Rejuvenation of extensively passaged human chondrocytes to engineer functional articular cartilage.

Human articular chondrocytes (hACs) are scarce and lose their chondrogenic potential during monolayer passaging, impeding their therapeutic use. This study investigated (a) the translatability of conservative chondrogenic passaging and aggregate rejuvenation on restoring chondrogenic properties of hACs passaged up to P9; and (b) the efficacy of a combined treatment of transforming growth factor-beta 1 (TGF-ß1) (T), chondroitinase-ABC (C), and lysyl oxidase-like 2 (L), collectively termed TCL, on engineering functional human neocartilage via the self-assembling process, as a function of passage number up to P11. Here, we show that aggregate rejuvenation enhanced glycosaminoglycan (GAG) content and type II collagen staining at all passages and yielded human neocartilage with chondrogenic phenotype present up to P7. Addition of TCL extended the chondrogenic phenotype to P11 and significantly enhanced GAG content and type II collagen staining at all passages. Human neocartilage derived from high passages, treated with TCL, displayed mechanical properties that were on par with or greater than those derived from low passages. Conservative chondrogenic passaging and aggregate rejuvenation may be a viable new strategy (a) to address the perennial problem of chondrocyte scarcity and (b) to successfully rejuvenate the chondrogenic phenotype of extensively passaged cells (up to P11). Furthermore, tissue engineering human neocartilage via self-assembly in conjunction with TCL treatment advances the clinical use of extensively passaged human chondrocytes for cartilage repair.

An Antibody Targeting ICOS Increases Intratumoral Cytotoxic to Regulatory T-cell Ratio and Induces Tumor Regression.

The immunosuppressive tumor microenvironment constitutes a significant hurdle to immune checkpoint inhibitor responses. Both soluble factors and specialized immune cells, such as regulatory T cells (Treg), are key components of active intratumoral immunosuppression. Inducible costimulatory receptor (ICOS) can be highly expressed in the tumor microenvironment, especially on immunosuppressive Treg, suggesting that it represents a relevant target for preferential depletion of these cells. Here, we performed immune profiling of samples from tumor-bearing mice and patients with cancer to demonstrate differential expression of ICOS in immune T-cell subsets in different tissues. ICOS expression was higher on intratumoral Treg than on effector CD8 T cells. In addition, by immunizing an Icos knockout transgenic mouse line expressing antibodies with human variable domains, we selected a fully human IgG1 antibody called KY1044 that bound ICOS from different species. We showed that KY1044 induced sustained depletion of ICOShigh T cells but was also associated with increased secretion of proinflammatory cytokines from ICOSlow effector T cells (Teff). In syngeneic mouse tumor models, KY1044 depleted ICOShigh Treg and increased the intratumoral TEff:Treg ratio, resulting in increased secretion of IFNγ and TNFα by TEff cells. KY1044 demonstrated monotherapy antitumor efficacy and improved anti-PD-L1 efficacy. In summary, we demonstrated that using KY1044, one can exploit the differential expression of ICOS on T-cell subtypes to improve the intratumoral immune contexture and restore an antitumor immune response.

Translating the application of transforming growth factor-ß1, chondroitinase-ABC, and lysyl oxidase-like 2 for mechanically robust tissue-engineered human neocartilage.

Strategies to overcome the limited availability of human articular chondrocytes and their tendency to dedifferentiate during expansion are required to advance their clinical use and to engineer functional cartilage on par with native articular cartilage. This work sought to determine whether a biochemical factor (transforming growth factor-ß1 [T]), a biophysical agent (chondroitinase-ABC [C]), and a collagen crosslinking enzyme (lysyl oxidase-like 2 [L]) are efficacious in forming three-dimensional human neocartilage from expanded human articular chondrocytes. Among the treatment regimens, the combination of the three stimuli (TCL treatment) led to the most robust glycosaminoglycan content, total collagen content, and type II collagen production. In particular, TCL treatment synergistically increased tensile stiffness and strength of human neocartilage by 3.5-fold and 3-fold, respectively, over controls. Applied to two additional donors, the beneficial effects of TCL treatment appear to be donor independent; tensile stiffness and strength were increased by up to 8.5-fold and 3-fold, respectively, over controls. The maturation of human neocartilage in response to TCL treatment was examined following 5 and 8 weeks of culture, demonstrating maintenance or further enhancement of functional properties. The present study identifies a novel strategy for engineering human articular cartilage using serially passaged chondrocytes.

