Furnishing Wound Repair by the Subcutaneous Fascia.

Mammals rapidly heal wounds through fibrous connective tissue build up and tissue contraction. Recent findings from mouse attribute wound healing to physical mobilization of a fibroelastic connective tissue layer that resides beneath the skin, termed subcutaneous fascia or superficial fascia, into sites of injury. Fascial mobilization assembles diverse cell types and matrix components needed for rapid wound repair. These observations suggest that the factors directly affecting fascial mobility are responsible for chronic skin wounds and excessive skin scarring. In this review, we discuss the link between the fascia's unique tissue anatomy, composition, biomechanical, and rheologic properties to its ability to mobilize its tissue assemblage. Fascia is thus at the forefront of tissue pathology and a better understanding of how it is mobilized may crystallize our view of wound healing alterations during aging, diabetes, and fibrous disease and create novel therapeutic strategies for wound repair.

Advances in Subcutaneous Delivery Systems of Biomacromolecular Agents for Diabetes Treatment.

Diabetes mellitus is a major threat to human health. Both its incidence and prevalence have been rising steadily over the past few decades. Biomacromolecular agents such as insulin and glucagon-like peptide 1 receptor agonists are commonly used hypoglycemic drugs that play important roles in the treatment of diabetes. However, their traditional frequent administration may cause numerous side effects, such as pain, infection or local tissue necrosis. To address these issues, many novel subcutaneous delivery systems have been developed in recent years. In this review, we survey recent developments in subcutaneous delivery systems of biomacromolecular hypoglycemic drugs, including sustained-release delivery systems and stimuli-responsive delivery systems, and summarize the advantages and limitations of these systems. Future opportunities and challenges are discussed as well.

Distribution of sole Pacinian corpuscles: a histological study using near-term human feet.

INTRODUCTION: Fast-adapting afferent input from the sole Pacinian corpuscles (PCs) is essential for walking. However, the distribution of PCs in the plantar subcutaneous tissue remains unknown. MATERIALS AND METHODS: Using histological sections tangential to the plantar skin of eight near-term fetuses, we counted 528-900 PCs per sole. RESULTS: Almost half of the sole PCs existed at the level of the proximal phalanx, especially on the superficial side of the long flexor tendons and flexor digitorum brevis. Conversely, the distribution was less evident on the posterior side of the foot. The medial margin of the sole contained fewer PCs than the lateral margin, possibly due to the transverse arch. In contrast to a cluster formation in the anterior foot, posterior PCs were almost always solitary, with a distance greater than 0.5 mm to the nearest PC. DISCUSSION AND CONCLUSION: Because a receptive field of PCs is larger than that of the other receptors, fewer solitary PCs might cover the posterior sole. In infants, the amount of anterior sole PCs seemed to determine the initial walking pattern using the anterior foot without heel contact. Anterior PCs concentrated along flexor tendons might play a transient role as tendon organs during the initial learning of walking. During a lesson in infants, mechanical stress from the tendon and muscle was likely to degrade the PCs. In the near term, the sole PCs seemed not to be a mini-version of the adult morphology but suggested an infant-specific function.

Perfusion bioreactor culture of human adipose-derived stromal cells on decellularized adipose tissue scaffolds enhances in vivo adipose tissue regeneration.

