Adipose-Derived Stem Cells Protect Skin Flaps against Ischemia/Reperfusion Injury via IL-6 Expression.

Flap necrosis is the most frequent postoperative complication encountered in reconstructive surgery. We elucidated whether adipose-derived stem cells (ADSCs) and their derivatives might induce neovascularization and protect skin flaps during ischemia/reperfusion (I/R) injury. Flaps were subjected to 3 hours of ischemia by ligating long thoracic vessels and then to blood reperfusion. Qtracker-labeled ADSCs, ADSCs in conditioned medium (ADSC-CM), or ADSC exosomes (ADSC-Exo) were injected into the flaps. These treatments led to significantly increased flap survival and capillary density compared with I/R on postoperative day 5. IL-6 levels in the cell lysates or in conditioned medium were significantly higher in ADSCs than in Hs68 fibroblasts. ADSC-CM and ADSC-Exo increased tube formation. This result was corroborated by a strong decrease in skin repair after adding IL-6-neutralizing antibodies or small interfering RNA for IL-6 ADSCs. ADSC transplantation also increased flap recovery in I/R injury of IL-6-knockout mice. IL-6 was secreted from ADSCs through signal transducer and activator of transcription phosphorylation, and then IL-6 stimulated angiogenesis and enhanced recovery after I/R injury by the classic signaling pathway. The mechanism of skin recovery includes the direct differentiation of ADSCs into endothelial cells and the indirect effect of IL-6 released from ADSCs. ADSC-CM and ADSC-Exo could be used as off-the-shelf products for this therapy.

Lentiviral transduction of face and limb flaps: implications for immunomodulation of vascularized composite allografts.

BACKGROUND: Ex vivo introduction of an immunomodulatory transgene into a face or hand allograft may improve the risk-to-benefit ratio of vascularized composite allografts. Abrogation of the immunogenicity of the skin component of a face or hand allograft may decrease alloreactivity and permit the induction of immunologic tolerance. Proof-of-principle demonstrations of transduction of composite tissue have been established using adenoviral vectors, producing transient gene expression. The authors hypothesized that transduction, integration, and long-term expression of transgenes in a vascularized composite allograft could be achieved using lentiviral vectors. METHODS: Ex vivo transduction of heterogeneous primary rat cell lines representative of a composite tissue flap's cellular architecture was performed using a luc-enhanced green fluorescent protein (eGFP) human immunodeficiency virus-1-based lentiviral vector. Ex vivo injections of rat superficial inferior epigastric artery flaps with the viral vector were performed intraarterially, intramuscularly, and intradermally. RESULTS: Quantifiable reporter expression by flow cytometry (fluorescence-activated cell sorting) analysis and in vitro bioluminescence was observed. The luc-eGFP vector exhibited broad tropism and allowed transgene expression in relevant cell lines and throughout the flaps. Ex vivo intradermal transfection resulted in genomic integration and long-term constitutive gene expression (>150 days). Similarly, efficient intradermal transfection of face and hand flaps in a rat model corroborated this approach. Ex vivo intravascular perfusion of the vector proved inferior to intradermal injection. CONCLUSIONS: Intradermal delivery of the transgenes proved superior to intravascular perfusion. Optimization of this gene-delivery approach may allow long-term, constitutive expression of immunomodulatory proteins in face and hand allografts. Future goals include replacement of the luciferase and eGFP reporter genes with key immunomodulatory proteins.

Short-term application of doxorubicin chemotherapy immunosuppressive side effects for composite tissue allotransplantation.

