United States

Background:
There is a strong desire in orthopaedics to have soft tissue, particularly tendon, grow into metallic implants. Extensor mechanism and abductor reconstructions in total joint arthroplasty are problematic, and current methods for repair are associated with substantial complications and failure rates. With the introduction of three-dimensional (3D) printed porous metal implants, we hypothesized that tendons could directly attach to the implants. However, the effects of the porous metal structure on tissue growth and penetration into the pores are unknown. In order to improve outcomes after tendon injury, a better understanding of the optimal conditions to promote healing and prevent fibrosis is needed. Using a rat model, we investigated the effect of pore size on tendon repair fixation using 3D printed titanium implants.

Methods:
There were three experimental groups of eight Sprague Dawley rats (n = 24) plus control (n = 3). Implants had defined pore sizes of 400μm (n = 8), 700μm (n = 8), and 1000μm (n = 8). A defect was created in the Achilles tendon and the implant positioned between cut ends and secured with suture. Specimens were harvested at twelve weeks. Half the specimens underwent mechanical testing to assess tensile load to failure. The remaining specimens were fixed and processed for hard tissue histological analysis.

Results:
The average load to failure was 72.6N for controls (SD 10.04), 29.95N for 400μm (SD 17.95), 55.08N for 700μm (SD 13.47), and 63.08N for 1000μm (SD 1.87). The load to failure was generally better in the larger pore sizes. The 700m and 1000m specimens performed similarly, while the 400m showed significant differences vs control (p = 0.039), vs 1000μm (p = 0.010), and approached significance vs 700μm (p = 0.066). There was increasing ingrowth as pore sized increased. Histology showed fibrous tendon tissue within and around the implants, with collagen fibers organized in bundles.

Conclusion:
Tendon repair utilizing implants with 700μm and 1000μm pores exhibited similar load to failure as controls. Using a defined pore structure at the attachment points of tendons to implants may allow predictable tendon ingrowth onto/into an implant at the time of revision arthroplasty.

AMA Research Challenge 2022

Orthopaedic Surgery - Influence of Porosities of Titanium Implants on Tensile Properties in a Rat Tendon Repair Model - Orthopaedic Surgery

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poster

Background
Postoperative complications are a concern for surgeons due to worse outcomes and increased cost. It has been shown that social determinants of health disparities (SDHD) are associated with higher rates of postoperative complications. Understanding the role SDHD plays in patient’s health can help prepare providers to optimize patients’ conditions prior to surgery and improve postoperative care. The purpose of this study was to compare the postoperative complication rates between patients undergoing total shoulder arthroplasty (TSA) with a past medical history of SDHD with a control group without SDHD diagnoses.
Methods
A retrospective cohort analysis was conducted using a national insurance claims database. Using International Classification of Diseases (ICD) and Current Procedural Terminology (CPT) codes patients who underwent primary TSA with at least two years of follow-up in the database were identified. The SDHD and control groups were matched and analyzed using multivariable analysis for 90-day medical complications and 2-year surgical complications.
Results
A total of 120,367 patients underwent TSA between 2010-2019: 110,822 patients in control group and 9,394 in SDHD group. SDHD group had significantly longer length of stay (mean [SD]; Control = 1.99 days [1.39] vs SDHD = 3.84 days [18.32]; p<0.001) along with significantly higher odds ratios for 90-day medical complications including heart failure (OR: 3.71; p<0.001), cerebrovascular accident (OR: 2.48; p<0.001), renal failure (OR: 2.91; p<0.001), death (OR: 3.32; p<0.001), deep vein thrombosis (OR: 3.16; p<0.001), pulmonary embolism (OR: 1.95; p<0.001), pneumonia (OR: 2.84; p<0.001), sepsis (OR: 4.94; p<0.001), and urinary tract infection (OR: 2.60; p<0.001). Additionally, the SDHD group had a significantly higher odds ratio for revision surgery within 2-years following TSA (OR: 1.45; p<0.001).
Conclusion
This study highlights the negative impact of SDHD on patient outcomes following TSA including a higher rate of postoperative complications within 90-days and revision surgery within 2-years. Additionally, patients with a history of SDHD had a significantly longer length of hospital stay than patients without SDHD. Quantifying the risk of complications and differences in length of stay for TSA patients with a history of SDHD are important in determining value-based payment models and risk stratification to optimize patient care.

