# Cell Supported Single Membrane Technique for the Treatment of Large Bone Defects: Depletion of CD8+ Cells Enhances Bone Healing Mechanisms During the Early Bone Healing Phase

## TL;DR

This study shows that removing CD8+ T cells from bone marrow cells improves early bone healing by altering the proteins in a membrane used for treatment.

## Contribution

The study reveals how CD8+ T cell depletion accelerates the shift from inflammation to regeneration in bone healing.

## Key findings

- CD8+ T cell depletion suppresses early inflammatory mediators while upregulating regenerative proteins.
- By day seven, regenerative mediators like CCL20 and RGM-A are enriched in CD8-depleted groups.
- MMP-9, Galectin-1, and GDF-15 increase exclusively in the CD8-depleted group.

## Abstract

Introduction: The one-step membrane technique, derived from the Masquelet induced membrane technique, uses human acellular dermal matrix (hADM) that is wrapped around the bone defect to bypass membrane induction, reducing treatment time. Pre-colonization of hADM with bone marrow cells (BMC), particularly after CD8+ T cell depletion, enhances bone regeneration. This study examined how CD8+ T cell depletion alters the proteins accumulated in the hADM during early healing. Materials and Methods: Eighteen male Sprague-Dawley rats received 5 mm femoral defects filled with autologous bone chips and wrapped with hADM, hADM + BMC, or hADM + BMC-CD8. hADMs were recovered on days 3 and 7 (n = 3/group/timepoint), incubated ex vivo, and conditioned medium analyzed with a proteome profiler detecting 79 proteins. Results: The protein content of the hADM evolved dynamically. At day three, 41 proteins were detected, rising to 47 by day seven, with RGM-A, osteoprotegerin, LIF, IL-6, CCL20, and CCL17 emerging late, consistent with increased regenerative activity. CD8+ T cell depletion suppressed early inflammatory and pro-osteogenic mediators (e.g., CCL2, IGF-I, IL-1RA) while upregulating LIX. By day seven, regenerative mediators (CCL20, GDF-15, RGM-A) were enriched, whereas inflammatory factors (CCL21, IL-1a, WISP-1) declined. MMP-9, Galectin-1, and GDF-15 increased exclusively in the CD8-depleted group. Conclusions: The hADM protein content transitions from pro-inflammatory to pro-regenerative within one week after surgery. CD8+ T cell depletion accelerates this shift, highlighting hADM as a dynamic scaffold that contributes to the immune–regenerative crosstalk in bone healing.