In vitro reconstitution of skin: fibroblasts facilitate keratinocyte growth and differentiation on acellular reticular dermis.

Extensive full-thickness burns require replacement of both epidermis and dermis. We have described a method in which allogeneic dermis from engrafted cryopreserved cadaver skin was combined with cultured autologous keratinocytes. In the present study we combined human keratinocytes and fibroblasts, and acellular human dermis in vitro and transplanted this "reconstituted skin" into athymic mice. Both human papillary dermis in which the basement membrane zone has been retained and human reticular dermis that has been repopulated with human dermal fibroblasts are good substrates for keratinocyte attachment, stratification, growth, and differentiation. Both of these dermal preparations can be lyophilized and stored at room temperature without losing their ability to support keratinocyte growth. In contrast, human papillary dermis that has been treated with trypsin lacks laminin and collagen type IV in the BMZ and supports keratinocyte attachment and differentiation less well.

Reconstitution of structure and cell function in human skin grafts derived from cryopreserved allogeneic dermis and autologous cultured keratinocytes.

Grafts of allogeneic dermis plus autologous epidermal cell cultures were used to replace extensively burned skin. Cryopreserved split-thickness cadaveric skin was grafted onto debrided burn wound, and autologous keratinocytes were cultured from uninjured donor sites. Several weeks later, allograft epidermis was abraded and replaced with the keratinocyte cultures. The final grafts were thus composites of autologous cultured epidermis and allogeneic dermis. In a case with 28 months follow-up, reconstitution of the dermal-epidermal (BMZ.1) and microvascular (BMZ.2) basement membrane zones was studied immunohistochemically and ultrastructurally. Immediately before grafting, thawed cryopreserved skin reacted with antibodies against laminin and type IV collagen in normal patterns. Twenty-nine days after grafting, BMZ.1 reacted weakly with both antibodies, and anticollagen type IV reactivity was absent from BMZ.2. Antilaminin reactivity of BMZ.2, however, was moderately intense, consistent with recent neovascularization. On day 29, the allograft epidermis was replaced with autologous keratinocyte cultures. Twenty-five days later (54 d after allografting), staining of both BMZs was intense with both antibodies. Ultrastructurally, at day 76 (47 d after culture placement) BMZ.1 revealed only small hemidesmosomes, few incipient anchoring fibrils, and a discontinuous lamina densa. BMZ.2, however, was fully reconstituted. By 124 d, both BMZs appeared normal. Observations in the dermis at 76 d included the presence of lymphocytes, organellar debris, and hyperactive collagen fibrillogenesis, all indicative of dermal remodelling. The microvasculature was well differentiated, but no elastic fibers or nerves were found. In the epidermis, melanocytes and evidence of melanosome transfer were seen at 5, 47, and 95 d after grafting of keratinocyte cultures. We conclude that the composite procedure reconstitutes skin with excellent textural and histologic qualities.

Augmentation of experimental skin flap survival via postoperative phosphocreatine replacement.

Despite improvements in our understanding of cutaneous vascular territories, clinical skin flap necrosis resulting from ischemic compromise is still a reality. Therapy with vasodilators has been generally unsuccessful, but replacement of high-energy phosphometabolites through the use of ATP-MgCl2 and fructose 1,6-diphosphate has been effective. Recently we reported that phosphocreatine is the major high-energy phosphometabolite in mammalian skin and that ATP levels and cellular well-being in skin flaps are dependent on adequate supply of this phosphometabolite. We report herein the successful augmentation of survival of ischemically compromised skin flaps through postoperative phosphocreatine infusion. This metabolite effectively circumvents the nonfunctioning mitochondrial creatine-phosphocreatine energy shuttle without disturbing the delicate [ATP]/[ADP] balance in the cytosol. In addition, phosphocreatine may favorably redistribute blood flow from muscle to the ischemically compromised skin.

Myocutaneous flaps for head and neck reconstruction.

The recent development of myocutaneous flaps has ushered in an era of one-stage reconstructions, which have changed the patterns of surgery for head and neck cancer. This article reviews the development of these flaps and the clinical applications at various sites of the head and neck. Personal experience with 31 consecutive myocutaneous flaps confirms the versatility and reliability of these new techniques.

Repair of lumbosacral myelomeningoceles with double Z-rhomboid flaps. Technical note.

