The Effect of BMP7 on Ectopic Calcification with and without Nanosilver Particles in Gluteofemoral Muscle of Rat
*1Hossein Shahoon 2Fateme Mashhadi Abbas 3Mahsa Nematollahi 4Shadi Shahoon
*1Corresponding Author: Assistant Professor, Dept. of Oral & Maxillofacial Surgery, Dental School, Shahed University, Tehran- Iran. E-mail: [email protected]
2Assistant Professor, Dept. of Oral & Maxillofacial Pathology, Dental School, Shahid Beheshti University of Medical Sciences, Tehran- Iran.
3Dentist
4Undergraduate student, Dental School, Szeged Medical University, Szeged-Hungary.
Abstract
Objective: Discovery, purification and synthesis of Bone Morphogenetic Proteins (BMP) were an important turning point in understanding of bone physiology for repair of bone defects. The present study aimed at eliminating the limitations caused by the application of bone auto-grafts and finding a proper substitute for bone grafts. This study was the first to evaluate the effect of BMP7 and nanosilver particles on the permeability of muscle cells for ectopic bone formation in Rat gluteofemoral muscle.
Methods: This experimental double blind study was conducted on 42 rats divided into 6 groups of 7 each. The first 3 groups received an injection of 200 ng BMP7 with 0.1 mg nanosilver particles and the remaining 3 groups received 200 ng BMP7 in 0.4 mg normal saline. Rats were euthanized 7, 14 and 21 days post-operation and evaluated in terms of presence of tissue changes like necrosis, inflammation, and formation of fibrous or calcified tissue under a light microscope with X40, X100, and X400 magnification. The obtained results were analyzed with Hosmer-Lemeshow, chi square and Wilcoxon tests. Results of the 2 groups were analyzed and compared using Mann Whitney U and Kruskal-Wallis tests.
Results: No statistically significant differences were detected between the subgroups of our 2 main understudy groups in terms of bone formation, distribution of inflammation (P=0.439), the amount of inflammation (P=0.743) and presence or absence of foreign body (P=0.381) over time. However, the two groups of BMP7 + nanosilver and BMP7 alone showed significant differences over time.
Conclusion: Rats do not have sufficient number of efficient receptors for BMP7 in muscle tissue.
Even by using nanosilver particles for increasing the permeability of cells, BMP7 was not able to induce muscle cell differentiation to initiate calcification process.
Key words: BMP7, Bone formation, Ectopic, Nanosilver particles Please cite this article as follows:
Shahoon H, Mashhadi Abbas F, Nematollahi M, Shahoon SH. The effect of BMP7 on ectopic clacification with and without nanosilver particles in Gluteofemoral muscle of rat. J Dent Sch 2013;30(4):224-232.
Received: 07.01.2011 Final Revision: 07.04.2012 Accepted: 13.05.2012
Introduction:
Tissue engineering covers a broad range of applications and discusses the use of engineering methods in biologic science to obtain a more accurate perception about structural interactions in healthy and pathological tissues of mammals.
It also addresses the advancements in discovery of new biologic substitutes to resume, preserve or improve the function of tissues (1-3). Bone is the most unique tissue among all tissue types
because when injury occurs, it heals by new bone formation. Another interesting characteristic would be the fact that cellular and molecular reactions responsible for development of skeletal tissues in fetus are very similar to the ones involved in the process of bone healing after injury (4). Discovery, purification and synthesis of Bone Morphogenetic Proteins (BMP) were an important turning point in understanding of bone physiology. BMPs belong to the group of Transforming Growth Factor-ß
(TGF-ß). TGF-ß superfamily also includes the TGF-ßs, activins, inhibins, and Müllerian- inhibiting substance but acts via a different intracellular pathway. BMPs are osteoinductive and are capable of rapid bone production by differentiating the mesenchymal cells into chondroblasts and osteoblasts (5, 6).
Nanoparticles can pass through the biological membrane and affect cell physiology. In order to do so, one mechanism is that the nanosilver particles can play a role in differentiation of these cells through increasing the permeability of cell membrane and subsequent cellular exchange with the outer environment (7-10).
