Evaluation of gelatin bloom strength on gelatin methacryloyl hydrogel properties.

Gelatin methacryloyl (GelMA) hydrogels are widely used for a variety of tissue engineering applications. The properties of gelatin can affect the mechanical properties of gelatin gels; however, the role of gelatin properties such as bloom strength on GelMA hydrogels has not yet been explored. Bloom strength is a food industry standard for describing the quality of gelatin, where higher bloom strength is associated with higher gelatin molecular weight. Here, we evaluate the role of bloom strength on GelMA hydrogel mechanical properties. We determined that both bloom strength of gelatin and weight percent of GelMA influenced both stiffness and viscoelastic ratio; however, only bloom strength affected diffusivity, permeability, and pore size. With this library of GelMA hydrogels of varying properties, we then encapsulated Swan71 trophoblast spheroids in these hydrogel variants to assess how bloom strength affects trophoblast spheroid morphology. Overall, we observed a decreasing trend of spheroid area and Feret diameter as bloom strength increased. In identifying clear relationships between bloom strength, hydrogel mechanical properties, and trophoblast spheroid morphology, we demonstrate that bloom strength should considered when designing tissue engineered constructs.

Evaluation of gelatin bloom strength on gelatin methacryloyl hydrogel properties.

Gelatin methacryloyl (GelMA) hydrogels are widely used for a variety of tissue engineering applications. The properties of gelatin can affect the mechanical properties of gelatin gels; however, the role of gelatin properties such as bloom strength on GelMA hydrogels has not yet been explored. Bloom strength is a food industry standard for describing the quality of gelatin, where higher bloom strength is associated with higher gelatin molecular weight. Here, we evaluate the role of bloom strength on GelMA hydrogel mechanical properties. We determined that both bloom strength of gelatin and weight percent of GelMA influenced both stiffness and viscoelastic ratio; however, only bloom strength affected diffusivity, permeability, and pore size. With this library of GelMA hydrogels of varying properties, we then encapsulated Swan71 trophoblast spheroids in these hydrogel variants to assess how bloom strength affects trophoblast spheroid morphology. Overall, we observed a decreasing trend of spheroid area and Feret diameter as bloom strength increased. In identifying clear relationships between bloom strength, hydrogel mechanical properties, and trophoblast spheroid morphology, we demonstrate that bloom strength should considered when designing tissue engineered constructs.

Tumor Spheroid Fabrication and Encapsulation in Polyethylene Glycol Hydrogels for Studying Spheroid-Matrix Interactions.

Three-dimensional (3D) encapsulation of spheroids is crucial to adequately replicate the tumor microenvironment for optimal cell growth. Here, we designed an in vitro 3D glioblastoma model for spheroid encapsulation to mimic the tumor extracellular microenvironment. First, we formed square pyramidal microwell molds using polydimethylsiloxane. These microwell molds were then used to fabricate tumor spheroids with tightly controlled sizes from 50-500 µm. Once spheroids were formed, they were harvested and encapsulated in polyethylene glycol (PEG)-based hydrogels. PEG hydrogels are a versatile platform for spheroid encapsulation, as hydrogel properties such as stiffness, degradability, and cell adhesiveness can be tuned independently. Here, we used a representative soft (~8 kPa) hydrogel to encapsulate glioblastoma spheroids. Finally, a method to stain and image spheroids was developed to obtain high-quality images via confocal microscopy. Due to the dense spheroid core and relatively sparse periphery, imaging can be difficult, but using a clearing solution and confocal optical sectioning helps alleviate these imaging difficulties. In summary, we show a method to fabricate uniform spheroids, encapsulate them in PEG hydrogels and perform confocal microscopy on the encapsulated spheroids to study spheroid growth and various cell-matrix interactions.

Hydrogel Delivery Device for the In Vitro and In Vivo Sustained Release of Active rhGALNS Enzyme.