Tissue-engineering approaches hold promise to address the need in plastic and reconstructive surgery for new therapies that promote stable adipose tissue regeneration. Previous studies have demonstrated the potential of combining decellularized adipose tissue (DAT) scaffolds with adipose-derived stromal cells (ASCs) for volume augmentation applications. With the goal of enhancing in vivo angiogenesis and adipogenesis, this study evaluated the effects of culturing human ASCs on DAT scaffolds within a perfusion bioreactor. Using this system, the impact of both dynamic culture and hypoxic preconditioning were explored in vitro and in vivo. Initial studies compared the effects of 14 days of culture within the perfusion bioreactor under hypoxia (2% O2 ) or normoxia (~20% O2 ) on human ASC expansion and expression of hypoxia inducible factor-1 alpha (HIF-1α) in vitro relative to static cultured controls. The findings indicated that culturing within the bioreactor under hypoxia significantly increased ASC proliferation on the DAT, with a higher cell density observed in the scaffold periphery. Subsequent characterization in a subcutaneous implant model in athymic nude mice revealed that in vivo angiogenesis and adipogenesis were markedly enhanced when the ASCs were cultured on the DAT within the perfusion bioreactor under hypoxia for 14 days prior to implantation relative to the other culture conditions, as well as freshly seeded and unseeded DAT control groups. Overall, dynamic culture within the perfusion bioreactor system under hypoxia represents a promising approach for preconditioning ASCs on DAT scaffolds to enhance their capacity to stimulate angiogenesis and host-derived adipose tissue regeneration.

Tissue Resistance during Large-Volume Injections in Subcutaneous Tissue of Minipigs.

PURPOSE: Injection devices for administration of biopharmaceuticals enable subcutaneous self-administration by patients. To meet patient specific capabilities, injection forces need to be characterized. We address the open question of whether tissue resistance significantly contributes to overall injection forces, especially for large injection volumes. METHODS: Subcutaneous tissue resistance was systematically quantified for injection volumes up to 11 mL depending on viscosity (1-20 mPa·s) and injection rates (0.025-0.2 mL/s) using Göttingen Minipigs as the animal model. The contribution of an artificially applied external force at the injection site simulating autoinjector needle cover depression was tested between 2.5-7.5 N. RESULTS: Tissue resistance reached average values of ~120 mbar for injection volumes up to 11 mL independent of viscosity and injection rate, and maximum values of 300 mbar were determined. Artificially applied external forces led to higher values, independent of the absolute applied force - maximum values of 1 bar were obtained when injecting 4.5 mL of the 20 mPa·s solution at an injection rate of 0.1 mL/s with the application of an artificial 5 N force, corresponding to ~450 mbar. All conditions yield defined injection sites suggesting tissue resistance is defined by mechanical properties of the subcutaneous tissue. CONCLUSIONS: We set our results in relation to overall injection forces, concluding that maximum values in tissue resistance may cause challenges during subcutaneous injection when using injection devices. Graphical abstract.

Semimembranosus muscle displacement is associated with movement of the superficial fascia: An in vivo ultrasound investigation.

The deep fascia enveloping the skeletal muscle has been shown to contribute to the mechanics of the locomotor system. However, less is known about the role of the superficial fascia (SF). This study aimed to describe the potential interaction between the Hamstring muscles and the SF. Local movement of the dorsal thigh's soft tissue was imposed making use of myofascial force transmission effects across the knee joint: In eleven healthy individuals (26.8 ± 4.3 years, six males), an isokinetic dynamometer moved the ankle into maximal passive dorsal extension (knee extended). Due to the morphological continuity between the gastrocnemius and the Hamstrings, stretching the calf led to soft tissue displacements in the dorsal thigh. Ultrasound recordings were made to dynamically visualize (a) the semimembranosus muscle and (b) the superficial fascia. Differences in and associations between horizontal movement amplitudes of the two structures, quantified via cross-correlation analyses, were calculated by means of the Mann-Whitney U test and Kendal's tau test, respectively. Mean horizontal movement was significantly higher in the muscle (5.70 mm) than in the SF (0.72 mm, p < 0.001, r = 0.82). However, a strong correlation between the tissue displacements in both locations was detected (p < 0.001, r = 0.91). A Direct mechanical relationship may exist between the SF and the skeletal muscle. Deep pathologies or altered muscle stiffness could thus have long-term consequences for rather superficial structures and vice versa.

Evidence of mesenchymal stromal cell adaptation to local microenvironment following subcutaneous transplantation.

