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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.

Obesity affects nearly one billion globally and can lead to life-threatening sequelae. Consequently, there is an urgent need for novel therapeutics. We have previously shown that laminin, alpha 4 (Lama4) knockout in mice leads to resistance to adipose tissue accumulation; however, the relationship between LAMA4 and obesity in humans has not been established. In this study we measured laminin-α chain and collagen mRNA expression in the subcutaneous white adipose tissue (sWAT) of mice placed on chow (RCD) or 45% high fat diet (HFD) for 8 weeks, and also in HFD mice then placed on a “weight loss” regimen (8 weeks HFD followed by 6 weeks RCD). To assess extracellular matrix (ECM) components in humans with obesity, laminin subunit alpha mRNA and protein expression was measured in sWAT biopsies of female control subjects (BMI35) both before and three months after surgery. Lama4 was significantly higher in sWAT of HFD compared to RCD mice at both the RNA and protein level (pLAMA4 mRNA (pLama4 or LAMA4 expression was detected following short-term weight loss in either mouse or human samples, respectively. From these results we propose a significant association between obesity and elevated LAMA4 expression in humans, as well as in mouse models of obesity. Further studies should clarify the mechanisms underlying this association to target LAMA4 effectively as a potential therapy for obesity.

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.

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 (

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.

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.

Delivery of compounds to the peripheral nervous system has the potential to be used as a treatment for a broad range of conditions and applications, including neuropathic pain, regional anesthesia, traumatic nerve injury, and inherited and inflammatory neuropathies. However, efficient delivery of therapeutic doses can be difficult to achieve due to peripheral neuroanatomy and the restrictiveness of the blood-nerve barrier. Depending on the underlying integrity of the blood-nerve barrier in the application at hand, several strategies can be employed to navigate the peripheral nerve architecture and facilitate targeted delivery to the peripheral nerve. This review describes different applications where targeted delivery to the peripheral nervous system is desired, the challenges that the blood-nerve barrier poses in each application, and bioengineering strategies that can facilitate delivery in each application.

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.

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.

The recent proliferation of three dimensional (3D) printing technologies has allowed the exploration of increasing complex designs, and, furthermore, the consideration of 3D printed constructs for biological applications. However, there is an unmet need for a consistent set of tools for the design and evaluation of these biological 3D printed constructs, particularly as they relate to engineered tissues. For example, identifying the most advantageous construct parameters for the rapid vascularization of an engineered tissue - a critical parameter in regenerative medicine - is difficult without a common group of measures. We demonstrate here a toolbox to design, characterize, and evaluate 3D printed scaffolds for vascularized tissue regenerative medicine. Our toolbox (1) identifies the range of design specifications using a modular design, (2) nondestructively compares the 3D printed scaffolds to the design, (3) evaluates biocompatibility and mechanical properties, and (4) predicts host vessel integration. As a case study, we designed, fabricated, and evaluated polymer scaffolds using a poly(propylene fumarate) based resin. Our work highlights the potential for these tools to be combined as a consistent methodology for the evaluation of porous 3D printed constructs for regenerative medicine.

Contains fulltext : 169834.pdf (Publisher’s version ) (Open Access) Contains fulltext : 169834pos.pdf (Author’s version postprint ) (Open Access) BACKGROUND: Neuroimaging studies have shown structural alterations in several brain regions in children and adults with attention deficit hyperactivity disorder (ADHD). Through the formation of the international ENIGMA ADHD Working Group, we aimed to address weaknesses of previous imaging studies and meta-analyses, namely inadequate sample size and methodological heterogeneity. We aimed to investigate whether there are structural differences in children and adults with ADHD compared with those without this diagnosis. METHODS: In this cross-sectional mega-analysis, we used the data from the international ENIGMA Working Group collaboration, which in the present analysis was frozen at Feb 8, 2015. Individual sites analysed structural T1-weighted MRI brain scans with harmonised protocols of individuals with ADHD compared with those who do not have this diagnosis. Our primary outcome was to assess case-control differences in subcortical structures and intracranial volume through pooling of all individual data from all cohorts in this collaboration. For this analysis, p values were significant at the false discovery rate corrected threshold of p=0.0156. FINDINGS: Our sample comprised 1713 participants with ADHD and 1529 controls from 23 sites with a median age of 14 years (range 4-63 years). The volumes of the accumbens (Cohen's d=-0.15), amygdala (d=-0.19), caudate (d=-0.11), hippocampus (d=-0.11), putamen (d=-0.14), and intracranial volume (d=-0.10) were smaller in individuals with ADHD compared with controls in the mega-analysis. There was no difference in volume size in the pallidum (p=0.95) and thalamus (p=0.39) between people with ADHD and controls. Exploratory lifespan modelling suggested a delay of maturation and a delay of degeneration, as effect sizes were highest in most subgroups of children (21 years): in the accumbens (Cohen's d=-0.19 vs -0.10), amygdala (d=-0.18 vs -0.14), caudate (d=-0.13 vs -0.07), hippocampus (d=-0.12 vs -0.06), putamen (d=-0.18 vs -0.08), and intracranial volume (d=-0.14 vs 0.01). There was no difference between children and adults for the pallidum (p=0.79) or thalamus (p=0.89). Case-control differences in adults were non-significant (all p>0.03). Psychostimulant medication use (all p>0.15) or symptom scores (all p>0.02) did not influence results, nor did the presence of comorbid psychiatric disorders (all p>0.5). INTERPRETATION: With the largest dataset to date, we add new knowledge about bilateral amygdala, accumbens, and hippocampus reductions in ADHD. We extend the brain maturation delay theory for ADHD to include subcortical structures and refute medication effects on brain volume suggested by earlier meta-analyses. Lifespan analyses suggest that, in the absence of well powered longitudinal studies, the ENIGMA cross-sectional sample across six decades of ages provides a means to generate hypotheses about lifespan trajectories in brain phenotypes. FUNDING: National Institutes of Health. 10 p.

