Fibroblast-Generated Extracellular Matrix Guides Anastomosis during Wound Healing in an Engineered Lymphatic Skin Flap.

A healthy lymphatic system is required to return excess interstitial fluid back to the venous circulation. However, up to 49% of breast cancer survivors eventually develop breast cancer-related lymphedema due to lymphatic injuries from lymph node dissections or biopsies performed to treat cancer. While early-stage lymphedema can be ameliorated by manual lymph drainage, no cure exists for late-stage lymphedema when lymph vessels become completely dysfunctional. A viable late-stage treatment is the autotransplantation of functional lymphatic vessels. Here we report on a novel engineered lymphatic flap that may eventually replace the skin flaps used in vascularized lymph vessel transfers. The engineered flap mimics the lymphatic and dermal compartments of the skin by guiding multi-layered tissue organization of mesenchymal stem cells and lymphatic endothelial cells with an aligned decellularized fibroblast matrix. The construct was tested in a novel bilayered wound healing model and implanted into athymic nude rats. The in vitro model demonstrated capillary invasion into the wound gaps and deposition of extracellular matrix fibers, which may guide anastomosis and vascular integration of the graft during wound healing. The construct successfully anastomosed in vivo, forming chimeric vessels of human and rat cells. Overall, our flap replacement has high potential for treating lymphedema.

Impact of spatial and temporal stability of flow vortices on vascular endothelial cells.

PURPOSE: Intracranial aneurysms (IAs) are pathological dilations of cerebrovascular vessels due to degeneration of the mechanical strength of the arterial wall, precluded by altered cellular functionality. The presence of swirling hemodynamic flow (vortices) is known to alter vascular endothelial cell (EC) morphology and protein expression indicative of IAs. Unfortunately, less is known if vortices with varied spatial and temporal stability lead to differing levels of EC change. The aim of this work is to investigate vortices of varying spatial and temporal stability impact on ECs. METHODS: Vortex and EC interplay was investigated by a novel combination of parallel plate flow chamber (PPFC) design and computational analysis. ECs were exposed to laminar (7.5 dynes/[Formula: see text] wall shear stress) or low (<1 dynes/[Formula: see text]) stress vortical flow using PPFCs. Immunofluorescent imaging analyzed EC morphology, while ELISA tests quantified VE-cadherin (cell-cell adhesion), VCAM-1 (macrophage-EC adhesion), and cleaved caspase-3 (apoptotic signal) expression. PPFC flow was simulated, and vortex stability was calculated via the temporally averaged degree of (volume) overlap (TA-DVO) of vortices within a given area. RESULTS: EC morphological changes were independent of vortex stability. Increased stability promoted VE-cadherin degradation (correlation coefficient r = [Formula: see text]0.84) and 5-fold increased cleaved caspase-3 post 24 h in stable (TA-DVO 0.736 ± 0.05) vs unstable (TA-DVO 0.606 [Formula: see text]0.2) vortices. ECs in stable vortices displayed a 4.5-fold VCAM-1 increase than unstable counterparts after 12 h. CONCLUSION: This work demonstrates highly stable disturbed flow imparts increased inflammatory signaling, degraded cell-cell adhesion, and increased cellular apoptosis than unstable vortices. Such knowledge offers novel insight toward understanding IA development and rupture.

Detergent-Based Decellularization for Anisotropic Cardiac-Specific Extracellular Matrix Scaffold Generation.

Cell-derived extracellular matrix (ECM) has become increasingly popular in tissue engineering applications due to its ability to provide tailored signals for desirable cellular responses. Anisotropic cardiac-specific ECM scaffold decellularized from human induced pluripotent stem cell (hiPSC)-derived cardiac fibroblasts (hiPSC-CFs) mimics the native cardiac microenvironment and provides essential biochemical and signaling cues to hiPSC-derived cardiomyocytes (hiPSC-CMs). The objective of this study was to assess the efficacy of two detergent-based decellularization methods: (1) a combination of ethylenediaminetetraacetic acid and sodium dodecyl sulfate (EDTA + SDS) and (2) a combination of sodium deoxycholate and deoxyribonuclease (SD + DNase), in preserving the composition and bioactive substances within the aligned ECM scaffold while maximumly removing cellular components. The decellularization effects were evaluated by characterizing the ECM morphology, quantifying key structural biomacromolecules, and measuring preserved growth factors. Results showed that both treatments met the standard of cell removal (less than 50 ng/mg ECM dry weight) and substantially preserved major ECM biomacromolecules and growth factors. The EDTA + SDS treatment was more time-efficient and has been determined to be a more efficient method for generating an anisotropic ECM scaffold from aligned hiPSC-CFs. Moreover, this cardiac-specific ECM has demonstrated effectiveness in supporting the alignment of hiPSC-CMs and their expression of mature structural and functional proteins in in vitro cultures, which is crucial for cardiac tissue engineering.

