Assembly of Building Blocks by Double-End-Anchored Polymers in the Dilute Regime Mediated by Hydrophobic Interactions at Controlled Distances.

Hierarchical assembly of building blocks via competing, orthogonal interactions is a hallmark of many of nature's composite materials that do not require highly specific ligand-receptor interactions. To mimic this assembly mechanism requires the development of building blocks capable of tunable interactions. In the present work, we explored the interplay between repulsive (steric and electrostatic) and attractive hydrophobic forces. The designed building blocks allow hydrophobic forces to effectively act at controlled, large distances, to create and tune the assembly of membrane-based building blocks under dilute conditions, and to affect their interactions with cellular membranes via physical cross-bridges. Specifically, we employed double-end-anchored poly(ethylene glycol)s (DEA-PEGs)-hydrophilic PEG tethers with hydrophobic tails on both ends. Using differential-interference-contrast optical microscopy, synchrotron small-angle X-ray scattering (SAXS), and cryogenic electron microscopy, we investigated the ability of DEA-PEGs to mediate assembly in the dilute regime on multiple length scales and on practical time scales. The PEG length, anchor hydrophobicity, and molar fraction of DEA-PEG molecules within a membrane strongly affect the assembly properties. Additional tuning of the intermembrane interactions can be achieved by adding repulsive interactions via PEG-lipids (steric) or cationic lipids to the DEA-PEG-mediated attractions. While the optical and electron microscopy imaging methods provided qualitative evidence of the ability of DEA-PEGs to assemble liposomes, the SAXS measurements and quantitative line-shape analysis in dilute preparations demonstrated that the ensemble average of loosely organized liposomal assemblies maintains DEA-PEG concentration-dependent tethering on defined nanometer length scales. For cationic liposome-DNA nanoparticles (CL-DNA NPs), aggregation induced by DEA-PEGs decreased internalization of NPs by cells, but tuning the DEA-PEG-induced attractions by adding repulsive steric interactions via PEG-lipids limited aggregation and increased NP uptake. Furthermore, confocal microscopy imaging together with colocalization studies with Rab11 and LysoTracker as markers of intracellular pathways showed that modifying CL-DNA NPs with DEA-PEGs alters their interactions with the plasma and endosomal membranes.

Competition of charge-mediated and specific binding by peptide-tagged cationic liposome-DNA nanoparticles in vitro and in vivo.

Cationic liposome-nucleic acid (CL-NA) complexes, which form spontaneously, are a highly modular gene delivery system. These complexes can be sterically stabilized via PEGylation [PEG: poly (ethylene glycol)] into nanoparticles (NPs) and targeted to specific tissues and cell types via the conjugation of an affinity ligand. However, there are currently no guidelines on how to effectively navigate the large space of compositional parameters that modulate the specific and nonspecific binding interactions of peptide-targeted NPs with cells. Such guidelines are desirable to accelerate the optimization of formulations with novel peptides. Using PEG-lipids functionalized with a library of prototypical tumor-homing peptides, we varied the peptide density and other parameters (binding motif, peptide charge, CL/DNA charge ratio) to study their effect on the binding and uptake of the corresponding NPs. We used flow cytometry to quantitatively assess binding as well as internalization of NPs by cultured cancer cells. Surprisingly, full peptide coverage resulted in less binding and internalization than intermediate coverage, with the optimum coverage varying between cell lines. In, addition, our data revealed that great care must be taken to prevent nonspecific electrostatic interactions from interfering with the desired specific binding and internalization. Importantly, such considerations must take into account the charge of the peptide ligand as well as the membrane charge density and the CL/DNA charge ratio. To test our guidelines, we evaluated the in vivo tumor selectivity of selected NP formulations in a mouse model of peritoneally disseminated human gastric cancer. Intraperitoneally administered peptide-tagged CL-DNA NPs showed tumor binding, minimal accumulation in healthy control tissues, and preferential penetration of smaller tumor nodules, a highly clinically relevant target known to drive recurrence of the peritoneal cancer.

