The correlation between neovascularization and macrophage populations under the influence of androgens.
Previous studies in our laboratory have quantified the tissue implant response surrounding tricalcium phosphate (TCP) bioceramic implants and have demonstrated how this response may be modified in the presence of androgens [1-3]. Subsequent studies revealed the extent to which androgens modify the tissue-implant response with respect to the presence of macrophages and neovascularization [4, 5]. To date, there is little data elucidating the relationship between vascularity and macrophages in the fibrous tissue capsule surrounding TCP bioceramic implants. The purpose of this study was to examine the relationship between macrophages and neovascularization in the fibrous tissue capsule surrounding TCP implants loaded with three androgenic hormones. We hypothesized that low levels of neovascularization would be reflected in low macrophage counts.
Sixteen Sprague-Dawley male albino rats weighing 280-300g were obtained (Holtz Company, Madison, WI), acclimatized in the animal care facility for two weeks prior to surgery, and randomly divided into three experimental groups (n=4/group) and 1 control group (n=4). The tricalcium-phosphate bioceramic implants were prepared according to standard laboratory protocol [1, 3-5]. Surgery on all animals was performed according to a standard laboratory protocol reviewed and approved by the University of Mississippi Medical Center Animal Care and Use Committee and has been described previously [1, 3-5]. Animals in Group I were designated for the implantation of the sham TCP ceramics (control), animals in Group II were designated for T-TCP (testosterone) implantation, animals in Group III were selected for the implantation of the D-TCP (dihydrotestosterone) ceramic devices, and animals designated in Group IV were implanted with the A-TCP (androstenedione) ceramic. All animals in this investigation were kept on a 12-hour day/night cycle and were fed Purina Rodent Chow 5001 (Ralston Purina, St. Louis, MO) and water ad libitum.
The TCP ceramic implants and their fibrous capsules were collected, observed grossly, photographed, and fixed in 10% neutral buffered formalin at 90 days post-implantation. Processing of these tissues included infiltration with paraffin and routine histologic processing.
Three inch glass microscope slides were coated with chrome-alum adhesive to aid in the tissue attachment to the glass slide . Three to five sections were cut at 5 um and mounted onto glass slides every 25 um throughout the entire depth of the fibrous tissue mounted in the paraffin blocks.
Tissues were stained with hematoxylin and eosin (H&E), Masson's trichrome, and Papanicolaou stains. Histologic sections prepared from the tissue directly surrounding the implants were evaluated using the light microscope and Image Pro Plus digital analysis software (Media Cybernetics, Silver Spring, MD) on captured images . Blood vessels in the fibrous tissue capsule were quantified by counting numbers of vessels per high power field (HPF) with the assistance of the digital analysis software using a semi-automated method [3, 5]. Macrophages were identified based on cellular morphology and imaging characteristics and reported as the number of cells/HPF . To minimize bias and variability, a minimum of 10 fields per slide were randomly examined throughout the depth of the tissue capsule on 125 randomly selected slides per animal to thoroughly examine the depth of the tissue.
Statistical analysis was conducted using Stata version 12 statistical software (StataCorp, College Station, TX). Quantification of neovascularity and macrophage counts throughout the different layers of fibrous tissue were expressed as mean [+ or -] sd. The number of blood vessels and macrophages in each group were compared using Pearson's correlation coeffecient (Pearson r, a=0.05).
Upon extraction of the implants from the peritoneal cavity, multiple stains revealed fibrous tissue of varying degrees of thickness for all four groups. Morphometric analysis revealed both macrophages and vascularity were highly variable among the four groups (Figure). The control group had the highest number of blood vessels/HPF, while Group III had the highest number of macrophages. The statistical analysis revealed macrophages and vascularity were highly and significantly correlated in each of the three experimental groups. The most interesting finding was the correlation of macrophages and vascularity in the control group. Analysis revealed these counts were highly correlated, but this group failed to demonstrate statistical significance (r=0.94, p=0.064). The number of blood vessels/HPF was two times higher compared to the number of macrophages/HPF. In the experimental groups, the number of macrophages/HPF more closely mirrored the number of blood vessels/HPF. To better demonstrate the positive linear relationship, the number of macrophages/HPF was plotted against the number of blood vessels/HPD for each of the four groups and a composite where data were combined. Overall, macrophages and vascularity were highly correlated when tested together as a composite (r=0.89, p < 0.0001). The data suggest that although androgenic hormones tend to suppress vascularity, vascularity was highly correlated to macrophage counts in all treatment groups. Correlation in the control group was not statistically significant, providing evidence that androgenic hormones may strongly influence neovascularization.
Figure 1 shows the correlation of macrophage counts and vascularity (y-axis) in fibrous tissue surrounding TCP ceramic implants loaded with testosterone, dihydrotestosterone, androstenedione, and control. Overall, macrophages and vascularity were highly correlated when tested together as a composite. Though androgenic hormones tend to suppress vascularity, vascularity was highly correlated to macrophage counts in all treatment groups. Correlation in the control group was not statistically significant.
In this investigation, we demonstrated macrophage counts and vascularity to be highly and significantly correlated in all three experimental groups treated with androgenic hormones. In the control group, though highly correlated, statistical significance was not obtained. This was a surprising finding indicating certain androgens, particularly testosterone and androstenedione, appear to have more of an effect on blood vessel development compared to dihydrotestosterone. Previous investigations support these findings [3-5].
