in situ bone tissue engineering via ultrasound-mediated ... · more than 2 million bone-grafting...

SC I ENCE TRANS LAT IONAL MED I C I N E | R E S EARCH ART I C L E

BONE REGENERAT ION

1Skeletal Biotech Laboratory, Hadassah Faculty of Dental Medicine, The HebrewUniversity of Jerusalem, Ein Kerem, Jerusalem 91120, Israel. 2Department of Sur-gery, Cedars-Sinai Medical Center, 8700 Beverly Boulevard, Los Angeles, CA90048, USA. 3Board of Governors Regenerative Medicine Institute, Cedars-SinaiMedical Center, Los Angeles, CA 90048, USA. 4Biomedical Imaging Research Insti-tute, Cedars-Sinai Medical Center, Los Angeles, CA 90048, USA. 5Department ofBiomedical Sciences, Cedars-Sinai Medical Center, Los Angeles, CA 90048, USA.6Department of Imaging, Cedars-Sinai Medical Center, Los Angeles, CA 90048,USA. 7Department of Biomedical Engineering and the Center for MusculoskeletalResearch, University of Rochester Medical Center, Rochester, NY 14642, USA.8Department of Orthopedics, Cedars-Sinai Medical Center, Los Angeles, CA 90048,USA. 9Department of Biomedical Engineering, University of California, Davis, 451Health Sciences Drive, Davis, CA 95616, USA.*These authors contributed equally to this work.†Corresponding author. Email: [email protected]

Bez et al., Sci. Transl. Med. 9, eaal3128 (2017) 17 May 2017

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In situ bone tissue engineering viaultrasound-mediated gene delivery toendogenous progenitor cells in mini-pigsMaxim Bez,1,2 Dmitriy Sheyn,2,3 Wafa Tawackoli,2,3,4,5 Pablo Avalos,3 Galina Shapiro,1

Joseph C. Giaconi,6 Xiaoyu Da,4 Shiran Ben David,2,3 Jayne Gavrity,7 Hani A. Awad,7 Hyun W. Bae,2

Eric J. Ley,2 Thomas J. Kremen,2,8 Zulma Gazit,1,2,3,8 Katherine W. Ferrara,9

Gadi Pelled,1,2,3,4,5* Dan Gazit1,2,3,4,5,8*†

More than 2 million bone-grafting procedures are performed each year using autografts or allografts. However,both options carry disadvantages, and there remains a clear medical need for the development of new therapies formassive bone loss and fracture nonunions. We hypothesized that localized ultrasound-mediated, microbubble-enhanced therapeutic gene delivery to endogenous stem cells would induce efficient bone regeneration andfracture repair. To test this hypothesis, we surgically created a critical-sized bone fracture in the tibiae of Yucatánmini-pigs, a clinically relevant large animal model. A collagen scaffold was implanted in the fracture to facilitaterecruitment of endogenous mesenchymal stem/progenitor cells (MSCs) into the fracture site. Two weeks later,transcutaneous ultrasound-mediated reporter gene delivery successfully transfected 40% of cells at the fracturesite, and flow cytometry showed that 80% of the transfected cells expressed MSC markers. Human bonemorphogenetic protein-6 (BMP-6) plasmid DNA was delivered using ultrasound in the same animal model, leadingto transient expression and secretion of BMP-6 localized to the fracture area. Micro–computed tomographyand biomechanical analyses showed that ultrasound-mediated BMP-6 gene delivery led to complete radiographicand functional fracture healing in all animals 6 weeks after treatment, whereas nonunion was evident in controlanimals. Collectively, these findings demonstrate that ultrasound-mediated gene delivery to endogenous mesenchy-mal progenitor cells can effectively treat nonhealing bone fractures in large animals, thereby addressing a majororthopedic unmet need and offering new possibilities for clinical translation.

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INTRODUCTIONFracture nonunion, when the normal process of fracture repair isthwarted and the fracture fails to heal, constitutes a complex medicalcondition with an estimated incidence of 100,000 new nonunioncases every year in the Unites States alone (1). These injuries not onlycause patients to suffer but also lead to long-term hospitalization anddisability, repeated surgeries, loss of working days, and considerablecosts to the health system (2). Various factors affect the incidence ofnonunion formation, including fracture pattern, anatomical location,degree of bone loss, and the quality of surgical treatment (3).
Currently, the gold-standard treatment for fracture nonunion isautologous bone grafting (autografts). However, autografts are notalways available and their harvest often leads to prolongedpostoperativepain and substantial donor sitemorbidity (4). Bone allografts are readilyavailable from tissue banks and, therefore, are an alternative to auto-grafts. Unfortunately, allografts have very low osteogenic potential,

leading to poor graft-host integration that results in numerous failuresdue to fractures and nonunion (5). In recent years, recombinant humanbone morphogenetic proteins (BMPs) have been introduced to theclinic, among other indications, for long-bone fracture repair. Localadministration of BMP-2 (6) and BMP-7 (7) in patients with tibialnonunions resulted in increased healing rates. However, BMPs arecostly and have been associated with a high incidence of side effectsincluding infection, heterotopic bone formation, and immunogenicreactions, possibly due to their use in megadoses (8). Thus, fracturenonunion remains a clear unmet clinical need.

