Experimental Research of Fibroblasts Rebuilding Decellularized
Dermal Matrix
Daofeng LIU 1,a , Xin Sheng HUANG1,band Jinhua ZUO2,c
1
Shengli oilfield central hospital, 2 Dept. of oral and maxillofacial surgery, affiliated hospital
of Binzhou medical college
No. 31, Jinan Road #1, Dongying City, Shandong Prov. 257034, China
Keywords: fibroblast; acellular
dermal matrix;
hydroxyproline;
matrix
metalloproteinase-1.
Abstract: purpose: Discuss the function of the fibroblasts in the process of
decellularized dermal matrix rebuilding. Method: The research team established the
animal model of ADM allograft, and cut the specimens to do HE dyeing, picric
acid-sirius red dyeing and matrix metalloproteinases 1 immunohistochemical dyeing
to test the hydroxyproline at the scheduled time. Results: Count the fibroblasts: the
number of the fibroblasts increased sharply within 2 weeks, and the number reached
the peak at 2 weeks , and it gradually decreased to the level of genuine leather. Ⅰ/Ⅲ
collagen ratio: The ratio declined from 1 to 4 weeks, and the ratio rised to the level of
genuine leather. Hydroxyproline: The Hydroxyproline increased within 4 weeks, and
the number increased to level of genuine leather. MMP-1 activity: The value of the
AOD decreased within 3 weeks, and the value decreased sharply from 2 to 3 weeks,
and the value of the AOD increased gradually to level of genuine leather. Conclusions:
The fibroblasts has the main function in the process of decellularized dermal matrix
rebuilding.
1 Instruction
Acellular dermal matrix was made of natural skin except epidermis and dermal cells.
And the ADM only retain the collagen and the extracellular matrix, and it is widely
used in the clinic [1-2]. The fibroblast is the most important cell in the connective
tissue cells. This study explained the function in the process of decellularized dermal
matrix rebuilding through observing the dynamic change of ADM allografts.
2 material and method
There were 70 male rats that were 2 months of age(license: SCXR(Lu)20060004),
and the weight was 250±25g. And the researchers collected 14 male rats to prepare
ADM. And the others was divided into 7 groups to allograft ADM. And in 1, 2, 3, 4,
8, 16, 24 weeks, the researchers drew materials. The disposal of the animals
accorded with 《Ethical issues in animal experimentation》.
2.1 reagent and instrument
DispaseⅡ(Invitrogen Co. USA), Triton X-100(Amresco Co. USA), Mobay
Chemical Co. USA, ECLIPSE E600 POL ( Nikon ) , ultraviolet visible
spectrophotometer, hydroxyproline checkerboard, rabbit anti rat MMP-1 PcAb.
2.2 preparation of ADM
We used the method of Dispase-Triton to prepare ADM.
2.3 animal model of ADM allograft
The rats was injected in the peritoneal with 3.6% chloral hydrate(1ml/100g), and
they were fixed in the anatomy after the success of the anesthesia. And the hair of the
back should be removed and sterilized. The researchers made a full-thickness
incision which is 2 cm long on the left of the centre line of the back to separate the
lacuna. And then ADM was inserted into the lacuna, and was fixed with line 1 after
flatting four corners. After the incision and bandage, the rats was fed in the single
cage.
2.4 animal model of ADM allograft
The rats was injected in the peritoneal with 3.6% chloral hydrate(1ml/100g), and they
were fixed in the anatomy after the success of the anesthesia. And the hair of the
back should be removed and sterilized. The researchers made a full-thickness
incision which is 2 cm long on the left of the centre line of the back to separate the
lacuna. And then ADM was inserted into the lacuna, and was fixed with line 1 after
flatting four corners. After the incision and bandage, the rats was fed in the single
cage.
2.5 results
At a predetermined time, the researchers executed the animals to get the specimens.
2.5.1 observation index
The size of ADM, color, texture and the relationship with the surrounding tissue, etc.
2.5.2 HE dyeing
We used the conventional method, and observed with the ordinary microscope. And
the researchers chose 10 slices at every time point, and randomly selected five high
vision(*400) from each slice.
