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Laminin-α4 (LAMA4) is an extracellular matrix protein implicated in the regulation of adipocyte differentiation and function. Prior research describes a role for LAMA4 in modulating adipocyte thermogenesis and uncoupling protein-1 (UCP1) expression in white adipose; however, the mechanisms involved are poorly understood. Here, we describe that Lama4 knockout mice (Lama4−/−) exhibit heightened mitochondrial biogenesis and peroxisome proliferator-activated receptor γ coactivator-1 (PGC-1) expression in subcutaneous white adipose tissue (sWAT). Furthermore, the acute silencing of LAMA4 with small interfering RNA in primary murine adipocytes was sufficient to upregulate the expression of thermogenic markers UCP1 and PR domain containing 16 (PRDM16). Silencing also resulted in an upregulation of PGC1-α and adenosine 5′-monophosphate–activated protein kinase (AMPK)-α expression. Subsequently, we show that integrin-linked kinase (ILK) is downregulated in the sWAT of Lama4−/− mice, and its silencing in adipocytes similarly resulted in elevated expression of UCP1 and AMPKα. Last, we demonstrate that treatment of human induced pluripotent stem cell–derived thermogenic adipocytes with LAMA4 (LN411) inhibited the expression of thermogenic markers and AMPKα. Overall, our results indicate that LAMA4 negatively regulates a thermogenic phenotype and pathways involving mitochondrial biogenesis in adipocytes through the suppression of AMPKα.

Obesity and the metabolic disease epidemic has led to an increase in morbidity and mortality. A rise in adipose thermogenic capacity via activation of brown or beige fat is a potential treatment for metabolic diseases. However, an understanding of how local factors control adipocyte fate is limited. Mice with a null mutation in the laminin α4 (LAMA4) gene (KO) exhibit resistance to obesity and enhanced expression of thermogenic fat markers in white adipose tissue (WAT). In this study, changes in WAT extracellular matrix composition in the absence of LAMA4 were evaluated using liquid chromatography/tandem mass spectrometry. KO-mice showed lower levels of collagen 1A1 and 3A1, and integrins α7 (ITA7) and β1 (ITB1). ITA7-ITB1 and collagen 1A1-3A1 protein levels were lower in brown adipose tissue compared to WAT in wild-type mice. Immunohistochemical staining confirmed lower levels and different spatial distribution of ITA7 in KO-WAT. In culture studies, ITA7 and LAMA4 levels decreased following a 12-day differentiation of adipose-derived stem cells into beige fat, and knock-down of ITA7 during differentiation increased beiging. These results demonstrate that extracellular matrix interactions regulate adipocyte thermogenic capacity and that ITA7 plays a role in beige adipose formation. A better understanding of the mechanisms underlying these interactions can be used to improve systemic energy metabolism and glucose homeostasis.

An accurate in vitro model of human adipose tissue could assist in the study of adipocyte function and allow for better tools for screening new therapeutic compounds. Cell culture models on two-dimensional surfaces fall short of mimicking the three-dimensional in vivo adipose environment, while three-dimensional culture models are often unable to support long-term cell culture due, in part, to insufficient mass transport. Microfluidic systems have been explored for adipose tissue models. However, current systems have primarily focused on 2D cultured adipocytes. In this work, a 3D human adipose microtissue was engineered within a microfluidic system. Human adipose-derived stem cells (ADSCs) were used as the cell source for generating differentiated adipocytes. The ADSCs differentiated within the microfluidic system formed a dense lipid-loaded mass with the expression of adipose tissue genetic markers. Engineered adipose tissue showed a decreased adiponectin secretion and increased free fatty acid secretion with increasing shear stress. Adipogenesis markers were downregulated with increasing shear stress. Overall, this microfluidic system enables the on-chip differentiation and development of a functional 3D human adipose microtissue supported by the interstitial flow. This system could potentially serve as a platform for in vitro drug testing for adipose tissue-related diseases.

