International Biopharmaceutical Association Publication
BISPHOSPHONATE DELIVERY TO LARGE SEGMENT BONE ALLOGRAFT
Anwaar H Naqvi, MS. Candidate
INTRODUCTION
Bone fractures and damage are serious health problems in all day clinical work. Common bone substitution materials are autografts, allografts, xenografts and various synthetic materials like polymers, metallic materials, composites and bioceramics. However, none of these materials provides a perfect solution for guided bone healing because there always remain questions about mechanical stability, long-term in vivo biocompatibility and biodegradability.
One of the most prominent
bone substitution materials is large
segment structural allografts to reconstruct bony defects created by the
resection of primary and metastatic bone tumors and in the removal of
prosthesis during revision joint replacement. Such allografts have been shown
to have a long-term fracture rate as high as 27% 2,3,6,17,20. In the
process of healing, the host osteoclasts resorb portions of the allograft to
initiate healing of the osteotomy site and remodeling at the junction site.
However, the resorption that occurs around allograft screw holes leads to the
development of stress risers that then weaken the bone and cause the fracture
of the allograft under certain loading conditions. We theorize that local
treatment of allograft bone with anti-resorptive agents will inhibit local
osteoclastic effects, prevent bone resorption and the development of stress
risers. This should reduce fatigue failure through the resorption holes and the
frequency of revision surgery.
How is the bone surface targeted for resorption?
The bone surface,
where resorption takes place, is covered by a layer of lining cells derived
from osteoblasts. These cells are separated from the mineralized matrix by a
thin layer of non-mineralized material which is removed prior to resorption,
probably by collagenase digestion. The lining cells move away, and the
mineralized surface is now exposed. Osteoclasts attach to this mineralized
surface through a specialized circular area of the membrane called the sealing
zone. Resorption occurs in this sealed space of about 1000 µm3
between the bone surface and the osteoclast membrane.
How Bisphosphonates work:
Bisphosphonates contain compounds that inhibit the function of the osteoclasts, sending a chemical message to the osteoclasts that instructs them to slow down on bone destruction. At the same time, bisphosphonates stimulate osteoblasts to secrete chemicals that discourage the formation of more osteoclasts. By inhibiting the osteoclasts and stimulating the osteoblasts, bisphosphonates help keep bones intact.
Pamidronate disodium, a nitrogenous
bisphosphonate, is used clinically to inhibit osteoclast mediated bone
resorption associated with many pathologic processes of bone 8,9,15,16,21. Bisphosphonates covalently bind to the
hydroxyapatite crystals in mineralized bone 12,19
and have a half life measured in years matching that of the bone 15.
Pretreatment of small cancellous bone chips by 10 minutes of simple soaking
with a pamidronate solution prevented allograft resorption in mouse model 1,11, this findings suggests that pretreating a
structural allograft with anti resorptive agents many inhibit local
osteoclast-mediated bone resorption.
Structural bulk allografts are not
vascularized, and thus systemically administered bisphosphonates must passively
diffuse into them from adjacent tissues. Similarly, soaking bone in solutions
containing anti-resorptive drugs, a passive process, is suitable for treating
small pieces of bone but will require many hours to achieve uniform
distribution of the anti-resorptive drug within large allografts. Since bone is
an open pore material,18 as an alternative
strategy, we propose to adapt a “water flooding” technique 4,5,10,14
to pump pamidronate containing fluids through the allograft. Water flooding, a
method typically used as part of secondary oil recovery from porous rock,
relies on the use of aqueous fluids to displace oil from open pore rock. We
compared pressurized pumping of a dye- disphosphonate solution through large
structural allografts with simple soaking of the allografts within the solution
to determine the more efficacious approach.
Since bisphosphonates are such new drugs, their usage has not been studied as extensively as other treatments. Some clinical trials have shown great success with bisphosphonate treatment. Others are inconclusive. More study is needed to show exactly how bisphosphonates help prevent bone destruction and act upon metastatic tumors. But for many patients, bisphosphonate can reduce the need for radiotherapy and surgery, reduce pain, reduce or delay the onset of broken bones and improve quality of life.
The long term goal of this research is the creation of an animal model
for in vivo testing of the bisphosphonates impregnated allografts. Ovine bone
allograft was used because of easily available and has some resemblance in
composition with Canine bone allograft which is considered the gold standard.
