Improved recovery of human islets from young donor pancreases utilizing increased protease dose to collagenase for digesting peri-islet extracellular matrix.

Human islet isolation from young donor pancreases (YDP) utilizing the current purified standard dose of collagenase-protease enzyme mixtures often results in the release of a high percentage of mantled islets. Mantled islets are those surrounded by exocrine tissue and are difficult to purify by density gradient centrifugation, leading to poor islet recovery. Based on difference in extracellular matrix, and total collagen content between YDP and old donor pancreas (ODP, > 35 Y) led us to compare results from islet isolation using increased collagenase combination (ICC) or increased protease combination (IPC), to the standard enzyme combination (SEC) in a "trisected" pancreas model to overcome the donor-to-donor variability. These results showed a reduced percentage of mantled islets (17% ± 7.5%) and higher postpurification islet recovery (83.8% ± 5.6%) with IPC. Furthermore, these results were confirmed in 13 consecutive whole pancreas islet isolations utilizing IPC from VitaCyte, Roche, or SERVA collagenase-protease enzyme mixtures. Results obtained from in vitro and in vivo islet functional assessment indicated that islets isolated using IPC retained normal islet morphology, insulin secretion, and the ability to reverse diabetes after transplantation in diabetic nude mice. This is the first report utilizing trisected pancreas to assess the effectiveness of different enzyme combinations to improve islet recovery from young donor pancreases.

Beneficial effect of recombinant rC1rC2 collagenases on human islet function: Efficacy of low-dose enzymes on pancreas digestion and yield.

A high number of human islets can be isolated by using modern purified tissue dissociation enzymes; however, this requires the use of >20 Wunsch units (WU)/g of pancreas for digestion. Attempts to reduce this dose have resulted in pancreas underdigestion and poor islet recovery but improved islet function. In this study, we achieved a high number of functional islets using a low dose of recombinant collagenase enzyme mixture (RCEM-1200 WU rC2 and 10 million collagen-degrading activity [CDA] U of rC1 containing about 209 mg of collagenase to digest a 100-g pancreas). The collagenase dose used in these isolations is about 42% of the natural collagenase enzyme mixture (NCEM) dose commonly used to digest a 100-g pancreas. Low-dose RCEM was efficient in digesting entire pancreases to obtain higher yield (5535 ± 830 and 2582 ± 925 islet equivalent/g, P < .05) and less undigested tissue (16.7 ± 5% and 37.8 ± 3%, P < .05) compared with low-dose NCEM (12WU/g). Additionally, low-dose RCEM islets retained better morphology (confirmed with scanning electron microscopy) and higher in vitro basal insulin release (2391 ± 1342 and 1778 ± 978 µU/mL; P < .05) compared with standard-dose NCEM. Nude mouse bioassay demonstrated better islet function for low-dose RCEM (area under the curve [AUC] 24 968) compared with low-dose (AUC-38 225) or standard-dose NCEM (AUC-38 685), P < .05. This is the first report indicating that islet function can be improved by using low-dose rC1rC2 (RCEM).

Adipose-derived cellular therapies in solid organ and vascularized-composite allotransplantation.