Advances in the engineering of comprehensive skeletal muscle models in vitro will improve drug screening platforms and can lead to better therapeutic approaches for the treatment of skeletal muscle injuries. To this end, a vascularized tissue-engineered skeletal muscle (TE-SkM) model that includes adipocytes was developed to better emulate the intramuscular adipose tissue that is observed in skeletal muscles of patients with diseases such as diabetes. Muscle precursor cells cultured with and without microvessels derived from adipose tissue (microvascular fragments) were used to generate TE-SkM constructs, with and without a microvasculature, respectively. TE-SkM constructs were treated with adipogenic induction media to induce varying levels of adipogenesis. With a delayed addition of induction media to allow for angiogenesis, a robust microvasculature in conjunction with an increased content of adipocytes was achieved. The augmentation of vascularized TE-SkM constructs with adipocytes caused a reduction in maturation (compaction), mechanical integrity (Young's modulus), and myotube and vessel alignment. An increase in basal glucose uptake was observed in both levels of adipogenic induction, and a diminished insulin-stimulated glucose uptake was associated with the higher level of adipogenic differentiation and the greater number of adipocytes. IMPACT STATEMENT: The findings of the current study represent the effectiveness of employing a combinatorial approach involving muscle precursor cells and microvascular fragments to create a vascularized tissue-engineered skeletal muscle model with adipocytes that induce structural and metabolic changes. This model is a platform to support the discovery of mechanisms underlying the phenomena of intramuscular adipose tissue that is characteristic of the skeletal muscles of patients with diseases.

Cancer is a heterogeneous and complex disease. Traditional cancer therapy is associated with low therapeutic index, acquired resistance, and various adverse effects. With the increasing understanding of cancer biology and technology advancements, more strategies have been exploited to optimize the therapeutic outcomes. The rapid development and application of nanomedicine have motivated this progress. Combinational regimen, for instance, has become an indispensable approach for effective cancer treatment, including the combination of chemotherapeutic agents, chemo-energy, chemo-gene, chemo-small molecules, and chemo-immunology. Additionally, smart nanoplatforms that respond to external stimuli (such as light, temperature, ultrasound, and magnetic field), and/or to internal stimuli (such as changes in pH, enzymes, hypoxia, and redox) have been extensively investigated to improve precision therapy. Smart nanoplatforms for combinational therapy have demonstrated the potential to be the next generation cancer treatment regimen. This review aims to highlight the recent advances in smart combinational therapy.

Skeletal muscle is a tissue that is directly involved in the progression and persistence of type 2 diabetes (T2D), a disease that is becoming increasingly common. Gaining better insight into the mechanisms that are affecting skeletal muscle dysfunction in the context of T2D has the potential to lead to novel treatments for a large number of patients. Through its ability to emulate skeletal muscle architecture while also incorporating aspects of disease, tissue-engineered skeletal muscle (TE-SkM) has the potential to provide a means for rapid high-throughput discovery of therapies to treat skeletal muscle dysfunction, to include that which occurs with T2D. Muscle precursor cells isolated from lean or obese male Zucker diabetic fatty rats were used to generate TE-SkM constructs. Some constructs were treated with adipogenic induction media to accentuate the presence of adipocytes that is a characteristic feature of T2D skeletal muscle. The maturity (compaction and creatine kinase activity), mechanical integrity (Young's modulus), organization (myotube orientation), and metabolic capacity (insulin-stimulated glucose uptake) were all reduced by diabetes. Treating constructs with adipogenic induction media increased the quantity of lipid within the diabetic TE-SkM constructs, and caused changes in construct compaction, cell orientation, and insulin-stimulated glucose uptake in both lean and diabetic samples. Collectively, the findings herein suggest that the recapitulation of structural and metabolic aspects of T2D can be accomplished by engineering skeletal muscle in vitro. IMPACT STATEMENT: The tissue engineering of skeletal muscle to model disease and injury has great promise to provide a tool to develop and/or improve therapeutic approaches for improved health care. A tissue-engineered skeletal muscle model of one of the most common and debilitating diseases, type 2 diabetes, has been developed in vitro as evidenced by the structural and metabolic alterations that are consistent with the disease phenotype in vivo.

In this article, we present an educational intervention that embeds ethics education within research laboratories. This structure is designed to assist students in addressing ethical challenges in a more informed way, and to improve the overall ethical culture of research environments. The project seeks (a) to identify factors that students and researchers consider relevant to ethical conduct in science, technology, engineering, and math (STEM) and (b) to promote the cultivation of an ethical culture in experimental laboratories by integrating research stakeholders in a bottom-up approach to developing context-specific, ethics-based guidelines. An important assumption behind this approach is that direct involvement in the process of developing laboratory specific ethical guidelines will positively influence researchers’ understanding of ethical research and practice issues, their handling of these issues, and the promotion of an ethical culture in the respective laboratory. The active involvement may increase the sense of ownership and integration of further discussion on these important topics. Based on the project experiences, the project team seeks to develop a module involving the bottom-up building of codes-of-ethics-based guidelines that can be used by a broad range of institutions and that will be distributed widely.

