An official website of the United States government
More than 54 million Americans have or are at high risk of developing a metabolic bone disease; disorders of bone strength that leave individuals with fragile bones and disabilities. The gold standard to evaluate these diseases is dual energy x-ray absorptiometry, but this only measures mineral content. These diseases, however, impact collagen and mineral integrity which impede the bone’s ability to store hormones, proteoglycans, and glycoproteins imperative to homeostasis. We have established a second harmonic generation (SHG) polarimetric assay that describes bone collagen organization. To further our analysis, we propose multimodal optical evaluation of bone quality with third harmonic generation (THG) to measure osteocyte dendritic processes. This method of analysis could be used to evaluate the disease state of bone and response to therapy.
more »
« less
Second harmonic generation characterization of collagen in whole bone
Bone is a unique biological composite material made up of a highly structured collagen mesh matrix and mineral deposits. Although mineral provides stiffness, collagen’s secondary organization provides a critical role in bone elasticity. Here, we performed polarimetric analysis of bone collagen fibers using second harmonic generation (SHG) imaging to evaluate lamella sheets and collagen fiber integrity in intact cranial bone. Our polarimetric data was fitted to a model accounting for diattenuation, polarization cross-talk, and birefringence. We compared our data to the fitted model and found no significant difference between our polarimetric observation and the representation of these scattering properties up to 70µm deep. We also observed a loss of resolution as we imaged up to 70µm deep into bone but a conservation of polarimetric response. Polarimetric SHG allows for the discrimination of collagen lamellar sheet structures in intact bone. Our work could allow for label-free identification of disease states and monitor the efficacy of therapies for bone disorders.
more »
« less
- Award ID(s):
- 1706916
- PAR ID:
- 10172730
- Publisher / Repository:
- Optical Society of America
- Date Published:
- Journal Name:
- Biomedical Optics Express
- Volume:
- 11
- Issue:
- 8
- ISSN:
- 2156-7085
- Format(s):
- Medium: X Size: Article No. 4379
- Size(s):
- Article No. 4379
- Sponsoring Org:
- National Science Foundation
More Like this
-
-
Abstract Bone is a dynamic mineralized tissue that undergoes continuous turnover throughout life. While the general mechanism of bone mineral metabolism is documented, the role of underlying collagen structures in regulating osteoblastic mineral deposition and osteoclastic mineral resorption remains an active research area, partly due to the lack of biomaterial platforms supporting accurate and analytical investigation. The recently introduced osteoid-inspired demineralized bone paper (DBP), prepared by 20-μm thin sectioning of demineralized bovine compact bone, holds promise in addressing this challenge as it preserves the intrinsic bony collagen structure and retains semi-transparency. Here, we report on the impact of collagen structures on modulating osteoblast and osteoclast-driven bone mineral metabolism using vertical and transversal DBPs that exhibit a uniaxially aligned and a concentric ring collagen structure, respectively. Translucent DBP reveals these collagen structures and facilitates longitudinal tracking of mineral deposition and resorption under brightfield microscopy for at least 3 wk. Genetically labeled primary osteogenic cells allow fluorescent monitoring of these cellular processes. Osteoblasts adhere and proliferate following the underlying collagen structures of DBPs. Osteoblastic mineral deposition is significantly higher in vertical DBP than in transversal DBP. Spatiotemporal analysis reveals notably more osteoblast adhesion and faster mineral deposition in vascular regions than in bone regions. Subsequent osteoclastic resorption follows these mineralized collagen structures, directing distinct trench and pit-type resorption patterns. In vertical DBP, trench-type resorption occurs at an 80% frequency, whereas transversal DBP shows 35% trench-type and 65% pit-type resorption. Our studies substantiate the importance of collagen structures in regulating mineral metabolism by osteogenic cells. DBP is expected to serve as an enabling biomaterial platform for studying various aspects of cellular and extracellular bone remodeling biology.more » « less
-
Not AvailaMineral imbalances in the body from chronic kidney disease can impact bone turnover and cause cortical bone loss. Synthetic salmon calcitonin is an FDA-approved treatment for bone fragility observed in diseases such as osteoporosis, and clinical trials have demonstrated a reduction in fractures compared to untreated individuals. This study documents the effects of calcitonin on cortical bone using an in vivo mouse model of chronic kidney disease. Serum BUN and PTH are reported. Calcitonin was found to impact at a dose of 50/IU/kg/day five times a week for five weeks. MicroCT was used to evaluate bone quantity measures, such as cortical porosity, thickness, bone area, and long bone structural geometric parameters. It was found that porosity, thickness, and bone geometry are affected by disease, but not by treatment at the specified regime. Small and wide-angle x-ray scattering (SAXS and WAXS) was used to obtain the nanostructure of the mineral-collagen-water composite, including mineral dimensions, -periodicity and collagen spacing. Data from thermogravimetric analysis (TgA) were used to quantify wt.% of the mineral, collagen, and bound water of each sample. Chronic kidney disease was found to decrease collagen spacing to increase mineral weight fractions, and to reduce loosely bound water. There were no changes from chronic kidney disease on the -Periodicity. Treatment increased the weight percent of collagen, with no effect on other bone quality parameters.more » « less
-
Intravital microscopy using multiphoton processes is the standard tool for deep tissue imaging inside of biological specimens. Usually, near-infrared and infrared light is used to excite the sample, which enables imaging several mean free path inside a scattering tissues. Using longer wavelengths, however, increases the width of the effective multiphoton Point Spread Function (PSF). Many features inside of cells and tissues are smaller than the diffraction limit, and therefore not possible to distinguish using a large PSF. Microscopy using high refractive index microspheres has shown promise to increase the numerical aperture of an imaging system and enhance the resolution. It has been shown that microspheres can image features ~λ/7 using single photon process fluorescence. In this work, we investigate resolution enhancement for Second Harmonic Generation (SHG) and 2-photon fluorescence microscopy. We used Barium Titanate glass microspheres with diameters ∼20–30 μm and refractive index ∼1.9–2.1. We show microsphere-assisted SHG imaging in bone collagen fibers. Since bone is a very dense tissue constructed of bundles of collagen fibers, it is nontrivial to image individual fibers. We placed microspheres on a dense area of the mouse cranial bone, and achieved imaging of individual fibers. We found that microsphere assisted SHG imaging resolves features of the bone fibers that are not readily visible in conventional SHG imaging. We extended this work to 2-photon microscopy of mitochondria in mouse soleus muscle, and with the help of microsphere resolving power, we were able to trace individual mitochondrion from their ensemble.more » « less
-
Near infrared and infrared multi-photon imaging through or inside bone is an emerging field that promises to help answer many biological questions that require minimally invasive intravital imaging. Neuroscience researchers especially have begun to take advantage of long wavelength imaging to overcome multiple scattering and image deep inside the brain through intact or partially intact bone. Since the murine model is used in many biological experiments, here we investigate the optical aberrations caused by mouse cranial bone, and their effects on light propagation. We previously developed a ray tracing model that uses second harmonic generation in collagen fibers of bone to estimate the refractive index structure of the sample. This technique is able to rapidly provide initial information for a closed loop adaptive optics system. However, the ray tracing method does not account for refraction or scattering. Here, we extend our work to investigate the wavefront aberrations in bone using a full electromagnetic model. We used Finite-Difference Time-Domain modeling of light propagation in refractive index bone datasets acquired with second harmonic generation imaging. In this paper we show modeled wavefront phase from different originating points across the field of view.more » « less