PURPOSE OF REVIEW: Controlling acute allograft rejection following vascularized composite allotransplantation requires strict adherence to courses of systemic immunosuppression. Discovering new methods to modulate the alloreactive immune response is essential for widespread application of vascularized composite allotransplantation. Here, we discuss how adipose-derived cellular therapies represent novel treatment options for immune modulation and tolerance induction in vascularized composite allotransplantation. RECENT FINDINGS: Adipose-derived mesenchymal stromal cells are cultured from autologous or allogeneic adipose tissue and possess immunomodulatory qualities capable of prolonging allograft survival in animal models of vascularized composite allotransplantation. Similar immunosuppressive and immunomodulatory effects have been observed with noncultured adipose stromal-vascular-fraction-derived therapies, albeit publication of in-vivo stromal vascular fraction cell modulation in transplantation models is lacking. However, both stromal vascular fraction and adipose derived mesenchymal stem cell therapies have the potential to effectively modulate acute allograft rejection via recruitment and induction of regulatory immune cells. SUMMARY: To date, most reports focus on adipose derived mesenchymal stem cells for immune modulation in transplantation despite their phenotypic plasticity and reliance upon culture expansion. Along with the capacity for immune modulation, the supplemental wound healing and vasculogenic properties of stromal vascular fraction, which are not shared by adipose derived mesenchymal stem cells, hint at the profound therapeutic impact stromal vascular fraction-derived treatments could have on controlling acute allograft rejection and tolerance induction in vascularized composite allotransplantation. Ongoing projects in the next few years will help design the best applications of these well tolerated and effective treatments that should reduce the risk/benefit ratio and allow more patients access to vascularized composite allotransplantation therapy.

Pig islet xenotransplantation.

PURPOSE OF REVIEW: The current article reviews the rationale, sources and preparation of pig islets for xenotransplantation, and presents current progress in solving the problems associated with establishing pig islet transplant as a clinical treatment for type 1 diabetes. SUMMARY: Islet transplantation is an effective treatment option for type 1 diabetes, but the available supply of human pancreases is insufficient to meet the need and demand for obtaining islets. Pig islets provide a readily available source for islet transplantation, with trials in non-human primates demonstrating their potential to reverse diabetes. The risk of zoonosis can be reduced by designated pathogen-free breeding of the donor pigs, but porcine endogenous retroviruses (PERVs) that are integrated into the genome of all pigs are especially difficult to eliminate. However, clinical trials have demonstrated an absence of PERV transmission with a significant reduction in the number of severe hypoglycemic episodes and up to 30% reduction in exogenous insulin doses. A number of methods such as production of various transgenic pigs to better xenotransplantation efficiency and the encapsulation of islets to isolate them from the host immune system are currently being tested to overcome the xenograft immune rejection. Furthermore, ongoing research is also shedding light on factors such as the age and breed of the donor pig to determine the optimal islet quantity and function.

Immunobiology in VCA.

Transplantation of vascularized composite tissue is a relatively new field that is an amalgamation of experience in solid organ transplantation and reconstructive plastic and orthopedic surgery. What is novel about the immunobiology of VCA is the addition of tissues with unique immunologic characteristics such as skin and vascularized bone, and the nature of VCA grafts, with direct exposure to the environment, and external forces of trauma. VCAs are distinguished from solid organ transplants by the requirement of rigorous physical therapy for optimal outcomes and the fact that these procedures are not lifesaving in most cases. In this review, we will discuss the immunobiology of these systems and how the interplay can result in pathology unique to VCA as well as provide potential targets for therapy.

Manipulating the microvasculature and its microenvironment.

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.

Determinants of microvascular network topologies in implanted neovasculatures.

OBJECTIVE: During neovascularization, the end result is a new functional microcirculation composed of a network of mature microvessels with specific topologies. Although 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 prepatterning on the final microvascular network topology using a model of implant neovascularization. METHODS AND RESULTS: We used 3D direct-write bioprinting or physical constraints in a manner permitting postangiogenesis vascular remodeling and adaptation to pattern angiogenic microvascular precursors (neovessels formed from isolated microvessel segments) in 3D collagen gels before implantation and subsequent network formation. Neovasculatures prepatterned into parallel arrays formed functional networks after 4 weeks postimplantation but lost the prepatterned architecture. However, maintenance of uniaxial physical constraints during postangiogenesis remodeling of the implanted neovasculatures produced networks with aligned microvessels, as well as an altered proportional distribution of arterioles, capillaries, and venules. CONCLUSIONS: Here we show that network topology resulting from implanted microvessel precursors is independent from prepatterning of precursors but can be influenced by a patterning stimulus involving tissue deformation during postangiogenesis remodeling and maturation.