Fabrication of a Completely Biological and Anisotropic Human Mesenchymal Stem Cell-Based Vascular Graft.

Tissue-engineered small-diameter vascular grafts are required to match mechanical properties as well as cellular and extracellular architecture of native blood vessels. Although various engineering technologies have been developed, the most reliable strategy highlights the needs for incorporating completely biological components and anisotropic cellular and biomolecular organization into the tissue-engineered vascular graft (TEVG). Based on the antithrombogenic, immunoregulatory, and regenerative properties of human mesenchymal stem cells (hMSCs), this chapter provides a step-by-step protocol for generating a completely biological and anisotropic TEVG that comprises of hMSCs and highly aligned extracellular matrix (ECM) nanofibers. The hMSCs were grown on an aligned nanofibrous ECM scaffold derived from an oriented human dermal fibroblast (hDF) sheet and then wrapped around a temporary mandrel to form a tubular assembly, followed by a maturation process in a rotating wall vessel (RWV) bioreactor. The resulting TEVG demonstrates anisotropic structural and mechanical properties similar to that of native blood vessels. A completely biological, anisotropic, and mechanically strong TEVG that incorporates immunoregulatory hMSCs is promising to meet the urgent needs of a surgical intervention for bypass grafting.

Enhancement of Lymphangiogenesis by Human Mesenchymal Stem Cell Sheet.

Preparation of human mesenchymal stem cell (hMSC) suspension for lymphedema treatment relies on conventional enzymatic digestion methods, which severely disrupts cell-cell and cell-extracellular matrix (ECM) connections, and drastically impairs cell retention and engraftment after transplantation. The objective of the present study is to evaluate the ability of hMSC-secreted ECM to augment lymphangiogenesis by using an in vitro coculturing model of hMSC sheets with lymphatic endothelial cells (LECs) and an in vivo mouse tail lymphedema model. Results demonstrate that the hMSC-secreted ECM augments the formation of lymphatic capillary-like structure by a factor of 1.2-3.6 relative to the hMSC control group, by serving as a prolymphangiogenic growth factor reservoir and facilitating cell regenerative activities. hMSC-derived ECM enhances MMP-2 mediated matrix remodeling, increases the synthesis of collagen IV and laminin, and promotes lymphatic microvessel-like structure formation. The injection of rat MSC sheet fragments into a mouse tail lymphedema model confirms the benefits of the hMSC-derived ECM by stimulating lymphangiogenesis and wound closure.

Engineering the Lymphatic Network: A Solution to Lymphedema.

Secondary lymphedema is a life-long disorder characterized by chronic tissue swelling and inflammation that obstruct interstitial fluid circulation and immune cell trafficking. Regenerating lymphatic vasculatures using various strategies represents a promising treatment for lymphedema. Growth factor injection and gene delivery have been developed to stimulate lymphangiogenesis and augment interstitial fluid resorption. Using bioengineered materials as growth factor delivery vehicles allows for a more precisely targeted lymphangiogenic activation within the injured site. The implantation of prevascularized lymphatic tissue also promotes in situ lymphatic capillary network formation. The engineering of larger scale lymphatic tissues, including lymphatic collecting vessels and lymph nodes constructed by bioengineered scaffolds or decellularized animal tissues, offers alternatives to reconnecting damaged lymphatic vessels and restoring lymph circulation. These approaches provide lymphatic vascular grafting materials to reimpose lymphatic continuity across the site of injury, without creating secondary injuries at donor sites. The present work reviews molecular mechanisms mediating lymphatic system development, approaches to promoting lymphatic network regeneration, and strategies for engineering lymphatic tissues, including lymphatic capillaries, collecting vessels, and nodes. Challenges of advanced translational applications are also discussed.

Preservation of microvascular integrity and immunomodulatory property of prevascularized human mesenchymal stem cell sheets.

