Treatment of Ligament Constructs with Exercise-conditioned Serum: A Translational Tissue Engineering Model.

In vitro experiments are essential to understand biological mechanisms; however, the gap between monolayer tissue culture and human physiology is large, and translation of findings is often poor. Thus, there is ample opportunity for alternative experimental approaches. Here we present an approach in which human cells are isolated from human anterior cruciate ligament tissue remnants, expanded in culture, and used to form engineered ligaments. Exercise alters the biochemical milieu in the blood such that the function of many tissues, organs and bodily processes are improved. In this experiment, ligament construct culture media was supplemented with experimental human serum that has been 'conditioned' by exercise. Thus the intervention is more biologically relevant since an experimental tissue is exposed to the full endogenous biochemical milieu, including binding proteins and adjunct compounds that may be altered in tandem with the activity of an unknown agent of interest. After treatment, engineered ligaments can be analyzed for mechanical function, collagen content, morphology, and cellular biochemistry. Overall, there are four major advantages versus traditional monolayer culture and animal models, of the physiological model of ligament tissue that is presented here. First, ligament constructs are three-dimensional, allowing for mechanical properties (i.e., function) such as ultimate tensile stress, maximal tensile load, and modulus, to be quantified. Second, the enthesis, the interface between boney and sinew elements, can be examined in detail and within functional context. Third, preparing media with post-exercise serum allows for the effects of the exercise-induced biochemical milieu, which is responsible for the wide range of health benefits of exercise, to be investigated in an unbiased manner. Finally, this experimental model advances scientific research in a humane and ethical manner by replacing the use of animals, a core mandate of the National Institutes of Health, the Center for Disease Control, and the Food and Drug Administration

Prevalence of amyloid PET positivity in dementia syndromes: a meta-analysis

IMPORTANCE: Amyloid-β positron emission tomography (PET) imaging allows in vivo detection of fibrillar plaques, a core neuropathological feature of Alzheimer disease (AD). Its diagnostic utility is still unclear because amyloid plaques also occur in patients with non-AD dementia. OBJECTIVE: To use individual participant data meta-analysis to estimate the prevalence of amyloid positivity on PET in a wide variety of dementia syndromes. DATA SOURCES: The MEDLINE and Web of Science databases were searched from January 2004 to April 2015 for amyloid PET studies. STUDY SELECTION: Case reports and studies on neurological or psychiatric diseases other than dementia were excluded. Corresponding authors of eligible cohorts were invited to provide individual participant data. DATA EXTRACTION AND SYNTHESIS: Data were provided for 1359 participants with clinically diagnosed AD and 538 participants with non-AD dementia. The reference groups were 1849 healthy control participants (based on amyloid PET) and an independent sample of 1369 AD participants (based on autopsy). MAIN OUTCOMES AND MEASURES: Estimated prevalence of positive amyloid PET scans according to diagnosis, age, and apolipoprotein E (APOE) ε4 status, using the generalized estimating equations method. RESULTS: The likelihood of amyloid positivity was associated with age and APOE ε4 status. In AD dementia, the prevalence of amyloid positivity decreased from age 50 to 90 years in APOE ε4 noncarriers (86% [95% CI, 73%-94%] at 50 years to 68% [95% CI, 57%-77%] at 90 years; n = 377) and to a lesser degree in APOE ε4 carriers (97% [95% CI, 92%-99%] at 50 years to 90% [95% CI, 83%-94%] at 90 years; n = 593; P < .01). Similar associations of age and APOE ε4 with amyloid positivity were observed in participants with AD dementia at autopsy. In most non-AD dementias, amyloid positivity increased with both age (from 60 to 80 years) and APOE ε4 carriership (dementia with Lewy bodies: carriers [n = 16], 63% [95% CI, 48%-80%] at 60 years to 83% [95% CI, 67%-92%] at 80 years; noncarriers [n = 18], 29% [95% CI, 15%-50%] at 60 years to 54% [95% CI, 30%-77%] at 80 years; frontotemporal dementia: carriers [n = 48], 19% [95% CI, 12%-28%] at 60 years to 43% [95% CI, 35%-50%] at 80 years; noncarriers [n = 160], 5% [95% CI, 3%-8%] at 60 years to 14% [95% CI, 11%-18%] at 80 years; vascular dementia: carriers [n = 30], 25% [95% CI, 9%-52%] at 60 years to 64% [95% CI, 49%-77%] at 80 years; noncarriers [n = 77], 7% [95% CI, 3%-18%] at 60 years to 29% [95% CI, 17%-43%] at 80 years. CONCLUSIONS AND RELEVANCE: Among participants with dementia, the prevalence of amyloid positivity was associated with clinical diagnosis, age, and APOE genotype. These findings indicate the potential clinical utility of amyloid imaging for differential diagnosis in early-onset dementia and to support the clinical diagnosis of participants with AD dementia and noncarrier APOE ε4 status who are older than 70 years

