Clinical Tests at Dermatologic Clinic.
Date: 15 July 1999
A clinical test which established that Crescina is an adjuvant in natural hair growth and is also a preparation that will help cut down scalp hair thinning. The tests were undertaken by the medical team of the Dermatology Clinic at a public hospital in July 1999.
The research took 6 months and there were thirty volunteers, 19 of whom were male and 11 were female, between 20 and 52 years of age. The males were affected by thinning up to level V on the Hamilton scale, and the females up to level III on the Ludwig scale. The right-hand side of the scalp was treated with Crescina lotion, whilst the left-hand side was treated with a placebo lotion.
After 2 months of treatment the clinic noted that regrowth on the part of the scalp treated with Crescina was: judged good for 13 subjects out of the 30, satisfactory for 13 subjects out of the 30, moderate for 4 out of the 30. This means that in 2 months, all subjects had some result. 86.6% judged the result satisfactory to good.
After four months, the clinical tests showed regrowth judged to be:
– good in 21 subjects out of the 30
– satisfactory in 7 subjects out of the 30
– moderate in 2 subjects out of the 30.
After four months, the regrowth was judged to be satisfactory to good in 93.3% of subjects subjected to treatment.
After six months the following results were achieved:
– good regrowth for 25 subjects out of the 30
– satisfactory regrowth in 5 subjects out of the 30
Which means that the entire panel of volunteers achieved satisfactory to good results. In the part of the scalp where a placebo was used, the result was judged as non-existent or moderate. However, the clinical tests did not just classify the regrowth results, but also those connected to hair strength and trophism, as well as the state of thinning.
– Hair strength after 6 months of treatment: good for 27 subjects out of the 30, satisfactory for 3 subjects out of the 30.
– Hair trophism after 6 months of treatment: good for 28 subjects out of the 30, satisfactory for 2 subjects out of the 30.
The feedback on thinning should be discussed separately, since it is more important (as the subjects in the tests felt it to be so) and because it was more diversified, given that subjects arrived at the tests with what can be termed basal situations that were very different from one another.
5 subjects out of the 30 were affected initially by extensive incipient baldness: after 6 months all 5 subjects showed an improvement that placed them in the incipient baldness category.
– 3 subjects out of the 30 were affected initially by incipient baldness (2 as Hamilton level IV, 1 as Ludwig level II): after 6 months all three had passed to a level of serious thinning.
– 10 subjects out of the 30 were affected initially by serious thinning (6 as Hamilton level III, 4 as Ludwig level II): after 6 months all had passed to a level of slight thinning.
– 9 subjects out of the 30 were affected initially by serious thinning (5 as Hamilton level II, 4 as Ludwig level I): after just 4 months all had passed to a level of slight thinning.
– 3 subjects out of the 30 were affected by slight thinning (Ludwig level I): after just 4 months all 3 subjects showed total regression of thinning.
Final overall opinion expressed by the Head Dermatologist after 6 months of treatment with regard to the state of thinning: good for 21 subjects, satisfactory for 9 subjects. It should also be noted that 50% of the sample population had found improvement in thinning after only 2 months of treatment.
Instrumental and clinical test at Institute for Chemical Researches and Bioingeneering, Milan
Date: 5 May 1999
The test, entitled “Clinical Tests for assessment of a trichological lotion (Crescina) in subjects suffering thinning due to androgenetic alopecia and/or hair loss”, was undertaken with a group of 25 willing, healthy volunteers, 14 women and 11 men, aged between 23 and 45, affected by androgenetic alopecia and/or hair loss at level I-II of the Ludwig scale, for women, and level II-III, of the Hamilton scale for men. The treatment, after decisions taken by the volunteers, was completed on 22 of the 25 initial subjects.
The objectives of the test included clinical assessment using diagnostic measurements, of Crescina’s effectiveness in hair regrowth (and its tolerability) in relation to different types of thinning caused by androgenetic alopecia and/or hair loss.
Skin area for the test: scalp.
Methodology: treatment every other day for 90 days.
Diagnostic measurement after one month, two months, three months (sebum, hydration, pH) to assess:
1) state of alopecia and/or loss of hair,
2) level of seborrhoea on scalp and hair,
3) appearance of dandruff,
4) pull test.
In basal conditions and at the end of the treatment the following were also performed:
the part of the skin to be subjected to electronic counting of growing hair was shaved and photographed
photograph of an area of alopecia
a hair sample was taken for morphological assessment under an optical microscope, checking the state of the bulb, the shaft, the thickness and the glossiness of the shaft.
Tests results Test results are supported by the enclosed graphs and tables.
* Treatment well-tolerated: no irritation or allergy phenomena.
** A statistically significant 13% increase in growing hair compared to the basal count, checked using an electronic counter.
