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

Abstract

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 β-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.

The recent advent of tissue engineering techniques, which combine scaffolding matrices and competent cells, offers tremendous perspectives for the fabrication of viable tissues in the laboratory. Recently, there has been extensive research on the use of bone marrow as a potential source of adult stem cells [mesenchymal stem cells (MSCs)] (Caplan 2005), which can be easily isolated and profoundly expanded in monolayers and retain their capacity to repair various tissues (Preston et al. 2003; Alison et al. 2004). Experimental studies indicate that MSCs loaded onto osteoconductive biomaterials can efficiently repair critical-size bone defects in small and large animals (Petite et al. 2000), and several authors have recently taken a step forward by applying this technology to humans in a clinical setting (Quarto et al. 2001; Vacanti et al. 2001). However, few data exist on the number of MSCs that survive after implantation and really contribute to the newly formed tissue, the specific role of the various subsets of cells contained in the bone marrow, and their mechanisms of differentiation and homing after systemic injection (Hassan and El-Sheemy 2004). Thus, the development of a reproducible method that would allow cell fate monitoring in transplant recipients is a pressing concern for the entire field of tissue engineering (Sharp et al. 2005).

Among the different techniques that have been proposed to follow the fate of implanted cells in vivo, only the transduction of detectable genetic markers, such as those encoding for β-galactosyltransferase (LacZ) and green fluorescent protein gene (GFP) allow stable and reliable long-term labeling of transplanted cells. To date, attempts to develop an easy, efficient, and reliable method to monitor genetically modified MSCs in a bone defect have had limited success, mainly because the label was not compatible with the specific procedures used for bone histology. GFP, from the jellyfish Aequorea victoria, offers a serious advantage over the other methods because it can be easily detected by UV light and fluorescence microscopy and can be monitored in living animals without the use of any substrate. With this label, successful MSC monitoring has been achieved in soft tissues in small animal studies, such as myocardium (Thompson et al. 2005), brain (Mothe et al. 2005), and liver (Anjos-Afonso et al. 2004), which are evaluated on frozen sections (Jiang et al. 2005) but could not be reliably done in skeletal tissues because of the important natural background autofluorescence. Therefore, despite substantial improvements in the histological procedures performed (Brazelton and Blau 2005; Jiang et al. 2005), visualization of GFP fluorescence in tissue-engineered bone must still be interpreted with great caution. lacZ is a widely employed reporter gene, encoding the bacterial Escherichia coli enzyme β-galactosidase (β-gal), because of its ease of use and availability of different detecting methods including fluorogenic and chromogenic agents (Tung et al. 2004). With the standard substrate X-Gal, β-gal is visualized in tissue sections as an intense blue intracellular precipitate. However, several authors have recently pointed out the need for optimizing the specificity and reliability of the X-Gal reaction to distinguish between β-gal and endogenous galactosidases activity (Nolan et al. 1988; Lal et al. 1994; Weiss et al. 1997; Sanchez-Ramos et al. 2000; Bell et al. 2005).

The aims of the study were (1) to optimize the histological procedure for LacZ staining of bone explants harvested from lacZ transgenic mice and embedded in MMA without prior decalcification and (2) to trace LacZ-labeled MSCs that were expanded in vitro, seeded onto collagen scaffolds, and implanted in vivo to repair a critical size bone defect. Tissues were fixed in different fixatives for different fixation times, dehydrated in acetone, and embedded in MMA. The procedure described here is suitable for conventional histology of bone, detection of enzyme activity, and immunohistochemistry.

