Divergent Spatial Learning, Enhanced Neuronal Transcription, and Blood-Brain Barrier Disruption Develop During Recovery from Post-Injury Sleep Fragmentation.

Traumatic brain injury (TBI) causes pathophysiology that may significantly decrease quality of life over time. A major propagator of this response is chronic, maladaptive neuroinflammation, which can be exacerbated by stressors such as sleep fragmentation (SF). This study determined whether post-TBI SF had lasting behavioral and inflammatory effects even with a period of recovery. To test this, male and female mice received a moderate lateral fluid percussion TBI or sham surgery. Half the mice were left undisturbed, and half were exposed to daily SF for 30 days. All mice were then undisturbed between 30 and 60 days post-injury (DPI), allowing mice to recover from SF (SF-R). SF-R did not impair global Barnes maze performance. Nonetheless, TBI SF-R mice displayed retrogression in latency to reach the goal box within testing days. These nuanced behavioral changes in TBI SF-R mice were associated with enhanced expression of neuronal processing/signaling genes and indicators of blood-brain barrier (BBB) dysfunction. Aquaporin-4 (AQP4) expression, a marker of BBB integrity, was differentially altered by TBI and TBI SF-R. For example, TBI enhanced cortical AQP4 whereas TBI SF-R mice had the lowest cortical expression of perivascular AQP4, dysregulated AQP4 polarization, and the highest number of CD45+ cells in the ipsilateral cortex. Altogether, post-TBI SF caused lasting, divergent behavioral responses associated with enhanced expression of neuronal transcription and BBB disruption even after a period of recovery from SF. Understanding lasting impacts from post-TBI stressors can better inform both acute and chronic post-injury care to improve long-term outcome post-TBI.

Application of a High Throughput and Automated Workflow to Therapeutic Protein Formulation Development.

Rapid and efficient formulation development is critical to successfully bringing therapeutic protein drug products into a competitive market under increasingly aggressive timelines. Conventional application of high throughput techniques for formulation development have been limited to lower protein concentrations, which are not applicable to late stage development of high concentration therapeutics. In this work, we present a high throughput (HT) formulation workflow that enables screening at representative concentrations via integration of a micro-buffer exchange system with automated analytical instruments. The operational recommendations associated with the use of such HT systems as well as the efficiencies gained (reduction in hands-on time and run time by over 70% and 30%, respectively), which enable practical characterization of an expanded formulation design space, are discussed. To demonstrate that the workflow is fit for purpose, the formulation properties and stability profiles (SEC and CEX) from samples generated by the HT workflow were compared to those processed by ultrafiltration/diafiltration, and the results were shown to be in good agreement. This approach was further applied to two case studies, one focused on a formulation screen that studied the effects of pH and excipient on viscosity and stability, and the other focused on selection of an appropriate viscosity mimic solution for a protein product.

Histological characterization of human breast implant capsules.

BACKGROUND: This study investigated the relationships between histomorphological aspects of breast capsules, including capsule thickness, collagen fiber alignment, the presence of α-smooth muscle actin (α-SMA)-positive myofibroblasts, and clinical observations of capsular contracture. METHODS: Breast capsule samples were collected at the time of implant removal in patients undergoing breast implant replacement or revision surgery. Capsular contracture was scored preoperatively using the Baker scale. Histological analysis included hematoxylin and eosin staining, quantitative analysis of capsule thickness, collagen fiber alignment, and immunohistochemical evaluation for α-SMA and CD68. RESULTS: Forty-nine samples were harvested from 41 patients. A large variation in histomorphology was observed between samples, including differences in cellularity, fiber density and organization, and overall structure. Baker I capsules were significantly thinner than Baker II, III, and IV capsules. Capsule thickness positively correlated with implantation time for all capsules and for contracted capsules (Baker III and IV). Contracted capsules had significantly greater collagen fiber alignment and α-SMA-positive immunoreactivity than uncontracted capsules (Baker I and II). Capsules from textured implants had significantly less α-SMA-positive immunoreactivity than capsules from smooth implants. CONCLUSION: The histomorphological diversity observed between the breast capsules highlights the challenges of identifying mechanistic trends in capsular contracture. Our findings support the role of increasing capsule thickness and collagen fiber alignment, and the presence of contractile myofibroblasts in the development of contracture. These changes in capsule structure may be directly related to palpation stiffness considered in the Baker score. Approaches to disrupt these processes may aid in decreasing capsular contracture rates. LEVEL OF EVIDENCE III: This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

Cartilage synthesis in hyaluronic acid-tyramine constructs.

The objective of this study was to determine the potential for cartilage production within a hyaluronic acid-tyramine (HA-Tyr) hydrogel scaffold. Chondrocytes were encapsulated within HA-Tyr hydrogels and subcutaneously implanted in mice. The HA-Tyr hydrogels were formed by the oxidative coupling of Tyr moieties catalyzed by hydrogen peroxide (H2O2) and horseradish peroxidase (HRP). Harvested constructs were shown to achieve a glycosaminoglycan (GAG) content of 1.2 wt%, and they demonstrated 40% of the collagen content of normal articular cartilage, including the presence of Type II collagen, which is the characteristic of articular cartilage. Matrix production was found to be influenced by the initial cell density, scaffold degradation rate and Type II collagen concentration. Injectability was also imparted to the system by delivering HRP through thermoresponsive liposomes. The method of HRP delivery, either by simple addition or through thermoresponsive liposomes, was not shown to have an effect on matrix production.

Liposomal delivery of horseradish peroxidase for thermally triggered injectable hyaluronic acid-tyramine hydrogel scaffolds.

A thermoresponsive injectable hydrogel scaffold for tissue engineering has been developed, whereby the scaffold was injected as a liquid at room temperature, and gelled at the target site in response to the change in body temperature. Our approach involved suspending thermoresponsive liposomes, which encapsulated horseradish peroxidase (HRP), in a hyaluronic acid-tyramine (HA-Tyr) conjugate and hydrogen peroxide (H2O2) solution. At room temperature, HRP was separated from the HA-Tyr conjugate by the lipid membrane, and hence the precursor solution remained as a liquid and was injectable. Upon injection and exposure to body temperature, the lipids experienced a phase transition, which significantly increased the membrane permeability and led to the release of HRP and the oxidative coupling of Tyr moieties with H2O2, forming a crosslinked hydrogel scaffold. It was shown in this study that the precursor solution remained as a liquid on the order of hours at 20 °C, yet gelation could be induced within minutes by heating to 37 °C. Furthermore, it was shown that HRP release could be controlled by various material and processing parameters, and that the gelation rate could be adjusted to meet various clinical needs by adjusting HRP encapsulation, liposome concentration and HA-Tyr concentration.