feasibility CLINICAL ORTHOPAEDICS AND RELATED by HC76e9e9f310aafbe4d8ddaa6bbf8ef0c7


									                                                                                                    CLINICAL ORTHOPAEDICS AND RELATED RESEARCH
                                                                                                    Number 425, pp. 237–243
                                                                                                    © 2004 Lippincott Williams & Wilkins

                   Feasibility of Percutaneous Gene Transfer to an
                            Atrophic Nonunion in a Rabbit
                   Christian Lattermann, MD*,†; Axel W. Baltzer, MD†,‡; Boris A. Zelle, MD*;
                  Janey D. Whalen, PhD*; Christopher Niyibizi, PhD*; Paul D. Robbins, PhD†;
                           Christopher H. Evans, PhD, DSc*†; and Gary S. Gruen, MD*

Treatment of atrophic nonunions is a challenge to orthopae-                          which 5–10% percent result in a delayed union or non-
dic surgeons. Growth factors potentially are valuable factors                        union.25 In addition, the costs for the treatment of a non-
for improvement of tissue healing. The use of growth factors,                        union range approximately between $10,000 and $50,000
however, is limited by their short half-lives. Gene therapy
                                                                                     per case and pose a substantial socioeconomic burden.3,13
has the potential to improve the treatment. This study aimed
to establish and validate an atrophic nonunion model in a
                                                                                         Although the introduction of intramedullary nailing re-
rabbit for the use of a percutaneous in vivo gene therapy                            duced surgical invasiveness and reduced the risk for hy-
protocol. An atrophic tibial nonunion was established in 24                          pertrophic nonunions, this improvement still has not found
New Zealand White rabbits. Radiologic and histologic fol-                            its match in atrophic nonunions. As a result, atrophic non-
lowup was for 64 weeks. The rabbit tibias showed no radio-                           unions are challenging for the orthopaedic surgeon be-
logic or histologic signs of healing. In addition, an adenoviral                     cause of their greatly impaired healing capacity and mar-
vector carrying a marker gene was injected percutaneously                            ginal vascularity. To treat an atrophic nonunion, restora-
into the nonunion site in 12 rabbits. Expression of the                              tion of vascularity to allow for a healing response to occur
marker gene was assessed for as many as 4 weeks. The per-
                                                                                     is necessary. Current treatment options therefore still in-
cutaneous gene delivery resulted in transgene expression in
the nonunion site for as many as 4 weeks. The described                              volve extensive surgical debridement of the nonunion site
model reliably leads to an atrophic tibial nonunion in rab-                          to reestablish vascularity to the site. After this occurs,
bits. Adenoviral percutaneous gene delivery into the non-                            growth factors, competent osteoprogenitor cells, and other
union site is feasible and leads to transgene expression locally                     blood-borne components of the healing cascade can gain
for at least 1 month. This study provides investigators with a                       access to the nonunion. However, extensive surgical ap-
reliable and reproducible model of an atrophic nonunion.                             proaches additionally endanger an already impaired blood
                                                                                     flow at the nonunion site. Additionally, there is a high risk
                                                                                     of infection associated with these surgical procedures.28
An impaired healing response leading to a delayed union                              Therefore, novel treatment approaches for atrophic non-
or nonunion after a fracture still poses a challenge to or-                          unions are desirable to reduce the morbidity and costs for
thopaedic surgeons. It is estimated that approximately 5.6                           patients. Experimental studies have reported the delivery
million fractures occur in the United States each year of                            of growth hormone (GH), transforming growth factor-
                                                                                     (TGF- ), or bone morphogenetic proteins (BMP) into the
Received: February 26, 2003
                                                                                     healing site of fresh fractures.4,6,22,32 The direct delivery
Revised: August 14, 2003                                                             of growth factors has major drawbacks because of their
Accepted: November 6, 2003                                                           short biologic half-lives, which typically are in the range
From the *Department of Orthopaedic Surgery and †Department of Molecu-
lar Genetics and Biochemistry, University of Pittsburgh, Pittsburgh, PA; and         of minutes to hours.7 To overcome this problem BMP-2
‡Department of Orthopaedic Surgery, Heinrich-Heine-University, Dussel-               and OP-1 have been delivered in a collagen sponge yield-
dorf, Germany.                                                                       ing very high local concentrations to the fracture site.8,9,12
This work was supported by a grant from the Albert B. Ferguson Orthopae-
dic Fund of the Pittsburgh Foundation.                                               Another option to overcome this problem may be to de-
Correspondence to: Christian Lattermann, MD, Department of Orthopaedic               liver the genes encoding these growth factors, rather than
Surgery, University of Pittsburgh Medical Center, Kaufmann Building, Suite           delivering the growth factors.
1010, 3471 Fifth Avenue, Pittsburgh, PA 15213. Phone: 412-648-1090; Fax:
412-648-8412; E-mail: lattermannc@msx.upmc.edu.                                          To study traditional and novel treatment options for
DOI: 10.1097/01.blo.0000137292.25504.22                                              atrophic nonunions it is essential to test these techniques in

