74 BIOTECHNOLOGIES
fragment rich in cell signaling domains that is a fraction of the size of the full protein. Herein we describe a series of in vitro, ex vivo and in vivo studies designed to demonstrate H21’s anti- ageing efficacy.
ECM stimulation - in vitro Fibroblasts are the most prevalent cell in the human dermis and are the main producers of ECM.
The effect of H21 on the expression of collagen
type 1 was evaluated by immunofluorescence microscopy in human dermal fibroblasts (Figure 1). Fibroblasts were cultured in media alone (control), or with 0.03%, 0.05%, or 0.1% H21 for 24 hours.
Fibroblasts were fixed, then stained using
antibodies that bind to collagen type I, thus allowing visualisation by fluorescence microscopy. Compared to cells treated with media alone, cells treated with H21 showed brighter fluorescence signal intensity (green), which indicates the presence of more collagen type I. ELISA experiments also confirmed dose
response increases of collagen type 1 and elastin from human fibroblasts at 24, 48 and 72 hours when stimulated with H21 compared to media control (data not shown). To evaluate changes in collagen type
1, collagen type III, fibronectin and elastin production, a full thickness reconstituted human skin tissue model was used. This cultured model consisted of a multilayered tissue forming a dermis constituted of fibroblasts in a collagen matrix and an epidermis constituted of keratinocytes. The epidermal and dermal layers were
mitotically and metabolically active and exhibited an in vivo-like morphological and growth characteristics. A well-developed basement membrane and stratum corneum was also present.10
A total of 50 µL of the test materials
diluted in phosphate-buffered saline were applied to the surface of the skin once per day. Untreated skin served as the negative control. ECM proteins were measured from collected tissue culture media at 48 and 96 hours using ELISA kits following manufacturer’s protocols (Figure 2). Topical treatments containing 0.1% H21
penetrated the in vitro skin equivalents upregulating the ECM proteins collagen type
Collagen Type I 96 hours
2500 2000 1500 1000 500 0
+61%
+68% *
+148% *
CONTROL 0.03% H21
0.05% H21
0.1% H21
Figure 1: Expression of collagen type I increases with H21 in human fibroblasts
I (+61%), type III (+86% *p<0.05), elastin (+70% *p<0.05), and fibronectin (+44% *p<0.05) versus untreated. H21 performed similarly to retinol. Interestingly, H21 had synergistic boosting benefits with low molecular weight HA for collagen type I production (patent pending).
ECM stimulation – ex vivo Collagen, elastin, fibronectin, and HA are key components of the ECM which undergo profound transformation both naturally with age and in response to photodamage. Given the promising in vitro capacity of ECM synthesis by H21, further testing was completed to determine whether this effect was seen in an ex vivo setting. Human skin explants from a 47-year-
old Caucasian woman were taken from an abdominoplasty, with an average diameter of 12mm. Explants were kept in culture at 37°C and 5% CO2
H21, retinol (1%), or left untreated. Experiments were performed in triplicates. Treatments were
Collagen Type III 48 hours
7000 5250 3500 1750 0
+86% *
*
7000 5250 3500 1750 0
+70% *
*
1200 900 600 300 0
+44% *
* in survival medium for five days with
topically applied to explants at day 0, day 1 and day 4. On day 5, the explants were prepared for immunohistochemistry and immunostained for collagen type I, type III, elastin, fibronectin, and HA. In Figure 3, representative images of collagen type I and HA staining in explant models are presented. Collagen type I staining was particularly elevated in the papillary dermis near the dermo-epidermal junction. Quantitative staining analysis showed there was a statistically significant increase in the total collagen score compared to control (+90% **p<0.01). Furthermore, staining of collagen type III (+10%), fibronectin (+60% **p<0.01) and elastin (+20%) in the papillary dermis were also increased compared to controls.
Endogenous production of HA HA, also known as hyaluronan, is a key component of the ECM. Involved in several processes including tissue hydration, repair, and fibroblast proliferation, HA helps maintain
Elastin 96 hours
Fibronectin 96 hours
* significant p<0.05 vs negative control Figure 2: H21 increases ECM production in full thickness skin equivalents PERSONAL CARE March 2023
www.personalcaremagazine.com
Type I C-peptide (ng/ml)
Untreated 0.1% H21 Retinol 10 µM 0.1% HA
0.1% H21+ 0.1% HA
Type III C-peptide (ng/ml)
Untreated 0.1% H21 Retinol 10 µM
Elastin (ng/ml)
Untreated 0.1% H21 Retinol 10 µM
Fibronectin (ng/ml)
Untreated 0.1% H21 Retinol 10 µM
Page 1 |
Page 2 |
Page 3 |
Page 4 |
Page 5 |
Page 6 |
Page 7 |
Page 8 |
Page 9 |
Page 10 |
Page 11 |
Page 12 |
Page 13 |
Page 14 |
Page 15 |
Page 16 |
Page 17 |
Page 18 |
Page 19 |
Page 20 |
Page 21 |
Page 22 |
Page 23 |
Page 24 |
Page 25 |
Page 26 |
Page 27 |
Page 28 |
Page 29 |
Page 30 |
Page 31 |
Page 32 |
Page 33 |
Page 34 |
Page 35 |
Page 36 |
Page 37 |
Page 38 |
Page 39 |
Page 40 |
Page 41 |
Page 42 |
Page 43 |
Page 44 |
Page 45 |
Page 46 |
Page 47 |
Page 48 |
Page 49 |
Page 50 |
Page 51 |
Page 52 |
Page 53 |
Page 54 |
Page 55 |
Page 56 |
Page 57 |
Page 58 |
Page 59 |
Page 60 |
Page 61 |
Page 62 |
Page 63 |
Page 64 |
Page 65 |
Page 66 |
Page 67 |
Page 68 |
Page 69 |
Page 70 |
Page 71 |
Page 72 |
Page 73 |
Page 74 |
Page 75 |
Page 76 |
Page 77 |
Page 78 |
Page 79 |
Page 80 |
Page 81 |
Page 82 |
Page 83 |
Page 84 |
Page 85 |
Page 86 |
Page 87 |
Page 88 |
Page 89 |
Page 90 |
Page 91 |
Page 92 |
Page 93 |
Page 94 |
Page 95 |
Page 96 |
Page 97 |
Page 98 |
Page 99 |
Page 100 |
Page 101 |
Page 102 |
Page 103
