Processing and Sectioning Undecalcified Murine Bone Specimens.

Preparation of mineralized tissue specimens for bone-specific staining encompasses a critical sequence of histological techniques that provides visualization of tissue and cellular morphology. Bone specimens are fixed in 10% neutral buffered formalin (NBF), dehydrated in graded ethanol (EtOH) solutions (and optionally cleared in xylene), infiltrated and embedded in polymethyl methacrylate (methyl methacrylate or MMA), classically sliced into 4-10 micrometer (µm) sections, and stained with bone-specific histological stains such as von Kossa (with either nuclear fast red solution counterstain or MacNeal's tetrachrome counterstain), modified Goldner's trichrome, Alizarin Red S, Safranin O, and tartrate-resistant acid phosphatase (TRAP) stain. Here, we describe the tissue processing of mineralized mouse bones from dissection to staining for histological analysis by light microscopy.

The effects of spaceflight and fracture healing on distant skeletal sites.

Spaceflight results in reduced mechanical loading of the skeleton, which leads to dramatic bone loss. Low bone mass is associated with increased fracture risk, and this combination may compromise future, long-term, spaceflight missions. Here, we examined the systemic effects of spaceflight and fracture surgery/healing on several non-injured bones within the axial and appendicular skeleton. Forty C57BL/6, male mice were randomized into the following groups: (1) Sham surgery mice housed on the earth (Ground + Sham); (2) Femoral segmental bone defect surgery mice housed on the earth (Ground + Surgery); (3) Sham surgery mice housed in spaceflight (Flight + Sham); and (4) Femoral segmental bone defect surgery mice housed in spaceflight (Flight + Surgery). Mice were 9 weeks old at the time of launch and were euthanized approximately 4 weeks after launch. Micro-computed tomography (µCT) was used to evaluate standard bone parameters in the tibia, humerus, sternebra, vertebrae, ribs, calvarium, mandible, and incisor. One intriguing finding was that both spaceflight and surgery resulted in virtually identical losses in tibial trabecular bone volume fraction, BV/TV (24-28% reduction). Another important finding was that surgery markedly changed tibial cortical bone geometry. Understanding how spaceflight, surgery, and their combination impact non-injured bones will improve treatment strategies for astronauts and terrestrial humans alike.

Cohousing Male Mice with and without Segmental Bone Defects.

Spaceflight results in bone loss like that associated with osteoporosis or decreased weight-bearing (for example, high-energy trauma such as explosive injuries and automobile accidents). Thus, the unique spaceflight laboratory on the International Space Station presents the opportunity to test bone healing agents during weightlessness. We are collaborating with NASA and the US Army to study bone healing in spaceflight. Given the unique constraints of spaceflight, study design optimization was required. Male mice were selected primarily because their femur is larger than females', allowing for more reproducible surgical outcomes. However, concern was raised regarding male mouse aggression. In addition, the original spaceflight study design included cohousing nonoperated control mice with mice that had undergone surgery to create a segmental bone defect. This strategy prompted the concern that nonoperated mice would exhibit aggressive behavior toward vulnerable operated mice. We hypothesized that operated and nonoperated male mice could be cohoused successfully when they were cagemates since birth and underwent identical anesthetic, analgesic, preoperative, and postoperative conditions. Using quantitative behavioral scoring, body weight, and organ weight analyses (Student t test and ANOVA), we found that nonoperated and operated C57BL/6 male mice could successfully be housed together. The male mice did not exhibit aggressive behavior toward cagemates, whether operated or nonoperated, and the mice did not show evidence of stress, as indicated by veterinary assessment, or change in body or proportional organ weights. These findings allowed our mission to proceed (launched February 2017) and may inform future surgical study designs, potentially increasing housing flexibility.

Tips and techniques for processing and sectioning undecalcified murine bone specimens.

Preparation of mineralized tissue specimens for bone-specific staining encompasses a critical sequence of histological techniques that provides visualization of tissue and cellular morphology. Bone specimens are fixed in 10 % neutral-buffered formalin, dehydrated in graded ethanol (EtOH) solutions (and optionally cleared in xylene), infiltrated and embedded in polymethyl methacrylate (methyl methacrylate), classically sliced into 4-10 micrometer (µm) sections, and stained with bone-specific histological stains such as von Kossa (with either nuclear fast red solution counterstain or MacNeal's tetrachrome counterstain), modified Goldner's trichrome, and alizarin red S stain. Here, we describe the tissue processing of mineralized mouse bones from dissection to staining for histological analysis by light microscopy.

A review of mouse critical size defect models in weight bearing bones.

Current and future advances in orthopedic treatment are aimed at altering biological interactions to enhance bone healing. Currently, several clinical scenarios exist for which there is no definitive treatment, specifically segmental bone loss from high-energy trauma or surgical resection - and it is here that many are aiming to find effective solutions. To test experimental interventions and better understand bone healing, researchers employ critical size defect (CSD) models in animal studies. Here, an overview of CSDs is given that includes the specifications of varying models, a discussion of current scaffold and bone graft designs, and current outcome measures used to determine the extent of bone healing. Many promising graft designs have been discovered along with promising adjunctive treatments, yet a graft that offers biomechanical support while allowing for neovascularization with eventual complete resorption and remodeling remains to be developed. An overview of this important topic is needed to highlight current advances and provide a clear understanding of the ultimate goal in CSD research--develop a graft for clinical use that effectively treats the orthopedic conundrum of segmental bone loss.