Orthopaedic Surgery - Social Determinants of Health Disparities Impact Postoperative Complications in Patients Undergoing Total Shoulder Arthroplasty - Orthopaedic Surgery

Background
Neurofibromatosis type 2 (NF2) is an autosomal dominant tumor predisposition syndrome resulting from mutations in the NF2 gene. NF2 patients are prone to developing schwannomas, which are benign tumors originating from Schwann cells of the peripheral nervous system. Though the hallmark of NF2 is the development of bilateral vestibular schwannomas, schwannomas can occur on any cranial or peripheral nerve. Facial nerve schwannoma (FNS) is the most common primary neoplasm of the facial nerve, although it is uncommon overall. Due to the rarity of FNS, animal models are imperative for therapeutic testing of pharmaceutical compounds. The Postn-Cre;Nf2flox/flox mouse model is known to develop cranial nerve schwannomas, including FNS. However, the nature of these tumors has not been robustly characterized.

Methods
Postn-Cre;Nf2flox/flox and control littermates were euthanized at 12 months of age. A novel method was employed for dissection of the intratemporal facial nerve. Whole heads or isolated facial nerves were formalin-fixed and paraffin embedded. Sections were stained by hematoxylin and eosin or immunohistochemistry. Schwann cell density and tumorlets were quantified in geniculate ganglia and peri-geniculate facial nerves. Comparative statistical analysis was performed using Student’s t-test (GraphPad Prism).

Results
In this study, we report the first thorough histopathological analysis of FNS development in the Postn-Cre;Nf2flox/flox mouse model. Schwann cell density is increased in mutant facial nerves and geniculate ganglia compared to control (p<0.001). Tumorlets are present only in Postn- Cre;Nf2flox/flox mutant mouse ganglia (p<0.01). Neuron density is decreased in mutant geniculate ganglia (p<0.001). Merlin, S100, SOX10, GFAP, FABP7, Neurofilament, and Ki-67 immunostaining confirmed a Schwann cell phenotype.

Conclusion
The Postn-Cre;Nf2flox/flox mouse model demonstrates consistent and predictable development of facial nerve schwannoma. While this model has been described to develop cranial nerve schwannomas, our study establishes the first quantifiable readout of tumor burden through measurement of Schwann cell and tumorlet density. These findings provide a novel endpoint for preclinical testing of targeted therapies for neurofibromatosis type 2-associated cranial nerve schwannomas in the Postn-Cre;Nf2flox/flox mouse model.

Facial Nerve Schwannoma Development in a Genetically Engineered Neurofibromatosis Type 2Mouse Model

Abstract Title:
Pancreatic Lipase Deficiency: Characterization of Novel PNLIP Mutations

Background:
Pancreatic triglyceride lipase (PNLIP) is the primary lipase responsible for dietary fat digestion in humans. Studies have reported that mutations in PNLIP lead to pancreatic lipase deficiency, steatorrhea, and fat-soluble vitamin deficiencies. Previously, we illustrated that a rare homozygous missense mutation (p.T221M) in PNLIP causes steatorrhea through its secretion defect. This led to a loss of digestive lipase function, insoluble aggregation via protein misfolding, and a gain of proteotoxic function increasing the risk for pancreatic diseases like pancreatitis. Recently, two more novel PNLIP mutations (p.R188C and p.W419X) have been identified and associated with congenital pancreatic lipase deficiency in symptomatic children. The aim of this study was to characterize the lipase function and proteotoxicity of these two PNLIP mutants in vitro.

Methods:
We generated a mammalian protein expression construct pcDNA3/PNLIP WT. Next, we made plasmid DNA constructs pcDNA3/PNLIP R188C or W419X by site-directed mutagenesis. We then transfected HEK293T cells to express PNLIP WT, R188C, or W419X. Three days post-transfection, we harvested conditioned media and cells. We assessed secretion of lipase in medium through lipase activity assay and immunoblot against PNLIP. We fractionated the cell lysates into detergent soluble and insoluble fractions to assess PNLIP misfolding and endoplasm reticulum (ER) stress by immunoblot against PNLIP and BiP (an ER stress marker), respectively. 

