| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
a First Department of Pathology,
b Department of Orthopedic Surgery,
c Transplantation Center, and
d Regeneration Research Center for Intractable Diseases, Kansai Medical University, Moriguchi City, Osaka, Japan;
e Laboratory of Immunobiology, Department of Animal Development and Physiology, Division of Systemic Life Science, Graduate School of Biostudies, Kyoto University, Kyoto, Japan
Key Words. Bone marrow transplantation • Osteoporosis • SAMP6 mouse
Correspondence:
Susumu Ikehara, M.D., Ph.D., First Department of Pathology, Kansai Medical University, 10-15 Fumizono-cho, Moriguchi City, Osaka 570-8506, Japan. Telephone: 81-6-6993-9429; Fax: 81-6-6994-8283; e-mail: ikehara{at}takii.kmu.ac.jp
 |
ABSTRACT |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
In addition to the above, BM stromal cells in the treated SAMP6 mice were replaced with donor stromal cells, and the message level of interleukin-11 (IL-11), which is produced by the BM stromal cells and is known as an important factor in the regulation of bone remodeling, was restored to a level similar to that observed in normal B6 mice. Furthermore, the message level of IL-6, which is known to enhance osteoclastogenesis, was also restored to normal. These results indicate that the BM microenvironment was normalized after IBM-BMT and that the increased production of IL-11 and IL-6 ameliorated the imbalance between bone absorption and formation, resulting in the prevention of osteoporosis in SAMP6 mice.
|
INTRODUCTION |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Osteoporosis might be attributable to an imbalance between bone absorption and formation, and primary age-dependent senile osteoporosis is thought to be due to a reduction in bone formation as a result of a decrease in the recruitment of osteoblasts, and also due to an elevation of bone absorption that might result from an increase in parathyroid hormone caused by the failure of vitamin D activation [2–4]. Numerous reagents have been developed over the past 30 years to treat osteoporosis, and antiresorptive reagents, such as calcium, vitamin D, estrogens, calcitonin, and bisphosphonates, have been used widely in clinical situations. However, no effective treatment for osteoporosis has been developed, and these reagents are primarily designed to prevent bone loss and consequent fractures. As such, osteoporosis is actually an intractable disease and, also, is a major health care problem because of the associated costs, morbidity, and mortality; direct financial expenditure for the treatment of osteoporotic fracture in the U.S. is estimated at $10-$15 billion per year [5].
The P6 substrain of senescence-accelerated mice (SAMP6) is an animal model for osteoporosis that exhibits age-dependent inhibition of osteoblastogenesis and osteoclastogenesis with enhanced adipogenesis, resulting in an early decrease in bone mass [6]. In this model mouse, it has been reported that interleukin-11 (IL-11) is essentially involved in the early onset of osteoporosis, where enhanced adipogenesis due to the decreased production of IL-11 impairs bone metabolism, resulting in osteoporosis [7]. In the bone marrow (BM), IL-11 is mainly produced by stromal cells [8, 9], and a lower production of IL-11 from BM stromal cells is found in SAMP6 mice [7]. Furthermore, IL-11 has been shown not only to inhibit adipogenesis but also to enhance the differentiation of osteoblasts [10]. It has been noted that some other cytokines (IL-6 [11], tumor necrosis factor alpha [TNF-
] [12], transforming growth factor beta [TGF-ß] [13]) might be involved in osteoporosis through the regulation of osteoblastogenesis and osteoclastogenesis. TNF- can stimulate the production of IL-6 by osteoblasts (originally derived from stromal cells), and IL-6 can regulate osteoclastogenesis, whereas TGF-, which is produced by osteoclasts, controls the osteoblastogenesis of stromal cells. Taking these findings into consideration, the reconstitution of abnormal stromal cells with normal stromal cells might be one step in developing a new strategy for the prevention and treatment of osteoporosis.
