United States

Abstract Title 
Hyperexcitation of Mediobasal Hypothalamic Glutamatergic Neurons Induces a Phenotype Resembling Anorexia Nervosa

Background
Anorexia Nervosa (AN) is an eating disorder characterized by a voluntary restriction in food intake resulting in a sustained critically low body weight, hyperactivity, and increased anxiety. AN is among the deadliest psychiatric disorders, and the primary method of assessing its neurobiological basis is with animal models. However, current animal models restrict feeding, exogenously induce stress, and/or show unrelated phenotypes like normoactivity, dissimilar from the hyperactivity and self-induced starvation seen in AN. 
The Mediobasal hypothalamus (MBH) is known to be involved in the regulation of feeding and anxiety-related behaviors. In particular, once activated, glutamatergic neurons within the MBH promote these behaviors. However, these approaches acutely activated the neurons, and the behavioral changes were usually transient. Whether chronic activation of glutamatergic MBH neurons is involved in AN is unknown. 
Previous studies identified a bacterial sodium channel (NachBac), which is more readily activated and takes more time to inactivate than mammalian counterparts. This leads to constantly increased activity when expressed in mammalian neurons. Recent treatment-based rationale suggested chronic glutamatergic signaling may contribute to AN. Based on these observations, we hypothesized that chronic activation of glutamatergic neurons in the MBH will recapitulate the self-induced starvation observed in AN.

Methods
We used the Cre-LoxP technique to achieve neuron-specific gene expression, where genes flanked with LoxP were expressed in the presence of Cre recombinase. We used mice expressing Cre driven by the promoter of vesicular glutamate transporter 2 (Vglut2, a marker gene for glutamatergic neurons), Vglut2-Cre, to target hypothalamic glutamatergic neurons. We stereotaxically injected an Adeno-Associated Virus with a FLip-EXcision vector, AAV-FLEX-NaChBac-GFP, into the MBH to express NaChBac in a Cre-dependent manner.

Results
Interestingly, not only did we observe decreased feeding behavior, but we also observed increased locomotion, increased anxiety, and drastically decreased body weight, classically resembling AN.

Conclusion
These results demonstrate that constitutive activation of glutamatergic MBH neurons recapitulates the AN phenotype. In contrast to the most-used AN mouse models, our model exhibits AN phenotype without feeding restriction or stress induction. Importantly, these results complement the growing rationale of increased glutamatergic signaling in the pathophysiology of AN, based on the use of the glutamate receptor antagonist, ketamine, in treating AN in mice and humans. These findings reveal neurobiological evidence of glutamatergic signaling hyperexcitation contributing to the AN phenotype and provide a more clinically relevant animal model to examine mechanistic insights on AN pathogenesis.

AMA Research Challenge 2024 

Chronic Activation of Ventral Hypothalamic Glutamatergic Neurons Induces Anorexia Nervosa Phenotype in Mice

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Background
Glaucoma is a group of optic neuropathies that affects approximately 2.7 million people in the United States and 76 million people worldwide. Glaucoma is irreversible and can result in permanent blindness if it is not treated. The main risk factor is elevated intraocular pressure (IOP) which predominantly affects retinal ganglion cells (RGC), resulting in cell death and permanent vision loss. Current therapeutics for glaucoma involve reducing IOP and halting progression of disease, but no current treatments can revive degenerated RGCs. Our project aims to evaluate the therapeutic effect of a neurotrophic factor, human Neuritin 1 (NRN1), in regenerating and protecting RGCs lost due to glaucomatous disease progression. Human NRN1 has been proven to enhance plasticity and maturation of RGC axons and dendrites in an acute glaucoma mouse model. The present study utilized non-glaucomatous and glaucomatous induced pluripotent stem cells (iPSC) derived RGCs seeded within in vitro collagen scaffolds. By administering NRN1 to the scaffolds, we hope to elucidate the ability of the therapeutic to increase survival of glaucomatous RGCs.