Morquio A disease is a genetic disorder resulting in N-acetylgalactosamine-6-sulfate sulfatase (GALNS) deficiency, and patients are currently treated with enzyme replacement therapy via weekly intravenous enzyme infusions. A means of sustained enzyme delivery could improve patient quality of life by reducing the administration time, frequency of hospital visits, and treatment cost. In this study, we investigated poly(ethylene-glycol) (PEG) hydrogels as a tunable, hydrolytically degradable drug delivery system for the encapsulation and sustained release of recombinant human GALNS (rhGALNS). We evaluated hydrogel formulations that optimized hydrogel gelation and degradation time while retaining rhGALNS activity and sustaining rhGALNS release. We observed the release of active rhGALNS for up to 28 days in vitro from the optimized formulation. rhGALNS activity was preserved in the hydrogel relative to buffer over the release window, and encapsulation was found to have no impact on the rhGALNS structure when measured by intrinsic fluorescence, circular dichroism, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). In vivo, we monitored the retention of fluorescently labeled rhGALNS in C57BL/6 albino mice when administered via subcutaneous injection and observed rhGALNS present for up to 20 days when delivered in a hydrogel versus 7 days in the buffer control. These results indicate that PEG hydrogels are suitable for the encapsulation, preservation, and sustained release of recombinant enzymes and may present an alternative method of delivering enzyme replacement therapies that improve patient quality of life.

Mechanobiology of cancer cell responsiveness to chemotherapy and immunotherapy: Mechanistic insights and biomaterial platforms.

Mechanical forces are central to how cancer treatments such as chemotherapeutics and immunotherapies interact with cells and tissues. At the simplest level, electrostatic forces underlie the binding events that are critical to therapeutic function. However, a growing body of literature points to mechanical factors that also affect whether a drug or an immune cell can reach a target, and to interactions between a cell and its environment affecting therapeutic efficacy. These factors affect cell processes ranging from cytoskeletal and extracellular matrix remodeling to transduction of signals by the nucleus to metastasis of cells. This review presents and critiques the state of the art of our understanding of how mechanobiology impacts drug and immunotherapy resistance and responsiveness, and of the in vitro systems that have been of value in the discovery of these effects.

Multimodal peptide ligand extracts parvovirus from interface in affinity aqueous two-phase system.

Aqueous two-phase systems (ATPS) have found various applications in bioseparations and microencapsulation. The primary goal of this technique is to partition target biomolecules in a preferred phase, rich in one of the phase-forming components. However, there is a lack of understanding of biomolecule behavior at the interface between the two phases. Biomolecule partitioning behavior is studied using tie-lines (TL), where each TL is a group of systems at thermodynamic equilibrium. Across a TL, a system can either have a bulk PEG-rich phase with citrate-rich droplets, or the opposite can occur. We found that porcine parvovirus (PPV) was recovered at a higher amount when PEG was the bulk phase and citrate was in droplets and that the salt and PEG concentrations are high. To improve the recovery, A PEG 10 kDa-peptide conjugate was formed using the multimodal WRW ligand. When WRW was present, less PPV was caught at the interface of the two-phase system, and more was recovered in the PEG-rich phase. While WRW did not significantly increase the PPV recovery in the high TL system, which was found earlier to be optimal for PPV recovery, the peptide did greatly enhance recovery at a lower TL. This lower TL has a lower viscosity and overall system PEG and citrate concentration. The results provide both a method to increase virus recovery in a lower viscosity system, as well as provide interesting thoughts into the interfacial phenomenon and how to recover virus in a phase and not at the interface.

Glioblastoma spheroid growth and chemotherapeutic responses in single and dual-stiffness hydrogels.

Glioblastoma (GBM) is the deadliest brain tumor for which there is no cure. Bioengineered GBM models, such as hydrogel-encapsulated spheroids, that capture both cell-cell and cell-matrix interactions could facilitate testing of much needed therapies. Elucidation of specific microenvironment properties on spheroid responsiveness to therapeutics would enhance the usefulness of GBM models as predictive drug screening platforms. Here, GBM spheroids consisting of U87 or patient-derived GBM cells were encapsulated in soft (∼1 kPa), stiff (∼7 kPa), and dual-stiffness polyethylene glycol-based hydrogels, with GBM spheroids seeded at the stiffness interface. Spheroids were cultured for 7 days and examined for viability, size, invasion, laminin expression, hypoxia, proliferation, and response to the chemotherapeutic temozolomide (TMZ). We noted excellent cell viability in all hydrogels, and higher infiltration in soft compared to stiff hydrogels for U87 spheroids. In dual gels spheroids mostly infiltrated away from the stiffness interface with minimal crossing over it and some individual cell migration along the interface. U87 spheroids were equally responsive to TMZ in the soft and stiff hydrogels, but cell viability in the spheroid periphery was higher than the core for stiff hydrogels whereas the opposite was true for soft hydrogels. HIF1A expression was higher in the core of spheroids in the stiff hydrogels, while there was no difference in cell proliferation between spheroids in the stiff vs soft hydrogels. Patient-derived GBM spheroids did not show stiffness-dependent drug responses. U87 cells showed similar laminin expression in soft and stiff hydrogels with higher expression in the spheroid periphery compared to the core. Our results indicate that microenvironment stiffness needs to be considered in bioengineered GBM models including those designed for use in drug screening applications. STATEMENT OF SIGNIFICANCE: Recent work on tumor models engineered for use in drug screening has highlighted the potential of hydrogel-encapsulated spheroids as a simple, yet effective platform that show drug responses similar to native tumors. It has also been shown that substrate stiffness, in vivo and in vitro, affects cancer cell responses to drugs. This is particularly important for glioblastoma (GBM), the deadliest brain cancer, as GBM cells invade by following the stiffer brain structures such as white matter tracks and the perivascular niche. Invading cells have also been associated with higher resistance to chemotherapy. Here we developed GBM spheroid models using soft, stiff and dual-stiffness hydrogels to explore the connection between substrate stiffness, spheroid invasion and drug responsiveness in a controlled environment.