The authors describe a double Z-rhomboid technique for the closure of large lumbosacral myelomeningocele defects. After the dorsally displaced neural tissue has been reduced into the vertebral canal and a watertight closure of the dura lining the sac has been achieved, the skin defect is surgically converted to the shape of a rhombus. Equilateral Z-plasty flaps are elevated at the sides of the rhombus and transposed across the defect to secure wound closure. A successful experience with 10 consecutive cases of lumbosacral myelomeningoceles repaired with this technique is reported.

Use of the pectoralis major myocutaneous flap for reconstruction of large cervical, facial or cranial defects.

The pectoralis major myocutaneous flap has been very reliable for reconstruction of large facial defects, orbital exenteration and radical temporal bone resection. Experience with this flap in such extensive resections in 12 consecutive patients in whom the flap was transferred successfully in one stage is reported.

Double Z-plasty repair of large and small rhombic defects: the double-Z rhomboid.

An alternative method for the repair of rhombic skin defects utilizing opposing Z-plasties has been developed and applied clinically. The design, orientation, and execution of this method share the simplicity of the Z-plasty. The technique has been employed successfully in over 100 cases during the past 5 years to resurface defects as large as 150 cm2, and it obviates many of the inherent limitations of the Limberg flap.

Immediate reconstruction of a composite mandibular defect with a regional osteomusculocutaneous flap.

This is a report of an immediate reconstruction of a composite mandibular defect, using a pectoralis major osteomusculocutaneous flap incorporating a segment of the fifth rib. Viability of the transferred rib segment was established by tetracycline labeling at 2 weeks and technetium scanning at 3 months postoperatively. This clinical example supports the experimental observations of a vascular network interconnecting muscle, periosteum, and compact bone.

The carpal boss: surgical treatment and etiological considerations.

We report on 30 symptomatic cases of carpal boss which were treated surgically. The condition represents a highly localized degenerative arthritis at the base of the middle metacarpal, and is seen primarily in relatively young patients. Symptomatic relief was obtained by excision of degenerated tissue in all these patients.

Double-Z rhombic technique for reconstruction of facial wounds.

The double-Z rhombic technique of repair of excisional defects is characterized by borrowing the required tissue from two nonadjacent opposite sides of the defect. Most other flaps borrow the required tissue from a single adjacent region or all adjacent directions. The "sharing" of tissue from two opposite regions minimizes tension in that direction, while not borrowing from the remaining regions prevents the distortion of anatomic landmarks located along that direction. The orientation of the final scar and direction of tissue tension can be controlled by rotating the rhombic defect about its central axis. This study was undertaken to assess the utility of the double-Z rhombic technique in terms of cosmesis and avoidance of displacement of mobile anatomic landmarks such as eyelids, eyebrows, nasal alae, and lips. Excisional defects resulting from removal of skin neoplasms in 30 patients in whom primary closure or reconstruction with direct tissue advancement was not feasible and displacement of facial landmarks was undesirable were reconstructed using the double-Z rhombic technique. No considerable asymmetry or facial anatomic landmark deformity was observed in any of the 30 patients. Our results are presented along with representative illustrations.

Versatility and safety of flap coverage for wide excision of cutaneous melanomas.

This study is a retrospective analysis of a consecutive series of 85 patients with primary melanomas of the trunk (51 patients) and extremities (34 patients) treated by wide excision and flap repair. Both the standard rhombic flap (39 cases) and the double-Z rhomboid flap (46 cases) were used. Postoperative follow-up was 11 to 49 months, with a mean of 20 months. Major postoperative flap necrosis was encountered in one case (1.1 percent). There were no local recurrences and only one instance of intransit metastasis. As compared with skin grafts, flap repairs were found to be free of the classic contour defect. In this series, flap repair also permitted earlier mobilization and resulted in shorter hospital stays.

Single-stage repair of complex scalp and cranial defects with the free radial forearm flap.

The free radial forearm flap provides a reliable single-stage method to cover difficult scalp and forehead defects where other local methods are unsuitable. Six patients have been successfully reconstructed utilizing seven free radial forearm flaps. A description of our surgical technique along with illustrative cases demonstrate the indications and advantages of this flap. Cranial soft-tissue deficits are primarily cutaneous; therefore, a more predictable reconstruction can be obtained utilizing a cutaneous flap with similar characteristics. Restoration of bony defects should be considered separately utilizing autogenous bone and/or alloplastic materials and not merely obliterated with a bulky soft-tissue flap. Therefore, if the local factors of a cranial wound require the utilization of a distant tissue transfer, we prefer the thin coverage of a well-vascularized free fasciocutaneous radial forearm flap over two-stage distant pedicle flaps or skin-grafted free flaps.

