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Lips form a structure that are difficult to reconstruct after a traumatic avulsion injury or cancer ablative surgery secondary to loss of volumetric muscle mass. Traditional tissue engineering approaches of in vitro fabrication of mature tissue constructs can supply an alternative to the current surgical standard of care for functional lip reconstruction. We demonstrate a hybrid approach that combines the advantages of in situ muscle flap prefabrication with in vitro fabrication of an autogenous mucocutaneous construct as the laminate for prelamination to form a designer microvascular muscle free flap for lip reconstruction.
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BACKGROUND: Pain is a complex experience that involves sensory-discriminative and cognitive-emotional neuronal processes. It has long been known across cultures that pain can be relieved by mindful breathing (MB). There is a common assumption that MB exerts its analgesic effect through interoception. Interoception refers to consciously refocusing the mind's attention to the physical sensation of internal organ function. OBJECTIVE: In this study, we dissect the cortical analgesic processes by imaging the brains of healthy subjects exposed to traditional MB (TMB) and compare them with another group for which we augmented MB to an outside sensory experience via virtual reality breathing (VRB). METHODS: The VRB protocol involved in-house-developed virtual reality 3D lungs that synchronized with the participants' breathing cycles in real time, providing them with an immersive visual-auditory exteroception of their breathing. RESULTS: We found that both breathing interventions led to a significant increase in pain thresholds after week-long practices, as measured by a thermal quantitative sensory test. However, the underlying analgesic brain mechanisms were opposite, as revealed by functional near-infrared spectroscopy data. In the TMB practice, the anterior prefrontal cortex uniquely modulated the premotor cortex. This increased its functional connection with the primary somatosensory cortex (S1), thereby facilitating the S1-based sensory-interoceptive processing of breathing but inhibiting its other role in sensory-discriminative pain processing. In contrast, virtual reality induced an immersive 3D exteroception with augmented visual-auditory cortical activations, which diminished the functional connection with the S1 and consequently weakened the pain processing function of the S1. CONCLUSIONS: In summary, our study suggested two analgesic neuromechanisms of VRB and TMB practices-exteroception and interoception-that distinctively modulated the S1 processing of the ascending noxious inputs. This is in line with the concept of dualism (Yin and Yang).
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Espectroscopia de Luz Próxima ao Infravermelho , Realidade Virtual , Encéfalo/diagnóstico por imagem , Humanos , Dor , Córtex Pré-FrontalRESUMO
Quantitative diffuse reflectance spectroscopy (DRS) was developed for label-free, noninvasive, and real-time assessment of implanted tissue-engineered devices manufactured from primary human oral keratinocytes (six batches in two 5-patient cohorts). Constructs were implanted in a murine model for 1 and 3 weeks. DRS evaluated construct success in situ using optical absorption (hemoglobin concentration and oxygenation, attributed to revascularization) and optical scattering (attributed to cellular density and layer thickness). Destructive pre- and postimplantation histology distinguished experimental control from stressed constructs, whereas noninvasive preimplantation measures of keratinocyte glucose consumption and residual glucose in spent culture media did not. In constructs implanted for 1 week, DRS distinguished control due to stressed and compromised from healthy constructs. In constructs implanted for 3 weeks, DRS identified constructs with higher postimplantation success. These results suggest that quantitative DRS is a promising, clinically compatible technology for rapid, noninvasive, and localized tissue assessment to characterize tissue-engineered construct success in vivo. Impact statement Despite the recent advance in tissue engineering and regenerative medicine, there is still a lack of nondestructive tools to longitudinally monitor the implanted tissue-engineered devices. In this study, we demonstrate the potential of quantitative diffuse reflectance spectroscopy as a clinically viable technique for noninvasive, label-free, and rapid characterization of graft success in situ.