Vascular networks provide nutrients, oxygen, and progenitor cells that are essential for bone function. It has been proposed that a preformed vascular network may enhance the performance of engineered bone. In this study vascular networks were generated from human umbilical vein endothelial cell and mesenchymal stem cell spheroids encapsulated in fibrin scaffolds, and the stability of preformed vascular networks and their effect on bone regeneration were assessed in an in vivo bone model. Under optimized culture conditions, extensive vessel-like networks formed throughout the scaffolds in vitro. After vascular network formation, the vascularized scaffolds were implanted in a critical sized calvarial defect in nude rats. Immunohistochemical staining for CD31 showed that the preformed vascular networks survived and anastomosed with host tissue within 1 week of implantation. The prevascularized scaffolds enhanced overall vascularization after 1 and 4 weeks. Early bone formation around the perimeter of the defect area was visible in X-ray images of samples after 4 weeks. Prevascularized scaffolds may be a promising strategy for engineering vascularized bone.

In this article, we summarize the key findings of an exploratory study in which students and faculty completed a survey that sought to identify the most important ethical issues in STEM fields, how often these issues are discussed in research groups, and how often these ethical issues come up in the daily practice of research. Participants answered a series of open-ended and Likert-scale questions to provide a detailed look at the current ethical landscape at a private research university in the Midwest. The survey also looked at potential differences between faculty and undergraduate and graduate students' perceptions in answering these questions. The results indicate that while all community members tended to view issues that can be classified as research misconduct as the most important activities to avoid in STEM-related research, the level of discussion and actual witnessing of these practices was relatively low. The study points to a consensus among students and faculty about the important ethical issues in STEM and the need for more discussion and attention to be paid to communication, collaboration, and interpersonal relationships in the research environment.

Laminins are extracellular matrix proteins that reside in the basement membrane and provide structural support in addition to promoting cellular adhesion and migration. Through interactions with cell surface receptors, laminins stimulate intracellular signaling cascades which direct specific survival and differentiation outcomes. In metabolic tissues such as the pancreas, adipose, muscle, and liver, laminin isoforms are expressed in discrete temporal and spatial patterns suggesting that certain isoforms may support the development and function of particular metabolic cell types. This review focuses on the research to date detailing the expression of laminin isoforms, their potential function, as well as known pathways involved in laminin signaling in metabolic tissues. We will also discuss the current biomedical therapies involving laminins in these tissues in addition to prospective applications, with the goal being to encourage future investigation of laminins in the context of metabolic disease.

The current standard treatment for Type 1 diabetes is the administration of exogenous insulin to manage blood glucose levels. Cellular therapies are in development to address this dependency and allow patients to produce their own insulin. Studies have shown that viable, functional allogenic islets can be encapsulated inside alginate-based materials as a potential treatment for Type 1 diabetes. The capability of these grafts is limited by several factors, among which is the stability and longevity of the encapsulating material in vivo. Previous studies have shown that multilayer Alginate-Poly-L-Ornithine-Alginate (A-PLO-A) microbeads are effective in maintaining cellular function in vivo. This study expands upon the existing encapsulation material by investigating whether covalent crosslinking of the outer alginate layer increases stability. The alginate comprising the outer layer was methacrylated, allowing it to be covalently crosslinked. Microbeads with a crosslinked outer layer exhibited a consistent outer layer thickness and increased stability when exposed to chelating agents in vitro. The outer layer was maintained in vivo even in the presence of a robust inflammatory response. The results demonstrate a technique for generating A-PLO-A with a covalently crosslinked outer layer.

Longitudinal in vivo monitoring is essential for the design and evaluation of biomaterials. An ideal method would provide three-dimensional quantitative information, high spatial resolution, deep tissue penetration, and contrast between tissue and material structures. Photoacoustic (PA) or optoacoustic imaging is a hybrid technique that allows three-dimensional imaging with high spatial resolution. In addition, photoacoustic imaging allows for imaging of vascularization based on the intrinsic contrast of hemoglobin. In this study, we investigated photoacoustic computed tomography (PACT) as a tool for longitudinal monitoring of an implanted hydrogel in a small animal model. Hydrogels were loaded with gold nanorods to enhance contrast and imaged weekly for 8 weeks. PACT allowed non-invasive three-dimensional, quantitative imaging of the hydrogels over the entire 8 weeks. Quantitative volume analysis was used to evaluate the in vivo degradation kinetics of the implants which deviated slightly from in vitro predictions. Multispectral imaging allowed for the simultaneous analysis of hydrogel degradation and local vascularization. These results provide support for the substantial potential of PACT as a tool for insight into biomaterial performance in vivo.

