Boosting angiogenesis experimentally in ovo by biofunctionalizing collagen membranes with platelet-rich fibrin and hyaluronic acid: implications for regenerative oral surgery?

Abstract

Purpose: Biocompatible collagen membranes (CM) are widely used in regenerative dentistry, particularly in guided tissue regeneration (GTR) and guided bone regeneration (GBR). While resorbable CMs offer advantages such as reduced patient morbidity and enhanced wound healing, their barrier function can impede vascularization, potentially compromising graft survival. Biofunctionalization of CMs with platelet-rich fibrin (PRF) and hyaluronic acid (HA) has shown promise in enhancing angiogenesis. This experimental study evaluates the pro-angiogenic effects of biofunctionalizing collagen membranes with advanced PRF (A-PRF), injectable PRF (i-PRF), and HA using the chorioallantoic membrane (CAM) assay in ovo.

Methods: Three porcine-derived collagen membranes (Mucoderm®, Bio-Gide®, and Smartbrane®) were biofunctionalized with A-PRF, i-PRF, or HA and applied to the CAM assay on day 7 of incubation. Afterwards, two experimental series were evaluated. The first series investigated Mucoderm (MM native, A-PRF, and MM combined with A-PRF) over an observation period ranging from 24 to 120 h (N = 135). The second series examined Bio-Gide® and Smartbrane (BM native, BM combined with i-PRF, BM combined with HA, and SM combined with HA) over a period from 24 to 72 h (N = 60). To assess impact on angiogenesis, vascularization was evaluated at multiple time points (24 h, 48 h, 72 h, 96 h, and 120 h) using immunohistochemical staining (hematoxylin-eosin, α-smooth muscle actin, CD105) and artificial intelligence (AI)-assisted image analysis (IKOSA® software).

Results: Biofunctionalization of Mucoderm® with A-PRF significantly enhanced angiogenesis up to 96 h, as evidenced by increased vessel area, length, and branching points (p < 0.05). I-PRF biofunctionalization of Bio-Gide® also promoted angiogenesis between 24 h (p = 0.036) and 72 h, showing significantly improved values for total area (p = 0.007), vessel length (p = 0.018), and vessel thickness (p = 0.008) compared with the native membrane. While HA biofunctionalization of Bio-Gide® and Smartbrane® resulted in significantly increased angiogenesis at 48 and 72 h (p < 0.05), its effects were less pronounced than those achieved with PRF variants. Native Bio-Gide® exhibited greater pro-angiogenic potential than native Mucoderm® at 24 h (p = 0.012); however, biofunctionalized membranes generally outperformed native variants.

Conclusions: Biofunctionalization of collagen membranes with A-PRF and i-PRF significantly enhances angiogenesis in ovo, with A-PRF showing sustained effects up to 96 h. HA also promotes angiogenesis and represents a viable, cost-effective alternative that does not require blood collection. Both PRF and HA biofunctionalization may offer potential benefits for enhancing vascularization in GBR/GTR applications. However, their pro-angiogenic potential and clinical relevance remain exploratory at this stage for pourely in-ovo results. The study is limited to an in-ovo setting, therefore the transferability to oral soft tissue and bone conditions is restricted. Further randomized clinical studies are needed to better understand and evaluate the possible advantages.

Keywords: Angiogenesis; Chorioallantoic membrane; Collagen; Fibrin hyaluronic acid; Neovascularization; Platelet-rich plasma.