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Corresponding author at: Department of Oral Biology, The Maurice and Gabriela Goldschleger School of Dental Medicine, Tel-Aviv University, Tel-Aviv 69978, Israel. Tel.: +972 3 6406430; fax: +972 3 6409250.
Bone formation is reduced in animals and humans with type 1 diabetes, leading to lower bone mass and inferior osseous healing. Since bone formation greatly depends on the recruitment of osteoblasts from their bone marrow precursors, we tested whether experimental type 1 diabetes in rats diminishes the number of bone marrow osteoprogenitors.
Diabetes was induced by 65 mg/kg streptozotocin and after 4 weeks, femoral bone marrow cells were extracted and cultured. Tibia and femur were frozen for further analysis.
The size of the osteoprogenitor pool in bone marrow of diabetic rats was significantly reduced, as evidenced by (1) lower (∼35%) fraction of adherent stromal cells (at 24 h of culture); (2) lower (20–25%) alkaline phosphatase activity at 10 days of culture; and (3) lower (∼40%) mineralized nodule formation at 21 days of culture. Administration of insulin to hyperglycemic rats normalized glycemia and abrogated most of the decline in ex vivo mineralized nodule formation. Apoptotic cells in tibial bone marrow were more numerous in hyperglycemic rats. Also, the levels of malondialdehyde (indicator of oxidative stress) were significantly elevated in bone marrow of diabetic animals.
Experimental type 1 diabetes diminishes the osteoprogenitor population in bone marrow, possibly due to increased apoptosis via Oxidative Stress. Reduced number of osteoprogenitors is likely to impair osteoblastogenesis, bone formation, and bone healing in diabetic animals.
]. Thus, reduced osteoblastogenesis and bone formation are prominent features of DM.
The actual mechanism of DM-associated osteopenia is yet unclear. Lack of insulin action and the presence of hyperglycemia per se are some obvious possibilities. Recently, attention has been drawn to AGEs (advanced glycation end products) and oxidative stress (OxS, exaggerated production of reactive oxygen species (ROS)) as major mechanisms of many diabetic complications [
]. The increased ROS levels result in cell death, tissue damage and impaired healing. As in many other DM-associated pathologies, OxS has been implicated in the pathogenesis of DM-related loss of bone mass [
] and a lower number of osteoprogenitors within the marrow stroma may impede osteoblast generation and bone formation and will compromise bone mass and healing.
The objective of this study was to test whether type 1 DM in rats, which is known to cause diminished bone formation and lower bone mass, results in a reduced number of osteoprogenitors in bone marrow.
2. Materials and methods
Four-month-old Wistar rats (8–10 per group) were used in all experiments in this study, except where noted otherwise. All animal procedures were approved by the animal use ethics committee of the Faculty of Medicine, Tel-Aviv University.
Chemicals for tissue culture were from Biological Industries (Beit Haemek, Israel), unless otherwise stated. Dexamethasone (DEX), streptozotocin (STZ), ascorbic acid, naphthol AS-MX phosphate, fast red violet B, phosphatase substrate, alkaline buffer solution, silver nitrate, sodium carbonate and formalin were from Sigma–Aldrich (Rehovot, Israel). Beta-glycerophosphate (β-GP) was from Calbiochem (La Jolla, CA, USA). Tissue culture dishes were from Nunc (Rosekilde, Denmark). HbA1c levels were measured with the Glyco-tek column kit from Helena Laboratories (Beaumont, TX, USA). Ketamine chlorhydrate was from Rhone-Mérieux (Lyon, France) and Xylazine from Vitamed (Bat-Yam, Israel). Sustained-release insulin implants were from LinShin (Toronto, ON, Canada). OXI-TEK TBARS assay kit was from Enzo Life Sciences (Lausen, Switzerland) and BCA protein determination kit was from Pierce (Rockford, IL, USA).
2.2 Induction of DM and insulin repletion
Diabetes in rats was induced with a single intra-peritoneal (IP) administration of streptozotocin (65 mg/kg of body weight) diluted in citrate buffer (0.01 M, pH = 4.3). Control animals receive the buffer alone. Animals were given food and water ad libitum and body weight was continuously monitored. Blood glucose level was evaluated at regular intervals using a glucometer (Accu-Check, Roche Diagnostics, Basel, Switzerland) and rats with blood glucose level >250 mg/dL were considered diabetic.