An attractive alternative to BMP protein therapy is a local targetedgene therapy (9). Such therapy could facilitate nonunion healing viatransient overexpression of an osteogenic BMP gene without requiringthe production of high-cost recombinant proteins or their administra-tion inmegadoses. Viral BMP gene delivery has been shown to inducenonunion healing in rodents and large animals (10–12). Although viralvectors are considered to be efficient gene delivery methods, they intro-duce risks of malignancy and immunogenic reactions, which greatlylimits their translational potential (13). Nonviral vectors are consideredsafer for human use, albeit much less efficient for gene expression (9).Our group showed that in vivo electroporation of aBMP gene to endog-enous mesenchymal stem cells (MSCs) within a nonunion fracture siteyielded efficient fracture healing in rodents (14). Although successful,this approach requires the insertion of electrodes and conduction ofpowerful electrical currents resulting in pain and tissue damage, limitingits use in clinical applications.

An alternative physical method of gene transfection, sonoporation—the use of ultrasound for gene delivery—is especially attractive (15).Ultrasound is widely used in medicine and considered one of the safest

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imaging modalities. In addition, ultra-sound can be applied noninvasively tomany different regions of interest andcan reach deep-lying tissues with minimalsystemic side effects. Sonoporation usesultrasonic waves to increase cell mem-brane permeability by forming transientnanosized pores in themembrane, subse-quently enhancing uptake of drugs andnucleic acids (16–18). Most studies havecombined ultrasoundwith the adminis-tration of formulated microbubble sus-pensions to enhance the delivery of nucleicacids to target cells (19–23). Such micro-bubble suspensions contain micrometer-sized bubbles filled with a low-diffusivitygas core and stabilized by a shell. Orig-inally designed and U.S. Food andDrugAdministration–approved foruse as bloodpool contrast agents, exposure to ultra-sound drives microbubbles to a forcedoscillation state in which the gas core isrepeatedly compressed and expanded.Within a range of ultrasound parameters,these microbubbles can deform the cellmembrane and form membrane pores,thus enhancing the cellular uptake ofdrugs and nucleic acids.

Recently, we showed that the combi-nation of ultrasound and microbubblescan be used to overexpress a reporter genein endogenous MSCs recruited to a frac-

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ture site in a large animal model (24). Although ultrasound-mediatedBMP gene delivery was shown to induce ectopic bone formation invivo (25–27), previous attempts at fracture repair were not successful(25). Here, we hypothesized that microbubble-enhanced, ultrasound-mediated gene delivery of a BMP gene would lead to efficient bone re-generation and fracture healing in a clinically relevant, large animalmodel (porcine). Multiple structural, biomechanical, and safety para-meters weremonitored to demonstrate the feasibility of this therapeuticapproach for human use.

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RESULTSSonoporation leads to reporter gene expression inendogenous MSCs recruited to fracture siteMini-pigs underwent surgery during which a 1-cm critical-sized tibialbone fracture was created, a biodegradable collagen scaffold was im-planted, and fractures were internally fixated with custom-made dy-namic compression plates (fig. S1). The fracture sites were treated withmicrobubble-enhanced, ultrasound-mediated gene delivery 14 daysafter surgery, when maximal cell recruitment of mesenchymal lineagewas noted (fig. S2). Plasmid DNA encoding a reporter gene [greenfluorescent protein (GFP) or luciferase] mixed with microbubbleswas transcutaneously injected into the fracture site, and half of theanimals were randomly assigned to receive ultrasound treatment (fig.S3). Ex vivo optical imaging of the fracture site revealed fluorescentsignal localized to the fracture site in GFP-treated animals (Fig. 1A).Flow cytometry revealed that 40% of cells collected from within the


tibial fracture of animals treated with ultrasound (US) expressed GFPsix times greater than that observed in untreated animals (NoUS, P=0.049; Fig. 1B). GFP-expressing cells from bone fractures in ultrasound-treated animals were also three times more fluorescent (P = 0.0013;Fig. 1C). In another experiment, cells isolated from bone fractures inanimals treated with luciferase and ultrasound demonstrated 80 timesmore luciferase activity than cells from fracture sites in animals notsubjected to ultrasound (P = 0.0001; Fig. 1D). An examination of thetransfected cells showed significantly more cells expressing MSCmarkers CD29 and CD90 in the ultrasound-treated group: 90% ofGFP-positive cells in the ultrasound-treated group were CD29-positiveand 80% of GFP-positive cells were CD90-positive, whereas only 30%of GFP-positive cells in the untreated group were CD29-positive (P =0.0001; Fig. 1E) and 40%were CD90-positive (P = 0.0057; Fig. 1F). Nosignificant differenceswere observed inCD44-positive cells, a cellmarkerof mesenchymal lineage (P = 0.0531; Fig. 1G). Overall, ultrasound-treated fracture sites exhibited significantly higher progenitor transfec-tion rates and stronger transgene expression.

Sonoporation induces transient BMP-6 expression at bonefracture siteEighteen pigs received tibial fractures as described above and weretreatedwith ultrasound after local injection ofBMP-6 andmicrobubbles(BMP-6 + US) or injection of BMP-6 and microbubbles without ultra-sound application (BMP-6 control). Local expression of BMP-6 geneafter sonoporation to tibial bone fractures was evaluated over time.Quantitative polymerase chain reaction (PCR) analysis of isolated

Fig. 1. Reporter gene expression in mini-pig tibial fractures after ultrasound-mediated gene delivery. (A) Ex vivofluorescence imagingof anexposedGFP-treated fracture5days after treatment. Fracturemargins are representedbyadashedrectangle. (B) Flow cytometry analysis of the percentage of GFP+ cells isolated from fracture sites 5 days after treatmentwith orwithout ultrasound (*P = 0.049). (C) Mean fluorescence intensity per cell isolated from fractures 5 days after treatment with orwithout ultrasound (**P=0.0013). (D) Luciferase enzyme activity in cells isolated from fracture sites 5 days after treatmentwithorwithout ultrasound (****P=0.0001). Percentage of (E) CD29+ (****P=0.0001), (F) CD90+ (**P=0.0057), and (G) CD44+ cells(P = 0.0531) out of GFP+ cells isolated from fractures 5 days after treatment with or without ultrasound. Data are means ±SEM; P values were determined by two-tailed Student’s t tests (No US, n = 3; US, n = 4; RLU, relative light units).