Methods
The iPSCs derived from non-glaucomatous and glaucomatous donor corneal fibroblasts were differentiated to retinal organoids (RO) to generate RGCs in vitro. After around 30 days of differentiation, the ROs were dissociated to isolate RGCs. The RGCs were seeded at one end of three different in vitro collagen scaffolds. The first scaffold received no NRN1 treatment, the second received NRN1 at the cell body, and the third received NRN1 at the opposite end of the cultured RGCs. Downstream immunofluorescence with the TRA-1-60 antibody was performed on iPSCs to confirm that they were successfully reprogrammed from donor fibroblasts. Additionally, gene expression experiments for iPSC markers were performed to further verify successful iPSC reprogramming. Karyotyping procedures were performed on iPSCs to ensure no reprogramming induced chromosomal aberrations were evident.

Results
We successfully generated iPSC-derived RGCs from both non-glaucomatous and glaucomatous donor eyes. These RGCs effectively integrated within the three collagen scaffolds. After NRN1 treatment, both cell types demonstrated differential expression of apoptotic markers. Downstream iPSC experimentation confirmed that iPSCs were successfully reprogrammed from donor fibroblasts with no chromosomal aberrations.

Conclusion
Our study demonstrates that NRN1 exhibits a therapeutic effect on glaucomatous RGCs. NRN1 could potentially be a useful intervention to restore vision in patients suffering from severe glaucoma. The results also indicate that iPSC-derived RGCs can successfully be obtained from donor eyes and used in collagen scaffolds for future research.

Determining the Restorative Role of Human Neuritin 1 in Protecting Retinal Ganglion Cells from Glaucoma Patients

Background
Glaucoma is a neurodegenerative ocular disorder involving the degeneration of retinal ganglion cells (RGCs), thinning of the retinal nerve fiber layer, and atrophy of the optic nerve. Disease progression is often characterized by increased intraocular pressure (IOP) and visual field loss. Currently, the only efficacious therapy for treating glaucoma is modification of IOP through pharmacological eye drops or surgical intervention. Even though these interventions slow disease progression, they do not prevent RGC death entirely thus emphasizing the need for targeting RGC survival. Decreased axonal transport of essential neurotrophic factors contributes to RGC death. Neurotrophic factor supplementation has shown to improve survival of RGCs and sustain retinal function. Neuritin-1 (NRN1), a neurotrophic protein downstream of the neurotrophin family, has shown to play a protective role in non-glaucomatous eyes exposed to high pressure via the ex vivo ocular perfusion model, Translaminar Autonomous System (TAS). We will investigate the effects of NRN1 in glaucomatous human donor eyes within the TAS to understand if NRN1 can protect RGC loss in diseased retinas.

Methods
Three sets of glaucomatous human donor eyes were obtained from eye banks in accordance with the Declaration of Helsinki. Eyes were dissected, and posterior cups were perfused using the TAS model for 6-7 days under high and normal pressure conditions with and without NRN1. We then assessed RGC survival by measuring apoptosis, inflammation, and retinal markers using qRT-PCR and immunohistochemistry. Retinal activity was compared between treated and untreated retinas using OcuScience® Ex Vivo electroretinogram.

Results
The posterior eye cups were successfully maintained in the TAS model under normal and high-pressure conditions for 6-7 days. NRN1 treated eyes showed differential expression of various inflammatory and apoptotic markers. NRN1 treated eyes showed no difference in extra cellular matrix deposition following perfusion. Improved retinal activity was seen within the treated glaucomatous eyes. 

Conclusion
The TAS model can mimic pressure induced pathogenesis in human glaucomatous eyes. Data from this study shows that NRN1 may serve as a potential therapeutic target by promoting RGC survival under glaucomatous conditions.