Results:
The conditioned media from cells expressing PNLIP WT had steady lipase activity whereas the R188C or W419X media lacked detectable lipase activity. Immunoblot revealed a strong PNLIP band in the WT media, but no visible bands in R188C or W419X media. These results suggest the defective secretion of both mutant PNLIP proteins. Moreover, a much higher proportion of PNLIP R188C and W419X was present in the detergent insoluble fraction compared with WT indicating increased misfolding of the PNLIP mutant proteins. Further immunoblot analysis of cell lysates indicated markedly up-regulated BiP in the mutant samples relative to WT controls.

Conclusion:
Our data illustrate that both R188C and W419X mutations in PNLIP result in loss of lipase function due to abolished secretion. Additionally, both mutations may increase the risk to develop chronic pancreatitis through its gain of proteotoxic function. 


Pancreatic Lipase Deficiency: Characterization of Novel PNLIP Mutations

Abstract Title: Dopaminergic contributions to evaluation and reevaluation of neuroeconomic decision making 

Background: Dopamine signaling in the nucleus accumbens core is an important neural substrate for neuroeconomic decision making. Dopamine dynamics reflect reward value, prediction error, and motivation, but have not been studied extensively under conditions of neuroeconomic foraging tasks known to show cognitive consideration of future and past events. Furthermore, decreases in dopamine in settings of internally-driven decision reevaluation have not been reported. 

Methods: Mice were trained on a neuroeconomic foraging task (Restaurant Row) to evaluate offers to earn food rewards. They were given a daily budget of one hour to forage for food rewards of four distinct flavors (restaurants). Upon entering a restaurant, mice first encountered an Offer Zone in which a tone sounded whose pitch signaled the delay the animal would have to wait before reward would be delivered (higher pitched tones signaled longer delays, lower pitched tones signaled shorter delays). After the tone presentation in the Offer Zone, the animal could either accept or reject the offer. To reject the offer, mice would continue to the next corner to receive a new offer for a different flavored pellet. To accept the offer, mice would advance into a Wait Zone, where the countdown to reward began. At any point during the countdown, the animal had the opportunity to quit the trial, and would only earn reward if it waited out the entire delay. 

Using fiber photometry to monitor dopamine dynamics in mice while they performed this neuroeconomic task, we investigated how dopamine encodes evaluation and reevaluation of neuroeconomic decisions. 

Results: Dopamine dynamics in the OZ scaled with the value of the offer in both groups, consistent with descriptions of dopamine’s role in encoding value. Notably, the depth of the dopamine decrease was greatest for worse value offers, reflecting a re-evaluation process. 

Conclusion: Dopamine dynamics during quits reflect a cognitive consideration of the past offer value. This work demonstrates that decreases in dopamine can track a retrospective process wherein mice reevaluate their decision to accept a bad offer. This suggests that dopamine may encode current as well as past value as agents reevaluate and update their decisions.


Dopaminergic contributions to evaluation and reevaluation during neuroeconomic decision making

BMX-001 prevents chemoradiotherapy-induced toxicity of healthy tissue in rectal cancer

Tensile strength of penile tunica albuginea in a human model

Background: At present, damage to nervous tissue is difficult to treat, often irreversible, and furthermore nerve growth to new sites is very limited. I hypothesize that teeth have a special capability to induce nerves and that we can isolate tissues and factors that could be used in a clinical setting to completely regenerate nerves. In human clinical studies where developing third molar teeth have been transplanted to donor sites, the teeth have continued to grow but more astoundingly have gained the ability for nociception in response to cold stimuli. This finding implies that developing teeth transplanted to a bony defect induce de-novo nerve growth at the recipient site. In this working study I utilize the rat model to demonstrate de-novo nerve growth by transplanting whole teeth to iatrogenic bony-defects created in autologous tibia and performing histological and biochemical analysis. Demonstrating innervation in transplanted teeth of post-natal rats would indicate that dental tissues are indeed capable of nerve growth and would greatly enhance our understanding of nerve induction.

