Use of Fluorochrome Labels inIn VivoBone Tissue Engineering Research

Abstract

The use of fluorochromes in bone research is a widely accepted technique that dates back to the 1950s. Several pioneers, such as Harold Frost, have thoroughly investigated the potential of fluorochrome use for the study on bone formation and bone remodeling dynamics. Since the development of bone tissue engineering, a renewed interest in the benefits of fluorochrome use was perceived. Fluorochrome use in animal models makes it possible to determine the onset time and location of osteogenesis, which are the fundamental parameters in bone tissue engineering studies. There is, however, a lack of standardized procedures for using this technique. In addition, many types of fluorochromes exist and one could be confused upon selecting the appropriate type, the appropriate concentration, the route of administration, and methods of visualization. All these variables can potentially affect the outcome during fluorescence microscopy. This work aims at providing the bone tissue engineering researcher with an overview of the history, working mechanism, and the potential pitfalls in the use of fluorochromes in animal studies. Experiments using some of the more frequently used fluorochromes are explained and illustrated.

Direct or indirect stimulation of adenosine A 2A receptors enhances bone regeneration as well as bone morphogenetic protein‐2

“…To detect new bone formation in a dynamic fashion XenoLight RediJect Bone Probe 680, a fluorescent conjugate that binds to hydroxyapatite in new bone, was injected intravenously as described under Materials and Methods (26)(27)(28). As shown in Fig.…”

“…This fluorescence reagent targets hydroxyapatite, and in combination with the IVIS imaging system (Caliper), allows in vivo detection and measurement of skeletal changes (26)(27)(28). Fluorescence images were taken with an excitation peak of 681 nm and an emission peak of 696 nm.…”

Preclinical animal models of multiple myeloma

“…B2 and 6 days prior to experimental end point. Different fluorophores such as calcein green, tetracycline yellow, xylenol orange and alizarin red can also be used to measure bone formation 10 The time between each fluorophore varies depending on the age of the mice used. 11,12 After mice are killed, collect the spines and then fix in 10% neutral-buffered formalin for at least 48 h at 4 1C.…”

Hydrogel-based Delivery of rhBMP-2 Improves Healing of Large Bone Defects Compared With Autograft

“…Two animals were administered a series of bone labels: calcein green (10 mg/kg; Sigma C0875) at 4 weeks, xylenol orange (100 mg/kg; Sigma 398187) at 8 weeks, and calcein blue (10 mg/kg; Sigma M1255) at 12 weeks postsurgery as subcutaneous injections to identify temporal mineralization patterns [15,29,33,42]. Fixed samples were processed into cryoblocks, and nondecalcified sections (5 lm) were obtained to qualitatively observe repair, the mineral deposition pattern visualized by bone labels, and alkaline phosphatase (AP) staining for osteoblastic activity [21,22,41,44].…”

“…Our primary questions were (1) whether the NMA-rhBMP-2 group would produce quantitatively similar levels of bone mineral volume (BV) and bone mineral density (BD) to freshly harvested autograft; and if so, (2) whether the biomechanical properties of the healed bones would be different between treatments. Using vital bone labels [42] (fluorochromes binding the mineralizing front of bone tissue) and histology, we also determined the quality and distribution of the regenerated bone.…”