Subcutaneous transplantation of mesenchymal stromal cells (MSC) emerged as an alternative to intravenous administration because it avoids the pulmonary embolism and prolongs post-transplantation lifetime. The goal of this study was to investigate the mechanisms by which these cells could affect remote organs. To this aim, murine bone marrow-derived MSC were subcutaneously transplanted in different anatomical regions and the survival and behaviour have been followed. The results showed that upon subcutaneous transplantation in mice, MSC formed multicellular aggregates and did not migrate significantly from the site of injection. Our data suggest an important role of hypoxia-inducible signalling pathways in stimulating local angiogenesis and the ensuing modulation of the kinetics of circulating cytokines with putative protective effects at distant sites. These data expand the current understanding of cell behaviour after subcutaneous transplantation and contribute to the development of a non-invasive cell-based therapy for distant organ protection.

Cooling of Lower Extremity Muscles According to Subcutaneous Tissue Thickness.

CONTEXT: When using an ice bag, previous researchers recommended cooling times based on the amount of subcutaneous tissue. Unfortunately, many clinicians are unaware of these recommendations or whether they can be applied to other muscles. OBJECTIVE: To examine if muscles of the lower extremity cool similarly based on recommended cooling times. DESIGN: Crossover study. SETTING: Athletic training laboratory. PATIENTS OR OTHER PARTICIPANTS: Fourteen healthy participants volunteered (8 men, 6 women; age = 21.1 ± 2.2 years, height = 174.2 ± 4.5 cm, weight = 74.0 ± 7.5 kg). INTERVENTION(S): Subcutaneous tissue thickness was measured at the largest girth of the thigh, medial gastrocnemius, and medial hamstring. Participants were randomized to have either the rectus femoris or medial gastrocnemius and medial hamstring tested first. Using sterile techniques, the examiner inserted a thermocouple 1 cm into the muscle after accounting for subcutaneous tissue thickness. After the temperature stabilized, a 750-g ice bag was applied for 10 to 60 minutes to the area(s) for the recommended length of time based on subcutaneous adipose thickness (0 to 5 mm [10 minutes]; 5.5 to 10 mm (25 minutes]; 10.5 to 15 mm [40 minutes]; 15.5 to 20 mm [60 minutes)]. After the ice bag was removed, temperature was monitored for 30 minutes. At least 1 week later, each participant returned to complete testing of the other muscle(s). MAIN OUTCOME MEASURE(S): Intramuscular temperature (°C) at baseline, end of treatment time (0 minutes), and posttreatment recovery (10, 20, and 30 minutes postintervention). RESULTS: At the end of treatment, temperature did not differ by subcutaneous tissue thickness (10 minutes = 29.0°C ± 3.8°C, 25 minutes = 28.7°C ± 3.2°C, 40 minutes = 28.7°C ± 6.0°C, 60 minutes = 30.0°C ± 2.9°C) or muscle (rectus femoris = 30.1°C ± 3.8°C, gastrocnemius = 28.6°C ± 5.4°C, hamstrings = 28.1°C ± 2.5°C). No significant interaction was present for subcutaneous tissue thickness or muscle (P ≥ .126). CONCLUSIONS: Lower extremity muscles seemed to cool similarly based on the recommended cooling times for subcutaneous tissue thickness. Clinicians should move away from standardized treatment times and adjust the amount of cooling time by ice-bag application based on subcutaneous tissue thickness.

Comprehensive Biomechanism of Impact Resistance in the Cat's Paw Pad.