Dihydrotestosterone and androstenedione appeared to inhibit macrophage migration and attraction to the implant compared to testosterone and control. This could be advantageous in extending the life of the implant. Fewer macrophages at the tissue implant interface could reduce the amount of degradative enzymes and other reactive oxygen intermediates as they act to dissolve the implant . Likewise, the reduced vascularity observed in the testosterone treated group was unexpected. Previous reports indicated the testosterone treated groups demonstrated fewer blood vessels/HPF, but the blood vessels present had greater diameter compared to control helping to sustain macrophage counts .
The strengths of this study include the use of digital analysis software to aid in data collection, the care taken to treat all animals in exactly the same manner, and the use of genetically related animals to minimize variation. This study is not without limitations. Previous experiments have demonstrated as few as three animals per group were adequate to determine significant differences among groups [2, 6-8]. While the number of animals per group was low, the number of observations per slide per animal was high to further minimize variability. Another limitation concerns the use of morphometry to identify the number macrophages and blood vessels/HPF. In doing so, without immunohistochemical staining, we may underestimate the number of proliferating or budding vessels or macrophages . However, this is not a major concern since the histomorphometric evaluation occurred 90-days post-implantation allowing the tissue-implant response to fully stabilize.
The findings from this study demonstrate that macrophage populations and vascularity are highly correlated. The number of macrophages present in the tissue-implant response appears to be directly proportional to the number of blood vessels per high power field. These findings suggest macrophages participating in the tissue implant response are dependent on the vascularity of the fibrous tissue capsule surrounding the implants. This information could be advantageous as sustained release technologies are further enhanced for potential use in humans.
The authors thank Ms. Gerri Wilson and Ms. Lisa McCammon, from the Department of Orthopedic Surgery and Rehabilitation for their technical and administrative support.
 K. Butler, H. Benghuzzi, P. Bajpai, A. Puckett, M. Tucci, Z. Cason, and B. England, "One year histopathological evaluation of fibrous tissue surrounding TCPL implants using adult rats as a model," Biomed Sci Instrum, vol. 33, pp. 233-9, 1997.
 K. Butler, A. Puckett, and H. Benghuzzi, "Quantitative analysis of the cellular components of the fibrous tissue matrix surrounding ALCAP, HA, and TCP bioceramics using adult male rats as a model," Biomed Sci Instrum, vol. 35, pp. 267-72, 1999.
 K. R. Butler, Jr. and H. A. Benghuzzi, "Morphometric analysis of the hormonal effect on tissue-implant response associated with TCP bioceramic implants," Biomed Sci Instrum, vol. 39, pp. 535-40, 2003.
 K. R. Butler, Jr., H. Benghuzzi, M. Tucci, and A. Puckett, "Androgen administration and macrophage behavior in the tissue-implant response - biomed 2011," Biomed Sci Instrum, vol. 47, pp. 228-33, 2011.
 K. R. Butler, H. Benghuzzi, M. Tucci, and A. Puckett, "Neovascularization is influenced by androgenic hormones in the tissue implant response," Biomed Sci Instrum, vol. 48, pp. 49-56, 2012.
 K. Butler, H. Benghuzzi, and A. Puckett, "Cytological evaluation of the tissue-implant reaction associated with S/C and I/P implantation of ALCAP and HA bioceramics in vivo," Pathol Res Pract, vol. 197, pp. 2939, 2001.
 K. Butler, H. Benghuzzi, M. Tucci, and Z. Cason, "A comparison of fibrous tissue formation surrounding intraperitoneal and subcutaneous implantation of ALCAP, HA, and TCP ceramic devices," Biomed Sci Instrum, vol. 34, pp. 18-23, 1997.
 K. R. Butler, H. A. Benghuzzi, and A. Puckett, "Morphometric evaluation of tissue-implant reaction associated with ALCAP and TCP bioceramics in vivo," J Invest Surg, vol. 14, pp. 139-52, May-Jun 2001.
 S. Henno, J. C. Lambotte, D. Glez, M. Guigand, G. Lancien, and G. Cathelineau, "Characterisation and quantification of angiogenesis in beta-tricalcium phosphate implants by immunohistochemistry and transmission electron microscopy," Biomaterials, vol. 24, pp. 3173-81, Aug 2003.
Kenneth R. Butler, PhD, Hamed A. Benghuzzi, PhD, Michelle Tucci, PhD, Aaron D. Puckett, PhD
University of Mississippi Medical Center Jackson, Mississippi--USA
Correlation of Macrophage Counts and Vascularity Group Variable Mean [+ or -] SD Pearson r Overall Macrophages 5.3 [+ or -] 2.2 .89 ** Vascularity 7.4 [+ or -] 4.8 Control Macrophages 6.8 [+ or -] 1.4 0.94 Vascularity 12.9 [+ or -] 1 Androstenedione Macrophages 3 [+ or -] 0.5 0.971 * Vascularity 3.5 [+ or -] 0.6 Dihydrotestosterone Macrophages 7.5 [+ or -] 1 0.989 * Vascularity 10.5 [+ or -] 3.3 Testosterone Macrophages 3.7 [+ or -] 1.4 .998 ** Vascularity 2.7 [+ or -] 0.5 Group Variable P-value Overall Macrophages <0.0001 Vascularity Control Macrophages 0.064 Vascularity Androstenedione Macrophages 0.029 Vascularity Dihydrotestosterone Macrophages 0.011 Vascularity Testosterone Macrophages 0.002 Vascularity
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|Author:||Butler, Kenneth R.; Benghuzzi, Hamed A.; Tucci, Michelle; Puckett, Aaron D.|
|Publication:||Journal of the Mississippi Academy of Sciences|
|Date:||Apr 1, 2014|
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