Comparative Efficacy of Dermal Fibroblast-Mediated and Direct
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Gene Therapy (2010) 17, 733–744 & 2010 Macmillan Publishers Limited All rights reserved 0969-7128/10 $32.00 www.nature.com/gt ORIGINAL ARTICLE Comparative efficacy of dermal fibroblast-mediated and direct adenoviral bone morphogenetic protein-2 gene therapy for bone regeneration in an equine rib model A Ishihara1,2, LJ Zekas3, SE Weisbrode2 and AL Bertone1,2,3 1Comparative Orthopedic Research Laboratories, Department of Veterinary Clinical Sciences, The Ohio State University, Columbus, Ohio, USA; 2Department of Veterinary Biosciences, The Ohio State University, Columbus, Ohio, USA and 3Department of Veterinary Clinical Sciences, The Ohio State University, Columbus, Ohio, USA Cell-mediated and direct adenoviral (Ad) vector gene were evident in the cortical and cancellous bone directly therapies can induce bone regeneration, including dermal adjacent to the healing drill defects treated with either fibroblasts (DFbs). We compared two effective therapies, DFb-BMP2 or Ad-BMP2. Using our cell/vector dosage and DFb-mediated and direct Ad vector delivery of bone model, BMP2, whether delivered by the DFb vector or direct morphogenetic protein-2 (BMP2), for relative efficacy in Ad vector, induced greater and robust bone regeneration. bone regeneration. Equine rib drill defects were treated by DFb-mediated BMP2 therapy promoted greater cortical percutaneous injection of either DFb-BMP2 or an Ad-BMP2 bone regeneration than did direct gene delivery, possibly vector. At week 6, both DFb-BMP2- and Ad-BMP2-treated rib because of an increased cellularity of the bone healing defects had greater bone filling volume and mineral density, site. BMP2 delivery, regardless of gene delivery method, with DFb-BMP2 inducing greater bone volume and maturity increased the mineral density of the neighboring bone, which in the cortical bone aspect of the defect than Ad-BMP2. may be beneficial clinically in repairing or weak bone. The transplantation of DFb alone induced modest bone Gene Therapy (2010) 17, 733–744; doi:10.1038/gt.2010.13; formation. Increased mineral density and bone turnover published online 11 March 2010 Keywords: bone morphogenetic protein; dermal fibroblasts; bone healing; cell-based gene therapy; adenoviral vectors Introduction relative potency between gene- and cell-mediated BMP2 deliveries is unknown. Regenerative strategies for inferior bone repair are a Although embryonic and adult mesenchymal stem growing need in aging people, in which our immediate cells have been extensively studied for cell-mediated population demands include slow healing fractures gene therapy,8,9 other cell sources, such as dermal (estimated 1 million patients annually), spine fusions fibroblasts (DFbs) may be an alternative candidate (300 000 surgeries and $18 billion annually) and osteo- because of their excellent reprogramming capacity.10 In porotic fractures (704 000 cases annually) (http:// fact, DFbs have been differentiated into bone-forming www.boneandjointdecade.org). At present, pharmaceu- cells by the transduction of osteogenic genes including ticals (such as osteoclast inhibitors) and bioactive BMP2,11–15 and such genetically modified DFbs have proteins (such as bone morphogenetic protein-2 been autologously transplanted to induce ectopic bone (BMP2)) are recognized advancements in bone repair; formation14,15 and promote bone healing in rodent however, the use of living cell vectors as regenerative models.12,13,15 In contrast to the use of stem cells, skin treatment for bony disorders offers the advantages of cell-mediated therapy is attractive for clinical use molecular engineering to release both paracrine and because of the relatively less painful harvest technique, autocrine bioactive factors, direct integration of the cells low risk of donor site infection or morbidity, less into the regenerative process and a broad diversity of fastidious culture procedure and rapid high cell medical applications.1–4 Although direct BMP2 gene yield.12–15 Therefore, the use of DFb may be considered delivery is shown to promote bone regeneration,5–7 the as one choice of cell type for ex vivo gene therapy, although the osteoinductive capacity may not be superior to stem cells. Correspondence: Dr AL Bertone, Department of Veterinary Clinical The authors’ laboratory has demonstrated in separate Sciences, The Ohio State University, 1900 Coffey Road, Columbus, studies that DFb-mediated16 and direct adenoviral (Ad) OH 43210, USA. 7 E-mail: [email protected] BMP2 gene therapy significantly accelerated cortical Received 20 July 2009; revised 13 September 2009; accepted 2 long-bone healing in an equine metacarpal/tarsal model, November 2009; published online 11 March 2010 with DFb functioning through endochondral