Cats are able to jump from a high-rise without any sign of injury, which is attributed in large part to their impact-resistant paw pads. The biomechanical study of paw pads may therefore contribute to improving the impact resistance of specific biomimetic materials. The present study is aimed at investigating the mechanics of the paw pads, revealing their impact-resistant biomechanism from macro- and microscopic perspectives. Histological and micro-CT scanning methods were exploited to analyze the microstructure of the pads, and mechanical testing was conducted to observe the macroscopic mechanical properties at different loading frequencies. Numerical micromodels of the ellipsoidal and cylindrical adipose compartments were developed to evaluate the mechanical functionality as compressive actions. The results show that the stiffness of the pad increases roughly in proportion to strain and mechanical properties are almost impervious to strain rate. Furthermore, the adipose compartment, which comprises adipose tissue enclosed within collagen septa, in the subcutaneous tissue presents an ellipsoid-like structure, with a decreasing area from the middle to the two ends. Additionally, the finite element results show that the ellipsoidal structure has larger displacement in the early stage of impact, which can absorb more energy and prevent instability at touchdown, while the cylindrical structure is more resistant to deformation. Moreover, the Von Mises of the ellipsoidal compartment decrease gradually from both ends to the middle, making it change to a cylindrical shape, and this may be the reason why the macroscopic stiffness increases with increasing time after contact. This preliminary investigation represents the basis for biomechanical interpretation and can accordingly provide new inspirations of shock-absorbing composite materials in engineering.

In Vitro Simulation of Tissue Back-Pressure for Pen Injectors and Auto-Injectors.

The aim of this project was to show that tissue back-pressure can be measured in vitro using a simple pneumatic model. A thorough literature study revealed 4 relevant papers all describing in vivo studies. One of these studies where the subcutaneous tissue back-pressure was determined in 11 patients was used as a reference for the present work. A pneumatic model capable of simulating the back-pressure and the diffusion of drug during subcutaneous injection was developed. The in vitro model was tested using the same type of pen injector as used in the reference study. Comparison of the results revealed that the measured pressure in the in vitro experiments was similar to the subcutaneous tissue back-pressure measured in vivo. G30 0.3 × 8.0 mm and G32 0.23/0.25 × 4.0 mm needles were used for the in vitro experiments, whereas a G31 0.25 × 6.0 mm needle was used for the in vivo experiments. This is one possible explanation of approximately 30 µL/s higher flow rates for the in vitro experiments compared to the in vivo experiments. The low-complexity model allows repeated measurements and provides a stable data output paving the way for measuring subcutaneous back-pressure in vitro.

Structural mechanics of 3D-printed poly(lactic acid) scaffolds with tetragonal, hexagonal and wheel-like designs.

While various porous scaffolds have been developed, the focused study about which structure leads to better mechanics is rare. In this study, we designed porous scaffolds with tetragonal, hexagonal and wheel-like structures under a given porosity, and fabricated corresponding poly(lactic acid) (PLA) scaffolds with three-dimensional printing. High-resolution micro-computed tomography was carried out to calculate their experimental porosity and confirm their high interconnectivity. The theoretical and experimental compressive properties in the longitudinal direction were characterized by finite element analysis method and electromechanical universal testing system, respectively. Thereinto, the scaffold with the tetragonal structure exhibited higher mechanical strength both theoretically and experimentally. Creep and stress relaxation behaviors of the scaffolds revealed that the tetragonal scaffold had less significant viscoelasticity. Immersion dynamic mechanical analysis was performed to test their cycle-loading fatigue behaviors in the simulated body fluid at 37 °C; the tetragonal scaffold exhibited the latest fatigue beginning point at 4400 cycles, which indicated a better anti-fatigue performance; the hexagonal and wheel-like ones exhibited the middle and earliest fatigue beginning points at 3200 and 2500 cycles, respectively. What is more, cytocompatibility and histocompatibility of the scaffolds with all of the structures were confirmed by cell counting kit-8 assay in vitro and three-month subcutaneous implantation in rats in vivo. Hence, the key property difference of the three examined structures comes from their mechanics; the tetragonal structure exhibited better mechanics in the longitudinal direction examined in this study, which could be taken into consideration in design of a porous scaffold for tissue engineering and regeneration.

Low-intensity current (LIC) stimulation of subcutaneous adipose derived stem cells (ADSCs) - A missing link in the course of LIC based wound healing.