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cells from the fracture site showed 850- and 400-fold higher BMP-6expression in ultrasound-treated animals compared to control animalson days 2 and 5 after treatment, respectively (Fig. 2A). On day 10,BMP-6 gene expression was undetectable. Similarly, BMP-6 proteinsecretion in the fracture site was 70- and 120-fold higher on days 2and 5 after treatment, respectively, compared to animals that werenot treated with ultrasound (Fig. 2B). Negligible amount of BMP-6protein was detected on day 10. In addition, no overexpression ofBMP-6 was found in off-target tissues (fig. S4).

BMP-6 sonoporation facilitates in vivo bone regenerationNext, the therapeutic effect of local BMP-6 gene delivery was evaluatedin the same pig model of tibial nonhealing fracture. Tibial fractureregeneration in BMP-6 + US–treated animals was compared to anontreated control group (Scaffold only), animals treated with plasmidpremixed withmicrobubbles without ultrasound (BMP-6 only) or anempty plasmid premixed with microbubbles and ultrasound (US only),and a gold-standard control group treatedwith an autograft (Autograft).Micro–computed tomography (mCT) analysis 8 weeks after surgeryrevealed that fractures treated with BMP-6 + US regenerated 75%of their volume with new-formed bone, which was equivalent toautograft transplantation (P = 0.102) and doubled the bone volumedensity seen in all other treatment groups (P = 0.0001; Fig. 3A). Nodifferences in healing were noted betweenmale and female pigs with-in the BMP-6 + US group (P = 0.3945; fig. S5). In addition, the new-formed bone in the BMP-6 + US group was significantly more calcifiedthan in all other groups (P= 0.0029; Fig. 3B) except the Autograft group(P = 0.068). Only fractures treated with BMP-6 + US or autograftshowed union, evident by the complete cortical continuity of the tibia(Fig. 3C). Histological analysis showed that there was a complete re-generation of the fracture with mature new-formed bone and thepresence of osteocytes in BMP-6 + US–treated pigs (Fig. 3D). All othergroups showed evidence of insufficient bone formation, abundant gran-ulation tissue deposition, and fibrosis (Fig. 3D).

BMP-6 gene delivery induces functional healing oftreated bonesTorsional testing was performed blindly on tibiae excised from theoperated animals to examine the mechanical properties of the treatedbones 8 weeks after surgery. In accordance with the mCT and histolog-


ical analysis results, tibial bones from the BMP-6 +US group showedat least twofold higher mechanical stiffness (P = 0.02), strength (P =0.0048), and toughness comparedwith tibial fractures from theBMP-6–only group (P= 0.0077; Fig. 4). No significant differences were observedbetween the BMP-6 + US and the Autograft groups (P = 0.1552).

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DISCUSSIONHere, we used ultrasound-mediated, microbubble-enhanced genedelivery for in situ bone engineering in a mini-pig model of tibialfracture repair. We showed that ultrasound-mediated BMP-6 deliveryto cells residing within the fracture site achieved transient local geneexpression, mainly targeting endogenousMSCs. Osteogenic gene de-livery resulted in a spatial and temporal expression pattern that isconsistent with nonviral gene delivery systems. Furthermore, it resultedin complete fracture union within 8 weeks with autograft-equivalentbiomechanical properties. No evidence of ectopic transgene expressionor inflammatory response was observed, suggesting a favorable safetyprofile. Using a clinical ultrasound system within the range of para-meters used in B-mode ultrasound imaging of microbubble contrastagent in humans, we saw no clinical or histological signs of heatingor heat-associated damage.

Almost 20 years ago, Bonadio et al. (28) showed for the first time thatnonviral gene delivery can achieve complete fracture repair in a largeanimal model (canine). Using gene-activated matrix implantscontaining plasmid DNA encoding human parathyroid hormone,complete critical-sized tibial fracture repair was achieved by 18 and8 weeks for 1.6- and 1.0-cm-sized fractures, respectively. Since then,other works involving large animals focusedmainly on viral gene deliv-ery. Egermann et al. (29) showed improved fracture repair after BMP-2adenoviral gene delivery in a tibial fracture in osteoporotic sheep.Dai et al. (30) showed improved fracture repair after implantation ofBMP-2 virally transduced bonemarrow–derivedMSCs into a goat tibialfracture. Despite these promising results, viruses have many safetyconcerns, limiting their use in humans. Xu et al. (31) showed thatadenoviral BMP-2 gene delivery to a fracture site resulted in temporarycellular and persistent humoral immune responses against the viralvector. In addition, clinical trials have shown that viral gene deliverycan evoke a systemic inflammatory response resulting in death (32)and may cause carcinogenesis by random gene insertions into thegenome (33, 34). Thus, efficient nonviral gene delivery methods mustbe developed for clinical use.