In one of the experiments, STZ-injected animals were treated with insulin via sustained-release implants or with identical implants without insulin (sham). Success of insulin repletion was monitored via blood glucose and HbA1c levels.
2.3 Isolation and enumeration of BMSCs
Four weeks after injection of STZ, rats were sacrificed with an overdose of ketamine chlorhydrate (90 mg/kg) and xylazine (10 mg/kg), followed by asphyxiation with carbon dioxide. Blood was collected from the tail vein for final glucose and HbA1C measurements. Femurs were retrieved and bone marrow was expelled and pooled from both femurs of each animal. Cells were counted with a hemocytometer and seeded in 6-well plates at 2 × 107 cells/well in a medium composed of Minimum Essential Medium-Alpha containing 5.5 mmol/L d-glucose in triplicates for each assay. This medium was supplemented with 13% fetal Calf Serum (FCS) + 2 mM glutamine + 100 μ/mL penicillin + 100 mg/mL streptomycin + 12.5 U/mL Nystatin (basic medium). After 24 h, cultures were washed with PBS to remove non adherent cells and attached cells were collected with 0.25 w/v trypsin/0.02 w/v EDTA, counted and their number calculated as percent of the cells seeded.
2.4 Osteoblastic differentiation
Explanted cells were seeded in 6-well plates as described, in basic culture medium supplemented with 10 mM β-GP + 50 μg/mL ascorbic acid + 10 nM DEX (osteogenic medium). Cultures were washed with PBS after 24 h to remove non-adherent cells and were cultured for 10 or 21 days at 5% CO2 and 37 °C in the same medium, which was changed twice a week. The number of osteoprogenitor cells was evaluated by measuring alkaline phosphatase (ALP) activity and mineralized nodule formation as described below.
2.5 ALP activity
For in situ histochemical assessment, cultures were fixed on day 10 in a 1:1:1.5 solution of 10% formalin/methanol/water for 2 h. Dishes were stained with naphthol AS-MX phosphate and fast red violet B solution and the area of positively stained (purple) nodules (representing CFU-ALP = ALP-positive colony forming units) was determined per dish using an image analysis system (NOVA, BIOQUANT Corporation, Nashville).
For biochemical measurement of ALP activity, cells were washed in PBS on day 10, scraped in 10 mmol/L Tris-HCl buffer (pH 7.6) containing 10 mmol/L MgCl2 and 0.1% Triton X-100, and stored at −20 °C until use. Protein content was measured using the BCA protein determination kit and lysates were incubated with phosphatase substrate and alkaline buffer solution for 15 min at 37 °C and then quenched by addition of 0.2 N NaOH. The resulting optical density was measured at 405 nm using a SpectraMax 190 Microplate reader (Molecular Devices, Sunnyvale, CA). ALP activity was expressed as International Units per milligram protein (IU/mg protein).
2.6 Mineralized nodule formation
On day 21, cultures were washed in PBS, fixed as indicated above and stained with the Von Kossa method [
]. Briefly, cells were washed with distilled water, treated with 5% silver nitrate for 15 min, washed again with distilled water, treated with 5% sodium carbonate in 25% formalin for 5 min and washed with tap water. The area of mineralized nodules (stained black, representing CFU-O = colony forming unit, osteoblastic) was determined per dish using the same image analysis system. Due to the vast number of dishes for BMSC cultures generated from each animal, the experiment described hitherto was divided into 2 similar parts. In each part, values belonging to control (normoglycemic) animals were transformed to 100%, and data from the 2 parts were thus merged.
2.7 In situ measurement of bone marrow cell apoptosis
Tibiae from 4 animals from each group were decalcified with 10% EDTA, embedded in paraffin and sectioned longitudinally at 5 μm. Apoptotic cells were detected using the DeadEnd Colorimetric TUNEL System (Promega, CA) as per the manufacturer's instructions. TUNEL positive cells in bone marrow were counted in the epiphyseal bone marrow in 6–8 adjacent fields under a 10× objective of a Zeiss Axioplan 2 microscope.