Millions of people die as a result of fatal injuries accounting for 9% of the total global annual deaths. Non fatal injuries generally result in variety of wounds. The normal wound healing process is slow and takes weeks to months, depending on the type of wound. In last two decades, electrotherapy called low-intensity currents (LIC) for the treatment became popular for faster wound healing, as well as in management of nonresponding and ulcerative wounds. It was reported that LIC mimics 'the current of injury' which is generated by body on wounding and helps in faster wound healing. Researchers have also studied the migration of localized cell and other bio-molecules under the influence of LIC helping the wound to heal faster. Literature review has also suggested that, electrical stimulation of isolated adipose tissue derived stem cells (ADSCs) releases growth factors and differentiates in to specialized cells like fibroblasts and keratinocytes in laboratory conditions. These research areas are well explored and emerged as independent state-of-the-arts therapies and technologies. Considering the fact, that adipose tissue (along with ADSCs) is present subcutaneously, a new hypothesis is proposed which states that 'low intensity current (LIC) stimulation of wound stimulates subcutaneous adipose tissue containing ADSCs which releases different growth factors and also differentiates into certain cells like fibroblasts, neurons and keratinocytes. These cells easily migrate to wound site due to lipolysis and loosening of fat tissue, resulting in faster wound healing'. Thus this hypothesis provides a missing link between two state of the art technologies; first one is 'LIC based electrotherapy' and second one is 'in-vitro LIC stimulation of ADCSs' where role and significance of in-situ ADCSs were never studied.

Structural Gender Dimorphism and the Biomechanics of the Gluteal Subcutaneous Tissue: Implications for the Pathophysiology of Cellulite.

BACKGROUND: This study was performed to investigate gender differences in gluteal subcutaneous architecture and biomechanics to better understand the pathophysiology underlying the mattress-like appearance of cellulite. METHODS: Ten male and 10 female body donors [mean age, 76 ± 16.47 years (range, 36 to 92 years); mean body mass index, 25.27 ± 6.24 kg/m (range, 16.69 to 40.76 kg/m)] were used to generate full-thickness longitudinal and transverse gluteal slices. In the superficial and deep fatty layers, fat lobule number, height, and width were investigated. The force needed to cause septal breakage between the dermis and superficial fascia was measured using biomechanical testing. RESULTS: Increased age was significantly related to decreased dermal thickness, independent of sex (OR, 0.997, 95 percent CI, 0.996 to 0.998; p < 0.0001). The mean number of subdermal fat lobules was significantly higher in male body donors (10.05 ± 2.3) than in female body donors (7.51 ± 2.7; p = 0.003), indicating more septal connections between the superficial fascia and dermis in men. Female sex and increased body mass index were associated with increased height of superficial fat lobules. The force needed to cause septal breakage in male body donors (38.46 ± 26.3 N) was significantly greater than in female body donors (23.26 ± 10.2 N; p = 0.021). CONCLUSIONS: The interplay of dermal support, septal morphology, and underlying fat architecture contributes to the biomechanical properties of the subdermal junction. This is influenced by sex, age, and body mass index. Cellulite can be understood as an imbalance between containment and extrusion forces at the subdermal junction; aged women with high body mass index have the greatest risk of developing (or worsening of) cellulite.

Whole-Tooth Regeneration by Allogeneic Cell Reassociation in Pig Jawbone.

IMPACT STATEMENT: The methods developed in this study to manipulate pig tooth germ cells in vitro and in vivo provide a reference for studying whole-tooth regeneration and tooth development in large animals. Of importance, compared with conventional ectopic tooth regeneration, conducted in the omentum, subcutaneous tissues, or kidney capsule (among other locations) with low with immune reactivity in rodent models, this study achieved orthotopic regeneration and development of whole teeth in a large mammal, representing a large stride toward the realization of tooth regenerative therapy for humans with missing teeth.

Osteochondral Differentiation of Fluorescent Multireporter Cells on Zonally-Organized Biomaterials.

