**Abstract**

Osteoporosis is a skeletal disorder characterized by decreased bone strength which affects the increased risk of fracture. Emerging evidence discovered that osteoporosis is associated with reduced bone density and bone quality. Therefore, analysis of bone morphology can afford insight into the characteristics and processes of osteoporosis. Electron microscopy, one of the best methods, can directly provide ultrastructure evidence for bone morphology. Here, we describe an experimental procedure for electron microscopy preparation and analysis of the resulting images, especially scanning and transmission electron microscopes, to analyze bone morphology in animal models of rats. Compared to other bone analyzers such as atomic absorption spectrophotometer, ultraviolet–visible spectroscopy, Fourier transform infrared spectroscopy, Raman spectroscopy, and X-ray diffraction, scanning and transmission electron microscopes are still important to strengthen visual analysis, and a better understanding of this method could be significant to examine bone morphology.

**Keywords:** osteoporosis, bone strength, fractures, electron microscopy, bone morphology

### **1. Introduction**

Osteoporosis is a metabolic disorder causing bone mineral density to decrease and changing the bone structure [1]. It is a degenerative disease whose initial symptoms are not known with certainty. Someone who suffers from osteoporosis will usually experience complaints if the stage is severe [2, 3]. Bones with osteoporosis will experience a decrease in mechanical strength so they are prone to fracture, and will easily crack or become brittle if exposed to a hard object. It is characterized by low bone mass and structural breakdown of bone tissue. Some parts of the body that are at risk for osteoporosis include the spine, pelvis, femur, tibia, pelvic bones, wrist bones, and other bone parts dominated by the trabecular bone [4–6].

Osteoporosis can be diagnosed clinically using bone mineral density measurements. At present, bone densitometry is the standard method for diagnosis and treatment monitoring. However, it still possesses significant drawbacks because it cannot give information about the structural manifestations of the disease. Frequently, bone mineral density is analyzed using x-ray or ultrasound imaging methods. In x-ray imaging such as dual-energy x-ray absorptiometry (DEXA) and quantitative computer tomography (QCT), the intensity of the image is correlated to the mineral density of

the tissue. In ultrasound, the intensity of the image reflects changes in the frequency and amplitude of sound waves traveling through tissue. X-ray procedures employ ionizing radiation, which can have a damaging impact in sufficient doses. Ultrasound, although harmless, offers only a small field of view, which can restrict measurement accuracy. In addition to bone density, bone quality which includes bone microarchitecture is also a concern. Recent developments in imaging, especially electron microscopy,

#### *Analysis of Osteoporosis by Electron Microscopy DOI: http://dx.doi.org/10.5772/intechopen.104582*

can now give detailed information about the effects of architecture on disease progression and regression in response to treatment. However, before the diagnosis is made, of course, it is necessary to study and research in a sample or biological material to determine the process of bone remodeling and osteoporosis. The samples analyzed generally use rats as animal models. It takes a long time to make rats osteoporosis naturally. Therefore, rats were given treatment to condition the occurrence of osteoporosis. Some of the common actions taken to condition osteoporosis rats are by giving them a calcium-deficient diet or by performing ovariectomy on these rats [7–10].

Several characterization tools that can be used to analyze mouse bones include X-ray Diffraction (XRD), Fourier Transform Infrared Spectroscopy (FTIR), Ultraviolet (UV)-visible Spectroscopy, or Atomic Absorption Spectroscopy (AAS). However, these tools provide information in the form of numbers or graphs. A promising imaging modality for morphological analysis of both cortical and trabecular bone is electron microscopy. The types of electron microscopes commonly used to analyze bone morphology are Scanning Electron Microscope (SEM) and Transmission Electron Microscope (TEM). **Figure 1** illustrates the different imaging modalities, between SEM and TEM, which were used to analyze the morphology of the rat femur bone.

This review focuses on the emerging methodology of quantitative electron microscopy to assess the bone structure and morphology of osteoporotic rats. For more than 10 years, numerous approaches have been investigated to obtain quantitative imagebased information on bone architecture, both trabecular bone, and cortical bone. An indirect method that does not require resolution at individual trabecular scales and can therefore be performed at any skeletal location, a recoverable component of the degree of total transverse relaxation. Therefore, electron microscopy-based structure analysis is technically demanding in terms of the required image acquisition. Other requirements that must be fulfilled involve motion correction and image registration, both of which are important to achieve the reproducibility required in repeated studies. The main targeted clinical application involves the prediction of fracture risk in femoral rats conditioned by osteoporosis due to ovariectomy.

## **2. Electron microscopy basics**

An electron microscope is a type of microscope in which the illumination source is an electron beam. Illumination itself is a process of light coming to an object. There are electron microscopes that have high image resolution, even magnifying objects on the nanometer scale, which are produced by the controlled use of electrons in a vacuum captured on a fluorescent screen. The first electron microscope was introduced by an engineer and professor from German, Ernst Ruska (1906-1988), in 1931, and the same principles behind his prototype still dominate modern Ems [11, 12].

#### **2.1 Principle**

Electron microscopy uses signals generated by the interaction of the electron beam with the sample to gain information about its structure, morphology, and composition. The process and major parts of an electron microscope are:

1.Electrons are produced by the electron gun

2.The electron beam is concentrated on the sample by condenser lenses.