Ultrasound-mediated BMP gene delivery had not been successfullyapplied for fracture repair previously. This is not to be mistaken withlow-intensity pulsed ultrasound, which uses different ultrasoundparameters, and is applied daily over a period of weeks to the fracture site(35). Feichtinger et al. (25) used microbubble-enhanced, ultrasound-mediated gene delivery of BMP-2 and BMP-7 in a femur nonunionmodel in rats. Despite successfully inducing gene overexpression andectopic bone formation using repetitive treatments, they could notinduce significant orthotopic bone formation in a fracture site. Thesedifferences can be attributed to diminished osteogenic potentials ofBMP-2 and BMP-7 within the fracture site microenvironment, asreported in other animal and clinical studies (36–41). The biological re-sponse to BMPs is dependent on the cell type and microenvironmentthat is present at the site of BMP delivery (42–44). BMP-6 has a distinctBMP receptor type 1A specificity, making it less affected by endogenousBMP inhibitors while strongly promoting osteogenesis by osteoblasts(45, 46). Previously, we showed that BMP-6 has better osteogenic

Fig. 2. BMP-6 expression after ultrasound-mediated gene delivery to tibiafractures in mini-pigs. (A) Gene [relative quantification (RQ); *P = 0.0111, ****P =0.0001] and (B) protein expression (**P = 0.0085, ****P = 0.0001) in tibial fracturesites 2, 5, and 10 days after ultrasound-mediated BMP-6 gene delivery. Data aremeans ± SEM; P values were determined by two-way analysis of variance (ANOVA)with multiple comparisons (n = 3 per experimental group).

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potential and induced more efficient bone formation than BMP-2 invitro and in vivo when overexpressed in MSCs (47). Some studieshave suggested that BMP-6 signaling works through mechanisms ofboth osteochondral and intramembranous ossifications, with the latterbeing the primary pathway, thus resulting in more rapid calcificationwithin the fracture site (44). Therefore, BMP-6 could serve as a morepotent orthotopic bone inducer.

In contrast to the study conducted by Feichtinger et al., our studyused a two-step procedure that included recruitment of endogenousprogenitor cells to the fracture site. We showed that by implanting acollagen scaffold into the fracture site, efficient endogenous progenitormigration and retention in the scaffold could be achieved within 14 days.Betz et al. (10) showed thatdelayed administrationof aBMP-2–encoding


adenoviral vector improved bone forma-tion in a critical-sized femoral fracture inrats. It is likely that delayed treatmentallows for more progenitors to populatethe fracture site, making the vector moreefficient in targeting cells that respond toBMP secretion and induce bone regen-eration. Overall, it seems that the combi-nation of a potent osteogenic proteintogether with an effective delivery strategyto a viable progenitor population is key toefficient fracture repair.

A modification of the method de-scribed here could include preloading ofmicrobubbles and plasmid into the scaf-fold, which would eliminate the need toinject these materials in a subsequentprocedure. A recent study applied ultra-sound to intramuscularly implanted fibrin/collagenhybridmatrices containingmicro-bubbles,BMP-2/7 coexpression plasmids,and C2C12 cells in mice (48). Althoughthat study combined exogenous cellswith-in the scaffold, only a small amount ofnew bone was generated in vivo. In ourtwo-step method, the recruitment of en-dogenous progenitor cells is an importantfactor that occurs over the course of atleast 2weeks, aswe have previously shownin rodents (14, 24). Hence, implantingscaffolds containing plasmids and micro-bubbleswould require long-termprotec-tion of these components because nakedDNA is degraded by deoxyribonucleases,andmicrobubbles have a short half-life ofabout 10 min when injected locally. Tothe best of our knowledge, there are tech-nological barriers that still need to be over-come togenerate suchacomposite scaffold.However, local injection, as applied here,is attractive for near-term studies becausethe properties can be well controlled andinjections can be repeated andmonitoredas needed.

The clinical gold standard for non-union treatment uses autografts (49).

As a living tissue, their osteoinductive and osteoconductive propertiesmake them far superior over other types of grafts (50). The mostcommon donor site for bone grafting is the patient’s own iliac crest.Because the autografts used here were the native osteotomized tibiae,they were far superior to autografts available in a clinical setting. Thisautograft group represents a hypothetically perfect case scenario, inwhich an exact autologous replica of the lost bone was implantedwithin the fracture site. The autografts used in this study were structur-ally identical to the fractured site in size, shape, and bone content. Thisallowed efficient bone formation and complete bridging of the defectin 8weeks, as seen in our results. In addition, their harvest did not resultin donor-site morbidities, shortening the surgical time and improvingrecovery. Moreover, these autografts were attached to a periosteal blood

Fig. 3. Ultrasound-mediated BMP-6 gene delivery to mini-pig tibial bone fractures. Quantitative analysis ofbone formation in the tibial fractures, including (A) bone volume density (****P = 0.0001) and (B) apparent density(**P = 0.0029). Data are means ± SEM; P values were determined by one-way ANOVA with multiple comparisons. (C) Rep-resentative mCT slices of the fractures 8 weeks after surgery. Asterisks represent new bone formation within the fracture.Arrows point to cortical discontinuity indicating nonunion within the fracture. Autograft margins are represented by adashed square. (D) Masson’s trichrome staining of tibial fractures 8 weeks after surgery at low magnification (uppersubfigures) and highmagnification of the yellow square (lower subfigures). Arrows point to the border between nativebone andnewly formedbone in the fracture. Scale bars, 1mm. (Autograft,n=7; BMP-6+US,n=8; BMP-6,n=6; USonly,n=3; Scaffold only, n = 4; HA, hydroxyapatite.)

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supply. These factors were shown to improve fracture healing (51, 52).Runyan et al. (53) showed that this approach resulted in superiorfracture repair relative to other types of grafts. The similarities foundbetween the Autograft and the BMP-6 + US groups serve as a proof ofconcept that our proposed therapy is as efficient as an “optimal” auto-graft for the treatment of critical-sized fractures.