Obesity is characterized by an increase in adipose mass and is the leading risk factor for type 2 diabetes. Increasing adipose thermogenic capacity, by activation of brown or beige fat, could be a treatment for metabolic diseases. Nevertheless, many approaches fail in maintaining transformed adipose tissue in vivo, in part, due to the limited understanding of how environmental factors control cell fate and maintenance. We have shown that mice with a null mutation in the laminin α4 exhibit resistance to obesity, enhanced expression of thermogenic fat markers (UCP1) in subcutaneous white adipose tissue (sWAT), increased energy expenditure and enhanced insulin sensitivity. The knockout (KO) of Lama4 results in complex changes in overall ECM composition. We used liquid chromatography/tandem mass spectrometry (LC/MS) to evaluate ECM composition in sWAT from wild type (WT) and KO mice. In addition to the expected absence of Lama4, collagen 1A1 (Col1A1) and collagen 3A1 (Col3A1) were significantly lower in KO mice. Also, integrins α7 (ITα7) and β1 (ITβ1) were dramatically reduced. Immunohistochemical staining confirmed lower levels of Itα7 in KO compared to WT mice (P Disclosure M.A. Gonzalez Porras: None. K. Stojkova: None. M.K. Vaicik: None. A.A. Goddi: None. R.N. Cohen: None. E. Brey: None.

The development of ectopic adipose tissue in skeletal muscle is associated with several skeletal muscle and metabolic pathologies, including Type II Diabetes Mellitus. The adipogenic differentiation of muscle precursor cells (MPCs) has been postulated to occur in skeletal muscle in vivo in a three-dimensional (3-D) configuration; therefore, it is appropriate to investigate this phenomenon using 3-D matrices in vitro. The capacity for MPC adipogenic differentiation in a 3-D environment was investigated in fibrin hydrogels by treating MPCs derived from healthy or diabetic animals with adipogenic induction medias that differed in their ability to increase lipid accumulation and activate the expression of genes associated with adipogenic differentiation (peroxisome proliferator-activated receptor gamma (PPARG), adiponectin (ADIPOQ), and fatty acid synthase (FAS)). The capacity for adipogenic differentiation was diminished, but not prevented, if myogenic differentiation preceded MPC exposure to adipogenic induction conditions. Conversely, adipogenic differentiation was greater in hydrogels containing MPCs from diabetic rats as compared to those derived from lean rats, as evidenced by an increase in lipid accumulation and adipogenic gene expression. Collectively, the data herein support a role for the MPCs in adipogenesis in a 3-D environment and that they may contribute to the ectopic accumulation of adipose tissue. The observation that the potential for adipogenic differentiation is maintained even after a period of myogenic differentiation alludes to the possibility that adipogenesis may occur during different phases of muscle development. Finally, the increase in adipogenic differentiation in hydrogels containing MPCs derived from diabetic animals provides strong evidence that a pathological environment in vivo increases their capacity for adipogenesis.

There is a need to overcome the donor-site morbidity and loss of volume over time that accompanies the current clinical approaches to treat soft tissue defects caused by disease and trauma. The development of bioactive constructs that can regenerate adipose tissue have made great progress toward addressing the limitations of current therapies, but their lack of vascularization and ability to meet the significant dimension requirements of tissue defects limit their clinical translatability. Microvascular fragments (MVFs) can form extensive vascular networks and contain resident cells that have the ability to differentiate into adipocytes. Therefore, the objective of this study was to determine if vascularized adipose tissue could be engineered using a fibrin-based hydrogel containing MVFs as the sole source of microvessels and adipocyte-forming cells. The potential for MVFs from different fat depots (epididymal, inguinal, and subcutaneous) to form microvascular networks and generate adipocytes when exposed to growth media (GM), adipogenic differentiation media (ADM), or when treated with GM before adipogenic induction (i.e., they were allowed to presprout before adipogenic induction) was evaluated. MVFs treated with adipogenic induction media, both with and without presprouting, contained lipid droplets, had an increase in expression levels of genes associated with adipogenesis (adiponectin and fatty acid synthase [FAS]), and had an increased rate of lipolysis. MVFs allowed to presprout before ADM treatment maintained their ability to form vascular networks while maintaining an elevated lipid content, adipogenic gene expression, and lipolysis rate. Collectively, these results support the contention that MVFs can serve as the sole source of biologic material for creating a vascularized adipose tissue scaffold. IMPACT STATEMENT: Microvascular fragments have both the ability to form extensive vascular networks and function as a source of adipocytes. These phenomena were exploited as vascularized adipose tissue was generated by first allowing for a period of angiogenesis before the adipogenic induction. This strategy has the ability to provide a means of both improving soft tissue reconstruction while also serving as a model to better understand adipose tissue expansion.