Therefore, nanoparticles are used for increasing the transfer of therapeutic agents and genes into the cells (11, 12). Nanosilver particles are used as an antibacterial agent in various medical domains like wound healing and bone cement production (13-16). These particles in low concentration and in-vitro conditions have no effect on MSC cells, fibroblasts or osteoblasts but are bactericidal to penicillin-resistant bacteria like Staphylococcus aureus, yeasts and fungi (11-13,17).
The aim of the present study was to eliminate the limitations caused by the application of bone autografts and find a suitable substitute for bone grafts. Considering the function of BMP7 in ectopic calcification in rat muscle, we decided to evaluate the role of nanosilver particles in increasing the possibility of ectopic calcification through increasing the permeability of muscle cells. This study was the first to assess the effect of BMP7 and nanosilver particles on the permeability of myoblast membrane for ectopic bone formation in rat gluteofemoral muscle.
Methods:
In this experimental double blind study data was collected through microscopic observation and completion of questionnaire in Shahid Beheshti and Shahed Schools of Dentistry. A total of 42 Sprague Dawley (SD) rats with an approximate weight of 200-220 g were bought from the Pasteur Institute of Iran. Rats were divided into 6 groups of 7 each. BMP7 was purchased from Bio Med Hans (Korea) and nanosilver colloidal solution was purchased from Plasmachem (Germany). For the first 3 groups (A, B, C), 200
ng BMP7 dissolved in 0.4 cc normal saline and mixed with 10 μg/ml (0.1 cc) nanosilver was injected by an insulin syringe and 18 gauge needle at the medial aspect of femoral muscle of rat and marked with a dye. Normal saline with a pH of 7.4 was injected into the contralateral muscle as the control. Selection of case and control muscles was random. For the remaining 3 groups (E, F, G), 200 ng BMP7 (in 0.4 cc normal saline) was injected. Case and control muscles in these groups were similar to the first three.
Groups A and E were sacrificed after one week, B and F after 2 weeks and C and G after 4 weeks and their case and control muscles were sent to the pathology department of Shahid Beheshti Dental School for histopathological examination. Samples were then fixed in 10%
formalin solution for 24 days and sectioned. For slide preparation, paraffin embedded blocks were prepared from the sectioned slices and after cutting micron-thin sections, they were stained with Hematoxylin and Eosin. The specimens were then evaluated for tissue changes like presence of necrosis, inflammation, fibrous tissue and calcified tissue formation under a light microscope with X40, X100 and X400 magnification.
a
b
c
Figure 1- BMP group: a- week 1, b- week 2, c- week 4
a
b
c
Figure 1- BMP+nanos group: a- week 1, b- week 2, c- week 4
The measuring index for the production of skeletal tissue was Photoshop 8 software.
Tolerance of the bone formation areas exhibiting distinct similar coloring characteristics was selected and by histogram command number of pixels in such areas was calculated and recorded.
The obtained results were analyzed with Hosmer-Lemeshow, chi square and Wilcoxon tests. Results of the 2 groups were compared using Mann Whitney U and Kruskal Wallis tests.
Results:
Similar changes were observed among the subgroups over time. In other words, no significant differences were detected among the subgroups in terms of bone formation (P=0.283), distribution of inflammation (P=0.439), the amount of inflammation (P=0.743) and presence or absence of foreign body (P=0.381) over time.
However, significant differences were found between the 2 groups of BMP7+nanosilver and BMP7 alone over time. The results were evaluated in 4 subgroups: 1- Presence or absence of foreign body, 2- Distribution of inflammation, 3- Amount of inflammation 4- Bone formation (Table 1).
1- Foreign body: As time passed from week 1 to week 4, presence of foreign body in both groups decreased from 71.4% to 35.7%.
This reduction was statistically significant (P=0.031). It means that in both groups of BMP7+nanosilver and BMP7 alone, the implanted materials had vanished from their initial location over time. The goodness of fit for logistic regression model was confirmed by Hosmer-Lemeshow test.
