Biomimetic matrices for rapidly forming mineralized bone tissue based on stem cell-mediated osteogenesis

Abstract

Bone regeneration, following fracture, relies on autologous and allogenic bone grafts. However, majority of fracture population consists of older individuals with poor quality bone associated with loss and/or modification of matrix proteins critical for bone formation and mineralization. Allografts suffer from same limitations and carry the risk of delayed healing, infection, immune rejection and eventual fracture. In this work, we apply a synergistic biomimetic strategy to develop matrices that rapidly form bone tissue - a critical aspect of fracture healing of weight bearing bones. Collagen matrices, enhanced with two selected key matrix proteins, osteocalcin (OC) and/or osteopontin (OPN), increased the rate and quantity of synthesized bone matrix by increasing mesenchymal stem/stromal cell (MSC) proliferation, accelerating osteogenic differentiation, enhancing angiogenesis and showing a sustained bone formation response from MSC obtained from a variety of human tissue sources (marrow, fat and umbilical cord). In vivo assessment of OC/OPN mineralized scaffolds in a critical sized-defect rabbit long-bone model did not reveal any foreign body reaction while bone tissue was being formed. We demonstrate a new biomimetic strategy to rapidly form mineralized bone tissue and secure a sustained bone formation response by MSC from multiple sources, thus facilitating faster patient recovery and treatment of non-union fractures in aging and diseased population. Acellular biomimetic matrices elicit bone regeneration response from MSC, obtained from multiple tissue sources, and can be used in variety of scaffolds and made widely available.

Figure 1

Design of the biomimetic OC/OPN-enhanced collagen matrices. (a) Schematic of OC/OPN-enhanced collagen gels. (b) Schematic of fabrication of OC/OPN-enhanced mineralized collagen scaffolds. (c) Different concentrations of OC and OPN incorporated into type I collagen gels at 3 mg/ml to create biomimetic matrices, based on the average of OC recovered from human bone. Cell proliferation, osteogenic differentiation, mineralization and angiogenesis were the different variables evaluated after cell culture on OC/OPN-enhanced collagen gels. (d) Left: Representation of the OC/OPN-enhanced mineralized collagen scaffold implanted into a rabbit tibia after 6 weeks of surgery. Right: OC/OPN-enhanced mineralized collagen scaffolds prior to implantation into a rabbit model.

Figure 2

Effects of OC/OPN-enhanced collagen gels on cell proliferation and osteogenic differentiation of BM MSC. (a) Fluorescence microscopy images of DAPI (blue) and Phalloidin (red) of BM MSC seeded on top of different substrates 24 h and 15 days after cell seeding (OC/OPN #1) under DMEM + 10% FBS. Scale bar, 100 µm. (b,c) Proliferation of BM MSC cultured on OC/OPN-enhanced collagen gels for 15 days under (b) DMEM + 10% FBS and (c) osteogenic differentiation medium. (d) Percentage of maximum gene expression (collagen I [ColI], runx2, osteopontin [OPN], osteocalcin [OC] and alkaline phosphatase [ALP]) by BM MSC upon culture for 7, 15 and 21 days on control collagen gels and OC/OPN-enhanced collagen gels. Data are expressed as mean ± s.e.m.; **P < 0.01; *P < 0.05.

Figure 3

Effects of OC/OPN-enhanced collagen gels on BM MSC in vitro angiogenic properties assessed by multiple assays. (a) Cell migration assay. Scratch at t = 0 h, t = 8 h and t = 24 h when HUVEC were treated with EGM-2 (positive control), conditioned medium from BM MSC cultured on control collagen gels and conditioned medium from BM MSC cultured on OC/OPN-enhanced collagen gels. Borders of the scratch at t = 0 are indicated with solid lines, borders after migration at t = 8 and t = 24 hours with dashed lines. (b) Percentage of migration distance quantification of HUVEC treated with conditioned medium from BM MSC cultured on control collagen gels, OC-collagen gels, OPN-collagen gels and OC/OPN-collagen gels. (c) VEGF relative expression of BM MSC cultured on OC/OPN–enhanced collagen gels, OC-enhanced collagen gels, OPN-enhanced collagen gels and control collagen gels after 21 days of culture under osteogenic differentiation, normalized to VEGF relative expression of undifferentiated BM MSC. (d–f) Endothelial cell tube formation assay: (d) Tube formation of HUVEC on a Matrigel substrate incubated with EGM-2 (positive control), EBM-2 (negative control) and conditioned medium from BM MSC cultured on collagen gels, OC-collagen gels, OPN-collagen gels and OC/OPN-collagen gels. (e,f) Number of tubes/field (e) and number of branch points/field (f). Scale bars, 100 µm. Data are expressed as mean ± s.e.m.; **P < 0.01; *P < 0.05.

