the carrier. © 1994 Wiley-Liss, Inc.

We report on the use of dental x-ray equipment for micro-arteriography in 18 rat arterial anastomoses. Our experience has shown that an exposure of 70 kV, at 7mA, for 0.17 seconds, gives excellent results. This is an easy and useful technique for evaluating the patency and stenosis of microvascular anastomoses. © 1993 Wiley-Liss Inc.

A case of successful microvascular replantation of a traumatically amputated ear is presented. The postoperative course was complicated by venous thrombosis requiring the use of medicinal leeches and systemic heparinization for salvage. This is the tenth successful microvascular ear replantation reported in the literature. © 1993 Wiley-Liss Inc.

Tissue expansion alters the function of skin cells. We studied the effects of expansion on the contractile function of dermal fibroblasts using an in vitro model, the fibroblast-populated collagen lattice (FPCL). Spherical expanders were placed dorsally in 30 Sprague-Dawley rats; one-half were serially inflated. One, 2, and 4 weeks later, 5 rats from each group were killed. Fibroblasts were cultured from dermis overlying the expanders and mixed with collagen, medium, and serum in petri dishes to form FPCL. Fibroblasts from 5 rats that had not undergone surgery were cultured to make control FPCL. Contraction was assessed by measuring the areas of the FPCL. At 48 hours, FPCL containing expanded fibroblasts had contracted significantly less than those containing unexpanded or control fibroblasts. Four weeks of expansion resulted in less contraction than 1 or 2 weeks. Tissue expansion inhibits the in vitro contractile function of dermal fibroblasts in the rat in a time-related fashion.

The omentum used as a vascular carrier is a new experimental model for prefabrication of free flaps in the rat. A 2.5 x 4 cm patch of omentum with gastroepiploic vessels and its rich vascular arcades was transferred under a bipedicled 2.5 x 6 cm right abdominal panniculocutaneous flap. At subsequent time intervals (postoperative days 0, 3, 5, 7, and 14), the skin pedicles were divided and the skin flap was raised as a composite island flap vascularized only by the underlying omental patch. The composite flap was then sutured back in place. Prefabricated flaps examined postoperatively demonstrated a dye fluorescence index (DFI) of 4.60% +/- 2.82% and 1.67% +/- 4.08% flap survival (FS) in the day 0 group; in the day 3 group, 9.83% +/- 1.47% of DFI and 15.00% +/- 10.35% of FS; in the day 5 group, 28.39% +/- 5.25% of DFI and 86.0% +/- 12.74% of FS (P less than 0.05); in the day 7 group, 38.19% +/- 7.52% of DFI and 98.13% +/- 3.72% of FS (P less than 0.05); and, in the day 14 group, 49.55% +/- 6.70% of DFI and 100% of FS (P less than 0.05). India-ink injection and histologic examination confirmed revascularization of the overlying skin by day 5.

Hyaluronan has long been associated with the remodelling extracellular matrix. Such remodelling occurs in development, growth and wound healing. This role has been thought to be related to the physical structure and chemical composition of the pure glycosaminoglycan chain. We question this proposition and present evidence which suggests that proteins associated with hyaluronan may be more critical determinants of tissue remodelling.

The use of High-Frequency Pulsed Doppler Ultrasound (HFPDU) for evaluation of microvascular hemodynamics is well established. Due to technical limitations of existing probes, quantitation of anastomotic stenoses and detailed waveform analyses are difficult to perform and impractical for clinical use. We present a new Tandem Doppler Probe (TDP) for simple and accurate study of blood flow in vessels smaller than 1 mm in diameter. Its unique ability to compare the pre- and post-anastomotic waveforms simultaneously allows for quantitative detection of anastomotic narrowing of as little as 5%, as well as identification of subtle pathologic anastomotic waveform changes not seen with single probes. Such early and precise delineation of anastomotic problems in the operating room can provide an invaluable, objective assessment of the technical adequacy of the anastomoses as well as an important baseline for later post-operative monitoring of free-tissue transfers with implantable Doppler devices.

This study describes our experience using an improved technique for microsurgical sleeve anastomosis of arteries. Modifications of previously described techniques include utilization of a sidecut and placement of the two stitches within separate vertical planes. In 40 arteries, we attained a patency rate of 97.5%. Histology, in vivo observation, and flow study using high-frequency pulsed ultrasound Doppler at 2 hr to 8 weeks demonstrate that the sidecut sleeve anastomosis shows patency and flow characteristics similar to those of standard end-to-end anastomosis, but with a dramatic decrease in operative time.