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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.


Orthopaedic Proceedings
Vol. 95-B, Issue SUPP_13 | Pages 24 - 24
1 Mar 2013
Hardwick T Kalia P Baawa-Ameyaw J Lozano D Sinha J Di Silvio L
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Introduction

Parathyroid hormone-related peptide (PTHrP) has been shown to be an important regulator of bone remodelling1. The aim of this study was to investigate the effect of the N-terminal domain of PTHrP (1–36) on osteogenic and angiogenic gene expression in human osteoblasts (HOB) and human bone marrow stromal cells (hBMSCs).

Materials and Methods

Primary hBMSC's and HOBs were cultured in standard or osteogenic media with different concentrations of PTHrP, either continuously for 8, 24, 48 h and 9 days, or with 3 cycles of intermittent exposure (24 h with PTHrP, 24 h without) over 6 days. Cell lysates were then processed for analysis of gene expression. Expression of the osteogenic markers runt-related transcription factor 2 (RUNX-2), alkaline phosphatase (ALP) and Collagen 1, and the angiogenic marker; vascular endothelial growth factor (VEGF), were measured.


Orthopaedic Proceedings
Vol. 90-B, Issue SUPP_I | Pages 13 - 14
1 Mar 2008
Di Silvio L Ali Z Narvani A Goodship A Bentley G Tsiridis E
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Current bone grafts include allograft and autografts, both of which have limitations. Tissue engineering biotechnology has shown considerable promise in improving grafts. A competent graft material should ideally have osteoconductive and osteoinductive properties and comprise of bone forming cells and osteoinductive growth factors. In this study, we have evaluated the in vitro formation of bone and have used human demineralised bone matrix [DBM] and human insoluble collagenous matric [ICM] as scaffolds for mesenchymal stem cells [MSCs] and osteogenic protein [OP-1]. The objective was to determine whether combined addition of OP-1 and MSCs resulted in a superior bone graft substitute by improving the inherent osteoinductive property.

DBM and ICM were prepared and combined with rhOP [1.4 mg/0.25 mg of bone] and MSCs [1 x 105/ ml]. Statistically significant differences in MSC proliferation were seen between materials with and without OP-1 [P< 0.05}, n=8] in DBM on day 1, and both DBM and ICM on day 7 and 14. Enhanced osteogenic differentiation was observed in the presence of OP-1 when compared to DBM alone and on DBM and ICM with OP-1. In conclusion MSCs and OP-1 can be seeded together on DBM and ICM and Von Kossa staining and X-ray analysis confirmed in vitro de novo bone formation, with DBM + MSCs + OP-1 being more successful in this regard.

Conclusion: To date, no other study, to the author’s knowledge, has used MSCs and OP-1 together on a graft material; this funding, therefore, has very important clinical implications.


Orthopaedic Proceedings
Vol. 90-B, Issue SUPP_I | Pages 14 - 14
1 Mar 2008
Tsiridis E Bhalla A Narvani A Goodship A Bentley G Di Silvio L
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Limitations of allografts and autografts for bone repair have increased the demand for a synthetic bone graft substitute for load-bearing and non-load bearing osseous defects. Tissue engineering of bone has thus been implicated to circumvent and eliminate the limitations of existing therapies, with living cell-scaffold constructs ultimately “integrating” with the patients own tissue. Bone engineering requires cells, growth inducing factors and a scaffold for delivery of cells to the anatomic site, creation of 3-D space for tissue formation and mechanical support. In this study, we investigated whether addition of osteogenic Protein-1 (OP-1) enhanced the osseoinductive properties of hydroxyapatite (HA) loaded with mesenchymal stem cells (MSCs). The study was conducted over a fourteen day period and the two groups HA/MSC and HA/MSC loaded with OP-1 were analysed qualitatively by SEM and quantitatively by assessment of proliferation (Alamar blue assay and total cellular DNA) and differentiation marker alkaline phosphatase activity (ALP). HA/MS/OP-1 showed a statistically significant (p< 0.05) increase in cell proliferation (286.52 ± 58.2) compared to the unloaded samples (175.62 ± 23.51). ALP activity (release) was also significantly enhanced (p < 0.05) in the loaded samples at day 14 (12.63 ± 1.58) compared to the control (2.73 ± 1.07).

Conclusion: the osseoinductive potential of HA was markedly improved by the incorporation of MSC’s and OP-1. This type of graft could provide improved mechanical stability at an earlier time point, and may influence future clinical application of HA for load bearing sites.


Orthopaedic Proceedings
Vol. 88-B, Issue SUPP_III | Pages 364 - 364
1 Oct 2006
Korda M Sharpe J Rust P Hua J Phipps K Di Silvio L Coathup M Goodship A Blunn G
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Introduction: Wear particle induced osteolysis is one of the main reasons for revision total hip replacements (THRs). Loss in bone stock as a result of aseptic loosening is responsible for inferior results in revision THRs. Results from impaction grafting to fill osteolytic defects are frequently inconsistent. Our hypothesis is that the combination of autologous mesenchymal stem cells (MSCs) and allograft will enhance bone regeneration. This study asks whether: MSCs with allograft scaffolds survive at a normal impaction force during revision THRs.