BACKGROUND: Adjuvant chemotherapy is often required for the treatment of bone cancers after tumor resection, which often results in a large continuity defect. The immunosuppressive side effects could instead be exploited to allow immediate reconstruction with a composite tissue allograft (CTA) that would provide for replacement of tissues. We used a short course of doxorubicin to achieve a novel method of immunosuppression in a rat model undergoing CTA to create an immunological environment for allograft survival. MATERIALS & METHODS: The Institutional Animal Care and Use Committee-approved protocol consisted of 3 experimental groups. Groups 2 and 3 consisted of Brown Norway rats (n = 5) as allograft donors and Lewis rats (n = 5) as transplant recipients. An abdominal wall CTA was harvested off the superficial inferior epigastric vessels. Doxorubicin therapy was administered in group 3 animals. Survival of the CTA was assessed by physical examination and histological analysis. RESULTS: Allotransplant without treatment showed complete clinical and histologic rejection by day 7. Allotransplant rats treated with doxorubicin had clinically and histologically normal grafts through day 10. Kaplan-Meier survival analysis showed a statistically significant difference, with increased CTA survival time to end point with doxorubicin treatment, from a mean of 8.8 days in group 2 to 16.4 days in group 3. CONCLUSIONS: Allotransplant flaps without treatment developed complete clinical and histological rejection. The allotransplant group which received doxorubicin showed a delay of allograft rejection with an 86% increased CTA graft survival time. This demonstrates the feasibility of the immunosuppression side effect caused by chemotherapy to prevent rejection of a CTA.

Vaginal reconstruction with two lower abdominal skin flaps in rabbits: histological and macroscopic evaluation.

OBJECTIVE: To evaluate the use of two skin flaps of the lower abdominal wall in the creation of a cylindrical conduit in vaginal reconstruction surgery in rabbits, through macroscopic and histological analysis. STUDY DESIGN: An experimental study was performed in 16 female New Zealand rabbits, consisting of the use of two rectangular-shaped skin flaps of the lower abdominal wall measuring 1cm longitudinally and 3 cm transversely anastomosed to each other through continuous suture of the edges of the two flaps to create a tube. Hysterectomy and excision of the vaginal vault were performed, and the skin tube was anastomosed to the remaining vaginal stump with separate points of polyglycolic acid 4.0. Animals were divided into 4 groups according to the euthanasia at 2, 4, 8 and 12 weeks, when after excision of the neovagina, macroscopic and histological evaluation with hematoxylin-eosin and Masson trichrome were performed. RESULTS: Of 16 operated rabbits, only 1 presented partial abdominal wall dehiscence, not compromising the flap viability. The macroscopic analysis of the vaginal conduit showed that it was kept open throughout the experimental steps, with a good patency and gauge, showing a slight retraction in the skin conduit length of no statistical significance. In the histopathological analysis, a local inflammatory process in the anastomosis was observed, which was larger in the early evaluation but decreased in late evaluations, as well as the local fibrosis process. Integration of the vaginal and skin epithelia was made with no alterations in their primary characteristics. CONCLUSION: The use of two skin flaps of the lower abdominal wall as a vaginal conduit presented good integration between skin and vaginal tissue with minimal length retraction, kept the patency during evaluations and did not show strictures, presenting good local healing and a low rate of complications.

Vascular and cellular events in post-mastectomy seroma: an immunohistochemical study.

This study aimed to describe the vascular and cellular histopathological changes that occurred in post-mastectomy seroma in an animal model. Unilateral mastectomies were conducted on 45 female albino rabbits. On day seven, the skin flap and the underlying tissues of the mastectomy regions were dissected and processed for histopathological examination using immunohistochemical staining of the T- and B-lymphocytes and macrophages (CD3, CD20, and CD68 respectively), and the vascular endothelia. The post-mastectomy regions in the seroma group showed a large number of inflammatory cells and newly formed blood vessels that lost the integrity of their endothelial cell linings, as revealed by the von Willebrand factor staining, as well the basement membrane, as revealed by the histochemical stain. The post-mastectomy seroma beds showed many CD3 and CD20+ve lymphocytes and CD68+ve macrophages. These macrophages were producing angiogenic factors, resulting in the persistent and continuous formation of new blood vessels. These new blood vessels were defective and represented an underlying cause of seroma formation.

Evidence that FoxP3+ regulatory T cells may play a role in promoting long-term acceptance of composite tissue allotransplants.

