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Department of Internal Medicine (S.W.C., E.S.O., K.W.O., J.M.L., K.H.Y., M.I.K., B.Y.C., K.W.L., H.Y.S., S.K.K., C.C.K.) and Hematopoietic Stem Cell Transplantation Center (C.C.K.), The Catholic University of Korea, College of Medicine; and Kangbuk Samsung Hospital (W.Y.L.), Sungkyunkwan University School of Medicine, Seoul, Korea
Address all correspondence and requests for reprints to: Moo-Il Kang, M.D., Division of Endocrinology and Metabolism, Department of Internal Medicine, St. Mary’s Hospital, The Catholic University of Korea, 62 Yoido-dong Youngdeungpo-Gu, Seoul 150-010, Korea. E-mail: mikang{at}cmc.cuk.ac.kr
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Abstract |
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In a clinical study of 67 patients undergoing BMT, the mean serum carboxy-terminal cross-linked telopeptide of type I collagen increased progressively until 4 wk after BMT. Thereafter, it began to decrease and reached basal values after 1 yr. Serum osteocalcin decreased progressively until 3 wk after BMT and reached basal values after 3 months. One year after BMT, lumbar spine BMD had decreased by 3.3% (P < 0.05), and total proximal femoral BMD had decreased by 8.9% (P < 0.001).
For the ex vivo study, bone marrow was obtained from healthy donors (n = 7) and transplant recipients (n = 7). Then, mononuclear cells including marrow stromal cells were isolated and cultured to osteoblastic lineage. Alkaline phosphatase activities of each group were measured by the time course of secondary culture, and the mineralizing potentials were compared between the two groups. Cells cultured in our system showed characteristics of osteoblast-like cells differentiated from marrow stromal cells. They were initially in a fibroblastic-like spindle shape and became cuboidal with the formation of nodules that were later confluent. The cells were stained to both alkaline phosphatase histochemistry and Von Kossa histochemistry, demonstrating that these cells were of osteoblastic lineage differentiating from marrow stromal cells. The mean time required for the near-confluence in the primary culture was 15 and 22.9 d in healthy donors and transplant recipients, respectively (P = 0.003). Alkaline phosphatase activity was significantly lower in the bone marrow recipients than in the healthy donors at d 7 and 10 of the secondary cultures. The period at which peak activity of alkaline phosphatase was reached was also delayed in the osteoblasts derived from BMT recipient bone marrow compared with those of healthy donors. Using Von Kossa histochemistry, much more mineralization was observed in osteoblasts of healthy donors than those of BMT recipients. After BMT, although the peripheral mononuclear cells in the recipients were of donor origin, the bone marrow stromal cells were of recipient origin according to the PCR analysis using YNZ 22 mini-satellite probe.
In conclusion, the differentiation of bone marrow stromal cells into osteoblasts was impaired after BMT, and this might contribute to post-BMT bone loss.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Previous studies have demonstrated a 30–35% increase in lumbar fracture after heart and liver transplantation and an 8–17% loss in bone mineral density (BMD) within 1 yr after kidney transplantation (2, 3, 4, 5, 6). However, little is known about the effect of BMT on the skeletal system. Kelly et al. (7) reported a significant decrease in bone density at the lumbar spine and femoral neck in a group of subjects after allogeneic BMT. Recently, Ebeling et al. (8) also found that postallogeneic BMT patients lost 11.7% of femoral neck BMD. Michelson et al. (9) reviewed pre- and posttransplantation iliac crest bone biopsies of lymphoma patients undergoing BMT that was performed after they received chemotherapy and total body irradiation (TBI), finding that marrow fibrosis was significantly increased and osteocyte viability was decreased.
There exist osteogenic precursor cells, that is, bone marrow stromal cells as well as hematopoietic stem cells in the normal bone marrow. Bone marrow stromal cells play an important role in the formation of the bone marrow microenvironment that influences hematopoiesis and immunological reconstitution by producing various cytokines. It has been generally reported that hematopoietic cells are of donor origin after BMT. However, the origin of bone marrow stromal cells is controversial. Although the origin of marrow stromal cells after allogeneic BMT is controversial, many studies indicate that bone marrow stromal cells are of host origin (10, 11). When we suppose that marrow stromal cells are derived from the patients themselves, osteogenic differentiation may be affected by high-dose chemotherapy and total body irradiation that have been administered before BMT and finally, bone formation may be decreased. Although there are reports that detail the differentiation of marrow stromal cells into osteoblasts by using normal bone marrow (12, 13), there has been no study concerning the differentiation of bone marrow stromal cells into the osteoblasts in BMT setting.