                                                                                                                    Clinical Orthopaedics
238     Lattermann et al                                                                                           and Related Research

a reliable animal model. No reliable animal model for an               fracture site to hold the silastic tubing in place (Fig 1D). After
atrophic nonunion has yet been reported and validated. We              routine irrigation, the wound was closed. After 4 weeks, the
therefore decided to investigate an observation of Oni,24              cerclage wires and the Silastic tubings were surgically removed
who studied fracture healing and observed that some rab-               through two small incisions.
                                                                           For radiologic analysis, anteroposterior and lateral views
bits did not achieve healing of a tibial fracture when the
                                                                       from each rabbit were immediately obtained postoperatively and
bone was devascularized and revascularization of the frac-             consecutively 1, 2, 3, 4, 8, 16, and 64 weeks after surgery. The
ture was prevented for 4 weeks. The first goal of the cur-             radiographs were analyzed for nonunion, callus, and ectopic
rent study therefore was to validate an atrophic nonunion              bone formation. At the time the radiograph was obtained, the
model in a rabbit tibia based on the observations of Oni.24            tibia was clinically assessed for rotational stability.
In the second part of this study we investigated the feasi-                At 8, 16, and 64 weeks after surgery, four rabbits each were
bility of delivering a marker gene percutaneously into the             euthanized for histologic analysis. The right tibia was harvested
nonunion site, as a prelude to developing a percutaneous in            and the muscle tissue was stripped. The fibrous capsule of the
vivo gene therapy approach to the treatment of atrophic                nonunion site was left in place. The tibia was cut 2 cm proximal
nonunions. Several methods are available for the delivery              and 2 cm distal from the nonunion site and the intramedullary
of transgenes into musculoskeletal tissues. We chose to                Steinmann pin was removed. The samples were fixed in 4%
                                                                       paraformaldehyde (pH 7.5) for 7–10 days. After fixation, the
test the feasibility of a percutaneous gene transfer to the
                                                                       tibial samples were decalcified for 4–6 weeks in 50 mL conical
atrophic nonunion site using an adenoviral vector carrying             tubes containing 20% EDTA. Paraffin sections were cut at a
a marker gene.                                                         thickness of 5–7 m and stained with hematoxylin and eosin.
                                                                           The remaining 12 rabbits were used for the gene transfer
MATERIALS AND METHODS                                                  feasibility study. The first generation adenoviral vector ( E1,
                                                                         E3) used in this study is replication deficient because of a
We used 24 skeletally mature New Zealand White rabbits weigh-          deletion of the E1 gene. The lacZ marker gene is inserted in
ing 4.5–5 kg. All experimental animals were housed and oper-           place of the E1 gene. Gene expression is driven by the human
ated in the Central Animal Facility of the University of Pitts-        cytomegalovirus early promoter. The Ad/CMV/lacZ virus is
burgh Medical Center, and the treatment protocol for animal            grown in 293 cells (ATCC, Bethesda, MD), a human embryonic
subjects was approved by the Institutional Animal Care and Use         kidney cell line that constitutively expresses the E1-encoded
Committee of the University of Pittsburgh Medical Center.              proteins E1a and E1b. Viral titers were determined by optical
    All 24 rabbits included in this study had the same surgical        density at 260 nm. Eight weeks after the initial surgery, these 12
procedure. The rabbits were tranquilized with an intramuscular         rabbits were tranquilized using 2 mg/kg xylazine and 20 mg/kg
dose of 4 mg/kg xylazine and 40 mg/kg ketamine preoperatively.         ketamine intramuscularly. The nonunion was observed fluoro-
Anesthesia was maintained using 1.5–2.5% isoflurane delivered          scopically. After sterilization of the tibia, a syringe with a 27-
through a mask. The animals were monitored with electrocardi-          gauge needle was inserted percutaneously into the fibrous gap
ography and pulse oximetry throughout the procedure. Postop-           under fluoroscopic control. After the needle was placed cor-
eratively, a dose of 0.1 mg/kg Torbugesic (Wyeth, Philadel-            rectly, 1 × 107 pfu (plaque forming units) of Ad/CMV-lacZ virus
phia, PA) was administered subcutaneously twice a day for 2            diluted in 50 L saline solution was injected into the nonunion
days. After sterilization and preparation of the right tibia, an       site. The rabbits were divided into four groups of three animals
anteromedial incision of approximately 7–8 cm was made. The            each. In the first group, the histologic analysis of the marker
tibia was exposed using a periosteal elevator. In rabbits, the         gene expression was done 1 week after viral injection; in the
distal fibulotibial insertion usually is located in the upper 1⁄2 of   second group, the histologic analysis was done 2 weeks after
the tibial shaft (Fig 1A). This structure was used as a bony           viral injection; in the third group, the histologic analysis was
landmark, and the tibia was cut using a high-speed dental burr         done 3 weeks after viral injection; and in the fourth group, the
with a 2-mm burr bit 1 cm distal from the fibulotibial insertion.      histologic analysis was done 4 weeks after viral injection. The
The periosteum was thoroughly stripped 1.5 cm proximal and             expression of the lacZ marker gene was detected histologically
distal from the fracture site. Then the marrow cavity was reamed       by X-gal staining.
using the dental burr and a 2.5-mm drill bit. A hole was drilled
retrogradely through the marrow cavity into the anteromedial
aspect of the tibial head. An exactly fitted Steinmann pin was         RESULTS
placed antegradely through this drill hole (Fig 1B). The pin           Twenty-three of the 24 rabbits had a radiologically docu-
diameter in our series ranged from 3–4.5 mm. Afterward, silastic
                                                                       mented nonunion (Fig 2). One rabbit had a soft tissue
tubing (Nalgene 180 clear PVC tubing; Fisher Scientific, Pitts-
burgh, PA) was placed over the proximal end of the fracture and
                                                                       infection after the primary procedure and had to be ex-
advanced proximally for 1 cm as described by Oni (Fig 1C).24           cluded from the study. Radiographic analysis showed no
The distal fracture end was reduced into the silastic tubing and       callus formation in the original fracture site for as many as
the Steinmann pin was advanced into the distal marrow cavity.          4 weeks when the silastic tubing was removed. After 2
The Steinmann pin was cut to its appropriate lengths and two           weeks, in three of the rabbits there was minor callus for-
cerclage wires were positioned proximally and distally to the          mation at the interface of the distal edge of the silastic
Number 425
August 2004                                                                                     Atrophic Nonunion in a Rabbit    239