Nuclear magnetic resonance spectroscopy of skin: predictive correlates for clinical application.

The first application of phosphorous 31 (31P) and proton (1H) nuclear magnetic resonance (NMR) spectroscopy to the analysis of the metabolic profiles of skin flaps in a rat model and of human skin grafts is presented. Resonances of adenosine triphosphate (ATP), phosphocreatine (PCr), and inorganic phosphate (Pi) were identified in 31P nuclear magnetic resonance spectra. Resonances of phosphocreatine, creatine (Cr), and lactate (Lac) were identified in 1H nuclear magnetic resonance spectra. The most significant finding was the substantial presence of phosphocreatine as the major high-energy phosphometabolite in mammalian skin, a finding which heretofore has not been widely recognized. An energy shuttle between phosphocreatine and ATP is operative in skin to buffer the fall in ATP during ischemic (anaerobic) insult. Inability to replenish exhausted phosphocreatine reserves predictively correlates with eventual flap necrosis. We have defined and analyzed temporal fluxes in the phosphocreatine-creatine and phosphocreatine plus creatine-lactate ratios by proton nuclear magnetic resonance. Both are sensitive, accurate, and unambiguous early prognostic indices of eventual flap outcome. These findings support the concept that the fate of a flap may be established as early as 3 hours after elevation and have laid the groundwork for development and application of noninvasive in vivo nuclear magnetic resonance spectroscopy to the study of skin flaps in animals and humans.

Composite autologous-allogeneic skin replacement: development and clinical application.

A major unsolved problem in skin restoration in severe burns is replacement of lost dermis. We report the development and clinical application of a composite grafting technique in which allogeneic skin is the source of dermis, and cultured autologous keratinocytes generate epidermis. Excised burn wounds are resurfaced with unmatched allograft. Immunosuppression from the burn and reduced immunoreactivity of the allograft permit successful allograft engraftment. Keratinocyte cultures are initiated from the patient. Allogeneic epidermis is removed, and the dermal bed is resurfaced with keratinocyte cultures. The allogeneic dermis promotes rapid (less than 7 days) stratification, maturation, and integration of the cultures and the synthesis of anchoring fibrils. One case followed 11 months has shown no evidence of rejection. We reason that removal of the epidermis from allograft eliminates the majority of cells constitutively expressing alloclass II antigens, leaving behind a viable allogeneic dermal bed that serves as an ideal substrate for engraftment and integration of keratinocyte cultures but does not initiate rejection.

Improved reliability in tumor diagnosis by fine needle aspiration.

A simple but useful modification of sample preparation of fine needle aspirates of soft tissue masses is presented, in conjunction with its assessment by a controlled study. The modification, employing routine histologic techniques, reduces the requirement for cytopathologic expertise. The potential utility of fine needle aspiration is thereby significantly expanded. In addition, this modification allows the pathologist to use special tissue strains or electron microscopy as options, when necessary, to enhance diagnostic accuracy.

Critical role of phosphagens in the energy cascade of cutaneous ischemia and protective action of phosphocreatine analogues in skin flap survival.

A general understanding of the pivotal role of phosphocreatine (PCr) as the principal determinant of skin flap survival is now emerging. Definitive metabolic investigations using phosphorus (31P) and proton (1H) magnetic resonance spectroscopy (MRS) have established that the inability to replenish metabolically exhausted PCr reserves predictably correlates with skin flap necrosis. Furthermore, postoperative parenteral administration of PCr has been shown to augment effectively skin flap survival. We hypothesized that creatine kinase, the enzyme controlling the utilization of the high-energy phosphate component of PCr, is a critical determinant of the tolerance of a skin flap to ischemic insult. In other words, if the rate of utilization of PCr is too rapid, PCr stores will rapidly deplete, and the flap will not be able to withstand a period of ischemia. Alternatively, if the rate of dephosphorylation of PCr is reduced, survival of skin flaps during periods of ischemia could be extended. To test this hypothesis, we investigated the metabolic distribution and fate of cyclocreatine (cCr), a competent creatine analogue with a lower affinity for the creatine kinase enzyme. When administered as 1.5 percent (w/w) of the normal diet of laboratory rats, cCr accumulates in skin as the competent phosphagen, phosphocyclocreatine (PcCr). Cutaneous flaps elevated in these animals, and studied by 31P and 1H MRS, demonstrate that once depletion of PCr has occurred, PcCr continues to sustain ATP levels. This results in significant enhancement of skin flap survival (p < 0.005). These observations confirm the importance of the creatine kinase enzyme in cutaneous flap ischemia and suggest new approaches to augment skin flap survival.