Cationic liposome-nucleic acid nanoparticle assemblies with applications in gene delivery and gene silencing.

Cationic liposomes (CLs) are synthetic carriers of nucleic acids in gene delivery and gene silencing therapeutics. The introduction will describe the structures of distinct liquid crystalline phases of CL-nucleic acid complexes, which were revealed in earlier synchrotron small-angle X-ray scattering experiments. When mixed with plasmid DNA, CLs containing lipids with distinct shapes spontaneously undergo topological transitions into self-assembled lamellar, inverse hexagonal, and hexagonal CL-DNA phases. CLs containing cubic phase lipids are observed to readily mix with short interfering RNA (siRNA) molecules creating double gyroid CL-siRNA phases for gene silencing. Custom synthesis of multivalent lipids and a range of novel polyethylene glycol (PEG)-lipids with attached targeting ligands and hydrolysable moieties have led to functionalized equilibrium nanoparticles (NPs) optimized for cell targeting, uptake or endosomal escape. Very recent experiments are described with surface-functionalized PEGylated CL-DNA NPs, including fluorescence microscopy colocalization with members of the Rab family of GTPases, which directly reveal interactions with cell membranes and NP pathways. In vitro optimization of CL-DNA and CL-siRNA NPs with relevant primary cancer cells is expected to impact nucleic acid therapeutics in vivo. This article is part of the themed issue 'Soft interfacial materials: from fundamentals to formulation'.

Rab11 and Lysotracker Markers Reveal Correlation between Endosomal Pathways and Transfection Efficiency of Surface-Functionalized Cationic Liposome-DNA Nanoparticles.

Cationic liposomes (CLs) are widely studied as carriers of DNA and short-interfering RNA for gene delivery and silencing, and related clinical trials are ongoing. Optimization of transfection efficiency (TE) requires understanding of CL-nucleic acid nanoparticle (NP) interactions with cells, NP endosomal pathways, endosomal escape, and events leading to release of active nucleic acid from the lipid carrier. Here, we studied endosomal pathways and TE of surface-functionalized CL-DNA NPs in PC-3 prostate cancer cells displaying overexpressed integrin and neuropilin-1 receptors. The NPs contained RGD-PEG-lipid or RPARPAR-PEG-lipid, targeting integrin, and neuropilin-1 receptors, respectively, or control PEG-lipid. Fluorescence colocalization using Rab11-GFP and Lysotracker enabled simultaneous colocalization of NPs with recycling endosome (Rab11) and late endosome/lysosome (Rab7/Lysotracker) pathways at increasing mole fractions of pentavalent MVL5 (+5 e) at low (10 mol %), high (50 mol %), and very high (70 mol %) membrane charge density (σM). For these cationic NPs (lipid/DNA molar charge ratio, ρchg = 5), the influence of membrane charge density on pathway selection and transfection efficiency is similar for both peptide-PEG NPs, although, quantitatively, the effect is larger for RGD-PEG compared to RPARPAR-PEG NPs. At low σM, peptide-PEG NPs show preference for the recycling endosome over the late endosome/lysosome pathway. Increases in σM, from low to high, lead to decreases in colocalization with recycling endosomes and simultaneous increases in colocalization with the late endosome/lysosome pathway. Combining colocalization and functional TE data at low and high σM shows that higher TE correlates with a larger fraction of NPs colocalized with the late endosome/lysosome pathway while lower TE correlates with a larger fraction of NPs colocalized with the Rab11 recycling pathway. The findings lead to a hypothesis that increases in σM, leading to enhanced late endosome/lysosome pathway selection and higher TE, result from increased nonspecific electrostatic attractions between NPs and endosome luminal membranes, and conversely, enhanced recycling pathway for NPs and lower TE are due to weaker attractions. Surprisingly, at very high σM, the inverse relation between the two pathways observed at low and high σM breaks down, pointing to a more complex NP pathway behavior.

Optimizing cationic and neutral lipids for efficient gene delivery at high serum content.