IMPACT STATEMENT: This study represents significant advancement in the use of biomimetic scaffolds to direct zonal osteochondral tissue formation. We describe the use of a novel fluorescent reporter system that enables the real-time evaluation of cellular differentiation in a nondestructive manner. In this study, we use this tool to confirm the osteogenic and chondrogenic capabilities of our scaffold alongside control scaffolds, and use cryohistological methods to probe zone-specific differences in cell and tissue quality. We believe this approach can be widely adopted by others for a variety of biomaterial and cell systems in the development of tissue engineered therapeutics.

Characterization of the Proliferating Layer Chondrocytes of Growth Plate for Cartilage Regeneration.

IMPACT STATEMENT: In recent years, cell-based therapy is a promising strategy for repairing defect cartilage. However, in vitro expansion of articular chondrocytes (ACs) for collecting enough cell numbers eventually develops cell de-differentiation. In the present study, we choose the proliferative layer chondroctytes (PLCs) of growth plate as new candidate. The novel findings include (1) the higher proliferation potential of PLCs in comparison with the ACs, (2) PLCs produced more GAG than ACs, (3) the increased in GAG matrix production, (4) and lower senescence in PLCs. From these results, we found PLCs might be suitable as cell source for cartilage regeneration.

Electron transfer processes occurring on platinum neural stimulating electrodes: pulsing experiments for cathodic-first, charge-imbalanced, biphasic pulses for 0.566 ⩽ k ⩽ 2.3 in rat subcutaneous tissues.

OBJECTIVE: Charge injection through platinum neural stimulation electrodes is often constrained by the Shannon limit (Shannon 1992 IEEE Trans. Biomed. Eng. 39 424-6) of k = 1.75. By leveraging the tools of electrochemistry to better understand the reactions at electrode-tissue interface, we endeavor to find a way to safely inject more charge than allowed if the traditional Shannon limit were followed. APPROACH: In previous studies on platinum electrodes using charge-balanced, cathodic-first, biphasic pulses, we noted that during the secondary anodic phase, the electrode potential moves into a range where platinum dissolution is possible when charge injection is greater than k = 1.75. Platinum dissolution products are known to be toxic to brain tissues. We hypothesize that by injecting less charge in the anodic phase than the cathodic phase, the anodic potential excursions will decrease, thereby avoiding potentials where platinum dissolution is more likely. MAIN RESULTS: Our findings show that using these charge-imbalanced pulses decreases the anodic potential excursions to a level where platinum oxidation and dissolution are less likely, and aligns the anodic potentials with those observed with charge-balanced stimulation at k < 1.75-a range widely accepted as safe for stimulation with platinum. SIGNIFICANCE: From these results, we further hypothesize that charge-imbalanced biphasic stimulation would permit more charge to be safely injected through platinum electrodes than would be permitted if the dogma of charge-balanced biphasic stimuli were followed. Testing this hypothesis in cat brain in the same manner as the experiments that formed the basis for the Shannon plot could open the door for safe charge injection through platinum electrodes at levels greater than k = 1.75.

Prefoldin 6 mediates longevity response from heat shock factor 1 to FOXO in C. elegans.

Heat shock factor 1 (HSF-1) and forkhead box O (FOXO) are key transcription factors that protect cells from various stresses. In Caenorhabditis elegans, HSF-1 and FOXO together promote a long life span when insulin/IGF-1 signaling (IIS) is reduced. However, it remains poorly understood how HSF-1 and FOXO cooperate to confer IIS-mediated longevity. Here, we show that prefoldin 6 (PFD-6), a component of the molecular chaperone prefoldin-like complex, relays longevity response from HSF-1 to FOXO under reduced IIS. We found that PFD-6 was specifically required for reduced IIS-mediated longevity by acting in the intestine and hypodermis. We showed that HSF-1 increased the levels of PFD-6 proteins, which in turn directly bound FOXO and enhanced its transcriptional activity. Our work suggests that the prefoldin-like chaperone complex mediates longevity response from HSF-1 to FOXO to increase the life span in animals with reduced IIS.