Evaluating the Therapeutic Effect of Neuritin 1 on Human Retinal Ganglion Cell Survival

Innate Immune Memory refers to the ability of innate immune cells to display altered responses upon secondary exposure to pathogens. This phenomenon is driven by dynamic and heritable histone protein modifications, which influence post-translational gene transcription. Currently, while the broader MAPK and PI3K/AKT pathways of α-MSH are well established, there is little existing research on the direct link between α-MSH and histone modifications. Our study aims to investigate the effects of alpha-melanocyte-stimulating hormone (α-MSH), a neuropeptide with notable anti-inflammatory properties, on Histone 3 (H3) post-translational markers and their epigenetic impact on mouse macrophages.

We conducted a comprehensive analysis of H3 modifications to identify key changes, focusing on H3K4 global methylation, H3K9 dimethylation, and H3K14 acetylation levels in response to varying concentrations of α-MSH. Methylation and acetylation of histone proteins, such as H3K4, H3K9, and H3K14, regulate the accessibility of transcriptional start sites and promoters, thereby influencing gene transcription. In immune cells, these histone modifications alter the transcription of genes associated with immune responses, particularly inflammation.

Utilizing enzyme-linked immunosorbent assays (ELISA) to quantify the H3 methylation and acetylation levels, the response of histone modifications to α-MSH treatment was measured, highlighting the potential of α-MSH as a modulator of epigenetic marks in innate immune cells. Such findings enhance our understanding of how neuropeptides like α-MSH can influence immune responses through epigenetic mechanisms, providing insights into new therapeutic strategies for controlling inflammation and enhancing immune memory.

Our results demonstrate significant effects of α-MSH on histone modifications, with notable demethylation of H3K4, increased dimethylation of H3K9, and elevated acetylation of H3K14 observed in treated macrophages. Specifically, significant changes in H3K4 global methylation and H3K14 acetylation were detected at 1 ng/mL and 10 ng/mL α-MSH, while H3K9 dimethylation was significantly affected at 30 pg/mL α-MSH. Additionally, low-dose α-MSH (30 pg/mL) induced demethylation and deacetylation of H3K4 and H3K14, respectively.

These findings suggest that α-MSH exerts anti-inflammatory effects by modulating histone methylation and acetylation patterns. Changes in H3 histone modifications decrease the expression of proinflammatory molecules and enhance the expression of anti-inflammatory molecules in macrophages. This epigenetic regulation may contribute to the innate immune memory, eliciting trained responses to subsequent exposures.

In summary, α-MSH modulates histone H3 post-translational modifications in a concentration-dependent manner, altering the epigenetic landscape of macrophages to promote anti-inflammatory responses. This research underscores the significance of histone modifications in the regulation of innate immune memory and the potential for targeting these pathways in immunotherapy. 



Histone Epigenetic Effects of α-MSH

Background
Age-related cataract affects 22% of individuals over the age of 75 years, and data suggest that delaying the progression rate by 10 years would decrease cataract surgery by 50%. When rodent lenses are incubated in culture medium, they lose their transparency after 24-48 hours due to protein aggregation, which changes the index of refraction resulting in light scattering and opacity. We hypothesized that finding ways to prevent or delay this process through manipulation of the culture medium would provide important clues toward cataract prevention and present a solid foundation for a drug testing model on specific mutated mice lenses for cataract treatment.
Methods
13 culture conditions, each comprising 4-6 adult C57BL6 mice lenses, were tested. Variations in glucose, pyruvate, fructose, antioxidant, osmolality (sorbitol), and crowding agent (polyethylene glycol) were used. The extent of opacification, the type of opacity (nuclear vs cortical), and the lens diameter were determined as a function of incubation time.
Results
Results show that compared to the baseline conditions (Medium TC199), basic nutrition materials that lenses need for survival and antioxidants that prevent oxidative stress in lenses show no improvement in preventing lens opacification. Next, we mimicked the lens environment in the eye and tested hyperosmolarity. PEG 1000 stood out among the rest by successfully preventing cataract formation in all of its lenses. We also noticed the lens size difference between the PEG 1000 condition and the rest. PEG 1000 successfully prevents the lens from swelling and keeps lens volume low. Thus, from our results, we tested another set of biopolymers which could prevent massive intake of medium to the lens that we saw in previous conditions. We saw that Lymphoprep successfully kept lenses clear until day 4 (96 hours), which was a major improvement compared to the rest of the conditions that we tested.
Conclusion
This project is novel in its approach as the first to develop a system of drug testing lens model for cataract by keeping mice lenses clear and transparent for 7-14 days. While we are still in the process of reaching 7-14 days of clarity in lenses, we have already tested some strong potential conditions that improved lens transparency in vitro incubations, such as PEG 1000 and Lymphoprep. We plan to further test possible conditions for optimizing lens culture medium for in vitro lens transparency, specifically focusing on mimicking the lens environment in the eye to maintain lens homeostasis.