BACKGROUND: Cationic liposome (CL)-DNA complexes are promising gene delivery vectors with potential application in gene therapy. A key challenge in creating CL-DNA complexes for application is that their transfection efficiency (TE) is adversely affected by serum. In particular, little is known about the effects of a high serum content on TE, even though this may provide design guidelines for application in vivo. METHODS: We prepared CL-DNA complexes in which we varied the neutral lipid [1,2-dioleoyl-sn-glycerophosphatidylcholine, glycerol-monooleate (GMO), cholesterol], the headgroup charge and chemical structure of the cationic lipid, and the ratio of neutral to cationic lipid; we then measured the TE of these complexes as a function of serum content and assessed their cytotoxicity. We tested selected formulations in two human cancer cell lines (M21/melanoma and PC-3/prostate cancer). RESULTS: In the absence of serum, all CL-DNA complexes of custom-synthesized multivalent lipids show high TE. Certain combinations of multivalent lipids and neutral lipids, such as MVL5(5+)/GMO-DNA complexes or complexes based on the dendritic-headgroup lipid TMVLG3(8+) exhibited high TE both in the absence and presence of serum. Although their TE still dropped to a small extent in the presence of serum, it reached or surpassed that of benchmark commercial transfection reagents, particularly at a high serum content. CONCLUSIONS: Two-component vectors (one multivalent cationic lipid and one neutral lipid) can rival or surpass benchmark reagents at low and high serum contents (up to 50%, v/v). We propose guidelines for optimizing the serum resistance of CL-DNA complexes based on a given cationic lipid.

Synthesis and characterization of degradable multivalent cationic lipids with disulfide-bond spacers for gene delivery.

Gene therapy provides powerful new approaches to curing a large variety of diseases, which are being explored in ongoing worldwide clinical trials. To overcome the limitations of viral gene delivery systems, synthetic nonviral vectors such as cationic liposomes (CLs) are desirable. However, improvements of their efficiency at reduced toxicity and a better understanding of their mechanism of action are required. We present the efficient synthesis of a series of degradable multivalent cationic lipids (CMVLn, n=2 to 5) containing a disulfide bond spacer between headgroup and lipophilic tails. This spacer is designed to be cleaved in the reducing milieu of the cytoplasm and thus decrease lipid toxicity. Small angle X-ray scattering demonstrates that the initially formed lamellar phase of CMVLn-DNA complexes completely disappears when reducing agents such as DTT or the biologically relevant reducing peptide glutathione are added to mimic the intracellular milieu. The CMVLs (n=3 to 5) exhibit reduced cytotoxicity and transfect mammalian cells with efficiencies comparable to those of highly efficient non-degradable analogs and benchmark commercial reagents such as Lipofectamine 2000. Thus, our results demonstrate that degradable disulfide spacers may be used to reduce the cytotoxicity of synthetic nonviral gene delivery carriers without compromising their transfection efficiency.

Facial augmentation with core fat graft: a preliminary report.

BACKGROUND: Facial rejuvenation with autologous fat has the advantage of replacing or augmenting tissue with like tissue. The results of injected fat are unpredictable because of cellular trauma and other factors. Excised whole or en bloc fat grafting has been shown experimentally to have a greater percentage of adipocyte survival when compared with blunt cannula delivery techniques. En bloc grafting, however, requires an incision for the harvesting and placement of the fat graft with visible scars. A novel, less traumatic cylinder core fat harvest and delivery technique for facial augmentation with minimal incisional access is reported here. METHODS: Twenty-six facial augmentation procedures were performed involving the malar area, buccal area, lips, nasolabial folds, and mental region. Fat transfer volume ranged between 1 and 4 cc per site. In this report, 16 patients' results were documented 6 to 16 months postoperatively (mean follow-up time, 9.5 months) with a postoperative questionnaire and photographs. RESULTS: Social recovery for patients was short, with a recovery time of 2 to 20 days (mean, 10.91 days). There were no procedure-related complications at the fat harvest or recipient graft sites. The need for overcorrection was minimal. Graft maintenance during the average follow-up at 9.5 months appeared excellent and without appreciable volume loss. The mean patient satisfaction score was 7.64 +/- 2.97 on a scale of 0 to 10. CONCLUSIONS: The authors' preliminary results have demonstrated that this technique is effective and highly predictable. The surgical time was short, and patient recovery time has been significantly reduced compared with recovery time after other fat injection techniques.