2.5.3 Picric acid-sirius red dying
Paraffin slice was dewaxed to water. And the picric acid-sirius red was inserted into
the paraffin slice for 1 hour. And then, we used distilled water to wash 5 min. We used
the hematoxylin to strain. And the researchers chose 10 slices at every time point,
and randomly selected five high vision (*400) from each slice to observe the collagen
distribution of Ⅰ(red) and Ⅲ(green). And we use IPP 6.0 software to analyze the
collagen distribution of Ⅰ(red) and Ⅲ(green).
2.5.4 hydroxyproline
We tested the absorbance through the specification.
We make the following rules. H is the content of the hydroxyproline. D is the
determination absorbance. B is blank pipe absorbance. S is standard pipe absorbance.
Sc is content of the standard pipe. Hv is the volume of the hydrolysate. W is the wet
weight of the organization.
D B
Hv
H 
 Sc 
S B
W
2.5.5 matrix metalloproteinase-1(MMP-1) immumohistochemical dyeing
The researchers chose 10 slices at every time point, and randomly selected five high
vision(*400) from each slice with IPP 6.0 software to test average optical density.
2.6 Statistics
The data was shown as mean±standard deviation, and we use SPSS 13.0 to analyze.
The statistics had meaning if the p<0.05.
3 Results
3.1 physical characteristics
ADM was smooth within 1 week. The color didn’t change within 2 weeks. The more
area increased more and more within 3 weeks. ADM was covered by the film which
was covered with blood-vessel network, and the color of the ADM was milk white,
and the area equal to the beginning of the transplant. ADM fused with the skin within
8 weeks. And the thickness of the ADM pinched after 16 weeks.
3.2 HE dyeing
FB around the ADM increased, and the shape was ball within 1 week, and we could
see the capillaries. There were more FB in the center of the ADM, and the shape
change into a long spindle within 2 weeks, and the collagen accumulated around the
ADM, and the blood vessels was rich.
3.3 picric acid-sirius red dyeing
ADM has strong sense of layer with polarization microscope, and red bulk fasciculate
I type collagen composes rack, and green thin Ⅲ type collagen distribute in it.
3.4 change of hydroxyproline content
OHP content increased within 4 weeks, and after 4 weeks, OHP content decreased
continually to normal level.
3.5 matrix metalloproteinase-1(MMP-1) immunohistochemical dyeing
MMP-1 positive locates in FB cell. And AOD has the negative correlation with
MMP-1 activity.
Table 1 dynamic change of ADM
Time
Normal
1W
2W
3W
4W
8W
16W
24W
26
±6
242
±22*
397
±28*
261
±23*
133
±21*
54
±9*
25
±5
18
±3
Ⅰ/Ⅲpercent
age
3.02
±0.35
2.81
±0.27
2.22
±0.28*
1.96
±0.17*
1.85
±0.23*
2.02
±0.22*
2.43
±0.27
2.73
±0.25
OHPcontent
15.87
±1.02
16.90
±1.29
19.32
±1.37*
21.17
±1.42*
22.33
±0.72*
17.11
±1.19
16.59
±1.03
15.73
±1.66
AOD
0.490
±0.011
0.405*
±0.015
0.380*
±0.019
0.264*
±0.016
0.309*
±0.012
0.465
±0.013
0.475
±0.010
0.486
±0.006
FB
4 Discussions
The reconstruction after ADM allografts is mainly collagen metabolism. Fibroblast is
the main cell which can synthetize and degrade collagen. The functions of FB are
chemotactic migration, proliferation and conversion, etc.
ADM contains a lot of arginine- glycine- aspartic acid. And FB can use the integrin
receptor lig combine with RGD, and migrate into ADM. This study observed the FB
was lack of the ability of migration when ADM was inserted into the skin because
the number of the cytoskeleton inhibition of movement and migration are less than
the receptors required. And the infiltration of FB couldn’t be seen in the microscope
in ADM. So FB change into the type of migration through the temptation of the
collagen along the direction of the collagen fiber bundle. The platelet derivation
growth factor (PDGF) combines with FB membrane when ADM was implanted 1
week, and mitosis signal which was produced by activation of receptor tyrosine
kinase lead to DNA synthesis and FB proliferation. And part of the FB transformed
into myofibroblast. And this is because of the collagen contraction, and it will
disappear through the cell apoptosis. The amount of FB rose around the ADM, and
the gap along the small resistance penetrate to the center, and there were a little of
MFB, and there were a lot of new collagen fibers around FB, and the old collagen
degrade into small pieces. It will be the peak that FB transforms into MFB after 2
weeks. FB strings the collagen matrix through its pseudopodia and retraction. The
cytoplasmic of FB will be stellate because of the diversion of the arrangement of
collagen fibers. The structure of collagen fibers will change as the activity between
FB and collagen fibers. Its arrangement turned into unidirectional from the diversity.