Adipose stromal vascular fraction cell construct sustains coronary microvascular function after acute myocardial infarction.

A three-dimensional tissue construct was created using adipose-derived stromal vascular fraction (SVF) cells and evaluated as a microvascular protection treatment in a myocardial infarction (MI) model. This study evaluated coronary blood flow (BF) and global left ventricular function after MI with and without the SVF construct. Fischer-344 rats were separated into four groups: sham operation (sham), MI, MI Vicryl patch (no cells), and MI SVF construct (MI SVF). SVF cells were labeled with green fluorescent protein (GFP). Immediately postinfarct, constructs were implanted onto the epicardium at the site of ischemia. Four weeks postsurgery, the coronary BF reserve was significantly decreased by 67% in the MI group and 75% in the MI Vicryl group compared with the sham group. The coronary BF reserve of the sham and MI SVF groups in the area at risk was not significantly different (sham group: 83 ± 22% and MI SVF group: 57 ± 22%). Griffonia simplicifolia I and GFP-positive SVF immunostaining revealed engrafted SVF cells around microvessels in the infarct region 4 wk postimplant. Overall heart function, specifically ejection fraction, was significantly greater in MI SVF hearts compared with MI and MI Vicryl hearts (MI SVF: 66 ± 4%, MI: 37 ± 8%, and MI Vicryl: 29 ± 6%). In conclusion, adipose-derived SVF cells can be used to construct a novel therapeutic modality for treating microvascular instability and ischemia through implantation on the epicardial surface of the heart. The SVF construct implanted immediately after MI not only maintains heart function but also sustains microvascular perfusion and function in the infarct area by sustaining the coronary BF reserve.

Microvascular repair: post-angiogenesis vascular dynamics.

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, AV 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 to develop effective therapeutic strategies for repairing compromised microcirculations as a means to treat disease.

Tissue engineering solutions to replace contractile function during pediatric heart surgery.

Pediatric heart surgery remains challenging due to the small size of the pediatric heart, the severity of congenital abnormalities and the unique characteristics of each case. New tools and technologies are needed to tackle this enormous challenge. Tissue engineering strategies are focused on fabricating contractile heart muscle, ventricles, Fontan pumps and whole hearts, and a transplantable tissue equivalent has tremendous implications in pediatric heart surgery to provide functional cardiac tissue. This technology will prove to be a game-changer in the field of pediatric heart surgery and provide a novel toolkit for pediatric heart surgeons. This review will provide insight into the potential applications of tissue engineering technologies to replace lost contractile function in pediatric patients with heart abnormalities.

3D bioprinting and its potential impact on cardiac failure treatment: An industry perspective.

3D printing technologies are emerging as a disruptive innovation for the treatment of patients in cardiac failure. The ability to create custom devices, at the point of care, will affect both the diagnosis and treatment of cardiac diseases. The introduction of bioinks containing cells and biomaterials and the development of new computer assisted design and computer assisted manufacturing systems have ushered in a new technology known as 3D bioprinting. Small scale 3D bioprinting has successfully created cardiac tissue microphysiological systems. 3D bioprinting provides an opportunity to evaluate the assembly of specific parts of the heart and most notably heart valves. With the continuous development of instrumentation and bioinks and a complete understanding of cardiac tissue development, it is proposed that 3D bioprinting may permit the assembly of a heart described as a total biofabricated heart.

3D Bioprinting the Cardiac Purkinje System Using Human Adipogenic Mesenchymal Stem Cell Derived Purkinje Cells.