2- Distribution of inflammation: Evaluation of inflammation distribution in all 6 subgroups indicated a decrease in inflammation over time from 92.9% to 42.9%. However, this reduction separately in the BMP7+
nanosilver and BMP7 alone groups was not statistically significant(P=0.067).Logistic regression analysis was used for this comparison and its goodness of fit was confirmed with Hosmer-Lemeshow test (P=0.918). Distribution of inflammation in
different time periods between the 2 main groups regardless of the subgroups was different as the distribution of inflammation was significantly different in week 1 and week 4 (P=0.01) and week 2 and week 4 (P=0.021). However, no such difference was detected between weeks 1 and 2 (P=0.536).
3- Amount of inflammation: Of 42 samples evaluated for amount of inflammation, a mean number of 11 specimens (26.2%) had no inflammation, 18 (42.9%) had 1-10%
inflammation, 8 (19%) had 10-30%
inflammation and 5 (11.9%) had 30-50%
inflammation. Amount of inflammation is a dependent ordinal variable and considering the 3 understudy time periods, Kruskal Wallis test was employed for its analysis.
The amount of inflammation was
significantly different in the 3 time periods (P<0.001). For paired comparison between the groups Bonferroni Mann Whitney U test was used which demonstrated a significant difference in the amount of inflammation between weeks 1 and 4 (P<0.001) and weeks 2 and 4 (P<0.001). However, the difference between weeks 1 and 2 in this respect was not statistically significant (P=0.114). In general, amount of inflammation in the group of BMP7+nanosilver was significantly higher than in BMP7 alone (P=0.009). Amount of inflammation in all 6 subgroups showed a decreasing trend post- operatively.
4- Bone formation: No sign of bone formation was observed in any of the 6 subgroups.
Table 1- Histological evaluation of the effect of BMP7 and nanosilver particles on 1- presence or absence of foreign body, 2- distribution of inflammation, 3- amount of inflammation and 4- bone
formation in the 2 groups of BMP7 + nanosilver and BMP7 alone
Variable
Presence of foreign body
Absence of
foreign body Test N % N %
Group Bmp+N 18 85.7 3 14.3
Logestic Regresion model (P=0.918) confirmed by Hosmer
& Lemeshow test
Bmp 13 61.9 8 38.1
Time
1st week A,E 13 92.9 1 7.1 2nd week B,F 12 85.7 2 14.3 4th week C,G 6 42.9 8 57.1 Variable
Distribution of inflammation
No
inflammation Test N % N %
Group Bmp+N 18 85.7 3 14.3
Logestic Regresion model (P=0.918) confirmed by Hosmer
& Lemeshow test
Bmp 13 61.9 8 38.1
Time
1st week A,E 13 92.9 1 7.1 2nd week B,F 12 85.7 2 14.3 4th week C,G 6 42.9 8 57.1 Variable
Amount of inflammation
0% 0-10% 10-30% 30-50%
N % N % N % N % Group Bmp+N 3 14.3 7 33.3 6 28.6 5 23.8
Bmp 8 38.1 11 52.4 2 9.5 0 0
Time
1stweek A,E 1 7.1 5 35.7 3 21.4 5 35.7
2nd week B,F 2 14.3 7 50 5 35.7 0 0
4th week C,G 8 57.1 6 42.9 0 0 0 0
Discussion:
Mesenchymal cells are considered as an important source for repair of calcified tissue
defects due to their abundance, availability, accessibility and multi-potential nature (18).
Bioactive molecules such as growth factors like TGFß, IGF, BMPs and cytokines play a very
important role in the process of osteogenic differentiation of these cells and proliferation and differentiation of these mesenchymal cells depend on these molecules (19). For in-vitro osteogenic differentiation of mesenchymal cells, simultaneous use of several synergistic growth factors and establishing a delivery system with nanoparticles that guarantees the long term stability and activity of biologic factors have been studied (20-23).
A suggested possible mechanism for increasing the stability and biologic activity of growth factors by nanoparticles in-vitro is changing the ionic properties of the environment by these materials which would prevent accumulation, aggregation and sedimentation of growth factors.