Glioma Through the Looking GLASS: Molecular Evolution of Diffuse Gliomas and the Glioma Longitudinal AnalySiS Consortium

Adult diffuse gliomas are a diverse group of brain neoplasms that inflict a high emotional toll on patients and their families. The Cancer Genome Atlas (TCGA) and similar projects have provided a comprehensive understanding of the somatic alterations and molecular subtypes of glioma at diagnosis. However, gliomas undergo significant cellular and molecular evolution during disease progression. We review the current knowledge on the genomic and epigenetic abnormalities in primary tumors and after disease recurrence, highlight the gaps in the literature, and elaborate on the need for a new multi-institutional effort to bridge these knowledge gaps and how the Glioma Longitudinal AnalySiS Consortium (GLASS) aims to systemically catalog the longitudinal changes in gliomas. The GLASS initiative will provide essential insights into the evolution of glioma toward a lethal phenotype, with the potential to reveal targetable vulnerabilities, and ultimately, improved outcomes for a patient population in need

Engineering Bone-to-Bone Ligaments and Their Use as a Physiological Model

Anterior cruciate ligament (ACL) injuries are one of most common musculoskeletal injuries and negatively affect mobility and quality of life. ACL rupture requires reconstruction to repair ligament at an estimated cost of $1.5 billion/year. Current surgical solutions invariably involve either donor site morbidity with the use of autografts or the risk of disease transmission and immune rejection with the use of allografts. Successful reconstruction requires the presence of an intact interface between ligament and bone, a transitional tissue called the enthesis. The enthesis is critical for the safe and effective transfer of force from the stiff bone to the more compliant ligament by providing a gradual transition of mechanical and biochemical properties to prevent the formation of stress concentrations. A tissue engineered ligament containing mature entheses is a promising alternative to autografts and allografts, especially since this interface does not normally regenerate. Toward this end, this dissertation sought to improve engineered fibrin-based bone-to-bone ligaments previously developed by our lab and to demonstrate their utility in understanding physiological processes through three specific aims: 1) optimize the environment for in vitro ligament function, 2) induce the formation of a fibrocartilaginous interface, and 3) demonstrate the utility of engineered ligaments as a physiological model. In Aim 1, the in vitro culture environment was investigated for engineered ligaments formed using human ACL fibroblasts. Using a DOE approach, we identified significant effects and interactions of soluble factors on the maximal tensile load (MTL) and collagen content of engineered human ACL. The DOE model was used to predict a maximal growth media which significantly improved the MTL and collagen content of engineered ligaments and can be combined with increases in the initial construct volume for 77% further improvement in MTL. In addition to the improvements in tissue function, these data suggest that a DOE approach can more efficiently optimize in vitro parameters including the dosage and timing of chemical and mechanical stimuli as well as any interactions. Aim 2 presented two strategies to improve of the engineered enthesis. First, the local release of bone morphogenetic protein (BMP)-4 at the enthesis of engineered ligaments demonstrated improved interface strength as well as the transition of cells at the enthesis towards an unmineralized fibrocartilage phenotype. Second, engineered ligaments formed in a modular fashion improved the mechanical function and the morphology of the engineered enthesis including the development of cell and soft tissue integration into the mineral phase, a tidemark between mineralized and unmineralized tissue, and the presence of a dense band of extracellular matrix (ECM) at the soft tissue-mineral interface. Importantly, this is the first demonstration of the in vitro formation of a functional interface between engineered ligament and mineral in a complete bone-to-bone ligament unit. Aim 3 demonstrated the use of our engineered ligament model as a physiological tool. During the estrogen surge in the menstrual cycle, there is an associated increase in the incidence of ACL ruptures as well as knee laxity. Using physiological levels of estrogen mimicking the estrogen surge in vitro, we determined that estrogen decreases the activity of the collagen crosslinker lysyl oxidase (LOX) with a subsequent decrease in tissue stiffness providing insight into why women have greater incidences of ACL rupture. We also examined the role of the exercise-induced biochemical environment on connective tissue using our in vitro model. Engineered ligaments cultured with serum obtained from human donors after exercise had significantly better mechanical strength and collagen content than those treated with serum obtained at rest. In 2D culture, we determined that this effect was likely a result of greater mTOR and ERK signaling. In summary, the work in this dissertation has made great strides in developing a more mature engineered bone-to-bone ligament. We have optimized a growth factor environment for their in vitro culture and created the most advanced engineered enthesis to date. We have also used these engineered tissues as a platform to mechanistically study the influence of hormones on connective tissue. With further advances in our understanding of the in vivo development of ligaments and their entheses, our bone-to-bone engineered ligaments can be improved making them more suited for clinical applications and for probing physiologically processes in a more controlled environment