Our study aim was to determine encapsulated islet graft viability in an omentum pouch and the effect of fibroblast growth factor 1 (FGF-1) released from our redesigned alginate microcapsules on the function of the graft.Isolated rat islets were encapsulated in an inner core made with 1.5% low-viscosity-high-mannuronic-acid alginate followed by an external layer made with 1.25% low-viscosity high-guluronic acid alginate with or without FGF-1, in microcapsules measuring 300 to 400 µm in diameter. The 2 alginate layers were separated by a perm-selective membrane made with 0.1% poly-L-ornithine, and the inner low-viscosity-high-mannuronic-acid core was partially chelated using 55 mM sodium citrate for 2 minutes.A marginal mass of encapsulated islet allografts (∼2000 islets/kg) in streptozotocin-diabetic Lewis rats caused significant reduction in blood glucose levels similar to the effect observed with encapsulated islet isografts. Transplantation of alloislets coencapsulated with FGF-1 did not result in better glycemic control, but induced greater body weight maintenance in transplant recipients compared with those that received only alloislets. Histological examination of the retrieved tissue demonstrated morphologically and functionally intact islets in the microcapsules, with no signs of fibrosis.We conclude that the omentum is a viable site for encapsulated islet transplantation.

Plaque vulnerability depends, in part, on composition. Imaging techniques are needed that can aid the prediction of plaque stability. High-contrast images of soft-tissue structure have been obtained with x-ray phase-contrast (PC) imaging. This research investigates multiple image radiography (MIR), an x-ray PC imaging technique, for evaluation of human carotid artery plaques.Carotid plaques were imaged with ultrasound and subsequently excised and formalin fixed. MIR imaging was performed. By using synchrotron radiation, conventional radiographs were acquired for comparison. Image texture measures were computed for soft-tissue regions of the plaques.Ultrasound evaluation identified plaques as homogeneous without calcifications. MIR images revealed complex heterogeneous structure with multiple microcalcifications consistent with histology, and possessed more image texture in specific regions than conventional radiographs (P.05). MIR refraction images allowed imaging of the geometric structure of tissue interfaces within the plaques, while scatter images contained more texture in soft-tissue regions than absorption or refraction images.X-ray PC imaging better depicts plaque soft-tissue heterogeneity than ultrasound or conventional radiographs. MIR imaging technique should be investigated further as a viable imaging technique to identify high-risk plaques.

The in vitro investigation of many optically opaque biological microstructures requires 3D analysis at high resolution over a large field of view. We demonstrate a new nondestructive volumetric imaging technique that eliminates the structural and computational limitations of conventional 2D optical microscopy by combining x-ray phase-contrast tomography with critical point drying sample preparation. We experimentally demonstrate the enhancement of small features afforded by phase-contrast imaging and show the contrast improvement afforded by the drying of a hydrated specimen. We further demonstrate the biological application of this technique by imaging the microstructure of the accommodative apparatus in a primate eye using a benchtop phase-contrast tomography system.

This prospective study was designed to compare the accuracy rate between remote smartphone photographic assessments and in-person examinations for free flap monitoring. One hundred and three consecutive free flaps were monitored with in-person examinations and assessed remotely by three surgeons (Team A) via photographs transmitted over smartphone. Four other surgeons used the traditional in-person examinations as Team B. The response time to re-exploration was defined as the interval between when a flap was evaluated as compromised by the nurse/house officer and when the decision was made for re-exploration. The accuracy rate was 98.7% and 94.2% for in-person and smartphone photographic assessments, respectively. The response time of 8 ± 3 min in Team A was statistically shorter than the 180 ± 104 min in Team B (P = 0.01 by the Mann-Whitney test). The remote smartphone photography assessment has a comparable accuracy rate and shorter response time compared with in-person examination for free flap monitoring.

