A growing number of fractures progress to delayed or nonunion, causing significant morbidity and socioeconomic impact. Localized delivery of stem cells and subcutaneous parathyroid hormone (PTH) has been shown individually to accelerate bony regeneration. This study aimed to combine the therapies with the aim of upregulating fracture healing. A 1.5 mm femoral osteotomy (delayed union model) was created in 48 female juvenile Wistar rats, aged six to nine months, and stabilized using an external fixator. At day 0, animals were treated with intrafracture injections of 1 × 106 cells/kg bone marrow mesenchymal stem cells (MSCs) suspended in fibrin, daily subcutaneous injections of high (100 μg/kg) or low (25 μg/kg) dose PTH 1-34, or a combination of PTH and MSCs. A group with an empty gap served as a control. Five weeks post-surgery, the femur was excised for radiological, histomorphometric, micro-CT, and mechanical analysis.Aims
Methods
For cementless implants, stability is initially attained by an interference fit into the bone and osteo-integration may be encouraged by coating the implant with bioactive substances. Blood based autologous glue provides an easy, cost-effective way of obtaining high concentrations of growth factors for tissue healing and regeneration with the intention of spraying it onto the implant surface during surgery. The aim of this study was to incorporate nucleated cells from autologous bone marrow (BM) aspirate into gels made from the patient’s own blood, and to investigate the effects of incorporating three different concentrations of platelet rich plasma (PRP) on the proliferation and viability of the cells in the gel. The autologous blood glue (ABG) that constituted 1.25, 2.5, and 5 times concentration PRP were made with and without equal volumes of BM nucleated cells. Proliferation, morphology, and viability of the cells in the glue was measured at days 7 and 14 and compared to cells seeded in fibrin glue.Aims
Methods
Mesenchymal stem cells (MSCs) are of growing interest in terms of bone regeneration. Most preclinical trials utilize bone-marrow-derived mesenchymal stem cells (bMSCs), although this is not without isolation and expansion difficulties. The aim of this study was: to compare the characteristics of bMSCs and adipose-derived mesenchymal stem cells (AdMSCs) from juvenile, adult, and ovarectomized (OVX) rats; and to assess the effect of human parathyroid hormone (hPTH) 1-34 on their osteogenic potential and migration to stromal cell-derived factor-1 (SDF-1). Cells were isolated from the adipose and bone marrow of juvenile, adult, and previously OVX Wistar rats, and were characterized with flow cytometry, proliferation assays, osteogenic and adipogenic differentiation, and migration to SDF-1. Experiments were repeated with and without intermittent hPTH 1-34.Objectives
Methods
This study aimed to assess the effect of age and osteoporosis on the proliferative and differentiating capacity of bone-marrow-derived mesenchymal stem cells (MSCs) in female rats. We also discuss the role of these factors on expression and migration of cells along the C-X-C chemokine receptor type 4 (CXCR-4) / stromal derived factor 1 (SDF-1) axis. Mesenchymal stem cells were harvested from the femora of young, adult, and osteopenic Wistar rats. Cluster of differentiation (CD) marker and CXCR-4 expression was measured using flow cytometry. Cellular proliferation was measured using Alamar Blue, osteogenic differentiation was measured using alkaline phosphatase expression and alizarin red production, and adipogenic differentiation was measured using Oil red O. Cells were incubated in Boyden chambers to quantify their migration towards SDF-1. Data was analyzed using a Student’s Objectives
Methods
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.
Osteoporosis is characterised by an uncoupling of bone formation and resorption resulting in net resorption. 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 (Figure 2). 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.
Intermittent parathyroid hormone (iPTH 1–34) increases bone formation via modelling and remodelling mechanisms and as such is used to treat osteoporosis. The actions of iPTH on mesenchymal stem cell (MSCs) may underpin a further treatment option. We isolated bone marrow derived MSCs from young (WT) and ovarectomized senile (OVX) rats, investigating the effect of intermittent and continuous PTH administration on migration to SDF-1, proliferation and osteogenic differentiation. MSCs were harvested from the femora of 6–10week old WT rats and 10–13month old OVX rats. Cells were cultured with 25,50 and 100nmMol of PTH 1–34 added to osteogenic media either continuously or intermittently for 6hours in every 72hour cycle. ALP and Alizarin Red assessed osteogenic differentiation, and Alamar Blue- proliferation. Cells were seeded in a Boyden chamber to quantify SDF-1 migration. A student t-test was used to analyse results, and a p value<0.05 considered significant. ALP and Alizarin Red were significantly increased for WT and OVX groups at 50nmMol of iPTH. Continuous administration at all concentrations reduced calcium phosphate deposition by day 21 in all groups. In comparison to cells cultured in osteogenic media, 50nmMol of iPTH led to significantly higher ALP and Alizarin Red measurements up to days 10 and 7 respectively (figure 1). There was no change in proliferation between the groups, and PTH had no effect (figure 2.) WT MSCs not only had improved osteogenic differentiation, but also showed increased migration to SDF-1 in comparison to OVX groups. iPTH led to further increases in migration of both OVX and WT cells. iPTH increases the osteogenic differentiation and migration of MSCs from both young and ovarectomised rats, though this effect is not dose dependent. Ultimately, the role of iPTH on MSCs may lead to improved bone formation and cell homing capacity-particularly in the context of osteoporosis.
