The effect of botulinum toxin type A in different dilution on the contraction of fibroblast—In vitro study

Abstract Background Botulinum toxin type A (BoNT‐A) may directly remodel dermal tissues or induce a loss of normal morphology and cytoplasmic retraction and spread. Intradermal injection was claimed to produce a dermo‐lifting effect, including midface lifting by using low concentration with variable dilution. Objective To understand how intradermal BoNT‐A achieves tissue lifting, we examined different type of BoNT‐A and their effects on dermal fibroblast contraction. Methods Normal human dermal fibroblasts were treated with onabotulinumtoxin (ONA), abobotulinumtoxin (ABO), prabotulinumtoxinA (PRABO), incobotulinumtoxinA (INCO), and letibotulinumtoxin A (LETI) in dilutions used in real‐world practice. Fifty fibroblasts per dilution were photographed and measured the length to demonstrate their contraction every 2 hours from baseline (0 hours) to 12 hours post‐treatment. Results ONA did not significantly decrease fibroblast lengths, at any timepoint or dilution. At 1:7 dilution ratios, ABO decreased fibroblast lengths after 2 hours and significantly after 10‐12 hours. At 1:7, 1:8, 1:9, and 1:10 dilution, PRABO decreased length, and most rapidly at 1:7 and 1:8. At 1:6, 1:8, 1:9, and 1:10 dilution, INCO decreased lengths almost immediately. At 1:6 dilution, INCO decreased lengths almost immediately. At 1:7 dilution, INCO decreased lengths after 2‐4 hours, while at 1:8, 1:9, and 1:10 dilution, INCO decreased lenghts nearly imediately. LETI decreased lengths at all dilutions except 1:9, with near‐immediate effects at 1:6, 1:7, 1:8, and 1:10. At 1:4 dilution, LETI decreased lengths from 1 hour. Conclusions Different commercial preparations of BoNT‐A toxins cause different fibroblast contractions in vitro. Product selection and dilution used may affect the clinical outcome of intradermal injection of BoNT‐A for face lifting.

