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Large animal in vivo evaluation of a binary blend polymer scaffold for skeletal tissue‐engineering strategies; translational issues
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Ferdous Khan Alexander Aarvold Richard B. Cook Allen Goodship Mark Bradley Richard O. C. Oreffo 《Journal of tissue engineering and regenerative medicine》2017,11(4):1065-1076
Binary blend polymers offer the opportunity to combine different desirable properties into a single scaffold, to enhance function within the field of tissue engineering. Previous in vitro and murine in vivo analysis identified a polymer blend of poly(l ‐lactic acid)–poly(ε‐caprolactone) (PLLA:PCL 20:80) to have characteristics desirable for bone regeneration. Polymer scaffolds in combination with marrow‐derived skeletal stem cells (SSCs) were implanted into mid‐shaft ovine 3.5 cm tibial defects, and indices of bone regeneration were compared to groups implanted with scaffolds alone and with empty defects after 12 weeks, including micro‐CT, mechanical testing and histological analysis. The critical nature of the defect was confirmed via all modalities. Both the scaffold and scaffold/SSC groups showed enhanced quantitative bone regeneration; however, this was only found to be significant in the scaffold/SSCs group (p = 0.04) and complete defect bridging was not achieved in any group. The mechanical strength was significantly less than that of contralateral control tibiae (p < 0.01) and would not be appropriate for full functional loading in a clinical setting. This study explored the hypothesis that cell therapy would enhance bone formation in a critical‐sized defect compared to scaffold alone, using an external fixation construct, to bridge the scale‐up gap between small animal studies and potential clinical translation. The model has proved a successful critical defect and analytical techniques have been found to be both valid and reproducible. Further work is required with both scaffold production techniques and cellular protocols in order to successfully scale‐up this stem cell/binary blend polymer scaffold. © 2015 The Authors. Journal of Tissue Engineering and Regenerative Medicine published by John Wiley & Sons, Ltd. 相似文献
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Morgan SM Tilley S Perera S Ellis MJ Kanczler J Chaudhuri JB Oreffo RO 《Biomaterials》2007,28(35):5332-5343
Strategies to expand human bone marrow stromal cells (HBMSC) for bone tissue engineering are a key to revolutionising the processes involved in three-dimensional skeletal tissue reconstruction. To facilitate this process we believe the use of biodegradable porous poly(DL-lactide-co-glycolide) (PDL LGA) hollow fibres as a scaffold used in combination with HBMSC to initiate natural bone repair and regeneration offers a potential solution. In this study, the biocompatibility of 75:25 PDL LGA fibres with HBMSC and the capacity of a PDL LGA fibre-associated HBMSC-monolayer to establish an osteogenic phenotype in vivo was examined. A high proportion of HBMSC survived when expanded on PDL LGA fibres for 6 days, with only 10% of the propidium iodide (pI)-labelled population represented in the sub-G1 DNA peak on analysis by flow cytometry. Tracking carboxy-fluorescein diacetate, succinimidyl ester (CFSE)-labelled HBMSC by flow cytometry indicated that HBMSC attachment to the P(DL)LGA fibres does not interfere with their rate of proliferation. Furthermore, in response to osteogenic stimuli, HBMSC expanded on PDL LGA fibres can differentiate, as expected, along the osteogenic lineage with associated alkaline phosphatase activity. Following implantation into SCID mice, osteogenic-conditioned PDL LGA fibre-HBMSC graft resulted in type I collagen deposition and associated bone mineralisation and osteoid formation, as evidenced by immunohistochemistry and histology. These studies provide evidence that porous PDL LGA hollow fibre-HBMSC graft is an innovative biomaterial that offers new approaches to mesenchymal cell expansion, which could be utilised as a scaffold for skeletal tissue generation. 相似文献
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James O. Smith Richard O.C. Oreffo Nicholas M.P. Clarke Helmtrud I. Roach 《Journal of orthopaedic science》2003,8(6):849-857
Avascularity is important for the unique biomechanical properties of articular cartilage, and normal cartilage actively repels vascular invasion. This study investigated whether the antiangiogenic properties changed in the presence of osteoarthritis (OA) by culturing explants of human articular cartilage on the chorioallantoic membrane (CAM) of chick embryos and investigating the incidence of vascular invasion and the effects of exogenous vascular endothelial growth factor (VEGF) and matrix metalloproteinase-9 (MMP-9). The results were compared with those of non-OA cartilage obtained after femoral neck fractures. Altogether, 72% of OA samples but only 5% of non-OA samples were invaded by the CAM, indicating that changes in the antiangiogenic properties represented a fundamental difference between control and OA cartilage. Exogenous VEGF or MMP-9 increased the frequency of invasion to 70%–100%. Invasion most frequently occurred into cartilage matrix from which proteoglycans had been lost, the latter being detectable by sirius red staining of cartilage collagen. VEGF was synthesized by chondrocytes in proportion to the severity of degradation and might exacerbate the loss of resistance to invasion. These results indicate that loss of resistance to vascular invasion distinguishes OA cartilage from normal articular cartilage, which may be important in the pathogenesis of OA. 相似文献
47.