Vascularization is critical for the survival of engineered tissues post implantation. It has been previously shown that biomaterials containing preformed networks can anastomose to host vasculature following implantation. However, the optimal source of cells for vascularization for clinical use remains elusive. In this study, vascular networks were generated from endothelial cells derived from human induced pluripotent stem cells (iPSCs). Network formation by iPSC-ECs within fibrin gels was investigated in a mesenchymal stem cell (MSC) co-culture spheroid model. Statistical design of experiments (DOE) techniques were applied to identify optimal conditions for vessel-like network formation. The prevascularized units were then combined with hydroxyapatite nanoparticles to develop a vascularized composite hydrogel that was implanted in a rodent critical sized cranial defect model. Immunohistological staining for human-specific CD31 at week 1 indicated the presence and maintenance of the implanted vessels. Erythrocytes in the vessel lumen further suggests anastomosis of vessels with host vasculature. At week 8, isolectin staining indicated functionality of the human implanted vessels. There was a slight increase in bone volume in prevascularized scaffolds compared to MSC-only scaffolds. However, a pronounced increased in bone regeneration with prevascularization was not observed. These results show that prevascularized scaffolds can be generated from ECs derived from iPSC and that the networks survive and inosculate with the host post implantation in a bone model.

Several imaging modalities are available for investigation of the morphological, functional, and molecular features of engineered tissues in small animal models. While research in tissue engineering and regenerative medicine (TERM) would benefit from a comprehensive longitudinal analysis of new strategies, researchers have not always applied the most advanced methods. Photoacoustic imaging (PAI) is a rapidly emerging modality that has received significant attention due to its ability to exploit the strong endogenous contrast of optical methods with the high spatial resolution of ultrasound methods. Exogenous contrast agents can also be used in PAI for targeted imaging. Applications of PAI relevant to TERM include stem cell tracking, longitudinal monitoring of scaffolds in vivo, and evaluation of vascularization. In addition, the emerging capabilities of PAI applied to the detection and monitoring of cancer and other inflammatory diseases could be exploited by tissue engineers. This article provides an overview of the operating principles of PAI and its broad potential for application in TERM. IMPACT STATEMENT: Photoacoustic imaging, a new hybrid imaging technique, has demonstrated high potential in the clinical diagnostic applications. The optical and acoustic aspect of the photoacoustic imaging system works in harmony to provide better resolution at greater tissue depth. Label-free imaging of vasculature with this imaging can be used to track and monitor disease, as well as the therapeutic progression of treatment. Photoacoustic imaging has been utilized in tissue engineering to some extent; however, the full benefit of this technique is yet to be explored. The increasing availability of commercial photoacoustic systems will make application as an imaging tool for tissue engineering application more feasible. This review first provides a brief description of photoacoustic imaging and summarizes its current and potential application in tissue engineering.

Despite the wide variety of tissue-engineered vascular grafts that are currently being developed, autologous vessels, such as the saphenous vein, are still the gold standard grafts for surgical treatment of vascular disease. Recently developed technologies have shown promising results in preclinical studies, but they still do not overcome the issues that native vessels present, and only a few have made the transition into clinical use. The endothelial lining is a key aspect for the success or failure of the grafts, especially on smaller diameter grafts (