MATERIALS AND METHODS
Specimens
Five matched pairs of Ovine tibia were obtained from Colorado State University College of Veterinary Medicine (CSU) under IACUC approved protocols from MSKCC and CSU to obtain necropsy tissue from experimental animal. The tissue was harvested from skeletally mature sheep that were free of metabolic disorders and cancer at necropsy.
Allograft Preparation
The tibias were stripped of
all soft tissues and were cut with a band-saw at the inferior aspect of the
lesser trochanter. The medullary canal was cleaned with distilled water
pressurized through a 3.5 inch spinal needle (Tyco Healthcare Group,
in 70% ethanol for one hour to kill any bacterial contaminant and to aid in removal of any residual medullary fat. Our preparation method is similar to the processing of human allograft with saline/distilled water irrigation and treatment with a sterilizing solution 7,22,13. The five pairs of ovine tibias were divided into two equal groups, left tibia and right tibia. Left tibia was treated using positive pressure pumping of the treatment solution while right tibia was soaked in the treatment solution. Each bone’s weight and displacement volume was measured to determine the bulk density. Each bone was weighed on METTLER TOLEDO (PG5002-S Delta Range) digital balance. Displacement volume was measured by placing each bone in know volume of distilled water in graduated cylinder and measured the change in volume. The bone was wrapped in saline soaked gauze and stored at -26 oC until the treatment experiments began.
Treatment Media
The treatment media
consisted of 0.1% (w/v) toluidine blue (MW=306)-saline solution (Aldrich
Chemical Company,
[T1/2=6.02hrs, 140.5keV γ] prepared by MSKCC Radiopharmacy.
The toluidine blue is a cationic stain routinely used in pathological processing of bone that binds to anionic moieties such as sulfates, phosphates and carbonates in the bony matrix. Toluidine blue has a molecular weight of 306 which makes its diffusion characteristics equivalent to pamidronate with a molecular weight of 369. Toluidine blue is easily visualized while Tc99m-ADP (molecular weight) is a bisphosphonates that binds to the hydroxyapatite crystals of bone and is routinely used as part of standard clinical bone scintigraphy. Prior to the start of each experiment, 25 mCi Tc99m-ADP was added to the solution to enable the non-invasive SPECT imaging of Tc99m-ADP distribution throughout the bone. Fresh solutions were prepared for each bone. The study was performed at room temperature.
Experimental Protocol
Left
tibia Pumping Protocol: The pumping apparatus used is shown in Figure 1.It
consisted of a Cole-Parmer Model 7020 Master Flex positive peristaltic pump
(Cole-Parmer Instrument CO,
Figure 1. Pump Apparatus.
Bones were thawed in distilled water; left tibia was selected for pumping and right tibia was treated by soaking. A rubber coupling connected the allograft (left tibia) to the fluid steam. Saline was pumped through the bone to identify a flow rate such that the back pressure did not exceed 4psi, the systolic arterial pressure. This resulted in a flow rate of 6 ml/sec.
The treatment solution of 1900ml (sufficient to cover the bone within chamber and fill all fluid lines) was infused through the left tibia for 20 minutes. The treatment solution was then replaced with 70% ethanol and pumped to remove unbound dye and Tc99m-APD.
Right tibia Pumping Protocol: Control tibia was submerged in treatment solution of 2000ml for one hour. After one hour replaced treatment solution with 70% ethanol and washed to remove unbound dye and Tc99m-APD.
Immediately
following either treatment, fluid within the canal was drained by gravity and
excess surface fluid was removed with pat drying. The bones were transferred to
plastic bags and placed within a dose calibrator (Squibb Model CRC 17
Radioisotope Calibrator, ER Squibb & Sons, Inc,
Micro SPECT Imaging Protocol: The bone was placed in the XSPECT (Gamma Medica, North ridge, CA), a dedicated small-animal SPECT-CT scanner. SPECT scanning times were selected such that 100k counts were acquired; a typical image session was 1hour. Registered CT and SPECT images were obtained at 3 cm and 8 cm from the proximal cut end and at 1 cm proximal to the trochlear groove. Following imaging, the Tibia were again stored at
-26 0 C 3 days (10 half lives of Tc99m) prior to histological preparation.