Follicular anatomy of the anterior temporal hairline and implications for rhytidectomy.

BACKGROUND: Incisions made perpendicular to the hair follicles during anterior frontal hairline brow lifts or forehead shortening procedures help produce an inconspicuous forehead scar. The success of this "hidden" incision relies on the anteriorly directed frontal hairline follicles and their growth vector. The authors hypothesized that a similar incision could be made perpendicular to the hair follicles in the temple region during rhytidectomy. A well-designed anterior hairline beveled incision over the temple would allow for improved leverage during soft-tissue repositioning and a concealed hairline incision in the temple region. METHODS: Anterior temporal hairline strips 4 cm in length at the level of the lateral canthus were excised from 16 fresh cadavers. Hairline follicles (n = 227) were assessed for direction and angle of growth after appropriate tissue preparation and staining (hematoxylin and eosin). The hair follicle angle was analyzed microscopically as it approached the epidermis. RESULTS: The anterior temporal hairline follicles were oriented at a mean angle with the epidermis of 16 +/- 3 degrees anteriorly and inferiorly. CONCLUSIONS: The anterior temporal hairline follicles of the scalp are oriented anteriorly and inferiorly with the epidermis, providing the surgical rational for using a beveled hairline incision angled 30 to 45 degrees to the external skin surface to undercut the distal flap. This incision is perpendicular to and transects the temporal hair follicles during rhytidectomy, permitting hair growth through and anterior to the scar. This modified anterior temporal hairline incision reduces visibility of the scar at the hairline for patients in whom scar show and hairstyle versatility are important concerns.

Electrically stimulated rectus abdominis muscle flap to achieve enterostomal continence: development of a functional canine model.

BACKGROUND: Dynamic myoplasty has many clinical applications and has proven to be a versatile surgical procedure with great promise. This procedure has been used to achieve fecal/urinary continence, as in the dynamic graciloplasty, and to augment cardiac ventricular function, as is commonly seen with dynamic latissimus cardiomyoplasty. In the present study, the authors describe a functional innovative island flap sphincter created from the rectus abdominis muscle in a large-animal model to provide stomal continence for future clinical studies. METHODS: The caudal region of the rectus abdominis muscle in eight mongrel canines (23 to 25 kg) was investigated through anatomical dissections during which the location of the neurovascular pedicles and the intersegmental muscle dimensions between the muscle inscriptions were noted. The rectus abdominis muscle was used to create functional dynamic stomal sphincters that were trained with subcutaneously implanted pulse stimulators. RESULTS: The neurovascular pedicles were consistently found in similar locations along the posterior medial aspect of the caudal portion of the canine's rectus abdominis muscle. The vertical height of the deep inferior epigastric pedicle and caudal intercostal nerve muscular mean entry points were 6.75 +/- 1.89 cm and 7.44 +/- 0.86 cm, respectively. The mean caudal intersegmental muscle length of the rectus abdominis muscle used to create the sphincter was 9.69 +/- 1.81 cm. CONCLUSIONS: The canine rectus abdominis muscle has reliable anatomical locations where the neurovascular pedicle may be found. This canine muscle may be used to create a continent island flap stomal sphincter. This large-animal sphincter model is versatile, durable, and easy to manipulate, with minimal morbidity to the animal.

Investigation of risk acceptance in facial transplantation.