As research into the adipocyte microenvironment has advanced, it is becoming more widely accepted that the extracellular matrix (ECM) contributes to adipocyte dysfunction. The majority of current published work focuses on the role of collagens in metabolic disease while less emphasis has been placed on the contribution of laminins, an important component of the adipocyte basement membrane. Laminins are trimeric ECM proteins composed of α, β, and γ chains. The α chains contain sites which can interact with cell surface receptors and is considered the driver of tissue-specific expression and specialized signaling. Our group has shown that the laminin-α4 (LAMA4) chain, which is highly expressed in mature adipocytes, plays a role in adipocyte function and thermogenesis in mice (1). In this study we investigate the relationship between laminin α chain expression and obesity by assessing gene expression of LAMA1-5 in subcutaneous white adipose tissue (sWAT) from mice fed chow (RCD) and 45% high fat diet (HFD) for 8 weeks. Expression of LAMA2 and LAMA4 was significantly increased in the HFD sWAT compared to chow (6.1 fold, p=0.01 and 4.9 fold, p=0.001 respectively), however LAMA4 displayed a much stronger positive correlation with weight (R2=0.697) than did LAMA2 (R2=0.382). In order to validate the relevance of these findings in human models of obesity, we evaluated gene expression of LAMA2, LAMA4, and LAMA5 in sWAT biopsies from non-diabetic adult females with obesity (class II or higher). sWAT samples from obese subjects exhibited 4.5 fold higher LAMA4 expression (p=0.0089) than samples from non-obese control subjects, suggesting that the LAMA4 chain may play an important role in human obesity. Lastly we examined changes in sWAT LAMA4 expression following a period of weight loss in obese mice and in human subjects after bariatric surgery, and found that LAMA4 expression levels remain largely unchanged in both cases. In this study we demonstrate the relationship between LAMA4 expression and obesity and present findings that can be extended to human models of obesity. Reference: (1) Vaicik et al., Endocrinology. 2018 Jan;159(1):356–67.

Laminin α4 (LAMA4) is located in the extracellular basement membrane that surrounds each individual adipocyte. Here we show that LAMA4 null (Lama4−/−) mice exhibit significantly higher energy expenditure (EE) relative to wild-type (WT) mice at room temperature and when exposed to a cold challenge, despite similar levels of food intake and locomotor activity. The Lama4−/− mice are resistant to age- and diet-induced obesity. Expression of uncoupling protein 1 is higher in subcutaneous white adipose tissue of Lama4−/− mice relative to WT animals on either a chow diet or a high-fat diet. In contrast, uncoupling protein 1 expression was not increased in brown adipose tissue. Lama4−/− mice exhibit significantly improved insulin sensitivity compared with WT mice, suggesting improved metabolic function. Overall, these data provide critical evidence for a role of the basement membrane in EE, weight gain, and systemic insulin sensitivity., Despite similar food consumption and activity, mice lacking basement membrane protein laminin α4 were resistant to obesity and had increased beige subcutaneous adipose tissue and energy expenditure.

Transplantation of functional islets encapsulated in stable biomaterials has the potential to cure Type I diabetes. However, the success of these materials requires the ability to quantitatively evaluate their stability. Imaging techniques that enable monitoring of biomaterial performance are critical to further development in the field. X-ray phase-contrast (XPC) imaging is an emerging class of X-ray techniques that have shown significant promise for imaging biomaterial and soft tissue structures. In this study, XPC imaging techniques are shown to enable three dimensional (3D) imaging and evaluation of islet volume, alginate hydrogel structure, and local soft tissue features ex vivo. Rat islets were encapsulated in sterile ultrapurified alginate systems produced using a high-throughput microfluidic system. The encapsulated islets were implanted in omentum pouches created in a rodent model of type 1 diabetes. Microbeads were imaged with XPC imaging before implantation and as whole tissue samples after explantation from the animals. XPC microcomputed tomography (μCT) was performed with systems using tube-based and synchrotron X-ray sources. Islets could be identified within alginate beads and the islet volume was quantified in the synchrotron-based μCT volumes. Omental adipose tissue could be distinguished from inflammatory regions resulting from implanted beads in harvested samples with both XPC imaging techniques. Individual beads and the local encapsulation response were observed and quantified using quantitative measurements, which showed good agreement with histology. The 3D structure of the microbeads could be characterized with XPC imaging and failed beads could also be identified. These results point to the substantial potential of XPC imaging as a tool for imaging biomaterials in small animal models and deliver a critical step toward in vivo imaging.