A final difference between our study and that of Feichtinger et al. isthe use of ultrasound imaging to monitor microbubble oscillationduring treatment. This is a key aspect of the protocol because, althoughultrasound therapy is highly promising, ultrasound wave propagationis limited by tissue properties. Both bone and fixation plates are highlyreflective of ultrasound waves. These reflections can significantly alterthe ultrasound field in the fracture site to lower transfection efficacy.This effect was observed in other works, requiring longer ultrasoundexposure time for effective gene delivery (54). For efficient gene deliv-ery using our proposed therapy, imaging provides the opportunity toensure sufficient sonoporation despite the fixation plates that have thepotential to impede the ultrasonic waves. More robust ultrasoundsystems can be implemented to bypass these hurdles in future humantherapies (55).

One of the parameters not tested in this study is the use of repeatedtreatments. Because of the therapy’s relative ease and low invasiveness,repeated treatments at various time points might further enhance theeffect of osteogenesis and promote faster and improved bone repair.


Thus, implementation of multiple treatments could be another ap-proach in future ultrasound-mediated gene delivery studies. However,note that prolonged ultrasound exposure could result in a variety of ad-verse effects, including excessive tissue heating and dystrophic calcifica-tions due to tissue damage (22). Using higher ultrasound intensities, thecombination of ultrasound and microbubbles can also result in cellularlysis due to jetting during microbubble collapse within the tissue (56).

The current study evaluated the effect of ultrasound-based BMPgene delivery for a short term. A long-term study is required to fullyassess the efficiency of the method in bone healing compared to controls.Here, all animals received collagen scaffold implants that have beenpreviously shown to have some osteoconductive properties (14, 57).However, even with the inclusion of the scaffold, and within a shortperiod of time, we were able to show significantly higher union rate,bone formation, and biomechanical properties among BMP-6 andultrasound–treated animals.We can conclude that the combined therapyof a collagen scaffold, BMP-6 plasmid, microbubbles, and ultrasoundis superior to the control groups and is similar to autograft-treatedanimals.

These results show great promise for future ultrasound-mediatedgene therapies to tissues. Because ultrasound technology is safeand widely used in the clinic, this approach can easily be translatedinto clinical practice. The introduction of microbubbles allows local-ization of the injected material and real-time monitoring of the sono-poration procedure in the treatment site. This method has the potentialto be used for many different applications, promoting in situ tissueengineering. In the context of bone fractures, this therapy can be appliedto a variety of orthopedic indications. It isminimally invasive, does notrequire ex vivo stem cell manipulation or the patient’s own bone har-vest for implantation, and avoids use of costly growth factors. Becauseno alternative efficient method of inducing bone regeneration in siteswith severe bone loss has yet been found, ultrasound-mediated genetherapy is a promising tool that might offer a positive response to thisunmet clinical need.

MATERIALS AND METHODSStudy designThe objective of our study was to develop a therapy for critical-sizedbone fracture repair consisting of ultrasound-mediated gene targetingto endogenousMSCs. Our prespecified hypothesis was that ultrasound-mediated, microbubble-enhanced gene delivery of BMP-6 would leadto efficient bone regeneration and fracture healing in a clinically rele-vant, large animal model. Male and female Yucatán mini-pigs (S&SFarms) were used in this study. The mean weight ± SD of the animalswas 37.0 ± 3.6 kg, and their mean age ± SD was 7.8 ± 1.2 months. Thesample size used was estimated to achieve a power of 0.8 and a = 0.05using one-way ANOVA.

Sonoporation was investigated for its capacity to regenerate a critical-sized tibial bone fracture. Pigs underwent surgery consisting of thecreation of a 1-cm critical-sized bone fracture in the tibia bone. Duringthe same surgery, a collagen scaffold was implanted within the fracturesite to recruit endogenous MSCs. Fourteen days later, the pigs wererandomly assigned either to receive the treatment, which consistedof an injection of BMP-6 plasmid premixed withmicrobubbles to thefracture site followed by ultrasound application (BMP-6 +US), or to oneof the control groups. The following control groups were used: (i) Notreatment (Scaffold only); (ii) BMP-6 plasmid premixed with micro-bubbles injection (BMP-6); or (iii) empty plasmid vector premixed

Fig. 4. Biomechanical properties of treated tibiae. Torsion testing was per-formed on tibiae harvested from treated mini-pigs. (A) Photograph of a tibiareaching its failure point during biomechanical testing. Analysis of load and rota-tion was performed to determine the (B) maximum torque (strength; **P =0.0048), (C) torsional rigidity (stiffness; *P = 0.02), and (D) energy to maximum(toughness; **P = 0.0077) of treated bones. Data are means ± SEM; P values weredetermined by one-way ANOVA with multiple comparisons (N·m, Newton meter;n = 5 per experimental group).

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with microbubbles injection followed by ultrasound application (USonly). In addition, a control group treated with an autograft (Autograft)was used to assess treatment efficacy compared to the gold-standardtreatment for large fractures. Sonoporation potencywas estimated usingreporter gene expression and tissue analysis forBMP-6 gene andproteinexpression. Bone formation was assessed using mCT, histology, andbiomechanical testing. Animals that developed acute procedural com-plications, such as bone fixation failure or signs of distress during follow-up that compromised animal welfare, were eliminated from the study.