Loss of adipose tissue can occur due to congenital and acquired lipoatrophies, trauma, tumor resection, and chronic disease. Clinically, it is difficult to regenerate or reconstruct adipose tissue. The extensive microvsacular network present in adipose, and the sensitivity of adipocytes to hypoxia, hinder the success of typical tissue transfer procedures. Materials that promote the formation of vascularized adipose tissue may offer alternatives to current clinical treatment options. A number of synthetic and natural biomaterials common in tissue engineering have been investigated as scaffolds for adipose regeneration. While these materials have shown some promise they do not account for the unique extracellular microenvironment of adipose. Adipose derived hydrogels more closely approximate the physical and chemical microenvironment of adipose tissue, promote preadipocyte differentiation and vessel assembly in vitro, and stimulate vascularized adipose formation in vivo . The combination of these materials with techniques that promote rapid and stable vascularization could lead to new techniques for engineering stable, vascularized adipose tissue for clinical application. In this review we discuss materials used for adipose tissue engineering and strategies for vascularization of these scaffolds. Clinical Relevance Materials that promote formation of vascularized adipose tissue have the potential to serve as alternatives or supplements to existing treatment options, for adipose defects or deficiencies resulting from chronic disease, lipoatrophies, trauma, and tumor resection.

Background Multilayered alginate microcapsules with a permselective poly-L-ornithine membrane can be used for the dual purpose of encapsulating cells in the inner core and sustained release of angiogenic proteins from the outer layer. The aim of this study was to examine the encapsulation and release of a novel chimeric form of fibroblast growth factor–1 (FGF-1) from the outer layer of alginate microcapsules. Methods Heparin-binding growth-associated molecule bound to FGF-1 (HB-GAM/FGF-1) was encapsulated in the outer layer of multilayered alginate microbeads constructed using varying alginate conditions. The encapsulation and release of the chimera was quantified. Results The outer layer was able to encapsulate and release HB-GAM/FGF-1 for up to 30 days. The outer layer made with 1% alginate of high mannuronic acid content provided the fastest release, while 1.25% high guluronic acid content alginate displayed the longest duration of release. Conclusions The outer layer of multilayered alginate microbeads can be used for the encapsulation and long-term release of HB-GAM/FGF-1.

The use of growth factors for the therapeutic stimulation of neovascularization in regenerative medicine has been extensively investigated, but the inability to control their temporal delivery may limit clinical success. A strategy that delivers continuous therapeutic concentrations of growth factors may increase the protein's efficacy. The present study investigates the ability of sustained delivery of fibroblast growth factor-1 (FGF-1), to induce neovascularization in vivo . Alginate microbeads were synthesized to release active FGF-1 for three weeks. Microbeads loaded with FGF-1 (total amount 150 ng) were implanted into a surgically created omentum pouch in rats and were compared to control empty microbead implants and a single bolus injection of 150 ng of FGF-1 with empty microbead implant. Animals were sacrificed at either 3 or 6 weeks post implantation and omenta were analyzed for vascular density and mural cell interactions. Vascular area for bolus FGF-1 and FGF-1 loaded microbeads was higher than control at 3 weeks. At 6 weeks, vascular density in the group with FGF-1 loaded microbeads was significantly higher than the group with bolus administration of FGF-1, primarily due to an increase in the number of vessels less than 20 µm in diameter. Vascular density in omenta of the group receiving the bolus FGF-1 returned to control levels by 6 weeks. Staining for smooth muscle actin showed that 50% of vessels had associated mural cells. There was a trend of increased mural cell staining at 6 weeks for the FGF-1 loaded beads compared to bolus FGF-1 and control levels. Results in these studies suggest that sustained release of FGF-1 increases the duration of the vascular response in contrast to a bolus injection of FGF-1.

Background: Large craniofacial skeletal defects require complex reconstruction. Vascularized tissue transfer is the current standard in treatment, but these operations are technically difficult and associated with donor-site morbidity. Guided flap prefabrication offers a technique for endogenously engineering vascularized composite tissues with complex three-dimensional structure. This study evaluates the relationship between implantation time and tissue structure for generating tissues of clinically relevant volume and structure. Methods: Twenty skeletally mature domestic sheep were implanted with poly-(methyl methacrylate) chambers designed to mimic the size and shape of the mental protuberance of the mandible. Each chamber was filled with morcellized bone graft and implanted with the open face apposed to the cambium layer of the rib periosteum. Chambers were harvested at 3, 6, 9, 12, and 24 weeks, and the tissue inside the chambers was analyzed for shape conformation to chamber geometry, gross tissue volume, and bone histomorphometric parameters. Results: Histologically, active endochondral, direct, and appositional bone formation was observed. Calcified tissue area and new bone formation increased for each time point up to 12 weeks of implantation. The tissues formed maintained volumetric and geometrical structure consistent with the chamber up to 9 weeks after implantation. Significant decreases in total volume and agreement with chamber geometry were observed at 12 and 24 weeks. Conclusions: Periosteum-guided tissue prefabrication was found to be an effective means of engineering three-dimensional vascularized bone of clinical size and shape. The optimal duration of incubation before significant volume loss occurs is 9 weeks in this large-animal model.