Background Cell-seeded biomaterial scaffolds have been proposed as a future option for reconstruction of bone tissue. The ability to generate larger, functional volumes of bone has been a challenge that may be addressed through the use of perfusion bioreactors. In this study, the authors investigated use of a tubular perfusion bioreactor system for the growth and differentiation of bone marrow stromal (mesenchymal stem) cells seeded onto fibrin, a highly angiogenic biomaterial. Methods Cells were encapsulated within fibrin beads and cultured either within a tubular perfusion bioreactor system or statically for up to 14 days. Scaffolds were analyzed for osteogenic differentiation. A rodent cranial defect model (8-mm diameter) was used to assess the bone regeneration of scaffolds cultured in the bioreactor, statically, or used immediately after formation. Immunohistochemistry was used to visualize CD31 vessel density. Micro-computed tomographic imaging was used to visualize mineral formation within the defect volume. Results Tubular perfusion bioreactor system-cultured samples showed significantly greater osteodifferentiation, indicated by an increase in VEGF expression and mineral deposition, compared with statically cultured samples. Increased expression of OPN, RUNX2, VEGF, and CD90 was seen over time in both culture methods. After implantation, bioreactor samples exhibited greater bone formation and vessel density compared with all other groups. Analysis of micro-computed tomographic images showed full union formation through the greatest diameter of the defect in all bioreactor samples and the highest levels of mineralized volume after 8 weeks. Conclusion Mesenchymal stem cells encapsulated in fibrin beads and cultured in the tubular perfusion bioreactor system resulted in increased vascularization and mineralized tissue formation in vivo relative to static culture.

Imaging soft biomaterials in vivo is a significant challenge, as most conventional techniques are limited by biomaterial contrast, penetration depth, or spatial resolution. Exogeneous contrast agents used to increase contrast may also alter material properties or exhibit local toxicity. The capability to observe biomaterial constructs in vivo without introducing exogenous contrast would improve preclinical testing and evaluation. Conventional X-Ray Computed Tomography (CT) allows fast, high-resolution imaging at high penetration depth, but biomaterial contrast is low. Previous studies employing X-Ray phase contrast (XPC) and utilizing a synchrotron source provided support for the significant potential of XPC in imaging biomaterials without contrast agents. In this study, XPC tomography was used to image alginate hydrogel microspheres within a small animal omental pouch model using a commercially available X-Ray source. Multilayer microbeads could be identified in the XPC images with volumetric and structural information not possible in histological analysis. The number of microbeads present and microbead volume and diameter could be quantified from the images. The results of this study show that XPC tomography can be a useful tool for monitoring of implanted soft biomaterials in small animal models.

This small-scale design study describes disciplinary learning in mathematical modeling and science from an authentic engineeringthemed module. Current research in tissue engineering served as source material for the module, including science content for readings and a mathematical modeling activity in which students work in small teams to design a model in response to a problem from a client. The design of the module was guided by well-established principles of model-eliciting activities (a special class of problem-solving activities deeply studied in mathematics education) and recently published implementation design principles, which emphasize the portability of model-eliciting activities to many classroom settings. Two mathematical modeling research questions were addressed: 1. What mathematical approaches did student-teams take when they designed mathematical models to evaluate the quality of blood vessel networks? and 2. What attributes of mature mathematical models were captured in the mathematical models that the student-teams designed? One science content research question was addressed: 1. Before and after the module, what aspects of angiogenesis did students describe when they were asked what they knew about the process of blood vessel growth from existing vessels? Participants who field-tested the module included high school students in a summer enrichment program and early college students enrolled in four general-studies mathematics courses. Data collected from participants included mathematical models produced by small teams of students, as well as students’ individual responses before and after the module to a prompt asking them what they knew about the process of new blood vessel growth from existing vessels. The data were analyzed for mathematical model type and science content by adopting methods of grounded theory, in which researchers suspend expectations about what should be in the data and, instead, allow for the emergence of patterns and trends. The mathematical models were further analyzed for mathematical maturity using an a priori coding scheme of attributes of a mathematical model. Analyses showed that student-teams created mathematical models of varying maturity using four different mathematical approaches, and comparisons of students’ responses to the science prompt showed students knew essentially nothing about angiogenesis before the module but described important aspects of angiogenesis after the module. These findings were used to set up an agenda for future research about the design of the module and the relationship between disciplinary learning and authentic engineering problems.