BACKGROUND: The surgical techniques necessary to transplant a human face are well established, and the early success of human hand transplants suggests that the immunological hurdles of transplanting human facial tissues have largely been overcome. Therefore, it is the ethical barriers that pose the greatest challenge to performing facial transplantation. At the center of the ethical debate is the question, "Do the risks posed by the life-long immunosuppression that a recipient would have to take justify the benefits of receiving a face transplant?" In this study, the authors answer this question by assessing the degree of risk individuals would be willing to accept to receive a face transplant. METHODS: To quantitatively assess risks versus benefits in facial transplantation, the authors developed the Louisville Instrument for Transplantation, or LIFT, which contains 237 standardized questions. Respondents in three study populations (healthy individuals, n = 150; organ transplant recipients, n = 42; and individuals with facial disfigurement, n = 34) were questioned about the extent to which they would trade off specific numbers of life-years, or sustain other costs, in exchange for receiving seven different transplant procedures. RESULTS: The authors found that the three populations would accept differing degrees of risk for the seven transplant procedures. Organ transplant recipients were the most risk-tolerant group, while facially disfigured individuals were the least risk tolerant. All groups questioned would accept the highest degree of risk to receive a face transplant compared with the six other procedures. CONCLUSIONS: This study presents an empirical basis for assessing risk versus benefit in facial transplantation. In doing so, it provides a more solid foundation upon which to introduce this exciting new reconstructive modality into the clinical arena.

Risk acceptance in composite-tissue allotransplantation reconstructive procedures.

Composite-tissue allotransplantation (CTA) is a new therapeutic modality to reconstruct major tissue defects of the face, larynx, and extremities. Unlike most life-saving organ-transplantation procedures, CTA is considered to improve quality of life. Therefore, the question arises, do the risks posed by the immunosuppression drugs that patients must take to prevent rejection justify the benefits of these procedures? The purpose of this study was to assess the relative risk that individuals are willing to accept in order to receive the benefits of CTA procedures. We used a psychometrically reliable and valid instrument to question two primary populations of individuals: those who live with the risks of immunosuppression, and healthy individuals. The level of risk acceptance for the seven transplant procedures tested (foot, single hand, double hand, larynx, kidney, hemiface, and full face) showed significant differences in research participants' risk acceptance for the different transplant procedures, but no significant differences between groups. Based on these findings, we conclude that certain CTA procedures convey benefits to recipients that are perceived by subjects, including individuals who live with the risks of immunosuppression, to warrant the risks of these procedures.

Investigation of risk acceptance in hand transplantation.

PURPOSE: Twenty-four hands have been transplanted in 18 individuals and clinicians continue to debate whether the risks justify the benefits in these procedures. To assess quantitatively the risks versus benefits in hand transplantation we used a validated instrument (Louisville Instrument for Transplantation) to measure the degree of risk individuals are willing to accept to receive different types of transplantation procedures. METHODS: The Louisville Instrument for Transplantation contains 237 standardized questions that incorporate modified standard gamble and time trade-off outcome measures and questions that assess body image perception, depression, self-esteem, optimism, socially desirable responding, and demographics. Respondents were questioned on the extent to which they would trade off specific numbers of life-years or sustain other costs in exchange for receiving 7 different transplantation procedures. For this study we questioned 227 individuals in 4 study populations with differing life experiences: healthy individuals, organ transplant recipients, upper-extremity amputee patients, and lower-extremity amputee patients. RESULTS: All populations questioned perceived risk similarly despite their differing life experiences and would accept differing degrees of risk for the different transplantation procedures. Organ transplant recipients were the most risk-tolerant group whereas upper-extremity amputee patients were the most risk adverse, even when considering a single hand transplant. All groups that were questioned would accept a higher degree of risk to receive a double versus a single hand transplant. CONCLUSIONS: By questioning individuals who have experienced directly the risks of immunosuppression and those who could benefit from hand transplantation, this study presents an empiric basis for assessing risk versus benefit in this procedure.

Functional outcome for children with thumb aplasia undergoing pollicization.

