We studied in vitro attachment, proliferation and survival of сadaver skin fibroblasts in collagen bands, dermal matrix and cancellous demineralized bone, enriched with platelet components. It was shown, that PRP enhanced revitalization of collagen grafts, especially followed by components of activated or freezed platelets. Fibroblasts had the best rate of proliferative activity in response to 150 pg platelet derived growth factor (PDGF), released from 100 million platelets with granules and measured for 100 × 10 3 cultivated cells. The use of platelet-derived material could increase fibroblast proliferation activity for 1.5-3 times in all types of collagen transplants without damage or decay of cell’s biological value.
Collagen matrixes are widely used in cell biology, including studies of proliferative activity, adhesion, locomotion of the cells [
The aim of this work is to study the biological effect of platelet components in collagen matrixes revitalization, applied in clinical practice.
We used collagen bands with human type 1 collagen, performed for burns wound treatment [
Platelet-rich plasma (PRP) was received from donor blood, preserved on citrate, after centrifugation at 350 g for 5 min. Using the method of assessing the functional status of human platelets [
Primary cultures of cadaver skin fibroblasts (CSF) obtained under the standard scheme from tissue donors [
Cultivated cells were studied with confocal microscope “Nikon Eclipse 80i” (Japan), using our method of vital-stained cells observation [
Autofluorescence ability is the natural feature of collagen, which allows visualizing the collagen-containing components in fluorescent microscope to assess architecture and density of collagen [
Before applying the PRP it was necessary to determine the optimal dose of platelet components for CSF culture. Our previous study of human fibroblasts M-22 found that the maximum rate of the fibroblasts proliferation observed at concentration 120 - 150 pg PDGF-BB per 100 × 103 cultivated cells without decay of their structural integrity [
Study of proliferative activity in CSF culture under influence of PDGF-BB from PRP showed that 60 pg PDGF-BB for 100 × 103 cells increased Ip3 to 1.7, 90 pg―to 2.2, 120 pg―to 2.7, 150 pg―to 3.0, 180 pg―to 2.4, 240 pg―to 1.5, whereas in control wells Ip3 did not exceed 1.4. Cell membranes integrity (CMI) through the 3 culturing days in control and experimental wells with 60 - 150 pg PDGF-BB amounted to 36.8 ± 1.4 points, with 180 pg―36.0 ± 1.7 points, experience with 240 pg―32.2 ± 2.3 points, comparing to 37.0 ± 2.5 points in control. Therefore, the concentration 150 pg PDGF-BB was non-toxic and most effective for stimulation of cell proliferation. In view of the fact that 150 pg PDGF-BB is released from 100 million platelets with granules (biologically high-grade platelets), it confirmed the effectiveness of this dose for 100 × 103 cells in CSF culture.
The next step of the research was saturating of the collagen matrixes with platelets components in vitro to give them growth-stimulating properties. It must be noticed, that the PDGF-BB output, as well as other growth factors, can be stimulated by different ways: 1) platelet degranulation during the process of adhesion; 2) degranulation under the influence of activation inductor; 3) degranulation during clot formation; 4) platelet destruction under the influence of a chemical or physical factors. In PRP-treated collagens bands and dermal matrix settlement of CSF was similar to the one recorded in the control wells. By the end of the 1th day number of cells on the surface of these grafts averaged 6.1 and 5.8 × 103/cm2. In wells with unactivated PRP one could observe total platelet activation, accompanied by their degranulation, but direct collagen adhesion was observed only in 10% of the total number of platelets and the remaining 90% platelets aggregated and effluxed granules in medium without collagen contact. Presence of platelet material did not significantly affect the adhesion of fibroblasts in wells with collagen bands and dermal matrix. In PRP-treated CDB process of cells settling from suspension was expressed weaker: after 1 day of culturing in wells with activated PRP and cryoPRP number of CSF amounted to (1.3 - 1.4) × 103/cm2, in wells with unactivated PRP―0.1 × 103/cm2, i.e. CSF adhesion was the same as in control. It is necessary to emphasize that the platelets did not completely set on the surface of CDB.
After 3 days of CSF cultivation in collagen bands and dermal matrix with the activated PRP and cryoPRP InP3 was on average 1.8 times higher than in control, in the CDB―more than 3 times (
Collagen matrix type | Collagen bands | Dermal matrix | Cancellous demineralized bone | |||
---|---|---|---|---|---|---|
number of adhesive cells, 103/cm2 | InP3 | number of adhesive cells, 103/cm2 | InP3 | number of adhesive cells, 103/cm2 | InP3 | |
control | 9.0 ± 0.5 | 1.5 ± 0.1 | 7.5 ± 0,5 | 1.2 ± 0.1 | 0.1 ± 0.1 | 1.0 ± 0.1 |
unactivated PRP | 16.2 ± 1.0* | 2.5 ± 0.2* | 11.4 ± 1.1* | 1.9 ± 0.1* | 0.1 ± 0.1 | 1.0 ± 0.2 |
activated PRP | 16.8 ± 1.1* | 2.7 ± 0.2* | 13.5 ± 1.3* | 2.2 ± 0.2* | 5.2 ± 2.2* | 3.6 ± 0.4* |
cryoPRP | 16.7 ± 1.0* | 2.6 ± 0.4* | 13.3 ± 1.6 | 2.1 ± 0.2* | 4.9 ± 1.1* | 3.4 ± 0.3* |
*Comparing to control type at p < 0.05.
on the bottom of the well (CMI = 36.6 ± 0.9 points). In experiments with CDB after 3 days monolayer did not form, but the proliferation of cells continued during the later cultivation in the presence of activated PRP or cryoPRP. As a result through 7 - 8 days confluent monolayer was observed on the surface of the CDB, maintaining the structural integrity of the cells (
Fibroblast migration in the internal area of collagen matrixes, saturated with platelet components during the whole period of cultivation was quite low and occured only through the activity of cells surface monolayer. After 7 days of cultivation maximum penetration depth of CSF samples in dermal matrix amounted to 20 µm, in CDB―10 µm, and did not depend on way of platelets processing. One could conclude, that total revitalization of biotransplants could estimate more than 30 days.
The efficacy of acellular dermal matrix was shown during treatment of full-thickness skin injuries [
Saturation of collagen matrixes with platelet components stimulated human fibroblast proliferation, undergone by platelet growth-stimulating factors output. Otherwise, it is shown that prolonged use of PRP in platelet quality-reducing conditions led to decay of reparative and regenerating effect. This points both importance of matrix preparation procedure and assessment of platelet biological integrity [
Maxim SergeevichMakarov,Natalya ValerjevnaBorovkova,Olga IvanovnaKonushko,Valery BorisovichKhvatov, (2015) Use of Platelet-Rich Plasma for Collagen Matrixes Revitalization with Human Fibroblast. Journal of Biosciences and Medicines,03,80-87. doi: 10.4236/jbm.2015.310011
PRP―platelet-rich plasma; CDB―cancellous demineralized bone; InP3―3d-day’s proliferative index; CSF― cadaver skin fibroblasts; CMI―cell’s membrane integrity; cryoPRP―PRP after cryodestruction; PDGF― platelet derived growth factor.