In temporal domains, agents need to actively gather information to make more informed decisions about both the present and the future. When such a domain is modeled as a temporal graphical model, what the agent observes can be incorporated into the model by setting the respective random variables as evidence. Motivated by a tissue engineering application where the experimenter needs to decide how early a laboratory experiment can be stopped so that its possible future outcomes can be predicted within an acceptable uncertainty, we first present a dynamic Bayesian network (DBN) model of vascularization in engineered tissues and compare it with both real-world experimental data and agent-based simulations. We then formulate the question of "how early an experiment can be stopped to guarantee an acceptable uncertainty about the final expected outcome" as an active inference problem for DBNs and empirically and analytically evaluate several search algorithms that aim to find the ideal time to stop a tissue engineering laboratory experiment.

Islet transplantation is currently in clinical use as a treatment for type I diabetes, but donor shortages and long-term immunosuppression limit broad application. Alginate microcapsules coated with poly-l-ornithine can be used to encapsulate islets in an environment that allows diffusion of glucose, insulin, nutrients, and waste products while inhibiting cells and antibodies. While clinical trials are ongoing using islets encapsulated in alginate microbeads, there are concerns in regards to long-term stability. Evaluation of the local tissue response following implantation provides insight into the underlying mechanisms contributing to biomaterial failure, which can be used to the design of new material strategies. Macrophages play an important role in driving the response. In this study, the stability of alginate microbeads coated with PLO containing islets transplanted in the omentum pouch model was investigated. Biomaterial structure and the inflammatory response were characterized by X-ray phase contrast (XPC) μCT imaging, histology, and immunostaining. XPC allowed evaluation of microbead 3D structure and identification of failed and stable microbeads. A robust inflammatory response characterized by high cell density and the presence of pro-inflammatory macrophages was found around the failed grafts. The results obtained provide insight into the local tissue response and possible failure mechanisms for alginate microbeads. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 1581-1590, 2016.

Stable and extensive blood vessel networks are required for cell function and survival in engineered tissues. A number of different strategies are currently being investigated to enhance biomaterial vascularization with screening primarily through extensive in vitro and in vivo experiments. In this article, we describe an agent-based model (ABM) developed to evaluate various strategies in silico, including design of optimal biomaterial structure, delivery of angiogenic factors, and application of prevascularized biomaterials. The model predictions are evaluated using experimental data. The ABM developed provides insight into different strategies currently applied for scaffold vascularization and will enable researchers to rapidly screen new hypotheses and explore alternative strategies for enhancing vascularization.

Human trials have demonstrated the feasibility of alginate-encapsulated islet cells for the treatment of type 1 diabetes. Encapsulated islets can be protected from the host's immune system and remain viable and functional following transplantation. However, the long-term success of these therapies requires that alginate microcapsules maintain their immunoprotective capacity and stability in vivo for sustained periods. In part, as a consequence of different encapsulation strategies, islet encapsulation studies have produced inconsistent results in regard to graft functioning time, stability, and overall metabolic benefits. Alginate composition (proportion of M- and G-blocks), alginate purity, the cross-linking ions (calcium or barium), and the presence or absence of additional polymer coating layers influence the success of cell encapsulation. This review summarizes the outcomes of long-term studies of alginate-encapsulated islet transplants in animals and humans and provides a critical discussion of the graft failure mechanisms, including issues with graft biocompatibility, transplantation site, and integrity of the encapsulated islet grafts. Strategies to improve the mechanical stability of alginate capsules and methods for monitoring graft survival and function in vivo are presented.

The importance of vascularization in the field of bone tissue engineering has been established by previous studies. The present work proposes a novel poly(propylene fumarate) (PPF)/fibrin composite scaffold for the development of vascularized neobone tissue. The effect of prevascularization (i.e., in vitro pre-culture prior to implantation) with human mesenchymal stem cells (hMSCs) and human umbilical vein endothelial cells (HUVECs) on in vivo vascularization of scaffolds was determined. Five conditions were studied: no pre-culture (NP), 1 week pre-culture (1P), 2 week pre-culture (2P), 3 week pre-culture (3P), and scaffolds without cells (control, C). Scaffolds were implanted subcutaneously in a severe combined immunodeficiency (SCID) mouse model for 9 days. During in vitro studies, CD31 staining showed a significant increase in vascular network area over 3 weeks of culture. Vascular density was significantly higher in vivo when comparing the NP and 3P groups. Immunohistochemical staining of human CD-31 expression indicated spreading of vascular networks with increasing pre-culture time. These vascular networks were perfused with mouse blood indicated by perfused lectin staining in human CD-31 positive vessels. Our results demonstrate that in vitro prevascularization supports in vivo vascularization in PPF/fibrin scaffolds.