Preformed Vascular Networks Survive and Enhance Vascularization in Critical Sized Cranial Defects.

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.

Large Animal Models of an In Vivo Bioreactor for Engineering Vascularized Bone.

Reconstruction of large skeletal defects is challenging due to the requirement for large volumes of donor tissue and the often complex surgical procedures. Tissue engineering has the potential to serve as a new source of tissue for bone reconstruction, but current techniques are often limited in regards to the size and complexity of tissue that can be formed. Building tissue using an in vivo bioreactor approach may enable the production of appropriate amounts of specialized tissue, while reducing issues of donor site morbidity and infection. Large animals are required to screen and optimize new strategies for growing clinically appropriate volumes of tissues in vivo. In this article, we review both ovine and porcine models that serve as models of the technique proposed for clinical engineering of bone tissue in vivo. Recent findings are discussed with these systems, as well as description of next steps required for using these models, to develop clinically applicable tissue engineering applications.

Inhibiting intimal hyperplasia in prosthetic vascular grafts via immobilized all-trans retinoic acid.

Peripheral arterial disease is a leading cause of morbidity and mortality. The most commonly utilized prosthetic material for peripheral bypass grafting is expanded polytetrafluoroethylene (ePTFE) yet it continues to exhibit poor performance from restenosis due to neointimal hyperplasia, especially in femoral distal bypass procedures. Recently, we demonstrated that periadventitial delivery of all-trans retinoic acid (atRA) immobilized throughout porous poly(1,8 octamethylene citrate) (POC) membranes inhibited neointimal formation in a rat arterial injury model. Thus, the objective of this study was to investigate whether atRA immobilized throughout the lumen of ePTFE vascular grafts would inhibit intimal formation following arterial bypass grafting. Utilizing standard ePTFE, two types of atRA-containing ePTFE vascular grafts were fabricated and evaluated: grafts whereby all-trans retinoic acid was directly immobilized on ePTFE (atRA-ePTFE) and grafts where all-trans retinoic acid was immobilized onto ePTFE grafts coated with POC (atRA-POC-ePTFE). All grafts were characterized by SEM, HPLC, and FTIR and physical characteristics were evaluated in vitro. Modification of these grafts, did not significantly alter their physical characteristics or biocompatibility, and resulted in inhibition of intimal formation in a rat aortic bypass model, with atRA-POC-ePTFE inhibiting intimal formation at both the proximal and distal graft sections. In addition, treatment with atRA-POC-ePTFE resulted in increased graft endothelialization and decreased inflammation when compared to the other treatment groups. This work further confirms the biocompatibility and efficacy of locally delivered atRA to inhibit intimal formation in a bypass setting. Thus, atRA-POC-ePTFE grafts have the potential to improve patency rates in small diameter bypass grafts and warrant further investigation.

Synthesis and evaluation of dual crosslinked alginate microbeads.

Alginate hydrogels have been investigated for a broad variety of medical applications. The ability to assemble hydrogels at neutral pH and mild temperatures makes alginate a popular choice for the encapsulation and delivery of cells and proteins. Alginate has been studied extensively for the delivery of islets as a treatment for type 1 diabetes. However, poor stability of the encapsulation systems after implantation remains a challenge. In this paper, alginate was modified with 2-aminoethyl methacrylate hydrochloride (AEMA) to introduce groups that can be photoactivated to generate covalent bonds. This enabled formation of dual crosslinked structure upon exposure to ultraviolet light following initial ionic crosslinking into bead structures. The degree of methacrylation was varied and in vitro stability, long term swelling, and cell viability examined. At low levels of the methacrylation, the beads could be formed by first ionic crosslinks followed by exposure to ultraviolet light to generate covalent bonds. The methacrylated alginate resulted in more stable beads and cells were viable following encapsulation. Alginate microbeads, ionic (unmodified) and dual crosslinked, were implanted into a rat omentum pouch model. Implantation was performed with a local injection of 100 µl of 50 µg/ml of Lipopolysaccharide (LPS) to stimulate a robust inflammatory challenge in vivo. Implants were retrieved at 1 and 3 weeks for analysis. The unmodified alginate microbeads had all failed by week 1, whereas the dual-crosslinked alginate microbeads remained stable up through 3 weeks. The modified alginate microbeads may provide a more stable alternative to current alginate-based systems for cell encapsulation. STATEMENT OF SIGNIFICANCE: Alginate, a naturally occurring polysaccharide, has been used for cell encapsulation to prevent graft rejection of cell transplants for people with type I diabetes. Although some success has been observed in clinical trials, the lack of reproducibility and failure to reach insulin dependence for longer periods of time indicates the need for improvements in the procedure. A major requirement for the long-term function of alginate encapsulated cells is the mechanical stability of microcapsules. Insufficient mechanical integrity of the capsules can lead to immunological reactions in the recipients. In this work, alginate was modified to allow photoactivatable groups in order to allow formation of covalent crosslinks in addition to ionic crosslinking. The dual crosslinking design prevents capsule breakdown following implantation in vivo.

Investigation of DNA damage in cells exposed to poly (lactic-co-glycolic acid) microspheres.

