# BIO3 Protocol: Clinical and In Vitro Evidence of Triple Biostimulation With CaHA and P (LA/CL) Threads

## TL;DR

This study shows that combining CaHA and P(LA/CL) threads improves facial rejuvenation by enhancing collagen and elastin production.

## Contribution

The novel contribution is demonstrating a synergistic effect of combining CaHA with P(LA/CL) threads for enhanced biostimulation.

## Key findings

- Combined treatment upregulated ECM-related genes more than monotherapies in vitro.
- Patients receiving CaHA followed by threads showed greater facial thickening and higher aesthetic scores.
- 100% patient satisfaction was achieved in both groups by day 30 with no adverse events.

## Abstract

Facial aging involves progressive deterioration of the extracellular matrix (ECM), volume loss, and structural sagging. Minimally invasive strategies like absorbable threads provide mechanical support and biostimulation, but their efficacy may be compromised in patients with poor dermal quality. Calcium Hydroxyapatite (CaHA) is a well‐documented biostimulator that enhances collagen and elastin synthesis. This study explored whether combining CaHA with P (LA/CL)‐based absorbable threads could synergistically improve ECM remodeling and clinical outcomes.

To evaluate the biological and clinical effects of the BIO3 protocol—sequential biostimulation with CaHA and P (LA/CL) threads—by integrating in vitro gene expression analysis and retrospectively collected clinical outcomes.

An in vitro model of full‐thickness human skin was used to assess COL1A1, COL3A1, and ELN expression after treatment with CaHA, P (LA/CL) threads, or both. A retrospective clinical study was also conducted with 11 female patients aged 35–65 undergoing facial rejuvenation with threads alone (n = 6) or preceded by CaHA (n = 5). Clinical outcomes were assessed using GAIS, WSRS, facial laxity scores, ultrasound imaging, and satisfaction questionnaires at multiple timepoints.

Combined in vitro treatment resulted in significantly higher upregulation of ECM‐related genes compared to monotherapies. Clinically, all patients showed aesthetic improvement. The CaHA/P (LA/CL) group demonstrated greater dermal and subcutaneous thickening and higher GAIS ratings at D120. Patient satisfaction reached 100% in both groups by D30 and remained stable. No adverse events were reported.

The BIO3 protocol—combining CaHA and P (LA/CL) threads—showed promising synergistic effects on ECM remodeling and aesthetic outcomes. These findings support the rationale for layered and sequential biostimulation strategies in clinical practice.