Plasmid DNA productionPlasmids encoding luciferase under the control of the ubiquitin promoter(pUb-Luc2) and enhanced GFP under the control of the cytomegalovi-ruspromoter (pCMV-EGFP-N1)wereused to study transgene expression.A plasmid encoding BMP-6 under the control of the cytomegaloviruspromoter (pCMV-BMP6) was used to induce bone regeneration. Plas-mid preparation was detailed elsewhere (58, 59). All plasmids wereexpanded using standard laboratory procedures and purified using anEndoFree Kit (Qiagen).

Critical-sized tibia fracture model: Surgical procedureAll animal procedures were approved by the Cedars-Sinai MedicalCenter institutional review board for animal experiments (IACUC#004740). A critical-sized circumferential bone defect of 1 cm in lengthwas created in the tibia bones of Yucatán mini-pigs per previouslydescribed method (60). A critical-sized defect in humans was definedas “involving 50% of the cortical diameter of the tibia and >1 cm inlength” (61). The average length of the tibia bone in the Yucatánmini-pig is 10 cm, and in an adult human it is 43 cm. Hence, a 1-cmdefect in the mini-pig can be compared to a 4.3-cm defect in an averageperson. After an 18-hour preoperative fast, each mini-pig was sedatedusing intramuscular acepromazine (0.25 mg/kg), ketamine (20 mg/kg),and atropine (0.02 to 0.05 mg/kg). The animal was then administeredpropofol (2mg/kg) intravenously, and endotracheal intubationwas per-formed. Anesthesia was maintained using 1 to 3.5% inhaled isofluranefor the duration of the procedure. A 10-cm anteromedial skin incisionwas made over the tibia. Soft tissue and periosteum were bluntlydissected to expose the tibial diaphysis. An oscillating saw (CONMED)was used to remove 1 cm of bone and create a segmental bone fracture.The fracture was internally fixated using a custom-made six-holelimited-contact dynamic compression plate (Veterinary OrthopedicImplants; fig. S1), and a flat sheet of biodegradable collagen scaffold(DuraGen Matrix, Integra LifeSciences) with a pore size of 100 mmwas shaped and implanted in the fracture site. For autograft-treatedanimals, the resected bone segment was grafted into the fracture andfixated using the fixation plate. Finally, the subcutaneous tissuewas closed with an absorbable subcutaneous suture, and the skin wasclosed using a nonabsorbable suture, which was removed 2 weeks aftersurgery. The animal received perioperative antimicrobial prophylaxis(ceftiofur, 0.05 ml/kg) and postoperative analgesia (buprenorphine,0.2 mg/kg).

Flow cytometry characterization of cells recruited tofracture siteNine animals underwent surgery and were anesthetized and sacrificedby injection of veterinary euthanasia solution (VetOne) on postoperativedays 7, 14, and 21 (three animals were euthanized per time point). Post-mortem tissue was extracted from the fracture site, washed withphosphate-buffered saline (PBS), digested using 0.1% collagenase (type


1A, Sigma-Aldrich) for 1 hour, filtrated using a 70-mm cell strainer, andcentrifuged at 2000 rpm for 7 min. Freshly isolated cells were analyzedforMSC surface markers, taking into consideration the limited avail-ability of anti-pig antibodies. The cells were stained with mouse anti-human (with cross-reactivity to pig) CD90, mouse anti-pig CD29(BD Biosciences Pharmingen), and rat anti-pig CD44 (FitzgeraldIndustries International). Primary antibodies were detected by applyingfluorochrome-conjugated secondary antibodies rat anti-mouse–PE(phycoerythrin) (BD Biosciences Pharmingen) and donkey anti-rat–PE(Imgenex Corp.) according to the manufacturer’s recommendations.The cells were analyzed and plotted using an LSR II flow cytometer,BD FACSDiva, and FCS Express software (BD). Nonspecific bindingof secondary antibodies was quantified, and the fluorescence signalwas subtracted from the detection values of the experimental group.

Ultrasound-mediated, microbubble-enhanced gene deliveryFourteen days after fracture creation, scaffold implantation, and bonestabilization, each animal was sedated in the manner described above.First, amicrobubble suspension (DEFINITY, LantheusMedical Imaging)composed of a gas core containing octafluoropropane and a lipid shellwas activated by 45 s of shaking using a VIALMIX shaker (LantheusMedical Imaging). Then, 107 microbubbles and 1-mg plasmid DNAwere mixed together to a final volume of 500 ml. To avoid microbubbleaggregation and adhesion to syringe walls, we manually rotated thesyringe containing the mixture periodically before injection. Next,the fracture was located using a Fluoroscan mini C-arm (Hologic),and an 18-gauge needle was inserted into the center of the fracture (fig.S3A). The mixture was injected while being visualized using a Sonos5500 (Philips Ultrasound) unit equipped with a focused S3 probeset to B-mode with its focal point matching the location of the defect(fig. S3, B and C). Then, a therapeutic ultrasonic pulse was appliedusing cadence contrast agent imaging mode at a transmission fre-quency of 1.3 MHz, a mechanical index of 0.6, and a depth of 4 cmfor about 2min until microbubble oscillation could no longer be visual-ized (fig. S3D). Repeated oscillation of the microbubbles eventuallyresults in dissolution as a result of either fragmentation or diffusion (62).

Transfection efficacy evaluation: GFP expressionSix animals underwent surgery. Fourteen days later, the mini-pigs wereinjected with GFP plasmid premixed with microbubbles. The animalswere randomly assigned so only half were treated with ultrasoundimmediately after the injection. Five days after transfection, the animalswere sacrificed. Ex vivo optical imaging of the fracture site was per-formed using a fluorescence imaging system (IVIS; PerkinElmer) tolocalize the signal within the treated limb. Then, cells from the fracturesites were isolated as described above. The cells were examined usingflow cytometry to detect GFP expression. The percentage of GFP-positive cells served as a measure of transfection rate.

