Characterization of immunologically detectable T-cell sensitization, Immunohistochemical detection of pro-inflammatory cytokines, and clinical parameters of patients after allogeneic intraoral bone grafting procedures: a prospective randomized controlled clinical trial in humans

Abstract

Background: The null hypotheses were tested that intraoral bone augmentation using two different allogeneic materials has no impact on the patient's blood levels of material-specific lymphocytes and on the immunohistochemical detection of pro-inflammatory cytokines IL-1α, IL1ß and TNF-α and T-cell markers CD4, CD8 in biopsies of the test groups.

Methods: In this prospective RCT, 60 systemically healthy participants were randomly assigned to two allogeneic test groups (1: Maxgraft®, freeze-dried, multiple donors, and 2: Puros®, solvent-dehydrated, single donor) and an autologous control group (10 patients). Plasma samples were collected pre-(T1) and postoperatively (2 weeks (T2) and 4 months (T3)). The Lymphocyte Transformation Test (LTT) was used for analyzing levels of transformed lymphocytes for type IV immune reactions by 3H-thymidine activity. Bone biopsies were harvested at T3 and immunohistochemically analyzed for IL-1α, IL1ß, TNF-α, CD4, CD8 and correlated with the immunological and clinical findings.

Results: A statistically significant difference between the tested materials was observed for LTT measurements at T3 (p = 0.033). Furthermore, three groups were identified: Group A (LTT negative T1-T3, n = 48), group B (LTT positive T1-T3, n = 7), group C (developing positive LTT at T2, n = 5). A highly significant elevation of IL-1α, IL1ß, TNF-α in patients of group C (p = 0.0001) and a significant elevation of CD4+ cells in patients of group B (p = 0.005) was shown.

Conclusion: Our data show that following allogeneic bone grafting, local and systemic immunological reactions can be detected in some patients. These findings were statistically significant for the timepoint T3 between the tested materials as well as for the groups B and C correlated with group A for both tested materials. Therefore, the null hypotheses were rejected. A preoperative compatibility test for allogeneic materials in order to improve patient safety and the predictability of these materials would be desirable.

Trial registration: Ethical commission of the Ärztekammer Hamburg, Germany (PV5211) as well as by the German Registry of Clinical Studies (DRKS00013010) on 30/07/2018 ( http://apps.who.int/trialsearch/ ).

Keywords: Allogeneic bone graft; Alloimmunization; Human histology; Immunohistochemistry; Lymphocyte transformation test.

Fig. 1

Flow diagram of study design. The flow-diagram shows the study design, the bone allograft material as well as the three different time points for blood sample collection and tissue biopsy as well as the subsequent laboratory experiments. Furthermore, the clinical procedure for the test and the control groups are shown

Fig. 2

The fig. 2c shows the mean value of the stimulation indices for the 25 patients in the test group 1 (Maxgraft®, red) and the 25 patients in the test group 2 (Puros®, blue) for all 3 time points.  For calculation and presentation, all patients with a stimulation index in the LTT < 2 were set to 1. At time point 3, T-cell sensitisation to the material used is significantly more frequent in the Maxgraft® group (p = 0.033)

Fig. 3

a-f Immunohistochemical analysis of IL-1α, IL-1ß and TNF-α. a, b Immunostaining for IL-1α. (A) Weak immunostaining pattern: staining of bone lining cells (arrow), b = newly formed bone, Maxgraft®, patient of group A, DAB, original magnification × 20); (B) Moderate immunostaining: Staining of a cluster of fibroblasts (f), b = newly formed bone, M = Maxgraft® granules, Maxgraft,® patient of group C, DAB, original magnification × 20. c, d Immunostaining for IL-1ß. (A) Weak immunostaining pattern: staining of bone lining cells and vessel walls (arrows), b = newly formed bone, Puros®, patient of group A; DAB, original magnification × 20); (B) Strong immunostaining: stronger extracellular immunoreactivity within connective tissue areas (asterisks), b = newly formed bone, Puros®, patient of group C, DAB, original magnification × 20. e, f Immunostaining for TNF-α. (A) Weak immunostaining pattern: staining of fibroblasts and vessel walls (arrows), b = newly formed bone, M = Maxgraft® granules, patient of group A DAB, original magnification × 10); (B) Strong immunostaining: Extracellular immunoreactivity and fibroblast staining (asterisks) in the connective tissue among Maxgraft® granules (M) with newly formed bone (b), patient of group C DAB, original magnification × 10