Hydrogel-based microfluidic device with multiplexed 3D in vitro cell culture.

Microfluidic devices that combine an extracellular matrix environment, cells, and physiologically relevant perfusion, are advantageous as cell culture platforms. We developed a hydrogel-based, microfluidic cell culture platform by loading polyethylene glycol (PEG) hydrogel-encapsulated U87 glioblastoma cells into membrane-capped wells in polydimethyl siloxane (PDMS). The multilayer microfluidic cell culture system combines previously reported design features in a configuration that loads and biomimetically perfuses a 2D array of cell culture chambers. One dimension of the array is fed by a microfluidic concentration gradient generator (MCGG) while the orthogonal dimension provides loading channels that fill rows of cell culture chambers in a separate layer. In contrast to typical tree-like MCGG mixers, a fractional serial dilution of 1, ½, », and 0 of the initial solute concentration is achieved by tailoring the input microchannel widths. Hydrogels are efficiently and reproducibly loaded in all wells and cells are evenly distributed throughout the hydrogel, maintaining > 90% viability for up to 4 days. In a drug screening assay, diffusion of temozolomide and carmustine to hydrogel-encapsulated U87 cells from the perfusion solution is measured, and dose-response curves are generated, demonstrating utility as an in vitro mimic of the glioblastoma microenvironment.

CCL21 and beta-cell antigen releasing hydrogels as tolerance-inducing therapy in Type I diabetes.

Type-I Diabetes (T1D) is caused by defective immunotolerance mechanisms enabling autoreactive T cells to escape regulation in lymphoid organs and destroy insulin-producing ß-cells in the pancreas, leading to insulin dependence. Strategies to promote ß-cell tolerance could arrest T1D. We previously showed that secretion of secondary lymphoid chemokine CCL21 by CCL21 transgenic ß-cells induced tolerance and protected non-obese diabetic (NOD) mice from T1D. T1D protection was associated with formation of lymph node-like stromal networks containing tolerogenic fibroblastic reticular cells (FRCs). Here, we developed a polyethylene glycol (PEG) hydrogel platform with hydrolytically degradable PEG-diester dithiol crosslinkers to provide controlled and sustained delivery of CCL21 and ß-cell antigens for at least 28 days in vitro and recapitulate properties associated with the tolerogenic environment of CCL21 transgenic ß-cells in our previous studies. CCL21 and MHC-II restricted antigens were tethered to gels via simple click-chemistry while MHC-I restricted antigens were loaded in PEG-based polymeric nanovesicles and incorporated in the gel networks. CCL21 and antigen release kinetics depended on the PEG gel tethering strategy and the linkers. Importantly, in vitro functionality, chemotaxis, and activation of antigen-specific T cells were preserved. Implantation of CCL21 and ß-cell antigen gels under the kidney capsule of pre-diabetic NOD mice led to enrichment of adoptively transferred antigen-specific T cells, formation of gp38 + FRC-like stromal cell networks, and increased regulation of specific T cells with reduced accumulation within pancreatic islets. Thus, our platform for sustained release of ß-cell antigens and CCL21 immunomodulatory molecule could enable the development of antigen-specific tolerance therapies for T1D.

Microfluidic fabrication of imageable and resorbable polyethylene glycol microspheres for catheter embolization.