Background Muscle fascia and periosteum have been used clinically to guide prefabrication of vascularized bone flaps for reconstruction of complex three-dimensional tissues. Although it seems that both locations have the capacity to generate vascularized bone, there have been no studies that directly compare different implantation sites. The authors performed a rigorous, quantitative, histomorphometric comparison of bone prefabrication in a large-animal model comparing graft implanted against muscle fascia and periosteum. Methods Twenty skeletally mature domestic sheep were implanted with rectangular chambers containing equal weights of morcellized bone graft. Two chambers were implanted into each sheep, one with the open face apposed to the cambium layer of the rib periosteum and the other with the open face apposed to the fascia of the latissimus dorsi muscle. Animals were euthanized at 3, 6, 9, 12, and 24 weeks and the chambers were harvested. Tissue inside the chambers was analyzed for shape conformation to chamber geometry, gross tissue volume, and bone histomorphology. Results There were no differences in volume or shape of tissue formed in the chambers. However, chambers in contact with fascia consisted almost entirely of fibrovascular tissue, with progressive resorption of the morcellized bone graft and little evidence of new bone. Chambers in contact with periosteum showed active endochondral, direct, and appositional bone formation over time, with increasing calcified tissue area and new bone formation. Conclusions Both periosteum and muscle fascia were able to vascularize bone grafts, but bone formation was higher in the periosteum. The periosteum appears to be a more suitable foundation from which to promote flap prefabrication.

Diffraction-enhanced imaging (DEI) is an analyser-based x-ray imaging method that produces separate images depicting the projected x-ray absorption and refractive properties of an object. Because the imaging model of DEI does not account for ultra-small-angle x-ray scattering (USAXS), the images produced in DEI can contain artefacts and inaccuracies in medical imaging applications. In this work, we investigate an extended DEI method for concurrent reconstruction of three images that depict an object's projected x-ray absorption, refraction and USAXS properties. The extended DEI method can be viewed as an implementation of the recently proposed multiple-image radiography paradigm. Validation studies are conducted by use of computer-simulated and synchrotron measurement data.

3D image reconstruction using light microscope modalities without exogenous contrast agents is proposed and investigated as an approach to produce 3D images of biological samples for live imaging applications. Multimodality and multispectral imaging, used in concert with this 3D optical sectioning approach is also proposed as a way to further produce contrast that could be specific to components in the sample. The methods avoid usage of contrast agents. Contrast agents, such as fluorescent or absorbing dyes, can be toxic to cells or alter cell behavior. Current modes of producing 3D image sets from a light microscope, such as 3D deconvolution algorithms and confocal microscopy generally require contrast agents. Zernike phase contrast (ZPC), transmitted light brightfield (TLB), darkfield microscopy and others can produce contrast without dyes. Some of these modalities have not previously benefitted from 3D image reconstruction algorithms, however. The 3D image reconstruction algorithm is based on an underlying physical model of scattering potential, expressed as the sample’s 3D absorption and phase quantities. The algorithm is based upon optimizing an objective function - the I-divergence - while solving for the 3D absorption and phase quantities. Unlike typical deconvolution algorithms, each microscope modality, such as ZPC or TLB, produces two output image sets instead of one. Contrast in the displayed image and 3D renderings is further enabled by treating the multispectral/multimodal data as a feature set in a mathematical formulation that uses the principal component method of statistics.

Brain tumors represent a leading cause of cancer death for people under the age of 40 and the probability complete surgical resection of brain tumors remains low owing to the invasive nature of these tumors and the consequences of damaging healthy brain tissue. Molecular imaging is an emerging approach that has the potential to improve the ability for surgeons to correctly discriminate between healthy and cancerous tissue; however, conventional molecular imaging approaches in brain suffer from significant background signal in healthy tissue or an inability target more invasive sections of the tumor. This work presents initial studies investigating the ability of novel dual-tracer molecular imaging strategies to be used to overcome the major limitations of conventional “single-tracer” molecular imaging. The approach is evaluated in simulations and in an in vivo mice study with animals inoculated orthotopically using fluorescent human glioma cells. An epidermal growth factor receptor (EGFR) targeted Affibody-fluorescent marker was employed as a targeted imaging agent, and the suitability of various FDA approved untargeted fluorescent tracers (e.g. fluorescein & indocyanine green) were evaluated in terms of their ability to account for nonspecific uptake and retention of the targeted imaging agent. Signal-to-background ratio was used to measure and compare the amount of reporter in the tissue between targeted and untargeted tracer. The initial findings suggest that FDA-approved fluorescent imaging agents are ill-suited to act as untargeted imaging agents for dual-tracer fluorescent guided brain surgery as they suffer from poor delivery to the healthy brain tissue and therefore cannot be used to identify nonspecific vs. specific uptake of the targeted imaging agent where current surgery is most limited.

