Mineralized Biomaterials Mediated Repair of Bone Defects Through Endogenous Cells

Abstract

Synthetic biomaterials that create a dynamic calcium (Ca²⁺)-, phosphate (PO₄^3-) ion-, and calcium phosphate (CaP)-rich microenvironment, similar to that found in native bone tissue, have been shown to promote osteogenic commitment of stem cells in vitro and in vivo. The intrinsic osteoconductivity and osteoinductivity of such biomaterials make them promising bone grafts for the treatment of bone defects. We thus aimed to evaluate the potential of mineralized biomaterials to induce bone repair of a critical-sized cranial defect in the absence of exogenous cells and growth factors. Our results demonstrate that the mineralized biomaterial alone can support complete bone formation within critical-sized bone defects through recruitment of endogenous cells and neo-bone tissue formation in mice. The newly formed bone tissue recapitulated many key characteristics of native bone such as formation of bone minerals reaching similar bone mineral density, presence of bone-forming osteoblasts and tartrate-resistant acid phosphatase-expressing osteoclasts, as well as vascular networks. Biomaterials that recruit endogenous cells and provide a tissue-specific microenvironment to modulate cellular behavior and support generation of functional tissues are a key step forward in moving bench-side tissue engineering approaches to the bedside. Such tissue engineering strategies could eventually pave the path toward readily available therapies that significantly reduce patient cost of care and improve overall clinical outcomes.

Keywords: bone repair; critical defect; mineralized biomaterial; osteoinductive.

FIG. 1.

Biomineralized matrix characterization. (A) SEM images of non-mineralized and mineralized macroporous cryogels. Scale bars: 100 μm (top panels) and 10 μm (lower panels). (B) EDS showing presence of calcium and phosphate ions within mineralized hydrogels. EDS, energy-dispersive spectra; SEM, scanning electron microscopy. Color images available online at www.liebertpub.com/tea

FIG. 2.

Calcified bone tissue formation within critical-sized cranial defects. (A) μCT images of mouse cranial defects treated with mineralized and non-mineralized cryogels, as well as sham groups, at 0 (day 0), and 2 and 8 weeks postimplantation. Scale bars: 1 mm. (B) Quantification of bone volume for sham, non-mineralized, and mineralized groups at 2 and 8 weeks posttreatment. Asterisks denote p values with statistical significance (***p < 0.001). μCT, Microcomputed tomography.

FIG. 3.

Morphological assessment of bone formation within critical-sized cranial defects. H&E staining of cranial sections following 8 weeks of implantation. High magnification images reveal bone tissue within the defect site (lower left panels) and at the interface between the defect site and native bone (lower right panels). A yellow dotted line was used to delineate the location of the interface between the neo-tissue/implant and native bone. Yellow asterisks denote bone tissue. Scale bars: 500 μm (upper panel) and 20 μm (lower panels). H&E, hematoxylin and eosin. Color images available online at www.liebertpub.com/tea

FIG. 4.

Bone-specific markers in newly formed tissue within cranial defects. (A) Immunohistochemical staining of osteocalcin for sham (S), non-mineralized (NM), and mineralized (M) groups and (B) mean histogram intensity of images following 8 weeks of implantation. Lower intensity values correspond to higher expression. (C) Histochemical staining for TRAP and (D) percent positive area of the cranial defect site for sham (S), non-mineralized (NM), and mineralized (M) treatment groups following 8 weeks of implantation. Arrow indicates TRAP-positive cells found within the constructs. (E) Histochemical staining for RANK and (F) percent positive area of the cranial defect site for sham (S), non-mineralized (NM), and mineralized (M) treatment groups following 8 weeks of implantation. Scale bars: 30 μm. Asterisks denote p values with statistical significance (*p < 0.05, ***p < 0.001). RANK, receptor activator of nuclear factor κB; TRAP, tartrate-resistant acid phosphatase; u.d, undetectable. Color images available online at www.liebertpub.com/tea

FIG. 5.

Vascularization of mineralized constructs. Immunofluorescent staining for platelet endothelial cell adhesion molecule (CD31) and Hoechst 33342 staining of cell nuclei within the defect site for sham, non-mineralized, and mineralized treatment groups following 8 weeks of implantation. Scale bars: 100 μm. Color images available online at www.liebertpub.com/tea