Bone marrow transplant (BMT) has been used to treat various intractable diseases, such as aplastic anemia and leukemia, and has now been applied to systemic and organ-specific autoimmune diseases [14–29]. It has now been widely accepted that BM cells (BMCs) contain not only hematopoietic stem cells but also mesenchymal stem cells (MSCs), which have the potential to differentiate into various tissues, such as bone, cartilage, tendon, muscle, and adipose tissue. Therefore, BMT can recruit a pluripotent MSC population that differentiates into bone and cartilage for the correction of skeletal-related diseases. Rodriguez et al. have demonstrated that MSCs from osteoporotic postmenopausal women show a lower proliferation rate and mitogenic response to osteogeneic growth factors, indicating that the MSCs from osteoporotic patients produce a deficient type I collagen and are somehow involved in the progression of osteoporosis in humans [30].
In the present study, using SAMP6 mice, we performed intrabone marrow BMT (IBM-BMT) from allogeneic normal mice to examine its effect on the prevention of osteoporosis, and show that IBM-BMT could be applicable for human patients with osteoporosis.
|
MATERIALS AND METHODS |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Preparation and Inoculation of BMCs
BMCs were collected from the femurs and tibias of B6 mice. The whole BMCs were directly injected into the BM cavity (IBM injection) to facilitate the early recovery of hematopoiesis and donor cell engraftment. IBM injection was carried out according to the method described previously [24]. In brief, the region from the thigh to the knee joint was shaved of hairs with a razor. The knee was flexed to 90°, and the proximal side of the tibia was drawn to the anterior. A 26-gauge needle was inserted into the joint surface of the tibia through the patellar tendon and then inserted into the BM cavity. Using a microsyringe (50 µl; Hamilton Co.; Reno, NV), the donor BMCs (3 x 107/30 µl) were injected into the BM cavity.
Experimental Protocols
SAMP6 mice (4 months of age) received fractionated irradiation (5.5 Gy x 2 = 11 Gy; 4-hour interval), and 1 day after the irradiation, the mice were transplanted with whole BMCs (3 x 107) via IBM injection (IBM-BMT), as previously described [24].
SAMP6 mice that had been irradiated and injected intravenously with 3 x 107 whole BMCs (i.v.-BMT) were also prepared. Two additional groups were prepared as controls: B6 mice were irradiated and transplanted with syngeneic B6 BMCs (3 x 107) by IBM-BMT [B6
B6] and SAMP6 mice were transplanted with syngeneic SAMP6 BMCs (3 x 107) by IBM-BMT [SAMSAM].
Surface Marker Analyses
Spleen cells and BMCs were prepared from the recipient mice. To detect donor- or recipient-derived cells, the cells were stained with fluorescein isothiocyanate (FITC)-conjugated anti-H-2Kd and phycoerythrin (PE)-conjugated anti-H-2Kb monoclonal antibodies (mAbs) (PharMingen; San Diego, CA; http://www.bdbiosciences.com/pharmingen). FITC- or PE-conjugated mAbs against CD45R (B220), CD4, CD8, CD11b, and Gr-1 (PharMingen) were used to analyze the cells with mature lineage markers. The cells were analyzed using a FACScan® fluorescence-activated cell sorter (Becton Dickinson & Co.; Mountain View, CA; http://www.bd.com).
Histological Findings
The lumbar spine and femur of each recipient mice were removed and fixed in 10% formalin and then decalcified. The sections were stained with hematoxylin and eosin (H-E).
Microdensitometry
Bone mass was roentgenologically assayed according to the method described elsewhere [6]. Soft x-ray photographs of the exact lateral view of the femur were taken under the following conditions: focus-film distance 118 cm, 100 mA, 46 kVp, and 0.16 seconds of exposure. An aluminum plate (2 mm thick) was placed beneath the bone in order to shift the density of bone and base to the most sensitive range of the film (Fuji Softex Film FG; Fuji Photo Film; Tokyo, Japan). A standard aluminum wedge was also placed on the border of the film. The film was developed and fixed in the usual manner. The film was then scanned at the midpoint of the femoral shaft at right angles to the shaft by microphotodensitometer (Sakura Densitometer PDS15; Konica; Japan). The width and height of the slit were 10 µm and 1 mm, respectively. Scanning speed was 0.04 mm/minute. The optical density of the bone was converted to the thickness of aluminum by the density patterns of the standard aluminum wedge (Figs. 1A, 1B, and 1C
), and an M-shaped bone pattern was obtained (Fig. 1D). The integration of the pattern (
GS) corresponds to the absolute mineral mass on the scanned plane (Fig. 1E). The absolute matrix mass (area of the cortex on the scanned plane) can be given as follows:
 |
GS/D2 and the cortical thickness index (CTI = [D - d]/D), which indicate mineral mass and matrix mass, respectively. Statistical analyses of the GS/D and the CTI of recipient mice were performed using t tests.