In vitro formation of cataract as a model for age-related cataract

Purpose: Infectious keratitis caused by Aspergillus species is difficult to treat with conventional therapies. Novel treatments have been used to treat these severe infections, such as Rose Bengal (RB) photodynamic antimicrobial therapy (PDAT), which uses a photosensitizer (PS) activated with light. This activation leads to the generation of reactive oxygen species, which can kill infectious organisms. Resistant fungal strains, like Aspergillus, are not inhibited by RB. Other green light activated photosensitizers, such as Erythrosin B (EB), may inhibit fungal isolates. This study will investigate singlet oxygen (1O2) generation and fungal growth inhibition in RB and EB. 

Methods: To quantify 1O2, one mL aliquots of RB and EB (1.0, 2.0, and 4.9 mM) were plated onto SabDex agar. The plates were then measured with an optical dosimeter to detect the 1O2 luminescence signal. To evaluate in vitro inhibition, two Aspergillus strains (A. fumigatus and A. glaucus) were isolated from patients with keratitis and prepared in solutions of 1x10E4 CFU/mL. Organisms were mixed with RB or EB at the same three concentrations as the 1O2 experiments. Organism/photosensitizer solutions were plated in triplicate and irradiated with a custom green (518 nm) LED light (5.4 J/cm2). Plates were then incubated for 72 hours and photographed to analyze fungal growth.

Results: For RB and EB,1O2 signal increased as the concentration increased. Also, EB had more 1O2 signal than RB at the highest concentration (4.9 mM) (Figure 1). The 1O2 findings were supported by the in vitro inhibition experiments which showed increased growth inhibition with increased PS concentration. Furthermore, EB had more inhibition of the Aspergillus organisms than RB (Figure 2).

Conclusions: This study shows 1O2 generation alongside strain specific inhibition of PDAT in support of high concentrations of EB to kill Aspergillus species. Increasing concentrations of RB and EB boosts the 1O2 signal and Aspergillus species growth inhibition. These findings support the idea that 1O2 dose is an important metric for evaluating the efficacy of PDAT. 


Inhibition of Fungal Isolates via Singlet Oxygen Generation from Erythrosin B and Rose Bengal PDAT

Purpose:
Corneal Schwann cells (cSCs) ensheath axons and are believed to play a vital role in the health and regeneration of the cornea, including its sensory functions. Here we have exploited a nitrogen mustard (NM) corneal injury model to examine cSC and axonal degeneration/regeneration post injury and also tested a novel drug micellar formulation RM4404 that targets cSCs as a potential therapeutic.

Methods:
Transgenic mice harboring the proteolipid protein 1 promoter enhanced green fluorescent protein (PLP-eGFP) gene construct that marks cSCs were used. Topical 0.2% NM solution was applied for 5 min under sedation followed by a phosphate buffered saline washing. Over days 8 to 14, mice were treated twice per day with a topical micellar formulation RM4404 while the control group received vehicle Kolliphor HS-15. Corneal mechanosensitivity scoring by esthesiometer was performed. Mice were euthanized and corneal flat-mounts were stained with axonal β3-tubulin antibody. Fluorescently stained tissues were imaged using epifluorescence microscopy. Tiled images of the corneal staining were assembled using Leica software. Axonal and cSC networks were measured using FIJI Neuroanatomy plugin.