*** 33% decrease in hair detaching with a Pull Test, an indication of increased traction resistance.
**** Observation of bulb under optical microscope: increase of bulbs in the anagen phase (when new hair begins to grow), from 3.7 to 25.9% of bulbs removed, reduction of bulbs in the telogen phase (hair loss phase) from 81.5% to 57.7% of bulbs removed.
Morphometric analysis: 11.2% increase in the average diameter of the hair compared to the basal measurement.
* Diagnostic measurements: no significant variations in average sebum values, significant increase in hydration.
* Clinical assessments – statistically significant quantity reduction of dandruff in 66.7% of cases and decreased greasiness in 57.1% . No significant sebum variations on skin and hair.
Observation of images of alopecic areas allows identification of the halting of the alopecic process (no subject presented a deterioration) and a regrowth in 45% of cases.
The results obtained after using Crescina lotion for 3 months on subjects suffering various stages of alopecia show that it was halted and that hair regrowth was brought about by the treatment itself. No alteration of skin surface parameters.
Instrumental and clinical tests at Institute for Chemical Researches and Bioingeneering, Milan Date: 20 February 2001.
The test, “Clinical tests for assessment of a trichological lotion (Crescina) in subjects suffering thinning due to androgenetic alopecia and/or hair loss”, was undertaken with a group of 42 volunteers, who made use of the Crescina product in the ambit of a more extensive study, conducted on 84 subjects, to compare Crescina and a reference product used in the same way.
Application: lotion applied directly (ml 2.5) to the scalp, on areas where thinning was most concentrated, rubbing slightly until completely absorbed. In basal conditions (T0), at two (T2) and three months (T3), a phototrichogram was performed. Moreover, for each of the three periods (0, 2, 3) an alopecic area was photographed. Sample population. 42 volunteers, 24 females, 18 males. Affected by varying degrees of alopecia: I-II Ludwig for the females, II-III-IV-V Hamilton for the males. 5 volunteers dropped out during the course of treatment. 37 subjects completed the protocol. – Test results Phototrichogram for subjects with diagnosed hair loss. * A statistically significant increase in the average percentage increase of hair in the anagen phase: from 27 to 61%. ** Female population enrolled independently of the inclusion diagnosis: highly significant increase in percentage of hair in the anagen phase even after two months (T2), from 20% to 55%, and after 3 months (T3) from 20% to 50%. *** Female population diagnosed with alopecia: statistically significant increase in percentage of hair in the anagen phase even after 2 months of treatment, from 17 to 48%. **** Male population with diagnosed hair loss/alopecia of Hamilton level II, III, IV: significant increase in percentage of hair in the anagen phase after 3 months of treatment: from 27 to 56%. More evident in males with level II and III diagnosed hair loss/alopecia. Male population diagnosed with Hamilton level II and III alopecia: increase in percentage of hair in the anagen phase after 2 months from 35% (T0) to 69% (T2), after 3 month to 83% (T3). * Hair diameter: statistically significant increase in hair diameter (from 0.080 mm, to 0.090 mm after two months, to 0.095 mm after 3 months) for subjects with diagnosed hair loss. * Visual frontal-parietal assessment: observation of photos of the frontal-parietal zone shows how Crescina brought about an improvement of basal conditions in 41% of treated cases. Analysis by gender confirmed the overall datum. No subject showed deterioration of the basal conditions. –
Crescina is able to perform trophic activity, which means that it stimulates the growth of hair in subjects suffering thinning due to various causes. In subjects with diagnosed hair loss: improvement after 2 months of treatment. In subjects with diagnosed alopecia: increase in the number of hairs in the anagen phase compared to the telogen/catagen phase. Noticeable effects both for men and women.
“IN VITRO” TEST
Effects of Crescina® on human primary epidermal and dermal cells – Investigators: V C Biotechnology
Aim of project
It has been tested a preparation formed by the nucleus of Crescina hair treatment active principles in an aqueous solution and with the following composition: aqua, glycerin, lysine hydrochloride, acetyl cysteine, glycoproteins. The objectives of this project were the following:
Objective 1 We investigated effects of Crescina on human primary keratinocytes and fibroblast cell proliferation
Objective 2 We investigated the effects of Crescina on protein synthesis in human in primary human keratinocytes and fibroblasts.