Fig 1A–D. The tibial bone was exposed and the tibia was cut 1 cm distal from the fibulotibial insertion. (A) The proximal stump
with the distal fibulotibial insertion is shown after periosteal stripping. (B) The marrow cavity was reamed and a Steinmann pin was
inserted. (C) Silastic tubing was placed over the proximal end of the fracture. The distal fracture end was reduced into the silastic
tubing and the Steinmann pin was advanced into the distal marrow cavity. (D) The silastic tubing was fixed and sealed using two
cerclage wires.

tubing to the distal tibia. However, this was a minor reac-         nonunion site. Within 200 m proximal to and distal from
tion that did not interfere with percutaneous removal of the        the nonunion site no viable osteocytes were detectable.
silastic tubing. After removal of the silastic tube, no ad-         From the periphery we detected a fibrous ingrowth into the
ditional callus formation was detected. Radiologic healing          nonunion site, which was intimately associated with the
was not seen in any of the rabbits at any time for as many          ends of the bone. Although scar tissue was found 8 and 16
as 64 weeks. The bone originally covered by the silastic            weeks after silastic tube removal, very little inflammatory
tubing showed a slightly reduced diameter after 16 weeks            activity was detected in the fibrous scar tissue.
in some animals. The clinical examination showed unre-                 Expression of the lacZ marker gene was detected his-
stricted rotational instability of the distal part of the right     tologically by X-gal staining at all times. The most striking
tibia in all 23 rabbits until euthanasia. Because of this           finding was distribution of the transfected cells in the non-
obvious clinical instability we did not do a biomechanical          union (Fig 4). We did not see lacZ + cells in the bony parts
analysis on the specimen.                                           of the nonunion. The fibrous scar tissue, however, showed
    The paraffin sections showed normal eosin staining of           marked expression of -galactosidase. The transfected
the bone with regular bony architecture and fibrous in-             cells were distributed throughout the fibrous scar with no-
growth into the nonunion site (Fig 3). There was progres-           ticeable accumulation in the fibrous capsule that had
sive loss of viable osteocytes in their osteons toward the          formed around the nonunion. We detected marker gene
                                                                                                        Clinical Orthopaedics
240    Lattermann et al                                                                                and Related Research