Materials and Methods

Transgenic Mice

Experiments were carried out using a transgenic line bearing Vim ¹ mutation, kindly supplied by Dr. Charles Babinet (Institut Pasteur; Paris, France). Vimentin is a class III intermediate filament expressed only in mesenchymal cells of mature mammals. In this line, the endogenous vimentin gene has been disrupted by an in-frame insertion of the lacZ coding sequences into exon 1 of vimentin gene plus a nuclear localizing sequence. Thus, under the control of vimentin regulatory sequences, a vimentin β-gal fusion protein is synthesized and targeted to the nucleus. To optimize the detection of β-gal activity, we tested several variables on cultured MSCs and bone explants harvested from Vim ¹/Vim ⁺ heterozygous lacZ transgenic adult mice. C57/BL6 mice served as control animals. Animals were cared for and operated on according to the European Guidelines for the Care and Use of Laboratory Animals (Directive du Conseil 24.11.1986, 86/609/CEE).

LacZ Staining

Staining Protocol for Detection of β-gal Activity. The conditions used to detect β-gal activity were adapted to achieve specific localization of prokaryotic β-gal and significantly reduce endogenous background staining. In this experiment, X-Gal was used as staining substrate, and pH of the solution was set at 7.4 to eliminate false positive activity due to the ubiquitous mammalian enzyme (Nolan et al. 1988; Bell et al. 2005). Cultured cells and sections were incubated for 16 hr in the dark at 29C with the X-Gal staining solution prepared in PBS. The staining solution contained 2 mM MgCl₂, 5 mM K3Fe(CN)₆ (potassium ferricyanide; Sigma, St Louis, MO), 5 mM K4Fe(CN)₆ (potassium ferrocyanide; Sigma), 0.2% Tergitol NP40 (Sigma), 0.01% Na-deoxycholate (Sigma), and 1 mg/ml 5 bromo-4-chloro-3-indoyl-β-D-galactopyranoside (X-Gal; Fisher Scientific, Pittsburgh, PA). On the X-Gal reaction thus performed, cells expressing β-gal turn blue. MSCs and tissues from control mice were processed to assess background staining.

Immunodetection of β-gal. For immunodetection of β-gal, cultured LacZ MSCs were tested with the primary rabbit polyclonal anti-β-galactosidase antibody (Tebu-bio Laboratories; Le Perray en Yvelines Cedex, France), using Tris-HCl buffer, pH 8.5, as the antibody diluent (1:100). Nonspecific binding sites were blocked with goat serum diluted 1:10 in Tris-buffered saline (TBS) for 15 min before incubation with primary antibody. Excess serum was drained and primary antibody applied for 20 min at 4C. Sections were then washed in TBS and incubated with biotinylated, secondary antibody using the streptavidin-biotin-peroxidase procedure (LSAB kit; Dako, Carpinteria, CA) with diaminobenzidine (LSAB Kit; Dako) as substrate. The secondary antibody was biotinylated goat anti-rabbit antibody (LSAB Kit; Dako). Non-immunoreactive antibody controls were included in all assays.

Fixation

Influence of Different Fixatives on LacZ Staining In Vitro. The effect of several chemicals on both β-gal activity and immunostaining of LacZ was first evaluated on cultured MSCs expressing β-gal. LacZ MSCs were plated on 24-well cell culture microplates (Fisher Scientific) at a density of 3 × 10⁴ cells/cm² and cultured in DMEM (Gibco BRL, Life Technologies; Grand Island, NY), supplemented with 10% FBS (Sigma) for 24 hr.

Cells were rinsed twice in PBS and fixed for 12 hr at 4C before staining. Four different fixatives were evaluated in this study: (a) 10% neutral-buffered formaldehyde, (b) 2% formaldehyde/0.2% glutaraldehyde (F/GTA), (c) Schaffer's solution (containing 700 ml methanol, 300 ml 37% formaldehyde, 20g CaCO₃, 10 mg MgCO₃, pH 7.2), and (d) Neo-fix fixative, a formalinfree fixative (Ref. 65035-75; Merck, Darmstadt, Germany).