|
Cultured Stromal Cells
Cultured stromal cells were obtained as previously described [21]. In brief, the femurs, tibias, and humeri, from which the BMCs had been extensively washed out, were cut into pieces, and the bone pieces were cultured in a flask. The medium (RPMI1640 with fetal bovine serum) in the flask was replaced weekly with the same volume of fresh culture medium. Three weeks later, nonadherent cells were extensively removed, and the adherent cells were then collected from the surface of the flasks by trypsin/EDTA treatment (Sigma Chemical Co.; St. Louis, MO; http://www.sigmaaldrich.com).
Reverse Transcription-Polymerase Chain Reaction (RT-PCR) Assay of IL-11 cDNA
The IL-11 cDNA sequence from cultured stromal cells was analyzed by direct sequencing of PCR products to examine the entire coding region of mouse IL-11 cDNA, as previously described [3]. We prepared two pairs of primers, F1: 5'-TGTCGCCTGGTCCTGGTGGT-3', R1: 5'-TGCACGGC GCAGCCA-TTGTA-3' and F2: 5'-GAGTAGACTT GATGT CCTAC-3', R2: 5'-TAAATAAATAAGATCTG GTT-3'.
IL-11 cDNA was amplified by two pairs of primers under the following conditions: 96°C for 1 minute, 64°C for 1 minute, 72°C for 2 minutes x 35 cycles, and a final extension at 72°C for 10 minutes. Primers for IL-6 (F: 5'-ATGA ACTC CTTCTCCACAAGCGC-3', R: 5'-GAAGAGCC CTCAG GCTGGACTG), TNF- (F: 5'-CTTCAGACCTT TCCAGA CTCTTCC-3', R: 5'-AGAGGTTCAGTGATGTAGCGAC AG-3'), and TGF-ß (F: 5'-TTTCGATTCAGCGCTCACTGC TCTTGTGAC-3', R: 5'-ATGTTGGACAACTGCTCCACCT TGGGCTTGC-3') were also prepared and used [31].
The PCR products were electrophoresed on a 1% agarose gel, stained with ethidium bromide (0.5 µg/ml), and visualized by a UV transilluminator.
Analyses for Immunological Functions
Antibody production against sheep red blood cells (SRBCs) and mixed leukocyte reaction (MLR) were analyzed as immunological functions of the treated mice. Anti-SRBC antibody response was evaluated as described previously [29]. In brief, the spleen cells (4 x 106) were cultured with the same number of SRBCs for 5 days, and anti-SRBC antibody production was measured by the modified Jerne’s plaque-forming cell (PFC) assay. MLR was performed as follows: splenic T cells (2 x 105) were cultured with 2 x 105 responder T cells and 2 x 105 irradiated (12 Gy) stimulator spleen cells for 72 hours and pulsed with 0.5 µCi of [3H]-thymidine for the last 16 hours of the culturing period.
|
RESULTS |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Analyses of Reconstitution and Cell Surface Antigens
We carried out flow cytometrical analyses of cells harvested from the recipients, and their immunological functions were examined to confirm donor cell engraftment. As shown in Table 1, the percentage of donor-derived cells (H-2b+) in the spleen was almost 100%. Donor-derived cells with mature lineage markers (B220, CD4, CD8, Mac-1, and Gr-1) had been generated to normal levels in the spleen (Table 1) and BM (data not shown) when assayed 6 months after IBM-BMT (data not shown). The recipients were completely reconstituted with donor-derived cells and survived more than 1 year after IBM-BMT.
