Bone Histomorphometry - A Method to Assess Bone Tissues

Introduction:

Diagnosis of disease whether a laboratory study or relating to an actual patient greatly depends on the methods like radiology, histology, blood analysis, and gene expression analysis. All these methods contribute to providing a detailed set of biological information. Diagnosis of an infectious disease greatly relies on the histological examination (studying the cellular structures and tissues under the microscope) and it is considered the gold standard method of investigation for infectious disease. However, in skeletal conditions, radiological investigations are considered the gold standard method. Though histological examination and histomorphometry are other reliable methods to evaluate and assess systemic skeleton conditions. This article will discuss bone histomorphometry in detail.

What Is Bone Histomorphometry?

Bone histomorphometry is a unique tool for assessing bone tissues in both clinical and preclinical studies. This technique involves quantitative analysis of the histological samples of the bone tissue for assessment of metabolic diseases in the bone and their treatments. Applying this technique along with data analysis in bioinformatics helps in a better understanding of the cellular level differences in skeletal diseases.

• Earlier the traditional histomorphometric analysis was carried out in two dimensions by histology, which involves measurement of the structural changes and bone remodeling on sections (the process of providing thin slices of specimens for microscopical study), and the third dimension is explored using the standard stereology theory (stereology determines the three-dimensional interpretation of a two-dimensional image).

• Currently, with recent advancements, histomorphometric analysis is carried out using semi-automated and automatic image analysis along with advanced stereology software.

• Other advancements in this technique involve the emergence of plastic embedding techniques to reproduce high-quality histologic sections of the bone and the process of tetracycline labeling which involves binding the tetracycline to the mineralizing surface of the bone to help in better understanding of the bone tissues.

What Are the Step-Wise Procedures of Histomorphometric Analysis?

• Bone Biopsy:

  • A bone biopsy procedure is carried out to obtain a bone tissue sample. This procedure is often preferred to confirm the cases of osteomalacia or to characterize the various forms of renal osteodystrophy. They are advantageous in patients with bone pain or fragility or in patients with unexplained decreased bone mass or fractures. However, in clinical practice, they are mostly limited although it is a valuable research tool.

• Tetracycline Labeling:

  1. The process of tetracycline labeling is necessary done before the biopsy procedure especially when estimates of bone formation or bone mineralization are required.

     • The schedules for tetracycline labeling differ in each laboratory.

     • Generally, they are scheduled three weeks before the biopsy procedure.

     • 250 milligrams of tetracycline is administered four times a day for three weeks with drug-free days in the interval of 10 to 14th days. In patients with an estimated glomerular filtration rate of fewer than 30 milliliters per minute and dialysis patients are administered demeclocycline 150 milligrams twice a day.

     • This helps in depositing the labels at the site of new bone formation allowing these sites to be visually and quantitatively analyzed.

• Biopsy Procedure:

  1. The most preferred site for biopsy is the iliac crest (curved part along the top of the hip bone) which is located posterior to the anterior superior iliac spine.

  2. Small or fragmented sample of bone tissue compromises the quantitative analysis. Hence an intact core of bone comprising inner and outer cortical plates with the mediating cancellous bone is necessary.

  3. Once the biopsy specimen is obtained it is sent to the laboratory for the preparation of the undecalcified bone specimen for histomorphometric analysis.

• Methods of Measurement:

  1. In the laboratory, the obtained tissue is dehydrated in ethanol and it is embedded in plastic by controlled temperature polymerization.

  2. They are cut into 5 microns sectioned, stained, and mounted. These sections are analyzed according to the stereological theory using computer-aided planimetry and visualized using the image analysis software program.

  3. Using the microscope the primary measurements are made such as the thickness, area, and perimeter.

  4. The parameters of the histomorphometric analysis are categorized into structural and remodeling. And the remodeling is further divided into dynamic and static parameters.

  5. The bone histomorphometric analysis is normally limited to cancellous bone however, It can also be performed in cortical bone.

• Structural Parameters:

  1. Evaluation of bone structure is important due to the close relationship with bone strength.

  2. They involve the three-dimensional geometry of the bone and are measured by calculating the total bone perimeter and total bone area.

• Remodeling Parameters:

  1. Bone remodeling involves the process of skeletal changes to preserve structural integrity and balance the metabolic levels of calcium and phosphorus in the skeletal system. However, the main responsibility of bone remodeling is to renew the old bone by deposition of new bone. This process occurs in two phases: resorption of the functioning mineralized bone matrix with the help of osteoclasts (bone-resorbing cells) and formation of a new bone matrix by osteoblasts (bone-synthesizing cells).

  2. Static parameters are necessary for the evaluation of unmineralized bone (osteoid) and the extent of resorption cavities.

  3. Dynamic parameters of the bone remodeling are essential for the evaluation of bone formation rate and performing tetracycline labeling on the patients prior to the biopsy procedure is necessary for the evaluation of dynamic parameters.

What Are the Histomorphometric Findings in Skeletal Disorders?

The reference value for bone histomorphometry from a healthy individual is difficult to be obtained for comparison due to the influence of various factors such as race, age, and gender.

• In conditions like osteomalacia where there is an impairment of bone mineralization. The bone histomorphometric findings show accumulation of osteoid due to an increase in thickness, volume, and surface area of osteoid. The turnover rate of the bone is decreased due to the increase in thickness of the osteoid making it harder for the osteoclast to reach the calcified bone surface.

• In postmenopausal women with osteoporosis, the bone histomorphometric analysis shows a decrease in cortical thickness due to the increase in remodeling activity and higher turnover rate.

• In conditions like hyperparathyroidism, it shows an increase in eroded, osteoid, and mineralizing surfaces and elevated osteoblast, osteoclast, and accelerated bone turnover rate.

Conclusion:

Bone Histomorphometry is a unique tool in the analysis of bone tissues that involve quantitative analysis of the bone metabolism that helps to provide information that can not be obtained from any other investigation method. Additionally, it helps in providing information on evaluating a growing individual and in evaluating the response to pharmacological interventions in clinical trials. Having better knowledge about the histomorphometric changes in metabolic disease helps in the better management of diseases like osteoporosis and osteomalacia.