Childhood obesity can lead to chronic skeletal disability. In West Virginia, over one third of children are overweight or obese by age 10. Obese children have higher rates of linear growth, accelerated skeletal maturation, and diminished bone quality. These factors can lead to the development of painful conditions such as limb bowing, joint instability, fractures, and early onset osteoarthritis. The major treatment obstacle is lack of knowledge of the underlying cause of obesity-induced growth acceleration. Paradoxically, obese children have low to normal levels of growth hormone and IGF-I, the two major hormones that stimulate growth. Our research couples in vivo imaging with a mouse model of juvenile obesity to determine how obesity alters IGF-I transport to cartilage plates of the growing skeleton. We use in vivo models to investigate the physiological regulation of bone elongation and the impact of environmental influences such as diet and temperature on the bone lengthening process.
The Serrat Laboratory specializes in growth and morphology of the postnatal skeleton. We use in vivo models to investigate the physiological regulation of bone elongation and the impact of environmental influences such as temperature and diet on the bone lengthening process. One major area of focus is on molecular delivery to skeletal growth plates, the bands of cartilage at the ends of bones where lengthening occurs (Fig. 1).
Figure 1. Schematic of a long bone growth plate and its principal blood supplies. Image at right shows a tibial growth plate from a mouse injected with oxytetracycline (OTC) to label newly formed bone. Orientation matches the schematic on the left. The growth plate appears as a dark band between epiphyseal and metaphyseal bone. Vascular access to the growth plate is through epiphyseal vessels, metaphyseal vessels, and a subperichondrial plexus arising from a ring vessel that surrounds the growth plate. Bone elongation occurs through a series of well-orchestrated events in which chondrocytes in columns divide, enlarge, and are replaced with mineral by bone-forming osteoblasts that invade from the metaphyseal vasculature (image from Serrat, 2014, Comprehensive Physiology, 4:621-55, Copyright 2014 American Physiological Society).
Growth plate disorders have many different underlying causes, ranging from injury and illness, to genetic bone diseases, to skeletal effects of childhood obesity. These conditions can lead to limb length inequality and a lifetime of serious orthopaedic problems such as scoliosis, limb bowing, chronic back pain, and osteoarthritis. Bone elongation disorders are challenging to treat because cartilage growth plates are not penetrated by blood vessels like typical vascularized organs (Fig. 1). Targeting systemic drugs into cartilage is extremely difficult, and treatment options are often limited to painful corrective surgeries and/or expensive drug regimens. Noninvasive heat therapy could be a novel and cost-effective way to offset linear growth impediments, since our lab has previously demonstrated that warm temperature can increase bone length in experimental animals (Fig. 2).
Figure 2. Line drawing depicting temperature effects on limb length in growing mice (original artwork by Tom Pickens and Matt Crutchfield, Graphic Designers, Marshall University
School of Medicine).
Research in our lab takes an integrated, whole animal approach to these problems by employing tools such as multiphoton-based live animal imaging. This technique allows us to evaluate heat-based therapies for augmenting delivery of systemic bone lengthening drugs across vascular-cartilage interfaces of the growing skeleton in vivo. Ongoing projects use real-time multiphoton microscopy and a unique limb-heating model to test the relationship between temperature, bone lengthening, and vascular access to growth plates. We are also investigating the impact of a high-fat diet on molecular delivery to growth plates and are characterizing IGF-I uptake in a mouse model of juvenile obesity. These projects have practical relevance for treating a spectrum of linear growth disorders in children. Our goal is to develop noninvasive alternatives that can be applied to wide range of pediatric skeletal conditions. Results could potentially lead to new treatment modalities with better outcomes by reducing amount, toxicity and costs of high-dose systemic pharmaceuticals.
Imaging skeletal growth plates using in vivo multiphoton microscopy
Multiphoton microscopy is an emerging technology for live animal imaging that offers exciting possibilities for the study of growth plate dynamics in vivo. In collaboration with colleagues at Cornell University, we developed a unique platform for imaging intact skeletal growth plates that we use to assess how systemic regulators arrive at and move within the cartilage matrix of the growth plate under various experimental conditions. This system provides a new mechanism for understanding the physiological regulation of bone growth through the ability to dynamically measure changes in molecular transport to the growth plate of a living animal (Fig. 3).
Figure 3. In vivo multiphoton image of blood vessels in the plexus surrounding the tibial growth plate of a live, anesthetized 5-week-old mouse. Vessels were visualized using a multiphoton microscope after an intravenous injection of fluorescein. Plasma is red and blood cells appear as dark shadows within the vessels. The collagen-rich perichondrium around the growth plate (green-gray pseudocolor) was visualized by second-harmonic generation (SHG), a robust signal from unstained collagen that is unique to multiphoton excitation. SHG allows collagenous structures to be identified without injecting stains or dyes. Imaging was done by Maria Serrat on a Leica TCS SP5 II Multiphoton Microscope housed in the Molecular and Biological Imaging Center at Marshall University (image modified from Serrat, 2014, Comprehensive Physiology, 4:621-55, Copyright 2014 American Physiological Society).
Using biologically-inert dextrans as size-proxies for systemic regulators, we have shown that heat can increase real-time entry of large molecules into growth plate cartilage in vivo (Serrat, Efaw, and Williams, 2014). We took this work a step further when we validated a model to measure uptake of fluorescently-labeled, biologically active IGF-I in growth plates of live, intact mice (Serrat and Ion, 2017) (Fig. 4). We are currently using this system to quantify IGF-I uptake in the growth plate under different environmental conditions (temperature and dietary influences).
Figure 4. Comparison of growth plate visualization using standard postmortem histology (left) and in vivo multiphoton imaging of fluorescein dye (middle) and fluorescently-labeled, biologically active IGF-I (right). We developed techniques to quantify the transport of IGF-I into the growth plate of a living mouse. Chondrocytes (white with black nuclei in left panel) are shown as dark shadows when the growth plate is illuminated with fluorescein dye (middle). In contrast, fluorescence appears in a distinct pattern in the labeled IGF-I image (right), suggesting receptor-mediated localization to chondrocytes. Image on right from Serrat and Ion (2017).
Unilateral heating to lengthen extremities of growing mice
We recently developed and validated a novel unilateral heating model to increase extremity length on the heat-treated side of young growing animals. Our experimental system uses weanling mice (normal and dwarf models) exposed to a daily heating regimen to induce unilateral (one-sided) extremity growth. Mild 40C heat is applied to one side of the body for 40 minutes per day for two weeks as a unique, clinically applicable method to increase growth on the heat-treated side. This project complements our short-term in vivo imaging studies to determine the mechanisms underlying heat-enhanced bone elongation. Techniques employed in this project include microdissection; histology and immunostaining for bone morphology; fluorochrome bone labeling to measure elongation rate; protein assays to assess activation of growth stimulating pathways, and quantitative thermal imaging to measure heat gradients in the extremities (Fig. 5).
Figure 5. Limb heating experiments are being done in the Serrat Lab to study mechanisms underlying heat-enhanced bone elongation. Thermal image of juvenile mice on a heating pad (left) shows the temperature differential between heat-treated and non-treated sides. Limbs are equilibrated to 40C during the daily treatments. Digital image (right) shows foam separators that are used to keep the non-treated side at a cooler 30C. Images captured June 2014 by Holly Tamski and Maria Serrat.
Allison L. Machnicki, PhD – Postdoctoral Associate
Cassaundra A. Song – PhD Student
Darby McCloud – Undergraduate