|
Histopathological Findings
The aged SAMP6 mice exhibited histopathological findings of osteoporosis. Figure 2 shows H-E staining of the lumbar spinal vertebral body. The untreated SAMP6 showed a significant loss of trabecular bone and cortical bone thickness at 12 months of age (Fig. 2B). This was more prominent when examined at the older age (24 months) (Fig. 2C). However, the SAMP6 mice treated with IBM-BMT showed a marked increase in the trabecular bones at 12 months of age, 8 months after the treatment (Fig. 2D). Osteoporosis was observed in SAMP6 mice that had received syngeneic SAM BMCs [SAMSAM] when assayed at 12 months of age (data not shown).
|
, normal B6 mice showed the highest bone mineral density (BMD) at 12 months of age, after which it gradually decreased. In contrast, in untreated SAMP6 mice, the highest BMD was observed at 7 months of age but thereafter rapidly decreased. After treating SAMP6 mice with IBM-BMT from B6 mice, the kinetical changes in BMD of SAMP6 mice were similar to those of normal B6 mice.
Urinary DPD
An increase in DPD is the result of bone absorption and is also known as a clinical sign of osteoporosis. The DPD in untreated SAMP6 mice rapidly increased after 7 months of age (Fig. 3). However, the DPD in the SAMP6 mice treated with IBM-BMT remained low until 10 months of age, and was similar to that observed in normal B6 mice, indicating the prevention of bone resorption by IBM-BMT. The increase in DPD in SAMP6 mice treated with IBM-BMT ([B6SAMP6] mice) might be due to the natural course of senescence.
|
Production of IL-11 and Other Cytokines in Stromal Cells
IL-11, produced from BM stromal cells, is known to inhibit adipogenesis and, conversely, to stimulate osteoclast formation. It is also considered to be one of the most important factors in the regulation of bone remodeling. Therefore, we next examined the level of IL-11 production in the BM stromal cells of mice treated with IBM-BMT.
Stromal cells collected from the BM of [B6SAMP6] mice treated with IBM-BMT were first analyzed by a FACScan. They were found to be of donor origin (H-2b+) and to be positive for stromal-cell-specific mAb, PA6 (Fig. 4) [32]. Stromal cells were also collected from control mice: untreated young and aged B6 or SAMP6 mice, B6 mice treated with IBM-BMT using B6 BMCs [B6B6], and SAMP6 mice treated with IBM-BMT using SAMP6 BMCs [SAMSAM]. The message level of IL-11 was assayed by RT-PCR. As shown in Figure 5, the expression of IL-11 was found to decrease in stromal cells derived from untreated SAMP6 mice at the ages of 3 and 9 months when compared with that from the same ages of normal B6 mice. It should be noted that the message level of IL-11 in stromal cells of [B6SAMP6] mice treated with IBM-BMT was restored to a level similar to that observed in normal B6 mice (Fig. 5A). The expression of IL-11 in the stromal cells from [B6B6] mice or [SAMSAM] mice was similar to that of untreated B6 or untreated SAMP6 mice of corresponding ages, respectively (data not shown). Furthermore, IL-6 is known to regulate osteoclastogenesis, and the expression of IL-6 was lower in untreated SAMP6 mice (9 months of age). After IBM-BMT (in [B6SAMP6] mice), the expression of IL-6 was also restored to a level similar to that of normal B6 mice (3 and 9 months of age) (Fig. 5B). Other cytokines, such as TGF-ß (which is involved in osteoblastogenesis [13]) and TNF- (which augments RANKL (receptor activators of NF-
B ligand)-induced osteoclastogenesis [12]), were also examined. TNF- and TGF-ß were uniformly expressed in stromal cells of untreated SAMP6 mice (3 and 9 months of age), normal B6 mice, and treated SAMP6 [B6SAMP6] mice. These results indicate that IBM-BMT from normal B6 mice can reconstitute recipients with normal BM stromal cells of donors, normalizing the BM microenvironment and restoring the production of IL-11 and IL-6, and resulting in an amelioration of the imbalance caused by the lower expressions of IL-11 and IL-6.
