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BACKGROUND: Nipple reconstruction is widely regarded as the final step in post-mastectomy breast reconstruction. While grafts, local flaps, or combination approaches have been employed in nipple reconstruction, none has been able to achieve reliable long-term projection preservation. In response, we have sought to bioengineer neo-nipples in situ via the implantation of processed, decellularized cartilage xenografts placed within 3D-printed polylactic acid (PLA) scaffolds. MATERIALS AND METHODS: External nipple scaffolds were designed in-house and 3D-printed with PLA filament. Decellularized ovine xenograft infill was prepared and processed by mincing or zesting. All nipple scaffolds were placed subcutaneously on the dorsa of Sprague-Dawley rats and explanted after 1, 3, and 6 months for analysis. RESULTS: Explanted nipple scaffolds demonstrated gross maintenance of scaffold shape, diameter, and projection with accompanying increases in tissue volume. Histologic analyses revealed preservation of native cartilage architecture after 6 months without evidence of degradation. Analysis of formed tissue within the scaffolds revealed a progressive invasion of fibrovascular tissue with identifiable vascular channels and adipose tissue after 6 months in vivo. Confined compression testing revealed equilibrium moduli of both minced and zested samples that were within the expected range of previously reported human nipple tissue, while these data revealed no differences in the mechanical properties of the neo-tissue between time points or processing techniques. CONCLUSIONS: These preliminary data support potential use of decellularized allograft to foster healthy tissue ingrowth within a PLA scaffold, thereby offering a novel solution to current limitations in nipple reconstruction.

Objectives: Anterior cruciate ligament (ACL) injury is one of the leading orthopedic injuries in young individuals and even with surgical repair it is reported that around 50% of patients develop post-traumatic osteoarthritis (PTOA). Although ACL reconstruction improves anterior-posterior knee joint stability and cartilage contact biomechanics, some studies have shown persistent abnormalities in the tibiofemoral kinematics. Extensive rehabilitation work is often required following both ACL tear and ACL reconstruction surgery. New bone formation (osteophyte) and synovial inflammation (synovitis) are often associated with the progression and severity of OA post-injury and surgery, however the effect of mechanical loading stimulus in musculoskeletal repair remains relatively unexplored. The role played by resident synovial stromal cells in the activation of their local inflammatory and regenerative programming is poorly understood. Work in our laboratory has shown that in vivo imaging of activated fibroblasts using 68Ga-FAP radiotracer may be a valuable tool to follow the progression of OA and the bone-tendon interface healing. This study aims to explore the therapeutic effect of exercise following ACL reconstruction on the progression or mitigation of PTOA following an ACL injury, evaluating the mechanism and contribution of synovial fibroblast activation using microPET/CT imaging. Methods: All study procedures were approved by Weill Cornell IACUC (#2019-0034). Thirty, 12 week old male C57BL/6 mice underwent a non-invasive ACL rupture induced by a single axial compressive load of the tibia at a rate of 1mm/s on the right knee (Fig. 1). One week following injury, the animals were allocated into 4 treatment groups (n=6 per group): non-treated (G1); non-treated with sham surgery (G2); treated with ACL reconstruction (G3); treated with ACL reconstruction and treadmill exercise starting 1 week post-operatively (G4). Six mice were used to harvest the ACL tendon graft (allograft) and their knees scanned in the microCT as healthy controls.Treadmill exercise was performed 5 days/week, at 10m/min for 40min. PET/CT (Inveon scanner - Siemens Medical Solutions) imaging was performed pre-injury (baseline) and at 1, 2, 3, 4 and 8 weeks post-injury to measure fibroblast activation in the joint. One hundred μl of a 7% v/v EtOH/saline solution containing our 68Gallium (Ga)68-labeled fibroblast activation protein alpha (FAP-α) inhibitor (3.7-18.5MBq) was administered by tail vein injection and the mice were scanned for 60 minutes under anesthesia. The knee joint was identified as the region of interest (ROI) and the radioactivity concentration (Bq/ml x %ID/g) within the ROI was calculated using the Siemens imaging software. All animals were euthanized at 8 week post-injury. ACL graft healing and OA development were evaluated by micro-CT, X-Ray and routine histology. Statistical analyses were performed by Two-way ANOVA and Tukey’s post hoc test analysis was used to compare means ± SD. Significance level was defined as p ≤ 0.05. Results: PET/CT scans demonstrated accumulation of 68Ga-FAP in the injured and surgical knees compared to the contralateral healthy control knees (Fig. 2). FAP signal intensity peaked at 1 week after injury and continued increasing at 2 weeks in the surgical groups, with significant differences demonstrated in the ACL reconstructed groups (G3 and G4, pConclusions: The intimal layer of the native synovium is composed of 2 main cell types: macrophages and fibroblast-like cells. Recent studies have shown a correlation between activated macrophages and their association with OA severity and joint symptoms; however, the cross-talk between these 2 types of cells and their contribution to PTOA following ACL injury and reconstruction surgery is poorly understood. Our study identified time-dependent changes in FAP activity following ACL reconstruction and mechanical loading stimulus that could be correlated to PTOA development. Our data demonstrates that ACL reconstruction followed by a low intensity exercise regimen leads to a decrease of FAP activity in the ACL injured knee. We also found that the ACL reconstruction groups had less degenerative changes in the posterior medial tibial plateau, which may be due to an improvement of anterior-posterior joint stability, and the exercise group showed a greater amount of bone formed at the tendon-bone interface of the tibia suggesting a superior graft healing process. Immunochemistry for FAP-α is currently being analyzed to confirm the spatial localization of FAP expression. [Figure: see text][Figure: see text][Figure: see text]

