Model Model Model
Computer tomography (CT) and magnetic resonance imaging (MRI) were utilized for implant evaluation 12 weeks after injection. As a surrogate parameter for bone formation processes, the calcium deposition at the implant site was monitored by means of optical imaging (OI) during the experimental period.
2.9.3.1 2.9.3.1 2.9.3.1
2.9.3.1 Optical Imaging Optical Imaging Optical Imaging Optical Imaging
Proof of Colocalization of Implant and Calcification Signal Proof of Colocalization of Implant and Calcification Signal Proof of Colocalization of Implant and Calcification Signal Proof of Colocalization of Implant and Calcification Signal
The seven mice from the explorative pilot study of gel position and stability (2.9.2.3.) were investigated for the colocalization of rhBMP-2 and the resulting calcification. After 24 days, the implants were assessed for signs of calcification by means of a fluorescence-labelled calcium chelating agent (BoneTag, Licor). 100 µl of dye solution, prepared according to the manufacturer’s instructions, were injected into the tail vein. One, five and eleven days after injection, fluorescence images were acquired using NIR settings of the Maestro equipment.
Proof of Proof of Proof of
Proof of C C C Calcification alcification alcification alcification
Forty in situ-forming implants were administered subcutaneously to twenty mice as described above (study #4). The investigated groups (n=5) included gels with dispersed, rhBMP-2-loaded microspheres of all three polymers. RhBMP-2, dispersed in sols, served as positive control.
Negative controls of the four investigated systems were prepared without addition of growth factor (n=4-6).
In a similar approach, the impact of the microsphere vehicle was investigated (study #5). In this study, the active ingredient was encapsulated in PEP 5 microspheres. The microspheres were dispersed in in situ-forming implants of chitosan or modified hyaluronic acid gels and administered to female mice (n=5 each).
In studies #4 and #5, signs of implant calcification were assessed 3, 8 and 12 weeks after injection non-invasively by OI as described above. Another image was recorded ex vivo when the skin around the implant region was abscised.
Two different ways of result evaluation were applied. In the ‘Implant Volume Calculation Method’ (I), calcified volumes were determined by
2 Experimental
41
measuring the patella spectra and by separating them from the background spectra found in soft tissue areas of the mice. After this unmixing process, ectopic sites of fluorescence were regarded as calcified tissue and defined as regions of interest (ROI). Their volumes were determined by measuring the length l and width w of the ROI (fig.
2.9-1) and calculating the ellipsoid volume according to equation 2.9-1.
(2.9-1)
For method (II), referred to as ‘Total Signal Evaluation Method’, the ROI were defined by a standard ellipse with an area covering 461 mm², assuring a full coverage of any ectopic signal. The total signal, scaled by the exposure time of the image, was utilized for result evaluation.
FiFi
FiFig. g. g. 2.9g. 2.92.92.9----1111: Two different ways of result evaluation for OI calcification : Two different ways of result evaluation for OI calcification : Two different ways of result evaluation for OI calcification : Two different ways of result evaluation for OI calcification signals. Method (I), on the left, is based on the measured length and width signals. Method (I), on the left, is based on the measured length and width signals. Method (I), on the left, is based on the measured length and width signals. Method (I), on the left, is based on the measured length and width (l and w) of the implant. The total calcification signal of sta
(l and w) of the implant. The total calcification signal of sta (l and w) of the implant. The total calcification signal of sta
(l and w) of the implant. The total calcification signal of standard ellipses, ndard ellipses, ndard ellipses, ndard ellipses, scaled by exposure time, was used in the second approach (II; right). For scaled by exposure time, was used in the second approach (II; right). For scaled by exposure time, was used in the second approach (II; right). For scaled by exposure time, was used in the second approach (II; right). For comparison, a threshold image of fluorescence intensity is provided in the comparison, a threshold image of fluorescence intensity is provided in the comparison, a threshold image of fluorescence intensity is provided in the comparison, a threshold image of fluorescence intensity is provided in the middle.
middle.
middle.
middle.
