scholarly journals Temperature alters solute transport in growth plate cartilage measured by in vivo multiphoton microscopy

2009 ◽  
Vol 106 (6) ◽  
pp. 2016-2025 ◽  
Author(s):  
Maria A. Serrat ◽  
Rebecca M. Williams ◽  
Cornelia E. Farnum
Keyword(s):  
Solute Transport ◽  
Growth Plate ◽  
Quantitative Methods ◽  
Multiphoton Microscopy ◽  
Growth Plates ◽  
Absolute Growth ◽  
Body Core ◽  
Intracardiac Injection ◽  
Bone Elongation

Solute delivery to avascular cartilaginous plates is critical to bone elongation, and impaired transport of nutrients and growth factors in cartilage matrix could underlie many skeletal abnormalities. Advances in imaging technology have revolutionized our ability to visualize growth plates in vivo, but quantitative methods are still needed. We developed analytical standards for measuring solute delivery, defined by amount and rate of intravenous tracer entry, in murine growth plates using multiphoton microscopy. We employed an acute temperature model because of its well-established impact on bone circulation and tested the hypothesis that solute delivery changes positively with limb temperature when body core and respiration are held constant (36°C, 120 breaths/min). Tibial growth plates were surgically exposed in anesthetized 5-wk-old mice, and their hindlimbs were immersed in warm (36°C) or cool (23°C) saline ( n = 6/group). After 30 min of thermal equilibration, we administered an intracardiac injection of fluorescein (50 μl, 0.5%) and captured sequentially timed growth plate images spanning 10 min at standardized depth. Absolute growth plate fluorescence was normalized to vascular concentrations for interanimal comparisons. As predicted, more fluorescein infiltrated growth plates at 36°C, with standardized values nearly double those at 23°C. Changing initial limb temperature did not alter baseline values, suggesting a sustained response period. These data validate the sensitivity of our system and have relevance to strategies for enhancing localized delivery of therapeutic agents to growth plates of children. Applications of this technique include assessment of solute transport in models of growth plate dysfunction, particularly chondrodysplasias with matrix irregularities.

scholarly journals Hindlimb heating increases vascular access of large molecules to murine tibial growth plates measured by in vivo multiphoton imaging

2014 ◽  
Vol 116 (4) ◽  
pp. 425-438 ◽  
Author(s):  
Maria A. Serrat ◽  
Morgan L. Efaw ◽  
Rebecca M. Williams
Keyword(s):  
Growth Plate ◽  
Vascular Access ◽  
Multiphoton Microscopy ◽  
Molecular Transport ◽  
Vessel Diameter ◽  
Cartilage Growth ◽  
Multiphoton Imaging ◽  
Growth Plates ◽  
Large Molecules

Advances in understanding the molecular regulation of longitudinal growth have led to development of novel drug therapies for growth plate disorders. Despite progress, a major unmet challenge is delivering therapeutic agents to avascular-cartilage plates. Dense extracellular matrix and lack of penetrating blood vessels create a semipermeable “barrier,” which hinders molecular transport at the vascular-cartilage interface. To overcome this obstacle, we used a hindlimb heating model to manipulate bone circulation in 5-wk-old female mice ( n = 22). Temperatures represented a physiological range of normal human knee joints. We used in vivo multiphoton microscopy to quantify temperature-enhanced delivery of large molecules into tibial growth plates. We tested the hypothesis that increasing hindlimb temperature from 22°C to 34°C increases vascular access of large systemic molecules, modeled using 10, 40, and 70 kDa dextrans that approximate sizes of physiological regulators. Vascular access was quantified by vessel diameter, velocity, and dextran leakage from subperichondrial plexus vessels and accumulation in growth plate cartilage. Growth plate entry of 10 kDa dextrans increased >150% at 34°C. Entry of 40 and 70 kDa dextrans increased <50%, suggesting a size-dependent temperature enhancement. Total dextran levels in the plexus increased at 34°C, but relative leakage out of vessels was not temperature dependent. Blood velocity and vessel diameter increased 118% and 31%, respectively, at 34°C. These results demonstrate that heat enhances vascular carrying capacity and bioavailability of large molecules around growth plates, suggesting that temperature could be a noninvasive strategy for modulating delivery of therapeutics to impaired growth plates of children.

scholarly journals Imaging IGF-I uptake in growth plate cartilage using in vivo multiphoton microscopy

