Intermittent parathyroid hormone 1–34 (teriparatide) is the N-fragment terminal of the intact hormone, currently in clinical use to treat osteoporosis. Unlike anti-catabolic agents such as bisphosphonates, PTH 1–34 not only affects the osteoclast, but also up regulates bone formation via both modelling and remodelling mechanisms. The actions of iPTH on mesenchymal stem cell differentiation (MSCs) may underpin a further method in the treatment of osteoporosis specifically, and for fracture healing in general. Stem cells from older female osteoporotic animals have reduced activity and poorer osteogenic potential; additionally, their migration to and retention at sites of increased bone turnover are reduced in comparison to cells from younger animals. The aim of this study was to isolate bone marrow derived MSCs from both young Wild Type (WT) and ovarectomized senile (OVX) rats, then to investigate and compare the effect of pulsatile and continuous PTH administration on migration to SDF-1, proliferation and osteogenic differentiation. MSCs were harvested from the femora of 6–9week Wistar rats, and from 10–13month ovarectomized rats with established osteopenia. Cells were cultured with 25, 50 and 100nmMol of PTH 1–34 added to osteogenic media either continuously or in a pulsatile fashion for 6 hours in every 72hour cycle. ALP and Alizarin Red were used to assess the optimal concentration of PTH for osteogenic differentiation. Subsequently, proliferation was assessed with Alamar Blue and cells were seeded in a Boyden chamber to quantify the migration to SDF-1. As the data was parametric a student t-test was used to analyse results, and a p value < 0.05 was considered significant. ALP and Alizarin Red parameters were significantly increased for both WT and OVX groups at 50nmMol of pulsatile PTH in comparison to groups cultured in 25 or 100nmMol. Continuous administration at all concentrations led to reduced calcium phosphate deposition by day 21 in all groups. Interestingly, in comparison to cells cultured in osteogenic media, 50nmMol of pulsatile PTH lead to statistically significant higher ALP and Alizarin Red measurements up to day 10 and 14 respectively in WT cells, and days 10 and 21 in OVX cells. The proliferation rate normalised against DNA was similar for both OVX and WT rats at all-time points. PTH administration did not effect cell proliferation in any group. WT MSCs not only had improved osteogenic differentiation, but also showed increased migration to SDF-1 in comparison to OVX groups. Pulsatile PTH led to further increases in migration of both OVX and WT cells. Intermittent PTH increases the osteogenic diffrentiation and migration of MSCs from both young and ovarectomised rats, though importantly this effect is not dose dependent. Ultimately, the role of PTH 1–34 on MSCs may lead to improved bone formation and cell homing capacity-particularly in the context of osteoporosis.
Osteoporosis is characterised by an uncoupling of bone formation and resorption resulting in a net reduction in bone density. Stem cells derived from bone marrow in osteoporotic patients typically contain more adipocytes,. Intermittent Parathyroid hormone (iPTH), has been shown to cause the preferential differentiation of mesenchymal stem cells (MSCs) to osteoblasts. We isolated rat bone marrow derived MSCs, investigating the effect of iPTH on adipocyte differentiation. MSCs were harvested from the femora of 6–10week oldWT rats and cultured to induce adipogenesis for 21 days. Subsequently, cells were continually cultured in adipogenic media, osteogenic media or in osteogenic media supplemented with PTH 1–34 either continuously or intermittently for 6hours in every 72hour cycle. ALP and Alizarin Red assessed osteogenic differentiation, and Oil Red O used to assess intracellular microdroplet formation. A student t-test was used to analyse results, and a p value<0.05 considered significant. Quantitatively measurements of Alizarin Red staining significantly increased in all adipocytes grown in osteogenic media compared to the cells continually cultured in adipogenic media. Calcium phosphate deposition continued to increase significantly in these groups up to day 14. At day 14, Alizarin Red staining from cells cultured in iPTH were significantly higher than osteogenic media alone. ALP expression was significantly higher for cells cultured in osteogenic media and iPTH compared to adipogenic media at days 3–14. Expression peaked at day 7, at this timepoint cells cultured in iPTH expressed significantly more ALP than other groups. Oil Red O measurements were significantly reduced from days 7–14 for all osteogenic groups, this significance was greatest for the iPTH group at day 7. iPTH increased the transdifferentiation of adipocytes derived from MSCs into osteoblasts, this effect was most significant after 7 days. Ultimately, the role of iPTH on adipocytes may lead to improved bone formation with many orthopaedic applications.