BACKGROUND: Hand function is difficult to evaluate in young patients. It is helpful to assess young children after surgery for trauma or congenital anomaly to see how they cope as they progress through their developmental milestones. METHODS: Functional outcome in 10 children (12 upper extremities) who had previous pollicization for a congenitally absent or severely hypoplastic thumb were evaluated by standard radiographs, thumb total active range of motion, grip and pinch strength, parent questionnaire, modified Jebsen functional testing, and a pegboard Functional Dexterity Test. RESULTS: Grip strength was significantly less (p = 0.008) in the hands that had been operated on (mean, 2 kg) compared with the hands that had not been operated on (mean, 5.6 kg). Pinch strength was also significantly less (p = 0.008) in the hands that had been operated on (mean, 1.0 kg) compared with those that had not been operated on (mean, 2.1 kg). In most, hands that had been operated on and those that had not been operated on tested outside the 2-SD range of age-matched normals for pinch and grip strength and also for the Functional Dexterity Test. In contrast, total Jebsen Hand Function Test time was not significantly different from hands that had not been operated on, except that some subtests were significantly different, such as checker stacking (p = 0.016; mean difference, 7.2 seconds) and page turning (p = 0.031; mean difference, -10 seconds). The total active range of motion in hands that had been operated on and those that had not been operated on was also not significantly different. All children used their reconstructed thumbs in a normal pattern. Parent questionnaires revealed satisfaction with appearance and good social interactions. Functional activities showed greatest difficulty handling small objects, especially when simultaneous pinch strength was required, such as fastening buttons and small snaps. CONCLUSIONS: This study demonstrates the importance of evaluating multiple aspects of functional outcome for congenital hand problems and of using comparative age-appropriate validated norms. Pollicization is a rewarding procedure for children with thumb aplasia.

Bone quality and healing in a swine vascularized bone allotransplantation model using cyclosporine-based immunosuppression therapy.

Although vascularized bone and joint allotransplantation is a promising new treatment option for reconstructing large bone defects, the need for immunosuppressive agents to prevent rejection in these procedures poses a major problem. This problem stems from the fact that several of these agents can cause harmful side effects, such as alterations in bone quality and healing. Therefore, the purpose of this study was to determine what effect the commonly used immunosuppressant regimen cyclosporine A-based combination therapy has on bone quality and healing. In 10 pigs, vascularized bone allografts with skin and muscle components (osteomyocutaneous free flaps) were transplanted from size-matched donor animals. Recipient animals received oral cyclosporine A/mycophenolate mofetil/prednisone therapy for 90 days. Bone quality was studied before and after transplantation by measuring the bone's acoustic velocity and density and calculating the bone's elastic coefficient. Bone healing was assessed using radiographic analysis. Four animals were lost as a result of graft rejection or immunosuppression-related complications before the 90-day endpoint of the study. Although bone specimens taken from the six animals that completed the 90-day protocol had histological signs of rejection, they all seemed to have normal bone healing. Posttransplant bone density values were significantly decreased (p < 0.05) (1544.7 +/- 47.5 kg/m3) as compared with pretransplant values (1722.7 +/- 44.1 kg/m3). Results of the acoustic velocity and elastic coefficients measurements showed a significant decrease (p < 0.05) in posttransplant values (from 3503.0 +/- 165.1 meters/sec to 2963.0 +/- 54.6 meters/sec and from 21.6 +/- 2.2 GPa to 13.6 +/- 0.5 GPa, respectively), indicating diminished bone quality. The findings indicate that cyclosporine A/mycophenolate mofetil/prednisone combination therapy is ineffective in preventing bone rejection, that it decreases bone quality, and that it is associated with systemic toxicity, suggesting that this immunosuppressive regimen at the doses used in this study is not ideal for vascularized bone allotransplantation procedures.

Composite tissue allotransplantation in chimeric hosts: part I. Prevention of graft-versus-host disease.