Transfection efficacy evaluation: Luciferase expressionSix animals underwent surgery and 14 days later were injected withluciferase plasmid premixed with microbubbles. The animals wererandomly assigned so only half were treated with ultrasound imme-diately after the injection. Five days after transfection, cells from thefracture sites were isolated as described above and incubated withluciferase lysis buffer (Promega). The resulting homogenate was cen-trifuged at 10,000g for 10min, and 100 ml of luciferase reaction buffer(Promega) was added to 20 ml of the clear supernatant. Light emissionwas measured by a luminometer (TD-20/20; Turner BioSystems) in

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RLU. The values were normalized for protein content, which wasmeasured using bicinchoninic acid assay (Pierce). Luciferase activitywas expressed as RLU per milligram of protein.

BMP-6 gene and protein expression analysisEighteen animals underwent surgery and 14 days postoperatively wereinjected with BMP-6 plasmid premixed with microbubbles. The animalswere randomly assigned so only half were treated with ultrasound imme-diately after the injection. Then, animals were randomly sacrificed 2,5, or 10 days after ultrasound transfection. Tissues were collectedpostmortem to characterize BMP-6 gene and protein expression.

Quantitative reverse transcriptionPCRs (RT-PCRs)were performedto quantify BMP-6 gene expression in various organs after ultrasoundtransfection. Tissues from the fracture site, brain, bonemarrow, lung,liver, heart, skeletalmuscle, and spleenwere harvested postmortem, andRNA was isolated using an RNeasy extraction kit (Qiagen). The RNAwas then reverse-transcribed using random primers and reverse tran-scriptase (Promega). Expression of BMP-6 gene was analyzed using theABI 7500 Prism system (Applied Biosystems) with Hs00233470_m1primer (ABI). 18S was used as a housekeeping gene control.

Enzyme-linked immunosorbent assay (ELISA) was used to estimateBMP-6 protein expression in fracture over time. Tissue from the frac-ture site was homogenized using tissue homogenizer (VWR) and in-cubated with proteinase inhibitors (Roche) at 4°C for 2 hours. Theresulting homogenatewas centrifuged at 12,000 rpm for 10min, and thesupernatant was collected for a BMP-6 ELISA assay (R&D Systems).Values were normalized for protein content, which wasmeasured usingbicinchoninic acid assay (Pierce).

Bone formation analysis using mCTTwenty-eight mini-pigs underwent surgery and were randomly as-signed to receive the following 14 days later: (i) No treatment (“Scaffoldonly” group); (ii) BMP-6 plasmid premixed with microbubbles (“BMP-6” group); (iii) empty plasmid vector premixed with microbubbles andultrasound (“US only” group); (iv) BMP-6 plasmid premixed withmicrobubbles and ultrasound (“BMP-6 + US” group); or (v) autograftimplantation (“Autograft” group). Eight weeks postoperatively, theanimals were euthanized and their tibiae were harvested for ex vivo,high-resolution mCT (vivaCT 40; SCANCOMedical AG), as previouslydescribed (63). Microtomographic slices were acquired using an x-raytube with a 55-kVp (kilovolt peak) potential and reconstructed at a vox-el size of 35 mm. The fracture was evaluated using histomorphometricthree-dimensional (3D) evaluation. A constrained 3D Gaussian filter(s = 0.8, support = 1) was used to partly suppress noise in the volumeof interest. The bone tissue was segmented from marrow and softtissue using a global thresholding procedure. A quantitative assess-ment of bone volume density and apparent density based on micro-tomographic data sets was created using direct 3D morphometry.

Histological evaluationOne specimen from each treatment group was used for histologicalevaluation. The specimens were cleaned and fixed in 4% formalinfor 3 days. Dehydration was accomplished using a graded series of ethylalcohols and three stages of xylene. Infiltration was performed usinga graded series of xylene and Osteo-Bed (Polysciences) resins, followedby a catalyzed mixture of Osteo-Bed resin containing benzoyl peroxide(1.40 g/100 ml). Embedding was performed using a final catalyzedresin mixture of Osteo-Bed resin solution containing benzoyl peroxide(2.5 g/100ml). Tissue sections were cut at a section thickness of 5.0 mm


onaRM2155 rotarymicrotome(Leica)byusing tungsten carbideD-profileknives. The sectionswere stainedusingmatrix-specificMasson’s trichromestaining. In brief, tissue sections were treated overnight in Bouin’s solutionat room temperature. Slides were rinsed for 10min under running waterand stained with Weigert’s hematoxylin for 5 min. The slides werethen rinsed and stained with scarlet-acid fuchsin for 5 min and rinsedagain, after which the slides were again stained with phosphomolybdic-phosphotungstic, aniline blue, and 2% acetic acid for 5 min each. Final-ly, the slides were rinsed, dried, and mounted. The slides were imagedusing a four-channel Laser Scanning Microscope 780 (Zeiss) with ×20magnification, z-stacking, and 5 × 5 tile scanning. For zoom-in images,a single z-stacked image was generated. All samples were scanned usingthe same gain and exposure settings.