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Engenharia Tecidual , Alicerces Teciduais , Animais , Contagem de Células , Humanos , Queratinócitos , CamundongosRESUMO
The cultivation of human living cells into scaffolding matrices has progressively gained popularity in the field of periodontal wound healing and regeneration. Living cellular constructs based on fibroblasts, keratinocytes alone or in combination have been developed and used as alternatives to autogenous soft tissue grafts in keratinized tissue augmentation and in root coverage procedures. Their promising advantages include reduced patient morbidity, unlimited graft availability, and comparable esthetics. This manuscript reviews soft tissue augmentation and root coverage procedures using bioengineered living cellular therapy and highlights their expected clinical, esthetic, and patient-related outcomes.
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Retração Gengival/cirurgia , Gengivoplastia , Tecido Conjuntivo , Estética Dentária , Gengiva , Humanos , Periodonto , Medicina RegenerativaRESUMO
The present article focuses on the properties and indications of scaffold-based extracellular matrix (ECM) technologies as alternatives to autogenous soft tissue grafts for periodontal and peri-implant plastic surgical reconstruction. The different processing methods for the creation of cell-free constructs resulting in preservation of the extracellular matrices influence the characteristics and behavior of scaffolding biomaterials. The aim of this review is to discuss the properties, clinical application, and limitations of ECM-based scaffold technologies in periodontal and peri-implant soft tissue augmentation when used as alternatives to autogenous soft tissue grafts.
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Gengiva , Procedimentos de Cirurgia Plástica , Materiais Biocompatíveis , Matriz Extracelular , CicatrizaçãoRESUMO
This state-of-the-art review presents the latest evidence and the current status of autogenous soft tissue grafting for soft tissue augmentation and recession coverage at teeth and dental implant sites. The indications and predictability of the free gingival graft and connective tissue graft (CTG) techniques are highlighted, together with their expected clinical and esthetic outcomes. CTGs can be harvested from the maxillary tuberosity or from palate with different approaches that can have an impact on graft quality and patient morbidity. The influence of CTGs on soft tissue thickness and keratinized tissue width are also discussed.
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Implantes Dentários , Retração Gengival/cirurgia , Procedimentos de Cirurgia Plástica , Tecido Conjuntivo , Estética Dentária , Gengiva/cirurgia , Humanos , PlásticosRESUMO
This manuscript provides a state-of-the-art review on the efficacy of biologics in root coverage procedures, including enamel matrix derivative, platelet-derived growth factor, platelet concentrates, and fibroblast-growth factor-2. The mechanism of action and the rationale for using biologics in periodontal plastic surgery, as well as their anticipated benefits when compared with conventional approaches are discussed. Although the clinical significance is still under investigation, preclinical data and histologic evidence demonstrate that biologic-based techniques are able to promote periodontal regeneration coupled with the provision of tooth root coverage.
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Produtos Biológicos , Proteínas do Esmalte Dentário , Retração Gengival/cirurgia , Regeneração Tecidual Guiada Periodontal , Humanos , Periodonto , Retalhos Cirúrgicos/cirurgia , Engenharia TecidualRESUMO
Many conventional methods to assess engineered tissue morphology and viability are destructive techniques with limited utility for tissue constructs intended for implantation in patients. Sterile label-free optical molecular imaging methods analyzed tissue endogenous fluorophores without staining, noninvasively and quantitatively assessing engineered tissue, in lieu of destructive assessment methods. The objective of this study is to further investigate label-free optical metrics and their correlation with destructive methods. Tissue-engineered constructs (n = 33 constructs) fabricated with primary human oral keratinocytes (n = 10 patients) under control, thermal stress, and rapamycin treatment manufacturing conditions exhibited a range of tissue viability states, as evaluated by quantitative histology scoring, WST-1 assay, Ki-67 immunostaining imaging, and label-free optical molecular imaging methods. Both histology sections of fixed tissues and cross-sectioned label-free optical images of living tissues provided quantitative spatially selective information on local tissue morphology, but optical methods noninvasively characterized both local tissue morphology and cellular viability at the same living tissue site. Furthermore, optical metrics noninvasively assessed living tissue viability with a statistical significance consistent with the destructive tissue assays WST-1 and histology. Over the range of cell viability states created experimentally, optical metrics noninvasively and quantitatively characterized living tissue viability and correlated with the destructive WST-1 tissue assay. By providing, under sterile conditions, noninvasive metrics that were comparable with conventional destructive tissue assays, label-free optical molecular imaging has the potential to monitor and assess engineered tissue construct viability before surgical implantation.
