Bone Protein’s Gene Expression in Humans – Pilot ex vivo Study

Objectives: Durability of ribose cross-linked collagens (RCLC) to withstand degradation process in tissues and to rather ossify instead was demonstrated. We hypothesized that long-standing RCLC materials used for reconstruction of alveolar bone deficiencies in oral cavity (GBR) may influence gene expression profile of active osteoblasts facilitating bone formation process de novo.
Methods: Three categories of ex-vivo human tissue samples were analyzed. All non-smoking donors signed informed consent (ethic approval 155/2015); each group complied of 6 soft tissue samples containing subepithelial connective tissue or connective tissue plus residual portions of RCLC material. Controls were allocated as healthy donors with tissues sampled from the palate at connective tissue graft surgery. Negative controls complied of tissue samples from periodontitis sites, obtained during periodontal surgery. Tests represented sites augmented with RCLC materials applied 4 to 8 months prior to implant placement. RUNX2, BGLAP, IBSP, Periostin and Connexin43 (Cx43) expression was assessed by RT-qPCR using SYBR green® after extraction of total mRNA from each sample and standardizing for the total protein content. Non-parametric statistics and Spearman correlations were calculated with △-CT scores after adjusting expression levels of the targets to GAPDH (SPSS 23.0, USA). Coefficient of determination (R²x100%) was calculated for statistically significant correlations.
Results: Statistically significant correlation was depicted for RUNX2 to Periostin (P=0.05; R²=66%) and IBSP to Periostin (P=0.008; R²=86%) in tests, as for RUNX2 to Periostin (P=0.042; R²=67%) in controls. Expression levels in negative controls were statistically non-significant (Spearman-Rho test). Group-wise comparison revealed significantly greater expression of Cx43 in tests compared to controls (P=0.01; P=0.03 according to Bonferroni adjustment). For RUNX2 and Periostin trend favoring tests was shown, however, missing level of significance (Kruskal-Wallis-Test).
Conclusions: From this pilot study we conclude that presence of RCLC material in deficient bone areas may support gene expression profile of bone proteins by osteoblasts thereby promoting process of membranous bone formation. Coefficient of determination for IBSP to Periostin may indicate greater activity in tests.