2.9.3.2 2.9.3.2 2.9.3.2
2.9.3.2 Computer Tomography Computer Tomography Computer Tomography Computer Tomography
The implant shape and morphology in week 12 was evaluated by means of CT (Somatron CT Scanner, Siemens). The instrument was designed for human use, so that 5 animals per run were investigated to obtain a sufficient signal. The animals were first scanned in upper the extremity mode for bone and soft tissue, then images were processed
² 6 1 lw V =
π
with Syngo software. HG 300 0.6 U70u pictures were used for data acquisition and processing. Slice thickness was 600 µm and pulse frequence was set to 70 u. Bone appears bright under the used conditions, values range from 200 to 1500 units for bone, whereas darker areas (below 150 units) depict soft tissues. Bone volumes were calculated from the brightness of investigated areas. ROI were defined manually around newly-formed bone. Ectopic and orthotopic sites could easily be distinguished from one another. Within the defined ROI, the volume fulfilling the defined brightness criteria was calculated by means of the Syngo software. The volume of every ROI was calculated using length l, depth d and width w of the ROI provided by the software and regarding the implant as an ellipsoid according to equation 2.9-2.
(2.9-2)
2.9.3.3 2.9.3.3 2.9.3.3
2.9.3.3 M M M Magnetic agnetic agnetic R agnetic R Resonance R esonance esonance IIIImaging esonance maging maging maging
For comparison, magnetic resonance images were also acquired in the 12th week after sol administration. The images were composed of data gained with a slice width of 3 mm, slice separation of 3.5 mm, a spin echo time of 8 ms and a repetition time of 172 ms. 32 averages were recorded on a 27 mm field of view with a resolution of 64 x 64 pixels. A blinded result evaluation of the acquired data for signs of implants or ectopic bone was performed by a specialist.
2.9.3.4 2.9.3.4 2.9.3.4
2.9.3.4 Histology Histology Histology Histology
All implants with in vivo residence times of 28 days or longer were subjected to histological evaluation. For a detailed overview of the different studies, see table 2.9-1. After excision, the implants of study
#3 were frozen to -20 °C. Frozen sections were cut by means of a microtome (Jung Biocut 2035, Leica) to a slice thickness of 10 µm.
During this process, photographs were taken to gain an overview of the sample composition. After the slicing process, the sections were fixed with neutrally buffered formalin solution (4 % v/v).
Implants of the studies #4 and #5 were directly immersed into the fixation solution. The specimen were rinsed in PBS-containing sucrose (6 % w/v) and dehydrated in an acetone series until the organic solvent remained clear. Subsequently, the samples were embedded in a cold polymerizing resin (Technovit 8100, Heraeus Kulzer, Wehrheim, Germany) according to the manufacturer’s instructions. Sections of 5 µm thickness were prepared with the microtome.
dlw
V
π
6
= 1
2 Experimental
43
All further processing of the samples of studies #3, #4 and #5 was accomplished in the same manner. At least two sections per sample were investigated by hematoxylin-eosin staining. The sections were rinsed in distilled water for 5 minutes and then immersed in dilute Mayer's hemalum solution (1:4, Merck, Darmstadt, Germany). After an incubation period of 60 minutes, the sections were treated with tap water for blueing. The second staining was performed by immersion of the samples in 0.1 % (w/v) eosin solution for three minutes. The sections were rinsed again with distilled water and covered with Entellan (Merck).
Calcification and bone formation were monitored in studies #4 and #5 by means of von Kossa staining. This method, first described in 1901, is based on the reaction of silver with the phosphate deposit. After washing with water, the sections were immersed in silver nitrate (5 % w/v) for one hour and rinsed with distilled water twice. The silver ions were reduced by addition of pyrogallol (1 % w/v for five minutes), subsequent fixation was accomplished by adding sodium thiosulfate solution (5 % w/v). After another rinsing step, nuclei were counter-stained for four minutes with nuclear fast red (1 % w/v in aluminium sulphate solution). Excess dye was removed with distilled water and the stained sections were covered with Entellan (Merck).
The histological evaluation of the stained sections from studies #4 and
#5 was independently performed by three individuals on the basis of a scoring scheme (table 2.9-2). The samples were evaluated at different magnifications and examined for signs of bone formation, i.e., calcification and the presence of bone marrow. Four sections of each implant were investigated to gain a good overview of the whole implant. Microscopic pictures were acquired at an Eclipse 80i microscope with a connected workstation (Nikon instruments, Düsseldorf).
Table Table Table
Table 2.92.92.9----22.9 222: Score system for the histological eval: Score system for the histological eval: Score system for the histological eval: Score system for the histological evaluation of the implants uation of the implants uation of the implants uation of the implants obtained after 12 weeks in the mouse ectopic model.
obtained after 12 weeks in the mouse ectopic model.
obtained after 12 weeks in the mouse ectopic model.
obtained after 12 weeks in the mouse ectopic model.