Ginty PJ Howard D Rose FR Whitaker MJ Barry JJ Tighe P Mutch SR Serhatkulu G Oreffo RO Howdle SM Shakesheff KM 《Proceedings of the National Academy of Sciences of the United States of America》2006,103(19):7426-7431
We demonstrate that mammalian cells can survive for 5 min within high-pressure CO(2)(.) Cell survival was investigated by exposing a range of mammalian cell types to supercritical CO(2) (scCO(2)) (35 degrees C, 74 bar; 1 bar = 100 kPa) for increasing exposure and depressurization times. The myoblastic C2C12 cell line, 3T3 fibroblasts, chondrocytes, and hepatocytes all displayed appreciable but varying metabolic activity with exposure times up to 1 min. With depressurization times of 4 min, cell population metabolic activity was >/=70% of the control population. Based on survival data, we developed a single-step scCO(2) technique for the rapid production of biodegradable poly(dl-lactic acid) scaffolds containing mammalian cells. By using optimum cell-survival conditions, scCO(2) was used to process poly(dl-lactic acid) containing a cell suspension, and, upon pressure release, a polymer sponge containing viable mammalian cells was formed. Cell functionality was demonstrated by retention of an osteogenic response to bone morphogenetic protein-2 in C2C12 cells. A gene microarray analysis showed no statistically significant changes in gene expression across 4,418 genes by a single-class t test. A significance analysis of microarrays revealed only eight genes that were down-regulated based on a delta value of 1.0125 and a false detection rate of 0. 相似文献
48.
Bolton SJ Pinnion K Marshall CV Wilson E Barker JE Oreffo V Foster ML 《Toxicologic pathology》2008,36(3):440-448
Clara cell 10 kDa protein (CC10) is the major secretory protein of Clara cells and is thought to play a protective role in the lung owing to its anti-inflammatory properties. There is little information on the anatomical distribution of CC10-positive cells in rat lung following lipopolysaccharide (LPS) challenge. We have determined the expression of CC10 along the tracheobronchial tree in saline-treated and LPS-treated rats. Saline-treated rats showed sporadic CC10 staining in central airways and abundant staining in bronchioles. In transitional airways, most cells were positive except for squamous cells. Following LPS challenge, there was a reduction in staining in the upper airways but little change within bronchioles. Squamous epithelia within the transitional airways now showed positive staining. These cells also co-stained for pancytokeratin and appeared to co-localize with surfactant D- and Ki67-positive cells, indicating the presence of a dedifferentiated cell type with both epithelial and pneumocyte phenotypes. These data show that diffuse inflammatory injury results in generalized loss of CC10 in central airways. Conversely, the transitional airways showed evidence of a dedifferentiated population of squamous cells that now stained for CC10. We hypothesize that this is an attempt by peripheral lung to maintain alveolar sac integrity during an inflammatory episode. 相似文献
49.
The application of human bone marrow stromal cells and poly(dl-lactic acid) as a biological bone graft extender in impaction bone grafting 总被引:2,自引:0,他引:2
Bolland BJ Kanczler JM Ginty PJ Howdle SM Shakesheff KM Dunlop DG Oreffo RO 《Biomaterials》2008,29(22):3221-3227
Concerns over disease transmission, high costs and limited supply have led to interest in synthetic grafts in the field of impaction bone grafting (IBG). Poly(DL-lactic acid) (PLA) grafts are attractive alternatives due to their biocompatibility, established safety and versatile manufacturing process. This study examined the potential of PLA scaffolds augmented with human bone marrow stromal cells (HBMSCs) in IBG. In vitro and in vivo studies were performed on impacted morsellised PLA seeded with HBMSC and compared to PLA alone. In vitro samples were incubated under osteogenic conditions and in vivo samples were implanted subcutaneously into severely compromised immunodeficient mice, for 4 weeks. Biochemical, histological, mechanical and 3D micro-computed tomography analyses were performed. HBMSC viability, biochemical activity and histological evidence of osteogenic cellular differentiation, post-impaction were observed in vitro and in vivo in PLA/HBMSC samples compared to impacted PLA alone. In vitro PLA/HBMSC samples demonstrated evidence of mechanical enhancement over PLA alone. In vivo studies showed a significant increase in new bone and blood vessel formation in the PLA/HBMSC constructs compared to PLA alone. With alternatives to allograft being sought, these studies have demonstrated PLA/HBMSC living composites, to be a potential prospect as a biological bone graft extender for future use in the field of IBG. 相似文献
50.
The effect of mesenchymal populations and vascular endothelial growth factor delivered from biodegradable polymer scaffolds on bone formation 总被引:4,自引:0,他引:4
Kanczler JM Ginty PJ Barry JJ Clarke NM Howdle SM Shakesheff KM Oreffo RO 《Biomaterials》2008,29(12):1892-1900
The capacity to deliver, temporally, bioactive growth factors in combination with appropriate progenitor and stem cells to sites of tissue regeneration promoting angiogenesis and osteogenesis offers therapeutic opportunities in regenerative medicine. We have examined the bone regenerative potential of encapsulated vascular endothelial growth factor (VEGF(165)) biodegradable poly(DL-lactic acid) (PLA) scaffolds created using supercritical CO(2) fluid technology to encapsulate and release solvent-sensitive and thermolabile growth factors in combination with human bone marrow stromal cells (HBMSC) implanted in a mouse femur segmental defect (5 mm) for 4 weeks. HBMSC seeded on VEGF encapsulated PLA scaffolds showed significant bone regeneration in the femur segmental defect compared to the scaffold alone and scaffold seeded with HBMSC as analysed by indices of increased bone volume (BV mm(3)), trabecular number (Tb.N/mm) and reduced trabecular separation (Tb.Sp.mm) in the defect region using micro-computed tomography. Histological examination confirmed significant new bone matrix in the HBMSC seeded VEGF encapsulated scaffold group as evidenced by Sirius red/alcian blue and Goldner's trichrome staining and type I collagen immunocytochemistry expression in comparison to the other groups. These studies demonstrate the ability to deliver, temporally, a combination of VEGF released from scaffolds with seeded HBMSC to sites of bone defects, results in enhanced regeneration of a bone defect. 相似文献