An Improved Methodology to Evaluate Cell and Molecular Signals in the Reparative Callus During Fracture Healing

Abstract

Approximately 5% to 10% of all bone fractures do not heal completely, contributing to significant patient suffering and medical costs. Even in healthy individuals, fracture healing is associated with significant downtime and loss of productivity. However, no pharmacological treatments are currently available to promote efficient bone healing. A better understanding of the underlying molecular mechanisms is crucial for developing novel therapies to hasten healing. The early reparative callus that forms around the site of bone injury is a fragile tissue consisting of shifting cell populations held together by loose connective tissue. The delicate callus is challenging to section and is vulnerable to disintegration during the harsh steps of immunostaining, namely, decalcification, deparaffinization, and antigen retrieval. Here, we describe an improved methodology for processing early-stage fracture calluses and immunofluorescence labeling of the sections to visualize the temporal (timing) and spatial (location) patterns of cellular and molecular events that regulate bone healing. This method has a short turnaround time from sample collection to microscopy as it does not require lengthy decalcification. It preserves the structural integrity of the fragile callus as the method does not entail deparaffinization or harsh methods of antigen retrieval. Our method can be adapted for high-throughput screening of drugs that promote efficacious bone healing:

Episodic alcohol exposure attenuates mesenchymal stem cell chondrogenic differentiation during bone fracture callus formation

“…While the design utilized does limit direct comparisons over time of healing within a genotype, the timepoints analyzed for evaluation of the effects of alcohol on PRX1, Col2a1, and Col10a1 expression in fracture callus were chosen based on the literature (Einhorn, 1998) and our own experience in histological evaluation of fracture callus formation in mice, which allowed us to examine the effects of alcohol on early and later MSC‐chondrogenic differentiation during fracture repair. Fracture callus area was quantified using H & E‐stained histological sections; future studies should include sections stained using a cartilage‐specific stain such as safranin‐O (Kambrath et al, 2020) to confirm the cartilaginous area of the callus.…”

A Review of Recent Developments in the Molecular Mechanisms of Bone Healing

“…Furthermore, the method preserves the structural integrity of the fragile callus, because it does not involve deparaffinization or harsh antigen retrieval methods. This method could be adapted for high-throughput screening of drugs that promote the healing of bone fractures [ 1 ].…”

“…It is estimated that between 5% and 10% of bone fractures do not heal properly [ 1 ]. The categories of bone fracture are the following: Closed or open fractures; complete fractures; displaced fractures; partial fractures; stress fractures.…”

“…There are, however, no current pharmacological treatments to enable effective bone consolidation. A better understanding of the molecular mechanisms underlying bone healing is therefore essential for developing new treatments to accelerate the process [ 1 ].…”

“…There are, however, no current pharmacological treatments to enable effective bone consolidation. A better understanding of the molecular mechanisms underlying bone healing is therefore essential for developing new treatments to accelerate the process [1]. A biological or mechanical deficiency, a lack of information regarding the host's comorbidities, and a lack of vascularization can all lead to nonunion.…”