A multi-layer agent-based model (ABM) of biomaterial scaffold vascularization is extended to consider the effects of scaffold degradation kinetics on blood vessel formation. A degradation model describing the bulk disintegration of porous hydrogels is incorporated into the ABM. The combined degradation-angiogenesis model is used to investigate growing blood vessel networks in the presence of a degradable scaffold structure. Simulation results indicate that higher porosity, larger mean pore size, and rapid degradation allow faster vascularization when not considering the structural support of the scaffold. However, premature loss of structural support results in failure for the material. A strategy using multi-layer scaffold with different degradation rates in each layer was investigated as a way to address this issue. Vascularization was improved with the multi-layered scaffold model compared to the single-layer model. The ABM developed provides insight into the characteristics that influence the selection of optimal geometric parameters and degradation behavior of scaffolds, and enables easy refinement of the model as new knowledge about the underlying biological phenomena becomes available. Statement of significance This paper proposes a multi-layer agent-based model (ABM) of biomaterial scaffold vascularization integrated with a structural-kinetic model describing bulk degradation of porous hydrogels to consider the effects of scaffold degradation kinetics on blood vessel formation. This enables the assessment of scaffold characteristics and in particular the disintegration characteristics of the scaffold on angiognesis. Simulation results indicate that higher porosity, larger mean pore size, and rapid degradation allow faster vascularization when not considering the structural support of the scaffold. However, premature loss of structural support by scaffold disintegration results in failure of the material and disruption of angiogenesis. A strategy using multi-layer scaffold with different degradation rates in each layer was investigated as away to address this issue. Vascularization was improved with the multi-layered scaffold model compared to the single-layer model. The ABM developed provides insight into the characteristics that influence the selection of optimal geometric and degradation characteristics of tissue engineering scaffolds.

Atherosclerotic cardiovascular disease is a leading cause of death worldwide. Despite recent improvements in medical, operative, and endovascular treatments, the number of interventions performed annually continues to increase. Unfortunately, the durability of these interventions is limited acutely by thrombotic complications and later by myointimal hyperplasia followed by progression of atherosclerotic disease over time. Despite improving medical management of patients with atherosclerotic disease, these complications appear to be persisting. Cardiovascular gene therapy has the potential to make significant clinical inroads to limit these complications. This article will review the technical aspects of cardiovascular gene therapy; its application for promoting a functional endothelium, smooth muscle cell growth inhibition, therapeutic angiogenesis, tissue engineered vascular conduits, and discuss the current status of various applicable clinical trials.

Purpose The purpose of this study was to test the hypothesis that spatial and temporal localization of growth factors FGF-2 and VEGF in a rabbit tooth extraction socket model correlate with the histologic events of healing. Materials and Methods Twenty-four male New Zealand white rabbits divided into 8 groups of 3 were used in the study. Incisor teeth were extracted from both jaws and the healing extraction socket with surrounding jaw bone was harvested at 48 hours, 4 days, 1, 2, 4, 8, 12, and 16 weeks. Tissues were fixed, decalcified, and processed for hematoxylin-eosin and immunohistochemical staining. The sections were stained to detect FGF-2 and VEGF. The stained sections were then imaged and an automated computer program was used to detect the brown diaminobenzidine stain that represented the growth factors of interest. Data was obtained in the form of percentage area and intensity of stain and analyzed using the analysis of variance (ANOVA - Tukey Kramer and Scheffe’s post-test). Results Spatial and temporal differences in localization of FGF-2 and VEGF were observed across all time frames in both jaws. Statistically significant differences in percentage area and intensity of brown diaminobenzidine stain were seen temporally between FGF-2 and VEGF (P Conclusion The results of this study showed positive correlation of histologic events to spatial and temporal localization of FGF-2 and VEGF in a rabbit tooth extraction model.

A number of pathological entities and surgical interventions could benefit from therapeutic stimulation of new blood vessel formation. Although strategies designed for promoting neovascularization have shown promise in preclinical models, translation to human application has met with limited success when angiogenesis is used as the single therapeutic mechanism. While clinical protocols continue to be optimized, a number of exciting new approaches are being developed. Bioengineering has played an important role in the progress of many of these innovative new strategies. In this review, we present a general outline of therapeutic neovascularization, with an emphasis on investigations using engineering principles to address this vexing clinical problem. In addition, we identify some limitations and suggest areas for future research.

Background Through site-directed mutagenesis we have created a favorable fibroblast growth factor-1 (FGF-1) mutant (S130K) and linked it to a heparin-binding growth-associated molecule (HBGAM) to form the chimera S130K-HBGAM creating a heparin-independent, endothelial cell (EC)-specific mitogen. Methods The proliferative responses of primary canine carotid artery smooth muscle cells (SMC) and jugular vein EC to FGF-1, S130K, or S130K-HBGAM, with and without heparin (5 U/mL), was quantitated by measuring tritiated thymidine uptake over 24 hours and expressing the results as percent of positive control (20% fetal bovine serum [FBS]) for group comparison. Results Unlike FGF-1, both S130K and S130K-HBGAM are heparin-independent mitogens for EC and SMC. S130K-HBGAM was equivalent to FGF-1 with heparin at 6 nmol/L. S130K-HBGAM did not demonstrate relative EC specificity in this assay. Conclusions At higher concentrations, S130K-HBGAM is a potent, heparin-independent EC and SMC mitogen. Co-culture assays and in vivo delivery models may demonstrate EC specificity not identified in this single cell type proliferation assay.