LacZ Staining in MMA-embedded Bone Explants. Because our ultimate goal is the development of a procedure that allows the detection of labeled cells in skeletal tissues without the need for prior decalcification, our specimens were embedded in MMA according to a previously described protocol (Lebeau et al. 1995). Long bones were collected from three Vim ¹/Vim ⁺ heterozygous lacZ transgenic adult mice that were sacrificed with an overdose of pentobarbital. Tissues were immediately transferred to the different fixatives and processed manually in 10-ml glass vials. Bone samples were fixed in the four different fixatives for 2, 4, 7, 14, and 21 days at 4C.

After fixation, tissue specimens were washed three times in PBS, gradually dehydrated in an acetone bath for 16 hr, and then soaked in xylene for 2 hr at 4C. Samples were then infiltrated for 6 hr with three changes of the resin solution (2 hr each) containing 10 ml of MMA with 50 mg benzoyl peroxide (Sigma), 2 ml of N-plastoid (nonylphenyl polyethyleneglycolacetate, Ref. 74432; Sigma-Fluka, Buchs, Switzerland), and 5% methylbenzoate (MMA solution I). Infiltrated tissues were placed in the bottom of each glass vial and immersed in a polymerization solution, readily prepared, containing 10 ml of MMA solution I and 50 μl of N,N-dimethyl aniline (Sigma). Polymerization was carried out at 19C for 19 hr. Polymerized blocks were stored at 4C until they were sectioned at room temperature with a tungsten blade microtome (Polycut Reichert Jung; Cambridge Instruments, Nussloch, Germany). Five-μm-thick sections were floated out on water, transferred onto glass slides pretreated with 3–amino-propyltriethoxysilane (Dako), stretched using water, pressed with a slide press, and air dried overnight at 37C. Sections were deplastified in five changes of acetone for 1 hr each, rehydrated in sequential washes of acetone, and processed for conventional histological staining and LacZ staining.

In Vivo Tracking of Murine LacZ MSCs Into Nude Rats

Isolation of Murine MSCs. MSCs were isolated from the marrow of 2-month-old lacZ transgenic mice as previously described (Mardon et al. 1987). Briefly, femurs and tibias were aseptically excised, cleaned of soft tissues, and cut at both epiphyseal extremities. Bone marrow was flushed out by pressure injection of DMEM. Expelled marrow was dispersed by repeated pipetting. Cell suspension was then centrifuged at 400 × g for 10 min. The resulting cell pellets were resuspended and seeded at a density of 3.2 × 10⁶ nucleated cells per cm² and cultured in medium consisting of DMEM supplemented with 10% FBS, 5 μg/ml ascorbic acid 2-phosphate (Sigma), and a solution containing 10,000 U/ml penicillin, 10 mg/ml streptomycin, and 25 μg/amphotericin B (Sigma). Following 2 to 3 days in culture, non-adherent hematopoietic cells were removed by changing the medium. The attached cells grew and developed colonies in 6 to 7 days. Culture medium was changed every 2 to 3 days until cells reached nearly 90% confluence. Two to three passages were necessary to obtain enough cells for preparing the constructs. In this study, up to 45% ± 5% of the cells expressed β-gal for at least 6 weeks in vitro (Figure 1).

Cell Construct Assembly. Murine LacZ MSCs were enzymatically detached with 0.05% trypsin (Sigma), counted, collected by centrifuging for 10 min at 400 × g, and diluted to 20 × 10⁶ cells/ml in DMEM. The scaffolds used in this study are biodegradable bovine collagen sponges containing a mixture of highly purified bovine protein (BP) extracts (Boden et al. 2004). BP contains several growth factors with a verifiable osteoinductive activity and was a gift from Sulzer Orthopedics Biologics Inc. (Denver, CO). BP was mixed with bovine type I tendon collagen carrier solution (14 mg carrier and 1 mg BP), lyophilized, and refrigerated at 2-8C. Immediately prior to use, the materials were reconstituted with 0.5 ml of sterile water. MSC suspensions were loaded onto collagen scaffolds (n=3), and the resultant collagen/MSCs constructs were incubated for 2 hr at 37C before implantation.