BACKGROUND: FoxP3/CD4/CD25 regulatory T cells (Treg) play an important role in maintaining peripheral tolerance and are potent suppressors of T-cell activation. In this study, we evaluated the role of Treg in peripheral tolerance to composite tissue allografts (CTA). METHODS: Mixed allogeneic chimeric rats were prepared by preconditioning recipients with anti-αß-T-cell receptor monoclonal antibody followed by total body irradiation. Animals received T-cell-depleted August Copenhagen Irish bone marrow cells followed by antilymphocyte serum and FK-506. A modified osteomyocutaneous hindlimb flap composed of bone and all limb tissue components was placed in animals with chimerism greater than or equal to 1% on day 28. Recipients with CTA surviving more than or equal to 6 months were evaluated for Treg. Skin samples from tolerant long-term allogeneic transplanted, syngeneic transplanted, rejected, and naïve animals were immunostained with fluorochrome-conjugated anti-FoxP3 and anti-CD4 monoclonal antibody and visualized under a laser confocal microscope. RESULTS: Significant CD4/FoxP3 Treg infiltrates were observed in tolerant donor-allograft skin samples. No graft infiltrating FoxP3 cells were observed in rejector, naïve, or skin from syngeneic CTA. In parallel experiments, mixed leukocyte reaction assays were performed to investigate the suppressor function of Treg cells. Splenocytes from tolerant, rejected, and naïve rats were sorted by flow cytometry for CD4/CD25 T cells. Treg demonstrated similar suppressive levels between the three groups. CONCLUSIONS: These data suggest that Treg may play an important role in maintenance of tolerance and promoting graft acceptance in long-term CTA acceptors and may explain the favorable outcomes observed in clinical CTA recipients.

In vivo observations of cell trafficking in allotransplanted vascularized skin flaps and conventional skin grafts.

The problem of allogeneic skin rejection is a major limitation to more widespread application of clinical composite tissue allotransplantation (CTA). Previous research examining skin rejection has mainly studied rejection of conventional skin grafts (CSG) using standard histological techniques. The aim of this study was to objectively assess if there were differences in the immune response to CSG and primarily vascularized skin in composite tissue allotransplants (SCTT) using in vivo techniques in order to gain new insights in to the immune response to skin allotransplants. CSG and SCTT were transplanted from standard Lewis (LEW) ad Wistar Furth (WF) to recipient transgenic green fluorescent Lewis rats (LEW-GFP). In vivo confocal microscopy was used to observe cell trafficking within skin of the transplants. In addition, immunohistochemical staining was performed on skin biopsies to reveal possible expression of class II major histocompatibility antigens. A difference was observed in the immune response to SCTT compared to CSG. SCTT had a greater density cellular infiltrate than CSG (p<0.03) that was focused more at the center of the transplant (p<0.05) than at the edges, likely due to the immediate vascularization of the skin. Recipient dendritic cells were only observed in rejecting SCTT, not CSG. Furthermore, dermal endothelial class II MHC expression was only observed in allogeneic SCTT. The immune response in both SCTT and CSG was focused on targets in the dermis, with infiltrating cells clustering around hair follicles (CSG and SCTT; p<0.01) and blood vessels (SCTT; p<0.01) in allogeneic transplants. This study suggests that there are significant differences between rejection of SCTT and CSG that may limit the relevance of much of the historical data on skin graft rejection when applied to composite tissue allotransplantation. Furthermore, the use of novel in vivo techniques identified characteristics of the immune response to allograft skin not previously described, which may be useful in directing future approaches to overcoming allograft skin rejection.

The effects of CD133-positive cells to a nonvascularized fasciocutaneous free graft in the rat model.

A fasciocutaneous free graft could prove the ideal free tissue graft to prevent contracture following adhesion of the graft to the recipient bed. However, for graft survival, vascular anastomosis involving complicated surgical techniques is necessary. In the present study, we focused on peripheral blood-derived CD133(+) cells, including the endothelial progenitor cell fraction. The purpose of this study is to investigate whether locally transplanted CD133(+) cells could increase revascularization and improve the quality of free and thick grafted tissue.On the abdomen of nude rats (F344/N Jcl rnu/rnu), a 5 x 2-cm fasciocutaneous graft was designed, which was grafted on to the back. After operation, rats received local injection of 1 x 10(5) human CD133(+) cells resuspended in 400 microL of phosphate-buffered saline (PBS) (CD133(+) group) or the same volume of PBS without cells (control group) (n = 10 in each group). In the CD133(+) group graft perfusion or graft survival was not improved significantly over controls. However, the capillary density increased markedly in the surrounding fascia and the dermis matured at an earlier stage in the CD133(+) group. In this study, we demonstrated that transplanted CD133(+) cells induced new blood vessel growth and improved dermis quality. Cell therapy with CD133(+) cells is already applied in a clinical setting because autologous cells can be used and therefore these procedures are not hampered by ethical concerns. CD133(+) cell transfer therefore has great potential as an adjunct therapy in the realm of microsurgery.