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Imagem Óptica , Engenharia Tecidual/métodos , Sobrevivência de Tecidos , Sobrevivência Celular , Humanos , Queratinócitos/citologia , Imagem Molecular , Coloração e Rotulagem , Alicerces Teciduais/químicaRESUMO
Primary keratinocytes derived from human epidermis are widely used in tissue engineering and regenerative medicine. An important aspect in clinical applications is the preservation of human skin keratinocyte stem cells. However, it is difficult to expand the number of human skin keratinocyte stem cells, which are undifferentiated and highly proliferative in culture by using standard cell culture methods. It is even more difficult to identify them, since universal specific markers for human skin keratinocyte stem cells have not been identified. In this paper, we show a method to produce a large number of primary progenitor human skin keratinocytes by using our novel culture techniques. Primary human skin keratinocyte monolayers are cultured using twice the volume of medium without serum and lacking essential fatty acids. Once the cells reach 70-80% confluence, they begin to float up into the overlying medium and are called "epithelial pop-up keratinocytes (ePUKs)" allowing the cells to be passaged without the use of trypsin. We analyzed the properties of ePUKs by cell size, cell viability, immunocytofluorescence biomarker staining, and cell cycle phase distribution by fluorescence-activated cell sorting (FACS). Our results showed that these ePUKs appear to be progenitor epithelial cells, which are small in size, undifferentiated, and have a high proliferative capacity. We believe that ePUKs are suitable for use in medical applications requiring a large number of primary human progenitor skin keratinocytes.
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Queratinócitos/citologia , Cultura Primária de Células/métodos , Células-Tronco/citologia , Adulto , Idoso , Ciclo Celular , Tamanho Celular , Sobrevivência Celular , Células Epidérmicas , Células Epiteliais/citologia , Feminino , Fator 7 de Crescimento de Fibroblastos/metabolismo , Citometria de Fluxo , Glucose/metabolismo , Humanos , Pessoa de Meia-IdadeRESUMO
This study validates the use of a full-thickness oral mucosa model for in vitro studies with a collagen type I matrix, by comparison of this model with two other 3D oral mucosa models: human-sourced and porcine acellular dermal matrices (AlloDerm®/Strattice®, respectively). For the collagen matrix model, gingival fibroblasts were seeded either onto the dermal side of the AlloDerm® and Strattice® matrices or within the collagen matrices in complete culture medium (DMEM). For all scaffolds, DMEM was replaced every 24â¯h up to 72â¯h. For the full-thickness oral mucosa models, 72â¯h after fibroblast seeding, oral keratinocytes were seeded on the epidermal sides of AlloDerm® and Strattice® matrices or collagen matrices. All matrices and models were subjected to histological analysis, complementing phenotypic characterization by evaluation of glucose consumption, cell proliferation, gene expression and synthesis of growth factors. A higher fibroblast ratio was observed for the collagen matrix, in which the distribution of gingival fibroblasts was also more homogeneous. Metabolism, proliferation, and gene expression and synthesis of VEGF of these cells were also increased for the collagen matrix. All matrices provided a suitable substrate for oral keratinocytes adhesion, proliferation, and phenotypic expression; however, higher proliferation, stratification, and differentiation were noted when oral keratinocytes were seeded on the dermal matrices.
