Eric M. Brey

Imaging challenges in biomaterials and tissue engineering

Biomaterials are employed in the fields of tissue engineering and regenerative medicine (TERM) in order to enhance the regeneration or replacement of tissue function and/or structure. The unique environments resulting from the presence of biomaterials, cells, and tissues result in distinct challenges in regards to monitoring and assessing the results of these interventions. Imaging technologies for three-dimensional (3D) analysis have been identified as a strategic priority in TERM research. Traditionally, histological and immunohistochemical techniques have been used to evaluate engineered tissues. However, these methods do not allow for an accurate volume assessment, are invasive, and do not provide information on functional status. Imaging techniques are needed that enable non-destructive, longitudinal, quantitative, and three-dimensional analysis of TERM strategies. This review focuses on evaluating the application of available imaging modalities for assessment of biomaterials and tissue in TERM applications. Included is a discussion of limitations of these techniques and identification of areas for further development.

The role of pore size on vascularization and tissue remodeling in PEG hydrogels

Vascularization is influenced by the physical architecture of a biomaterial. The relationship between pore size and vascularization has been examined for hydrophobic polymer foams, but there has been little research on tissue response in porous hydrogels. The goal of this study was to examine the role of pore size on vessel invasion in porous poly(ethylene glycol) (PEG) hydrogels. Vascularized tissue ingrowth was examined using three-dimensional cell culture and rodent models. In culture, all porous gels supported vascular invasion with the rate increasing with pore size. Following subfascial implantation, porous gels rapidly absorbed wound fluid, which promoted tissue ingrowth even in the absence of exogenous growth factors. Pore size influenced neovascularization, within the scaffolds and also the overall tissue response. Cell and vessel invasion into gels with pores 25-50 μm in size was limited to the external surface, while gels with pores larger pores (50-100 and 100-150 μm) permitted mature vascularized tissue formation throughout the entire material volume. A thin layer of inflammatory tissue was present at all PEG-tissue interfaces, effectively reducing the area available for tissue growth. These results show that porous PEG hydrogels can support extensive vascularized tissue formation, but the nature of the response depends on the pore size.

The effect of FGF-1 loaded alginate microbeads on neovascularization and adipogenesis in a vascular pedicle model of adipose tissue engineering

Engineered vascularized adipose tissue could serve as an alternative to traditional tissue reconstruction procedures. Adipose formation occurs in a coordinated fashion with neovascularization. Previous studies have shown that extracellular matrix-based materials supplemented with factors that stimulate neovascularization promote adipogenesis in a number of animal models. The present study examines the ability of fibroblast growth factor (FGF-1) delivered from alginate microbeads to induce neovascularization and adipogenesis in type I collagen gels in a vascular pedicle model of adipose tissue engineering. FGF-1 loaded microbeads stimulated greater vascular network formation in an in vitro 3D co-culture model than a single bolus of FGF-1. In in vivo studies, FGF-1 loaded beads suspended in collagen and implanted in a chamber surrounding the exposed femoral pedicle of a rat resulted in a significant increase in vascular density at 1 and 6 weeks in comparison to bolus administration of FGF-1. Staining for smooth muscle actin showed that over 48% of vessels had associated mural cells. While an increase in neovascularization was achieved, there was less than 3% adipose under any condition. These results show that delivery of FGF-1 from alginate beads stimulated a more persistent neovascularization response than bolus FGF-1 both in vitro and in vivo. However, unlike previous studies, this increased neovascularization did not result in adipogenesis. Future studies need to provide a better understanding of the relationship between neovascularization and adipogenesis in order to design advanced tissue engineering therapies.

Three-dimensional modeling of angiogenesis in porous biomaterial scaffolds

Vascularization of biomaterial scaffolds is essential for the successful clinical application of engineered tissues. Experimental studies are often performed to investigate the role of scaffold architecture on vascularized tissue formation. However, experiments are expensive and time-consuming and synthesis protocols often do not allow for independent investigation of specific scaffold properties. Computational models allow for rapid screening of potential material designs with control over scaffold properties that is difficult in laboratory settings. We have developed and tested a three-dimensional agent-based framework for investigating the effect of scaffold pore architecture on angiogenesis. Software agents represent endothelial cells, interacting together and with their micro-environment, leading to the invasion of blood vessels into the scaffold. A rule base, driven by experimental findings, governs the behavior of individual agents. 3D scaffold models with well-defined homogeneous and heterogeneous pore architectures were simulated to investigate the impact of various design parameters. Simulation results indicate that pores of larger size with higher interconnectivity and porosity support rapid and extensive angiogenesis. The developed framework can be used to screen biomaterial scaffold designs for optimal vascularization and investigate complex interactions among invading blood vessels and their micro-environment.

Fibroblast Growth Factor-1 (FGF-1) Loaded Microbeads Enhance Local Capillary Neovascularization

BACKGROUNDnGrowth of new blood vessels (neovascularization) occurs naturally in the body, but the slow rate of the process may not be sufficient for survival of engineered tissues and transplanted cells, such as pancreatic islets. For transplanted islets, it is crucial that the transplantation site has sufficient vasculature to support the needs of the islets. Therefore, the specific aim of this research was quantify the effect of FGF-1 incorporation into alginate microbeads on neovascularization of such capsules in an in vivo rat transplant model.nnnMATERIALS AND METHODSnMicrobeads loaded with FGF-1 or control beads (beads without FGF-1) were implanted in the rat omental pouch model. Animals were sacrificed 7 d post-implantation.nnnRESULTSnMicrobeads loaded with FGF-1 stimulated a significant increase in vascular density compared with control rats implanted with control beads.nnnCONCLUSIONSnThese results indicate that alginate microbeads loaded with FGF-1 enhance local neovascularization around implanted microbeads. These data provide a compelling impetus for experimental pursuit of FGF-loaded alginate microcapsules for vascularization of transplanted islets.

