Surgical suction filter-derived bone graft displays osteogenic miRNA and mRNA patterns

Abstract

Purpose: Recently, a surgical suction filter device was introduced which aims at generating a suction filter-derived bone grafting substitute (SF-BGS). The osteogenic capacity of this grafting material, however, is unclear. MicroRNAs (miRNAs) and osteogenic mRNAs may influence these processes. The aim of this study was therefore to investigate the quality of the SF-BGS by determining the expression of miRNAs and osteogenic mRNAs.

Methods: Samples were collected during non-union surgery. Upon exposure of the intramedullary canal, the surgical vacuum system was fitted with the suction filter device containing collagen complex and synthetic β-TCP: (Ca₃(PO₄)₂, granule size 5-8 mm, total volume 10 mL (Cerasorb Foam^®, Curasan AG, Kleinostheim, Germany). As a control, venous blood was used as in current clinical practice. Samples were snap-frozen and mechanically disrupted. MiRNAs and mRNAs were isolated, transcribed, and pooled for qPCR analysis. Lastly, mRNA targets were determined through in silico target analyses.

Results: The study population consisted of seven patients with a posttraumatic long bone non-union (4♀; mean age 54 ± 16 years). From the array data, distinct differences in miRNA expression were found between the SF-BGS and control samples. Osteogenic marker genes were overall upregulated in the SF-BGS. Qiagen IPA software identified 1168 mRNA targets for 43 of the overall deregulated miRNAs.

Conclusion: This study revealed distinctly deregulated and exclusively expressed osteogenic miRNAs in SF-BGS, as well as overall enhanced osteogenic marker gene expression, as compared to the venous blood control group. These expression profiles were not seen in control samples, indicating that the derived material displays an osteogenic profile. It may therefore be a promising tool to generate a BGS or graft extender when needed.

Keywords: Bone grafting substitute; Bone regeneration; Graft extender; Non-union; Stem cells; microRNAs.

Fig. 1

Intraoperative workflow of harvesting the suction filter-derived bone grafting substitute. The device (A) is loaded with the filter material (B): collagen complex and synthetic β-TCP: (Ca₃(PO₄)₂, granule size 5–8 mm, total volume 10 mL (Cerasorb Foam®, Curasan AG, Kleinostheim, Germany). Harvesting was performed upon exposure of the intramedullary canal (C), and the harvested filter material (D) was then processed for further analyses. All procedures were performed by two senior consultants (MP & TB)

Fig. 2

Deregulated microRNAs from the pooled sample group (n = 7) in Qiagen's fibrosis array. Results are normalised to the mean of six pre-defined housekeeper genes, whole blood from healthy volunteers (n = 5) was used as a control. All shown miRNAs displayed a fold regulation of ≥ 2 or ≤ – 2

Fig. 3

Specific microRNAs that were only expression in the SF-BGS sample group (n = 7). Qiagen’s fibrosis array was used, results are normalised to the mean of six pre-defined housekeeper genes. Black dotted lines and a diagonal fill pattern display that the miRNA was considered deregulated, showing an absolute ∆Cq ≥ 2. Positive ∆Cq values depict upregulation, negative ∆Cq values depict downregulation

Fig. 4

Schematic overview of the role of the most deregulated miRNAs in the suction filter-derived bone grafting substitute (SF-BGS). MiRNAs that are exclusively expressed in SF-BGS are depicted in grey boxes, deregulated miRNAs in SF-BGS as compared to the control group are depicted in white boxes. Green boxes depict gene targets with a stimulatory effect on osteogenic differentiation, red boxes depict targets with an inhibitory effect on osteogenic differentiation. Mesenchymal stem cell (MSC), Bone Morphogenic Protein (BMP), Runt related transcription factor 2 (RUNX2), Odd Skipped Related 1 (OSR1), Sirtuin 1 (SIRT 1), LDL receptor related protein 5 (LRP5), Alkaline Phosphatase (ALP), Osteopontin (OPN), Casitas B-lineage lymphoma (cCBL), Phosphatidylinositol-3-kinase (PI3K), Protein kinase B (AKT), Extracellular signal-Regulated Kinases (ERK), Suppressor of Mothers against Decapentaplegic (SMAD), Krüppel‐like factor 5 (KLF5)

Fig. 5

Mean relative gene expression alkaline phosphatase (ALP), osterix (OSX), runt related transcription factor 2 (RUNX2), and osteopontin (OPN) in surgical suction filter-derived bone grafting substitute

Fig. 6

In silico target analysis, performed with Qiagen Ingenuity Pathway Analysis (IPA) software, of 3 miRNAs whose expression was upregulated in the SF-BGS group as compared to the control group. Green colour represents upregulated miRNA expression, dotted arrows indicate a relationship to bone regeneration

Fig. 7

In silico target analysis, performed with Qiagen Ingenuity Pathway Analysis (IPA) software, of 3 miRNAs whose expression was downregulated in the SF-BGS group as compared to the control group. Red colour represents downregulated miRNA expression, dotted arrows indicate a relationship to bone regeneration

Fig. 8

In silico target analysis, performed with Qiagen Ingenuity Pathway Analysis (IPA) software, of 3 miRNAs which were exclusively expressed in the SF-BGS group. Green colour represents upregulated miRNA expression as compared to the housekeeper genes, dotted arrows indicate a relationship to bone regeneration

Fig. 9

In silico target analysis, performed with Qiagen Ingenuity Pathway Analysis (IPA) software, of 3 miRNAs which were exclusively expressed in the SF-BGS group. Red colour represents downregulated miRNA expression as compared to the housekeeper genes, dotted arrows indicate a relationship to bone regeneration