In this work we sought to understand the effect of biomaterial properties upon healing bone tissue. We hypothesized that a hydrophilic polymer gel implanted into a bone tissue defect would impede the healing process owing to the biomaterial's prevention of protein adsorption and thus cell adhesion. To test this hypothesis, healing bone was investigated within a rabbit incisor extraction socket, a subcritical size bone defect that resists significant soft tissue invasion by virtue of its conformity. After removal of the incisor teeth, one tooth socket was left as an empty control, one was filled with crosslinked polymer networks formed from the hydrophobic polymer poly(propylene fumarate) (PPF), and one was filled with a hydrogel formed from the hydrophilic oligomer oligo(poly(ethylene glycol) fumarate) (OPF). At five different times (4 days as well as 1, 2, 4, and 8 weeks), jaw bone specimens containing the tooth sockets were removed. We analyzed bone healing by histomorphometrical analysis of hematoxylin and eosin stained sections as well as immunohistochemically stained sections. The proposed hypothesis, that a hydrophilic material would hinder bone healing, was supported by the histomorphometrical results. In addition, the immunohistochemical results reflect molecular signaling indicative of the early invasion of platelets, the vascularization of wound-healing tissue, the differentiation of migrating progenitor cells, and the formation and remodeling of bone tissue. Finally, the results emphasize the need to consider biomaterial properties and their differing effects upon endogenous growth factors, and thus bone healing, during the development of tissue engineering devices.

Purpose: The purpose of this study was to test the hypothesis that spatial and temporal localization of growth factors transforming growth factor (TGF)-β1, bone morphogenetic protein (BMP)-2, and platelet derived growth factor (PDGF)-A in a rabbit tooth extraction model correlate with the histologic events contributing toward healing. Materials and Methods: Twenty-four male New Zealand White rabbits were used in the study. Incisor teeth were extracted from both jaws, and the healing extraction socket with the surrounding jaw bone was harvested at 48 hours, 4 days, and 1, 2, 4, 8, 12, and 16 weeks. Tissues were fixed, decalcified, and processed for hematoxylin and eosin and immunohistochemical staining. The sections were stained to detect TGF-β1, BMP-2, and PDGF-A. Stained sections were then imaged, and an automated computer program was used to detect the brown 3,3′-diaminobenzidine regions representing the location of growth factors of interest. The data were collected in terms of percentage area and intensity of stain, and an analysis of variance was conducted (Tukey-Kramer and Scheffe’s test) for statistical comparison between different time points, jaws, and growth factors. These results were also compared with hematoxylin and eosin-stained histologic specimens obtained at similar time points. Results: Spatial and temporal differences in localization of TGF-β1, BMP-2, and PDGF-A were observed across all time frames in both jaws. Statistically significant differences in percentage area and intensity of brown diaminobenzidine stain were detected temporally between TGF-β1, BMP-2, and PDGF-A (P ≤ .0001). Conclusion: The results of this study showed positive correlation between histologic events and the spatial and temporal localization of TGF-β1, BMP-2, and PDGF-A in a rabbit tooth extraction model.

Principles of tissue engineering are now being applied in the induction and development of microvascular networks as well as vessel conduits, including the in vitro and in vivo biologic modification of synthetic vascular grafts and the generation of tissue-engineered blood vessels. Yet 100 years since the development of vascular anastomoses, small diameter vascular grafts continue to encounter significant translational barriers to widespread clinical application. Here we discuss the current limitations of clinical application and ways which bioengineering approaches can be utilized to improve clinical applicability and drive future work that may avoid or address current limitations in blood vessel creation, augmentation, or substitution.

Carotid artery plaque instability can result in rupture and lead to ischaemic stroke. Stability of plaques appears to be a function of composition. Current non-invasive imaging techniques are limited in their ability to classify distinct histological regions within plaques. Phase-contrast (PC) X-ray imaging methods are an emerging class of techniques that have shown promise for identifying soft-tissue features without use of exogenous contrast agents. This is the first study to apply analyser-based X-ray PC imaging in CT mode to provide three-dimensional (3D) images of excised atherosclerotic plaques. The results provide proof of principle for this technique as a promising method for analysis of carotid plaque microstructure. Multiple image radiography CT (MIR-CT), a tomographic implementation of X-ray PC imaging that employs crystal optics, was employed to image excised carotid plaques. MIR-CT imaging yields three complementary images of the plaque's 3D X-ray absorption, refraction and scatter properties. These images were compared with histological sections of the tissue. X-ray PC images were able to identify the interface between the plaque and the medial wall. In addition, lipid-rich and highly vascularized regions were visible in the images as well as features depicting inflammation. This preliminary research shows MIR-CT imaging can reveal details about plaque structure not provided by traditional absorption-based X-ray imaging and appears to identify specific histological regions within plaques. This is the first study to apply analyser-based X-ray PC imaging to human carotid artery plaques to identify distinct soft-tissue regions.