Prevascularization is essential to ensure the viability, functionality, and successful integration of tissue-engineered three-dimensional (3D) constructs with surrounding host tissues after transplantation. Human mesenchymal stem cell (hMSC) sheet can be prevascularized by coculturing with endothelial cells (ECs), and then be further used as building blocks for engineering 3D complex tissues. In addition, predifferentiation of hMSCs into a tissue-specific lineage in vitro has been proven to promote graft engraftment and regeneration. However, it is unclear if the prevascularized hMSC sheets can still maintain their microvascular integrity as well as the immune-regulatory properties after their tissue-specific differentiation. The objective of this study was to investigate the effects of differentiation cues on the microvascular structure, angiogenic factor secretion, and immunogenic responses of prevascularized hMSC sheets. The results showed that upon coculturing with ECs, hMSC sheets successfully formed microvascular network, while maintaining hMSCs' multi-lineage differentiation capability. The next step, osteogenic and adipogenic induction, damaged the preformed microvascular structures and compromised the angiogenic factor secretion ability of hMSCs. Nonetheless, this effect was mitigated by adjusting the concentration of differentiation factors. The subcutaneous transplantation in an immunocompetent rat model demonstrated that the osteogenic differentiated prevascularized hMSC sheet preserved its microvascular structure and immunomodulatory properties comparable to the undifferentiated prevascularized hMSC sheets. This study suggested that a balanced and optimal differentiation condition can effectively promote the tissue-specific predifferentiation of prevascularized hMSC sheet while maintaining its immunomodulatory and tissue integration properties.

Upgrading prevascularization in tissue engineering: A review of strategies for promoting highly organized microvascular network formation.

Functional and perfusable vascular network formation is critical to ensure the long-term survival and functionality of engineered tissues after their transplantation. Although several vascularization strategies have been reviewed in past, the significance of microvessel organization in three-dimensional (3D) scaffolds has been largely ignored. Advances in high-resolution microscopy and image processing have revealed that the majority of tissues including cardiac, skeletal muscle, bone, and skin contain highly organized microvessels that orient themselves to align with tissue architecture for optimum molecular exchange and functional performance. Here, we review strategies to develop highly organized and mature vascular networks in engineered tissues, with a focus on electromechanical stimulation, surface topography, micro scaffolding, surface-patterning, microfluidics and 3D printing. This review will provide researchers with state of the art approaches to engineer vascularized functional tissues for diverse applications. STATEMENT OF SIGNIFICANCE: Vascularization is one of the critical challenges facing tissue engineering. Recent technological advances have enabled researchers to develop microvascular networks in engineered tissues. Although far from translational applications, current vascularization strategies have shown promising outcomes. This review emphasizes the most recent technological advances and future challenges for developing organized microvascular networks in vitro. The next critical step is to achieve highly perfusable, dense, mature and organized microvascular networks representative of native tissues.

Engineering stem cell cardiac patch with microvascular features representative of native myocardium.

The natural myocardium is a highly aligned tissue with an oriented vasculature. Its characteristic cellular as well as nanoscale extracellular matrix (ECM) organization along with an oriented vascular network ensures appropriate blood supply and functional performance. Although significant efforts have been made to develop anisotropic cardiac structure, currently neither an ideal biomaterial nor an effective vascularization strategy to engineer oriented and high-density capillary-like microvessels has been achieved for clinical cardiovascular therapies. A naturally derived oriented ECM nanofibrous scaffold mimics the physiological structure and components of tissue ECM and guides neovascular network formation. The objective of this study was to create an oriented and dense microvessel network with physiological myocardial microvascular features. METHODS: Highly aligned decellularized human dermal fibroblast sheets were used as ECM scaffold to regulate physiological alignment of microvascular networks by co-culturing human mesenchymal stem cells (hMSCs) and endothelial cells (ECs). The influence of topographical features on hMSC and EC interaction was investigated to understand underlying mechanisms of neovasculature formation. RESULTS: Results demonstrate that the ECM topography can be translated to ECs via CD166 tracks and significantly improved hMSC-EC crosstalk and vascular network formation. The aligned ECM nanofibers enhanced structure, length, and density of microvascular networks compared to randomly organized nanofibrous ECM. Moreover, hMSC-EC co-culture promoted secretion of pro-angiogenic growth factors and matrix remodeling via metalloprotease-2 (MMP-2) activation, which resulted in highly dense vascular network formation with intercapillary distance (20 µm) similar to the native myocardium. CONCLUSION: HMSC-EC co-culture on the highly aligned ECM generates physiologically oriented and dense microvascular network, which holds great potential for cardiac tissue engineering.