BACKGROUND: Mixed allogeneic chimerism (MAC) has been shown to induce tolerance to composite tissue allografts (CTA). However, transplantation of unmanipulated donor-specific limbs results in severe graft-versus-host disease (GVHD). This suggests that nontolerant mature donor-derived cells in the CTA may affect the stability of chimerism, potentially resulting in GVHD. The aim of this study was to develop an approach to study and prevent GVHD in a mixed chimeric-rat hind-limb transplantation model. METHODS: [ACI-->WF] chimeras received a limb from Wistar Furth (WF) (syngeneic), Fisher (third-party), or ACI (irradiated [1,050 cGy] or nonirradiated) rats. In vitro tolerance was assessed using mixed lymphocyte reactivity (MLR) assays at the time the animals were killed. RESULTS: [ACI-->WF] chimeras with greater than 85% chimerism exhibited rejection-free survival of donor-specific hind limbs. However, 100% of these animals developed lethal GVHD 22.4+/-2.8 days after limb transplantation. [ACI-->WF] chimeras that underwent transplantation with irradiated ACI or syngeneic WF limbs showed no signs of rejection or GVHD at 5 months. Nonchimeric and third-party controls rejected limbs within 10 days. CONCLUSIONS: Conditioning of the host WF rats with 950 cGy of irradiation (sublethal, myeloablative) led to high levels of MAC without GVHD. The mature T-cell content of nonirradiated donor (ACI) limbs was sufficient to induce lethal GVHD in 100% of tolerant mixed chimeric [ACI-->WF] hosts. Irradiation of donor limbs before transplantation resulted in long-term donor-specific tolerance and prevented GVHD. These data demonstrate that (1) established chimeras could be susceptible to GVHD caused by immunocompetent donor cells transferred with the hind limb, and (2) inactivating these cells with irradiation prevents GVHD and destabilization of chimerism, and permits rejection-free graft acceptance.

Analysis of fiber type transformation and histology in chronic electrically stimulated canine rectus abdominis muscle island-flap stomal sphincters.

Dynamic skeletal muscle flaps are designed to perform a specific functional task through contraction and relaxation of their muscle fibers. The most commonly used dynamic skeletal flaps today are for cardiomyoplasty and anal or urinary myoplasty. Low-frequency chronic stimulation of these flaps enables them to use their intrinsic energy stores in a more efficient manner through aerobic metabolic pathways for increased endurance and improved work capacity. The purpose of this study was to (1) determine whether fiber type transformation from fatigue-prone (type II) muscle fibers to fatigue-resistant (type I) muscle fibers could be demonstrated in the authors' chronic canine stomal sphincter model where the rectus abdominis muscle was used to create a functional stomal sphincter, (2) assess whether there is any correlation between the degree of muscle fiber type transformation and the continence times, and (3) examine the long-term effects of the training regimens on the skeletal muscle fibers through histologic and volumetric analysis. Eight dynamic island-flap sphincters were created from a part of the rectus abdominis muscle in mongrel dogs by preserving the deep inferior epigastric vascular pedicle and the most caudal investing intercostal nerve. The muscular sphincters were wrapped around a blind loop of distal ileum and trained with pacing electrodes. Two different training protocols were used. In group A (n = 4), a preexisting anal dynamic graciloplasty training protocol was used. A revised protocol was used in group B (n = 4). Muscle biopsy specimens were obtained before and after training from the rectus abdominis muscle sphincter. Fiber type transformation was assessed using a monoclonal antibody directed against the fatigue-prone type II fibers. Pretraining and posttraining skeletal muscle specimens were examined histologically. A significant fiber type conversion was achieved in both group A and group B animals, with each group achieving greater than 50 percent conversion from fatigue-prone (type II) muscle fibers to fatigue-resistant (type I) muscle fibers. The continence time was different for both groups. Biopsy specimens 1 cm from the electrodes revealed that fiber type transformation was uniform throughout this region of the sphincters. Skeletal muscle fibers within both groups demonstrated a reduction in their fiber diameter and volume. Fiber type transformation is possible in this unique canine island-flap rectus abdominis sphincter model. The relative design of the flap with preservation of the skeletal muscle resting length and neuronal and vascular supply are important characteristics when designing a functional dynamic flap for stomal continence.