Diabetes mellitus (diabetes) is a devastating disease that affects millions of people globally and causes a myriad of complications that lead to both patient morbidity and mortality. Currently available therapies, including insulin injection and beta cell replacement through either pancreas or pancreatic islet transplantation, are limited by the availability of organs. Stem cells provide an alternative treatment option for beta cell replacement through selective differentiation of stem cells into cells that recognize glucose and produce and secrete insulin. Embryonic stem cells, albeit pluripotent, face a number of challenges, including ethical and political concerns and potential teratoma formation. Adipose tissue represents an alternative source of multipotent mesenchymal stem cells, which can be obtained using a relatively simple, non-invasive, and inexpensive method. Similarly to other adult mesenchymal stem cells, adipose-derived stem cells (ADSCs) are capable of differentiating into insulin-producing cells. They are also capable of vasculogenesis and angiogenesis, which facilitate engraftment of donor pancreatic islets when co-transplanted. Additionally, anti-inflammatory and immunomodulatory effects of ADSCs can protect donor islets during the early phase of transplantation and subsequently improve engraftment of donor islets into the recipient organ. Although ADSC-therapy is still in its infancy, the potential benefits of ADSCs are far reaching.
Diabetes mellitus; Diabetes; Insulin; Stem cells; Adipose; Pancreas; Beta-cells; Differentiation
The study of tissue function in vitro has been aided by the development of three-dimensional culture systems that more accurately duplicate the complex cell components of tissues and organs. Bioprinting of cells provides a rapid tissue fabrication technique that can be used to evaluate normal and pathologic conditions in vitro as well as to construct complex three-dimensional tissue structures for implantation in regenerative medicine therapies. Studies were performed using a direct write three-dimensional bioprinting system to fabricate adipose-derived stromal vascular fraction cell spheroids. Human fat–derived stromal vascular fraction cells were mixed in 1.5% (w/v) alginate solutions, and fabrication conditions were varied to produce an array of spheroids. The spheroids were placed in spinner culture, and spheroid integrity and encapsulated cell viability were assessed for 16 days. Results establish the ability to tightly control adipose SVF spheroids in the range of 800–1500 μm. Fabrication conditions were used to control spheroid size, and the results illustrate the ability to construct spheroids of precise size and shape. The adipose SVF cell population remains viable and the spheroid integrity was maintained for 16 days in suspension culture. The direct-write printing of adipose stromal vascular fraction cell containing spheroids provides a rapid fabrication technology to support in vitro microphysiologic system studies.
cell culture; stem cells; tissue engineering
The microvasculature is a dynamic cellular system necessary for tissue health and function. Therapeutic strategies that target the microvasculature are expanding and evolving, including those promoting angiogenesis and microvascular expansion. When considering how to manipulate angiogenesis, either as part of a tissue construction approach or a therapy to improve tissue blood flow, it is important to know the microenvironmental factors that regulate and direct neovessel sprouting and growth. Much is known concerning both diffusible and matrix-bound angiogenic factors, which stimulate and guide angiogenic activity. How the other aspects of the extravascular microenvironment, including tissue biomechanics and structure, influence new vessel formation is less well known. Recent research, however, is providing new insights into these mechanisms and demonstrating that the extent and character of angiogenesis (and the resulting new microcirculation) is significantly affected. These observations and the resulting implications with respect to tissue construction and microvascular therapy are addressed.
angiogenesis; microvessels; microvascular orientation; microvascular remodeling; microvessel guidance; three-dimensional (3D) vascular constructs; matrix mechanics
This study investigated the cardiac recovery potential of a stromal vascular fraction (SVF) construct implanted onto an established infarct. Findings suggest that SVF treatment of an established infarct stabilizes the heart at the time point of intervention by preventing a worsening of cardiac performance and infarcted volume and increasing perfusion of the microcirculation.