The purpose of the present study was to compare the osteogenic potential of bone marrow-derived stromal cells from healthy bone marrow donor and recipient. Using a DNA fingerprinting method, we also analyzed the origin of bone marrow stromal cells that were harvested 3–4 wk after BMT. In a prospective clinical study, we also observed the changes of bone turnover markers and the degree of bone loss after BMT.
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Materials and Methods |
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We prospectively investigated 67 patients (27 females and 40 males) undergoing BMT (8 autologous and 59 allogeneic) as treatment for hematologic diseases (50 leukemia, 13 severe aplastic anemia, and 4 myelodysplastic syndrome). The patients’ mean age was 30.7 yr (range, 15–47 yr) at the time of BMT. Preparative regimens consisted of several combinations of chemotherapeutics (high-dose cyclophosphamide, procarbazine, busulfan, melphalan, anti-lymphocyte globulin). Thirty-seven patients (15 females and 22 males) received additional TBI (10–13.2 Gy) as a conditioning regimen. This study was approved by the Institutional Review Board of St. Mary’s Hospital (Seoul, Korea), and informed consent was obtained from all participants.
Biochemical markers
Blood was sampled to determine serum levels of calcium, phosphorus, creatinine, total alkaline phosphatase (747 Automatic Analyzer, Hitachi, Tokyo, Japan), osteocalcin, and carboxy-terminal cross-linked telopeptide of type I collagen (ICTP) before BMT and at 1, 2, 3, 4, and 12 wk, 6 months, and 1 yr after BMT. Blood samples were taken between 0700 and 0900 h after an overnight fast to measure the biochemical markers. Serum creatinine levels were determined using an autoanalyser (747 Automatic Analyzer, Hitachi). Serum osteocalcin (N-tact osteo SP, INCSTAR Corp., Stillwater, MN) and ICTP (Telopeptide ICTP, Orion Diagnostica, Finland) were determined by RIA. The maximum inter- and intra-assay coefficients of variation for the range of concentrations evaluated were 7.7 and 5.4% for osteocalcin and 10.7 and 3.6% for ICTP, respectively.
Bone densitometry
BMD of the lumbar spine (lumbar vertebrae L2–L4) and of the proximal femur (femoral neck, trochanter, Ward’s triangle) was measured by dual-energy x-ray absorptiometry using a Lunar Expert (Lunar Corp., Madison, WI) before BMT and 12 months after BMT. The precision of the method (coefficient of variation) was 1.0% at both locations.
Subjects for the ex vivo study
Details for bone marrow donors and bone marrow recipients included in the ex vivo study are summarized in Table 1
. Three patients with acute myelogenous leukemia, two patients with chronic myelogenous leukemia, one patient with myelodysplastic syndrome, and one patient with acute biphenotypic leukemia underwent BMT. Six patients underwent allogeneic BMT, and one patient underwent unrelated BMT.
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Dexamethasone, ß-glycerophosphate, vitamin K (menadione sodium bisulfite), 
-MEM, naphthol AS-MX phosphate disodium salt, Fast Red TR salt, silver nitrate, p-nitrophenyl phosphate, p-nitrophenol, copper sulfate, and bicinchoninic acid were purchased from Sigma Chemicals (St. Louis, MO). FCS, penicillin-streptomycin, and trypsin/EDTA were obtained from Life Technologies, Inc. (Grand Island, NY). We used a DNA thermal cycler 480 (Perkin-Elmer Corp., Wellesley, MA). YNZ mini-satellite primer was purchased from Bioneer (Cheong Won, Korea).
Culture of human bone marrow stromal cells
Bone marrow was harvested from iliac crest. Mononuclear cells were separated using Ficoll-Hypaque, seeded in culture flasks including -MEM at a density of 4 x 105 cells per milliliter, and incubated at 37 C in a humidified atmosphere containing 95% air and 5% CO2 incubator (14). The medium was composed of 20% heat-inactivated FCS, 100 U/ml penicillin-streptomycin, and 10-8 M vitamin K. The next day, 10-8 M dexamethasone and 50 µg/ml ascorbic acid were added without media change. Subsequently, the culture flasks were left alone without disturbance to promote cell attachment for 4–5 d. After the attachment period, the culture medium was replaced with fresh -MEM including 20% heat-inactivated FCS, 100 U/ml penicillin-streptomycin, 10-8 M vitamin K, 10-8 M dexamethasone, and 50 µg/ml ascorbic acid, and then the cells were fed at a 2-d interval thereafter. At near-confluence, human bone marrow stromal cells were subcultured after 0.25% trypsin/1 mM EDTA digestion and seeded in six-well culture plates at a density of 5 x 104 cells per milliliter. Thereafter, 10-7 M dexamethasone and 10 mM ß-glycerophosphate were added in culture media (14). Alkaline phosphatase activities were measured at 3- to 4-d intervals for more than 2 wk in the secondary culture period. Von Kossa histochemical staining was performed at d 22 in the secondary culture. We also obtained bone marrow specimens from the BMT recipients at 3–4 wk after BMT and proceeded in the same manner.
