Utilization of a genetically modified muscle flap for local BMP-2 production and its effects on bone healing: a histomorphometric and radiological study in a rat model
© Momeni et al.; licensee BioMed Central. 2015
Received: 4 December 2014
Accepted: 10 March 2015
Published: 29 April 2015
Aim of the study
We developed an experimental rat model to explore the possibility of enhancing the healing of critical-size bone defects. The aim of this study was to demonstrate the feasibility of this concept by achieving high local BMP-2 expression via a transduced muscle flap that would facilitate bony union while minimizing systemic sequelae.
The transduction potential of the adenoviral vector encoding for BMP-2 was tested in different cell lines in vitro. In vivo experiments consisted of harvesting a pedicled quadriceps femoris muscle flap with subsequent creation of a critical-size defect in the left femur in Sprague-Dawley rats. Next, the pedicled muscle flap was perfused with high titers of Ad.BMP-2 and Ad.GFP virus, respectively. Twelve animals were divided into three groups comparing the effects of Ad.BMP-2 transduction to Ad.GFP and placebo. Bone healing was monitored radiologically with subsequent histological analysis post-mortem.
The feasibility of this concept was demonstrated by successful transduction in vitro and in vivo as evidenced by a marked increase of BMP-2 expression. The three examined groups only showed minor difference regarding bone regeneration; however, one complete bridging of the defect was observed in the Ad.BMP-2 group. No evidence of systemic viral contamination was noted.
A marked increase of local BMP-2 expression (without untoward systemic sequelae) was detected. However, bone healing was not found to be significantly enhanced, possibly due to the small sample size of the study.
A critical-size bone defect is, by definition, unable to regain bony union without intervention. The gold standard treatment of such defects is transplantation of autologous bone . However, donor-site morbidity and limited availability represent drawbacks of this approach. In contrast, allogenic bone grafts are readily available; however, they have a considerably lower osteoinductive potential and bear the risk of transplant rejection as well as transmission of infectious diseases . Bone regeneration through topical application of synthetic growth factors has already been demonstrated. In particular, administration of BMP-2 has been used with considerable success in orthopedic surgery and neurosurgery [3-6]. Limitations of this approach, however, include 1) high cost and 2) need for repeated applications secondary to the short half-life of the protein. Other experimental therapies include the development of injectable biological bone substitutes as well as exposure of human mesenchymal stem cells to BMP-2 ex vivo, thus, inducing their osteoblastic differentiation [7,8]. Transduction of target cells with a replication-deficient adenovirus, designed to carry cDNA encoding for BMP-2, represents a novel approach for local delivery of BMP-2 to the region of interest. Previous studies have shown significant improvement of bone regeneration after local application of BMP-2 carrying adenoviruses to the bone defect in a rat model [9-11]. Injecting very high virus titers directly into the bone defect, however, can lead to systemic spreading of the virus, with a resultant negative impact on other organs, along with initiation of potentially severe or even lethal immunological reactions . In order to minimize the risk of systemic contamination of the replication-deficient adenovirus used in the present study, a pedicled quadriceps muscle flap was harvested and selectively infused with the virus. The concept of using genetically altered soft tissue flaps as biologic pumps has been demonstrated by Liu et al. and Michaels et al. in a rat model [13-15]. The present study represents the first attempt of assessing whether muscle flaps can be altered genetically so as to not only provide soft tissue coverage but also enhance osseous healing by local production of growth factors, as muscle tissue is known to possess an ample quantity of mesenchymal stem cells capable of osteoblastic differentiation . Furthermore, BMP-2 has an angiogenetic potential, which could aid in facilitating the healing process .
Materials and methods
MSC - human mesenchymal stem cells (obtained by bone marrow biopsy at the University of Freiburg Medical Center)
Cal-72 human osteosarcoma cells (ACC 439 Deutsche Sammlung von Mikroorganismen und Zellkulturen Braunschweig, Germany)
SaOs-2 human osteosarcoma cells (ACC 243 Deutsche Sammlung von Mikroorganismen und Zellkulturen Braunschweig, Germany)
HUVEC - human umbilical vein endothelial cells (Promocell, Cat. No. C-12250, Heidelberg, Germany)
Sprague Dawley rat myocyte cell culture isolated from the quadriceps muscle
The cultured cells were incubated on six-well plates at 37°C/5% CO2 until confluence was reached, then 1 ml of virus suspension (Ad.GFP or Ad.BMP-2) was added to the medium. Five days after transduction, the efficiency of transduction was evaluated. BMP-2 levels in the culture medium were measured with a human BMP-2-specific ELISA assay (DBP 200). The cultures transfected with GFP-2 were examined microscopically for UV fluorescence.