In Vivo Implantation. Three rnu/rnu nude rats (Harlan; Gannat, France) were kept in a controlled environment and given free access to food and water. On average, the host nude rats were 6 weeks old and weighed an average of160 g. Animals were anesthetized by an IP injection of ketamine hydrochloride (25 mg/kg, Ketalar; Roche, Basel, Switzerland), and xylazine (3 mg/kg; Rompum, Bayer AG, Leverkusen, Germany). Surgical procedure was carried out as previously described (Marden et al. 1994). A sagittal incision over the scalp from the nasal bone to the middle sagittal crest was made. The periosteum was elevated. An 8-mm bone defect was created using a dental surgical drilling unit (Microfrance; Bourbon L'Archambault, France) with a trephine constantly cooled with sterile saline. As a strict inclusion criterion, the dura was not violated. The defect was thoroughly rinsed with physiological saline, and bone fragments were washed out. Defects were filled with the readily prepared collagen/MSC constructs. The periosteum and scalp were closed in layers with interrupted resorbable sutures. X-rays were taken immediately after fracture and at 1–week intervals thereafter. Animals were sacrificed at 4 weeks after surgery. Transplanted constructs were carefully dissected free of surrounding soft tissue, and microradiographs of the skull were performed. The specimens were fixed in F/GTA for 4 days at 4C, dehydrated in acetone, and embedded in MMA without prior decalcification. Tissue sections were collected, deplastified in acetone, rehydrated, and processed for conventional histological staining and the detection of β-gal activity. Positive and negative staining controls were included in each run.

Figure 1

After 12 hr of fixation at 4C, higher staining intensity was obtained with 2% formaldehyde/0.2% glutaraldehyde (F/GTA) fixative for β-gal activity (A) and Schaffer's solution for immunodetection of LacZ (B). Importantly, cell morphology did not differ between LacZ-expressing and non-expressing cells. Bar = 10 μm.

Results

Influence of Different Fixatives on LacZ Staining

As a preliminary step, we tested several fixatives for an incubation time of 12 hr on cell cultures (Table 1) and 2 to 21 days on bone explants harvested from LacZ mice (Table 2). Among all the fixatives tested, F/GTA at 4C for 4 days gave the best results for X-gal staining on both cell cultures (Figure 1) and bone explants (Figure 2). The use of Neo-fix fixative (Merck) led to a significant decrease in X-Gal staining after 2 days of fixation of bone samples when compared with the use of F/GTA. After 12 hr of fixation with Schaffer's solution and 10% formaldehyde, β-gal activity could not be detected on cell cultures or on bone specimens.

For immunodetection of β-gal with the primary rabbit polyclonal antibody, the influence of the different fixatives on the intensity of the reaction was more subtle (Table 1). The best performance was achieved with Schaffer's solution at 4C (Figure 1B). Negative immunostaining controls (control specimens and secondary antibody stainings) did not show any false positive binding (data not shown).

Visualization of LacZ Cells After Implantation In Vivo in a Bone Defect

Given a rate of β-gal expression of 45%, examination of LacZ cells must be interpreted with caution because β-gal-expressing cells may not be representative of the whole cell population. However, LacZ-positive cells plated on plastic did not show any difference in cell morphology as compared to non-labeled cells, thus reducing the possibility of a selective tracking of a subpopulation of MSCs (Figure 1).

Table 1

Influence of different fixatives on LacZ staining in vitro

	10% Formaldehyde	Schaffer's solution	F/GTA	Neo-fix
β-gal activity	−	−	+++	+++
Immunodetection of LacZ++	+++	++	++

−, no staining; +, mild staining; ++, moderate staining; +++, intense staining. β-gal, β-galactosidase; F/GTA, 2% formaldehyde/2% glutaraldehyde.

