Embedding of Bone Samples in Methylmethacrylate: A Suitable Method for Tracking LacZ Mesenchymal Stem Cells in Skeletal Tissues

Abstract

S U M M A R Y Considerable research has been focused on the use of bone marrow-derived mesenchymal stem cells (MSCs) for the repair of non-unions and bone defects. To date, the question of whether transplanted MSCs survive and engraft within newly formed tissue remains unresolved. The development of an easy and reliable method that would allow cell fate monitoring in transplant recipients is a pressing concern for the field of tissue engineering. To demonstrate the presence of transplanted cells in newly formed bone, we established a xenograft nude rat model allowing the detection of murine LacZ MSCs in vivo. MSCs were isolated from transgenic lacZ mice, seeded onto bioabsorbable collagen sponges, and transplanted to repair a calvarial defect in nude rats. As a preliminary step, the histological procedure was adapted to optimize the detection of LacZ cells in bone tissue embedded in methylmethacrylate (MMA). Four fixatives and four fixation times were evaluated. Among all the fixatives tested, 2% formaldehyde/0.2% glutaraldehyde at 4C for 4 days gave the best results for X-gal staining at pH 7.4 on both cell cultures and bone explants. All fixatives were effective for imunodetection of b-gal. In the chimeric LacZ/nude rat animal model, MSCs were detected in vivo for up to 4 weeks after implantation and contributed to the repair and the neovascularization of the bone defect. LacZ is a suitable phenotypic marker to track MSCs in skeletal tissues embedded in MMA. (J Histochem Cytochem 55:255-262, 2007)

Fate of Bone Marrow Stromal Cells in a Syngenic Model of Bone Formation

“…Some authors reported longer survival, up to 5 months in Nude mice, when the cells were associated with matrices such as Matrigel or type I collagen, which are thought to trigger lower inflammatory reaction than calcium phosphate biomaterials. 22,28,[45][46][47] The results obtained by Tasso et al, which show identical survival times (1 week) when C57BL/6 mouse BMSCs were grafted in syngenic or nude recipients, suggest that species specificity of donor cells is also probably involved. 22 In conclusion, using quantitative methods based on the detection of the Y chromosome, we show here in a syngenic model of SC bone formation that BMSCs die within 3 weeks after implantation, whereas host cells colonize massively the biomaterial, leading to the centripetal formation of new bone.…”

“…Most of the studies dedicated to analyze the destiny of the donor cells have been conducted using animal or human MSCs implanted into immunodeficient recipients such as nude mice, 1,20-23 NIH-Bg-nu-xid, 5,[24][25][26] and nude rats. 2,27,28 Several of these studies have shown that part of the donor cells can survive into the host for several weeks, leading to the conclusion that new bone originates from these surviving cells. 25,26 Other authors have suggested that the implanted cells could also, and perhaps principally, play a trophic role and increase local bone formation through the recruitment of hostderived stem cells.…”

Successful transplant of mesenchymal stem cells in induced osteonecrosis of the ovine femoral head: preliminary results

“…The methods used to tracking modified MSC in a bone defect did have obtained limited success, mainly because the label was not compatible with the specifics procedures used for bone histology 19 .In this study the bone samples used for evaluate CD+oMSC group were lost in procedures cause by bone histology preparation. However, the macroscopic findings after X-Gal staining gave us positive results, confirming the success of transplant.…”

“…Among the different techniques that have been proposed to track the fate of implanted cells in vivo, methods involving transduction of detectable genetic markers, such as those encoding for ß-galactosyltransferase (LacZ) and green fluorescent protein gene (GFP), are the only ones that allow stable and reliable long-term tracking of transplanted cells 19 .…”

Informed choices for challenging specimens when choosing methacrylate resin systems for histology

“…However, when specific needs arise, as, for example, when immunostaining fibrillar collagens in bone embedded in an MMA resin system, primary fixation in 70% ethanol provides greater immuno-sensitivity over formalin (Wittenburg et al, 2009). Additionally, Hannouche et al (2007) found that a mixture of 2% formaldehyde and 0.2% glutaraldehyde gave superior results when detecting the LacZ gene expression in cells and sections of mineralized tissue embedded in MMA using the X-gal detection method.…”