2017 ◽  
Vol 123 (5) ◽  
pp. 1101-1109 ◽  
Author(s):  
Maria A. Serrat ◽  
Gabriela Ion
Keyword(s):  
Real Time ◽  
Growth Plate ◽  
Multiphoton Microscopy ◽  
Biologically Active ◽  
Growth Plate Chondrocytes ◽  
Growth Plates ◽  
Metatarsal Bones ◽  
Wide Range ◽  
Igf I

Bones elongate through endochondral ossification in cartilaginous growth plates located at ends of primary long bones. Linear growth ensues from a cascade of biochemical signals initiated by actions of systemic and local regulators on growth plate chondrocytes. Although cellular processes are well defined, there is a fundamental gap in understanding how growth regulators are physically transported from surrounding blood vessels into and through dense, avascular cartilage matrix. Intravital imaging using in vivo multiphoton microscopy is one promising strategy to overcome this barrier by quantitatively tracking molecular delivery to cartilage from the vasculature in real time. We previously used in vivo multiphoton imaging to show that hindlimb heating increases vascular access of large molecules to growth plates using 10-, 40-, and 70-kDa dextran tracers. To comparatively evaluate transport of similarly sized physiological regulators, we developed and validated methods for measuring uptake of biologically active IGF-I into proximal tibial growth plates of live 5-wk-old mice. We demonstrate that fluorescently labeled IGF-I (8.2 kDa) is readily taken up in the growth plate and localizes to chondrocytes. Bioactivity tests performed on cultured metatarsal bones confirmed that the labeled protein is functional, assessed by phosphorylation of its signaling kinase, Akt. This methodology, which can be broadly applied to many different proteins and tissues, is relevant for understanding factors that affect delivery of biologically relevant molecules to the skeleton in real time. Results may lead to the development of drug-targeting strategies to treat a wide range of bone and cartilage pathologies. NEW & NOTEWORTHY This paper describes and validates a novel method for imaging transport of biologically active, fluorescently labeled IGF-I into skeletal growth plates of live mice using multiphoton microscopy. Cellular patterns of fluorescence in the growth plate were completely distinct from our prior publications using biologically inert probes, demonstrating for the first time in vivo localization of IGF-I in chondrocytes and perichondrium. These results form important groundwork for future studies aimed at targeting therapeutics into growth plates.

scholarly journals Exercise mitigates the stunting effect of cold temperature on limb elongation in mice by increasing solute delivery to the growth plate

2010 ◽  
Vol 109 (6) ◽  
pp. 1869-1879 ◽  
Author(s):  
Maria A. Serrat ◽  
Rebecca M. Williams ◽  
Cornelia E. Farnum
Keyword(s):  
Growth Plate ◽  
Bone Growth ◽  
Wheel Running ◽  
Multiphoton Microscopy ◽  
Tail Length ◽  
Limb Lengthening ◽  
Cold Temperature ◽  
Bone Lengthening ◽  
Bone Elongation

Ambient temperature and physical activity modulate bone elongation in mammals, but mechanisms underlying this plasticity are a century-old enigma. Longitudinal bone growth occurs in cartilaginous plates, which receive nutritional support via delivery of solutes from the vasculature. We tested the hypothesis that chronic exercise and warm temperature promote bone lengthening by increasing solute delivery to the growth plate, measured in real time using in vivo multiphoton microscopy. We housed 68 weanling female mice at cold (16°C) or warm (25°C) temperatures and allowed some groups voluntary access to a running wheel. We show that exercise mitigates the stunting effect of cold temperature on limb elongation after 11 days of wheel running. All runners had significantly lengthened limbs, regardless of temperature, while nonrunning mice had shorter limbs that correlated with housing temperature. Tail length was impacted only by temperature, indicating that the exercise effect was localized to limb bones and was not a systemic endocrine reaction. In vivo multiphoton imaging of fluoresceinated tracers revealed enhanced solute delivery to tibial growth plates in wheel-running mice, measured under anesthesia at rest. There was a minimal effect of rearing temperature on solute delivery when measured at an intermediate room temperature (20°C), suggesting that a lasting increase in solute delivery is an important factor in exercise-mediated limb lengthening but may not play a role in temperature-mediated limb lengthening. These results are relevant to the study of skeletal evolution in mammals from varying environments and have the potential to fundamentally advance our understanding of bone elongation processes.