Cells were fixed at 4C for 12 hr. For β-gal activity, X-Gal was used as staining substrate, and pH of the solution was set at 7.4

Table 2

Influence of different fixatives on β-gal activity in MMA-embedded bone explants

Time of fixation (days)	10% formaldehyde	Schaffer's solution	F/GTA	Neo-fix
2	−	−	+	−
4	−	−	+++	+
7	−	−	++	+
14	−	−	+	−
21	−	−	−	−

−, no staining; +, mild staining; ++, moderate staining; +++, intense staining. MMA, methylmethacrylate.

Fixation was performed at 4C. For β-gal activity, X-Gal was used as staining substrate, and pH of the solution was set at 7.4

The embedding procedure described in this study was successful and reliable. Thin sections of 5-μm thickness could be performed, even several months after polymerization of the blocks. MMA embedding allowed optimal tissue preservation for morphology assessment. Standard stainings could be applied on acetone-deplastified sections without modification of the histological protocols. Bone formation occurred within the defect and was predominantly present at the periphery of the lesion (Figure 3A). Histological examination revealed extracellular matrix formation with an important neovascu-larization. The predominant type of bone observed was woven bone. Standard X-Gal staining showed evidence of integration of bone marrow-derived MSCs in the newly formed tissue (Figures 3B and 3C). Blue staining was uniform and mainly localized in the medullary spaces along bone trabeculae (Figure 3B) and within vascular walls (Figure 3C). No evidence of enchondral ossification was observed at the different endpoints. Nucleated cells expressing LacZ were occasionally detected in peripheral blood of transplanted animals (Figure 3D).

Discussion

The aim of the present study was to optimize LacZ staining in bone specimens embedded in MMA and to apply this protocol for the detection of MSCs transplanted in bone defects. The rapid embedding procedure for MMA described in this study allowed detailed visualization of LacZ cells in bone tissue embedded in MMA with high morphological preservation. This shortened embedding method was originally designed to alleviate time-consuming steps during the processing of bone marrow biopsies (Lebeau et al. 1995). It is reliable and can be done with commercially available methacrylates. It relies on the use of a low amount of benzoylperoxide as initiator and N,N-dimethyl aniline as accelerator for chemical polymerization at 19C, to allow the detection of enzyme activity and protein antigens (Hantschick and Stosiek 1998). With the adjunction of N-plastoid as a plasticizer, thin sections of 2- to 3-μm thickness are easier to obtain. A great advantage of MMA embedding over cryostat sectioning is the histological appearance of morphological details and the ability to analyze all types of tissues including cartilage and bone without the need for prior decalcification (Jiang et al. 2005). With this procedure, hard tissues can be examined with conventional histology stainings and immunohistochemistry. A large panel of antibodies can be tested on MMA sections, and staining intensity can be greatly improved after antigen retrieval pretreatment methods (proteolytic digestion, microwave antigen retrieval), depending upon the antibody investigated (Shi et al. 1997).

Figure 2

Tissue section of a femoral bone explant harvested from a lacZ transgenic mice and embedded in methylmethacrylate. X-gal staining was performed at 4C, pH 7.4. Among all the fixatives tested, F/GTA for 4 days gave the best results. Blue cells were observed within the medullary space and along the walls of bone trabeculae (white arrows). Picrofuschin staining. Bar = 100 μm.

In regard to β-gal detection in embedded tissues, the major problem seems to be the fixation step, which must be adapted to the hardness of the tissue and the size of the samples examined. In this study, sensitivity of β-gal detection by X-Gal staining was closely dependent on the nature of the fixative used and duration of fixation, which corroborates with previous reports (Ma et al. 2002; Bell et al. 2005). After 12 hr of fixation at 4C, β-gal activity decreased dramatically in 10% formaldehyde and Schaffer's solution and could only be detected in 0.2% glutaraldehyde containing fixative and Neo-fix fixative (Merck). Higher staining intensity was obtained after 4 days of fixation at 4C with F/GTA fixative. Similar results were reported by Ma et al. (2002) in kidney tissue sections with 0.2% glutaraldehyde, but could not be reproduced after 8 hr of fixation, which is not sufficient for the processing of bony specimens (Lebeau et al. 1995; Mueller et al. 2000). Skeletal tissues usually require longer times of fixation, which may impair tissue reactivity in histochemical and immunohistochemical investigations. Our results emphasize the importance of performing a test battery to optimize the main parameters (nature of the fixative, duration, and temperature for tissue preparation) that influence the detection of β-gal activity in the tissue examined and avoid invalid conclusions when evaluating the in vivo fate of LacZ cells.