Vitamin C–enriched gelatin supplementation before intermittent activity augments collagen synthesis 1 , 2

BackgroundMusculoskeletal injuries are the most common complaint in active populations. More than 50% of all injuries in sports can be classified as sprains, strains, ruptures, or breaks of musculoskeletal tissues. Nutritional and/or exercise interventions that increase collagen synthesis and strengthen these tissues could have an important effect on injury rates.ObjectiveThis study was designed to determine whether gelatin supplementation could increase collagen synthesis.DesignEight healthy male subjects completed a randomized, double-blinded, crossover-design study in which they consumed either 5 or 15 g of vitamin C-enriched gelatin or a placebo control. After the initial drink, blood was taken every 30 min to determine amino acid content in the blood. A larger blood sample was taken before and 1 h after consumption of gelatin for treatment of engineered ligaments. One hour after the initial supplement, the subjects completed 6 min of rope-skipping to stimulate collagen synthesis. This pattern of supplementation was repeated 3 times/d with ≥6 h between exercise bouts for 3 d. Blood was drawn before and 4, 24, 48, and 72 h after the first exercise bout for determination of amino-terminal propeptide of collagen I content.ResultsSupplementation with increasing amounts of gelatin increased circulating glycine, proline, hydroxyproline, and hydroxylysine, peaking 1 h after the supplement was given. Engineered ligaments treated for 6 d with serum from samples collected before or 1 h after subjects consumed a placebo or 5 or 15 g gelatin showed increased collagen content and improved mechanics. Subjects who took 15 g gelatin 1 h before exercise showed double the amino-terminal propeptide of collagen I in their blood, indicating increased collagen synthesis.ConclusionThese data suggest that adding gelatin to an intermittent exercise program improves collagen synthesis and could play a beneficial role in injury prevention and tissue repair. This trial was registered at the Australian New Zealand Clinical Trials Registry as ACTRN12616001092482

Vitamin C-enriched gelatin supplementation before intermittent activity augments collagen synthesis

Background: Musculoskeletal injuries are the most common complaint in active populations. More than 50% of all injuries in sports can be classified as sprains, strains, ruptures, or breaks of musculoskeletal tissues. Nutritional and/or exercise interventions that increase collagen synthesis and strengthen these tissues could have an important effect on injury rates. Objective: This study was designed to determine whether gelatin supplementation could increase collagen synthesis. Design: Eight healthy male subjects completed a randomized, double-blinded, crossover-design study in which they consumed either 5 or 15 g of vitamin C–enriched gelatin or a placebo control. After the initial drink, blood was taken every 30 min to determine amino acid content in the blood. A larger blood sample was taken before and 1 h after consumption of gelatin for treatment of engineered ligaments. One hour after the initial supplement, the subjects completed 6 min of rope-skipping to stimulate collagen synthesis. This pattern of supplementation was repeated 3 times/d with ≥6 h between exercise bouts for 3 d. Blood was drawn before and 4, 24, 48, and 72 h after the first exercise bout for determination of amino-terminal propeptide of collagen I content. Results: Supplementation with increasing amounts of gelatin increased circulating glycine, proline, hydroxyproline, and hydroxylysine, peaking 1 h after the supplement was given. Engineered ligaments treated for 6 d with serum from samples collected before or 1 h after subjects consumed a placebo or 5 or 15 g gelatin showed increased collagen content and improved mechanics. Subjects who took 15 g gelatin 1 h before exercise showed double the amino-terminal propeptide of collagen I in their blood, indicating increased collagen synthesis. Conclusion: These data suggest that adding gelatin to an intermittent exercise program improves collagen synthesis and could play a beneficial role in injury prevention and tissue repair