Supplemental Digital Content is available in the text., Background: The application of bone tissue engineering for repairing bone defects has gradually shown some satisfactory progress. One of the concerns raising scientific attention is the poor supply of growth factors. A number of growth factor delivery approaches have been developed for promoting bone formation. However, there is no systematic comparison of those approaches on efficiency of neobone formation. In this study, the approaches using periosteum, direct supply of growth factors, or gene transfection of growth factors were evaluated to determine the osteogenic capacity on the repair of bone defect. Methods: In total, 42 male 21-week-old Sprague-Dawley rats weighing 250 to 400 g were used as the bone defect model to evaluate the bone repair efficiency. Various tissue engineered constructs of poly(ethylene glycol)-poly(l-lactic acid) (PEG-PLLA) copolymer hydrogel with periosteum, with external supply of bone morphogenetic protein-2 (BMP2), or with BMP2-transfected bone marrow–derived mesenchymal stem cells (BMMSCs) were filled in a 7-mm bone defect region. Animals were euthanized at 3 months, and the hydrogel constructs were harvested. The evaluation with histological staining and radiography analysis were performed for the volume of new bone formation. Results: The PEG-PLLA scaffold with BMMSCs promotes bone regeneration with the addition of periosteum. The group with BMP2-transfected BMMSCs demonstrated the largest volume of new bone among all the testing groups. Conclusions: Altogether, the results of this study provide the evidence that the combination of PEG-PLLA hydrogels with BMMSCs and sustained delivery of BMP2 resulted in the maximal bone regeneration.

Alginate-based materials have received considerable attention for biomedical applications because of their hydrophilic nature, biocompatibility, and physical architecture. Applications include cell encapsulation, drug delivery, stem cell culture, and tissue engineering scaffolds. In fact, clinical trials are currently being performed in which islets are encapsulated in PLO coated alginate microbeads as a treatment of type I diabetes. However, large numbers of islets are required for efficacy due to poor survival following transplantation. The ability to locally stimulate microvascular network formation around the encapsulated cells may increase their viability through improved transport of oxygen, glucose and other vital nutrients. Fibroblast growth factor-1 (FGF-1) is a naturally occurring growth factor that is able to stimulate blood vessel formation and improve oxygen levels in ischemic tissues. The efficacy of FGF-1 is enhanced when it is delivered in a sustained fashion rather than a single large-bolus administration. The local long-term release of growth factors from islet encapsulation systems could stimulate the growth of blood vessels directly towards the transplanted cells, potentially improving functional graft outcomes. In this article, we outline procedures for the preparation of alginate microspheres for use in biomedical applications. In addition, we describe a method we developed for generating multilayered alginate microbeads. Cells can be encapsulated in the inner alginate core, and angiogenic proteins in the outer alginate layer. The release of proteins from this outer layer would stimulate the formation of local microvascular networks directly towards the transplanted islets.

Biocompatible, degradable polymer scaffolds combined with cells or biological signals are being investigated as alternatives to traditional options for tissue reconstruction and transplantation. These approaches are already in clinical use as engineered tissues that enhance wound healing and skin regeneration. The continued enhancement of these material strategies is highly dependent on the ability to promote rapid and stable neovascularization (new blood vessel formation) within the scaffold. Whereas neovascularization therapies have shown some promise for the treatment of ischemic tissues, vascularization of polymer scaffolds in tissue engineering strategies provides a unique challenge owing to the volume and the complexity of the tissues targeted. In this article, we examine recent advances in research focused on promoting neovascularization in polymer scaffolds for tissue engineering applications. These approaches include the use of growth factors, cells, and novel surgical approaches to both enhance and control the nature of the vascular networks formed. The continued development of these approaches may lead to new tissue engineering strategies for the generation of skin and other tissues or organs.

Background Chimeric proteins may be used to direct cell-specific activity. Heparin-binding growth-associated molecule (HBGAM) binds to cell receptors that are relatively more robust on endothelial cells, and it may confer endothelial cell selectivity to potent angiogens such as fibroblast growth factor-1 (FGF-1). Methods By ligating fibroblast growth factor or its potent mutant, S130K, to HBGAM, we tested their effect on re-endothelialization after angioplasty injury by using a canine model. Results Both HBGAM/S130K- and HBGAM/FGF-1–treated arteries had increased neointimal mitotic index and re-endothelialization rates at 30 days compared with control arteries without inducing a significant increase in the neointimal thickness or the ratio of neointimal to medial thickness between treatment and control groups. Conclusion HBGAM/S130K and HBGAM/FGF-1 facilitates endothelial healing without myointimal thickening after canine carotid artery balloon angioplasty injury. Application of these growth factors in fibrin glue may improve endothelialization clinically after angioplasty or endarterectomy.

Background Therapeutic neovascularization using high growth factor concentrations may lead to transient vessel formation and abnormal microvascular structure. The goal of this study was to quantify temporal and concentration effects of fibroblast growth factor-1 (FGF-1) on the persistence and morphology of microvascular networks. Methods Endothelial cells were incubated in suspension culture forming aggregates that were embedded in fibrin glue (FG) and stimulated with varying concentrations of FGF-1 with of heparin. Capillary networks formed were quantified for 21 days. Results High FGF-1 concentrations resulted in rapid and intense sprout formation, with excessive branching. At later times, these vessels regressed, with cellular debris in former vessel locations. At later times, the 1-ng/mL group surpassed the high concentration groups with continuous sprout growth and complete FG vascularization by 23 days. Conclusion Sustained low levels of FGF-1 maintained a persistent microvascular network response, whereas higher levels resulted in abnormal phenotype followed by vessel regression.

