Type I Collagen is known to play a major role in cellular adhesion and migration within the extracellular matrix. At least 12 unique transmembrane receptors have been found including integrins, discoidin domain receptors, and mannose receptor family proteins. The amino acid sequences on collagen molecules that bind to these receptors are all some distance away from the N and C terminal domains of the collagen molecule. Thus, if a densely packed fibril brush structure could be synthesized by grafting collagen fibrils end-on to a surface, these binding sites could be made inaccessible to approaching cells. This would provide the ability to modulate cellular activity at a surface based only on orientation of the protein structures at that surface. We will present evidence that collagen nanofibrils can be produced with an end-on geometry on titanium and glass coverslips. XPS, fluorescent antibody labelling and SEM have been used to characterize the surfaces. Mechanical testing together with previous work using MEMS devices to test single fibrils provides an estimate of the spatial density of the fibrils on the surfaces.

Tissue vascularization remains a major tissue engineering challenge. While natural hydrogels are conducive to vasculogenic assembly, they are not suitable for producing permanent tissue grafts as they rapidly resorb after implantation. Synthetic hydrogels are more readily modifiable to better suit tissue grafting, but they typically slow or inhibit vasculogenic assembly due to their nanoporous nature. Here, we explored the potential for vascular network formation in granular hydrogel composites (GHCs), a method of scaffold fabrication that incorporates microscale porosity orthogonal to hydrogel stiffness and degradability. Dextran vinyl sulfone (DexVS) microgels (60 µm diameter) were fabricated via microfluidics and suspended with human lung microvascular endothelial cells (LMVECs) (6M/mL) in EGM2-MV or fibrinogen solution (5mg/mL) to form microporous annealed particle (MAP) or GHC constructs, respectively. Microgels were packed via centrifugation and annealed via poly(ethylene glycol) dithiol (15 mM) with/without inclusion of fibrinogen/thrombin (1 U/mL) to fill void spaces. Samples were gelled at 37 ºC, cultured with EGM2-MV supplemented with FBS, VEGF, and phorbol 12-myristate 13-acetate (PMA), and assessed by nuclear/F-actin staining and immunostaining. Our studies suggest that commonly utilized MAP hydrogels do not support vascular network assembly. LMVECs in MAP gels adopted a monolayer-like morphology, while LMVECs in GHCs formed cord-like structures comparable to those seen in pure fibrin hydrogels. This was supported by morphometric analysis of formed endothelial structures, indicating that LMVECs in MAP gels had larger average cell surface areas and thus were more spread out and monolayer-like than LMVECs in GHC and pure fibrin gel conditions. This is non-ideal as monolayer formation opposes vascular network formation. LMVECs typically adopt a monolayer-like morphology when adhering to 2D surfaces, suggesting that LMVECs in MAP gels mainly adhered to microgel surfaces. In contrast, the formation of cord-like structures in GHCs suggests the fibrin encapsulating LMVECs enabled 3D matrix interactions. These structures also appeared to be lumenized, with podocalyxin staining confirming proper apical-basal polarization of their constituent LMVECs. Taken together, these studies demonstrate the potential of GHCs to support the formation of microvasculature. These findings will guide future research directions towards integrating capillary networks in GHCs with host vasculature.

In previous work, we developed a composite midurethral sling (MUS) from bovine electrocompacted collagen (ELAC) thread to address stress urinary incontinence (SUI). An ovine feasibility study showed the sling was biocompatible. Currently, we have braided ELAC thread into pure-collagen, macroporous slings, CollaSlings, and implanted them in sheep. We hypothesized the CollaSling would produce a lasting, ligamentous tissue band around the midurethra. The objective was to determine the tissue response to and mechanical properties of a novel pure collagen sling in an ovine model prior to human clinical trials. CollaSlings were fabricated by braiding ELAC threads into a 12mm wide ribbon, segmenting into 50cm sections, and crosslinking with genipin. Ten adult Dorset ewes were implanted with the CollaSling using a standard retropubic approach. Ewes were sacrificed at 3 months (n=3), 6 months (n=3), 9 months (n=4). Lengths of CollaSling passing through the space of Retzius were mechanically tested for ultimate tensile strength (UTS), strain, and modulus. Histology sections were taken in the midurethral and muscle regions and scored according to ISO10993-6. No urinary retention or other adverse effects were observed. Grossly, the CollaSling integrated with the surrounding tissues at harvest with no fibrous encapsulation at any site. The sling formed a ligament that was distinct and palpable through the vagina, which felt supple at harvest. Histologically, there was no acute inflammatory response. There was minimal-mild dense fibrosis with low clusters of infiltrating lymphocytes, plasma cells, macrophages, and multi-nucleate giant cells, and evidence of neovascularization for all time points, and a trend towards slightly greater fibrosis present within the skeletal muscle site as compared to the midurethral site across all time points. Explants were significantly stronger at all time points compared to hydrated, naïve CollaSling due to tissue ingrowth and de novo collagen deposition. Strain and modulus were consistent over time, with values similar to native tissue. CollaSling is a pure collagen MUS deployable using a standard retropubic procedure. The sling is biocompatible and promotes new fibrous tissue deposition. Mechanical properties indicate that the sling can support the midurethra. Clinical studies will be run to determine efficacy in treating SUI.