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Técnicas de Cultura de Células , Queratinócitos/citologia , Mucosa Bucal , Animais , Proliferação de Células , Colágeno , Colágeno Tipo I/genética , Cadeia alfa 1 do Colágeno Tipo I , Fator de Crescimento Epidérmico/genética , Fator 7 de Crescimento de Fibroblastos/genética , Fibroblastos/citologia , Expressão Gênica , Gengiva/citologia , Glucose/metabolismo , Humanos , Queratinócitos/metabolismo , Fenótipo , Biossíntese de Proteínas , Suínos , Fator A de Crescimento do Endotélio Vascular/genéticaRESUMO
OBJECTIVE: To develop a bioreactor for automated culture, maintenance, and collection of normal human keratinocytes using an enzyme-free propagation method. METHODS: The culture of normal human epithelial keratinocytes was compared in two culture methods - a study team-developed automated bioreactor utilising an enzyme-free passage method, and a manual culture method. Cell size, glucose utilisation, and the proliferative capacity of the two cultures were evaluated. RESULTS: An automated bioreactor, not using enzymes for passage, but instead using the novel Epithelial Pop Up Keratinocytes (ePUK)1 culture technique, resulted in an extended culture longevity and proliferative capacity in normal primary human keratinocytes. Daughter cells were collected up to three times per day utilising the bioreactor. The daughter cells produced by the bioreactor were smaller than daughter cells produced by the manual culture method. The proliferative capacity and health of the parent monolayer within both the bioreactor and the manual culture flask was dependent upon sufficient glucose availability. Due to the contact inhibition nature of epithelial keratinocytes, the bioreactor enabled the study of an adherent cell type soon after cytokinesis and before the cell has integrated as part of an adherent matrix. CONCLUSION: The study demonstrates that increasing the number of media changes per day as necessary, based on glucose utilisation, is necessary for prolonged longevity and functional productivity of ePUK cultures.
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Reatores Biológicos , Técnicas de Cultura de Células/métodos , Proliferação de Células , Tamanho Celular , Células Epiteliais/citologia , Queratinócitos/citologia , Automação , Células Epiteliais/metabolismo , Glucose/metabolismo , Humanos , Queratinócitos/metabolismoRESUMO
Fluorescence lifetime sensing has been shown to noninvasively characterize the preimplantation health and viability of engineered tissue constructs. However, current practices to monitor postimplantation construct integration are either qualitative (visual assessment) or destructive (tissue histology). We employed label-free fluorescence lifetime spectroscopy for quantitative, noninvasive optical assessment of engineered tissue constructs that were implanted into a murine model. The portable system was designed to be suitable for intravital measurements and included a handheld probe to precisely and rapidly acquire data at multiple sites per construct. Our model tissue constructs were manufactured from primary human cells to simulate patient variability based on a standard protocol, and half of the manufactured constructs were stressed to create a range of health states. Secreted amounts of three cytokines that relate to cellular viability were measured in vitro to assess preimplantation construct health: interleukin-8 (IL-8), human ß-defensin 1 (hBD-1), and vascular endothelial growth factor (VEGF). Preimplantation cytokine secretion ranged from 1.5 to 33.5 pg/mL for IL-8, from 3.4 to 195.0 pg/mL for hBD-1, and from 0.1 to 154.3 pg/mL for VEGF. In vivo optical sensing assessed constructs at 1 and 3 weeks postimplantation. We found that at 1 week postimplantation, in vivo optical parameters correlated with in vitro preimplantation secretion levels of all three cytokines (p < 0.05). This correlation was not observed in optical measurements at 3 weeks postimplantation when histology showed that the constructs had re-epithelialized, independent of preimplantation health state, supporting the lack of a correlation. These results suggest that clinical optical diagnostic tools based on label-free fluorescence lifetime sensing of endogenous tissue fluorophores could noninvasively monitor postimplantation integration of engineered tissues.