Methods: Post-natal rats (Sprague-Dawley) aged 28-30 days were anesthetized and intact tooth germs with fully developed crowns undergoing root development were isolated. A bony defect was then created in the ipsilateral anterior tibia and the extracted tooth was transplanted to the defect. The wound was closed and hemostasis established. Hematoxylin and Eosin staining, and fluorescent immunohistochemistry were performed for nerve specific markers including anti-acetylated tubulin III antibodies. 

Results: Histology reveals a tooth well integrated into the tibia and a significant reduction in inflammation and bony remodeling, with dental specific enamel and dentin present in the leg. Moreover, the transplanted tooth is vascularized and early immunohistochemistry indicates evidence of innervation of transplanted teeth in line with the hypothesis that teeth have nerve inductive capacity postnatally.

Conclusion: Teeth transplanted autologously to tibia can integrate into the leg, survive as independent organs with their own blood supply, and early histology indicates innervation of the transplanted tooth. These data are applicable to patients’ needing re-innervation of eye, spine, extremity, teeth and many other organs. The implications are far-reaching and may lead to novel nerve regeneration therapeutics.




De-Novo Innnervation of a Transplant Organ

Background: Obesity, metabolic syndrome, and type 2 diabetes have dramatically grown in prevalence over the last decade. Weight loss and lifestyle modifications are often prescribed, as adherence to a resistance exercise training program reduces adiposity and improves glucose sensitivity. Despite the popularity of high protein diets for weight loss and muscle building, prospective and retrospective population-based studies as well as randomized clinical trials have shown that reduced protein intake is associated with better metabolic health in humans, the majority of whom are sedentary. Similar results are observed in sedentary rodents, where reduced protein intake improves metabolic health and body composition. We hypothesized that a unifying explanation of these results is that exercise is protective against high dietary protein intake. Here, we investigate how dietary protein intake interacts with resistance exercise training in mice to determine outcomes related to metabolic health. 

Methods: Male mice were assigned a group with either a high (36%) or low (7%) protein diet with or without a previously validated 12-week progressive overload resistance training program. Food consumption, body composition, and fitness were measured along with metabolic phenotyping outcomes. 

Results: Similar to previous studies, the low protein diets improved body composition in male mice when compared to high protein. However, high protein-fed sedentary animals accumulated significantly more fat mass than high protein-fed exercising mice. Low protein consumption significantly improves blood sugar control compared to high protein diets independently of resistance exercise, though resistance exercise itself may also promote glucose tolerance. Increasing dietary protein intake leads to more rapid strength and lean muscle gain in response to resistance exercise training. However, exercising low protein-fed mice eventually gained similar strength to high protein-fed exercised mice. 

Conclusions: Our results suggest that while low protein diets maximize metabolic health for sedentary mice, resistance exercise and muscle growth may be protective against some, but not all, negative metabolic effects of high protein diets and lead to greater strength and muscle mass. Future studies will be needed to determine if resistance or other forms of exercise are protective against the negative metabolic effects of high protein diets in humans, and to examine if resistance exercise allows high-protein-fed animals to live long, healthy lifespans.

Dietary protein restriction promotes leanness while enabling strength gain in mice