Evaluation of Physical and Mechanical Properties of Porous Poly (Ethylene Glycol)-co-(L-Lactic Acid) Hydrogels during Degradation

Porous hydrogels of poly(ethylene glycol) (PEG) have been shown to facilitate vascularized tissue formation. However, PEG hydrogels exhibit limited degradation under physiological conditions which hinders their ultimate applicability for tissue engineering therapies. Introduction of poly(L-lactic acid) (PLLA) chains into the PEG backbone results in copolymers that exhibit degradation via hydrolysis that can be controlled, in part, by the copolymer conditions. In this study, porous, PEG-PLLA hydrogels were generated by solvent casting/particulate leaching and photopolymerization. The influence of polymer conditions on hydrogel architecture, degradation and mechanical properties was investigated. Autofluorescence exhibited by the hydrogels allowed for three-dimensional, non-destructive monitoring of hydrogel structure under fully swelled conditions. The initial pore size depended on particulate size but not polymer concentration, while degradation time was dependent on polymer concentration. Compressive modulus was a function of polymer concentration and decreased as the hydrogels degraded. Interestingly, pore size did not vary during degradation contrary to what has been observed in other polymer systems. These results provide a technique for generating porous, degradable PEG-PLLA hydrogels and insight into how the degradation, structure, and mechanical properties depend on synthesis conditions.

Stability of alginate microbead properties in vitro.

Alginate microbeads have been investigated clinically for a number of therapeutic interventions, including drug delivery for treatment of ischemic tissues, cell delivery for tissue regeneration, and islet encapsulation as a therapy for type I diabetes. The physical properties of the microbeads play an important role in regulating cell behavior, protein release, and biological response following implantation. In this research alginate microbeads were synthesized, varying composition (mannuronic acid to guluronic acid ratio), concentration of alginate and needle gauge size. Following synthesis, the size, volume fraction, and morphometry of the beads were quantified. In addition, these properties were monitored over time in vitro in the presence of varying calcium levels in the microenvironment. The initial volume available for solute diffusion increased with alginate concentration and mannuronic (M) acid content, and bead diameter decreased with M content but increased with needle diameter. Interestingly, microbeads eroded completely in saline in less than 3xa0weeks regardless of synthesis conditions much faster than what has been observed in vivo. However, microbead stability was increased by the addition of calcium in the culture medium. Beads synthesized with low alginate concentration and high G content exhibited a more rapid change in physical properties even in the presence of calcium. These data suggest that temporal variations in the physical characteristics of alginate microbeads can occur in vitro depending on synthesis conditions and microbead environment. The results presented here will assist in optimizing the design of the materials for clinical application in drug delivery and cell therapy.

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 inxa0vitro, but methods that enable the study of spatially distributed soluble factors and multi-cellular processes inxa0vivo are limited. Here, we report on a method for the generation of persistent inxa0vivo 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 inxa0vivo using near-infrared fluorescence imaging and gradients were present for more than 3 weeks. The diffusion of PDGF-BB was modeled and verified with inxa0vivo 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 inxa0vivo.

Materials for engineering vascularized adipose tissue

UNLABELLEDnLoss 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.nnnCLINICAL RELEVANCEnMaterials 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.

A study of the intrinsic autofluorescence of poly (ethylene glycol)-co-(L-lactic acid) diacrylate.

Poly (ethylene glycol)-co-(L-Lactic acid) diacrylate (PEG-PLLA-DA) copolymers have been extensively investigated for a number of applications in medicine. PEG-PLLA-DA is biodegradable and the human body can process its degradation products. In this study, we describe the autofluorescence of PEG-PLLA-DA copolymers and compared it to the fluorescence of poly(ethylene glycol) diacrylate (PEG-DA) and the precursor molecules used for their synthesis. In addition, we examined the influence of pH on the fluorescence spectra. We found that PEG-PLLA-DA exhibits higher fluorescence than PEG-DA and all reagents involved in the synthesis of PEG-PLLA-DA. The fluorescence of PEG-PLLA-DA was affected by pH with fluorescence decreasing at high pH values. At high pH, PEG-PLLA-DA could not polymerize into hydrogels and exhibited a dramatic decrease in autofluorescence, suggesting that hydrolysis of the ester bond affected its autofluorescence. At low pH, PEG-PLLA-DA exhibited higher fluorescence and it was able to form crosslinked hydrogels. The autofluorescence of PEG-PLLA-DA could be exploited to monitor polymer degradation and material structure without the need to introduce exogenous fluorescent probes. The origin of fluorescence is not clear at this point in time but it appears to result from a synergetic effect of both lactate units and diacrylate groups in the PEG-PLLA-DA backbone. The observed autofluorescence of PEG-PLLA-DA persists after reaction of the acrylate groups in the polymerization reaction. This autofluorescence is advantageous because it could assist in the study of polymers used for drug delivery and tissue engineering applications.