Despite the number of research papers reporting the effectiveness of tissue engineering strategies for the repair of massive bone defects in animal models, very few studies have so far assessed the exact fate of implanted cells in vivo, mainly because of technical limitations during the histological procedure (Oshima et al. 2005). Whether implanted cells differentiate and incorporate into newly formed bone or simply interact with the host environment to initiate and promote the repair process is not well known. We have focused on tracking MSCs because these adult, autologous stem cells are excellent candidates for the engineering of several tissues including bone, cartilage, vessels, myocardium, and brain (Gregory et al. 2005). MSCs can easily and reliably be isolated from bone marrow aspirates and can maintain their growth potential through many cell divisions in vitro while at the same time retaining their capacity to differentiate along different lineages after their implantation in vivo (Caplan 2005). The lacZ gene has been widely used as a genetic marker of cell tracking and has several advantages over currently available exogenous tools such as radioactive probes (Slezak and Muirhead 1991) and fluorescent dyes (Hoechst 33342, PKH26, CM-Di1) (Zhang et al. 2002a; Hoffman 2004). Because lacZ gene is integrated in the genome of transplanted MSCs, β-gal activity is not affected by cell division. With the use of fluorescent dyes, fluorescence intensity decreases parallel to the number of cell divisions (Ferrari et al. 2001), long-term cell viability might be compromised (Mosahebi et al. 2000), and dye leaching may occur with a subsequent labeling of surrounding host cells (Iwashita et al. 2000). Recently, non-invasive techniques have been explored for in vivo cell monitoring after cellular implantation (Ahrens et al. 2005). They rely on cell tagging with nanoparticles of superparamagnetic iron oxide, gadolinium-based T1 substances, or perfluoropolyether particles that can be tracked with magnetic resonance imaging. These innovating, technically demanding and expensive technologies still need to be evaluated and used with caution, as they may alter MSCs differentiation down a specific pathway, as recently demonstrated by Kostura et al. (2004).

Figure 3

(A) Radiographic film of representative defect after 4 weeks of treatment. Defect was filled with a biodegradable bovine collagen sponge containing a mixture of bone proteins and LacZ MSCs. Bone formation occurred within the defect and was predominantly present at the periphery of the lesion. (B,C) Representative sections of defects after 4 weeks. Standard X-Gal staining showed evidence of integration of bone marrow-derived mesenchymal stem cells within newly formed tissue, along bone trabeculae (B) and within vascular walls (C). Picrofuschin staining. (D) Nucleated cells expressing LacZ were occasionally detected in peripheral blood of transplanted animals. Hematoxylin-eosin staining. Bars: B = 5 μm; C = 100 μm.