Fig 2A–D. (A) This anteroposterior radio-
graph shows establishment of the atrophic
nonunion. (B) There was no ectopic bone
formation around the silastic tubing after 2
weeks. At 4 weeks, the Silastic tube was
removed. After (C) 8 and (D) 16 weeks
there was no callus formation. The bone
ends round up and a visible gap persists.

expression for as many as 28 days. There was no sign of       cularized providing an ideal environment for bone healing.
monocyte or neutrophil infiltration, hyperemia, new blood     In contrast, an osseous nonunion in humans usually is
vessel formation, or any other indications of an inflamma-    caused by hypermobility of the fracture or decreased blood
tory reaction directed against virally infected cells. The    supply to the fracture.21 From the biologic standpoint, the
samples taken from the lung, liver, and spleen did not        segmental defect mimics a different biologic situation than
show any cells with -galactosidase activity at any time.      a common hypertrophic or atrophic nonunion. Therefore,
                                                              we tested a new approach to establish an atrophic tibial
DISCUSSION                                                    nonunion in a rabbit model. Our procedure was based on
                                                              the observations of Oni, who reported that tibial fractures
Throughout the last decade, numerous animal studies have      in a rabbit eventually fail to heal in a devascularized situ-
been done which assessed the healing potential of non-        ation.24 An additional focus of this study was to test the
unions after certain operative and nonoperative pro-          feasibility of a novel therapeutic approach to promote
cedures.14,17,20,22,27,29,30,32 All of these studies used a   healing of atrophic nonunions using a percutaneous gene
critical-size defect in long bones of different animal spe-   transfer technique. Atrophic nonunions usually are treated
cies (segmental defect) to mimic a nonunion. The segmen-      with techniques involving extensive surgery.21,28 How-
tal defect model creates a gap in a long bone, which cannot   ever, atrophic nonunions frequently are the result of an
be bridged by fracture callus and therefore the fracture      unfavorable soft tissue envelope which may complicate
cannot heal.26 At the fracture ends, however, a normal        additional invasive procedures. The available nonopera-
fracture healing response showing granulation tissue and      tive or minimally invasive techniques have not produced
formation of a fracture callus, with spontaneous filling of   reliable results in the treatment of atrophic nonunions.5,31
15–20% of the defect can be observed.14 In addition, there    Bone growth factors, such as BMP-2, have been shown to
is no increased micromotion between the bone ends and         increase fracture healing significantly in animal mod-
the surrounding healthy soft tissue usually is highly vas-    els16,32,33 and in human clinical trials.8,12 One of the major
Number 425
August 2004                                                                                 Atrophic Nonunion in a Rabbit   241