Involvement of matrix metalloproteinases in the growth plate response to physiological mechanical load

2010 ◽  
Vol 108 (1) ◽  
pp. 172-180 ◽  
Author(s):  
Adi Reich ◽  
Stav Simsa Maziel ◽  
Ziv Ashkenazi ◽  
Efrat Monsonego Ornan
Keyword(s):  
Growth Plate ◽  
Mechanical Loading ◽  
Mechanical Load ◽  
Tissue Growth ◽  
Matrix Assembly ◽  
Growth Plates ◽  
Load Release ◽  
Catch Up

Enzymes from the matrix metalloproteinase (MMP) family play a crucial role in growth-plate vascularization and ossification via proteolytic cleavage and remodeling of the extracellular matrix. Their regulation in the growth plate is crucial for normal matrix assembly. Endochondral ossification, which takes place at the growth plates, is influenced by mechanical loading. Using an in vivo avian model for mechanical loading, we have found increased blood penetration into the growth plates of loaded chicks. The purpose of this work was to study the involvement of MMP-2, -3, -9, -13, and -16 in the growth plate's response to loading and in the catch-up growth resulting from load release. We found that mechanical loading, as well as release from load, upregulated MMP-2, -9, and -13 expressions. In contrast, MMP-3, associated with cartilage injuries, and its associated protein connective tissue growth factor (CTGF), were downregulated by the load. However, after release from load, MMP-3 was upregulated and CTGF levels were elevated and caught up with the control. MMP-3 and CTGF were also downregulated after 60 min of mechanical stretching in vitro. These results demonstrate the central role of MMPs in the growth plate's response to mechanical loading, as well as in the catch-up growth followed load release.

scholarly journals Solute Transport in Growth Plate Cartilage: In Vitro and In Vivo

2007 ◽  
Vol 93 (3) ◽  
pp. 1039-1050 ◽  
Author(s):  
Rebecca M. Williams ◽  
Warren R. Zipfel ◽  
Michelle L. Tinsley ◽  
Cornelia E. Farnum
Keyword(s):  
Solute Transport ◽  
Growth Plate ◽  
Growth Plate Cartilage

Altered IGF-I activity and accelerated bone elongation in growth plates precedes excess weight gain in a mouse model of juvenile obesity

2022 ◽  
Author(s):  
Allison L. Machnicki ◽  
Cassaundra A. White ◽  
Chad A. Meadows ◽  
Darby McCloud ◽  
Sarah Evans ◽  
...  
Keyword(s):  
Mouse Model ◽  
Growth Plate ◽  
High Fat Diet ◽  
The United States ◽  
Growth Patterns ◽  
High Fat ◽  
Multiphoton Imaging ◽  
Growth Plates ◽  
Igf I ◽  
Bone Elongation

Nearly one-third of children in the United States are overweight or obese by their pre-teens. Tall stature and accelerated bone elongation are characteristic features of childhood obesity, which co-occur with conditions such as limb bowing, slipped epiphyses, and fractures. Obese children paradoxically have normal circulating IGF-I, the major growth-stimulating hormone. Here we describe and validate a mouse model of excess dietary fat to examine mechanisms of growth acceleration in obesity. We used in vivo multiphoton imaging and immunostaining to test the hypothesis that high-fat diet increases IGF-I activity and alters growth plate structure before the onset of obesity. We tracked bone and body growth in male and female C57BL/6 mice (N = 114) on high-fat (60% kcal fat) or control (10% kcal fat) diets from weaning (3-weeks) to skeletal maturity (12-weeks). Tibial and tail elongation rates increased after brief (1-2 week) high-fat diet exposure without altering serum IGF-I. Femoral bone density and growth plate size were increased, but growth plates were disorganized in not-yet-obese high-fat diet mice. Multiphoton imaging revealed more IGF-I in the vasculature surrounding growth plates of high-fat diet mice, and increased uptake when vascular levels peaked. High-fat diet growth plates had more activated IGF-I receptors and fewer inhibitory binding proteins, suggesting increased IGF-I bioavailability in growth plates. These results, which parallel pediatric growth patterns, highlight the fundamental role of diet in the earliest stages of developing obesity-related skeletal complications and validate the utility of the model for future studies aimed at determining mechanisms of diet-enhanced bone lengthening.

scholarly journals Temperature effects on solute transport: In vivo imaging of the growth plate

2009 ◽  
Vol 23 (S1) ◽  
Author(s):  
Maria A. Serrat ◽  
Rebecca M. Williams ◽  
Cornelia E. Farnum
Keyword(s):  
Solute Transport ◽  
Growth Plate ◽  
In Vivo Imaging ◽  
Temperature Effects
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