Abbreviations used NHEK: Normal human epidermal keratinocytes NHDF: Normal human dermal fibroblasts SD: Standard deviation MV: Mean value Study protocol Objective 1 Cell Cultures. Primary human keratinocytes and fibroblasts were obtained from Promocell (Heidelberg, Germany). Cells were maintained in specific growth medium supplemented with growth factors and seeded in 96 well plates (10 000 cells/well) for determination of cell proliferation and in 24 well plates for protein synthesis. Cells were treated for different period of time (24, 48 and 72 hours) with increasing concentrations (0.031, 0.062, 0.125, 0.25, 0.5% w/v = 310 µg/ml, 620.5 µg/ml, 1.25 mg/ml, 2.5 mg/ml, 5 mg/ml provided as 0.31 µl, 0.62 µl, 1.25 µl, 2.5 µl, 5 µl) of a solution of Crescina. Medium was removed before stimulation and replaced by fresh medium without growth factors. The growth factor supplements, as well as the growth factors PDGF (platelet derived growth factor) and FGF (fibroblast growth factor), were used as positive controls. Not-treated cells have been used as negative control. Determination of cell proliferation. Fibroblasts or keratinocytes were incubated with Crescina for 24 h, 48 h, and 72h. Each dose (see above) was tested 16 times (8 wells on 2 plates with two different methods). Proliferation of primary fibroblasts/keratinocytes was determined by: 1) Determination of DNA incorporation (dBrU) using a cell proliferation assay from Amersham-Pharmacia . Incorporation of 5-bromo-2′-deoxyuridine (BrdU) into newly synthesized DNA permits indirect detection of proliferating cells with labelled anti-BrdU antibodies thereby facilitating the identification of cells that have progressed through the S-phase of the cell cycle during the BrdU labelling period. The BrdU labelling method is a widely used method to measure proliferation of keratinocytes (Yano et al., 2004) and fibroblasts (Nasonova et al., 2004). 2) AlamarBlue staining (Biosource). AlamarBlue is a redox indicator that yields a colorimetric change and a fluorescent signal in a response to a metabolic activity. The compound is non-toxic, soluble and stable in tissue culture medium, and is used for the quantitative measurement of proliferation and viability of cells. The AlamarBlue assay incorporates a fluorometric/colorimetric growth indicator based on detection of metabolic activity. Specifically, the system incorporates an oxidation-reduction (REDOX) indicator that both fluoresces and changes colour in response to chemical reduction of growth medium resulting from cell growth. As cells grow, innate metabolic activity results in a chemical reduction of AlamarBlue. Continued growth maintains a reduced environment while inhibition of growth maintains an oxidized environment. Reduction related to growth causes the REDOX indicator to change from oxidized (non-fluorescent, blue) form to reduced (fluorescent, red) form. This colorimetric reaction was determined by an ELISA reading measuring the intensity of the red colour as marker of the increased AlamarBlue reaction. This colorimetric determination of colour intensity is expressed as arbitrary units, since there is no direct transaction to other metric systems possible. The increase of cell proliferation or metabolism has then to be expressed in comparison to untreated control cells. The AlamarBlue method is commonly used to determine proliferation (Voytik-Harbin et al., 1998) and cell viability (Ihalin et al., 2003) of keratinocytes and fibroblasts. Objective 2: Determination of protein content. Human primary keratinocytes or fibroblasts from human skin origin were seeded in 24 or 6 well plates and incubated with the Crescina (5 doses see above) for different times (24, 48 and 72 hours) with n = 4. After cell treatment, cells will be washed with phosphate buffered saline (PBS) and lysed in 1.3 x SDS (sodium dodecyl sulfate)-containing sample buffer without DTT containing 100 µM orthovanadate (Laemmli, 1970). Lysates will be homogenized by repeated passage through a 26-gauge-needle. Protein contents will be measured using the bicinchoninic acid method (BCA protein determination kit from Pierce, distributed by KFC Chemikalien, München, Germany) according to the manufacturer’s instructions (standards (bovine serum albumine, Sigma) ranging form 0.2 µg/µl to 4 µg/µl; optical density read at 570 nm). This method is a well established method to determine protein content and was part of at least 20 high impact publications from our group (e.g. Akundi et al., 2004). Deviations: To obtain more valid data in fibroblasts, additional experiments were performed using less cell numbers (1 000 and 5 000), and two additional time points. Results and Discussion Determination of cell proliferation. On human keratinocytes experiments, at all tested time points 24, 48 and 72 h, Crescina had no effect on DNA synthesis as measured by dBrdU incorporation. In contrast, Crescina revealed to increase the reaction of AlamarBlue. This effect was significant, suggesting that Crescina has an effect on cell viability (underlining values > 100% of metabolic activity) compared to non-treated cells (negative control) which cell vitality was considered as 100%. The effects were especially evident with 0.5% of the product Crescina with a maximum value of + 55% at 24 h (Fig. 4) and with 0.25% of the product Crescina with maximum values of + 56.3% at 48 h (Fig. 5) and + 61.6% at 