Micro SPECT Image Analysis: Using LumaGEM-P software on SPECT, export the image. Exported file showed up with s sings in the Inter files folder. Renamed and opened the same file in ASI proVM v6.0 on CT. Changed the parameters of “Raw data format” as Xdim=56, Ydim=56, Zdim=56, X-Y pixel size=2.232, Slice thickness=2.232 and selected 2-byte integer-γ ax/int style.
Adjust the number of pixels in the range of 100-50 to obtain clear image. Change color to view activity area clearly.
Opened tool menu, selected ROI, then selected Draw, Trace and 3D options. Chose range of interested region such that 10 slices could be taken.. After taking 10 readings, clicked stats to get the analysis .Saved the analysis as excel sheet. The same file would be seen on SPECT computer.
Histological Preparation and Image
Analysis: Bone sections were
prepared by cutting the frozen tibia on band saw (Hospital for Special Surgery,
Research Division) 1 cm and 2 cm proximal to the trochlear groove and again at
3,4,15 and 16 cm from the proximal cut end. The most distal segment (cancellous
metaphyseal bone) was labeled section 3, the middle section at the
meta-diaphyseal junction was labeled section 2, and the proximal diaphyseal
section was labeled section 1.
For image analysis, proximal and distal face
of section 1, 2, 3 was digitally photographed. Each face was segmented into
four regions (anterior, lateral, posterior and medial) giving 8 separate areas
per segment for subsequent color image analysis.Photographs
were acquired using Cool pix 880 camera (Nikon, Inc, Japan) mounted to tripod
set to high quality image.
Image Analysis Protocol: Determination
of bone area receiving dye was analyzed from the digital photographs using a
manual, sequential color image analysis scheme with Adobe Photo shop v7 (Adobe
Systems Inc.) and Sigma Scan Pro v5.0 (SPSS, Inc). Using Photo shop, the purple
and black pixels within each image were converted to absolute black and then
the image to identify the bone boundary for determination of total area. This
image was then transferred to Sigma Scan Pro where total bone area was determined
by pixel count. The absolute black area
was determined next by thresholding the image using intensity control and
registering this pixel count. Red overlay was used to determine total area
while blue overlay was used to determine dyed area. Pixel count was then
measured to determine total bone area and dye area.
Calibration distance was
determined by pixel count per mm then calibration area was determined by taking
square of it. Dye area (mm2) and total area (mm2) were
determined by dividing pixel count with calibration area. Total area (mm2)
should be equal to the sum of section area(mm2),
similarly dye area(mm2).Percent dye distribution within bone was
determined from the quotient of the black (dye) and total bone areas.
Statistical Analysis: All statistical analysis was performed using a paired two-tailed Student t-test as implemented by Microsoft Excel version XP (Microsoft Inc). Significance was set at a p value of less than .05.
Micro SPECT Image Analysis.
1) Using LumaGEM-P chooses the image and exports it.
2) Use F9 to minimize the screen
3) Open Inter files folder, export files will show up with S signs.
4) Rename these two files
5) Open the image file in CT computer.
6) Set the parameters as Xdim=56, Ydim=56, Zdim=56. X-Y pixel size to (2.232) and enable 2-byte integer-rax/intel style.
7) Open Tool menu and open ROI,and check Draw, Trace and 3D options.
8) Choose range of interested Region by selecting minimum and maximum range.
9) Take 10 readings.
10) Select Stat option to get the results
11) Save it as Excel file
12) The same file should be saved in SPECT computer.
RESULTS
MicroSPECT-CT Images
SPECT-CT images were used to non-invasively
assess the gross distribution of the Tc99m-ADP within the ovine allograft bone.
The pumped bone had a more evenly distributed pattern of medullary and cortical
bone penetration of the Tc99m-ADP treatment solution while the soaked bone had
a peripheral distribution of the radioactive tracer on the outer layers of the
cortical bone. Figures 3 and 4 present an image montage created from the
SPECT-CT scans of the pumped and soaked bones, respectively. Within each
montage, panel A is the metaphyseal CT image; B is the metaphyseal SPECT image;
C is the metaphyseal registered CT-SPECT image; D is the diaphyseal CT image; E
is the diaphyseal SPECT image; and F is the diaphyseal registered CT-SPECT
image.