The current treatment for osteoporosis such as bisphosphonates inhibits the catabolic activity of osteoclasts and subsequent bone resorption, but does not increase bone formation. There is therefore interest in using anabolic factors such as stem cells to augment fracture repair. The poor bone formation in postmenopausal women could be due to poor retention and function of Mesenchymal stem cells (MSCs) resulting into delayed unions. Another factor associated with fracture healing is the retention and migration of stem cells to the site of injury (1–3). The aim of this study was to isolate stem cells from osteopenic rats and investigate and compare the CD marker expression, proliferation, migration, osteogenic and adipogenic differentiation. The hypothesis of this study is that the migration of MSCs from young, adult and ovariectomised (OVX) rats will have different proliferation, differentiation and migratory abilities. Ovariectomy was performed in 6–9 month old Wistar rats and osteopenia developed over a 4 month post-op period. MSCs were harvested from the femora of young, adult and osteopenic Wistar rats. Proliferation of the these MSCs from the three group of rats was measured using Alamar blue, osteogenic differentiation was measured using ALP expression at day 0, 7, 14 and 21 and alizarin red at day 21. Adipogenic differentiation was measured at day 7, 14 and 21 using Oil red O. Cells were incubated in Boyden chambers to quantify their migration towards SDF1. For analysis, the number of cells migrating across the membrane was expressed as a percentage of the cells remaining on the upper membrane surface. Data was analysed using a Student t-test where p values < 0.05 were considered significant. The stem cells from all 3 groups of rats expressed on average the same amount of CD29 (>90%), CD90 (>96%), CD34 (<5%) and CD45 (approx 10%). The proliferation rate measured by Alamar blue normalised against DNA was also similar at day 3, 7, 10 and 14. However, interestingly the migration and differentiation ability was significantly different between the MSCs from the 3 groups of rats. The young MSCs were not only better at differentiating into bone and fat as well, but they also migrated significantly more towards SDF1. The migration of SDF-1 doubled with young rats compared to the adult rats (p = 0.023) and it was four times higher when compared to cells isolated from OVX rats (p = 0.013). MSCs from OVX rats are similar to MSCs from young rats. However when induced to turn into bone, fat and migrate towards SDF1, young MSCs are significantly more responsive than MSCs from OVX and adult control rats. The poor homing ability and differentiation of the stem cells and their retention may result in a reduction in bone formation leading to delayed union in fractures of osteoporotic patients(4).
Intermittently administered parathyroid hormone (PTH 1-34) has been shown to promote bone formation in both human and animal studies. The hormone and its analogues stimulate both bone formation and resorption, and as such at low doses are now in clinical use for the treatment of severe osteoporosis. By varying the duration of exposure, parathyroid hormone can modulate genes leading to increased bone formation within a so-called ‘anabolic window’. The osteogenic mechanisms involved are multiple, affecting the stimulation of osteoprogenitor cells, osteoblasts, osteocytes and the stem cell niche, and ultimately leading to increased osteoblast activation, reduced osteoblast apoptosis, upregulation of Wnt/β-catenin signalling, increased stem cell mobilisation, and mediation of the RANKL/OPG pathway. Ongoing investigation into their effect on bone formation through ‘coupled’ and ‘uncoupled’ mechanisms further underlines the impact of intermittent PTH on both cortical and cancellous bone. Given the principally catabolic actions of continuous PTH, this article reviews the skeletal actions of intermittent PTH 1-34 and the mechanisms underlying its effect.
There is increasing interest in using anabolic factors such as stem cells to augment fragility fracture repair. One of the factors associated with fracture healing is the retention and migration of stem cells to the site of injury (1–3). The aim of this study was to isolate stem cells from osteopenic rats and investigate and compare the CD marker expression, proliferation, migration, osteogenic and adipogenic differentiation. The hypothesis of this study is that the migration of MSCs from young, adult and ovariectomised (OVX) rats will have different proliferation, differentiation and migratory abilities. CD marker expression of MSCs from young, adult and osteopenic rats was measured using flow cytometry. Proliferation, osteogenic differentiation and adipogenic differentiation was measured using Alamar Blue, ALP expression and Alizari n Red and quantitative Oil red O respectively. Cells were incubated in Boyden chambers to quantify their migration towards SDF1. Data was analysed using a Student t-test where p values < 0.05 were considered significant. MSCs from all 3 groups of rats had similar proliferation and expression of CD29(>90%), CD90(>96%), CD34(<5%) and CD45(approx 10%). The proliferation rate was also similar. However, interestingly the migration and differentiation ability was significantly different between the MSCs from the 3 groups of rats. The young MSCs were not only better at differentiating into bone and fat, but they also migrated significantly more towards SDF1. MSCs from OVX rats are similar to MSCs from young rats. However when induced to turn into bone, fat and migrate towards SDF1, young MSCs are significantly more responsive than MSCs from OVX and adult control rats. The poor homing ability and differentiation of the stem cells and their retention may result in a reduction in bone formation leading to delayed union in fractures of osteoporotic patients(4).
