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Orthopaedic Proceedings
Vol. 99-B, Issue SUPP_8 | Pages 37 - 37
1 Apr 2017
Sanghani A Coathup M Samazideh S Kalia P Di Silvio L Blunn G
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Background

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.

Methods

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.


Aims

The Intraosseous Transcutaneous Amputation Prosthesis (ITAP) may improve quality of life for amputees by avoiding soft-tissue complications associated with socket prostheses and by improving sensory feedback and function. It relies on the formation of a seal between the soft tissues and the implant and currently has a flange with drilled holes to promote dermal attachment. Despite this, infection remains a significant risk. This study explored alternative strategies to enhance soft-tissue integration.

Materials and Methods

The effect of ITAP pins with a fully porous titanium alloy flange with interconnected pores on soft-tissue integration was investigated. The flanges were coated with fibronectin-functionalised hydroxyapatite and silver coatings, which have been shown to have an antibacterial effect, while also promoting viable fibroblast growth in vitro. The ITAP pins were implanted along the length of ovine tibias, and histological assessment was undertaken four weeks post-operatively.


Orthopaedic Proceedings
Vol. 99-B, Issue SUPP_4 | Pages 61 - 61
1 Feb 2017
Khan H Riva F Pressacco M Meswania J Panagiotidou A Coathup M Blunn G
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Background

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.

Methods

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.


Orthopaedic Proceedings
Vol. 99-B, Issue SUPP_4 | Pages 60 - 60
1 Feb 2017
Khan H Meswania J Riva F Pressacco M Panagiotidou A Coathup M Blunn G
Full Access

Background

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.

Methods

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.


The Bone & Joint Journal
Vol. 99-B, Issue 2 | Pages 276 - 282
1 Feb 2017
Mumith A Coathup M Chimutengwende-Gordon M Aston W Briggs T Blunn G

Aims

Massive endoprostheses rely on extra-cortical bone bridging (ECBB) to enhance fixation. The aim of this study was to investigate the role of selective laser sintered (SLS) porous collars in augmenting the osseointegration of these prostheses.

Materials and Methods

The two novel designs of porous SLS collars, one with small pores (Ø700 μm, SP) and one with large pores (Ø1500 μm, LP), were compared in an ovine tibial diaphyseal model. Osseointegration of these collars was compared with that of a clinically used solid, grooved design (G). At six months post-operatively, the ovine tibias were retrieved and underwent radiological and histological analysis.


Orthopaedic Proceedings
Vol. 99-B, Issue SUPP_2 | Pages 18 - 18
1 Jan 2017
Palanca M Cristofolini L Pani M Kinene E Blunn G Madi K Tozzi G
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DVC allowed measurements of displacement and strain distribution in bone through the comparison of two, or more, 3D images. Hence, it has a potential as a diagnostic tool in combination with clinical CT. Currently, traditional computed tomography (CT) allows for a detailed 3D analysis of hard tissues, but imaging in a weight-bearing condition is still limited. PedCAT-CT (Curvebeam, USA) emerged as a novel technology allowing, for the first time, 3D imaging under full-weight bearing (Richter, Zech et al. 2015). Specifically, a PedCAT-CT based DVC was employed to establish its reliability through the strain uncertainties produced on bone structure targets, preliminarily to any further clinical studies. In addition, a reverse engineering FE modeling was used to predict possible force associated to displacement errors from DVC.

Three porcine thoracic vertebrae were used as bone benchmark for the DVC (Palanca, Tozzi et al. 2016, Tozzi, Dall'Ara et al. 2016). The choice of using porcine vertebrae (in a CT designed for foot/ankle) was driven by availability, as well as similar dimensions to the calcaneus. Each vertebra was immersed in saline solution and scanned twice without any repositioning (zero-strain-test) with a pedCAT-CT (Curvebeam, USA) obtaining an isotropic voxel size of 370 micrometers. Volumes of interest of 35 voxel were cropped inside the vertebrae. Displacement and strains were evaluated using DVC (DaVis-DC, LaVision, Germany), with different spatial resolution. The displacement maps were used to predict the force uncertainties via FE (Ansys Mechanical v.14, Ansys Inc, Canonsburg, PA). Each element was assigned a linear elastic isotropic constitutive law (Young modulus: 8 GPa, Poisson's ratio: 0.3, as in (Follet, Peyrin et al. 2007)). Overall, the precision error of strain measurement was evaluated as the average of the standard deviation of the absolute value of the different component of strain (Liu and Morgan 2007).

