Periprosthetic joint infection (PJI) is a debilitating condition with a substantial socioeconomic burden. A novel autologous blood glue (ABG) has been developed, which can be prepared during surgery and sprayed onto prostheses at the time of implantation. The ABG can potentially provide an antimicrobial coating which will be effective in preventing PJI, not only by providing a physical barrier but also by eluting a well-known antibiotic. Hence, this study aimed to assess the antimicrobial effectiveness of ABG when impregnated with gentamicin and stem cells. Gentamicin elution from the ABG matrix was analyzed and quantified in a time-dependent manner. The combined efficiency of gentamicin and ABG as an anti-biofilm coating was investigated on titanium disks.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
Limb salvage in bone tumour patients replaces the bone with massive segmental prostheses where achieving bone integration at the shoulder of the implant through extracortical bone growth has been shown to prevent loosening. This study investigates the effect of multidrug chemotherapy on extracortical bone growth and early radiological signs of aseptic loosening in patients with massive distal femoral prostheses. A retrospective radiological analysis was performed on adult patients with distal femoral arthroplasties. In all, 16 patients were included in the chemotherapy group with 18 patients in the non-chemotherapy control group. Annual radiographs were analyzed for three years postoperatively. Dimensions of the bony pedicle, osseointegration of the hydroxyapatite (HA) collar surface, bone resorption at the implant shoulder, and radiolucent line (RLL) formation around the cemented component were analyzed.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
During remodelling, osteoclasts produce discrete bone cavities filled with bone and this is associated with the dimensions of the cavity. The aim of this study is to investigate the effect of pores of similar size to those produced by osteoclasts on the morphology, proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) in vitro. The hypothesis is that a porous surface similar in morphology to a bone surface prepared by osteoclasts will increase cell proliferation and osteogenic differentiation of MSCs. Sheep BMSCs were seeded onto plain titanium surfaces and 100µm, 250µm and 500µm discrete pores surfaces. Cell metabolic activity was investigated using Presto Blue on days 3, 7 and 10. Bone mineralisation was quantified by Alizarin red staining at days 3, 7 and 14. Cell morphology was observed by scanning electron microscopy (SEM). Data was statistically analysed using one-way analysis of variance and a Bonferroni correction method. Cells on porous discs had a three dimensional phenotype and aligned on the circumference of each pore. Metabolic activity was significantly higher by day 10 on plain discs compared to all porous discs. Bone mineralization was significantly higher on 100µm pores by day 3 (0.545mM±0.66; p=0.047) than plain discs and significantly higher on both 100µm and 250µm pores by day 7(p=0.000 and p=0.005) than plain discs. Substantial mineralised bone matrix was found on 100µm discs without being treated with osteogenic supplements, compared to other control disc types (p=0.043, p=0.003, p=0.000). The different topographies altered cell behaviour and migration.100µm pores demonstrated earlier and enhanced bone mineralisation even in the absence of osteogenic supplements. This pore size is aligned to the size of individual resorption bays that osteoclasts produce on bone surfaces and is considerably lower than the pore sizes used to enhance osteo-integration of implant surfaces.
A significant number of fractures develop non-union. Stem cell therapy may be beneficial in their treatment, however this requires acquisition, culture and delivery of stem cells. Stem cell homing and migration is regulated through SDF-1 and its receptor CXCR4. Studies have demonstrated endogenous mobilisation of different populations of stem and progenitor cells by administering growth factors with a pharmacological antagonist of CXCR4, AMD3100. This may therefore be a means to improve compromised fracture healing. A 1.5mm femoral osteotomy in adult female Wistar rats was stabilised with an external skeletal fixator. After osteotomy, saline/PBS (P) VEGF (V), IGF-1 (I) or GCSF (G) (100ug/kg, 0.5ml/100g i.p.), were administered daily for 4 days. On day 5, a single 5mg/kg i.p. dose of AMD3100 was given. Control group (C) did not receive growth factors or AMD 3100. At 5 weeks, the femur was retrieved and microCT scanned. Compared to group C (n=7), group P (n=5) had a significant increase in bone volume (P=0.01) 8.9±2.2um∧3 (control 4.3±3.1um∧3) and trabecular thickness (P=0.03). Group I (n=6) also had a significant increase in bone volume (P=0.035) 5.1±4.2um∧3 and trabecular thickness 0.062±0.008um (control 0.042±0.01um) (P=0.01). Group V (n=8), showed a non-significant increase in bone volume; 5.22±1.7um∧3 and trabecular thickness 0.048±0.007um. Group G (n=5) showed a significant decrease in bone volume (2.5±2.6um∧3) (P=0.048). AMD3100 alone and IgF1-AMD3100, showed the greatest increase in bone formation, presumably through mobilisation of beneficial combinations of stem and progenitor cells. GCSF-AMD3100, which is expected to mobilise hematopoietic progenitors inhibited bone healing.