|
|
|
DISCUSSION |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Using the SAMP6 mouse, an animal model for senile osteoporosis, we have shown here a new strategy for the prevention of age-related osteoporosis. Much evidence has accumulated that allogeneic BMT has a curative effect on various diseases, including organ-specific and systemic autoimmune diseases [14–18]. In a series of our experiments on allogeneic BMT, the importance of stromal cells for the success of allogeneic BMT has been progressively clarified; stromal cells have the capacity to support the early engraftment of donor-derived progenitor cells and also have the capacity to retain hematopoietic stem cells to maintain long-term hematopoiesis by donor-derived cells [24–29]. Based on these findings, we have established a powerful new strategy for allogeneic BMT: whole BMCs, including MSCs, are directly injected into the BM cavity (the tibia) of recipient mice to promote the proliferation of donor-derived hematopoietic stem cells in collaboration with donor-derived stromal cells [24]. After the IBM-BMT, the engraftment of donor-derived hematopoietic cells is easier than after i.v.-BMT or portal venous BMT (PV-BMT), even when radiation doses are lower. Autoimmune diseases in MRL/lpr mice are completely cured by IBM-BMT, although MRL/lpr mice are relatively radiosensitive in comparison with normal mouse strains, especially after the onset of autoimmune diseases. The advantage of IBM-BMT is that the radiation doses for conditioning can be reduced. In this study, recipients treated with i.v.-BMT developed osteoporosis, as did untreated SAMP6 mice, although the onset was delayed. This might be due to inefficient engraftment of MSCs, which differentiate into stromal cells and participate in osteoporosis through the production of cytokines. Furthermore, it is of importance that IBM-BMT is effective in the engraftment of donor-derived cells and that no immunosuppressive drugs are required to maintain donor-cell engraftment (full chimerism).
Various indices related to osteoporosis, such as BMD (Fig. 2), DPD (Fig. 3) in urine, and histological changes (Fig. 1) in the lumbar spine, were progressively aggravated in untreated SAMP6 mice, whereas those factors were retained at normal levels (similar to those observed in normal mouse strains) for more than 1 year after the treatment with IBM-BMT from B6 mice. Furthermore, it should be noted that the stromal cells harvested from the treated SAMP6 mice treated with IBM-BMT from B6 mice were reconstituted by cells with donor-type MHC determinants (Fig. 4) and that the expression of IL-6 and IL-11 in these stromal cells increased to a normal level (Fig. 5), although stromal cells from the age-matched untreated SAMP6 mice showed a lower level of these cytokines. One of the biological functions of IL-11 is to inhibit adipogenesis and, thereby, to enhance osteoblastogenesis in the BM, and the lower expression of IL-11 in SAMP6 mice, therefore, leads to a lower rate of osteoblastogenesis and, reciprocally, a greater rate of adipogenesis. IL-6 is also important in the regulation of osteoclastogenesis [11], and the normalization of IL-6 after IBM-BMT can help maintain the balance between bone formation and degradation and prevent osteoporosis. TNF- can stimulate the production of IL-6 [11] and can augment RANKL, which induces osteoclastogenesis [12]. TGF-ß might be involved in osteoblastogenesis [13]. Therefore, IL-6 and TGF-ß would be involved in the onset of osteoporosis through the regulation of osteoclastogenesis. However, in this study, the expression levels of TNF- and TGF-ß in stromal cells were unchanged among untreated SAMP6 mice, normal B6 mice, and treated SAMP6 mice (IBM-BMT). Thus, the low rate of bone formation and lower BMD [33] observed in SAMP6 mice might be partly attributable to the lower level of IL-11 and IL-6 production by the stromal cells. Though the production of IL-11 from BM stromal cells has not yet been compared between patients with osteoporosis and normal volunteers, the importance of this cytokine is evident and may be extrapolated to humans.