Background: Gestational diabetes causes placental lipid accumulation and essential long-chain polyunsaturated fatty acid (LCPUFA) deficiency in infants which signals poorer outcomes for both mother and baby. Physiologic LCPUFA biomagnification in the fetus suggests placental fatty acid transport occurs by both passive and active mechanisms. This study’s objective was to determine whether differential fatty acid uptake in human trophoblasts is mitochondrial-mediated.
Methods: Using MitoTracker fluorescent tagging of mitochondria and 4,4-difluoro-3a,4a-diaza-s-indacene, BODIPY, a fluorophore attached to fatty acids of variable lengths, we tracked live-cell uptake in BeWo and isolated cytotrophoblasts (CTB) from consenting mothers. Experiments were repeated in the presence of oligomycin (OM), which inhibits ATP-synthase, 2-deoxyglucose (2dG) which competitively inhibits glycolysis, etomoxir (Eto) which inhibits fatty acid oxidation, and carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP) which uncouples respiration. Statistical analysis was conducted using Student’s T-test per drug or 2-way ANOVA to determine fatty acid-specific, drug-based, and interaction effects. Significance was set at p<0.05.
Results: Live-cell imaging shows rapid uptake of both BODIPY-C12, which mimics a 16-carbon saturated fatty acid like palmitate, and BODIPY-C16, which mimics a 20-carbon LCPUFA occurs in BeWo and CTB. The time to peak uptake was longer for BODIPY-C16 than BODIPY-C12 (p<0.0001). Intracellularly, both fatty acids colocalize with mitochondria. Interestingly, both BODIPY-C12 and C16 stimulate visible cytoplasmic projections that facilitate peripheral uptake, followed by perinuclear accumulation. Treatment with 1uM OM, 50 mM 2dG, or 150 uM Eto significantly decreased ATP levels in BeWo while 0.3 uM FCCP did not (p=0.017, 0.002, 0.012, 0.458 respectively; n=3). ATP-inhibitors, slowed the rate of BODIPY-C16 uptake (p<0.0001 with 2dG and Eto; n=4). Conversely, 2dG and Eto increased the rate of BODIPY-C12 uptake (p=0.007, p=0.012 respectively; n=4). Primary cytotrophoblasts follow a similar trend for fatty acid uptake and response to ATP-inhibitors, taking up the BODIPY-C16 slower than BODIPY-12 (p<0.05 with OM and Eto; untreated control includes average of 4 patients while treatment was replicates from a single patient).
Conclusion: Differential fatty acid uptake in human trophoblasts is ATP-dependent which may explain placental lipotoxicity and LCPUFA deficiency associated with poorer pregnancy outcomes in GDM.

Differential fatty acid uptake in human trophoblast cells is ATP-dependent

Abstract Title: 
Dual role of CASP8AP2/FLASH in regulating EMT plasticity

Background
Metastasis and chemoresistance remain major challenges and the leading cause of cancer-associated deaths in most tumors. During metastasis cells undergo epithelial-to-mesenchymal transition (EMT), a process characterized by loss of epithelial cell markers, increased invasive potential and chemoresistance. EMT plasticity characterized by a seemingly back-and-forth transition between an epithelial and a mesenchymal phenotype plays a fundamental role in cancer progression. EMT occurs through broad transcriptome reprogramming mediated by key regulators, the EMT transcription factors (EMT-TFs). We identified FLASH (or CASP8AP2) as a critical novel EMT-TFs regulator. FLASH regulates a diverse set of cellular processes including apoptosis, histone mRNA processing and cell cycle S-phase progression. Here we show that FLASH is critical for EMT plasticity in multiple cancer cell lines due to its dual role in regulating EMT-TFs. We uncover that FLASH acts a repressor of SNAIL, but an activator of ZEB1, two critical EMT-TFs. Importantly, FLASH is highly expressed in tumors associated with early and aggressive metastasis such as small cell lung carcinoma. 

Methods
To address the role of FLASH in EMT we employ whole-genome NGS analysis, gene expression quantification by Q-PCR and computational EMT scoring using sets of established gene lists that are expressed in multiple cancers and have been validated as markers of the EMT phenotype.

Results
Our investigation reveals that: (1) the role of FLASH in EMT is independent of its role in cell cycle progression; (2) loss of FLASH in cancer cells drives a hybrid EMT phenotype with high epithelial scores; (3) FLASH expression inversely correlates with multiple EMT markers in cell lines from the Cancer Cell Line Encyclopedia (CCLE) dataset, owing to its dual role in EMT plasticity. 

Conclusion
The function of FLASH as a master regulator of pathways involved in tumor progression is novel. Here we show that FLASH regulates both epithelial and mesenchymal markers, thus controls EMT plasticity independent of its function in cell-cycle progression. Because FLASH function is conserved in a number of cancer cell lines including cervical, breast, pancreatic, prostate and lung cancer, FLASH expression modulation in cancer cells may play a critical role in EMT-driven metastasis and subsequent therapeutic resistance.


Next from AMA Research Challenge 2022

Orthopaedic Surgery - Social Determinants of Health Disparities Impact Postoperative Complications in Patients Undergoing Total Shoulder Arthroplasty - Orthopaedic Surgery

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