Facet Joints of the Spine: Structure-Function Relationships, Problems and Treatments, and the Potential for Regeneration.

The zygapophysial joint, a diarthrodial joint commonly referred to as the facet joint, plays a pivotal role in back pain, a condition that has been a leading cause of global disability since 1990. Along with the intervertebral disc, the facet joint supports spinal motion and aids in spinal stability. Highly susceptible to early development of osteoarthritis, the facet is responsible for a significant amount of pain in the low-back, mid-back, and neck regions. Current noninvasive treatments cannot offer long-term pain relief, while invasive treatments can relieve pain but fail to preserve joint functionality. This review presents an overview of the facet in terms of its anatomy, functional properties, problems, and current management strategies. Furthermore, this review introduces the potential for regeneration of the facet and particular engineering strategies that could be employed as a long-term treatment.

Characterization of facet joint cartilage properties in the human and interspecies comparisons.

The facet joint, a synovial joint located on the posterior-lateral spine, is highly susceptible to degenerative changes and plays a significant role in back-related morbidities. Despite its significance, the facet is rarely studied and thus current treatment strategies are lacking. This study aimed to characterize, for the first time, the properties of human, pig, monkey, and rabbit lumbar facet cartilage providing much-needed design criteria for tissue engineering approaches. In this study, where possible, the facet's morphological, histological, mechanical, and biochemical properties were evaluated. Comparisons between the properties of the inferior and superior facet surfaces, as well as among spinal levels were performed within each species. In addition, interspecies comparisons of the properties were determined. The human facet joint was found to be degenerated; 100% of joint surfaces showed signs of pathology and approximately 71% of these were considered to be grade 4. Joint morphology varied among species, demonstrating that despite the mini-pig facet being closest to the human in terms of width and length, it was far more curved than the human or any of the other species. No notable differences were found in the mini-pig, monkey, and rabbit mechanical and biochemical properties, suggesting that these species, despite morphological differences, may serve as suitable animal models for studying structure-function relationships of the human facet joint. The characterization data reported in this study may increase our understanding of this ill-described joint as well as provide the foundation for the development of new treatments such as tissue engineering. STATEMENT OF SIGNIFICANCE: This work provides the first comprehensive description of the properties of lumbar facet joint cartilage. Importantly, this work establishes that histological, biochemical, and mechanical properties are comparable between bipedal and quadrupedal animals, helping to guide future selection of appropriate animal models. This work also suggests that the human facet joint is highly susceptible to pathology. The mechanical properties of facet cartilage, found to be inferior to those of other synovial joints, provide a greater understanding of the joint's structure-function relationships as well as the potential etiology of facet joint pathology. Lastly, this work will serve as the foundation for the development of much-needed facet joint treatments, especially those based on tissue engineering approaches.

Determining the influence of calcification on the failure properties of abdominal aortic aneurysm (AAA) tissue.

Varying degrees of calcification are present in most abdominal aortic aneurysms (AAAs). However, their impact on AAA failure properties and AAA rupture risk is unclear. The aim of this work is evaluate and compare the failure properties of partially calcified and predominantly fibrous AAA tissue and investigate the potential reasons for failure. Uniaxial mechanical testing was performed on AAA samples harvested from 31 patients undergoing open surgical repair. Individual tensile samples were divided into two groups: fibrous (n=31) and partially calcified (n=38). The presence of calcification was confirmed by fourier transform infrared spectroscopy (FTIR). A total of 69 mechanical tests were performed and the failure stretch (λf), failure stress (σf) and failure tension (Tf) were recorded for each test. Following mechanical testing, the failure sites of a subset of both tissue types were examined using scanning electron microscopy (SEM)/energy dispersive X-ray spectroscopy (EDS) to investigate the potential reasons for failure. It has been shown that the failure properties of partially calcified tissue are significantly reduced compared to fibrous tissue and SEM and EDS results suggest that the junction between a calcification deposit and the fibrous matrix is highly susceptible to failure. This study implicates the presence of calcification as a key player in AAA rupture risk and provides further motivation for the development of non-invasive methods of measuring calcification.