Biomechanical analysisFifteen samples from the “Autograft,” “BMP-6 + US,” and “BMP-6only” groups (n = 5 per group) were used for biomechanical analysis.After harvest, the samples were wrapped in saline-soaked gauze, sealed,and frozen until analysis. Before analysis, the samples were thawedfor about 1 hour and then allowed to rehydrate in PBS for 2 hours. Acustom-made alignment jig was used to fix the proximal and distalends of the tibia in two aluminum square pots by submerging 2.5 cmof each end in Bosworth Fastray Cement (Midway Dental Supply).The gauge length of the exposed tibia between the two fixed ends wasmeasured using calipers before testing. Torsion testing was performedusing a custom-designed jig on an Instron ElectroPuls E10000 (Instron).The rotational actuator was rotated at a rate of 1 deg/s until failure. Loadand rotation data were continuously recorded during testing. Thecollected data were analyzed using a custom MATLAB code (datafile S1) to determine the torsional rigidity, yield torque, ultimate torque,rotation-to-yield torque, rotation-to-ultimate torque, work-to-yieldtorque, and work-to-ultimate torque.

Statistical analysisGraphPad Prism 5.0f software (GraphPad Prism) was used to analyzethe data. Results are presented as means ± SEM. Data analysis was con-ducted using Student’s t test, one-way ANOVA, or a two-way ANOVAwith Tukey’s multiple comparison post hoc test. To assess significance,we considered P < 0.05 statistically significant. Individual-level data areshown in table S1.

SUPPLEMENTARY MATERIALSwww.sciencetranslationalmedicine.org/cgi/content/full/9/390/eaal3128/DC1Fig. S1. Critical-sized bone fracture model in Yucatán mini-pigs’ tibia.Fig. S2. Endogenous mesenchymal progenitor cell recruitment to mini-pig tibial fracture site.Fig. S3. Ultrasound treatment setup.Fig. S4. Biodistribution of BMP-6 expression after ultrasound-mediated gene delivery.Fig. S5. Comparison of bone formation between male and female mini-pigs.Table S1. Primary data.Data file S1. MATLAB code of biomechanical analysis.

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Acknowledgments: We thank S. De Mel for the dedicated laboratory work during this study.Funding: We acknowledge funding from California Institute for Regenerative Medicine (CIRM)grant TR4-06713, a grant from IDF Medical Corps, a grant from Milgrom Family (D.G.), NIHR01CA112356 (K.W.F.), and NIH P30AR069655 (H.A.A.). Author contributions: G.P., Z.G.,E.J.L., K.W.F., and D.G. contributed to the concept of the study and revised the manuscript. G.P.,M.B., and D.G. designed the experiments. M.B., W.T., P.A., T.J.K., and H.W.B. performed large-animal surgical procedures. M.B., D.S., W.T., and J.C.G. performed ultrasound-mediated genedelivery experiments. M.B., D.S., and W.T. extracted bone and soft tissue samples. M.B., D.S.,and S.B.D. performed flow cytometry data analysis. S.B.D. performed RT-PCR and ELISA assays.J.G. and H.A.A. performed biomechanics testing. M.B. and G.S. performed the statisticalanalyses. M.B., W.T., and X.D. performed mCT imaging and biomechanical data analysis.M.B., G.S., and G.P. wrote the manuscript. Competing interests: T.J.K., G.P., and D.G. areinventors on patent application (PCT/US2017/020033) submitted by Cedars-Sinai MedicalCenter that “covers the use of ultrasound for endogenous stem cell activation.” G.P., Z.G.,and D.G. are shareholders in GamlaStem Medical Inc., which did not provide funds for thisstudy. All other authors declare that they have no competing interests. Data and materialsavailability: All materials are available from commercial sources or can be derived usingmethods described in this study. All relevant data are reported in the article.

Submitted 3 November 2016Resubmitted 8 February 2017Accepted 14 April 2017Published 17 May 201710.1126/scitranslmed.aal3128

Citation: M. Bez, D. Sheyn, W. Tawackoli, P. Avalos, G. Shapiro, J. C. Giaconi, X. Da, S. B. David,J. Gavrity, H. A. Awad, H. W. Bae, E. J. Ley, T. J. Kremen, Z. Gazit, K. W. Ferrara, G. Pelled, D. Gazit,In situ bone tissue engineering via ultrasound-mediated gene delivery to endogenousprogenitor cells in mini-pigs. Sci. Transl. Med. 9, eaal3128 (2017).

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progenitor cells in mini-pigsIn situ bone tissue engineering via ultrasound-mediated gene delivery to endogenous

Gadi Pelled and Dan GazitDavid, Jayne Gavrity, Hani A. Awad, Hyun W. Bae, Eric J. Ley, Thomas J. Kremen, Zulma Gazit, Katherine W. Ferrara, Maxim Bez, Dmitriy Sheyn, Wafa Tawackoli, Pablo Avalos, Galina Shapiro, Joseph C. Giaconi, Xiaoyu Da, Shiran Ben

DOI: 10.1126/scitranslmed.aal3128, eaal3128.9Sci Transl Med

critical-sized tibial nonunions in mini-pigs.. This therapy led to transient BMP-6 secretion, bone regeneration, and fracture healing over 6 weeks inBMP-6

and ultrasound was applied to oscillate the microbubbles, which helped the recruited progenitors take up the ) plasmid DNA and microbubbles were injected into nonunions,BMP-6 (bone morphogenetic protein-6weeks later,

stem/progenitor cells were recruited to the bone nonunion by implanting a collagen sponge in the defect site. Two . developed a two-step therapy. First, endogenous mesenchymalet alalternative to viral gene delivery, Bez

Treatments for bone nonunions (fractures that fail to heal) include surgery and bone grafting. As anBubbles and BMP-6 for bone repair

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REFERENCES

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