72 h. Furthermore, our data suggest, that Crescina increases the metabolic activity of cells by increasing cellular energy levels. At all tested time points fibroblastic DNA synthesis was not affected by Crescina, even if it’s possible to notice a quite moderate effect on DNA synthesis as measured by dBrdU incorporation (maximum value + 5.1% with 0.25% of the product Crescina after 48 h) in comparison with non-treated cells. In the fibroblast experiments using the AlamarBlue method, additional experiments were performed using an inferior number of cells (1 000 and 5 000 cells/well) and two additional time points (4 and 6 h) just to obtain data of major validity. The use of 10 000 cells as starting point did not gain significant data since the fibroblasts are rapid proliferating cells and therefore cell proliferation and metabolism was probably maximal and not further increasable with this amount of cells suggesting that cell metabolism was already decreased due to cell to cell interaction lowering the proliferation rate of the cells. In the AlamarBlue assays with 1 000 cells/well, Crescina showed a quite moderate increase of metabolic activity, compared to the negative control, after 4 and 6 h with maximal values of + 14.5 % and + 20.5% respectively, both with 0.5% of Crescina. Very potent effects were obtained after 24, 48, and 72 h (Figs. 13-14) with maximal increase in the metabolic activity of + 58.5% with 0.125% of Crescina at 24 h, + 111.4% with 0.25% of Crescina at 48 h and + 195.7% with 0.5% of Crescina at 72 h, compared to negative control. Furthermore, in the AlamarBlue assays determining the Crescina effects on cell proliferation with 5 000 cells/well, Crescina showed an increase in the metabolic activity, compared to non-treated cells (negative control), 4 and 6 h with maximal values of + 33.1% and + 36.5% both with 0.5% of Crescina. Very potent effects were detected after 24, 48, and 72 h (Figs. 18-19) showing an increase in metabolic activity of + 80.3% with 0.0625% of Crescina at 24 h, + 119.3% with 0.5% of Crescina at 48 h, and + 91.4% with 0.5% of Crescina at 72 h, all of them compared to negative control. In this way, Crescina increased AlamarBlue reactivity when 5 000 cells were seeded but not as potent as with 1 000 cells per well. Only slight effects were visible after 24 h, when 10 000 cells where seeded (+ 20.2% with 0.5% of Crescina), and no effects after 48 and 72 h (Fig. 20), suggesting that the proliferation and the viability of cells is not further increasable after later time points. This indicated, that the number of cells is critical for obtaining effects of Crescina and that the number of 10 000 cells is to high to induce cell viability by possible cell to cell interactions occurring in confluent cell layers. Determination of protein content. Determination of protein content performed using keratinocytes revealed that, compared to non-treated cells, Crescina increased protein content (+ 160.3%) at the dose of 0.125% of Crescina at 24 h, at the doses of 0.0625% (+ 242.1%) and 0,125% (+ 206.8%) after 48 h and, at least, after 72 h with 0.25% of Crescina it’s possible to notice an increase of protein content of + 73.9% (Fig. 23, 24). No increase in protein content was visible in fibroblasts after 24 h, but a slight increasing effect after 72 h: + 51.4% with the exposition at 0.125% of Crescina. Conclusions Determination of cell proliferation. Using human keratinocytes, the preparation Crescina had no effects on DNA synthesis as measured by dBrdU incorporation at all tested time points 24, 48 and 72 h. However, Crescina increased the metabolic activity of cells by increasing cellular energy levels as revealed by the reaction of AlamarBlue. Using human fibroblasts, Crescina had no effect on DNA synthesis as measured by dBrdU incorporation at all tested time points 24, 48 and 72 h. In the AlamarBlue assays, very potent effects were obtained after 24, 48, and 72 h with an increase in the metabolic activity compared to non-treated cells . Our data suggest that due to its positive effects on cell viability and metabolism as shown by the AlamarBlue assays, Crescina should also increase the cell number. Furthermore, we conclude that the treatment with Crescina increases cell life, finally resulting in increased cell numbers, if compared to untreated cells, which have a more rapid cell turnover. This increase in cell numbers in Crescina treated cells, will not increase DNA synthesis as observed in this study. Determination of protein content. Determination of protein content in keratinocytes revealed that Crescina increased protein content considerably after 24 h (+ 160.3%) and 48 h (+ 242.1%) compared to negative control. In fibroblasts, a slight increasing effect was visible after 72 h (+ 51.4%) compared to non-treated cells. The difference in the total protein content between keratinocytes and fibroblasts is due to the differences in the total cell volume between the epithelial keratinocytes and the smaller fibroblasts. Therefore, the protein content has not increased in consequence of the pro-proliferative effect of Crescina, but it might be increased due to increased cell viability. This fits to the hypothesis that Crescina promotes cellular metabolism. Finally, we demonstrated that preparation Crescina increased cell metabolism and therefore might act as a cell protective and energizing hair treatment.