In situ synthesis of biocompatible imidazolium salt hydrogels with antimicrobial activity.

Infection with antibiotic-resistant bacteria is becoming a significant public health risk. In this study, we synthesized a series of imidazolium salt (IMS)-containing polymers and hydrogels and tested their antimicrobial properties against both gram-positive (Staphylococcus aureus and MRSA) and gram-negative (Escherichia coli and PA01) bacteria. IMSs were either grafted as side chains or functionalized in the main chain of linear polymers, which demonstrated antimicrobial properties with minimum inhibitory concentrations as low as 2 µg/mL. Similarly, the optimized IMS-containing hydrogel effectively killed MRSA with a 96.1% killing efficiency and inhibited the growth of PA01. These hydrogels also demonstrated high performance in terms of mechanical property (compressive strength >2 MPa) and were noncytotoxic toward human dermal fibroblasts. STATEMENT OF SIGNIFICANCE: A series of polyimidazolium hydrogels were fabricated with acrylamide monomer and poly(ethylene glycol) dimethacrylate by thermal-initiated polymerization. These hydrogels completely killed methicillin-resistant Staphylococcus aureus and inhibited the growth of Pseudomonas aeruginosa. More importantly, these hydrogels demonstrated adequate mechanical property and biocompatibility. These antimicrobial hydrogels have the potential as biomaterials for preventing infections associated with multidrug-resistant bacteria.

Polydopamine and collagen coated micro-grated polydimethylsiloxane for human mesenchymal stem cell culture.

Natural tissues contain highly organized cellular architecture. One of the major challenges in tissue engineering is to develop engineered tissue constructs that promote cellular growth in physiological directionality. To address this issue, micro-patterned polydimethylsiloxane (PDMS) substrates have been widely used in cell sheet engineering due to their low microfabrication cost, higher stability, excellent biocompatibility, and most importantly, ability to guide cellular growth and patterning. However, the current methods for PDMS surface modification either require a complicated procedure or generate a non-uniform surface coating, leading to the production of poor-quality cell layers. A simple and efficient surface coating method is critically needed to improve the uniformity and quality of the generated cell layers. Herein, a fast, simple and inexpensive surface coating method was analyzed for its ability to uniformly coat polydopamine (PD) with or without collagen on micro-grated PDMS substrates without altering essential surface topographical features. Topographical feature, stiffness and cytotoxicity of these PD and/or collagen based surface coatings were further analyzed. Results showed that the PD-based coating method facilitated aligned and uniform cell growth, therefore holds great promise for cell sheet engineering as well as completely biological tissue biomanufacturing.

Bioactive polydimethylsiloxane surface for optimal human mesenchymal stem cell sheet culture.

Human mesenchymal stem cell (hMSC) sheets hold great potential in engineering three-dimensional (3D) completely biological tissues for diverse applications. Conventional cell sheet culturing methods employing thermoresponsive surfaces are cost ineffective, and rely heavily on available facilities. In this study, a cost-effective method of layer-by-layer grafting was utilized for covalently binding a homogenous collagen I layer on a commonly used polydimethylsiloxane (PDMS) substrate surface in order to improve its cell adhesion as well as the uniformity of the resulting hMSC cell sheet. Results showed that a homogenous collagen I layer was obtained via this grafting method, which improved hMSC adhesion and attachment through reliable collagen I binding sites. By utilizing this low-cost method, a uniform hMSC sheet was generated. This technology potentially allows for mass production of hMSC sheets to fulfill the demand of thick hMSC constructs for tissue engineering and biomanufacturing applications.

Tissue Engineering at the Blood-Contacting Surface: A Review of Challenges and Strategies in Vascular Graft Development.