2.8 Measurement of lipid peroxidation in bone
Tibiae (5–6 per group) were cleaned from the adhering muscles; the proximal metaphysis was frozen at −70 °C until use (4–6 weeks). Then, thawed bones were homogenized with a polytron® tissue disperser (Kinematica, Luzern, Switzerland) in PBS, and centrifuged at 8000 × g for 8 min at 4 °C to remove cortical bone and epiphyseal cartilage. The levels of malondialdehyde (MDA), an indicator of lipid peroxidation and hence tissue oxidative stress, were determined in supernatants by the thiobarbituric acid-reacting substances (TBARS) assay (OXI-TEK kit) according to the manufacturer's instructions. The intensity of fluorescence was read using a SpectraMax M5 Microplate reader (Molecular Devices) at 530 nm excitation and 550 nm emission. Protein content was measured using BCA protein determination kit. Values of TBARS fluorescence were expressed as nmol MDA per mg of protein.
2.9 Statistical analysis
Means and standard deviations were calculated for all parameters and analyzed with non-paired t tests using the SPSS (IBM) software.
Administration of STZ successfully induced diabetes in all animals, as evidenced by increased mean blood glucose at sacrifice (588.2 ± 19.3 mg/dL vs. 128.3 ± 29.7 mg/dL in control rats (P < 0.001)). HbA1c levels were elevated accordingly (11.54 ± 1.33% (102 mmol/mol) vs. 5.25 ± 0.96% (34 mmol/mol), respectively, P < 0.001).
The size of the osteoprogenitor pool in bone marrow was estimated by 3 parameters: the relative number (%) of adherent (stromal) cells (CFU-F), the extent of CFU-ALP (ALP-positive colonies) and the extent of CFU-O (mineralized tissue forming colonies). Fig. 1 shows that the fraction of stromal bone marrow cells is significantly reduced (∼35% lower) in hyperglycemic rats compared with normoglycemic animals. When these cells were induced to differentiate into osteoblasts for 10 days, the area of CFU-ALP was significantly (>20%) lower in hyperglycemic, compared with normoglycemic animals (Fig. 2A ). In support of this finding, we used an alternative biochemical method to measure ALP activity in BMSC cultures and found it to be significantly (>20%) lower in hyperglycemic rats (Fig. 2B). When osteoblastic differentiation was induced for 21 days, and mineralized bone-like tissue was formed in culture, the extent of CFU-O (Von-Kossa positive colonies) was significantly (∼40%) smaller in hyperglycemic animals (Fig. 3A ). These data support the notion that STZ-induced hyperglycemia is associated with a reduction in the osteoprogenitor population in bone marrow.
Administration of insulin to STZ-injected animals via sustained-release implants normalized glycemia (i.e. returned blood glucose and HbA1C levels to normal values (data not shown) and abrogated most of the decline in CFU-O colonies (Fig. 3B).
In a follow-up experiment, we found that apoptotic cells were more numerous (>2-fold) in the tibial bone marrow of hyperglycemic, compared with normoglycemic rats (Fig. 4).
Finally, to test the possible involvement of OxS in the STZ-induced reduction in the bone marrow osteoprogenitor population, the levels of malondialdehyde (MDA), an indicator of lipid peroxidation and hence tissue oxidative stress, were found to be significantly (∼35%) elevated in the proximal tibial metaphysis (which comprises mainly of bone marrow) of diabetic, compared with normoglycemic, animals (Fig. 5).
Thus, bone marrow of hyperglycemic rats contains fewer osteoprogenitors, a higher number of apoptotic cells and increased levels of an oxidative stress biomarker.
