Diagnostic Test System to detect resorption activity of osteoclasts in vitro just using small blood samples

Introduction:

Osteoclasts (OC) evolve from hematopoietic stem cells of blood and differentiate to form multinucleated cells being able to hydrolyze inorganic hydroxyapatite and degrade organic bone matrix resulting in bone resorption. Established methods to measure the resorption activity of OC rely on non-reproducible isolation of OC precursors (monocytes) requiring larger quantities of human blood before their differentiation to OC in vitro. This procedures influence the activity of the differentiated osteoclasts depending on isolation methods and blood donors. The aim of our project was therefore to develop a reproducible method for measuring the resorption activity of osteoclasts as a personalized parameter influenced only by individual bone health settings.

Methods:

Human peripheral blood mononuclear cells (PBMC's) were isolated from 9 ml human blood (German Red Cross blood donors) by removing the erythrocytes (ACK-Lysis). Defined cell numbers (PBMCs counts delivered by a routine diagnostic laboratory) were seeded on 24-well plates covered with an in vitro manufactured bone-like matrix (osteoblast-derived extracellular matrix, ODEM[1]). The differentiation of OC was initiated via addition of MCSF and RANKL in the culture medium. Resorption areas of OC on ODEM were visualized via von Kossa staining and quantified densitometrically. To further characterize OC actin ring formation (phalloidine staining), TRAP activity (acid phosphatase kit), and the number of nuclei per cell (DAPI staining) were analyzed. [1] Lutter AH et al. 2010. A Novel Resorption Assay for Osteoclast Functionality Based on an Osteoblast-Derived Native Extracellular Matrix. J Cell Biochem 109:1025 – 1032.

Results:

We developed a quick and easy procedure to isolate and differentiate OC from small human blood samples with reproducible resorption values. Based on our data (60 analyzed blood samples from healthy donors) we postulate a reference range for osteoclast resorption activity of healthy blood donors.

Discussion:

The development of this novel test procedure could be the basis to design a patient-specific diagnostic test system – especially for the prognostic identification of bone degradation.