Development of Neovasculature in Axially Vascularized Calcium Phosphate Cement Scaffolds

Abstract

Augmenting the vascular supply to generate new tissues, a crucial aspect in regenerative medicine, has been challenging. Recently, our group showed that calcium phosphate can induce the formation of a functional neo-angiosome without the need for microsurgical arterial anastomosis. This was a preclinical proof of concept for biomaterial-induced luminal sprouting of large-diameter vessels. In this study, we investigated if sprouting was a general response to surgical injury or placement of an inorganic construct around the vessel. Cylindrical biocement scaffolds of differing chemistries were placed around the femoral vein. A contrast agent was used to visualize vessel ingrowth into the scaffolds. Cell populations in the scaffold were mapped using immunohistochemistry. Calcium phosphate scaffolds induced 2.7-3 times greater volume of blood vessels than calcium sulphate or magnesium phosphate scaffolds. Macrophage and vSMC populations were identified that changed spatially and temporally within the scaffold during implantation. NLRP3 inflammasome activation peaked at weeks 2 and 4 and then declined; however, IL-1β expression was sustained over the course of the experiment. IL-8, a promoter of angiogenesis, was also detected, and together, these responses suggest a role of sterile inflammation. Unexpectedly, the effect was distinct from an injury response as a result of surgical placement and also was not simply a foreign body reaction as a result of placing a rigid bioceramic next to a vein, since, while the materials tested had similar microstructures, only the calcium phosphates tested elicited an angiogenic response. This finding then reveals a potential path towards a new strategy for creating better pro-regenerative biomaterials.

Keywords: NLRP3; angiogenesis; axial vascularization; bioceramic; bioinorganic; calcium phosphate; inflammation.

Figure 1

(A) Schematic diagram of scaffold geometry and (B) positioning of the vein axially within implant. (C) Photographs of the surgical procedure showing stages in scaffold assembly.

Figure 2

Micro-CT images of perfused blood vessels in decalcified scaffolds showing development of vasculature inside monetite scaffolds.

Figure 3

Decalcified immunohistochemistry (IHC) staining of alpha-smooth muscle cell expression at low and high magnification, outside and within the scaffold. Scale bars on low and high magnification represent 1 mm and 400 µm, respectively.

Figure 4

(A) Decalcified immunohistochemistry (IHC) staining of Calponin-1 expression at low and high magnification, outside and within the scaffold. Scale bars on low and high magnification represent 1 mm and 400 µm, respectively. (B) Quantification of Calponin-1 staining after different implantation times. Bars represent means ± std. dev. (n = 3).

Figure 5

Decalcified immunohistochemistry (IHC) staining of F4/80-expressing cells at low and high magnification, outside and within the scaffold. The black material inside the vessels at 4 weeks is Microfil contrast agent. Scale bars on low and high magnification represent 1 mm and 400 µm, respectively.

Figure 6

(A) Decalcified immunohistochemistry (IHC) staining of NLRP3 inflammasome, and (B) Il-1 beta expression at low and high magnification, outside and within the scaffold. Scale bars on low and high magnification represent 1 mm and 100 µm, respectively. (C) Quantitative analysis of NLRP3 and IL-1β staining after different implantation times. Bars represent means ± std. dev (n = 3).

Figure 7

(A) X-ray diffraction patterns of the set cements after setting. (B) Scanning electron micrographs of fracture surfaces of (i) MgPO₄, (ii) CaSO₄, (iii) monetite, (iv) brushite. Calcium sulphate and brushite had similar microstructures whose cementitious matrix was made up of at least one population of platy crystals up to ~5 µm in dimension, whereas monetite and magnesium phosphate were more crystalline with rhomboid crystals >10 µm in dimension. (C) Mercury porosimetry showed the modal pore sizes lay in the range ¼ to 5 µm.

Figure 8

Results of weight loss of the four experimental materials used as scaffold in the present study: MgP, CaSO₄, monetite, and brushite. The bars represent the standard deviation values for each measure (n = 5).

Figure 9

(A) Two-dimensional cross sections and 3D reconstructed images of vascular networks developed within and around scaffolds after 4 weeks of implantation, as shown by micro-CT. (B) Mean and standard deviation values of blood vessel density within scaffolds after 4 weeks of implantation (n = 4). There were neither statistically significant differences between brushite and monetite, nor between calcium sulphate and magnesium phosphate; however, these two groups were significantly different (* p < 0.05).