Radiopaque and degradable hydrogel microspheres have a range of potential uses in medicine including proper placement of embolic material during occlusion procedures, acting as inherently embolic materials, and serving as drug carriers that can be located after injection. Current methods for creating radiopaque microspheres are either unable to fully and homogeneously incorporate radiopaque material throughout the microspheres for optimal imaging capabilities, do not result in degradable or fully compressible microspheres, or require elaborate, time-consuming preparation. We used a simple one-step microfluidic method to fabricate imageable, degradable polyethylene glycol (PEG) microspheres of varying sizes with homogenous dispersion of barium sulfate-a biocompatible, high-radiopacity contrast agent. The imageability of the microspheres was characterized using optical microscopy and microcomputed tomography as a function of barium sulfate loading. Microspheres with 20% wt/vol barium sulfate had a mean CT attenuation value of 1,510 HU, similar to that of cortical bone, which should enable visualization with soft tissue. Compared with unloaded microspheres, barium sulfate-loaded ones saw an increase in gelation and degradation times and storage modulus and decrease in swelling. Imageable microspheres retained compressibility and were injectable via catheter. The developed radiopaque, degradable PEG microspheres have various potential uses for interventional radiologists and imaging laboratories.

Synthesis and Characterization of BaSO4-CaCO3-Alginate Nanocomposite Materials as Contrast Agents for Fine Vascular Imaging.

Microcomputed tomography is an important technique for distinguishing the vascular network from tissues with similar X-ray attenuation. Here, we describe a composite of barium sulfate (BaSO4) nanoparticles, calcium carbonate (CaCO3) nanoparticles, and alginate that provides improved performance over microscale BaSO4 particles, which are currently used clinically as X-ray contrast agents. BaSO4 and CaCO3 nanoparticles were synthesized using a polyol method with tetraethylene glycol as solvent and capping agent. The nanoparticles show good colloidal stability in aqueous solutions. A deliverable nanocomposite gel contrast agent was produced by encapsulation of the BaSO4 and CaCO3 nanoparticles in an alginate gel matrix. The gelation time was controlled by addition of d-(+)-gluconic acid δ-lactone, which controls the rate of dissolution of the CaCO3 nanoparticles that produce Ca2+ which cross-links the gel. Rapid cross-linking of the gel by Ba2+ was minimized by producing BaSO4 nanoparticles with an excess of surface sulfate. The resulting BaSO4-CaCO3 nanoparticle alginate gel mechanical properties were characterized, including the gel storage modulus, peak stress and elastic modulus, and radiodensity. The resulting nanocomposite has good viscosity control and good final gel stiffness. The nanocomposite has gelation times between 30 and 35 min, adequate for full body perfusion. This is the first nanoscale composite of a radiopaque metal salt to be developed in combination with an alginate hydrogel and designed for medical perfusion and vascular imaging applications.

Stability of proteins encapsulated in Michael-type addition polyethylene glycol hydrogels.

Degradable polyethylene glycol (PEG) hydrogels are excellent vehicles for sustained drug release due to their biocompatibility, tunable physical properties, and customizable degradation. However, protein therapeutics are unstable under physiological conditions and releasing degraded or inactive therapeutics can induce immunogenic effects. While controlling protein release from PEG hydrogels has been extensively investigated, few studies have detailed protein stability long-term or under stress conditions. Here, lysozyme and alcohol dehydrogenase (ADH) stability were explored upon encapsulation in PEG hydrogels formed through Michael-type addition. The stability and structure of the two model proteins were monitored by measuring the free energy of unfolding and fluoresce quenching when confined in a hydrogel and compared to PEG solution and buffer. Hydrogels destabilized lysozyme structure at low denaturant concentrations but prevented complete unfolding at high concentrations. ADH was stabilized as the confining mesh size approached the protein radius of gyration. Both proteins retained enzymatic activity within the hydrogels under stress conditions, including denaturant, high temperature, and agitation. Conjugation between lysozyme and PEG-acrylate was identified at long reaction times but no conjugation was observed in the time required for complete gelation. Studies of protein stability in PEG hydrogels, as the one detailed here, can lead to designer technologies for the improved formulation, storage, and delivery of protein therapeutics.

Microfluidic Chip Device for In Situ Mixing and Fabrication of Hydrogel Microspheres via Michael-Type Addition.