Tissue engineered vascular grafts (TEVGs) are beginning to achieve clinical success and hold promise as a source of grafting material when donor grafts are unsuitable or unavailable. Significant technological advances have generated small-diameter TEVGs that are mechanically stable and promote functional remodeling by regenerating host cells. However, developing a biocompatible blood-contacting surface remains a major challenge. The TEVG luminal surface must avoid negative inflammatory responses and thrombogenesis immediately upon implantation and promote endothelialization. The surface has therefore become a primary focus for research and development efforts. The current state of TEVGs is herein reviewed with an emphasis on the blood-contacting surface. General vascular physiology and developmental challenges and strategies are briefly described, followed by an overview of the materials currently employed in TEVGs. The use of biodegradable materials and stem cells requires careful control of graft composition, degradation behavior, and cell recruitment ability to ensure that a physiologically relevant vessel structure is ultimately achieved. The establishment of a stable monolayer of endothelial cells and the quiescence of smooth muscle cells are critical to the maintenance of patency. Several strategies to modify blood-contacting surfaces to resist thrombosis and control cellular recruitment are reviewed, including coatings of biomimetic peptides and heparin.

Natural Extracellular Matrix for Cellular and Tissue Biomanufacturing.

Natural extracellular matrices (ECM) derived from native tissues or cultured cells are extensively employed to fabricate biocompatible scaffolds or living tissue constructs for the application in cellular and tissue engineering. The composition and structure of ECM are not only heterogeneous, but also tissue or cell specific. Recapitulating the unique cell or tissue niche, ECM-based products are promising to quickly integrate with host tissues and accelerate restoration of tissue function. A variety of natural ECM-based scaffolds and tissue constructs have been biomanufactured using different approaches. Native tissue derived ECM is typically grounded into powders that can be further processed into hybrid composites in the form of hydrogels, foams, nanofibers, and 3D-printed complex constructs. Cell-derived ECM follows different biomanufacturing methods. Usually, cells are seeded on a scaffold to deposit ECM resulting in ECM-ornamented materials. The employment of resolvable scaffolds and cell sheet engineering technique enables production of complex 3D constructs exclusively composed of ECM with/without cells. In order to enhance mechanical strength, in vivo stability, and biological performance of ECM-based products, cross-linking reagents or bioactive factors are often used for modification. The major focus of this article is to provide an overview of current biomanufacturing approaches that utilize either native tissue or cell-derived natural ECM in the field of cellular and tissue engineering. Furthermore, the existing challenges for translational application of ECM-based products and the potential resolutions are discussed.

Osteogenic differentiation of MC3T3-E1 cells on poly(L-lactide)/Fe3O4 nanofibers with static magnetic field exposure.

Proliferation and differentiation of bone-related cells are modulated by many factors such as scaffold design, growth factor, dynamic culture system, and physical simulation. Nanofibrous structure and moderate-intensity (1 mT-1 T) static magnetic field (SMF) have been identified as capable of stimulating proliferation and differentiation of osteoblasts. Herein, magnetic nanofibers were prepared by electrospinning mixture solutions of poly(L-lactide) (PLLA) and ferromagnetic Fe3O4 nanoparticles (NPs). The PLLA/Fe3O4 composite nanofibers demonstrated homogeneous dispersion of Fe3O4 NPs, and their magnetism depended on the contents of Fe3O4 NPs. SMF of 100 mT was applied in the culture of MC3T3-E1 osteoblasts on pure PLLA and PLLA/Fe3O4 composite nanofibers for the purpose of studying the effect of SMF on osteogenic differentiation of osteoblastic cells on magnetic nanofibrous scaffolds. On non-magnetic PLLA nanofibers, the application of external SMF could enhance the proliferation and osteogenic differentiation of MC3T3-E1 cells. In comparison with pure PLLA nanofibers, the incorporation of Fe3O4 NPs could also promote the proliferation and osteogenic differentiation of MC3T3-E1 cells in the absence or presence of external SMF. The marriage of magnetic nanofibers and external SMF was found most effective in accelerating every aspect of biological behaviors of MC3T3-E1 osteoblasts. The findings demonstrated that the magnetic feature of substrate and microenvironment were applicable ways in regulating osteogenesis in bone tissue engineering.

Ultrasound guided neural stem cell transplantation through the lateral ventricle for treatment of cerebral palsy in children.

A total of 24 children with cerebral palsy were enrolled in this study and underwent ultrasound guided transplantation of neural stem cells through the lateral ventricle. Neural stem cells (3.8 × 10(6)-7.3 × 10(7)) were injected into the lateral ventricles. Mild injury of lateral ventricular blood vessels occurred in only two cases (8.3%). Seven cases (29.2%) experienced a fever. Clinical manifestations were improved to varying degrees in eight cases (28.0%) within 3 months after transplantation. Patient condition did not worsen, and no patient experienced severe adverse reactions.