The biaxial biomechanical behavior of abdominal aortic aneurysm tissue.

Rupture of the abdominal aortic aneurysm (AAA) occurs when the local wall stress exceeds the local wall strength. Knowledge of AAA wall mechanics plays a fundamental role in the development and advancement of AAA rupture risk assessment tools. Therefore, the aim of this study is to evaluate the biaxial mechanical properties of AAA tissue. Multiple biaxial test protocols were performed on AAA samples harvested from 28 patients undergoing open surgical repair. Both the Tangential Modulus (TM) and stretch ratio (λ) were recorded and compared in both the circumferential (Ï´) and longitudinal (L) directions at physiologically relevant stress levels, the influence of patient specific factors such as sex, age AAA diameter and status were examined. The biomechanical response was also fit to a hyperplastic material model. The AAA tissue was found to be anisotropic with a greater tendency to stiffen in the circumferential direction compared to the longitudinal direction. An anisotropic hyperelastic constitutive model represented the data well and the properties were not influenced by the investigated patient specific factors however, a future study utilizing a larger cohort of patients is warranted to confirm these findings. This work provides further insights on the biomechanical behavior of AAA and may be useful in the development of more reliable rupture risk assessment tools.

The impact of long term freezing on the mechanical properties of porcine aortic tissue.

BACKGROUND: Preservation of the native artery׳s functionality can be important in both clinical and experimental applications. Although, simple cryopreservation techniques offer an attractive solution to this problem, the extent to which freezing affects the tissue׳s properties is widely debated. Earlier assessments of the mechanical properties post-freezing have been limited by one or more of the following: small sample numbers, uncontrolled inter-specimen/animal variability, failure to account for the impact of potential errors in thickness measurements, short storage times and uniaxial test methods. MATERIAL AND METHODS: Biaxial mechanical tests were performed on porcine aortic samples (n=89) extracted from superior, middle and inferior regions of five aortas, stored in isotonic saline at -20°C for 1 day, 1 week, 1, 6 and 12 months, thawed and retested. The sample׳s weight and thickness were also measured pre and post-freezing. A total of 178 tests were performed and elastic modulus was assessed by calculating the slope of the Cauchy stress-stretch curve at the low and high stretch regions in both the circumferential (θ) and longitudinal (L) directions. RESULTS: The weight of the samples increased post-freezing. However, in general, no significant difference was found between the elastic modulus of porcine aortic tissue before and after freezing at -20°C and was unaffected by storage time. Although more accurate measuring instruments are warranted to confirm this finding, minor changes to the elastic modulus as a result of freezing were negatively correlated with regional variances i.e. changes in the elastic modulus decreased from the superior to the inferior region. CONCLUSIONS: These results indicate that for applications which require preservation of the gross mechanical properties, storing the tissue at -20°C in isotonic saline, for an extended period of time, is acceptable.

Complete humanization of the mouse immunoglobulin loci enables efficient therapeutic antibody discovery.

If immunized with an antigen of interest, transgenic mice with large portions of unrearranged human immunoglobulin loci can produce fully human antigen-specific antibodies; several such antibodies are in clinical use. However, technical limitations inherent to conventional transgenic technology and sequence divergence between the human and mouse immunoglobulin constant regions limit the utility of these mice. Here, using repetitive cycles of genome engineering in embryonic stem cells, we have inserted the entire human immunoglobulin variable-gene repertoire (2.7 Mb) into the mouse genome, leaving the mouse constant regions intact. These transgenic mice are viable and fertile, with an immune system resembling that of wild-type mice. Antigen immunization results in production of high-affinity antibodies with long human-like complementarity-determining region 3 (CDR3H), broad epitope coverage and strong signatures of somatic hypermutation. These mice provide a robust system for the discovery of therapeutic human monoclonal antibodies; as a surrogate readout of the human antibody response, they may also aid vaccine design efforts.