Adult male Sprague Dawley rats weighing 400–450 g (Charles River, Sulzfeld, Germany) were used for in vivo experiments. All animals were kept in an approved animal care facility with 12-h light/dark cycles and were allowed food and water ad libitum and unrestricted activity after surgery. All animal studies were approved by the ethical review board of Baden-Württemberg (G 07/33) and conducted in compliance with the guidelines specified in German legislation concerning animal experiments (Tierschutzgesetz, §§ 7 to 9).
Group 1: BMP group (n = 4): muscle flap infused with 1-ml Ad.BMP-2 suspension (8 × 109 PFU/ml),
Group 2: GFP group (n = 4): muscle flap infused with 1-ml Ad.GFP suspension (6 × 109 PFU/ml),
Group 3: control group (n = 4): muscle flap infused with 1 ml of sterile normal saline.
We used a BMP-2 ELISA kit (R&D Systems, DBP-200 Quantine) to quantify BMP-2 expression in both in vitro and in vivo experiments analyzing cell culture supernatants and tissue sample lysates, respectively. The samples were normalized to equal protein quantities, either 100 or 300 μg in different experiments, using a BCA protein Assay Reagent Kit® (Pierce, Thermo Scientific) and added to each well of the ELISA kit. Absorbance was measured at 450 nm and correlated to a standard curve obtained by measuring pre-equilibrated standard samples. The mean minimum detectable dose was 11 pg/ml. All test samples were examined in duplicates or triplicates.
Quantitative real-time RT-PCR
TaqMan RT-PCR was carried out as previously described . Total RNA was isolated from tissue samples using the TRIzol method . Total RNA (0.5 μg) was treated with 3 units of deoxyribonuclease I (DNase I, Invitrogen, Karlsruhe, Germany) to digest genomic DNA contamination. Random-primed cDNA synthesis was performed using 0.5 μg of DNase I-treated total RNA and 50 units of AffinityScript reverse transcriptase according to the manufacturer’s instructions (Stratagene, La Jolla, USA). TaqMan PCR assays were performed in 384-well optical plates on a LightCycler (Roche, Mannheim, Germany) using Absolute QPCR ROX Mix (Abgene, Hamburg, Germany) according to the manufacturer’s instructions. The thermal cycling conditions were 95°C for 15 min followed by 50 cycles at 95°C for 15 s and at 60°C for 1 min. Oligonucleotide primers and probes for human GAPDH (GADPH forward: 5′-TGGGCTACACTGAGCACCAG-3′; GAPDH reverse: 5′-CAGCGTCAAAGGTGGAGGAG-3′, GAPDH probe: 5′-FAM- TCTCCTCTGACTTCAACAGCGACACCC-TAMRA-3′) were designed using Primer Express (Applied Biosystems, Foster City, USA) according to company guidelines. Oligonucleotide primers and TaqMan probe for human BMP-2 (Cat. Nr. Hs00154192) were purchased from Applied Biosystems (Foster City, CA, USA). Assays were performed in triplicate. Data were analyzed using the relative standard curve method, with each sample being normalized to the housekeeping gene GAPDH.
Determining systemic spread of the viral vector
Samples from the spleen, liver, and lung from all animals in the Ad.GFP group were cut into 10-μm cryo-sections and examined using fluorescence microscopy.
All data are presented as mean ± standard error of the mean. Due to the small sample size, no further statistical analysis was performed.
An increase of high-density tissue, corresponding to mineralized bone between the first and last x-ray, was seen in all three groups. The increase was highest in the Ad.BMP-2 group showing an average of 53% high-density tissue in the region of interest vs. 42% and 37% in the GFP and control group, respectively (Figure 3B).
Although nonunions were seen in the majority of the animals, pronounced inter-individual differences within the groups were detected regarding bone formation. A comparison between the three groups showed little difference regarding the area of mineralized bone compared to the entire region of interest (Figure 4B). One case of mineralized bony union was seen in group 1.
Systemic spread of the viral vector (i.e., systemic contamination)
Samples from the spleen, liver, and lung from all animals in the Ad.GFP group were cut into 10-μm cryo-sections and microscopically examined under UV light. Fluorescence was not observed in any of the sections.
In the course of experiments, we lost five animals during the operation in the 24-h period after the operation. Four animals were excluded from the study and underwent euthanasia after a dislocation of the osteosynthesis, two due to wound dehiscence and one due to an ischemia of the operated limb.