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
Cellular movement and relocalisation are important for many physiologic properties. Local mesenchymal stem cells (MSCs) from injured tissues and circulating MSCs aid in fracture healing. Cytokines and chemokines such as Stromal cell-derived factor 1(SDF-1) and its receptor chemokine receptor type 4 (CXCR4) play important roles in maintaining mobilisation, trafficking and homing of stem cells from bone marrow to the site of injury. We investigated the differences in migration of MSCs from the femurs of young, adult and ovariectomised (OVX) rats and the effect of CXCR4 over-expression on their migration. MSCs from young, adult and OVX rats were put in a Boyden chamber to establish their migration towards SDF-1. This was compared with MSCs transfected with CXCR4, as well as MSCs differentiated to osteoblasts.Objectives
Methods
Children suffering from primary bone cancer necessitating resection of growth plates, may suffer progressive leg length discrepancy, which can be attenuated with extendable prostheses. A serious complication is catastrophic implant failure. Over time, bone will remodel, altering the stress pattern in the implant. By using finite element analysis we can model different bone remodeling conditions to ascertain the effect that this will have on stress distribution and magnitude. A finite element analysis was performed. Simplified computer generated models were designed of a cemented femoral Stanmore growing massive endoprosthesis. Three scenarios were designed, modelled on post-operative radiographs. Scenario 1 had a gap between the end of the femur and the implant collar, scenario 2 had no gap, but with no bone attachment into the collar, and scenario 3 had growth of the bone over the length of the collar with attachment. Physiological loading conditions were applied. The resultant stress in the implant for each scenario was measured, and compared to the strength of the material. Peak stresses were recorded at the stem-collar junction. The maximum stress recorded in the implant in scenario 1 was 3104.2Mpa, compared to 1054.4Mpa in scenario 2, and 321.2Mpa in scenario 3. Both accurate reduction and bone growth with attachment to the stem of a massive endoprosthesis will greatly reduce the resultant stress in the implant under loading conditions. The load is redistributed throughout the length of the bone. This may help to prevent catastrophic failure in the implant under loading conditions. Further investigations of patient findings are needed to ensure the model findings are verified.Background
Conclusions
Stress shielding and wear induced aseptic loosening cause failure in arthroplasty surgery. To improve survivorship, the use of a low modulus, low wearing biomaterial may be a suitable alternative to hard bearing prostheses, such as cobalt chromium (CoCr). There has been considerable research interest in the use of polyetheretherketone (PEEK) based on observed clinical success especially in spinal surgery. This study investigated the wear performance of PEEK, carbon reinforced PEEK (CFR-PEEK) and acetal as bearing materials in an all polymer total knee arthroplasty (TKA) using a unidirectional pin on plate test. The following material combinations were tested: PEEK vs. UHMWPE, CFR-PEEK vs. UHMWPE, PEEK vs. PEEK, CFR-PEEK vs. PEEK, CoCr vs. UHMWPE, PEEK vs. XLPE, CFR-PEEK vs. CFR-PEEK, PEEK vs. Acetal, Acetal vs. XLPE and CoCr vs. XLPE.Tribological couples tested (Pin vs. Plate) Using a previously validated modification of ASTM F732, 20mm diameter spherically ended pins with a radius of 25mm were articulated against 40mm diameter plates. A load of 1000N was applied to generate a contact stress of about 70MPa similar to contact stresses previously reported in the knee. The lubricant used was 25% newborn calf serum containing 0.3% sodium azide to retard bacteria growth and 20mM EDTA to prevent calcium deposition. Three repeats of pin on plate combinations (including 2 passive soak controls) were tested for 2 million cycles at a cycle frequency of 1Hz and a stroke length of 10 mm. Gravimetric wear was analysed every 250,000 cycles and results converted to volumetric wear using material density.Background
Methods
Osteoporosis and bone fractures lead to immobility, chronic pain and high patient care costs. Mesenchymal stem cells (MSCs) from postmenopausal women have a slower growth rate and osteogenic differentiation ability causing lower bone density and reduced fracture healing capacity compared to MSCs from premenopausal women. Cellular movement and relocalisation are necessary for many physiologic properties. Local MSCs from injured tissues and circulating MSCs are involved in fracture healing. Cytokines and chemokines such as SDF-1 and its receptor CXCR4 play important roles in maintaining mobilisation, trafficking and homing of stem cells from bone marrow to the site of injury. This study investigated the effect of CXCR4 over-expression on the migration of MSCs from ovariectomised, normal and young rats. MSCs were harvested from femora of young, normal and OVX rats, genetically modified to over-express CXCR4and put in a Boyden chamber to establish their migration towards SDF-1. This was compared to the non-transfected stem cells.Background