PURPOSE: The objective of this study was to reprogram human adipogenic mesenchymal stem cells (hADMSCs) to form Purkinje cells and to use the reprogrammed Purkinje cells to bioprint Purkinje networks. METHODS: hADMSCs were reprogrammed to form Purkinje cells using a multi-step process using transcription factors ETS2 and MESP1 to first form cardiac progenitor stem cells followed by SHOX2 and TBX3 to form Purkinje cells. A novel bioprinting method was developed based on Pluronic acid as the sacrificial material and type I collagen as the structural material. The reprogrammed Purkinje cells were used in conjunction with the novel bioprinting method to bioprint Purkinje networks. Printed constructs were evaluated for retention of functional protein connexin 40 (Cx40) and ability to undergo membrane potential changes in response to physiologic stimulus. RESULTS: hADMSCs were successfully reprogrammed to form Purkinje cells based on the expression pattern of IRX3, IRX5, SEMA and SCN10. Reprogrammed purkinje cells were incorporated into a collagen type-1 bioink and the left ventricular Purkinje network was printed using anatomical images of the bovine Purkinje system as reference. Optimization studies demonstrated that 1.8 mg/mL type-I collagen at a seeding density of 300,000 cells per 200 µL resulted in the most functional bioprinted Purkinje networks. Furthermore, bioprinted Purkinje networks formed continuous syncytium, retained expression of vital functional gap junction protein Cx40 post-print, and exhibited membrane potential changes in response to electric stimulation and acetylcholine evaluated by DiBAC4(5), an electrically responsive dye. CONCLUSION: Based on the results of this study, hADMSCs were successfully reprogrammed to form Purkinje cells and bioprinted to form Purkinje networks.

Modified Heterotopic Hindlimb Osteomyocutaneous Flap Model in the Rat for Translational Vascularized Composite Allotransplantation Research.

Vascularized composite allotransplantation (VCA) is a relatively new field in the reconstructive surgery. Clinical achievements in human VCA include hand and face transplants and, more recently, abdominal wall, uterus, and urogenital transplants. Functional outcomes have exceeded initial expectations, and most recipients enjoy an improved quality of life. However, as clinical experience accumulates, chronic rejection and complications from the immunosuppression must be addressed. In many cases where grafts have failed, the causative pathology has been ischemic vasculopathy. The biological mechanisms of the acute and chronic rejection associated with VCA, especially ischemic vasculopathy, are important areas of research. However, due to the very small number of VCA patients, the evaluation of proposed mechanisms is better addressed in an experimental model. Multiple groups have used animal models to address some of the relevant unsolved questions in VCA rejection and vasculopathy. Several model designs involving a variety of species are described in the literature. Here we present a reproducible model of VCA heterotopic hindlimb osteomyocutaneous flap in the rat that can be utilized for translational VCA research. This model allows for the serial evaluation of the graft, including biopsies and different imaging modalities, while maintaining a low level of morbidity.

Infrared imaging of lymphatic function in the upper extremity of normal controls and hand transplant recipients via subcutaneous indocyanine green injection.

OBJECTIVES: The goal of this study was to define the parameters of movement of indocyanine green in the upper extremity of normal control and hand transplant recipients. The purpose was to establish a non-invasive method of determining the level of lymphatic function in hand transplant recipients. In hand transplantation (and replantation), the deep lymphatic vessels are rarely repaired, resulting in altered lymphatic connections. In most cases, the relatively rapid inosculation of superficial lymphatic networks and drainage via the venous systems results in sufficient interstitial fluid and lymph drainage of the graft to prevent edema. However, our group and others have determined that some transplant recipients demonstrate chronic edema which is associated with lymphatic stasis. In one case, a patient with chronic edema has developed chronic rejection characterized by thinning of the skin, loss of adnexal structures, and fibrosis and contracture of the hand. METHODS: Lymphatic function was evaluated by intradermal administration of near-infrared fluorescent dye, indocyanine green, and dynamic imaging with an infrared camera system (LUNA). To date, the assessment of lymphatic drainage in the upper extremity by clearance of indocyanine green dye has been studied primarily in oncology patients with abnormal lymphatic function, making assessment of normal drainage problematic. To establish normal parameters, indocyanine green lymphatic clearance functional tests were performed in a series of normal controls, and subsequently compared with indocyanine green clearance in hand transplant recipients. RESULTS: The results demonstrate varied patterns of lymphatic drainage in the hand transplant patients that partially mimic normal hand lymphatic drainage, but also share characteristics of lymphedema patients defined in other studies. The study revealed significant deceleration of the dye drainage in the allograft of a patient with suspected chronic rejection and edema of the graft. Analysis of other hand transplant recipients revealed differing levels of dye deceleration, often localized at the level of surgical anastomosis. CONCLUSION: These studies suggest intradermal injection of indocyanine green and near-infrared imaging may be a useful clinical tool to assess adequacy of lymphatic function in hand transplant recipients.