Results:
We found that NM injury causes degeneration of cSCs and associated axons which remained significantly reduced during the 14-day post injury period (P=0.0025; P=0.0283). RM4404 treatment supported the regeneration of cSCs at 14 days post injury (dpi), showing significantly greater numbers in drug treated over vehicle-treated groups (P=0.0012), and RM4404 also supported the regeneration of axons at 14 dpi (P=0.0006). Comparisons of central versus peripheral corneal regions revealed that regeneration was greater at the peripheral regions for both cSCs and axons in RM4404 treated corneas compared to vehicle controls (P=0.0002; P=0.0002). Mechanosensory testing revealed sensory reduction at 7 dpi that remained low in vehicle treated mice at 14 dpi (P=0.0015), while RM4404 treated mice at 14 dpi showed significant restoration achieving corneal sensitivity of uninjured mice (P=0.7627).

Conclusions:
cSCs are vulnerable to DNA targeting agents, such as NM. Surviving cSCs in the corneal periphery can regenerate and promote trophic support for injured axons to regeneration. For the first time, we provide evidence that pharmacological targeting of cSCs can be exploited for axonal regeneration and restoration of corneal sensory function.

Sights Set on Corneal Schwann Cells: Restoring Corneal Sensory Function Post Nitrogen Mustard Injury


Abstract Title
Synergistic effect of silver nanoparticles and antibiotics in enhancing antimicrobial activity against ocular pathogens

Background
Ocular infections, such as endophthalmitis and keratitis, remain one of the leading causes of irreversible blindness, worldwide. Despite advances in antibiotic therapy, increasing antimicrobial resistance among pathogens limit treatment options. We sought to develop dual antibacterial nanoformulations using silver and antibiotics and evaluated their antimicrobial efficacy against ocular pathogens.

Methods
Silver nanoparticles were synthesized via a chemical reduction reaction, then loaded with vancomycin and coated with chitosan. Minimum inhibitory concentration (MIC) of silver nanoparticles against staphylococcus aureus (SA) and methicillin-resistant staphylococcus aureus (MRSA) were determined via double dilution antibacterial assay with optical density measured at 600 nanometers. Ex vivo porcine eyes, ex vivo human vitreous, cultured human retinal Müller glial (MIO-M1) cells, and in vivo mice eyes were then intravitreally infected with SA. Chitosan-silver-nanoparticles (CAgNPs), chitosan-silver-vancomycin-nanoparticles (CAgVNPs), and vancomycin alone were separately administered to the above testing groups post-6 hours of infection. The samples were harvested and plated for colony-forming units (CFUs). Statistical significance was performed using one-way ANOVA. Lastly, the inflammatory response and cytotoxicity of the nanoformulations were assessed: inflammatory markers of SA-infected mice eyes were evaluated via ELISA, cytotoxicity of nanoformulations in SA-infected MIO-M1 cells were evaluated quantitatively via LDH assay and qualitatively via Calcein assay, and cytotoxicity of nanoformulations in SA-infected mice eyes were evaluated via TUNEL assay and H&E staining.

Results
CAgVNPs had a two-fold reduction in MIC compared to vancomycin. Comparing CAgVNP to vancomycin, there was an 8-fold reduction in CFUs in ex vivo porcine eyes, a 3.5-fold reduction in ex vivo human vitreous, and a 103-fold reduction in in vivo mice eyes. Levels of inflammatory markers IL-1β, IL-6, and KC were decreased in SA-infected mice eyes treated with CAgVNPs compared to vancomycin. LDH values were the same as the control group without SA infection. Calcein assay showed that CAgVNPs were able to save MIO-M1 cells from SA infection. TUNEL assay of mice eye sections that were treated with CAgVNPs were histologically the same as the control group without SA infection. 