Poly (lactic-co-glycolic acid) (PLGA)-based materials are widely investigated for drug delivery and tissue engineering applications. Despite their popularity the genotoxic potential of PLGA has not been investigated. In this study, the comet assay, a sensitive assay for DNA damage, was used to evaluate potential genotoxicity in model cell types exposed to PLGA microspheres. Human umbilical vein endothelial cells (HUVECs) and mesenchymal stem cells (MSCs) cells were exposed to PLGA microspheres (0.4-6 mg/mL) and DNA damage assessed at 24 h, 4 days, and 7 days. DNA damage was not identified after 24 h. However, after 4 and 7 days of exposure to 2 and 6 mg/mL of PLGA microspheres a significant elevation of DNA damage in both cell types was observed. The PLGA microspheres did not exhibit any cytotoxic effects on the cells under the conditions tested. Our results suggest that PLGA may have a genotoxic effect on cells. A broader investigation of the PLGA genotoxic profile in biological systems is needed. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 284-291, 2017.

Computational Model-Based Analysis of Strategies to Enhance Scaffold Vascularization.

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.

Agent-based modeling of porous scaffold degradation and vascularization: Optimal scaffold design based on architecture and degradation dynamics.

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 angiogenesis. 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.

Biomaterials with persistent growth factor gradients in vivo accelerate vascularized tissue formation.

Gradients of soluble factors play an important role in many biological processes, including blood vessel assembly. Gradients can be studied in detail in vitro, but methods that enable the study of spatially distributed soluble factors and multi-cellular processes in vivo are limited. Here, we report on a method for the generation of persistent in vivo gradients of growth factors in a three-dimensional (3D) biomaterial system. Fibrin loaded porous poly (ethylene glycol) (PEG) scaffolds were generated using a particulate leaching method. Platelet derived growth factor BB (PDGF-BB) was encapsulated into poly (lactic-co-glycolic acid) (PLGA) microspheres which were placed distal to the tissue-material interface. PLGA provides sustained release of PDGF-BB and its diffusion through the porous structure results in gradient formation. Gradients within the scaffold were confirmed in vivo using near-infrared fluorescence imaging and gradients were present for more than 3 weeks. The diffusion of PDGF-BB was modeled and verified with in vivo imaging findings. The depth of tissue invasion and density of blood vessels formed in response to the biomaterial increased with magnitude of the gradient. This biomaterial system allows for generation of sustained growth factor gradients for the study of tissue response to gradients in vivo.

Pore Interconnectivity Influences Growth Factor-Mediated Vascularization in Sphere-Templated Hydrogels.

Rapid and controlled vascularization within biomaterials is essential for many applications in regenerative medicine. The extent of vascularization is influenced by a number of factors, including scaffold architecture. While properties such as pore size and total porosity have been studied extensively, the importance of controlling the interconnectivity of pores has received less attention. A sintering method was used to generate hydrogel scaffolds with controlled pore interconnectivity. Poly(methyl methacrylate) microspheres were used as a sacrificial agent to generate porous poly(ethylene glycol) diacrylate hydrogels with interconnectivity varying based on microsphere sintering conditions. Interconnectivity levels increased with sintering time and temperature with resultant hydrogel structure showing agreement with template structure. Porous hydrogels with a narrow pore size distribution (130-150 µm) and varying interconnectivity were investigated for their ability to influence vascularization in response to gradients of platelet-derived growth factor-BB (PDGF-BB). A rodent subcutaneous model was used to evaluate vascularized tissue formation in the hydrogels in vivo. Vascularized tissue invasion varied with interconnectivity. At week 3, higher interconnectivity hydrogels had completely vascularized with twice as much invasion. Interconnectivity also influenced PDGF-BB transport within the scaffolds. An agent-based model was used to explore the relative roles of steric and transport effects on the observed results. In conclusion, a technique for the preparation of hydrogels with controlled pore interconnectivity has been developed and evaluated. This method has been used to show that pore interconnectivity can independently influence vascularization of biomaterials.

Agent-based modeling of osteogenic differentiation of mesenchymal stem cells in porous biomaterials.

Mesenchymal stem cells (MSC) have shown promise in tissue engineering applications due to their potential for differentiating into mesenchymal tissues such as osteocytes, chondrocytes, and adipocytes and releasing proteins to promote tissue regeneration. One application involves seeding MSCs in biomaterial scaffolds to promote osteogenesis in the repair of bone defects following implantation. However, predicting in vivo survival and differentiation of MSCs in biomaterials is challenging. Rapid and stable vascularization of scaffolds is required to supply nutrients and oxygen that MSCs need to survive as well as to go through osteogenic differentiation. The objective of this study is to develop an agent-based model and simulator that can be used to investigate the effects of using gradient growth factors on survival and differentiation of MSCs seeded in scaffolds. An agent-based model is developed to simulate the MSC behavior. The effect of vascular endothelial growth factor (VEGF) and bone morphogenic protein-2 (BMP-2) on both survival and osteogenic differentiation is studied. Results showed that the survival ratio of MSCs can be enhanced by increasing VEGF concentration. BMP-2 caused a slight increase on survival ratio. Osteogenesis strongly depends on the VEGF concentration as well because of its effect on vascularization. BMP-2 increased the osteogenic differentiation of MSCs.