Hydrogel microspheres are sought for a variety of biomedical applications, including therapeutic and cellular delivery, sensors, and lubricants. Robust fabrication of hydrogel microspheres with uniform sizes and properties can be achieved using microfluidic systems that rely on droplet formation and subsequent gelation to form microspheres. Such systems work well when gelation is initiated after droplet formation but are not practical for timed gelation systems where gelation is initiated prior to droplet formation; premature gelation can lead to device blockage, variable microsphere diameter due to viscosity changes in the precursor solution, and limited numbers of microspheres produced in a single run. To enable microfluidic fabrication of microspheres from timed gelation hydrogel systems, an in situ mixing region is needed so that various hydrogel precursor components can be added separately. Here, we designed and evaluated three mixing devices for their effectiveness at mixing hydrogel precursor solutions prior to droplet formation and subsequent gelation. The serpentine geometry was found to be the most effective and was further improved with the inclusion of a pillar array to increase agitation. The optimized device was shown to fully mix precursor solutions and enable the fabrication of monodisperse polyethylene glycol microspheres, offering great potential for use with timed gelation hydrogel systems.

Co-delivery of fibrin-laminin hydrogel with mesenchymal stem cell spheroids supports skeletal muscle regeneration following trauma.

Volumetric muscle loss (VML) is traumatic or surgical loss of skeletal muscle with resultant functional impairment. Skeletal muscle's innate capacity for regeneration is lost with VML due to a critical loss of stem cells, extracellular matrix, and neuromuscular junctions. Consequences of VML include permanent disability or delayed amputations of the affected limb. Currently, a successful clinical therapy has not been identified. Mesenchymal stem cells (MSCs) possess regenerative and immunomodulatory properties and their three-dimensional aggregation can further enhance therapeutic efficacy. In this study, MSC aggregation into spheroids was optimized in vitro based on cellular viability, spheroid size, and trophic factor secretion. The regenerative potential of the optimized MSC spheroid therapy was then investigated in a murine model of VML injury. Experimental groups included an untreated VML injury control, intramuscular injection of MSC spheroids, and MSC spheroids encapsulated in a fibrin-laminin hydrogel. Compared to the untreated VML group, the spheroid encapsulating hydrogel group enhanced myogenic marker (i.e., MyoD and myogenin) protein expression, improved muscle mass, increased presence of centrally nucleated myofibers as well as small fibers (<500 µm2 ), modulated pro- and anti-inflammatory macrophage marker expression (i.e., iNOS and Arginase), and increased the presence of CD146+ pericytes and CD31+ endothelial cells in the VML injured muscles. Future studies will evaluate the extent of functional recovery with the spheroid encapsulating hydrogel therapy.

Hydrogel matrix presence and composition influence drug responses of encapsulated glioblastoma spheroids.

Glioblastoma multiforme (GBM) is the most aggressive brain tumor with median patient survival of 12-15 months. To facilitate treatment development, bioengineered GBM models that adequately recapitulate the in vivo tumor microenvironment are needed. Matrix-encapsulated multicellular spheroids represent such model because they recapitulate solid tumor characteristics, such as dimensionality, cell-cell, and cell-matrix interactions. Yet, there is no consensus as to which matrix properties are key to improving the predictive capacity of spheroid-based drug screening platforms. We used a hydrogel-encapsulated GBM spheroid model, where matrix properties were independently altered to investigate their effect on GBM spheroid characteristics and drug responsiveness. We focused on hydrogel degradability, tuned via enzymatically degradable crosslinkers, and hydrogel adhesiveness, tuned via integrin ligands. We observed increased cellular infiltration of GBM spheroids and increased resistance to temozolomide in degradable, adhesive hydrogels compared to spheroids in non-degradable, non-adhesive hydrogels or to free-floating spheroids. Further, a higher infiltration index was noted for spheroids in adhesive compared to non-adhesive degradable hydrogels. For spheroids in degradable hydrogels, we determined that infiltrating cells were more susceptible to temozolomide compared to cells in the spheroid core. The temozolomide susceptibility of the infiltrating cells was independent of integrin adhesion. We could not attribute differential drug responses to differential cellular proliferation or to limited drug penetration into the hydrogel matrix. Our results suggest that cell-matrix interactions guide GBM spheroid drug responsiveness and that further elucidation of these interactions could enable the engineering of more predictive drug screening platforms. STATEMENT OF SIGNIFICANCE: Glioblastoma multiforme (GBM) multicellular spheroids hold promise for drug screening and development as they better mimic in vivo cellular responses to therapeutics compared to monolayer cultures. Traditional spheroid models lack an external extracellular matrix (ECM) and fail to mimic the mechanical, physical, and biochemical cues seen in the GBM microenvironment. While embedding spheroids in hydrogel matrices has been shown to better recapitulate the tumor microenvironment, there is still limited understanding as to the key matrix properties that govern spheroid responsiveness to drugs. Here we decoupled and independently altered matrix properties such as degradability, via an enzymatically degradable peptide crosslinker, and cell adhesion, via an adhesive ligand, giving further insight into what matrix properties contribute to GBM chemoresistance.