Conclusion
This study demonstrates that silver nanoparticles have potent antimicrobial activity against ocular pathogens ex vivo, in vitro, and in vivo. More importantly, silver nanoparticles have synergistic effects with antibiotics to reduce MIC as well as enhance antimicrobial activity. Lastly, silver nanoparticles do not demonstrate any cytotoxicity, indicating their potential use for treatment of ocular infections. 


Synergistic effect of silver nanoparticles and antibiotics in enhancing antimicrobial activity against ocular pathogens

Background: Infectious keratitis (IK) is a sight-threatening corneal infection that causes inflammation, pain, and in severe cases blindness. Risk factors include contact lens use, trauma, surgery, and systemic disease. A recent outbreak of IK highlighted the potential danger of contaminated artificial tears. This incident involved a specific strain of drug-resistant Pseudomonas aeruginosa transmitted to patients. This critical event led to Food and Drug Administration (FDA) recalls of various artificial tear products. The need to understand microbial growth in artificial tears is clear. While preservatives are used to control contamination, their effectiveness varies, and the underlying mechanisms remain unclear. Understanding how the composition of artificial tears influences the growth rate of microbes causing IK is crucial. This study investigates the in vitro growth kinetics of IK isolates within artificial tears. 

Methods: Twenty different artificial tears were chosen for this study to cover a broad range of common ingredients (i.e. viscosity enhancing agents, electrolytes, buffers, osmoprotectants, etc.). These tears fell into three categories: preservative-free single-dose vials, preservative-free multidose bottles, and preservative-containing tears. Artificial tears were inoculated with a variety of microbes isolated from patients with IK including: bacterial (Staphylococcus spp., Mycobacterium spp., Corynebacterium spp., Serratia spp., Pseudomonas spp.), fungal (Candida spp., Fusarium spp., Aspergillus spp.), and protozoal (Acanthamoeba spp.) isolates. Artificial tear solutions were aliquoted in quadruplicate into a 96-well microplate and inoculated with microbial suspension. Microbial controls in tryptic soy broth and saline, as well as saline-only controls, were included. The turbidity of the samples was recorded periodically for 24 hours in a microplate reader at 37°C (Varioskan, ThermoFisher Scientific) to determine growth kinetics. Cell counts for each group were also performed using serial dilutions on Muller Hinton agar.

Results: Microbial growth varied across artificial tear formulations and microbe types. Preservative-free solutions generally supported higher bacterial growth compared to preservative-containing tears. Cell count data confirmed the trends observed through turbidity measurements. Artificial tear only and saline only samples did not show any microbial growth.

Conclusion: Preservative-free formulations appear to be particularly susceptible to supporting bacterial growth. This in vitro data highlights the importance of understanding how artificial tear composition influences microbial proliferation. Further research is warranted to explore the specific mechanisms by which tear ingredients promote or inhibit microbial growth. These findings can inform the development of safer artificial tear formulations with improved resistance to microbial contamination, ultimately contributing to the prevention and management of IK.

Tears For Fears? Investigating The Link Between Artificial Tears And Infection Risk