Effect of platelet glycoprotein IIb/IIIa receptor antagonist on survival of epigastric island flaps in rats with total venous occlusions.

We evaluated the effect of tirofiban hydrochloride on the survival of epigastric island flaps in rats that had had all the veins occluded. Male Wistar Albino rats were randomly assigned to control (treated with sterile saline) and experimental (treated with tirofiban hydrochloride 1 mg/kg intravenously) groups. An epigastric island skin flap 3x6 cm was raised in each rat. All veins that drained the flap were ligated to give total venous occlusion. Blood flow was recorded by laser Doppler preoperatively (baseline), immediately after the flap had been sutured back to its original position (acute) and on postoperative days 1 and 3. The degree of necrosis was evaluated on day 3. Mean percentage necrosis and minimum laser Doppler values were compared in the two groups. Total necrosis was evident on day 1 in the control group and on day 3 in the experimental group. Macroscopic evidence was confirmed by histopathological examination. There were appreciable differences in blood flow and in the necrotic area of the flap in the experimental group compared with the control group on both days 1 and 3. Tirofiban hydrochloride might be effective in this flap model.

A modified model of hindlimb osteomyocutaneous flap for the study of tolerance to composite tissue allografts.

Composite tissue allotransplantation (CTA) is the new frontier in transplantation. More than 25 hand allograft transplants have been performed worldwide, and the feasibility has been well established. The classical experimental model of CTA involves rat orthotopic hindlimb transplantation, a time-consuming procedure associated with high mortality and morbidity. We describe a rat heterotopic osteomyocutaneous flap that serves as a nonfunctional CTA, allowing the study of tolerance induction to a highly antigenic vascularized allograft of bone, muscle, and skin while minimizing the morbidity and mortality of full hind limb transplantation. In the present studies, we explored whether establishing chimerism by nonmyeloablative conditioning would induce tolerance to CTA. When compared with the classic hind limb transplantation model, these results demonstrate that our heterotopic hind limb flap is less morbid and as an effective experimental model for the study of CTA tolerance.

Extracorporeal shock wave enhanced extended skin flap tissue survival via increase of topical blood perfusion and associated with suppression of tissue pro-inflammation.

OBJECTIVE: Distal skin flap ischemic necrosis is a significant challenge in reconstructive surgery. This study assessed whether extracorporeal shock wave (ESW) treatment rescues compromised flap tissue by enhancing tissue perfusion and is associated with suppression of inflammatory response. METHODS: This study used the dorsal skin random flap model in a rodent. Thirty-six male Sprague Dawley rats were divided into three groups. Group I, a control group, received no treatment. Group II was administrated 500 impulses of ESW treatment at 0.15 mJ/mm(2) as a single treatment immediately postoperatively. Group III received 500 impulses of ESW at 0.15 mJ/mm(2) applied immediately postoperatively and the day following surgery. Flap blood perfusion was detected by laser Doppler. Flap survival/necrosis area and histological staining of flap ischemia zone was performed on day 7 postoperatively. The tumor necrosis factor alpha, vascular endothelial growth factor, and proliferating cell nuclear antigen expression were evaluated with immunohistochemical staining. RESULTS: Experimental results indicated that the necrotic area of the flaps in Group II was significantly reduced compared with that in the control group (13 +/- 2.6% versus 42 +/- 5.7%, P < 0.01). There was small and insignificant reduction in the necrotic area in Group III compared with the controls. Flap tissue blood perfusion was significantly increased postoperatively in Group II. Histological staining indicated that ESW treatment substantially increased vascular endothelial growth factor and proliferating cell nuclear antigen expressions, reduced leukocyte infiltration, and suppression of tumor necrosis factor alpha expression in flap tissue ischemic zones in Group II compared with that in controls. CONCLUSION: Optimal dosage of ESW treatment has a positive effect in rescuing ischemic zone of flap by increasing tissue perfusion and is associated with suppressing inflammatory response.