We have previously shown that myocardial infarction (MI) immediately treated with an epicardial construct containing stromal vascular fraction (SVF) from adipose tissue preserved microvascular function and left ventricle contractile mechanisms. In order to evaluate a more clinically relevant condition, we investigated the cardiac recovery potential of an SVF construct implanted onto an established infarct. SVF cells were isolated from rat adipose tissue, plated on Vicryl, and cultured for 14 days. Fischer-344 rats were separated into MI groups: (a) 6-week MI (MI), (b) 6-week MI treated with an SVF construct at 2 weeks (MI SVF), (c) 6-week MI with Vicryl construct at 2 weeks (MI Vicryl), and (d) MI 2wk (time point of intervention). Emax, an indicator of systolic performance and contractile function, was lower in the MI and MI Vicryl versus MI SVF. Positron emission tomography imaging (18F-fluorodeoxyglucose) revealed a decreased percentage of relative infarct volume in the MI SVF versus MI and MI Vicryl. Total vessel count and percentage of perfusion assessed via immunohistochemistry were both increased in the infarct region of MI SVF versus MI and MI Vicryl. Overall cardiac function, percentage of relative infarct, and percentage of perfusion were similar between MI SVF and MI 2wk; however, total vessel count increased after SVF treatment. These data suggest that SVF treatment of an established infarct stabilizes the heart at the time point of intervention by preventing a worsening of cardiac performance and infarcted volume, and is associated with increased microvessel perfusion in the area of established infarct.
Angiogenesis; Adipose stem cells; Stromal cells; Cardiac; Microvasculature
Vascular compromise and the accompanying perfusion deficits cause or complicate a large array of disease conditions and treatment failures. This has prompted the exploration of therapeutic strategies to repair or regenerate vasculatures thereby establishing more competent microcirculatory beds. Growing evidence indicates that an increase in vessel numbers within a tissue does not necessarily promote an increase in tissue perfusion. Effective regeneration of a microcirculation entails the integration of new stable microvessel segments into the network via neovascularization. Beginning with angiogenesis, neovascularization entails an integrated series of vascular activities leading to the formation of a new mature microcirculation and includes vascular guidance and inosculation, vessel maturation, pruning, arterio-venous specification, network patterning, structural adaptation, intussusception, and microvascular stabilization. While the generation of new vessel segments is necessary to expand a network, without the concomitant neovessel remodeling and adaptation processes intrinsic to microvascular network formation, these additional vessel segments give rise to a dysfunctional microcirculation. While many of the mechanisms regulating angiogenesis have been detailed, a thorough understanding of the mechanisms driving post-angiogenesis activities specific to neovascularization has yet to be fully realized, but is necessary in order to develop effective therapeutic strategies for repairing compromised microcirculations as a means to treat disease.
Regenerative medicine seeks to repair or replace dysfunctional tissues with engineered biological or biohybrid systems. Current clinical regenerative models utilize simple uniform tissue constructs formed with cells cultured onto biocompatible scaffolds. Future regenerative therapies will require the fabrication of complex three-dimensional constructs containing multiple cell types and extracellular matrices. We believe bioprinting technologies will provide a key role in the design and construction of future engineered tissues for cell-based and regenerative therapies. This review describes the current state-of-the-art bioprinting technologies, focusing on direct-write bioprinting. We describe a number of process and device considerations for successful bioprinting of composite biohybrid constructs. In addition, we have provided baseline direct-write printing parameters for a hydrogel system (Pluronic F127) often used in cardiovascular applications. Direct-write dispensed lines (gels with viscosities ranging from 30 mPa*s to greater than 600×106 mPa*s) were measured following mechanical and pneumatic printing via three commercially available needle sizes (20ga, 25ga, and 30ga). Example patterns containing microvascular cells and isolated microvessel fragments were also bioprinted into composite 3D structures. Cells and vessel fragments remained viable and maintained in vitro behavior after incorporation into biohybrid structures. Direct-write bioprinting of biologicals provides a unique method to design and fabricate complex, multi-component 3D structures for experimental use. We hope our design insights and baseline parameter descriptions of direct-write bioprinting will provide a useful foundation for colleagues to incorporate this 3D fabrication method into future regenerative therapies.
Bioprinting; Regenerative Medicine; Tissue Engineering; Hydrogels; Biohybrid Devices
During neovascularization, the end result is a new functional microcirculation comprised of a network of mature microvessels with specific topologies. While much is known concerning the mechanisms underlying the initiation of angiogenesis, it remains unclear how the final architecture of microcirculatory beds is regulated. To begin to address this, we determined the impact of angiogenic neovessel pre-patterning on the final microvascular network topology using an implant model of implant neovascularization.
Methods and Results
To test this, we used 3-D direct-write bioprinting or physical constraints in a manner permitting post-angiogenesis vascular remodeling and adaptation to pattern angiogenic microvascular precursors (neovessels formed from isolated microvessel segments) in 3-dimensional collagen gels prior to implantation and subsequent network formation. Neovasculatures pre-patterned into parallel arrays formed functional networks following 4 weeks post-implantation, but lost the pre-patterned architecture. However, maintenance of uniaxial physical constraints during post-angiogenesis remodeling of the implanted neovasculatures produced networks with aligned microvessels as well as an altered proportional distribution of arterioles, capillaries and venules.