The biaxial mechanical behaviour of abdominal aortic aneurysm intraluminal thrombus: classification of morphology and the determination of layer and region specific properties.

Intraluminal thrombus (ILT) is present in 75% of clinically-relevant abdominal aortic aneurysms (AAAs) yet, despite much research effort, its role in AAA biomechanics remains unclear. The aim of this work is to further evaluate the biomechanics of ILT and determine if different ILT morphologies have varying mechanical properties. Biaxial mechanical tests were performed on ILT samples harvested from 19 patients undergoing open surgical repair. ILT were separated into luminal, medial and medial/abluminal layers. A total of 356 tests were performed and the Cauchy stress (σ) and tangential modulus (TM) at a stretch ratio (λ) of 1.14 were recorded for each test in both the circumferential (θ) and longitudinal (L) directions. Our data revealed three distinct types of ILT morphologies, each with a unique set of mechanical properties. All ILT layers were found to be isotropic and inhomogeneous. Type 1 (n=10) was a multi-layered ILT (thick medial/abluminal layer) whose strength and stiffness decreased gradually from the luminal to the medial/abluminal layer. Type 2 (n=6) was a multi-layered ILT (thin/highly degraded medial/abluminal layer) whose strength and stiffness decreased abruptly between the luminal and medial/abluminal layer and Type 3 (n=3) is a single layered ILT with a lower strength and stiffness than Types 1 and 2. In a sub-study, we found the luminal layer to be stronger and stiffer in the posterior than the anterior region. This work provides further insights to the biomechanical behaviour of ILT and the use of our ILT classification may be useful in future studies.

Comparison of methods used to measure the thickness of soft tissues and their influence on the evaluation of tensile stress.

Measuring the physical dimensions of soft tissue is difficult due to its deformable nature. Such measurements are used to evaluate the tissue's mechanical properties. Imprecise measurements of the tissue's thickness can alter the assessment of tensile stress which may have significant clinical relevance when used as a diagnostic tool. The performance of routinely used measurement methods including a (i) vernier calipers, (ii) micrometer, (iii) thickness gauge, (iv) glass slide technique coupled with (i) and (ii) and a (v) laser displacement sensor were assessed by comparing them to a photogrammetric technique which was considered to be the measurement standard. All measurements were performed on two tissue types: porcine aorta and human intraluminal thrombus from an abdominal aortic aneurysm (AAA) and results were compared against predetermined criteria whose limits represented a 10% change in experimentally derived tensile stress. The inter-rater and retest reliability of the vernier calipers, micrometer and thickness gauge were also investigated. The thickness gauge was shown to be the most reliable and could accurately measure the thickness of aortic tissue. The conditions of the criteria were not met by any instrument used to measure the thickness of the AAA intraluminal thrombus, however, the micrometer, which proved highly reliable, was considered the most suitable (effects on tensile stress: +14.7%). For both tissues the glass slide and laser techniques significantly over estimated the thickness measurement altering the tensile stress by up to -29.6%. This study highlights the effects of inaccurate measurements on the assessment of tensile stress and recommends a thickness gauge be used to measure structured tissue (aorta) and a micrometer for unstructured tissue (AAA intraluminal thrombus).

An open trial of a comprehensive anger treatment program on an outpatient sample.