FB cytoplasmic projections gradually showed the dual polarity, and the cell turn the
appearance of long spindle. FB regulate the collagen contraction through integrins,
and it can promote the cells which be planted migrate into the center of ADM. And
the amount of FB increased around ADM in the microscope, and a part of FB turn
into MFB, and the new collagen accumulated around ADM, and the old collagen
degraded dramatically. A part of FB turned into the mature fiber cells after ADM was
implanted 3 weeks, and the MFB enter the cell apoptosis. The amount of FB
decreased in the microscope, and the number of the elongated cells increased,
especially in the center, and the degradation area of the old collagen reduced. The
function of FB was almost end after 4 weeks, and there were not FB in the
microscope, and the state of the cells was similar with the fiber cells in the dermis.
The state when the time was 8, 16, 24 weeks was similar with 4 weeks. It was
complied with the law of wound repair.
This study found that the content of OHP and Ⅰ/Ⅲ percentage in ADM was closely
related with FB. The number of FB increased within 2 weeks after ADM was
implanted. And the amount of FB increased, and the new collagen was synthesized
and the old collagen was degraded, and the content of OHP increased, the Ⅰ/Ⅲ
percentage reduced. The synthesis of FB declined between 2 and 4 weeks, but the
new collagen increased. The activity of degradation was strong, and the content of
OHP increased a little, and the Ⅰ/Ⅲ percentage continued to fall. MMP-1 was
synthesized with FB, and was the core of Ⅰ and Ⅲ collagen. The activity of MMP-1
had the regularity, and was linked with FB. FB supplied the migration pathways for
cells, space for the new collagen more regularity.
There were FB, vascular endothelial cells, macrophages, etc. And FB accounted for
absolute advantage in them. This study found that the fibroblasts has the main
function in the process of decellularized dermal matrix rebuilding through secreting
collagen matrix and the synthesis and degradation of ADM collagen controlled by
MMP-1. The quantity and the form can guarantee the full reconstruction of ADM
and avoid excessive renovation, and maintain the stability of ADM, improve the
quality of wound, and make the repaired tissue be close to the original tissue. So we
can see that FB has an important function for maximizing the clinical value of ADM.
Reference
[1] Gaspar K, Erdei I, Peter Z, et al. Role of acellular dermal matrix allograft in
minimal invasive coverage of deep burn wound with bone exposed--case report and
histological evaluation .International Wound Journal, 2006, 3 (1): 51-8.
[2] Sallum EA, Nogueira-Filho GR, Casati MZ, et al. Coronally positioned flap with
or without acellular dermal matrix graft in gingival recessions: a histometric study.
American Journal of Dentistry, 2006, 19 (2): 128-32.
[3] Walter RJ, Matsuda T, Reyse HM, et a1. Characterization of acellular dermal
matrices(ADMS) prepared by two different methods[J].Burns.1998,24(2):104~13.
[4] Sushunqing, Yuzhi, Zhangyiming. Reaction of Fiber Cell and Extracellular
Matrix in Repairing Skin Injury. Chinese Journal of Rrauma, 2000, 16(6):345-6.
[5] Prasertsung I, Kanokpanont S, Bunaprasert T, et al. Development of acellular
dermis from porcine skin using periodic pressurized technique.J Biomed Mater Res
B Appl Biomater. 2008, 85 (1):210-9.
[6] Piazuelo E, Lanas A, Jimenez P, et al. In vitro wound repair by human gastric
fibroblasts: implications for ulcer healing. Dig Dis Sci, 1998, 43(6):1230-40.
7. Shin D, Minn KW. The effect of myofibroblast on contracture of hypertrophic scar.
[J] Plast Reconstr Surg ,2004,113(2):633-64.
Figure1 two weeks after ADM was implanted
Figure2 eight weeks after ADM was implanted
Figure3 sixteen weeks after ADM was implanted
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Experimental Research of Fibroblasts Rebuilding Decellularized