Ductal Cell Reprogramming to Insulin-Producing Beta-Like Cells as a Potential Beta Cell Replacement Source for Chronic Pancreatitis.

Islet cell auto-transplantation is a novel strategy for maintaining blood glucose levels and improving the quality of life in patients with chronic pancreatitis (CP). Despite the many recent advances associated with this therapy, obtaining a good yield of islet infusate still remains a pressing challenge. Reprogramming technology, by making use of the pancreatic exocrine compartment, can open the possibility of generating novel insulin-producing cells. Several lineage-tracing studies present evidence that exocrine cells undergo dedifferentiation into a progenitor-like state from which they can be manipulated to form insulin-producing cells. This review will present an overview of recent reports that demonstrate the potential of utilizing pancreatic ductal cells (PDCs) for reprogramming into insulin- producing cells, focusing on the recent advances and the conflicting views. A large pool of ductal cells is released along with islets during the human islet isolation process, but these cells are separated from the pure islets during the purification process. By identifying and improving existing ductal cell culture methods and developing a better understanding of mechanisms by which these cells can be manipulated to form hormone-producing islet-like cells, PDCs could prove to be a strong clinical tool in providing an alternative beta cell source, thus helping CP patients maintain their long-term glucose levels.

An automatic algorithm for detecting stent endothelialization from volumetric optical coherence tomography datasets.

Recent research has suggested that endothelialization of vascular stents is crucial to reducing the risk of late stent thrombosis. With a resolution of approximately 10 microm, optical coherence tomography (OCT) may be an appropriate imaging modality for visualizing the vascular response to a stent and measuring the percentage of struts covered with an anti-thrombogenic cellular lining. We developed an image analysis program to locate covered and uncovered stent struts in OCT images of tissue-engineered blood vessels. The struts were found by exploiting the highly reflective and shadowing characteristics of the metallic stent material. Coverage was evaluated by comparing the luminal surface with the depth of the strut reflection. Strut coverage calculations were compared to manual assessment of OCT images and epi-fluorescence analysis of the stented grafts. Based on the manual assessment, the strut identification algorithm operated with a sensitivity of 93% and a specificity of 99%. The strut coverage algorithm was 81% sensitive and 96% specific. The present study indicates that the program can automatically determine percent cellular coverage from volumetric OCT datasets of blood vessel mimics. The program could potentially be extended to assessments of stent endothelialization in native stented arteries.

Vasculogenic and angiogenic potential of adipose stromal vascular fraction cell populations in vitro.

Adipose-derived stromal vascular fraction (SVF) is a heterogeneous cell source that contains endothelial cells, pericytes, smooth muscle cells, stem cells, and other accessory immune and stromal cells. The SVF cell population has been shown to support vasculogenesis in vitro as well vascular maturation in vivo. Matrigel, an extracellular matrix (ECM) mixture has been utilized in vitro to evaluate tube formation of purified endothelial cell systems. We have developed an in vitro system that utilizes freshly isolated SVF and ECM molecules both in pure form (fibrin, laminin, collagen) as well as premixed form (Matrigel) to evaluate endothelial tip cell formation, endothelial stalk elongation, and early stages of branching and inosculation. Freshly isolated SVF rat demonstrate cell aggregation and clustering (presumptive vasculogenesis) on Matrigel ECM within the first 36 h of seeding followed by tip cell formation, stalk cell formation, branching, and inosculation (presumptive angiogenesis) during the subsequent 4 days of culture. Purified ECM molecules (laminin, fibrin, and collagen) promote cell proliferation but do not recapitulate events seen on Matrigel. We have created an in vitro system that provides a functional assay to study the mechanisms of vasculogenesis and angiogenesis in freshly isolated SVF to characterize SVF's blood vessel forming potential prior to clinical implantation.