Pro-inflammatory cytokines in elective flap surgery.

BACKGROUND: Surgical trauma releases inflammatory mediators such as pro-inflammatory cytokines. In this prospective, controlled, randomized trial we investigated the release of pro-inflammatory cytokines and monocyte/macrophage activation in patients scheduled for breast reconstruction after mastectomy. Patients were allocated to one of three surgical procedures, differing in complexity and in the need for implants used for reconstruction. METHODS: Thirty mastectomized women underwent delayed breast reconstruction with the lateral thoracodorsal flap (LTD), the latissimus dorsi flap (LD), or the pedicled transverse rectus abdominis muscle flap (TRAM). Blood samples for TNF, IL-6, IL-8, neopterin, C-reactive protein (CRP), and leukocyte determination were drawn pre-operatively, 24 h, and 2 weeks post-operatively. RESULTS: All groups had significantly elevated IL-6 levels 24 h after surgery. The levels were significantly higher in the TRAM group compared to the LTD and LD groups. IL-8 levels were increased in all groups 2 weeks after surgery (P < 0.05), the LTD (83 pg/mL) and LD (84 pg/mL) group having higher mean IL-8 levels than the TRAM patients (48 pg/mL) (ns). TNF and leukocyte counts were within the normal range. CRP levels were elevated in all groups one day after surgery (P < 0.05). CONCLUSION: Flap procedures for breast reconstruction stimulate the pro-inflammatory response. IL-6 levels were highest in patients with TRAM operations, being the most extensive procedure studied, whereas the highest IL-8 levels were seen in women with a saline filled silicone implant suggesting immunomodulation by foreign material. Although all three investigated procedures are major operations in the field of plastic surgery, according to the inflammatory response to trauma they should be regarded as minor procedures.

Anti-TGFbeta1 antibody for modulation of expression of endogenous transforming growth factor beta 1 to prevent fibrosis after plastic surgery in rats.

The cytokine transforming growth factor beta 1 (TGFbeta1) substantially influences synthesis of extracellular matrix, fibrosis, and neoangiogenesis during wound healing in a dose dependent manner. We carried out experiments in rats to measure the degree of reduction of disorders of wound healing and fibrosis produced by inhibition of endogenous TGFbeta1 by polyclonal antibodies (poAB). A free myocutaneous gracilis flap was transplanted from the groin to the neck region in 30 Wistar rats (300-450 g body weight). In 15 animals intraoperatively and daily from days 3 to 7 postoperatively, 1 microg anti-TGFbeta1 poAB in 500 microl of phosphate buffered saline (PBS) were injected into the neck region. Fifteen animals served as controls. On postoperative days 3, 4, 5, 7, 14, and 28 the expression of endogenous TGFbeta1 in cytoplasm was analysed by immunohistochemistry (ABC-POX; AEC), in situ hybridisation of TGFbeta1-mRNA, and Sirus Red staining of collagen matrix; it was quantified using labelling indices. Neutralisation of the TGFbeta1 activity by specific poAB resulted in inhibition of the cytoplasmatic expression compared with untreated animals. In the transition area between grafted tissue and graft bed, a significant reduction of TGFbeta1 expression (mean (S.D.) 34.7 (6.5)) was found from day 5 in the group treated with anti-TGFbeta1 poAB compared with the control group (mean (S.D.) 48.1 (6.6)) (P <0.03). Up to day 14 the endogenous expression of TGFbeta1 (mean (S.D.) 30.0 (2.8)) was reduced after the application of TGFbeta1 poAB compared with the control group (mean (S.D.) 44.0 (12.3)). Sirus Red staining indicated a more complex packed structure and generally more prominent collagen types I-IV fibres in untreated animals than in animals that were given anti-TGFbeta1 poAB. Expression of TGFbeta-mRNA by in situ hybridisation was reduced in fibroblasts in animals that were given anti-TGFbeta1 poAB. The results indicate that anti-TGFbeta1 might improve the healing of free flaps in the graft beds of patients who are prone to excessive fibrosis.

Osteomyocutaneous flap as a preclinical composite tissue allograft: swine model.

