ABSTRACT
ALKALINE PHOSPHATASE ACTIVITY IN BONE REGENERATION
PART II
Objectives:
1.Regulate activity of Alkaline Phosphatase (AP) as marker in bone regeneration process. 2.Identify cell proliferation in presence of different electric fields. 3.Characterize electric fields to apply in connective cell cultures to regulate AP-activity and proliferation of bony precursor cells.
Methods:
Human fibroblasts “American Type Culture Collection (ATCC)” Ref.CRL-2014(HGF-1) were stimulated with Altern(AC) or Direct Current(DC). Stimulation time was 3, 6 and 12 hours/day, during four days. Electrical system adapted inside CO2 camera maintaining cell conditions in vitro. Fields were generated using aluminum plates, culture flasks were positioned inside avoiding edge effects. Faraday isolation blocked external interferences. Based on results obtained in Part I of this study, applied variables were: Current, AC-DC. Amplitude, 10mV and 20mV. Fields were controlled with “Kethley®Mod-6514” Electrometer. AP-activity measurement was made with Sigma® Kit “Catalog/Ref.104”. Positive control used for AP is “2-N enzyme control Sigma® “Catalog/Ref.S-2005”. System calibration made referenced on measurements of: 1.Proliferation. 2.AP-activity, by Elisa-technique. 3.Morphology. 4.Cellular viability. Controls were taken in cell culture flasks without stimulation.
Results:
|
AP-Activity |
Proliferation(x103) |
|||||
|
|
3h(X±S) |
6h(X±S) |
12h(X±S) |
3h(X±S) |
6h(X±S) |
12h(X±S) |
|
10mV-AC |
70,14±10,43 |
92,4±21,1 |
77,37±31,87 |
1473±81 |
947±340 |
1813±422 |
|
20mV-AC |
86,28±28,4 |
62,34±4,2 |
66,35±28,34 |
1333±153 |
1580±322 |
2033±140 |
|
10mV-DC |
8,9±2,55 |
9,46± 6,32 |
8,9±0,96 |
560±264 |
747±255 |
613±264 |
|
20mV-DC |
8,35±2,89 |
11,13±3,47 |
15,03±2,89 |
607±130 |
733±166 |
640±211 |
|
AP-Positive Control =45.09 |
AP-Negative Control =6.68 |
Control =647 |
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Conclusions:
AC is the election current in regeneration processes when bone needs precursor cells proliferation. Also can be used to regulate AP-activity, when extracelular matrix needs mineralization. Electric field with AC that shows the higher value in AP-activity reflects the lowest proliferation value, this could be meaning an inverse proportion between this two processes. This results, are short term applicable in solution of clinical problems related with handling of mineralized tissues of human body.