Consequently, increased stability, biologic activity and controlled release of these materials along with their long term presence in the area will boost the induction of osteogenic differentiation in multi-potential mesenchymal cells (24).
The present study used an animal model for the first time for in-vitro evaluation of the effect of nanosilver particles as carrier and booster of the effect of BMP7 to study the course of osteogenic differentiation in muscle tissue mesenchymal cells.
In order to evaluate the effect of TGFß on inducing chondrogenesis, Joyce and colleagues (1990) injected 200 ng of this material into subperiosteal region of rat femur on consecutive days. On day 4 (after injection of 3 consecutive doses of 200 ng daily), they reported the differentiation of chondroblasts (25).
Horisaka et al, in their study in 1991 prepared a mixture of 1 mg BMPs and 1.5 mg bovine collagen, pressed them into discs and implanted them under the fascia of the rectus abdominus muscle of rats. Bovine serum albumin discs were implanted in the control group. Histological evaluation of the implant site revealed initial mineral deposition followed by chondrogenesis and subsequent osteogenesis at day 14. No cartilage or bone formation occurred in the control group.
Horisaka et al, in 1991 also extracted bovine demineralized bone and implanted it along with collagen type I subcutaneously in rat’s chest area. Histological assessment of the implant site after a few days revealed lots of chondroblasts
and osteoblasts. Laboratory examinations detected elevated level of calcium and increased activity of alkaline phosphatase (26, 27).
Nakagawa and Tagawa (2000) evaluated the mechanism of direct bone formation induced by BMPs-collagen complex . They implanted partially purified BMPs combined with AC collagen into the calf muscles of 20 rats. Tissue specimens were histopathologically examined on days 7, 10, 14, and 21 after implantation. Early calcification was evident on day 10. In the uncalcified regions, mitochondrial calcification was seen in mesenchymal cells near AC fibers.
This study demonstrated that the mesenchymal cells invading BMPs-AC complex closely contacted the AC fibers, directly differentiated into osteogenic cells resulting in direct osteoinduction without cartilage formation (28). Levander in 1930 mentioned that injection of alcohol along with bone matrix into the muscle tissue results in new bone formation at the site of injection (29, 30). Lack of formation of calcified tissue in the present study may be due to the lack of an adequate delivery system for transportation of nanosilver particles and maintenance of BMP7 at the site of implant compared to the collagen carrier in Horisaka (1994)(27) and Tagawa (2000)(28) studies. Li et al, in 2009 used two cell types (C2C12, NIH/3T3), the former would express BMP4 only and the latter both BMP4
and VEGF in order to demonstrate the synergistic effect of vascular endothelial growth factor (VEGF) on BMP2 and BMP4-induced ectopic endochondral bone formation . These cells were cultured in-vitro and implanted into the gluteofemoral muscle in mice. Results showed that VEGF inhibited the in vitro calcification of C2C12 and NIH/3T3 cells. High ratio of VEGF to BMP4 induces ectopic bone formation.
However, when this ratio is kept low and constant over time VEGF loses its synergistic effect on ectopic bone formation. They proved that VEGF's synergistic role in BMP4-induced ectopic bone formation is dose and cell-type dependent. Based on the mentioned study, type of delivery system is of utmost importance in transport of growth factors for ectopic bone formation which further confirms the inefficient delivery system in the present study (31).
Lim and colleagues in 2010 aimed to develop a delivery system to increase the efficacy of
growth factors in an alginate solution culture medium. In order to do so, they used polyion complex nanoparticles in an alginate sodium solution culture medium containing BMP7 and TGFβ2. They mixed polyion complex nanoparticles with TGFβ2 and BMP7 and incubated in plates containing sodium alginate.
The results demonstrated that in the nanoparticle/hydrogel system at the end of an incubation period (day 20), the release of growth factors was 4 times the rate in the control group (80% versus 20%). The speed of release of growth factors was lower in the nanoparticle/hydrogel system than in the control group. In the dual growth factors-loaded hydrogel (without the nanoparticles) the highest rate of growth factor release was within the first 4 days and reached a plateau afterwards.