In this study we established a xenograft nude rat model allowing the detection of murine LacZ MSCs in tissue-engineered bone. To distinguish bone marrow-derived cells from host cells, murine LacZ MSCs were seeded onto a bioabsorbable collagen sponge and transplanted to repair a calvarial defect in nude rats. A non-transgenic background strain of mice could have been used to distinguish LacZ cells from host cells. However, the 8-mm rat craniotomy is a well-established intraosseous wound model that has been used by a number of authors to evaluate potential bone-healing materials including growth factors and cell-containing scaffolds. For reproducibility reasons, the craniotomy is performed with a cylindrical low-speed bur without breaching the dura. When performed in mice, this may damage the superior sagittal sinus located at the inner surface of the skull, resulting in a high animal mortality rate. Such damage is less likely to occur when performed in rats. Using the xenograft nude rat model, LacZ-labeled MSCs were detected in vivo for up to 4 weeks after implantation and contributed to the repair of the bone defect. After 4 weeks, the defect was partially filled with bone, which was predominant at the periphery of the defect. The short period of implantation in this study may account for the lack of osteogenesis in the middle of the defect. In a previous study performed in the same animal model, we showed a statistical increase in bone formation between 1 and 2 months when defects were filled with MSCs and BP (Arnaud et al. 1999). Moreover, bone healing after bone grafting involves a sequence of dynamic events that usually occurs in five phases: inflammation, microrevascularization, osteoinduction, osteoconduction, and osteoremodeling. Osteoinduction is largely controlled by growth factors, which induce migration, proliferation, and differentiation of non-committed mesenchymal cells in the fracture site. In this study, the scaffolds used were biodegradable bovine collagen sponges containing a mixture of BP that may have elicited new bone formation by the recruitment of cells migrating from the adjacent host bone marrow cavity. Although data from this study were only qualitatively evaluated without determining cell number per area, over time they gave important information on the fate of implanted cells. To the authors' current knowledge, this is the first time the lacZ gene was successfully used to track MSCs in skeletal tissues embedded in MMA. Using this chimeric animal model, we ascertained that the newly formed tissue contained cells derived from the transplanted LacZ MSCs. Histological examination revealed extracellular matrix formation and active osteoblastic cell rimming, but osteocytic cells could not be seen within bone tissue. Despite the use of BP, no enchondral ossification was observed in any specimens, which is consistent with previous reports (Arnaud et al. 1999) and is explained by the intramembranous ossification process that occurs in the calvaria. Interestingly, LacZ cells could be seen in the medullary spaces of recovered implants. In this study, MSCs were generated by a standard selection method based on plastic adhesion of bone marrow stromal cells. The proportion of hematopoietic cells in cultured cells is reduced from ∼11% of the total cell number at passage number 2 to <5% at passage 10 (Krebsbach et al. 1997). Therefore, it is not entirely clear whether LacZ-positive cells located in the bone marrow originated from the stromal or the hematopoietic fraction of bone marrow. This study also demonstrates that MSCs incorporate into the vasculature of newly formed bone and differentiate into endothelial cells. The neovascularization process, which occurs within a bone defect or at the fracture site, is of paramount importance to achieve bone healing. It supplies oxygen, nutrients, and growth factors that induce migration, proliferation, and differentiation of an appropriate subset of cells in the fracture site (Einhorn 1998). In bone marrow transplant studies, the contribution of MSCs to the formation of growing vessels in adult organisms remains controversial. Although there is increasing evidence that endothelial cells may originate from bone marrow-derived precursors (Takahashi et al. 1999; Hess et al. 2002; Zhang et al. 2002b), some authors failed to identify circulating GFP-labeled bone marrow-derived cells in vascular walls of growing vessels (Ziegelhoeffer et al. 2004). Here we demonstrate that the neovascularization that occurs within the defect not only depends on the sprouting of new vessels from a preexisting network (angiogenesis), but also on the contribution of implanted or circulating endothelial progenitors (postnatal vasculogenesis) contained in the bone marrow stroma. This study, however, did not permit us to identify the subpopulation of bone marrow-derived cells that are specifically involved in vasculogenesis. Further studies with rigorous quantification of cells are certainly warranted to clearly address this issue.

In conclusion, a rapid procedure for MMA embedding allows for both precise morphological analysis and histochemistry. This technique provides a unique tool for in vivo detection of β-gal-expressing cells incorporated into all types of tissues including undecalcified bone and cartilage. The present study provides irrefutable evidence that LacZ-labeled MSCs can be detected with the standard X-Gal staining in skeletal tissues embedded in MMA. LacZ still represents a useful phenotypic marker in cell-tracking studies and may provide important information on cell fate and relationships of cells to the different biomaterials investigated. Transplanted MSCs participated in the repair of a calvarial bone defect and contributed to the neovascularization of the lesion.