Objectives: Rotator cuff tears are one of the most prevalent musculoskeletal injuries with over 250,000 rotator cuff repairs performed annually in the Unites States. While surgical techniques have advanced, incomplete/failed healing after rotator cuff repair is still relatively common with rates ranging from 11-95%. Extrinsic factors such as excessive early activity and patient comorbidities such as smoking and diabetes have been previously identified as factors which may contribute to decreased rates of rotator cuff repair healing. However, there has been a recent focus on the intrinsic biologic factors which affect repair healing at the tendon-bone interface. CCR2 is a chemokine receptor that has been linked to the recruitment of monocytes into wound sites in early inflammatory stages and is associated with an increase in “pro-inflammatory” macrophages. The purpose of this study was to evaluate the role of CCR2 on rotator cuff tendon healing by evaluating relative gene expression and biomechanics following rotator cuff repair in wildtype (WT) and CCR2-/- knockout (KO) mice in a chronic rotator cuff injury model. We hypothesized that CCR2 KO would lead to decreased inflammatory cell recruitment and increased biomechanical properties after rotator cuff repair compared to WT mice. Methods: All procedures were approved by our Institutional Animal Care and Use Committee (Protocol# 2019-0021). A total of 28 12-week-old male mice were utilized for this study and divided into 2 groups (wildtype C57BL/6J and CCR2 KO). All mice underwent unilateral supraspinatus tendon detachment at the initial surgery, followed by delayed supraspinatus repair 2 weeks following the initial procedure. They were sacrificed at the 4-week time point following the second procedure (Fig 1). The primary outcome measures included biomechanical testing and gene expression analysis. Biomechanical testing was performed on the repaired rotator cuff tendons on a custom-designed materials testing system. Contralateral intact tendons were also tested as controls. Specimens were prepared and loaded to failure at a rate of 1mm/min. Load-to-failure data were recorded and stiffness was calculated from the load-deformation curves. Following total RNA isolation from both the control and repaired tendons (50ng) and muscles (100ng), gene expression was measured using the NanoString nCounter® Fibrosis Panel. Genes were assessed based on fold change and significance level, determined by adjusted p-values using the Benjamini-Yekutieli procedure. The significance level was set at p=0.05 for all statistical analyses. Results: All repaired tendons remained intact at the time of sacrifice 4 weeks following repair. The CCR2 KO group had thicker, more robust tendons on gross inspection than the WT controls. Biomechanical analysis demonstrated a significantly increased load-to-failure and stiffness of the supraspinatus tendon repair in the CCR2 KO group compared to the WT group. Mean load-to-failure in the WT group was 1.64 N ± 0.41 N versus 2.50 N ± 0.42 N in the CCR2 KO group (pConclusions: In this study, we utilized a delayed rotator cuff repair model, which is more clinically relevant than previously established models of acute tendon transection and repair. Utilizing CCR2 KO mice, we found that there were significant differences in biomechanical properties – both load-to-failure and stiffness – between the WT mice and the CCR2 KO mice. Given the role of CCR2 in recruitment and accumulation of pro-inflammatory macrophages, these data suggest that excessive or unresolved inflammation may hinder tendon healing. Our results suggest that CCR2 KO leads to improved biomechanical properties in a mouse model of delayed rotator cuff repair. Additional studies are necessary to further elucidate the role of CCR2 in inflammation in the setting of chronic rotator cuff disease; however, CCR2 may be a promising target for novel therapeutics which aim to improve tendon healing and decrease re-tear rates following rotator cuff repair. [Figure: see text][Figure: see text][Figure: see text][Figure: see text]