Methods
Complications of metal-on-metal hip resurfacing, leading to implant failure, include femoral notching, neck fracture, and avascular necrosis. Revision arthroplasty options include femoral-only revision with a head, however mis-matching radial clearance could accelerate metal ion release. Alternatively, revision of a well-fixed acetabular component could lead to further bone loss, complicating revision surgery. We have developed a ceramic hip resurfacing system with a titanium-ceramic taper junction; taking advantage of the low frictional torque and wear rates that ceramic affords. Taking a revision scenario into account, the ceramic head has a deep female taper for the resurfacing stem, but also a superficial tapered rim. Should revision to this resurfacing be required, any femoral stem with a 12/14 taper can be implanted, onto which a dual taper adaptor is attached. The outer diameter of the taper adaptor then becomes the male taper for the superficial taper of the ceramic head; ultimately allowing retention of the acetabular component. In an in-vitro model, we have compared the fretting corrosion of this taper adaptor to existing revision taper options: a titanium-cobalt chrome (Ti-CoCr) taper junction, and a titanium-titanium sleeve-ceramic (Ti-Ti-Cer) taper junction. To simulate gait, sinusoidal cyclical loads between 300N-2300N, at a frequency of 3Hz was applied to different neck offsets generating different bending moments and torques. Bending moment and frictional torque were tested separately. An electrochemical assessment using potentiostatic tests at an applied potential of 200mV, was used to measure the fretting current (μA) and current amplitude (μA). In a short term 1000 cycle test with bending moment, four neck lengths (short to x-long) were applied. For frictional torque, four increments of increasing torque (2-4-6-8Nm) were applied. In a long-term test using the taper adaptor, the combination of worst-case scenario of bending and torque were applied, and fretting currents measured every million cycles, up to 10 million cycles.Background
Methods
Hip resurfacing has advantages for the young active patient with arthritis; maintaining a large range of motion, preserving bone stock, and reduced dislocation risk. However high serum metal ion levels with metal-on-metal resurfacing, and their clinical implications, has led to a decline in the use of hip resurfacing. Ceramic bearing surfaces display the lowest frictional torque and excellent wear rates. Recent developments have enabled large, strong ceramic materials to be used as resurfacing components. Any wear debris that is generated from these articulations is inert. However an all-ceramic hip resurfacing could be at risk of fracture at the head-stem junction. A new ceramic hip resurfacing system with a titanium-ceramic modular taper junction has been developed. The introduction of a taper introduces the potential for fretting corrosion; we sought to determine the extent of this in an in-vitro model, and compared this prosthesis to the conventional 12/14 titanium-cobalt chrome (Ti6Al4V-CoCr) taper junction. To simulate the gait cycle, sinusoidal cyclical loads between 300N-2300N, at a frequency of 3Hz, were applied to different head-neck offsets generating different bending moments and torques. The effect of increasing the bending moment and frictional torque were tested separately. Furthermore, the resurfacing head was mounted in a fixture held with just the stem, thus representing complete bone resorption under the head. An electrochemical assessment using potentiostatic tests at an applied potential of 200mV, was used to measure the fretting current (μA) and current amplitude (μA). In a short-term 1000 cycle test, six neck lengths (short to xxx-long) of the Ti6Al4V-CoCr taper were compared to the standard neutral (concentric), and 3mm A/P offset stem options for the resurfacing design. To represent frictional torque, four increments of increasing torque (2-4-6-8Nm) were applied to both tapers. In a long term test with the resurfacing stem, the worst-case scenario of the eccentric offset option and 8Nm of torque were applied, and potentiostatic measurements were taken every million cycles, up to 10 million cycles.Background
Methods
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).