Composite tissue allotransplantation (CTA) constitutes one of the last frontiers of microsurgery. Prior to its clinical application, the long-term efficacy of modern immunotherapy must be tested in a pre-clinical CTA model. Based on the concept of osteomyocutaneous forearm flap, we developed a CTA flap model in swine. After identifying the vascular territory of the flaps in six pigs (vascular casting), flaps were transplanted from mismatched donor to recipient pigs (n = 6). Rejection was assessed daily by visual inspection and histopathology of biopsy specimens. Recipient pigs were able to ambulate immediately following surgery. There were no flap failures owing to technical or surgical complications. Rejection occurred over a period of 7 days as manifested by edema, cellular infiltration, epidermalysis, and thrombosis. This pre-clinical flap model is excellent for evaluating the effectiveness of modern immunotherapy because it is anatomically and immunologically relevant and because the minimal morbidity caused to the animal permits long-term studies.

Prolonged survival of musculoskeletal xenografts with combined cyclosporine and 15-deoxyspergualin.

This study was undertaken to evaluate the feasibility of performing vascularized musculoskeletal xenografts between mice and rats using immunosuppression. Vascularized musculoskeletal grafts were harvested from the hind limb of C57BL/6J (B6) mice, transplanted heterotopically into Lewis rats, and revascularized by microanastomoses of the graft artery and the recipient femoral artery and the graft vein to the recipient femoral vein. Recipient rats were divided into four groups. Group 1 received no immunosuppression (n = 10), group 2 was treated with cyclosporine (10 mg/kg/day; n = 10), group 3 was treated with 15-deoxyspergualin (5 mg/kg/day; n = 10), and group 4 received both cyclosporine and 15-deoxyspergualin (n = 10). Graft survival was directly examined on postoperative days 4, 7, and 14. In vitro assays were performed using mixed lymphocyte reactions and anti-donor cytotoxic antibody assays to assess the recipient's immune response. Grafts were examined by histology and immunohistochemistry. All grafts in group 1 were rejected by day 4. In groups 2 and 3, all grafts were rejected by day 7. In group 4, however, 8 of 10 recipients had viable grafts on day 14. Data from mixed lymphocyte reactions showed that cell-mediated immune responses were uniformly suppressed in groups 2, 3, and 4 compared with group 1. However, anti-donor antibody production was only partly suppressed in groups 2 and 3, suggesting that graft rejection was primarily caused by circulating cytotoxic anti-donor antibodies in groups 1, 2, and 3. Histologic observations in groups 1, 2, and 3 confirmed the important role of the humoral mechanism in xenograft rejection. Furthermore, immunohistochemical results demonstrated that the small vessels in the rejected grafts showed anti-rat immunoglobulin and complement depositions. Only a combination therapy of cyclosporine and 15-deoxyspergualin attenuated the rejection of xenografts.

Effects of calcitonin gene-related peptide on tissue survival, blood flow and neutrophil recruitment in experimental skin flaps.

Local administration of calcitonin gene-related peptide (CGRP) has been shown to improve tissue survival in surgical skin flaps. Moreover, topical CGRP has been demonstrated to exert anti-inflammatory effects in different animal models of skin inflammation. The aim of the present study was to establish whether systemic treatment with low doses of CGRP may improve survival and reduce neutrophil accumulation in surgical skin flaps. Using a well-established dorsal skin-flap model in the rat, we found that intraperitoneal (i.p.) pretreatment with low doses of CGRP dose-dependently increased flap survival. Thus, in untreated animals flap survival at day 7 after surgery was 42%, as compared to 44%, 60%, 69% and 73% survival after a single preoperative i.p. injection of 10(-15), 10(-12), 10(-9) and 10(-6) mol CGRP, respectively (P < 0.05 versus control for the three highest doses). The three effective doses had no detectable effects on either flap blood flow (laser Doppler) or mean arterial blood pressure. On the other hand, 5 x 10(-9) mol CGRP i.p. significantly reduced the marked surgery-induced accumulation of flap myeloperoxidase (a marker for neutrophil recruitment) without affecting the circulating neutrophil count. Taken together, our findings demonstrate that low systemic doses of CGRP can cause a major improvement in skin-flap survival in the rat, possibly via inhibition of surgically induced neutrophil recruitment.

The inflammatory reaction in elective flap surgery.