1. In this study, we tested the hypothesis that betaine’s methyl-donor properties would reduce homocysteine concentrations, which was recognised in a previous genetics study to be linked to bone quality, and improve bone quality. This was combined with phytase treatment, as phosphorus is critical for bone mineralisation. 2. Using a 2 X 2 arrangement, a total of 1920 Lohmann LSL-lite chickens in 24 replicates of 20 chickens were fed one of 4 diets containing dietary betaine (0 or 1000 mg/kg) and phytase (300 or 1000 FTU/kg) from one day old until end-of-lay. Blood and bone samples were collected at 45 and 70 weeks of age. 3. Hens that were fed betaine had lower plasma homocysteine level (P < 0.05), higher tibia breaking strength (P < 0.05), and higher tibia density (P < 0.05). 4. Egg production and egg quality were excellent throughout the study and were not affected by the dietary treatments. 5. In summary, the addition of dietary betaine was successful at reducing plasma homocysteine concentrations and improving bone strength in laying hens which could be used as an intervention to alleviate welfare concerns.

Purpose: While tissue engineering offers the promise of revolutionary innovation, scalable three-dimensional tissue culture is limited by the diffusion of nutrients and oxygen making media perfusion obligatory. Unfortunately, the cost of bioreactors for large construct tissue culture can be prohibitive, with a typical perfusion chamber costing several thousand dollars, and even small petri-dish-sized devices costing hundreds of dollars each. We have developed a low-cost perfusion setup that seals collagen-based perfusable cellular constructs within a sterile PDMS well between coverslips, allowing for repeated live-imaging of perfused 3D engineered tissues. Herein we describe fabrication of this novel system and validate its utility. Methods: Molds and frames were designed on 3D-modeling software (Fusion 360) and printed on a Prusa i3 MK3S 3D printer in poly(lactic acid) (PLA). Molds were filled with poly(dimethyl siloxane) (PDMS), which was cured to form chambers, bubble traps, mason jar lid chambers, and media reservoir lid adapters. In total, the tissue culture chamber device, mason jar lid inset, media reservoir lid, and bubble trap require 4, 1, 2, and 4 unique printed components, respectively. Results: Each perfusion chamber can be assembled for under 8 USD per device and reused repeatedly. The current model has a tissue chamber custom-built with 18x10x4 (LxWxH)mm3 dimensions, but this chamber can be readily customized to experiment-specific dimensions. These devices allow cellular hydrogel constructs to be maintained in a sterile environment after assembly, perfused at varying rates to expose cells to different levels of shear stress, and the cells can be intermittently imaged with light, fluorescent or confocal microscopy - an unparalleled benefit for monitoring of experiments and collection of timepoint imaging data. The perfusion circuit consists of autoclavable glass and PDMS components, including a bubble trap, a crucial component of the circuit for preventing air bubbles that can damage cells and block microchannels, and a lid adapter, which allows 50 mL conical tubes to serve as self-oxygenating media reservoirs. Media changes can be performed via peristaltic pump perfusion or with syringe-based cell culture techniques for static culture. Constructs have been perfused within standard incubators for up to 14 days demonstrating normal cell viability without contamination or evidence of infection, with longer perfusion culture intervals (>1 month) currently being tested. Conclusion: The increasing accessibility of 3D-modeling and 3D-printing has enabled rapid prototyping of devices to address the problems that we face as surgeon-scientists. We have developed a low-cost tissue-engineering perfusion circuit that facilitates 3D-tissue-culture while allowing for repeated live-imaging as the cultured tissue develops. The instructions for our setup can be utilized to replicate our devices in other labs, and these designs can be readily customized to meet the needs of specific experimental aims.