The force uncertainties obtained with the FE analysis produced magnitudes ranging from 231 to 2376 N. No clear trend on the force was observed in relation to the spatial resolution. Precision errors were smaller than 1000 microstrain in all cases, with the lowest ranging from 83 microstrain for the largest spatial resolution. Full-field strain on the bone tissue did not seem to highlight a preferential distribution of error in the volume.

The precision errors showed that the pedCAT-CT based DVC can be sufficient to investigate the bone tissue failure (7000–10000 microstrain) or, physiological deformation if well-optimized. FE analysis produced important force uncertainties up to 2376 N. However, this is a preliminary investigation. Further investigation will give a clearer indication on DVC based PedCAT-CT, as well as force uncertainties predicted. So far, the DVC showed its ability to measure displacement and strain with reasonable reliability with clinical-CT as well.


Orthopaedic Proceedings
Vol. 99-B, Issue SUPP_2 | Pages 84 - 84
1 Jan 2017
Osagie L Sanghani-Kerai A Coathup M Briggs T Blunn G
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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.


Orthopaedic Proceedings
Vol. 99-B, Issue SUPP_1 | Pages 28 - 28
1 Jan 2017
Osagie L Sanghani-Kerai A Coathup M Briggs T Blunn G
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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.


Orthopaedic Proceedings
Vol. 99-B, Issue SUPP_1 | Pages 59 - 59
1 Jan 2017
Sanghani Kerai A Osagie L Coathup M Blunn G
Full Access

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).


Orthopaedic Proceedings
Vol. 99-B, Issue SUPP_2 | Pages 81 - 81
1 Jan 2017
Cheng Y Samizadeh S Coathup M Blunn G
Full Access

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.


Bone & Joint Research
Vol. 6, Issue 1 | Pages 14 - 21
1 Jan 2017
Osagie-Clouard L Sanghani A Coathup M Briggs T Bostrom M Blunn G

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.

Cite this article: L. Osagie-Clouard, A. Sanghani, M. Coathup, T. Briggs, M. Bostrom, G. Blunn. Parathyroid hormone 1-34 and skeletal anabolic action: The use of parathyroid hormone in bone formation. Bone Joint Res 2017;6:14–21. DOI: 10.1302/2046-3758.61.BJR-2016-0085.R1.


Orthopaedic Proceedings
Vol. 98-B, Issue SUPP_23 | Pages 59 - 59
1 Dec 2016
Hislop S McKenna D Coathup MJ Blunn G Briggs T
Full Access

Aim

Photodynamic therapy (PDT) requires a photosensitiser, a light source of an appropriate wavelength, and the presence of molecular oxygen. Once stimulated to its excited phase by the light, the photosensitiser reacts with oxygen to form free radicals of ‘singlet oxygen’ which is cytotoxic to microorganisms.

We aim to demonstrate the effectiveness of PDT as an in-vitro antimicrobial technique against Staphylococcus aureus, Methicillin resistant Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa and Acinetobacter bauminii. This will form the scientific basis for further animal and human studies assessing PDT for treatment of periprosthetic infections, septic arthritis, and open fractures.

Method

A PDT treatment protocol was devised using lawns of bacteria on agar plates. PDT was targeted towards the bacteria and the remaining microorganisms were quantified using a serial dilution technique. In order to assess the ability of photodynamic therapy to target biofilms on metallic implants, biofilms were cultured on polished titanium and hydroxyapatite-coated titanium discs and subjected to PDT.


Orthopaedic Proceedings
Vol. 98-B, Issue SUPP_16 | Pages 2 - 2
1 Oct 2016
Clouard L Sanghani-Kerai A Coathup M Briggs T Blunn G
Full Access

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.


Orthopaedic Proceedings
Vol. 98-B, Issue SUPP_16 | Pages 4 - 4
1 Oct 2016
Cheng Y Sorousheh S Coathup M Blunn G
Full Access

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.