Gene therapy is an innovative approach for treatment of large bone defects. Previous studies demonstrated that the adenoviral vector is particularly well equipped for utilization in this regard [9-11]. Its favorable characteristics include easy handling and ability to transduce target tissue effectively. Furthermore, being replication deficient, it does have a favorable safety profile. In order to decrease technical complexity, yet continuing to minimize systemic toxicity and immune response, we modified the concept introduced by Michaels et al. by using the quadriceps femoris muscle as a pedicled flap [14,15]. The current model was chosen, as it resembles the clinical setting, in which muscle flaps are used to cover bone defects. The concept of being able to use a muscle flap not only as soft tissue coverage but also as a biologic pump that secretes growth factors stimulating bone regeneration is intriguing. While a quadriceps femoris free flap has been previously described , we employed its modification as a pedicled flap, thus, reducing technical complexity, ischemia time, and overall risk of flap failure. In none of the animals in group 1 did we detect an increase of BMP-2 expression in tissues other than the transduced muscle flap. PCR showed human BMP-2 mRNA in the transduced flap and absence thereof in the contralateral quadriceps. Samples from the spleen, liver, lung, and contralateral quadriceps of animals in group 2 were examined microscopically and did not display any fluorescence, thus confirming indirectly the lack of undesired systemic transduction.
In the present study, we were able to prove the concept that delivery of BMP-2 via a genetically altered muscle flap is possible without any measurable systemic contamination. Unfortunately, a significant difference between the groups with respect to ossification of the critical-size defects could not be observed. An explanation for this observation is the small sample size with resultant limited power in the present study. We achieved an overwhelming increase of BMP-2 expression in human and rat cell types in vitro, which remained stable on a high level in the first week after transduction. The time kinetics of BMP-2 expression paralleled reports of transgene expression being highest between the 5th and 14th day post-transduction . The fact that even in untreated cell culture supernatants and tissue samples, the BMP-2 ELISA kit detected BMP-2 levels higher than the minimum detectable concentration suggests some cross-reactivity with rodent proteins. An additional effect could be the induction of endogenous BMP-2 expression as a result of bone injury, i.e., creation of the critical-size femoral defect. This phenomenon has already been shown for a myriad of growth factors including PDGF, FGF, IGF, TGF-β, and VEGF [23-27].
In vivo BMP-2 expression was comparatively lower. Human BMP-2 mRNA was however detected in the muscle flaps of all Ad.BMP-2 transduced animals, thus, proving that transduction was successful.
Follow-up x-ray images revealed that the Ad.BMP-2 group showed the most pronounced improvement in the radiographical score for high mineralization. Due to the small sample size and considerable inter-individual differences, however, statistical significance was not achieved. The same applies for the histomorphometric data of the BMP-2 group. In conclusion, we were able to demonstrate that continuous local expression of BMP-2 is possible by means of muscle transduction. Shortcomings of topical BMP-2 application, such as associated cost and necessity for repeated applications secondary to the short half-life of the protein, can, thus, be addressed successfully. Most notably, this approach does not result in systemic contamination with viral particles. Clinical application would be particularly attractive as the need for harvesting autologous bone for reconstruction of composite defects could be reduced or even averted. While this development may not seem to be in reach at present, further development of this model employing larger groups and later larger animal model would provide important clinical data regarding applicability in a clinical setting.
Limitations of the study include the small sample size. The perioperative loss of 12 animals was likely caused by the relatively long, complex, and invasive surgical procedure consisting of microsurgical flap dissection, osteosynthesis, and creation of the critical-size defect as well as the incubation period of 1 h. The loss of animals was evenly spread in all three groups.
Compared to other BMP-2 application models such as direct recombinant BMP injections in the fracture site or the use of biodegradable BMP carrier , the use of an adenovirus as a vehicle of gene delivery has the disadvantage of a possible immune response which can lead to a shorter duration of BMP-2 expression . The results of the present study correspond to data published by Willett et al. showing the reduced osteogenic potential of BMP-2 in vivo after a composite muscle-bone injury compared to osseous injury alone, making further investigation in this topic desirable . Additional studies need to be carried out with larger sample size to address limitation of the present study. These experiments then need to be followed by large animal studies to confirm the effects seen in the rat model. As stated above, a larger animal model would also be appropriate to reduce the observed high dropout rate, since the grade of invasiveness and duration of the procedure are substantial in a small rodent model. Further modifications could include a transient immunosuppression shortly before and after surgery as proposed by Okubo  which may improve the osteogenetic potential of the present model.
Gene therapy is a promising option for the treatment of critical-size bony defects. We explored the effects of adenovirus-mediated, self-sustained BMP-2 production by a transduced pedicled quadriceps muscle flap in a critical-size femoral defect in a rat model. Our aim was to achieve high local BMP-2 expression while simultaneously minimizing the risks of systemic contamination and initiation of an adverse immune response. While statistical significance was not observed with respect to enhanced bone formation, we were able to demonstrate effective transduction and increased local BMP-2 expression by transduced flaps, both without evidence of systemic viral contamination.
The article processing charge was funded by the open access publication fund of the Albert Ludwigs University Freiburg.
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