relax facial muscles and improve cutaneous elasticity, pliability, and viscoelasticity, and to re-organize and re-orientate facial collagen fibers. 5 For dermal indications, BoNT-A has been used to change skin texture and sebum production at the site of injection 6,7 to resolve severe cystic acne, 8 and reduce sebum production and pore size in patients with oily skin. 9 Several studies have demonstrated the biological impact of BoNT-A in directly targeting nonneuronal cell types 10 including skin cells and tissues that express at least one BoNT-A-binding proteins, such as the SV2 vesicular protein or FGFR3. 11 Some dermal cells may also express the BoNT-A cleavage target, SNAP-25, including epidermal keratinocytes and subcutaneous adipose tissue mesenchymal stem cells. 12 BoNT-A remodels connective dermal tissue and its cutaneous effects are exploited in cutaneous flaps where it produces specific biological responses in dermal fibroblasts. This includes the expression of cytokines and growth factors such as vascular endothelial growth factor, platelet endothelial cell adhesion molecule 1, CD31, CD34, interleukin (IL)-1, and tumor necrosis factor-a. [13][14][15] In dermal fibroblasts, BoNT-A may directly facilitate tissue remodeling, wound closure, and scar formation. BoNT-A induced a loss of normal fibroblast morphology and cytoplasmic retraction and spread in experiments on cultured 3T3 fibroblasts. 16 It decreased senescence-related proteins in human dermal fibroblasts exposed to ultraviolet B radiation and induced premature senescence in antiphotoaging studies. These fibroblasts subsequently had less matrix metalloproteinase (MMP)-1 and MMP-3 but more collagen types I (Col-I) and III (Col-III). 17 In wound healing experiments on cultured human fibroblasts, BoNT-A prevented Col-I and Col-III expressions but improved MMP-2 and MMP-9 expressions, 18 although this observation requires clarification as others have shown the opposite that BoNT-A upregulated Col-I expression but decreased MMP expression. 19 BoNT did not, however, stimulate dermal fibroblast proliferation or cause inflammatory effects.
BoNT-A also inhibited Smad2 phosphorylation during silicone implant capsule formation, while inhibited TGF-1 signaling to disrupt fibroblast-to-myofibroblast differentiation. 20 Rat injury models demonstrated reduced wound and graft contraction following BoNT-A treatment, and improved and faster healing, and less severe scarring of burn wounds, with faster regeneration, less inflammation, and more fibroblasts. 21,22 Animal studies showed reduced hypertrophic scars thickness 23 due to BoNT-A modifying fibroblast growth and differentiation. In human fibroblasts, BoNT-A upregulated Rac1, Cdc42, and RhoA 24 ; inhibited fibroblast proliferation and fibroblast-to-myofibroblast differentiation 25 ; and stimulated apoptosis, but reduced myosin expression. It also regulated Col-I but downregulated TGF-b1, VEGF, MMP-1, and PDGFA and other genes involved in invasive proliferation of keloid fibroblasts. [26][27][28] At a clinical level, a slight increase in type I procollagen has been observed following the use of abobotulinumtoxin (ABO). 29 In the cosmetic treatment of hyperfunctional facial lines induced by muscle hyperactivity, BoNT-A is typically delivered by intramuscular injection. 30 However, reports of intradermal BoNT-A inducing a dermo-lifting effect, such as midface lifting, have surfaced 31 even with the use of different forms of the toxin (ABO and onabotulinumtoxin (ONA)). 32 Intradermal BoNT-A injections in facial rejuvenation can correct the downward pull of midfacial depressor, 29 platysmal, and lateral orbicularis oculi muscles. Interestingly, toxin interventions in platysmal and lateral orbicularis oculi muscles also increase the lifting effect of the levators to produce the visible midface lift. Our group has also previously demonstrated a significant face-lifting effect following the use of ABO in a split-face investigation. 33 This lifting effect was not physically induced by the pricking of the toxin-delivery needle 34 as more recent investigations indicate better clinical improvements with BoNT-A than with normal saline and an effect at the dermal level. As stated above, one potential mechanism for this lifting may have been the stimulation of collagen production. 5,7,35 Frontalis lifting has also been achieved by injecting ABO in the hair-bearing areas of the scalp, in the origin of the frontalis and in the glabella. 36  However, the mechanism by which intradermal BoNT-A achieves this lifting effect, aside from its capacity to block muscle-contracting nerves, remains unclear. Several investigators have proposed mechanisms including the paralysis of depressor muscles, the increase in collagen synthesis, and fibroblast contraction. 2,19 Whether and how intradermal BoNT-A injections induce fibroblast contraction, and which toxin types or dilutions potentially achieve this effect, remain to be seen. We therefore set out to examine the effect of BoNT-A on dermal fibroblast contraction.  could be captured per field of view (see Figure 1).

| D ISCUSS I ON
To the best of our knowledge, this is the first report that demon- and expected to be clinically effective only in the short-term.
We therefore conclude that different BoNT-A types induce fibroblast contraction to different extents and at different speeds.
Whether this was a consequence of the particular toxin type or whether the nontoxin components of the commercial preparations were responsible for this disparity, remains to be established. Of clinical significance was our finding that, while the fibroblasts displayed a measurable decrease in length, their overall size did not change and they did not disappear from the field of view. We therefore believe that BoNT-A had no cytotoxic effect on fibroblasts, in agreement with the observations by other investigators. 36 Due to these varying outcomes observed here, physicians should carefully consider the speed at which they hope to achieve an outcome, especially if fibroblast contraction may produce a visible tissue "lifting." It would also be interesting to determine and compare the tran-  As such, further work is needed to directly compare between toxins.
Finally, it was also challenging to collect measurements of the same fibroblast over a 24-hour period due to its proliferation.

| CON CLUS ION
To our knowledge, this is the first demonstration of different effects on fibroblast contraction by different commercial preparations of BoNT-A toxins. We have shown that different types and dilution of BoNT-A provided variable degree of fibroblast contraction in vitro.
Therefore, product selection and dilution used may affect the clinical outcome of intradermal injection of BoNT-A for face lifting.