Orthopaedic Proceedings
Vol. 98-B, Issue SUPP_16 | Pages 50 - 50
1 Oct 2016
Clouard L Sanghani-Kerai A Coathup M Briggs T Blunn G
Full Access

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.


Orthopaedic Proceedings
Vol. 98-B, Issue SUPP_16 | Pages 49 - 49
1 Oct 2016
Kerai AS Coathup M Osagie L Samizadeh S Blunn G
Full Access

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).


Orthopaedic Proceedings
Vol. 98-B, Issue SUPP_9 | Pages 83 - 83
1 May 2016
Christiansen J Nielsen P Laursen M Blunn G
Full Access

Introduction

The Primoris® femoral stem was designed to preserve bone and maintain normal stress to the proximal femur, thereby minimizing stress-shielding. The implant is anchored in the femoral neck and metaphysis without diaphysial involvement and differs from other neck prothesis by: a) Elliptical shape to fit the inner neck dimensions. b) On top of Ti– porous-coating electrochemically deposited hydroxy apatite (Bonemaster®) c) The surgical technique aims to enhance initial implant stability by compaction of neck and metaphyseal cancellous bone.

Objectives

As part of stepwise introduction to monitor bone remodeling, RSA data and clinical results.


Orthopaedic Proceedings
Vol. 98-B, Issue SUPP_7 | Pages 8 - 8
1 May 2016
Adesina T Ajami S Coathup M Blunn G
Full Access

Introduction

Stress shielding and wear induced aseptic loosening cause failure in total joint arthroplasty. To improve long-term outcomes in total knee arthroplasty (TKA), the use of a low modulus, low wearing biomaterial may be a suitable alternative to cobalt chromium (CoCr) femoral components. Based on its favorable mechanical properties and observed clinical success especially in spinal surgery, polyetheretherketone (PEEK) is investigated as a candidate material for a metal free TKA. An all polymer TKA has several theoretical advantages, these include a more physiological stress in the distal femur, elimination of biological reaction to metal, better radiographic visualisation of the bone implant interface especially with CT and MRI. In addition, polymers afford a cheaper option for the manufacture of prostheses.

Aims and Hypothesis

This study investigated the wear performance of PEEK and carbon reinforced PEEK (CFR-PEEK) as bearing materials in an all polymer TKA using a unidirectional pin on plate test. Our hypothesis was that reduced wear is generated from PEEK or CFR-PEEK bearings when compared with metal on polyethylene (MoP) bearings and that this combination may provide a suitable alternative in TKA.


Orthopaedic Proceedings
Vol. 98-B, Issue SUPP_7 | Pages 10 - 10
1 May 2016
Ajami S Coathup M Khoury J Blunn G
Full Access

Background

Polyetheretherketone (PEEK) may be advantageous as an alternative material to metal alloys in some orthopaedic applications. However, it is bioinert and does not osseointegrate1. A novel accelerated neutral atom beam technique (ANAB) has been developed to improve the bioactivity of PEEK where the surface is modified to a depth of 5 nm without affecting the integrity of the underlying PEEK structure2.

Aim

The aim of this study was to investigate the growth of human Mesenchymal Stem Cells (hMSCs), adult human Osteoblasts (hOB) and skin Fibroblasts (BR3G) on PEEK and ANAB treated PEEK.


The Bone & Joint Journal
Vol. 98-B, Issue 5 | Pages 579 - 584
1 May 2016
Osman K Panagiotidou AP Khan M Blunn G Haddad FS

There is increasing global awareness of adverse reactions to metal debris and elevated serum metal ion concentrations following the use of second generation metal-on-metal total hip arthroplasties. The high incidence of these complications can be largely attributed to corrosion at the head-neck interface. Severe corrosion of the taper is identified most commonly in association with larger diameter femoral heads. However, there is emerging evidence of varying levels of corrosion observed in retrieved components with smaller diameter femoral heads. This same mechanism of galvanic and mechanically-assisted crevice corrosion has been observed in metal-on-polyethylene and ceramic components, suggesting an inherent biomechanical problem with current designs of the head-neck interface.

We provide a review of the fundamental questions and answers clinicians and researchers must understand regarding corrosion of the taper, and its relevance to current orthopaedic practice.

Cite this article: Bone Joint J 2016;98-B:579–84.