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Citocinas/metabolismo , Processamento de Imagem Assistida por Computador/métodos , Queratinócitos/transplante , Microscopia de Fluorescência/métodos , Mucosa Bucal/transplante , Engenharia Tecidual/métodos , Animais , Sobrevivência Celular , Feminino , Humanos , Queratinócitos/citologia , Queratinócitos/metabolismo , Camundongos SCID , Mucosa Bucal/citologia , Mucosa Bucal/metabolismo , Alicerces Teciduais , Transplante HeterólogoRESUMO
Immunologically inert allogeneic acellular dermal scaffolds provide a matrix with molecular architecture close to native tissues, which synthetic scaffolds cannot. Not all nature-derived scaffolds possess the same biological and physical properties. The different properties of scaffolds supporting cellular growth used for manufacturing tissue engineered grafts could lead to different implantation results. The scaffold properties should be carefully considered in order to meet the expected outcomes of tissue engineered grafts. In this report, we evaluated the cellular growth on AlloDerm® and Allopatch, 2 acellular scaffolds derived from human cadaver skin, using a fabricated 3D organotypic culture with primary human oral keratinocytes to produce an ex vivo produced oral mucosa equivalent (EVPOME). A well stratified epithelium could be constructed on both scaffolds. AlloDerm® and Allopatch EVPOMEs were also implanted into severe combined immunodeficiency mice to compare the ingrowth of blood vessels into the dermal component of the two EVPOMEs. Blood vessel counts were 3.3 times higher (p = .01) within Allopatch EVPOMEs than within AlloDerm® EVPOMEs. An oral and skin keratinocyte co-culture, separated by a physical barrier to create a cell-free zone, was used to evaluate cell migration on AlloDerm® and Allopatch. Slower cell migration was observed on Allopatch than on AlloDerm®.
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Derme/química , Queratinócitos/metabolismo , Mucosa Bucal/química , Engenharia Tecidual , Alicerces Teciduais/química , Animais , Humanos , Queratinócitos/citologia , Camundongos , Camundongos SCIDRESUMO
In reconstructive surgery, the ability to detect blood flow interruptions to grafted tissue represents a critical step in preventing postsurgical complications. We have developed and pilot tested a compact, fiber-based device that combines two complimentary modalities-diffuse correlation spectroscopy (DCS) and diffuse reflectance spectroscopy-to quantitatively monitor blood perfusion. We present a proof-of-concept study on an in vivo porcine model (n=8). With a controllable arterial blood flow supply, occlusion studies (n=4) were performed on surgically isolated free flaps while the device simultaneously monitored blood flow through the supplying artery as well as flap perfusion from three orientations: the distal side of the flap and two transdermal channels. Further studies featuring long-term monitoring, arterial failure simulations, and venous failure simulations were performed on flaps that had undergone an anastomosis procedure (n=4). Additionally, benchtop verification of the DCS system was performed on liquid flow phantoms. Data revealed relationships between diffuse optical measures and state of occlusion as well as the ability to detect arterial and venous compromise. The compact construction of the device, along with its noninvasive and quantitative nature, would make this technology suitable for clinical translation.
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Retalhos de Tecido Biológico/irrigação sanguínea , Monitorização Hemodinâmica/instrumentação , Dispositivos Ópticos , Anastomose Cirúrgica , Animais , Artérias/diagnóstico por imagem , Artérias/patologia , Suínos , Veias/diagnóstico por imagem , Veias/patologiaRESUMO
In reconstructive surgery, tissue perfusion/vessel patency is critical to the success of microvascular free tissue flaps. Early detection of flap failure secondary to compromise of vascular perfusion would significantly increase the chances of flap salvage. We have developed a compact, clinically-compatible monitoring system to enable automated, minimally-invasive, continuous, and quantitative assessment of flap viability/perfusion. We tested the system's continuous monitoring capability during extended non-recovery surgery using an in vivo porcine free flap model. Initial results indicated that the system could assess flap viability/perfusion in a quantitative and continuous manner. With proven performance, the compact form constructed with cost-effective components would make this system suitable for clinical translation.
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In reconstructive surgery, impeded blood flow in microvascular free flaps due to a compromise in arterial or venous patency secondary to blood clots or vessel spasms can rapidly result in flap failures. Thus, the ability to detect changes in microvascular free flaps is critical. In this paper, we report progress on in vivo pre-clinical testing of a compact, multimodal, fiber-based diffuse correlation and reflectance spectroscopy system designed to quantitatively monitor tissue perfusion in a porcine model's surgically-grafted free flap. We also describe the device's sensitivity to incremental blood flow changes and discuss the prospects for continuous perfusion monitoring in future clinical translational studies.