Method: MSCs were isolated from a sheep iliac crest aspirate, expanded in culture and seeded onto irradiated sheep allografts (n=9). Viability of MSCs was assayed with alamar blue with absorbance measured on day 4 (before impaction). The constructs were then impacted using forces 3, 6, and 9 kN extrapolated in surgery then assayed daily for 6 days. The control was 0 kN. Samples were resin embedded after 10 days for histology and pieces of graft were taken for scanning electron microscopy (SEM).

Results: The 0KN control shows an MSC growth curve with a lag period and log phase. Compared with the control, the 3 and 6 kN showed initial reduction in cell proliferation measured by alamar blue (^p=0.015, ^p=0.002) but recovered by day 8, while 9kN showed a significant reduction (^p=0.011) over the time (Figure 1).

For cell proliferation over time, 3 and 6 kN showed no differences, but 9 kN showed a significant difference between day 4 and day 8 (^p=0.031). SEM and histological analysis showed a network of cuboidal cells on the allograft surface.

Conclusions: The results showed that MSCs recovered from impaction of 3 and 6 kN after an initial reduction in metabolism and exceeded original cell seeding densities with no significant difference in proliferation. Viability of MSCs were not effected by impaction forces up to 6 kN. This study shows that stem cells mixed with allograft are a potential method for repairing bone defects in revision total hip replacements.


Orthopaedic Proceedings
Vol. 86-B, Issue SUPP_III | Pages 351 - 351
1 Mar 2004
Tsiridis E Bhalla A Ali Z Roushdi I Goodship A Di Silvio L
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Aim: The biological activity of demineralised bone matrix (DBM) led to the discovery of bone morphogenetic proteins (BMP). OP-1 (BMP 7) is an osteoinductive protein and has been demonstrated to be capable of inducing new bone formation in rat subcutaneous tissue and in both orthotopic and heterotopic sites in primates. In this study we have investigated whether demineralisation and addition of osteogenic. protein 1 (OP-1) improves osteoinductive properties of allograft. Methods: A randomised controlled blind trial was performed in 16 rats. One group received two pellets of fresh frozen allograft; the other received two pellets of demineralised bone (DBM) intramuscularly. In each rat one pellet was treated with OP-1 (2mg/25mg of graft). The rats were sacriþced at 28 days and tissue þxed and processed for sectioning with haematoxylin and eosin for morphology and Alcian blue and Sirrus red for collagen types I, II. Qualitative observations were made and each specimen graded 0–5 on the degree of new bone formation and integration by two blind observers. Results & Conclusions: DBM with OP-1 yielded optimal results, being signiþcantly superior to allograft alone and allograft with OP-1. DBM alone was shown to be more effective compared to the allograft preparations. Hence we have shown that demineralization and OP-1 signiþcantly improve the osteoinductive properties of allograft


Orthopaedic Proceedings
Vol. 86-B, Issue SUPP_III | Pages 351 - 351
1 Mar 2004
Di Silvio L Ali Z Goodship A Tsiridis E
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Aim:. The objective of this study was to improve the osseoinductive capacity of human demineralised bone matrix (DBM) and human insoluble collagenous matrix (ICM), following incorporation of recombinant human osteogenic protein 1 (rhOP-1) and human mesenchymal stem cells (MSCs). Ethical Committee approval has been obtained by our Institution. Methods: Recombinant human osteogenic protein Ð 1 (400ng/0.25g of bone) was seeded onto DBM and ICM together with human MSCs (1 x 105). Cellular proliferation was quantitatively evaluated in vitrousing Alamar Blue and 3H-TdR assays. Quantitative cellular differentiation was assessed using the alkaline phosphatase assay. Von Kossa staining, X-ray analysis, and PCR were used for qualitative evaluation of cellular differentiation. Qualitative analysis of proliferation and differentiation was assessed using scanning electron microscopy (SEM). Results: MSC proliferation and differentiation down the osteogenic lineage was observed on DBM and ICM in the presence of OP-1, and also on DBM alone. Alamar blue and 3H-TdR assays conþrmed that MSC proliferation occurred on both DBM and ICM, with the values being signiþcantly greater with addition of OP-1. The ALP activity showed that MSCs differentiated into osteo-blasts on DBM alone, and on DBM and ICM with OP-1. In all cases, OP-1 had a signiþcant effect on MSCs. Discussion: DBM and ICM when seeded with MSCs and OP-1 provide an improved osteoconductive and osteoinductive graft material resulting in de novo-bone formation.