Hyaluronic Acid/Bone Substitute Complex Implanted on Chick Embryo Chorioallantoic Membrane Induces Osteoblastic Differentiation and Angiogenesis, but not Inflammation

Abstract

Microscopic and molecular events related to alveolar ridge augmentation are less known because of the lack of experimental models and limited molecular markers used to evaluate this process. We propose here the chick embryo chorioallantoic membrane (CAM) as an in vivo model to study the interaction between CAM and bone substitutes (B) combined with hyaluronic acid (BH), saline solution (BHS and BS, respectively), or both, aiming to point out the microscopic and molecular events assessed by Runt-related transcription factor 2 (RUNX 2), osteonectin (SPARC), and Bone Morphogenic Protein 4 (BMP4). The BH complex induced osteoprogenitor and osteoblastic differentiation of CAM mesenchymal cells, certified by the RUNX2 +, BMP4 +, and SPARC + phenotypes capable of bone matrix synthesis and mineralization. A strong angiogenic response without inflammation was detected on microscopic specimens of the BH combination compared with an inflammatory induced angiogenesis for the BS and BHS combinations. A multilayered organization of the BH complex grafted on CAM was detected with a differential expression of RUNX2, BMP4, and SPARC. The BH complex induced CAM mesenchymal cells differentiation through osteoblastic lineage with a sustained angiogenic response not related with inflammation. Thus, bone granules resuspended in hyaluronic acid seem to be the best combination for a proper non-inflammatory response in alveolar ridge augmentation. The CAM model allows us to assess the early events of the bone substitutes⁻mesenchymal cells interaction related to osteoblastic differentiation, an important step in alveolar ridge augmentation.

Keywords: BMP4; RUNX 2; SPARC; bone substitutes; chorioallantoic membrane.

Figure 1

Stereomicroscopic view of CAM treated with hyaluronic acid (H), saline solution (S), bone substitute resuspended in H (BH), or in a mixture of S and H (BSH). A slightly vascular reaction was induced by H on day 1 and 2 (a,b), which was persistent until the end of the experiment (c,d). In contrast, S did not induce an increase in CAM blood vessel density (e–h). The BH complex applied on day 1 (i) dynamically changed its structure during the experiment. Small bone particles dispersed on H on day 2 (j) became structured in bigger bone lamellae on day 4 (k), and formed a compact mass by the end of the experiment (l). Compared with the BH complex, bone substitutes resuspended in a 1:1 mixture of H and S showed a less compact structure on CAM, with a restorative process starting from day 2 (n), and showing discohesive particles on day 4 and 7 (o,p).

Figure 2

Microscopic view of CAM with a BH implant. There is an obvious integration of the BH implant inside CAM mesenchyme, with a clear stratification of the implant. The BH complex had a stratified appearance (a) below the CAM (blue arrow), the outer layer of BH (yellow arrow), and the internal area of BH (marked by red star). A well defined vascular network of small blood vessels penetrating the implant was observed (b) (yellow arrow). Stromal densification (c) compared to normal CAM (d). Mitotic activity of stromal mesenchymal cells was often seen amongst cells of CAM chorion, as marked in (c) (blue circle).

Figure 3

RUNX2 (a) and BMP4 (b) in hyaluronic acid (H) treated CAM specimens were negative. RUNX2 with moderate and heterogeneous expression in the BSH implant (c) and the implant adjacent to the CAM stroma (d). Moderate reaction for SPARC with an intense positive reaction in endothelial cells associated with inflammation adjacent to the implant (e). BMP4 is strongly positive in the vessels that invaded the BSH implant, but absent in the resuspended cells (f).

Figure 4

RUNX2 expression in BH treated samples, showing clearly the stratification of RUNX2 positive cells on distinct layers inside the CAM (a). RUNX2 positive cells (with nuclear pattern) had the highest density in the superficial layer (b) (with an average of 100 positive nuclei/field ×400 magnification), while layers below showed medium (c) (average of 70 positive nuclei/field ×400 magnification) and low density (d) (average of 50 positive nuclei/field ×400 magnification). Sharp limits in between layers can be shown based on the density of positive nuclei (e) between the superficial and middle layer, and (f) between the middle and the deep layer. Blue arrows indicate the detailed images from each three layers of BH treated samples. Yellow arrows show the limits between two consecutive layers and also highlighting differences regarding density of positive signals in between layers.

Figure 5

SPARC (a) and BMP4 (d) expression inside cellular components of CAM in BH treated specimens. Blue square indicates a suggestive zone for SPARC immunostaining, selected to be shown in detail in figure (b). Note that, stratification was also present for SPARC (b,c), as we have previously shown for RUNX2 in BH treated specimens. BMP4 was less pronounced in the densified chorion around the implant (d), and heterogeneously distributed in the nucleus (e) and cytoplasm ((f), cells inside blue circle) of CAM mesenchymal cells for BH treated specimens where inflammation was absent.