Purpose The purpose of this study was to look at the spatial and temporal localization of secretory IgA in healing tooth extraction sockets in a rabbit model. Materials and methods Twenty-four male New Zealand White rabbits were used in the study. Incisor teeth were extracted from both jaws, and the healing extraction socket with the surrounding jaw bone was harvested at 48 hours, 4 days, and 1, 2, 4, 8, 12, and 16 weeks. Tissues were fixed, decalcified, and processed for hematoxylin and eosin and immunohistochemical staining. The sections were stained to detect secretory IgA. The stained sections were then imaged, and an automated computer program was used to detect the brown 3,3′-diaminobenzidine stain that represented the secretory IgA. The data were obtained in the form of percentage area and intensity of stain and analyzed using analysis of variance (Tukey-Kramer and Scheffe’s tests). Results Spatial and temporal differences in localization of secretory IgA were observed across time frames in both jaws. Conclusion The results of this study showed definite trends in the spatial and temporal localization of secretory IgA in healing tooth extraction sockets in a rabbit model.

The increased use of immunohistochemistry (IHC) in both clinical and basic research settings has led to the development of techniques for acquiring quantitative information from immunostains. Staining correlates with absolute protein levels and has been investigated as a clinical tool for patient diagnosis and prognosis. For these reasons, automated imaging methods have been developed in an attempt to standardize IHC analysis. We propose a novel imaging technique in which brightfield images of diaminobenzidene (DAB)-labeled antigens are converted to normalized blue images, allowing automated identification of positively stained tissue. A statistical analysis compared our method with seven previously published imaging techniques by measuring each one's agreement with manual analysis by two observers. Eighteen DAB-stained images showing a range of protein levels were used. Accuracy was assessed by calculating the percentage of pixels misclassified using each technique compared with a manual standard. Bland-Altman analysis was then used to show the extent to which misclassification affected staining quantification. Many of the techniques were inconsistent in classifying DAB staining due to background interference, but our method was statistically the most accurate and consistent across all staining levels.

This article is a review of the book “Imaging in Cellular and Tissue Engineering” (ISBN-13: 978-1439848036, $149.95, 298 Pages, 114 Illustrations) edited by Y Hanry Yu and Nur Aida Abdul Rahim published by the CRC Press (Taylor&Francis) in 2013. The contents of the book and its relevance to tissue engineering and regenerative medicine are discussed in this invited review.

Tissue engineering has the potential to change the way reconstructive surgery is practised as well as increased patient quality of life. This review illustrates a small fraction of the areas in reconstructive surgery that can benefit from tissue engineering. The authors, at the University of Texas M.D. Anderson Cancer Center, believe that the best way to bring this potential to fruition is by bringing together experts from the operating room, engineering, acedemia, and industry, and combining the abilities and knowledge of each to obtain results that could not be accomplished independently.

Background: Early division of the pedicled groin flap can be achieved by using ischemic preconditioning. The goal of this study was to investigate the devices available for ischemic preconditioning and determine which device is the most effective and results in lowest patient discomfort. Methods: Rubber bands, custom-made Orthoplast sheets, long-nose locking pliers, intestinal clamps, and a pneumatic tourniquet device were used for ischemic preconditioning on 13 patients who sustained severe hand injuries with reconstruction of pedicled groin flaps. The devices were compared by using laser Doppler flowmetry and the patient's local pain levels. Results: Twelve of 13 flaps were successfully divided at a mean period of 8.3 days by using a custom-made Orthoplast sheet or a pneumatic tourniquet device. All devices except the rubber bands could result in a biologic zero flow level. Conclusion: The pneumatic tourniquet device is the most desirable ischemic preconditioning device, having the advantages of excellent ischemic effect, easy application, and minimal discomfort.

Introduction: R136K is a protease-resistant mutant of the angiogen FGF-1 that was bound to a CBD to localize delivery and prolong bioavailability onto collagen matrices. The clinical aim is to induce re-endothelialization of injured intima. In vitro, R136K-CBD was added to canine jugular vein EC and canine carotid artery smooth muscle cell (SMC) cultures to evaluate mitogenic response. Methods: R136K was ligated to the CBD’s amino terminus and inserted as a plasmid into BL21 cells for induction. The chimera was isolated with chromatography and verified by amino acid sequencing. Using our standard proliferation assay cells were grown to 80% confluence, quiesced by serum starvation, and stimulated with FGF-1, R136K, or R136K-CBD in serial concentrations with and without heparin. H3-thymidine uptake over 24 hours was quantified and results were normalized as percent of positive control (20% FBS). The assay was repeated in duplicate for SMC and triplicate for EC. Results: EC response was significantly greater than SMCs at 0.6M with heparin (p 0.05). Conclusions: R136K-CBD retains its mitogenicity for EC and SMCs. Its EC specificity may decrease myointimal hyperplasia while promoting re-endothelialization. This chimera may induce EC proliferation on exposed vascular collagen after intimal injury and on bioengineered scaffolds that utilize collagen matrices.