Mesenchymal stem cells (MSCs) are believed to be immune-privileged due to lack of antigen-presenting-cell related markers, however, evidence suggests that MSCs are immunogenic and are attacked by the immune system. Our research investigates the hypothesis that there are differences between MSC clones from the same individual in terms of their morphology, proliferation, differentiation and immune profile. Our goal is to discover immune-privileged stem cells, which can act as a universal allogenic mesenchymal stem cell donor to facilitate bone ingrowth for osteosarcoma patients status post tumor excision and prosthesis implantation. Serial dilutions of bone-marrow derived (BMMSCs) and adipose derived mesenchymal stem cells (ADMSCs) from same animal were carried out in order to isolate single-cell clones. From a single animal we obtained 3 clones from BMMSCs and 3 from ADMSCs. This procedure was repeated for another other 2 animals. The proliferation rate and cell doubling time of each clonal culture was measured. The proliferation rate of mixed clonal cultures was also measured. The tri-differentiation potential of the clonal cultures was compared and a comparison was also made with the original isolates from bone marrow and fat. The immune-privileged properties were measured by flow cytometry and immuno-staining for the major histocompatibility complex (MHC) antigens. To measure the immune response a mixed leucocyte reaction was used but where leucocytes from a different individual were mixed with the clonal MSC cells. All isolates were able to differentiate into osteoblasts, chondrocytes and adipocytes. All clonal cultures revealed significantly different proliferation rates and doubling times when compared with each other and with mixed cultures. All clonal cultures showed different surface marker presentations, which included differences in the expression of MHC antigens. One clone isolated from ADMSCs showed lack of MHCI and MHCII. Our mixed leucocyte reaction and MHC staining showed variety of immune-modulation and this was related to the expression of the MHC antigens. All clones tri-differentiated and therefore show a degree of ‘stemness’. MSCs are generally are believed not to express MHC II and to be immune-privileged. However, this study shows that the expression of these antigens in clones isolated from bone marrow and from fat is variable. A heterogeneous result indicates individual differences between MSCs, even from same origin. The immune response elicited by MSCs is complicated. MSCs have been shown to release interleukin 10, which could inhibit the immune response but on the other hand interferon-gamma could enhance MHCII presentation in some MSCs. Our results confirmed our hypothesis because clonal cultures isolated from different sources of MSCs in the same animal showed significant differences in proliferation rate, morphology and surface marker presentation. Mesenchymal stem cells are not immunogenic or immune-privileged. Individual differences highlighted through single-cell clonal cultures may be the key to finding a universal immune-privileged MSCs for allogeneic transplantation.
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.
Mesenchymal stem cells (MSCs) are usually believed to be immune-privileged. However, immunogenic MSCs were also reported. We hypothesize that there are differences between MSC clones from the same individual in terms of their morphology, proliferation, differentiation and immunogenicity. Our goal is to discover immune-privileged stem cells for universal allogenic MSCs transplantation. Serial dilutions of bone-marrow derived (BMMSCs) and adipose derived mesenchymal stem cells (ADMSCs) from same animal were carried out to isolate single-cell clones. From a single animal we obtained 3 clones from BMMSCs and 3 from ADMSCs. The proliferation rate of each clonal culture and mixed clonal culture were measured. The tri-differentiation potential of the clonal cultures was compared, as well as with the original isolates from bone marrow and fat. The immune-privileged properties were measured by flow cytometry and immuno-staining for the major histocompatibility complex (MHC) antigens. Mixed leucocyte reaction (MLR) were also performed to investigate immunogenicity. Tri-differentiation was confirmed in all isolates. All clonal cultures revealed significant different morphology and proliferation rates, compared with each other and mixed cultures. All clonal cultures showed different surface markers, inclusive of MHC antigens. One clone from ADMSCs showed lack of MHC antigens. Our MLR and MHC staining disclosed variety of immune properties. All clones tri-differentiated which indicated a degree of ‘stemness’. MSCs are generally believed not to express MHC II, resulting in immune-privileged. Our results confirmed our hypothesis because clonal cultures isolated from different origins of same animal show differences in morphology, proliferation rate, and surface marker presentation. Individual immune differences highlighted through single-cell clonal cultures may be crucial to find universal immune-privileged MSCs as universal allogeneic donor.