Alkaline phosphatase and Von Kossa histochemical staining
Alkaline phosphatase histochemistry was performed on d 14 of secondary culture. The medium was removed, and the cell layers were rinsed with 4 C Tris-buffered saline (pH 7.4) and fixed with 4 C 2% paraformaldehyde/0.1 M cacodylic buffer. Then, the medium was incubated with Tris-maleate buffer (pH 8.4) containing 20 mg/ml naphthol AS-MX phosphate disodium salt and 40 mg/ml Fast Red TR salt. After 30 min at 37 C, the cell layers were washed with 0.1 M cacodylic buffer and observed both grossly and with the light microscope.
Using Von Kossa histochemistry, the cultures were stained on d 14 for assessing the mineralized matrix. The medium was removed, and the cell layers were rinsed with 4 C Tris-buffered saline (pH 7.4), fixed with 4 C 2% paraformaldehyde/0.1 M cacodylic buffer, and then stained with 3% silver nitrate. After 15–30 min under the UV light, the cell layers were washed with 0.1 M cacodylic buffer and observed both grossly and with the light microscope.
Alkaline phosphatase activity
Human bone marrow stromal cells in six-well plates were washed three times with PBS. The cell layers were lysed with 0.1% Triton X-100, 1 ml per well. Tris-HCl (1 M, 500 µl), MgCl2 (5 mM, 100 µl), and p-nitrophenyl phosphate (5 mM, 100 µl) were added in the 300 µl lysate. The samples were incubated for 30 min at 37 C. Absorbance was read immediately after incubation at 410 nm in a plate reader (15). BSA was used as a standard for the measurement of protein concentration using bicinchoninic acid method (16). The enzyme activities were expressed as U/mg protein.
DNA fingerprinting
Peripheral blood was obtained from bone marrow donors and recipients before BMT. Heparinized bone marrow samples were obtained from recipients at 3–4 wk after BMT, cultured primarily in the culture plates as previously described until near-confluence, and then subcultured for about 2 wk. Subsequently, attached osteoblast-like cells were separated from the samples using the Ficoll-Hypaque gradient method, and genomic DNA was extracted using a modified salting-out method (17). Extracted DNA was quantified by the spectrophotometer and amplified with YNZ 22 mini-satellite primer. The PCR included early DNA denaturation at 94 C for 5 min, early 5 cycles, late 30 cycles, and elongation at 72 C for 5 min. Each cycle in the early 5 cycles consisted of DNA denaturation at 94 C for 1 min, annealing at 63 C for 1 min, and elongation at 72 C for 6 min. Each cycle in the late 30 cycles consisted of DNA denaturation at 94 C for 30 sec, annealing at 60 C for 30 sec, and elongation at 72 C for 1 min. The sequence of YNZ 22 mini-satellite primers was as follows: 5'-CGAAGAGTGAAGTGCACAGG-3', 5'-CACAGTCTTTATTCTTCAGCG-3'.
Five to 10 µl aliquots of the amplified products were subjected to electrophoresis on 0.8% agarose gel containing ethidium bromide (18).
Statistics
Values are expressed as the mean values ± SEM. Statistical analysis was performed using the t test. A difference of P < 0.05 was regarded as significant.
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Results |
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Following BMT, the marker of bone formation, osteocalcin, progressively decreased and reached its nadir at 3 wk. Thereafter, osteocalcin recovered back to its initial baseline level by 12 months (Fig. 1). The marker of bone resorption, ICTP, progressively increased and reached its maximum at 4 wk. Thereafter, it declined to baseline at 12 months. Serum creatinine levels were in the normal range throughout the entire observation period.
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The mean bone loss in the lumbar spine, calculated as the percentage change from the baseline (1.137 ± 0.020 g/cm2) to the level at 12 months after BMT (1.100 ± 0.022 g/cm2), was 3.3% (P < 0.05). In the proximal femur, bone loss was calculated as 8.9% from the baseline (1.092 ± 0.020 g/cm2) to the level at 12 months after BMT (0.995 ± 0.019 g/cm2; P < 0.001) (Fig. 2).