ossification Cell- and gene-based therapy for bone healing A Ishihara et al 734 and direct Ad-BMP2 delivery functioning through direct Ad-GFP, Ad-BMP2, DFb alone, DFb-GFP and DFb-BMP2 bone formation. Equine bone healing models allowed (Figure 2a). All injections were administered at 2 weeks the comparison of the efficacy of therapeutic methods after rib drilling, and all rib specimens were harvested at at multiple sites within the same animal to reduce 6 weeks after the injections (Figure 2b). During this individual variation, and mimic human osseous injuries period, all six horses showed good incisional and and demonstrate the practicality in cost and time surgical site healing, and there were no differences in efficiency for cell transplantation using large numbers healing of skin, edema and inflammation in subcuta- of genetically engineered cells. The study reported herein neous or muscular layers between the treatment groups. was designed to use drill hole defects within equine ribs to directly compare the efficacies of these two strategies Computed tomography that were successful in the long bone. Unlike other Quantitative computed tomography (qCT) at 6 weeks animal models such as slit osteotomy and critical-size after injection showed an induction of bone regeneration gap ostectomy,17–19 the rib drill defect model provides a in equine rib drill defects by direct gene delivery and well-contained area for percutaneous injection, a well- DFb-mediated BMP2 therapy (Figure 3a). In the cortical confined filling defect to quantify bone formation filling zone, bone volume was significantly greater in the with high resolution, both cortical and cancellous Ad-BMP2, DFb-GFP and DFb-BMP2 groups (Figure 3c), bone healing environments, and permits multiple and cortical mineral density was significantly greater in bone defects per animal for appropriate control groups the Ad-BMP2 group (Figure 3d). In the medullary filling within the same animal. In addition, the rib drill defect zone, bone volume was significantly greater in the model with a rich cancellous bone environment may be a DFb-BMP2 groups (Figure 3e). Bone density was not more receptive environment to exhibit differences in these changed in any group (Figure 3f). In the adjacent cortex two methods of BMP2 gene delivery mechanisms, zone, mineral density was significantly greater in the because of the potentially greater numbers of osteopro- Ad-BMP2 and DFb-BMP2 groups (Figure 3g). In the genitor cells as receptive targets for paracrine molecular adjacent medullary zone (Figure 3b), mineral density was signals of BMP2. significantly greater in the DFb-BMP2 group (Figure 3h). The goal of our study was to use an equine rib Of the 12 ribs used in 6 horses (3 holes per rib, 36 total drill defect model to evaluate the relative efficacy of holes), 5 ribs were fractured at 1 of the 3 drill holes. The DFb-mediated BMP2 and Ad-BMP2 gene delivery to treatment assignments of these five fractured drill holes promote bone regeneration. We compared the effects were GBSS (2), Ad-GFP (2) and Ad-BMP2 (1), and none of cell-mediated and direct BMP2 gene delivery using of the drill holes with DFb injections were fractured a percutaneous injection of DFbs or viral vectors regardless of the GFP/BMP2 gene transduction. Thus, the with and without BMP2 gene transduction. In this risk of fractures in DFb-injected defects (0 out of 18 model, we demonstrated successfully that an autologous defects) was significantly lower than in non-DFb-injected transplantation of BMP2-expressing DFbs and direct defects (5 out of 18 defects) (P ¼ 0.045). Owing to the Ad-BMP2 induced bone regeneration, and DFb-BMP2 fracture confounding our bone regeneration outcomes, vectors were more potent than a direct vector injection these five fractured drill defects were not included in for cortical bone regeneration. Cell- and gene-mediated qCT and histological evaluation and were treated as BMP2 delivery showed equivalent increased bone missing data points in data analysis. Despite this data regeneration in the cancellous bone region and increased exclusion, the computed powers were 40.8 in all bone density in adjacent osseous tissues. outcomes of qCT and histology. Histological evaluation Results Histomorphometry at 6 weeks after treatments showed denser and more mature bone formation and greater In vitro gene transduction and osteogenic differentiation mineralizing activity in equine rib drill defects by direct Equine DFbs were shown to be permissive to Ad gene gene delivery and DFb-mediated BMP2 therapy (Figures transduction, efficiently induced BMP2 protein and 4 and 5). In the cortical filling zone (Figure 4a), the successfully differentiated into bone-forming cells in vitro. %porosity and %mineralizing area was significantly In six horses, DFbs isolated from skin punch biopsy greater