Endothelial cell regulation through epigenetic mechanisms: Depicting parallels and its clinical application within an intra-islet microenvironment.

The intra-islet endothelial cells (ECs), the building blocks of islet microvasculature, govern a number of cellular and pathophysiological processes associated with the pancreatic tissue. These cells are key to the angiogenic process and essential for islet revascularization after transplantation. Understanding fundamental mechanisms by which ECs regulate the angiogenic process is important as these cells maintain and regulate the intra-islet environment facilitated by a complex signaling crosstalk with the surrounding endocrine cells. In recent years, many studies have demonstrated the impact of epigenetic regulation on islet cell development and function. This review will present an overview of the reports involving endothelial epigenetic mechanisms particularly focusing on histone modifications which have been identified to play a critical role in governing EC functions by modifying the chromatin structure. A better understanding of epigenetic mechanisms by which these cells regulate gene expression and function to orchestrate cellular physiology and pathology is likely to offer improved insights on the functioning and regulation of an intra-islet endothelial microvascular environment.

Microvascular transplantation after acute myocardial infarction.

The primary objective of this study was to evaluate epicardial transplantation of an intact microvascular network for treatment of myocardial ischemia in a murine model of acute myocardial infarction. We describe transplantation of an intact microvascular network constructed from isolated microvascular segments stabilized in a 3-dimensional matrix to the epicardial surface after acute myocardial infarction. This microvascular graft was implanted as a patch on the epicardium of mice after left coronary artery ligation. After 14 and 28 days of implantation, left ventricular (LV) function was assessed and grafts evaluated via histology and cytochemistry. Inosculation of microvessels within the graft with host coronary microcirculation occurred as early as 7 days after initial tissue grafting. Morphologic evaluation of the grafts revealed arterioles, venules, capillaries, and erythrocytes within vascular lumina. Control grafts of collagen alone remained avascular. LV infarct size was smaller, and LV function improved in treated animals. Engraftment of whole microvascular units can be achieved to support cell-assisted vascular remodeling. Microvascular grafts may provide therapeutic benefit as a primary treatment or serve as a microvascular platform for cardiac repair and regeneration.

Characterizing environmental factors that impact the viability of tissue-engineered constructs fabricated by a direct-write bioassembly tool.

Tissue engineering combines the fields of medicine and engineering to build replacement tissue capable of restoring, maintaining, or improving damaged tissue. Researchers have recently developed techniques to fabricate tissue in which both the cells and matrix have a carefully defined architecture. This report details studies evaluating the use of a direct-write, 3-dimensional (3D) bioassembly tool (BAT) capable of extruding cells and matrix into spatially organized, 3D constructs. This system has been characterized by its ability to fabricate viable 2-dimensional and 3D constructs containing up to 2 separate cellular solutions suspended in type I collagen. The effects of various environmental factors, such as extrusion pressure, humidity, and stage heating, were examined with respect to the viability of the extruded cells. The data indicate that the system parameters required to extrude cells suspended in collagen do not adversely affect the viability of those cells. Maintaining a high humidity, especially when stage heat was applied, is critical in maintaining the viability of the printed cells. These results demonstrate that the BAT is capable of spatially organizing separate cellular solutions into a defined architecture; however, when cells were extruded in a supporting matrix of 3.0 mg/mL type I collagen, it was not possible to consistently generate adjacent, touching, but nonoverlapping lines of separate solutions. Thus, when a fabrication system such as BAT is used to generate complex, 3D viable constructs, the supporting matrix for the cells should be carefully chosen on the basis of such characteristics as its rate of polymerization and stiffness.