Figure 3Pumped Tibia Figure 4 Soaked Tibia
Dye Distribution analysis
On visual analysis of the histological
specimens, positive pressurize pumping delivered the treatment solution along
the endosteal surface and the haversian system with in the cortical bone and
had deeper penetration into the metaphyseal
region of the bone. The diffusion pattern of the dye appears to decrease
radially from the endosteal surface. The outer cortex has the least dye
deposition. Simple soaking of the bone for 1 hour allowed the dye to penetrate
through the outer cortical bone to a depth of 0.14 mm(SE.01)
and the endosteal bone to a depth of 0.21 mm
(SE .01). The central portion of the cortical bone was
completely undyed. Representative dye distribution photographs used with the
image analysis scheme are presented in Figures 5 and 6.
Figure 5. Section 1: From a matched pair of femurs. A. Pumped Bone B. Soaked Bone
Figure 6. Section 3 of a matched femoral pair: A. Pumped Bone B. Soaked Bone
The results of the histological image
analysis are summarized within Figure 7. The findings demonstrated a
significant difference in dye penetration between pumping and soaking in the
three zones. The diaphyseal and the meta-diaphyseal junction of the pumped
cortical bone demonstrated a 200% greater uptake in dye penetration throughout
the cortical ring than comparable regions within the soaked bone. The most
striking difference was seen in the distal metaphysic where a greater than 600%
increase in dye penetration was measured between the pumped and soaked bones.
Dye distribution in all zones was statistically improved with pumping over
soaking.
Figure 7. Dye Distribution on Histological Analysis.
DISCUSSION
Various drug
treatments have been delivered to allograft bone by simple soaking. Preliminary
studies in rats 1, 11 have soaked small morsels of cancellous bone
(6mm x 2mm) with bisphosphonate solutions to inhibit allograft resorption and
to assess the affects of bisposphonates on allograft incorporation. This bone
preparation has a high surface to volume ratio and surface to mass ratio. These
rat bone conduction chamber studies showed a significant reduction in allograft
resorption without an effect on incorporation in alendronate1 treated
grafts, but clodronate11 treated grafts demonstrated a diminished
incorporation distance with a diminished resorption rate. Canine structural
allografts (4 cm long) 25 have been treated by simple soaking for
one hour in recombinant human osteogenic protein-1(rhOP-1) to improve allograft
incorporation. The rhOP-1 treated allograft had a significant increase in
porosity and density of active osteons. Recently, cancellous human allograft chips
impregnated with antibiotics 26 were clinically to prevent infection
in revision hip replacement with success. None of these studies however,
quantified the drug distribution within their bone specimens. Our findings
suggest that simple soaking may be an inefficient method for treating large
allografts because drug penetration is slow and limited to a narrow band near
the surface.
In this project, ovine tibia large
segment allografts were used to compare the delivery methods of soaking versus
positive pressure pumping. The pumping provided at least a 200 fold
increase in distribution in a time efficient manner. The average pumping time
was 2.8 minutes versus 60 minutes for the soaking. The dye distribution data
suggests that pressurized flooding provides a superior distribution of dye as
compared to soaking in a significantly shorter time. Therefore, a pamidronate
solution delivered in the same manner would deposit a greater concentration of
the drug more evenly throughout the allograft.
The microSPECT images
provide a non-invasive representation of the distribution of Tc99m-ADP
throughout the bone and anticipate the distribution of pamidronate bound to
bone. The SPECT images corroborate the dye distribution images, although the
resolution of this scintigraphic technique is on the order of 2-3 mm3.
Although the pressurized pumping
had a more even dye distribution throughout the bone, the outer cortical
surface did not receive an even coating. The soaked bone, on the other hand,
has consistent deposition along the endosteal and outer cortical surfaces to an
average depth of 0.14 mm, but the cortical bone was poorly penetrated. A
bisphosphonate coating on the outer surface would protect the area of greatest
contact against the host osteoclasts and an even distribution within the
cortical bone would protect the screw holes from resorption and prevent
creeping substitution with enlargement of the Haversian canal during
revascularization. We are currently evaluating a technique that incorporates
features from both methods.
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