As has been recently reported, BMCs contain not only hematopoietic stem cells but also stromal cells that have mesenchymal stem cell activity [28]. BM stromal cells can differentiate into chondrocytes, adipocytes, osteoblasts, cardiomyocytes [34, 35], and even neurons [36]. After IBM-BMT, stromal cells from recipient SAMP6 mice were completely replaced by cells of donor origin (Fig. 4). This indicates that normal MSCs were transplanted and that they generated osteoblasts and produced IL-11 at a normal level. Although there is no direct evidence for the generation of osteoblasts after IBM-BMT, it is highly likely that osteoblasts of donor origin can differentiate from donor stromal cells. Furthermore, IBM-BMT can facilitate the early engraftment of hematopoietic cells of donor origin, indicating that it can generate normal osteoclasts in the BM. Therefore, after IBM-BMT, the normal BM microenvironment for bone formation should be reconstituted, which would result in prevention of the early onset of osteoporosis in SAMP6 mice. We are now in the process of examining whether IBM-BMT can be used to treat osteoporosis in SAMP6 mice.
|
ACKNOWLEDGMENT |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
This work was supported by a grant from Haiteku Research Center of the Ministry of Education, a grant from the Millennium program of the Ministry of Education, Culture, Sports, Science and Technology, a grant from the Science Frontier program of the Ministry of Education, Culture, Sports, Science and Technology, a grant-in-aid for scientific research (B) 11470062, grants-in-aid for scientific research on priority areas (A)10181225 and (A)11162221, and also a grant from Japan Immunoresearch Laboratories Co., Ltd. (JIMRO).
|
REFERENCES |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
This article has been cited by other articles:
|
|
 |
|
  M. Dominici, R. Marino, V. Rasini, C. Spano, P. Paolucci, P. Conte, T. J. Hofmann, and E. M. Horwitz Donor cell-derived osteopoiesis originates from a self-renewing stem cell with a limited regenerative contribution after transplantation Blood, April 15, 2008; 111(8): 4386 - 4391. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Inaba, Y. Adachi, H. Hisha, N. Hosaka, M. Maki, Y. Ueda, Y. Koike, T. Miyake, J. Fukui, Y. Cui, et al. Extensive Studies on Perfusion Method Plus Intra-Bone Marrow-Bone Marrow Transplantation Using Cynomolgus Monkeys Stem Cells, August 1, 2007; 25(8): 2098 - 2103. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Ueda, M. Inaba, K. Takada, J. Fukui, Y. Sakaguchi, M. Tsuda, M. Omae, T. Kushida, H. Iida, and S. Ikehara Induction of Senile Osteoporosis in Normal Mice by Intra-Bone Marrow-Bone Marrow Transplantation from Osteoporosis-Prone Mice Stem Cells, June 1, 2007; 25(6): 1356 - 1363. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Fukui, M. Inaba, Y. Ueda, T. Miyake, N. Hosaka, A-H. Kwon, Y. Sakaguchi, M. Tsuda, M. Omae, Y. Kamiyama, et al. Prevention of Graft-Versus-Host Disease by Intra-Bone Marrow Injection of Donor T Cells Stem Cells, June 1, 2007; 25(6): 1595 - 1601. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Q. Li, H. Hisha, R. Yasumizu, T.-X. Fan, G.-X. Yang, Q. Li, Y.-Z. Cui, X.-L. Wang, C.-Y. Song, S. Okazaki, et al. Analyses of Very Early Hemopoietic Regeneration After Bone Marrow Transplantation: Comparison of Intravenous and Intrabone Marrow Routes Stem Cells, May 1, 2007; 25(5): 1186 - 1194. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Koike, Y. Adachi, Y. Suzuki, M. Iwasaki, N. Koike-Kiriyama, K. Minamino, K. Nakano, H. Mukaide, A. Shigematsu, Y. Kiyozuka, et al. Allogeneic Intrabone Marrow-Bone Marrow Transplantation plus Donor Lymphocyte Infusion Suppresses Growth of Colon Cancer Cells Implanted in Skin and Liver of Rats Stem Cells, February 1, 2007; 25(2): 385 - 391. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Takada, M. Inaba, N. Ichioka, Y. Ueda, M. Taira, S. Baba, T. Mizokami, X. Wang, H. Hisha, H. Iida, et al. Treatment of Senile Osteoporosis in SAMP6 Mice by Intra-Bone Marrow Injection of Allogeneic Bone Marrow Cells Stem Cells, February 1, 2006; 24(2): 399 - 405. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| STEM CELLS | THE ONCOLOGIST | CME | ALPHAMED PRESS JOURNALS |