Figure 4

Effects of OC/OPN-enhanced collagen gels on BM MSC mineralization and ALP activity. (a) Calcium content quantification of BM MSC on OC/OPN-enhanced collagen gels cultured for 21 days compared to control collagen gels. (b) Mineralized nodules of MSC after 21 days of culturing in osteogenic differentiation medium on OC/OPN-enhanced collagen gels and control collagen gels without OC/OPN incorporation. The nodules were visualized by Xylenol orange staining. Scale bars, 100 µm. (c–e) SEM images of BM MSC cultured on OC/OPN-enhanced collagen gels. (c) OC/OPN-enhanced collagen gels before cell culture; (d,e) BM MSC mineralization of OC/OPN-enhanced collagen gels after 21 days of osteogenic differentiation. (f) EDS spectrum of mineral deposition of BM MSC cultured on OC/OPN-enhanced collagen gels. (g) Alkaline phosphatase (ALP) activity of BM MSC cultured on OC/OPN-enhanced collagen gels for 15 days and 21 days of differentiation. Data are expressed as mean ± s.e.m.; **P < 0.01; *P < 0.05.

Figure 5

Bone regeneration in a critical sized-defect rabbit long-bone model using OC/OPN-enhanced mineralized collagen scaffolds. (a) Representative histological images of the rabbit critical sized-defect at 6 weeks postimplantation. Hematoxylin and Eosin (H&E), Goldner’s Trichrome (GT), Von Kossa (VK), Toluidine Blue (TB) and Tartrate-resistant acid phosphatase (TRAP) stainings. Panels on the first row are at 100x magnification. Black squares represent the area of new bone formed. Dashed squares represent the area of the implanted scaffold. Scale bars, 100 µm. Panels on the second row are at 200x magnification and represent the area of new bone formation (black squares). Scale bars, 50 µm. Panels on the third row are at 200x magnification and represent the area nearby the implanted scaffold (dashed squares). Black arrows outline the osteoblasts covering the new bone formed. Black arrowheads represent the osteoclasts by TRAP staining, indicating a possible remodeling of bone. White circle represents new vessels formed. Scale bars, 50 µm (b) Representative microcomputed tomography images of bone regeneration in the rabbit model at 6 weeks postimplantation. Top row represents a tibial defect, bottom row represents a femoral defect. Scale bars, 1 mm. (c) Goldner’s trichrome staining – detailed investigation. Detailed description of new bone formation within the OC/OPN-enhanced mineralized collagen scaffolds. NB – new bone; CT – connective tissue; S – scaffold. Scale bars, 100 µm.

Figure 6

Proliferation and mineralization of human MSC from different tissue sources cultured on OC/OPN-enhanced collagen gels. (a) Proliferation of MSC from BM, AT and UCM on OC/OPN-collagen gels after 15 days of culture. **P < 0.01; *P < 0.05 relative to the control group for each cell source. (b) Calcium quantification of MSC from BM, AT and UCM cultured on OC/OPN-enhanced collagen gels after 21 days of osteogenic differentiation. Data are expressed as mean ± s.e.m, **P < 0.01; *P < 0.05 relative to the control group for each cell source. (c) Summary of statistically significant differences between each different composition of OC/OPN and control collagen gels, OC-collagen gels and OPN-collagen gels for each cell source (BM, AT, UCM), regarding cell number and calcium quantification after 15 days of culture. **P < 0.01, *P < 0.05 relative to control collagen gel; ++P < 0.01, +P < 0.05 relative to OC-collagen gel; ^##P < 0.01, ^#P < 0.05 relative to OPN- collagen gel; n.s. – not significant.