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Establishment of culture system for human bone marrow stromal cells
The general characteristics of bone marrow donors and recipients are listed in Table 1
. There is no difference in the mean age between the groups. When the mononuclear cells obtained from bone marrow donors were plated in culture flasks, cells began to attach to the plastic surface after a mean 4–5 d. Most of the attached cells exhibited a fibroblast-like spindle shape. These stromal cells proliferated quickly to form colonies and eventually merged to form a confluent multicellular layer at the end of the primary culture (a mean period of 15 d from seeding). Subsequently, cells were subcultured and also proliferated quickly to exhibit a spindle shape. After secondary culture over 2 wk, stromal cells began to mineralize matrix and stained positively for alkaline phosphatase and Von Kossa histochemical activities. These findings indicated that bone marrow stromal cells differentiated into the osteoblastic lineage cells (Fig. 3). In contrast, a mean period of 22.9 d was required for the cultures to reach near-confluence in the bone marrow recipients. It took significantly more time to achieve near-confluence in the bone marrow recipients than in the bone marrow donors (P = 0.003). Stromal cells were stained positively for alkaline phosphatase and Von Kossa histochemical activity in normal bone marrow donors, but mineralization was significantly less in bone marrow recipients than in bone marrow donors (Fig. 4).
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Alkaline phosphatase activity reached a maximal value on d 10 of the secondary culture in bone marrow donors and on d 17 of the secondary culture in bone marrow recipients (Fig. 5). Thus, it showed significant differences in the mean period reaching maximal activity between two groups (P = 0.031). Alkaline phosphatase activity also was lower in bone marrow recipients than in bone marrow donors during the whole period of secondary culture and showed a significant difference between the two groups on d 7 and 10 of the secondary culture (P = 0.014 and 0.022, respectively) (Table 2).
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After BMT, peripheral mononuclear cells of recipients were of donor origin. However, bone marrow stromal cells of recipients were of recipient origin after BMT according to the PCR analysis using YNZ 22 mini-satellite probe (Fig. 6).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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It is interesting that skeletal site-selective differences between lumbar spine and femoral BMD change are found and their exact mechanism needs to be elucidated through additional basic studies. Similar results were found in other studies on post-BMT bone metabolism (8). It may be due to the differences in the tissue expression of several proteins related in bone metabolism such as BMP-2, several growth factors, and their receptors. Similarly, the BMD changes in response to therapeutic agent (e.g. HRT, alendronate) are different between spine and femur, but the exact mechanism is currently not well known. The exact mechanism should be elucidated in further studies.
Our clinical study demonstrates that bone resorption increased progressively and bone formation decreased during the early period after BMT, and bone loss was significant after BMT in both lumbar spine and total proximal femur. Our results on the change of bone turnover markers are exactly the same as those of Carlson et al. (19), obtained during the first 12 wk after BMT. These changes of bone formation and resorption may play a possible role in post-BMT bone loss. It is difficult to weigh the effects of bone formation and bone loss on post-BMT bone loss, and we think both are important. We agree that biochemical markers selected in clinical study might not be ideal, and bone- specific alkaline phosphatase was not assayed. However, both osteocalcin and serum ICTP are widely used as markers of bone formation and bone resorption, especially in the studies of transplant-related bone metabolism. We previously reported short-term changes of bone mineral metabolism and BMD in 31 patients undergoing BMT and extended the study to include more patients. Our results in this prospective study also are consistent with a previous report (1). Among the possible causes for the inhibition of osteoblast function, shown by the decrease of osteocalcin during the first few weeks after BMT, one hypothesis is that a substantial number of osteoprogenitor cells are probably damaged by the myeloablative therapy. We intended to prove this hypothesis by using human bone marrow cell culture and demonstrated that osteoblastic activities of marrow stromal cells from BMT recipients were significantly impaired compared with those from healthy donors. We also think that there may be additional explanations for the low level of osteoblastic cellular activity in recipients, including GVHD, poor nutritional status posttransplant, the use of immunosuppressive therapy, and the possible loss of stromal cells during the pretransplant active phase of diseases.