The role of topical treatment as a determinant of infection in outpatient burns.

Up to 95% of all burned patients are treated as outpatients. While uniform guidelines exist to evaluate the severity of an outpatient's burn, treatment forms vary greatly. Furthermore, the relative merits of one treatment modality over another have not been demonstrated. This study reviews 262 patients with uncomplicated, thermal partial-thickness burns. All of these patients were treated with either a petrolatum fine-mesh gauze (FMG = 102), a topical penetrating antibacterial agent (TPA = 58), or a topical nonpenetrating antibacterial agent (TNA = 102). The size of the injury along with its location and etiology, the age of the patient, the time from injury to treatment, and comorbid factors were comparable among the major treatment groups. On follow-up, a wound was classified as infected if it exhibited erythema, tenderness, increased warmth, and edema (cellulitis). Infection rates were 2.0% (2/102) in the FMG group, 5.2% (3/58) in the TPA group, and 2.0% (2/102) in the TNA group. The differences among the rates did not reach statistical significance (P = 0.41). Given the cost of treatment, frequency of dressing changes and overall patient comfort, we advocate FMG as the optimal method for the care of uncomplicated partial-thickness outpatient burns.

Immunologic alterations following excisional wounding and immediate repair with syngeneic or allogeneic skin grafts.

Immunosuppression and its associated infectious complications have long been recognized as consequences of major thermal trauma, though the factors that mediate this suppression remain unclear. A murine split-thickness skin graft model was developed to investigate the role of a large surface area wound in the initiation of immunosuppression in the absence of burn injury. Significant T cell-mediated immunosuppression was demonstrated following wounding and immediate repair with either syngeneic or allogeneic split-thickness skin grafts. These results are consistent with previous experiments in a murine burn model treated by escharectomy and resurfacing with syngeneic composite full-thickness skin. Data also supports the concept that mediators of inflammation at the wound site play an important role in postburn immunosuppression. Furthermore, these results suggest that the use of skin allografts during the early postburn period does not adversely affect cell-mediated immunity in any way that could be abrogated by primary autografting.

A reliable and cost-effective in vitro assay of skin viability for skin banks and burn centers.

The tetrazolium salt, MTT (3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide), was used to evaluate the viability of fresh native human skin and cryopreserved human skin under a wide variety of conditions. Viability of fresh and cryopreserved skin was determined with our modification of the MTT assay, and compared with the well-described tetrazolium reductase assay. The MTT assay provides a precise and reproducible index of viability for both fresh and cryopreserved skin. In comparison, the tetrazolium reductase assay (1) is subject to higher levels of variability and, (2) underestimates the viability of fresh native skin. The precision, simplicity, and economy of the MTT assay support its utility in the routine assessment of skin viability in skin banks, burn centers, and skin biology research units.

Effects of cryopreservation and deconstruction on the dermal glycosaminoglycan content of human skin.

Since the concept of a fabricated skin replacement was first proposed, it has been recognized that a permanent skin replacement must contain a functional complex structure consisting of epidermis integrated with dermis. Although a practical solution for the replacement of missing epidermis exists through the culture expansion of the autologous epidermis, a practical solution for permanently replacing missing dermis has not been achieved. While it is generally recognized that the insoluble matrix components--largely collagen and elastin--are essential, the role of other matrix components such as glycosaminoglycans (GAGs) and proteoglycans remains undefined. This article describes both the qualitative and quantitative GAG composition of fresh and cryopreserved human dermis. Through the use of 2 different colorimetric assays and cellulose acetate electrophoresis, we found the following: 1) the principal dermal GAGs are those of the heparin family; 2) dermatan sulfate is the second most predominant GAG component; 3) chondroitin-6-sulfate is found at concentrations of 2 orders of magnitude less than the heparins; and 4) hyaluronan and keratan sulfate were both found as only minor constituents. When the GAG composition of fresh skin was compared with that of cryopreserved skin, no significant differences were observed. This study also examined the time course of GAG leaching during the preparation of deconstructed human dermis, which is human dermis reduced to the native insoluble matrix components by exhaustive saline soaking. We found that GAG leaching was readily detectable even within the first day. Sixty percent of total GAG leaching occurred by day 7. These investigations establish a benchmark for the reproduction of GAGs in synthetic dermal constructs. Further, the results of the leaching study generate important considerations for short-term skin storage and long-term skin banking. Because GAG leaching commences immediately, appropriate precautions must be taken to minimize the potential functional compromise of cryopreserved human dermis.