Badreldin, Amr A.

Background: Previous studies report elevated serum titanium (Ti) levels in children with spinal implants. To provide additional data on this topic, we sought to assess serum ion levels at multiple timepoints in pediatric patients with growing spine devices, spinal fusion instrumentation, and extremity implants placed for fracture treatment. We hypothesized that serum Ti, cobalt (Co), and chromium (Cr) levels would be elevated in pediatric patients with growing spine devices compared with patients with extremity implants. Methods: Pediatric patients undergoing any primary spine implant placement, those with spine implant revision or removal surgery and patients with other appendicular implant removal had serum Ti, Co, and Cr ion levels drawn at the time of surgery. Fifty-one patients (12 growing spine devices, 13 fusions, and 26 extremity implants) had one set of labs, 31 of whom had labs drawn both preoperatively and postoperatively. Biopsies obtained from tissue specimens at the time of implant revision were analyzed histologically for the presence of metal debris and macrophage activity. Results: Patients with growing spine implants had elevated serum Ti (3.3 vs. 1.9 ng/mL, P=0.01) and Cr levels (1.2 vs. 0.27 ng/mL, P=0.01) in comparison to patients with fusion rods or extremity implants. With respect to patients with extremity implants, patients with growing spine devices had elevated serum Ti (3.3 vs. 0.98 ng/mL, P=0.013), Co (0.63 vs. 0.26 ng/mL, P=0.017), and Cr levels (1.18 vs. 0.26 ng/mL, P=0.005). On matched pairs analysis, patients who had labs drawn before and after spine implantation had significant increase in serum Ti levels (0.57 vs. 3.3 ng/mL, P=0.02). Histology of tissue biopsies adjacent to growing spine implants showed presence of metal debris and increased macrophage activity compared with patients with extremity implants. Conclusion: Serum Ti, Co, and Cr levels are elevated in children with spinal implants compared with those with extremity implants, particularly in those with growing spine devices. However, the clinical significance of these findings remains to be determined. Level of Evidence: Level II—prospective comparative study.

Skeletal development is controlled by epigenetic mechanisms that regulate chromatin compaction and suppress access to gene regulatory sequences. Post‐translational modifications in the N‐termini of histone 3 (H3), including tri‐methylated H3 lysine 9 (H3K9Me3) or lysine 27 (H3K27Me3) represent repressive chromatin marks that are recognized by a family of heterochromatin‐associated Chromobox (Cbx) proteins (i.e., Cbx1 to Cbx8). We show using RNA‐seq analysis that each of these Cbx isoforms is actively expressed in bone and osteoblasts with variable expression of distinct members (between 1 and 100 reads per million). The most prominently expressed Cbx member is Cbx3, which encodes a member of the heterochromatin protein 1 (HP1) family that interacts with H3K9me3. Cbx3 is known to be important for normal development while promoting neurogenesis, as well as renal and smooth muscle development. Therefore, we postulated that CBx3 may perform a similar role in osteoblastogenesis. We tested this hypothesis by both transient and stable knock‐down of Cbx3 by respectively siRNA or shRNA. RNA interference in MC3T3 osteoblasts effectively decreases Cbx3 mRNA levels by 2 to 5 fold, while increasing expression of bone‐related markers by RT‐qPCR (e.g., Alpl/alkaline phosphatase, Ibsp/bone sialoprotein, Bglap/osteocalcin, and Col1a1/Collagen Type I α1 chain). In addition, Cbx3 depletion increases mineral deposition as measured by Alizarin Red staining. Thus, Cbx3 normally appears to suppress osteoblastic differentiation, unlike its essential function in the neuronal differentiation. Because Cbx3 recognizes H3K9me3, we examined whether Cbx3 controls the levels of this histone mark. Loss of Cbx3 did not affect total H3K9me3 levels unless Cbx1 and Cbx5 were simultaneously depleted by siRNA. We propose that Cbx3 may suppress osteoblastogenesis by controlling select loci with H3K9me3 marks.