Whereas, in the nanoparticle/hydrogel system the release rate of growth factors kept a relatively constant gradient till the last day of the incubation period (day 21). The possible mechanism suggested by Lim and colleagues was that the presence of polyion complex particles in the system changes the ionic characteristics of the system and inhibits the aggregation and sedimentation of growth factors resulting in their constant release in the system (24). In the present study, presence of foreign body in both groups decreased from 71.4% in week one to 35.7% in week 4 and this decrease was statistically significant (P=0.031<0.05) which is indicative of the elimination of implanted materials in both groups of BMP7 alone and BMP7+nanosilver and is due to the absence of a delivery system that is required for maintaining the implanted materials at their respective site and for the desired time period.
BMP7 is the only member of TGFβ family that plays a role in inhibition of differentiation in mice (32-34). Aspenberg et al, in 1996 implanted type 1 collagen discs containing 0, 10, 40 or 200 microgram rhBMP-2 in monkeys’
muscles and observed bone induction only in half of the BMP-2 implants. They concluded that there may be too few cells expressing BMP-2 receptors in these monkeys (35). Therefore, we can conclude that rat model has the lowest value of BMP7 and this animal presumably lacks the necessary receptors for uptaking this factor in uncalcified tissues like muscle for induction of
ectopic bone formation. In contrast to rats, in humans BMP signaling plays no role in self- reproducibility and expedites the down regulation of Nagin and Oct ¾ genes and differentiation to trophoblast (36-41). In rats, BMP increases the expression of genes responsible for osteoporosis in humans.
Alt and coworkers in 2003 assessed the antibacterial properties and cytotoxicity of nanoparticulate silver bone cement. They studied the cytotoxicity of these nanoparticles on L929 fibroblasts through assessing the number of living cells. They concluded that this type of cement and its nanosilver particles are free of in- vitro cytotoxicity against fibroblasts and osteoblasts but are highly effective against penicillin-resistant bacteria especially staphylococcus aureus (16). The results of the mentioned study confirms our study finding regarding the lack of toxicity and antibacterial property of nanosilver particles since inflammation significantly decreased in all samples over time (P<0.05). Acute or chronic infection did not occur in any of the samples and antibiotic therapy was not required at all.
Nanoparticles are capable of passing through the biological membrane and affecting cell physiology. One mechanism is that the nanoparticles may play a role in cell differentiation through increasing the permeability of cell membrane and elevating cellular exchange rate with the outer-cell environment (7-10). Therefore, nanoparticles are used for increasing the delivery of therapeutic agents and transfer of genes into the cells (11- 13). Chen et al, in 2003 used sodium chloride modified silica nanoparticles as a vector for DNA transfer into HT1080 cells purified from mice. Electron microscopy demonstrated the adhesion of nanoparticles with cell surface and their entry into the cell. DNA transfer into the cell also increased from 35% to 75% (12).
Panyam et al, in 2003 used ammonium salt with poly isohexyl cyanoacrylate (PiHCA) nanoparticles to increase uptake and delivery of oligonucleotides into the cells in a culture medium. They concluded that nanoparticles bond with oligonucleotides enhancing their delivery into the cell (42).
Conclusion:
This study was the first in the field of osteology to determine the effect of nanosilver particles and growth factors on the course of ectopic calcification. The effect of BMP7 on muscle cells was not observed in any of the histological samples in the present study. Based on the results of other researchers, rat animal model does not have sufficient number of efficient BMP7 receptors in muscle tissue. Apart from the factors addressed in the discussion section, lack of the effect of nanosilver particles on cell
membrane permeability can also be considered as a reason for unsuccessful induction of ectopic calcification in muscle. However, this issue is still a matter of controversy and in order to make a definite statement further investigations are required to evaluate the biomolecular effects of nanosilver particles on ectopic osteogenesis.
Acknowledgement:
The authors would like to express their gratitude for the financial support provide by the Research Deputy of Shahed University for the conduction of this study.
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