BACKGROUND: This pilot study was designed to investigate the efficacy of a cognitive behavioral treatment for anger. METHOD: Twelve (5 men and 7 women) outpatient adults completed 2-hour group sessions for 16 sessions. Participants were diagnosed with 29 Axis I and 34 Axis II disorders with high rates of comorbidity. Empirically supported techniques of skills training, cognitive restructuring, and relaxation were utilized. In this protocol, cognitive restructuring emphasized the use of the ABC model to understand anger episodes and the Rational Emotive Behavior Therapy (REBT) techniques of disputing irrational beliefs and rehearsing rational coping statements, but additional cognitive techniques were used, e.g. self-instructional training (SIT). Skills training included problem-solving and assertiveness. Relaxation training was paced respiration. Motivational interviewing, imaginal exposure with coping, and relapse prevention were also included. RESULTS: Significant improvements were found from pre- to post-treatment on the following measures: the Trait Anger Scale of the State-Trait Anger Expression Inventory-II; and Anger Disorder Scale total scores; idiosyncratic anger measurements of situational intensity and symptom severity; and the Beck Depression Inventory-II. CONCLUSIONS: In order to extend the significant research findings of this pilot study, future investigations should involve larger sample sizes, populations drawn from various settings, and contact control groups.

A placebo controlled, double-blind study of mecamylamine treatment for cocaine dependence in patients enrolled in an opiate replacement program.

A placebo controlled, double-blind trial of mecamylamine treatment of cocaine dependence was performed in methadone or LAAM maintained subjects who met DSM-IV criteria for cocaine dependence. After an eight-week placebo run-in screening period, 35 subjects were randomly assigned to receive either mecamylamine (6 mg/day) or placebo transdermal patches for a 16-week treatment period. Outcome measures included quantitative urine benzoylecognine (BE) levels, self-report of cocaine use, cocaine craving, global impression scores, mood, retention, and safety. Mecamylamine was well tolerated, and study retention did not differ by treatment group. Evidence for cocaine use, based on urine BE levels and cocaine abstinence rates, did not differ by treatment group. Self reported cocaine use, cocaine craving, and global impression scores showed moderate improvement in both groups, with a significantly greater reduction in cocaine craving (p < 0.05) and self-rated severity of cocaine dependence (p < 0.05) in the placebo group. This pilot study does not support the effectiveness of mecamylamine for the treatment of cocaine dependence in methadone or LAAM maintained patients.

Sensitization to the psychosis-inducing effects of cocaine compared with measures of cocaine craving and cue reactivity.

A previous study has suggested that sensitization to the psychosis-inducing effects of cocaine may be a marker of vulnerability to relapse in cocaine addiction. In this report, cocaine-dependent subjects participating in a study on naturally occurring and cue-induced cocaine craving were interviewed about prior experience of cocaine-induced psychosis and the degree to which this effect had become more frequent or severe or had occurred at lower cumulative doses. Sensitization to cocaine-induced psychosis was negatively correlated with baseline measures of drug dependence severity and indices of cocaine craving over the preceding 24 hours but not with measures of cocaine cue reactivity.

Quantitative electroencephalographic studies of cue-induced cocaine craving.

Quantitative electroencephalographic (qEEG) profiles were studied in cocaine dependent patients in response to cocaine cue exposure. Using neurometric analytical methods, the spectral power of each primary bandwidth was computed and topographically mapped. Additional measures of cue-reactivity included cocaine craving, anxiety and related subjective ratings, and physiological measures of skin conductance, skin temperature, heart rate, and plasma cortisol and HVA levels. Twenty-four crack cocaine-dependent subjects were tested for their response to tactile, visual and audio cues related to crack cocaine or neutral items. All measures were analyzed for significant difference by comparing cocaine versus neutral cue conditions. An increase in cocaine craving, anxiety and related subjective ratings, elevated plasma cortisol levels, and a decrease in skin temperature, were induced by cocaine cue exposure. Distinct qEEG profiles were found during the paraphernalia handling and video viewing (eyes-open), and guided imagery (eyes-closed), phases of cocaine cue exposure. During paraphernalia handling and video viewing, there was an increase in beta activity accompanied by a drop in delta power in the frontal cortex, and an increase in beta mean frequency in the occipital cortex. In contrast, during guided imagery there was an increase in theta and delta power in the frontal cortex, and an increase in beta power in the occipital cortex. Correlation analyses revealed that cue-induced anxiety during paraphernalia handling and video viewing was associated with reduced high frequency and enhanced low frequency EEG activity. These findings demonstrated that EEG activation during cue-induced cocaine craving may be topographically mapped and subsequently analyzed for functional relevance.