                                                                  investigated the feasibility of percutaneous gene delivery
                                                                  into the nonunion site, using an adenoviral vector carrying
                                                                  the lacZ marker gene.
                                                                      The current study has strengths and limitations. Our
                                                                  operative technique created an atrophic nonunion as de-
                                                                  termined by radiologic and histologic criteria. The oppos-
                                                                  ing ends of the bone seemed atrophic with focal necrosis
                                                                  and no radiologic signs of healing were recorded. How-
                                                                  ever, the validity of our data is limited since the radiologic
                                                                  and histologic evaluations were qualitative and therefore
                                                                  subject to interobserver and intraobserver variations.
                                                                  Moreover, no detailed investigation of the immunologic
                                                                  response to the adenoviral gene transfer was done. Al-
                                                                  though the histologic sections did not show any significant
                                                                  neutrophil or lymphocyte infiltration, we cannot com-
                                                                  pletely rule out an early inflammatory response because
                                                                  we did not do immunohistochemical analysis for
                                                                  CD4/CD8 or antibody titer measurements for specific neu-
                                                                  tralizing antibodies against the adenoviral vector or the
                                                                  transgene. At distant organ sites, we did not find any cells
                                                                  with -galactosidase activity at any time. However, a real-
                                                                  time PCR was not done and therefore we are unable to
                                                                  determine whether adenoviral particles were present at dis-
                                                                  tant organ sites. In addition, no quantification of new ves-
                                                                  sel formation has been done in the current study. We at-
                                                                  tempted to establish an atrophic nonunion model using
                                                                  several steps including periosteal stripping, intramedullary
                                                                  reaming, and application of a silastic tube for 4 weeks.
                                                                  Because no control groups were used, we are unable to
                                                                  determine whether it was the periosteal stripping, the in-
                                                                  tramedullary reaming, the silastic tube, or the combination
Fig 3A–B. (A) The nonunion site was repopulated by fibrous        of these agents that led to the atrophic nonunion. Using
scar tissue that aligned intimately with the bone ends (arrow).   this suggested model, it was shown that healing of the
(B) At 8 weeks after removal of the silastic tubing the bone      fracture did not occur in any animal in our study, therefore,
ends seemed atrophic with empty osteocyte lacunae (arrows).       study provides investigators with a reliable animal model
                                                                  of an atrophic nonunion. Experimental methods for heal-
                                                                  ing of atrophic nonunions can be tested, using our sug-
drawbacks of single delivery of growth factors is the short       gested nonunion model. The study showed that percuta-
biologic half-life of most growth factors. We know from           neous in vivo gene delivery into a nonunion site is fea-
work on chronic skin wounds that one application of               sible. Additional studies should focus on gene transfer of
growth factors in a fibrous scar tissue is not sufficient to      bone growth factors or angiogenic factors to test their
reestablish a healing response.18 Sustained delivery of           healing potential.
growth factors, such as BMP-2, may be necessary for                   In contrast to segmental defect models described ear-
treatment of atrophic nonunions, and a delivery system            lier,14,17,20,22,27,29,30,32 this model is based on periosteal
that can provide a continuous local release of growth fac-        stripping, intramedullary reaming, and decreased vascular
tors is desirable. The suggested delivery of BMP-2 or             supply to the fractured bone. These etiologic factors are
OP-1 in a collagen sponge8,9,12 still requires an extensive       consistent with the clinical risk factors for development of
debridement and surgical approach for growth factor de-           an atrophic nonunion.21,24,28
livery. Using adenoviral vectors, gene expression has been            Novel treatment approaches in orthopaedic surgery in-
seen for as many as 6 weeks in various soft tissues.10,11,15      clude application of bone growth factors. Several growth
In addition, it was shown that gene delivery to bone and          factors, such as TGF- , BMP-2, and BMP-7 have been
fractures using viral vectors is feasible and leads to an         tested in different animal models and have been reported
accelerated healing response.1,2,19,23 Therefore, we also         to enhance fracture healing.2,6,17,19,32 The treatment of
                                                                                                                              Clinical Orthopaedics
242     Lattermann et al                                                                                                     and Related Research

Fig 4A–B. (A) After 1 week, a lon-
gitudinal section (magnification ×
100) showed transgene expression
in the surrounding tissue of the non-
union (arrows). (B) In a cross section
(magnification × 60), gene expres-
sion can be seen 3 weeks after gene
delivery (arrows).

atrophic fracture nonunions may benefit from such ap-                            of an adenovirus expressing BMP7. J Cell Biochem 78:476–486,
proaches. We suggest a reliable and reproducible atrophic                   8.   Friedlaender GE, Perry CR, Cole JD, et al: Osteogenic protein-1
nonunion model in a rabbit. Novel treatment approaches,                          (bone morphogenetic protein-7) in the treatment of tibial nonunions.
such as injection of bone growth factors or gene transfer,                       J Bone Joint Surg 83A(Suppl):S151–S158, 2001.
                                                                            9.   Geesink RG, Hoefnagels NH, Bulstra SK: Osteogenic activity of
can be tested using our suggested nonunion model.                                OP-1 bone morphogenetic protein (BMP-7) in a human fibular de-
                                                                                 fect. J Bone Joint Surg 81B:710–718, 1999.
                                                                           10.   Gerich TG, Kang R, Fu FH, Robbins PD, Evans CH: Gene transfer
Acknowledgments                                                                  to the patellar tendon. Knee Surg Sports Traumatol Arthrosc 5:118–
We thank Kurt Weiss and Joan Rosenberger for help with animal                    123, 1997.
care and surgery, and Dr. Jonny Huard for use of his imaging               11.   Ghivizzani SC, Lechman ER, Tio C, et al: Direct retrovirus-
                                                                                 mediated gene transfer to the synovium of the rabbit knee: Impli-
facilities. We thank Dr. Freddie H. Fu for support of this project.              cations for arthritis gene therapy. Gene Ther 4:977–982, 1997.
                                                                           12.   Govender S, Csimma C, Genant HK, et al: Recombinant human
                                                                                 bone morphogenetic protein-2 for treatment of open tibial fractures:
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