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It is essential to monitor tissue perfusion during and after reconstructive surgery, as restricted blood flow can result in graft failures. Current clinical procedures are insufficient to monitor tissue perfusion, as they are intermittent and often subjective. To address this unmet clinical need, a compact, low-cost, multimodal diffuse correlation spectroscopy and diffuse reflectance spectroscopy system was developed. We verified system performance via tissue phantoms and experimental protocols for rigorous bench testing. Quantitative data analysis methods were employed and tested to enable the extraction of tissue perfusion parameters. This design verification study assures data integrity in future in vivo studies.
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PURPOSE: We present a straightforward and reproducible technique to create, in vitro, a construct containing a mucocutaneous junction (MCJ) with a transitional zone (vermilion) for fabrication of a microvascular prelaminated flap for use in lip reconstruction. MATERIALS AND METHODS: Cultured primary human skin keratinocytes and oral mucosal epithelial cells at premixed ratios of 50% skin cells to 50% oral cells, 25% skin cells to 75% oral cells, and 75% skin cells to 25% oral cells were grown on an AlloDerm dermal equivalent (LifeCell, Branchburg, NJ) to create an MCJ equivalent with a lip or transitional zone (vermilion) using a novel 3-dimensional (3D) culture device with a barrier to separate co-cultured skin and oral cells. The 3 different cell ratios were compared by staining for the following specific differentiation markers to define the different areas of skin and mucosal keratinocytes: filaggrin, cytokeratin 10, cytokeratin 19, and small proline-rich protein 3. RESULTS: Immunohistochemical results showed that MCJ equivalents seeded with premixed cells were similar to the differentiation patterns of tissue-engineered 3D cultures using 100% oral mucosal epithelial cells or skin keratinocytes. The engineered MCJ-equivalent constructs, grown in the 3D device specifically constructed with a cell-free gap at the barrier site, formed a transitional zone (vermilion) at the barrier site with intermingling of the skin and oral keratinocytes. The results showed different and unique expression patterns of filaggrin, cytokeratin 10, cytokeratin 19, and small proline-rich protein 3 by those cells migrating into the gap, which were similar to those seen in human lip tissue. This pattern was not seen in MCJ equivalents created using premixed skin and oral cells. CONCLUSIONS: Using a device to separately co-culture human oral and skin keratinocytes to allow the cells to migrate into a cell-free zone resulted in phenotypic expression closer to what is seen in native tissue, in comparison to premixing the skin and oral cells before seeding.
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Lábio/cirurgia , Procedimentos de Cirurgia Plástica/métodos , Engenharia Tecidual/métodos , Diferenciação Celular , Células Cultivadas , Técnicas de Cocultura , Colágeno , Proteínas Filagrinas , Humanos , Técnicas In Vitro , Queratinócitos , Mucosa Bucal/citologia , Alicerces TeciduaisRESUMO
The vascularization and resulting perfusion of transferred tissues are critical to the success of grafts in buried free flap transplantations. To enable long-term clinical monitoring of grafted tissue perfusion during neovascularization and endothelialization, we are developing an implantable instrument for the continuous monitoring of perfusion using diffuse correlation spectroscopy (DCS), and augmented with diffuse reflectance spectroscopy (DRS). This work discusses instrument construction, integration, and preliminary results using a porcine graft model.
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Stable, relative localization of source and detection fibers is necessary for clinical implementation of quantitative optical perfusion monitoring methods such as diffuse correlation spectroscopy (DCS) and diffuse reflectance spectroscopy (DRS). A flexible and compact device design is presented as a platform for simultaneous monitoring of perfusion at a range of depths, enabled by precise location of optical fibers in a robust and secure adhesive patch. We will discuss preliminary data collected on human subjects in a lower body negative pressure model for hypovolemic shock. These data indicate that this method facilitates simple and stable simultaneous monitoring of perfusion at multiple depths and within multiple physiological compartments.