Here we show that network topology resulting from implanted microvessel precursors is independent from pre-patterning of precursors but can be influenced by a patterning stimulus involving tissue deformation during post-angiogenesis remodeling and maturation.
microcirculation; regeneration; bioprinting; vascular engineering; neovascularization
We have developed a 2.1 mm outer diameter optical coherence tomography endoscope that provides radial scans of luminal structures. The endoscope consists of three elements: (1) a stationary central core containing the fibers and focusing elements, (2) a rotating intermediate tube with a distal rod prism, and (3) a stationary sterilized glass cover. This design enables radial and spiral scanning and allows adjustment of the axial focal distance. Additionally, this design is capable of focusing light from multiple fibers into tissue. The performance of the endoscope was demonstrated in a study of tissue engineered blood vessels imaged at various time points during development.
Development of a small diameter (<6 mm) synthetic vascular graft with clinically acceptable patency must overcome the inherent thrombogenicity of polymers and the development of neointimal thickening. Establishment of an endothelial cell lining on the lumenal surface has been hypothesized as a mechanism to improve the function of vascular grafts. The major aim of this study was to evaluate the use of laminin type 1, covalently bound to all surfaces of ePTFE grafts on neovascularization of the interstices and lumenal surface endothelialization.
One mm i.d. vascular grafts were surface modified through covalent attachment of laminin type 1. Grafts were subsequently implanted as interpositional aortic grafts in rats. Following five weeks implantation the grafts were explanted and morphologically evaluated using scanning electron microscopy and light microscopy.
Scanning electron microscopy identified an extensive coverage of antithrombogenic cells on the lumenal flow surface of laminin type 1 modified grafts. Histological evaluation confirmed the presence of endothelial cells on the mid-graft lumenal surface of laminin 1 modified grafts. Extensive neovascularization of the interstices of the laminin modified grafts occurred as compared to control grafts. We conclude that surface modification using laminin type 1 accelerates both the neovascularization and endothelialization of porous ePTFE vascular grafts.
ePTFE; vascular graft; endothelial cells; extracellular matrix; laminin
During the typical healing response to an implanted biomaterial, vascular-rich granulation tissue forms around the implant and later resolves into a relatively avascular, fibrous capsule. We have previously shown that a microvascular construct (MVC) consisting of isolated microvessel fragments suspended in a collagen I gel forms a persistent microcirculation in lieu of avascular scar when implanted. The current study evaluated the potential for microvascular constructs to maintain a vascularized tissue environment around an implanted biomaterial. An analysis of the peri-implant tissue around bare expanded polytetrafluoroethylene (ePTFE), ePTFE embedded within a microvascular construct, or ePTFE embedded within collagen alone revealed that the presence of the MVC, but not collagen alone, promoted vascular densities comparable to that of the granulation tissue formed around bare ePTFE. The vessels within the microvascular construct surrounding the ePTFE were perfusion competent, as determined by India ink perfusion casting, and extended into the interstices of the polymer. In contrast to bare ePTFE, the presence of the MVC or collagen alone significantly reduced the number of activated macrophages in association with ePTFE. Similar results were observed for ePTFE modified to increase cellularity and prevent the formation of an avascular scar. The microvascular construct may prove effective in forming vascularized tissue environments and limiting the number of activated macrophages around implanted polymers thereby leading to effective implant incorporation.
granulation tissue; fibrous capsule; structural adaptation; microvascular networks
Effective tissue prevascularization depends on new vessel growth and subsequent progression of neovessels into a stable microcirculation. Isolated microvessel fragments in a collagen-based microvascular construct (MVC) spontaneously undergo angiogenesis in static conditions in vitro but form a new microcirculation only when implanted in vivo. We have designed a bioreactor, the dynamic in vitro perfusion (DIP) chamber, to culture MVCs in vitro with perfusion. By altering bioreactor circulation, microvessel fragments in the DIP chamber either maintained stable, nonsprouting, patent vessel morphologies or sprouted endothelial neovessels that extended out into the surrounding collagen matrix (i.e., angiogenesis), yielding networks of neovessels within the MVC. Neovessels formed in regions of the construct predicted by simulation models to have the steepest gradients in oxygen levels and expressed hypoxia inducible factor-1α. By altering circulation conditions in the DIP chamber, we can control, possibly by modulating hypoxic stress, prevascularizing activity in vitro.