The bone marrow microenvironment is a complex network of cells and extracellular matrix that maintains the hematopoietic system throughout the life of the individual. Bone marrow stromal cells have long been recognized as the source of osteoprogenitor cells. Pluripotential stromal cells in the bone marrow can differentiate into fibroblasts, adipocytes, chondrocytes, or osteocytes under the appropriate conditions (21, 22, 23). Bone marrow stromal cells play an important role in the formation of the bone marrow microenvironment that influences hemopoiesis and immunological reconstitution (24). We induced the differentiation of bone marrow stromal cells to osteoblastic lineage by culturing the bone marrow cells in an osteogenic media including dexamethasone and serum. A mean period of 15 d was required for bone marrow stromal cells obtained from bone marrow donors to reach near-confluence, similar to the report of Majors et al. (23), whereas a mean of 22.9 d was required for those from bone marrow recipients. In this study, cultured marrow stromal cells that were grown in osteogenic medium secreted high concentrations of alkaline phosphatase and were stained to both alkaline phosphatase and Von Kossa histochemistry, all of which demonstrated that these cells were of osteoblastic lineage. We found that alkaline phosphatase activity was significantly lower at d 7 and 10 of secondary culture and the reaching of peak activity was significantly delayed in the bone marrow recipients compared with that of bone marrow donors. These findings indicate that the proliferation and differentiation of bone marrow stromal cells are slower in bone marrow recipients than in bone marrow donors. If we had performed cell staining for osteocalcin and measured calcium levels in the cell layer for the evaluation of mineralization, this ex vivo study could be more complete. Little has been studied concerning the factors that affect the differentiation of bone marrow stromal cells into osteoblastic lineage cells in conditions such as BMT. It is probable that the combined effects of previously described factors (immunosuppressants, hypogonadism, TBI, etc.) may be related to bone loss after BMT.
It is well known that hematopoietic cells grafted after BMT are of donor origin. However, the origin of the marrow stroma is uncertain. Most studies examining marrow stroma after BMT generally indicate that stroma is of host origin (10, 11). Although the origin of bone marrow-derived fibroblastic stromal cells (BMF) after allo-BMT is difficult to determine, Perkins and Fleischmann (25) reported that stromal endothelial cells and adipocytes were of host origin, whereas the macrophage component of adherent layers was of donor origin. In contrast, Keating et al. (26) showed that the stromal cells from long-term marrow culture generated from patients undergoing allogeneic marrow transplantation became progressively donor-derived after a marrow transplant. Several murine studies support the contention of a donor origin for marrow stroma after a transplant (27). Keating et al. (26) explained that these conflicting results may be caused by differences in culture conditions, by different definitions of marrow stromal cells, or by different doses of mature marrow stromal and/or stromal precursor cells in the graft. Agematsu and Nakahori (28) showed that bone marrow stromal cells were of the recipient origin after allogeneic BMT by Southern blot analysis and PCR method. They reported that the recipient’s BMF might survive under conventional BMT conditioning and engraftment of the donor’s BMF precursors might not occur. In our study, as demonstrated by the result of DNA fingerprinting, long-term cultured bone marrow stromal cells showed the recipient’s DNA patterns in all of seven patients. These data indicate that bone marrow stromal cells are of recipient origin. Therefore, bone marrow stromal cells originated from bone marrow recipients may be damaged by pretransplant conditioning chemotherapy, TBI, steroid, and cyclosporine used for the prevention of posttransplant GVHD and may be affected for differentiation into the osteoblastic lineage cells. Such inhibition for osteoblastic differentiation may cause the decrease in bone formation.
In this report, we cultured bone marrow stromal cells obtained from bone marrow donors and bone marrow recipients, comparing the differentiation patterns between two groups. According to our results, bone marrow stromal cells were of recipient origin, and osteoblastic differentiation from bone marrow stromal cells was delayed in the bone marrow recipients. Our clinical study also implies that bone loss is a clinically significant problem after BMT. The rapid impairment of bone formation and the increase in bone resorption, as mirrored by the biochemical markers in this study, may play a role in post-BMT bone loss. On the basis of the above results, further studies are needed on measures to enhance osteoblastic differentiation and proliferation and for the prevention and treatment of bone mineral loss that can occur after BMT.
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Acknowledgments |
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Footnotes |
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Part of these results were presented in abstract form at the 21st Annual Meeting of the American Society for Bone and Mineral Research, St. Louis, Missouri, 1999.
Abbreviations: BMD, Bone mineral density; BMF, bone marrow- derived fibroblastic stromal cells; BMT, bone marrow transplantation; GVHD, graft vs. host disease; ICTP, carboxy-terminal cross-linked telopeptide of type I collagen; TBI, total body irradiation.
Received April 12, 2001.
Accepted September 24, 2001.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, P < 0.01 against the basal value.