Design of Hydrolytically Degradable Polyethylene Glycol Crosslinkers for Facile Control of Hydrogel Degradation.

Hydrogels, whose degradability can be controlled while also preserving cell viability or biomolecule stability, are in demand. Degradable polyethylene glycol crosslinkers are hydrolytically designed for use in hydrogels. Degradation is controlled by crosslinker chemical structure, such as introducing local hydrophobicity, steric hindrance, or electron-withdrawing moieties near a degradable ester moiety. Hydrogels made using these crosslinkers have gelation times from 1 to 22 min, storage moduli from 3 to 10 kPa, mesh sizes from 10 to 13 nm, and degradation times from 18 h to 16 d. However, when reaction conditions are modified to achieve similar gelation time, hydrogels have similar initial properties but preserve the wide range of degradation times. All crosslinkers support high cell viability upon hydrogel encapsulation or exposure to leachables and degradation products. This innovation in controlling degradation can help realize the hydrogels' potential for drug delivery or as matrices for cell encapsulation and transplantation.

Biodegradable polyethylene glycol hydrogels for sustained release and enhanced stability of rhGALNS enzyme.

Mucopolysaccharidosis IVA (Morquio A disease) is a genetic disorder caused by deficiency of N-acetylgalactosamine-6-sulfate-sulfatase (GALNS), leading to accumulation of keratan sulfate and chondroitin-6-sulfate in lysosomes. Many patients become wheelchair-dependent as teens, and their life span is 20-30 years. Currently, enzyme replacement therapy (ERT) is the treatment of choice. Although it alleviates some symptoms, replacing GALNS enzyme poses several challenges including very fast clearance from circulation and instability at 37 °C. These constraints affect frequency and cost of enzyme infusion and ability to reach all tissues. In this study, we developed injectable and biodegradable polyethylene glycol (PEG) hydrogels, loaded with recombinant human GALNS (rhGALNS) to improve enzyme stability and bioavailability, and to sustain release. We established the enzyme's release profile via bulk release experiments and determined diffusivity using fluorescence correlation spectroscopy. We observed that PEG hydrogels preserved enzyme activity during sustained release for 7 days. In the hydrogel, rhGALNS diffused almost four times slower than in buffer. We further confirmed that the enzyme was active when released from the hydrogels, by measuring its uptake in patient fibroblasts. The developed hydrogel delivery device could overcome current limits of rhGALNS replacement and improve quality of life for Morquio A patients. Encapsulated GALNS enzyme in a polyethylene glycol hydrogel improves GALNS stability by preserving its activity, and provides sustained release for a period of at least 7 days.

MicroCT vascular network analysis program: Development, validation, and comparison to manufacturer software.

The dependence on angiogenesis for bone repair makes accurate measuring of vascular networks of great importance to orthopedic researchers. A three-dimensional imaging modality like microcomputed tomography (µCT) would better capture these networks than histology. There are commercially available programs to analyze vessel networks in three dimensions, but these may be too costly for laboratories. Alternatively, µCT trabecular software could be used but may not be appropriate. The goal of this project was to develop a vascular network analysis protocol based on freely or commonly available software and compare its performance to that of a µCT trabecular analysis software. The protocol developed, called vascular network analysis or VNA, relies on two modules in Fiji ImageJ and a custom MATLAB program. We validated the software and compared it to a µCT trabecular analysis program (MicroCT) using in silico models of increasing complexity and differing homogeneity. In general, VNA outcomes were significantly different from true values, but most were within an acceptable percent error (<10%). VNA and MicroCT performed almost identically for volume but significantly differently for average vessel diameter. For the homogenous models, the average diameters differed only slightly but were starkly different for the heterogeneous models. In the most heterogeneous system, the MicroCT software overestimated average diameter by about 650% from true. VNA was within 1% of true for the same model. In conclusion, we have developed a program to analyze vascular networks from MicroCT scans which is easily accessible, insensitive to network homogeneity, and of higher accuracy compared to a µCT trabecular analysis software.