Comparison of Two Methods, the Sponge Method and Young's Modulus, for Evaluating Stiffness of Skin or Subcutaneous Tissues in the Extremities of Patients with Lymphedema: A Pilot Study.

The purpose of this study was to compare two methods, the sponge method and SOFTMEASURE, to evaluate the stiffness in lymphedema extremities. We investigated 12 lower extremities of six patients with lymphedema using the methods and compared the results with International Society of Lymphedema classification and lymphoscintigraphic staging. The average age was 53.8 years (43-70 years). We prepared three sponges with different hardness, each of them was numbered as 2, 4, or 6, for the sponge method. We compared the stiffness of the lymphedema extremities and recorded the number of the sponge that was most comparable in stiffness. SOFTMEASURE is a portable device used to measure Young's modulus of an object. Measurement was performed thrice for each extremity. We excluded the most extreme value and the average of the other two values was used for evaluation. The correlation coefficient between SOFTMEASURE and the International Society of Lymphology (ISL) stage was 0.79, and between SOFTMEASURE and the lymphoscintigraphic stage was 0.85 (indentation force of 0.5 N). The correlation coefficient between the sponge method and the ISL stage was 0.57, and between the sponge method and the lymphoscintigraphic stage was 0.54, which indicated an intermediate correlation. In conclusion, evaluation of the stiffness of the skin or subcutaneous tissue in the lymphedema extremities using SOFTMEASURE or the sponge method to assess lymphedema severity was easy and minimally invasive. Sponge method seemed to be more useful than SOFTMEASURE because it had higher correlation with the ISL staging and lymphoscintigraphic staging. Recognizing the change in stiffness is important for determining the therapeutic strategy of lymphedema.

Fabrication of heterogeneous porous bilayered nanofibrous vascular grafts by two-step phase separation technique.

Innterconnected porous architecture is critical for tissue engineering scaffold as well as biomimetic nanofibrous structure. In addition, a paradigm shift is recently taking place in the scaffold design from homogeneous porous scaffold to heterogeneous porous scaffold for the complex tissues. In this study, a versatile and simple one-pot method, dual phase separation, is developed to fabricate macroporous nanofibrous scaffold by phase separating the mixture solutions of immiscible polymer blends without using porogens. The macropores in the scaffold are interconnected, and their size can be tuned by the polymer blend ratio. Moreover, benefiting from the easy operation of dual phase separation technique, an innovative, versatile and facile two-step phase separation method is developed to fabricate heterogeneous porous layered nanofibrous scaffolds with different shapes, such as bilayered tubular scaffold and tri-layered cylindrical scaffold. The bilayered tubular nanofibrous scaffold composed of poly(l-lactic acid) (PLLA)/poly(l-lactide-co-ε-caprolactone) (PLCL) microporous inner layer and PLLA/poly(ε-caprolactone) (PCL) macroporous outer layer matches simultaneously the functional growth of endothelial cells (ECs) and smooth muscle cells (SMCs), and shows the favorable performance for potential small diameter blood vessel application. Therefore, this study provides the novel and facile strategies to fabricate macroporous nanofibrous scaffold and heterogeneous porous layered nanofibrous scaffold for tissue engineering applications. STATEMENT OF SIGNIFICANCE: The fabrication of porous tissue engineering scaffold made of non-water-soluble polymer commonly requires the use of porogen materials. This is complex and time-consuming, resulting in greater difficulty to prepare heterogeneous porous layered scaffold for multifunctional tissues repair, such as blood vessel and osteochondral tissue. Herein, a novel, versatile and simple one-pot dual phase separation technique is developed for the first time to fabricate porous scaffold without using porogens. Simultaneously, it also endows the resultant scaffold with the biomimetic nanofibrous architecture. Based on the easy operation of this dual phase separation technique, a facile two-step phase separation method is also put forward for the first time and applied in fabricating heterogeneous porous layered nanofibrous scaffold for tissue engineering applications.