The inflammatory response in three different flap procedures was investigated by measuring the preoperative and postoperative levels of C-reactive protein, leukocyte count, and body temperature. Patients scheduled for delayed breast reconstruction were operated on with the lateral thoracodorsal flap, the latissimus dorsi flap, or the pedicled TRAM flap. All patients received 2 gm of intravenous cloxacillin for antibiotic prophylaxis and 1 gm of paracetamol four times a day as basic treatment for postoperative pain. Within each treatment group, significant postoperative changes in C-reactive protein levels, leukocyte count, and body temperature were noted when compared with preoperative values. The highest C-reactive protein level (130 mg/ml) was found in the TRAM group on the third postoperative day. The kinetic pattern of C-reactive protein was similar for the latissimus dorsi flap and lateral thoracodorsal flap procedures, but the maximum C-reactive protein levels were significantly lower, 74 and 44 mg/ml respectively. Small (0.5 to 0.9 degrees C) but significant differences in body temperature were also noted on the second and third postoperative day. The TRAM flap group had the highest, the latissimus dorsi flap group intermediate, and the lateral thoracodorsal flap group the lowest value. The postoperative C-reactive protein levels seem to reflect the extent of the surgical trauma.

Augmentation of random-flap survival by implantation of vascularized fascia allografts and temporary immunosuppression: implications for flap fabrication.

The purpose of this study was to explore the possible use of an allogeneic vascular source for flap fabrication. Epigastric fascia with its superficial epigastric vessel pedicle was harvested from the donor rat and microsurgically revascularized in the recipient rat across a major histocompatibility barrier. ACI rats (Rtl-a) served as donors, and Lewis rats (Rtl-1) served as recipients. The recipient rat was immunosuppressed with a daily dose of 2 mg/kg cyclosporin A plus 5 mg/kg prednisone for 4 weeks. Three experiments were performed for skin, muscle, and bone studies. In experiment 1 (20 Lewis rats), placement of the allotransplanted fascia underneath the epigastric skin significantly improved the survival of a random epigastric skin flap raised 3 weeks later (7.35 +/- 0.65 cm2 versus 6.09 +/- 0.90 cm2, p < 0.05). Immunosuppression was discontinued 10 days after flap elevation with no observable additional skin necrosis. In experiment 2 (13 Lewis rats) and experiment 3 (14 Lewis rats), segments of isogeneic muscle and bone were grafted successfully on the allotransplanted fascia, respectively. The survival of these grafts was confirmed by metabolic bone activity, bone labeling, microangiography, and histologic studies and further confirmed 2 weeks after cessation of immunosuppression. An allotransplanted fascia as a vascular source proved in this model its capability to improve the survival of a random skin flap and to accept a free bone or muscle graft with temporary immunosuppression. These findings hold promise for possible use of an allogeneic vascular source in flap fabrication.

Myocutaneous flaps in patients with head and neck cancer retain their immunological capacities in an activated functional state.

Free/pedicled myocutaneous flaps used as functional replacement after radical dissection of advanced stage squamous cell carcinomas of the oral cavity/oropharynx were examined by immunohistochemistry (APAAP-technique). Biopsies from eight patients were taken at the time of surgery and at 3 and 5 months post-operatively. Fifteen monoclonal antibodies were used to detect surface antigens as markers of phenotypic changes of immune competent cells. In post-operative biopsies all antigens investigated increased significantly. Significantly higher numbers of CD45RO+ (P < 0.01) and CD45RA+ (P < 0.001) leukocytes were detectable. The majority of these leukocytes were TcR alpha/beta +/CD3+ T-cells, which increased in the CD4 (P < 0.05) and the CD8 (P < 0.001) subset. In addition, B-cells (P < 0.05), granulocytes (P < 0.05), NK cells (CD16+ lymphocytic cells; P < 0.05) and mature macrophages (25F9+cells; P < 0.01) were increased. Intra- and subepidermally a significantly (P < 0.01) higher number of dendritic-/Langerhans cells (CD1a+) was detectable. In post-operative biopsies, the activation-associated antigens ICAM-1, VCAM and HLA-DR were expressed on significantly more mononuclear-/endothelial cells and on keratinocytes. Our findings indicate that the myocutaneous flaps still contained cells with immunological capacities.