Safety Of Botulinum Toxin And Hyaluronic Acid Combination Therapy And The Effect On The Collagen Content Of The Dermis

BackgroundHyaluronic acid (HA) and botulinum toxin type A (BTX-A), both of which prove to be safe when singly used, are now two of the most widely used products in facial rejuvenation. Also, the combined injection of HA and BTX-A has been commonly applied in clinical practice, with optimal outcomes in individualized treatment. However, there is relatively little scientific evidence available on application safety of this generally-accepted combination. In addition, recently in vitro studies have shown that BTX-A would effectively inhibit the growth of fibroblasts and in turn cause a decrease in collagen deposition, yet no studies have been performed to verify the same inhibition effect in vivo.Objectives(1) To assess the safety of combined subcutaneous injection of BTX-A and HA.(2) To explore the effect of BTX-A, HA and their combined treatment on fibroblasts proliferation and collagen deposition in dermal layer.MethodsMale New Zealand rabbits were selected as animal model and subcutaneous injections were carried out under separate skin with saline (control group), HA (HA group), BTX-A (BTX-A group) and combination of BTX-A and HA (combination group). And the observation of differences between these four treatment groups was conducted on day7, day14, and day28respectively after the experimental treatment (day0), including (1) observation of local skin reactions with naked eyes.(2) observation of foreign body reaction and inflammation response with HE staining under light microscope.(3) observation of number and morphology of fibroblasts with HE staining under light microscope.(4) observation of deposition of type Ⅰ collagen and type Ⅲ collagen with immunohistochemical staining under light microscope.Results(1) Local skin reactions:No significant difference in local reaction positive rate was found among four treatment groups. Also no significant change in local reaction positive rate was shown while time changing.(2) Foreign body reaction grading:The combination group and the HA group had similar score in foreign body reaction grading on day7and day14after treatment, and both of them scored significantly higher than the control group. No significant difference was found between each treatment group on day28. All groups scored significantly lower on day14and day28than on day7.(3) Inflammation reaction grading:The combination group and the HA group had similar score in inflammation reaction grading on day7and day14after treatment, and both of them scored significantly higher than the control group. There was no significant difference between each treatment group on day28. All groups scored significantly lower on day14and day28than on day7.(4) Number of fibroblasts:Compared to the control group, the BTX-A group had significant smaller number of fibroblasts on day7and day14after treatment, and the HA group and combination group had significant larger number at all the three observation time points.(5) Production of type I collagen and type Ⅲ collagen:In terms of average IOD from immunohistochemistry section, the BTX-A group was significant lower than the control group on day7and day14after treatment. On day7, day14and day28, the HA group was significant higher than the control group while there was no significant difference between the combination group and the control group. All groups had significant higher average IOD on day14and day28than on day7.Conclusion(1) The combined subcutaneous injection of BTX-A and HA has similar foreign body reaction and inflammation response with the single application of HA, indicating its relatively acceptable safety.(2) Compared to subcutaneous injection of saline, the single injection of BTX-A will cause a decrease in the number of fibroblasts in dermal layer. However, the single injection of HA and the combined treatment will effectively promoting the proliferation of fibroblasts, indicating the advantage of combination in shunning the reduction of fibroblasts caused by BTX-A.(3) Compared to subcutaneous injection of saline, the single injection of BTX-A will decrease the deposition of type Ⅰ collagen and type Ⅲ collagen, while the single injection of HA has the reverse effect. The BTX-A in conjunction with HA will to some degree offset its inhibition effect on production of collagen.