Histologic Analysis of Undecalcified Bone. Because the differentiation between the two major metabolic bone diseases, osteoporosis and osteomalacia, is based in part on the quantity and quality of bone mineral, the ability to distinguish between calcified and uncalcified bone matrix (osteoid) is critical. The traditional procedures for processing bone for histology requires decalcification to facilitate sectioning. To preserve the distinction between mineralized bone and osteoid, tissue can be processed without decalcification, embedded in plastic, sectioned on a heavy-duty microtome, and stained with a trichrome connective tissue stain to produce thin histologic sections suitable for microscopic examination.

Osteomalacia is the consequence of insufficient mineralization, so undecalcified sections show an increase in the proportion of unmineralized bone compared with normal bone. The overall bone volume (density) may be normal but is often decreased. The overall amount of osteoid can also be increased in metabolic states characterized by increased bone turnover in general. Differentiation between these states is based on the determination of mineralization rates, using tetracycline as an in vivo bone marker.

Dynamic Tetracycline Labeling. Tetracycline antibiotics are autofluorescent and bind to immature bone mineral. These properties can be used to quantify the rate of bone mineralization, an index that can help determine if the increased osteoid in a biopsy is due to a mineralization defect (osteomalacia) or increased bone remodeling in general. One dose of a tetracycline based on the rate of bone mineralization is administered for 3 days, followed by a second 3-day dose about 14 days later. The bone biopsy is obtained 3 to 4 days after the last dose of tetracycline, and tetracycline fluorescence is evaluated on unstained, nondecalcified tissue sections by ultraviolet light. The first course of tetracycline appears as a discrete fluorescent band within the mineralized bone. The second, more recently administered course of tetracycline is located at the current mineralization front.

The mean distance between the midpoints of the tetracycline labels is measured, and this divided by the number of days between the two courses of tetracycline indicates the mineral appositional rate, which normally ranges from 0.4 to 0.9 µm/day (mean = 0.65 µm/day). This represents the amount of new bone synthesized and mineralized over the tetracycline-free interval.

When the mineral appositional rate increases, the distance between the labels grows wider. In contrast, with a reduced mineralization rate, the parallel bands become narrow and may fuse to produce single labels.

Abnormal patterns of fluorescent label deposition are the hallmark of osteomalacia. The amount of tetracycline fluorescence is proportional to the amount of immature amorphous calcium phosphate deposits in the mineralizing foci of the osteoid seam. If osteoid seams are deficient in mineral, the osteoid is incapable of binding tetracycline, leading to an absence of fluorescence. The mineralization front activity (percentage of osteoid seams bearing normal tetracycline labels) is therefore reduced.

Bone Histomorphometry. This technique is the quantitative analysis of undecalcified bone, in which the parameters of skeletal remodeling are expressed in terms of volumes, surfaces, and cell numbers. To obtain these data from a two-dimensional section, the principles of stereology are used to reconstruct the third dimension. This statistical principle states that if measurements are made at random, the ratio of areas is equal to the ratio of volumes. Although qualitative features of a bone biopsy can usually distinguish conditions of increased bone remodeling such as hyperparathyroidism from osteomalacia, histomorphometry can be helpful in individual cases as well as when following populations of patients as part of a prospective study.

Adynamic Bone. Some patients with osteoporosis, and on long-term therapy with antiresorptive agents, as well as some patients with chronic renal failure in whom PTH has been suppressed develop bone with a reduced rate of remodeling, a condition called “adynamic bone.” Patients with adynamic bone may be at increased risk of fracture. Undecalcified biopsies in patients with adynamic bone show features of low remodeling, including minimal osteoid, few osteoclasts, and low bone formation and mineralization rates.