Hyaluronic Acid with Bone Substitutes Enhance Angiogenesis In Vivo

Solomiya Kyyak, Sebastian Blatt, Nadine Wiesmann, Ralf Smeets and Peer W. Kaemmerer
Materials 2022, 15(11), 3839

What was examined? Why is it relevant?

Blood supply by forming blood vessels is fundamental for the regeneration of hard and soft tissue defects. It provides the required nutritional elements, oxygen, immune cells, mesenchymal stem cells, and growth factors for the successful integration and/or remodelling of bone grafting materials. Thus, the effective induction of angiogenesis, i.e. the growth of blood vessels from the residual tissue’s pre-existing vascular network is directly related to the success of bone substitute materials for maxillofacial osseous regeneration.

Based on its importance in early wound healing, stimulation of angiogenesis has been an active field of research in tissue regeneration. Hyaluronic acid as an essential element of the extracellular matrix has been demonstrated to play an important role in wound repair, also based on its regulatory activity on blood vessel formation apart from many others. In cerabone® plus the bovine bone graft is combined with hyaluronic acid to provide a unique application comfort, however whether the bone substitute has also proangiogenic effects is unknown. It was therefore the objective of the study to analyze blood vessel formation in response to cerabone® plus.

How was it analyzed?

Blood vessel formation was studied using the chicken chorioallantoic membrane (CAM) assay. Serving as a gas exchange surface, the CAM is an extraembryonic membrane that develops in a short time from a small avascular membrane into a structure that covers the entire inner surface of the shell displaying a densely organized vascular network. The test is easy to use, allows real time visualization with various imaging techniques and is an excellent model for more complex systems.

More information about advantages and limitations are provided in a review published in the journal Angiogenesis (

Recently, the CAM assay was shown to be suitable to study angiogenic properties of different bone-substitute materials. In this study, cerabone® plus or cerabone® were applied inside a silicon ring onto the CAM. After six days of incubation blood vessel formation was analysed qualitatively and quantitatively using microscopic images and immunohistochemical methods in defined regions of interest (ROI) near the silicon ring.

What are the results?

Almost every investigated parameter showed significantly higher values for cerabone® plus.

Results from the microscopic analysis:
– higher vessel number with a median of 34.5 vessels from branch to branch per ROI (cerabone®: 7 per ROI; p < 0.001)
– more vessel branching points with a median of 20 (cerabone®: 4; p < 0.001)
– larger total vessel area with 7.19 × 105 ± 0.5 × 105 pixels (cerabone®: 4.07 × 105 ± 0.57 × 105 pixels; p < 0.001)
– higher total vessel length of 914.17 ± 42.08 pixels (cerabone®: 560.0 ± 70.58 pixels; p < 0.001).

Results from the immunohistochemical analyses:
– higher total vessel area for cerabone® plus with 24.43 x 105 ± 6.32 x 105 pixel in the H&E staining (cerabone®: 8.24 x 105 ± 1.91 x 105; p < 0.001) and 42.0 x 105 ± 6.91 x 105 pixel in the alphaSMA staining (cerabone®: 16.27 x 105 ± 5.56 x 105 ;p = 0.037)
-higher numbers of brightness integration (8.09 x 104 ±3.24 x 104) indicating “young” vessels (cerabone®: 2.57 x 104 ± 0.37 x 104; p < 0.05)

No significant differences between the groups were found in the vessel mean thickness and in the average vessel area in HE- and alphaSMA staining.

What is the mechanism behind?

Hyaluronic acid has been demonstrated to orchestrate events leading to new blood vessel formation both in a physical and mechanical way by providing a cell supportive matrix, and on the cellular level by direct cell stimulation. However, according to many scientists, among them Prof. Anton Sculean who recently gave a botiss webinar on this topic (Link), hyaluronic acid is not a growth factor itself, but indirectly helps growth factors that are already available to be active. In addition, the effect of hyaluronic acid on cells depends on its molecular size and the interaction with specific cell surface receptors, and this way regulating cellular pathways. The same applies to proliferation of endothelial cells, which line the inner layer of blood vessel and thus are a key element of angiogenesis. Small hyaluronic acid fragments have been noted to stimulate endothelial cell proliferation and promote angiogenesis.

How can the results help me in treating patients?

The study by Kyyak and colleagues demonstrated, that the biofunctionalization of cerabone® with hyaluronic acid, i.e. cerabone® plus, leads to beneficial effects on angiogenesis in the used in vivo model. As a result, it can be assumed that, starting from the pre-existing vascular network, a faster vascularization of the biomaterial will also take place in the patient. More branching points and a higher number of blood vessels may lead to an increased vascular innervation of the biomaterial. Longer blood vessels indicate faster growth and presumably reach areas in the biomaterial that may be perfused less efficient depending on the size of the grafted bone defect. This leads to the following potential benefits:

– more efficient and reliable graft integration
– optimal conditions for the formation of new bone
– optimal support of osseous regeneration
– a better prognosis for larger augmentations
– improved healing of bone defects with limited blood supply

Conclusively, the proangiogenic effects of hyaluronic acid in cerabone® plus‚ can widen the indication for osseous regeneration procedures and may lead to faster and more efficient hard tissue regeneration.