This study seeks to advance cardiac tissue engineering technology by mapping the molecular components of donor decellularized heart matrix (DHM) and downstream signaling in recipient mice. We previously demonstrated mechanosensitive signaling contributes to the DHM induced therapeutic response in mouse recipients with myocardial infarction (MI). Here, we employ competing DHM treatments from fetal and exercise donors to define protein signature patterns in DHM and further evaluate the molecular response in MI recipients for converging downstream signaling pathways driving cardiac repair. Our work demonstrates DHM treatment at MI improved ejection fraction 3 weeks after injury approaching sham with DHM derived from fetal and adult-exercise hearts but not sedentary as well as other histological metrics. Proteomic analysis resolved distinct proteins between exercise and sedentary donor hearts. In addition, we defined overlapping signaling pathways activated in recipient mice with fetal and exercise DHM that are distinct from sedentary hearts. The proteomic analysis results will inform important signaling nodes for cardiac repair towards designing new therapies.

The evolving drug discovery landscape requires efficient methods for evaluating candidate drugs, especially in high-content and high-throughput screening (HCHTS). Traditional HCHTS based on 2D cell culture may face challenges in faithfully reproducing in-vivo conditions, leading to limited accuracy in predicting therapeutic and side effects. Here, we introduce a hydrogel-based scaffold with an inverted colloidal crystal (ICC) geometry designed for uniform and high-yield organoid culture. This scaffold is fabricated from an assembly of uniform alginate microgels within an agarose-based bulk backbone, exhibiting a hexagonal crystalline packing (HCP) structure with consistent void spaces (e.g., ~250 µm in diameter) and interconnecting channels (e.g., <50 µm). The ICC scaffold, a versatile platform for high-yield organoid culture, achieves remarkable yields of over 3,000 organoids per well in a standard 96-well plate and facilitates efficient transport of suspension cells, nutrients, waste, and drugs through interconnected channels, ensuring cost-effectiveness and time-saving. With high biocompatibility, customizable features, and the ability to replicate complex cell-to-cell interactions, this platform is well-suited for creating HCHTS models mimicking tissue and disease in vitro. Moreover, the unique HCP-arranged geometry and the transparency of the hydrogel materials seamlessly integrate with in-situ imaging and automated data processing, significantly enhancing our understanding of drug therapeutics through high-content assessments (e.g., >100 organoids∙analysis-1; >300 times faster), thereby accelerating therapeutic discovery and drug development. Eventually, this biomimetic ICC scaffold will represent a significant advancement in 3D cell culture as a high-yield organoid culture platform, offering enhanced biological relevance in tissue engineering, disease modeling, drug development, and biomedical research. 

Numerous studies indicate a strong correlation between cerebrovascular impairment and the development of Alzheimer’s Disease (AD). These cerebrovascular alterations precede the formation of amyloid plaques, tangles, and the onset of cognitive decline, suggesting that impaired cerebrovascular function plays a key role in the onset and/or progression of AD neuropathology. In consequence, therapeutic interventions aiming to restore cerebrovascular function constitute crucial strategies to attenuate the progression of the disease. Cell-based therapies constitute promising strategies to address vascular deficiencies in the brain. To implement safe and efficient cell therapies in AD, we used electroporation, to deliver 3 pro-vasculogenic transcription factors to reprogram mouse primary embryonic fibroblasts (pMEFs) into induced endothelial cells (iECs). To evaluate their therapeutic potential, pMEFs pre-labeled with 5-bromo-2’-deoxyuridine (BrdU) and transfected with Etv2, Foxc2, Fli1 (EFF) or a control empty plasmid were delivered with 3 intracranial injections into the lateral ventricles (LV) of females from the triple transgenic murine model of AD (3xTg-AD) or Wild-Type Controls. Within each cage mice with the same genotype were randomly assigned to either vasculogenic or control cell injections. Two weeks after the last injection spatial memory was analyzed with the Barnes Maze. Subsequently, brain tissue was processed for immunostaining and biochemical analysis. Our results indicate that pMEFs pre-programmed into vasculogenic iECs and delivered to the LVs induce an increase in global cerebral blood flow (CBF) as early as 7 days post-injection. Vasculogenic cells also lead to a reduction of spatial memory deficits in the 3xTg-AD mice. Histological analysis shows that the injected cells were able to migrate to multiple brain regions and survive for at least 4 weeks in close contact with brain blood vessels. Notably, animals injected with vasculogenic cells show an increase in the total vascular area in the cortex and a reduced amyloid-beta load. Together our results indicate that the development of electroporation-based cell therapies by direct reprogramming constitutes a promising approach to treat AD and AD-related dementias.