The animal model we used, i.e. 4 weeks post-diabetes induction with STZ in rats, has been repeatedly reported to result in significant bone loss [
] is likely to be the cause of osteopenia. This suggests that a decrease in osteoblast number and/or activity is the major mechanism for the DM-related reduction in bone formation. While systemic bone remodeling requires constant recruitment of new osteoblasts, trabecular bone formation in response to “acute” healing models is even more dependent on rapid recruitment of osteoblasts from their stromal precursors. Thus, according to our working hypothesis, it is not surprising that bone formation in response to femoral fractures [
Taken together, these studies imply that the decreased bone formation in type 1 DM may stem from deficit in the recruitment of osteoblasts from their marrow osteoprogenitors. Our results in this study, showing reduced numbers of osteoprogenitor cells in bone marrow of hyperglycemic rats (also seen in diabetic mice [
]) could point to this direction. Repletion of insulin in diabetic animals in this study normalized glycemia and abrogated most of the decline in ex vivo mineralized nodule formation, suggesting causal relationship between the diabetic condition and decreased OP number within the bone marrow. Our finding of reduced size of the OP pool four weeks after induction of DM agrees well with many reports documenting reduced bone formation as early as 4 weeks after injection of STZ [
], suggesting a reciprocal relationship between adiposity and bone formation. Therefore, our hypothesis that diabetes reduces the number of osteoprogenitors in bone marrow, certainly fits within this paradigm. Interestingly, metformin, a well-known anti-diabetic drug, exerts concomitant pro-osteogenic as well as anti-adipogenic effects on rat mesenchymal stem cells [
Regarding the mechanism for fewer osteoprogenitors in bone marrow, our data of increased number of apoptotic cells in bone marrow of diabetic rats suggests (although it does not prove definitively) that apoptosis of BMSCs/OPs is a likely explanation for the reduction in their number, induced by STZ. There are many studies showing that diabetic conditions have detrimental effects (including apoptosis) on osteoblastic cells in vivo (e.g. [
]. Although very little is known about the possible apoptogenic effect of the diabetic conditions on osteoprogenitors within the bone marrow, this possibility seems likely since AGEs and high concentrations of glucose in vitro inhibit the proliferation and/or osteogenic differentiation of mesenchymal stem cells [
]. In clear support for our hypothesis that diabetic conditions induce apoptosis of osteoprogenitors, we recently demonstrated directly that AGE-BSA increases apoptosis of rat BMSCs in vitro 2- to 3-fold in a caspase-dependent manner (Weinberg et al., [
The most prominent mechanisms proposed for the impairment of bone formation in diabetes include hyperglycemia per se, lack of insulin signaling, accumulation of AGEs and oxidative stress (OxS). Recently, mounting evidence focuses on the latter two as plausible mechanisms to many of the DM-associated pathologies such as nephropathy, retinopathy, neuropathy, and impaired dermal healing [
Specifically, the search for the intracellular pathways in which diabetic conditions induce apoptotic cell loss in various tissues, has highlighted the participation of oxidative stress (OxS), which may be associated with the pathogenesis of diabetic bone disorder. In vitro data confirm that OxS can cause death of many cell types [
]. We found that the levels of MDA, an indicator of tissue OxS, were significantly elevated in bone marrow of diabetic, compared with normoglycemic animals, suggesting that the local involvement of OxS is a strong possible explanation for osteoprogenitor apoptosis and reduced bone formation in DM. Our finding is in agreement with those of Mordwinkin et al. [
], who documented increased ROS (reactive oxygen species) levels in bone marrow of diabetic mice. In clear support for our hypothesis that OxS is involved in apoptosis of osteoprogenitors in diabetes, we recently demonstrated directly that rat BMSC apoptosis induced by AGE-BSA involves excessive ROS production and is inhibited by the antioxidant NAC (N-acetylcysteine) (Weinberg et al., [
This study reports that streptozotocin-induced type 1 diabetes in rats results in a diminution in the size of the osteoprogenitor pool in bone marrow, possibly due to oxidative stress-mediated apoptosis. This detrimental effect of hyperglycemia on bone marrow osteoprogenitors could help explain the reduction in bone formation and bone mass and healing, which are constant features of this disease in humans and animals.
Conflict of interest
The authors declare that they have no conflict of interest.
This work was performed in partial fulfillment of the requirements for a Ph.D. degree of E. Weinberg, Sackler Faculty of Medicine, Tel Aviv University, Israel. This study was supported by grant no. 4824 from the Chief Scientist Office of the Ministry of Health, Government of Israel .