Background 
This study aims to investigate how the anterior cruciate ligament (ACL) compensates for the change in knee joint stability and contact pressures due to partial horizontal cleavage tears (HCTs) in the meniscus and subsequent surgical interventions, such as partial meniscectomy and partial transplantation on knee joint stability and contact pressures. 
Methods 
Seven freshly frozen human cadaveric knees were used in a study to investigate the effects of different meniscal conditions and surgical interventions on the meniscus itself. The knees were carefully thawed, examined, and prepared for experimentation. Four testing scenarios were established: intact knees, knees with partial horizontal cleavage tears (HCT) of the meniscus, knees with partial meniscectomy, and knees with partial transplantation. Axial loading was applied to simulate single-leg standing, and contact pressures of the medial meniscus were measured using digital sensors. An axial load was applied at 0° and 30° for each testing condition. Additionally, a mathematical 3D finite element model was created to evaluate the behavior of the ACL under different meniscus scenarios, which could not have been measured experimentally. This model was used to assess the ACL's response and validate its contact and pressure response at the meniscus level. 
Results 
ACL contact pressure and stress analysis across various meniscal conditions demonstrated substantial variability. Horizontal cleavage tears (HCTs) resulted in heightened contact pressures and diminished joint stability, as evidenced by increased ACL stress attributed to compensatory mechanisms in the presence of meniscal tears. Conversely, transplantation procedures exhibited a mitigating effect, maintaining joint mechanics closer to intact conditions and minimizing alterations in ACL forces. These trends persisted at 30 degrees of knee flexion, where significant increases in ACL forces were observed in partial and complete HCT conditions, contrasting with minimal changes observed in transplantation scenarios. 
Conclusion 
This study uncovers the biomechanical impacts of meniscal injuries and surgical conditions on the 
ACL, demonstrating how the ACL compensates for various meniscus conditions. Due to the ACL’s role in providing stability, it takes on additional stress in conditions of increased contact pressure on the meniscus, as seen in HCT conditions. In contrast, transplantation and repair conditions only slightly increase the stress on the ACL, putting much less strain on the ACL and supporting structures of the knee joint than an unrepaired tear. It is important to note that the contact area and stress measure vary depending on the degree of flexion in the knee.

Anterior Cruciate Ligament Mechanical Response to Load in the Setting of Changes to the Medial Meniscus 


Background
The architecture of the native enthesis (tendon/bone junction), consists of an organized gradient of tendon, cartilage, and bone tissues. This complex tissue structure effectively transmits the interactions between dynamic muscle tissues and the rigid skeleton. Healed tendon repairs fail to regenerate the appropriate tissue architecture and composition present in the native enthesis. The objective of this study was to create a biologically compatible scaffold that recapitulates the transition of nano-mechanical properties found in the native enthesis.
Methods
Wild-type mouse Achilles tendon-bone composite tissues were harvested and flash frozen. The modulus of elasticity for each sample was evaluated using atomic force microscopy (AFM). Force curves were generated, and Young’s modulus values calculated using a Hertz-Sneddon model. Biocompatible inks (bio-inks) composed of gelatin methacryloyl (GelMA), and other physiologically relevant additives, such as alginate, hydroxyapatite, and collagen I were fabricated to generate hydrogels of different nano-mechanical properties as assessed using AFM. The gels were crosslinked using Lithium Phenyl (2,4,6-Trimethylbenzoyl) Phosphinate and UV light. The various concentrations of GelMA, additives, and photo cross-linking parameters were then formulated to more closely replicate the transition of nano-mechanical properties found in native enthesis tissues.
Results
The native mouse enthesis modulus values (n=3) for tendon, cartilage and bone tissues were 111.8 +/- 47.7 MPa, and 36.7 +/- 29.2 MPa, 6.5 +/- 2.6 GPa respectively. Several different formulations of bio-inks were tested to best replicate the modulus values of the native enthesis. Final bio-ink concentrations were created which mimic the nanomechanical properties of tendon, cartilage, and bone. The GelMA concentrations ranged from 10-30%. Hydroxyapatite content ranged from 0-10%. Alginate content ranged from 0-4%. The scaled nanomechanical modulus values for the synthesized hydrogels in each of the different tissue types were 17 +/- 6 KPa, 280 +/- 190 Pa, 87 +/- 28 KPa respectively. 
Conclusion
Three distinct bio-inks were created to form three distinct hydrogels with different concentrations of GelMA and other physiologically-relevant additives. These hydrogels differ in both composition and nano-mechanical properties, analogous to a native enthesis tissue gradient. Future directions include the 3D-printing of these novel bio-inks to create a biomimetic scaffold with a gradient of distinct layers. Each layer will have specific nano-mechanical properties and extracellular matrix components that support the differentiation of mesenchymal progenitor cells into distinct phenotypes reflective of the different tissue types making up the native enthesis.


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Determining the Restorative Role of Human Neuritin 1 in Protecting Retinal Ganglion Cells from Glaucoma Patients

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