Hypertension is linked with an increased risk of white matter hyperintensities; however, recent findings have questioned this association. We examined whether hypertension and additional cerebrovascular risk factors impacted on white matter integrity in an inducible hypertensive rat. No white matter hyperintensities were observed on magnetic resonance imaging either alone or in conjunction with ageing and high-fat diet. Aged hypertensive rats that were fed a high-fat diet had moderately reduced fractional anisotropy in the corpus callosum with no overt pathological features. Herein we show that moderate hypertension alone or with additional risk factors has minimal impact on white matter integrity in this model.

Maternal aggressive behaviour, which protects the offspring from harm, is one component of maternal behaviour. Not only maternal aggression, but also maternal care and social behaviour in general, is regulated by the brain oxytocin (OXT) and vasopressin (AVP) systems. In the present study, we quantified the intensity of maternal aggression using the maternal defence test at key time points throughout pregnancy, parturition and lactation. Furthermore, we quantified changes in central OXT and arginine AVP V1a receptor (V1a-R) binding in brain regions known to be important in regulating maternal aggression, aiming to investigate whether central changes coincide with the intensity of this behaviour. The intensity of aggression was found to dramatically change over the peripartum period, with its first appearance on the day before parturition. Aggression intensity fell immediately after parturition, although it increased during days 4-7 of lactation, before almost disappearing at weaning. OXT receptor (OTR) and V1a-R binding also showed changes through the peripartum period. OTR binding was highest at parturition within the bed nucleus of the stria terminalis and medial preoptic area and on days 4-7 of lactation in the lateral septum (LS) compared to any other time point during the peripartum period. OTR binding positively correlated with the peak of maternal aggression, suggesting that OXT may act in the LS to facilitate the expression of aggressive behaviour. At parturition, V1a-R binding was at its highest levels in the paraventricular nucleus and central amygdala (CeA) and, in the LS, V1a-R binding positively correlated with aggressive behaviour. V1a-R mRNA expression was also increased within the CeA at parturition. Taken together, the observed fluctuations in OTR and V1a-R binding in the neural circuitry important for regulating maternal behaviour may ensure that maternal aggression is expressed at the correct time during the peripartum period.

High glucocorticoid levels induced by stress enhance the memory of fearful events and may contribute to the development of anxiety and posttraumatic stress disorder. In contrast, elevated glucocorticoids associated with ageing impair spatial memory. We have previously shown that pharmacological inhibition of the intracellular glucocorticoid-amplifying enzyme 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) improves spatial memory in aged mice. However, it is not known whether inhibition of 11β-HSD1 will have any beneficial effects on contextual fear memories in aged mice. Here, we examined the effects of UE2316, a selective 11β-HSD1 inhibitor which accesses the brain, on both spatial and contextual fear memories in aged mice using a vehicle-controlled crossover study design. Short-term UE2316 treatment improved spatial memory in aged mice, an effect which was reversed when UE2316 was substituted with vehicle. In contrast, contextual fear memory induced by foot-shock conditioning was significantly reduced by UE2316 in a non-reversible manner. When the order of treatment was reversed following extinction of the original fear memory, and a second foot-shock conditioning was given in a novel context, UE2316 treated aged mice (previously on vehicle) now showed increased fear memory compared to vehicle-treated aged mice (previously on UE2316). Renewal of the original extinguished fear memory triggered by exposure to a new environmental context may explain these effects. Thus 11β-HSD1 inhibition reverses spatial memory impairments with ageing while reducing the strength and persistence of new contextual fear memories. Potentially this could help prevent anxiety-related disorders in vulnerable elderly individuals., Highlights • Aged mice were treated with UE2316 using a vehicle-controlled crossover design. • Short-term UE2316 treatment improves spatial memory in a reversible manner. • Contextual fear memory retention was impaired with UE2316. • Contextual fear memory effects persisted following reversal of treatment.