Micro-Nanostructures of Cellulose-Collagen for Critical Sized Bone Defect Healing

Abstract

Bone tissue engineering strategies utilize biodegradable polymeric matrices alone or in combination with cells and factors to provide mechanical support to bone, while promoting cell proliferation, differentiation, and tissue ingrowth. The performance of mechanically competent, micro-nanostructured polymeric matrices, in combination with bone marrow stromal cells (BMSCs), is evaluated in a critical sized bone defect. Cellulose acetate (CA) is used to fabricate a porous microstructured matrix. Type I collagen is then allowed to self-assemble on these microstructures to create a natural polymer-based, micro-nanostructured matrix (CAc). Poly (lactic-co-glycolic acid) matrices with identical microstructures serve as controls. Significantly higher number of implanted host cells are distributed in the natural polymer based micro-nanostructures with greater bone density and more uniform cell distribution. Additionally, a twofold increase in collagen content is observed with natural polymer based scaffolds. This study establishes the benefits of natural polymer derived micro-nanostructures in combination with donor derived BMSCs to repair and regenerate critical sized bone defects. Natural polymer based materials with mechanically competent micro-nanostructures may serve as an alternative material platform for bone regeneration.

Keywords: bone; micro-nanostructures; regeneration; stem cells; tissue engineering.

Figure 1. Surgical procedure and schematic of the study groups

(A) Steps in the surgical implantation: (1) Creation of two circular 3.5mm defects on the two sides of mouse calvaria, (2) removal of calvarial bone, (3) Implantation of the scaffolds into the defects, (4) Closure of the implants by suturing the skin. (B) Groups 1: left side defect was filled with PLGA and the right side defect with CA, group 2: left side defect was filled with PLGA and the right side was filled with CAc, group 3: left side was filled with CA and the right side was filled with CAc. In study 1, the materials alone were used. In study 2, 1×10⁶ Col3.6-Cyan BMSCs from donor mice were seeded on to each scaffold before implantation into host mice with Col3.6-Tpz BMSCs.

Figure 2. Examination of scaffold structure

Photographic images of 3D microporous scaffolds used (A) PLGA, (B) CA, (C) CAc, scale bar= 1mm; SEM images at 100× magnification (D) PLGA, (E) CA, (F) CAc, scale bar= 500μm; SEM images at 500× magnification (G) PLGA, (H) CA, (I) CAc, scale bar= 100μm; SEM images at 1000× magnification (J) PLGA, (K) CA, (L) CAc, scale bar= 50μm.

Figure 3. Whole calvaria and X-ray radiograph of defects implanted with materials and cells

(A) Top panel: 1. Group 1-PLGA vs CA, 2. Group 2-PLGA vs CAc, 3. Group 3-CA vs CAc.; X-ray radiographs of 4. Group 1-PLGA vs CA, 5. Group 2-PLGA vs CAc, 6. Group 3-CA vs CAc. (B) Quantitative radio opacity of defect area normalized to radio opacity of host bone, both measured per unit area; PLGA, n=6; CA, n=6; CAc, n=7. One-way ANOVAwith Tukey post-test, with 95% confidence intervals, *P < 0.001.

Figure 4. Deposition of ECM proteins with implantation of materials with donor cells

Fluorescent histological cross sectional images of calvaria implanted with material and cells at 8 weeks, A-D-Group 1, PLGA vs CA; E-H-Group 2, PLGA vs CAc; I-L-Group 3, CA vs CAc; Bone Sialoprotein (BSP) (red), Collagen 1 (yellow) –Bone ECM proteins.

Figure 5. Quantification of Bone Sialoprotein (BSP) and Collagen I (Coll 1) deposited by materials with cells

(A) BSP-Bone sialoprotein (red stain in Figure 4), (B) Coll 1-Collagen 1 (yellow stain in Figure 4). One-way ANOVAwith Tukey post-test, with 95% confidence intervals, *P < 0.001.

Figure 6. Fluorescent histological images of calvaria implanted with material and BMSCs at 8 weeks

Group 2: PLGA vs CAc, Top row: DIC-Differential interference channel image to visualize bone and microspheres with AC-red labeled alizarin complexone marking new mineral deposition, Donor cells-Col3.6-Cyan (blue), Host cells-Col3.6-Tpz (green) (PLGA inset A, CAc inset B); Second row: DIC-Differential interference channel image to visualize bone and microspheres with AC-red labeled alizarin complexone marking new mineral deposition (PLGA inset C, CAc inset D). Third row: DIC-Differential interference channel image to visualize bone and microspheres with Donor cells-Col3.6-Cyan (blue) (PLGA inset E, CAc inset F). Fourth row: DIC-Differential interference channel image to visualize bone and microspheres with Host cells-Col3.6-Tpz (green) (PLGA inset G, CAc inset H).

Figure 7. Quantification of Bone defect closure, mineralization and number of host and donor cells

(A) Bone area fraction-DIC quantification, (B) New mineral formation-AC quantification, (C) Donor cells-Col3.6-Cyan quantification, (D) Host cells-Col3.6-Tpz quantification, (E) Host (Green bars) and Donor (Blue bars) cells in each scaffold group. One-way ANOVAwith Tukey post-test, with 95% confidence intervals, *P < 0.001, #P < 0.01, @P < 0.05.

Figure 8. Fluorescent histological images of osteoblastic activity and cellularity on the calvaria implanted with material and BMSCs at 8 weeks

In each image set, First row: AP (red)-Alkaline phosphatase activity for osteoblastic activity. Second row : DAPI (blue) -cell nuclei. A-D-Group 1, PLGA vs CA (PLGA-inset A, C; CA -inset B, D); E-H-Group 2, PLGA vs CAc (PLGA –inset E. G; CAc –inset F, H); M-R-Group 3, CA vs CAc (CA –inset I, K; CAc –inset J, L).

Figure 9. Quantification of Cellularity, osteoblastic and osteoclastic activity on the calvaria implanted with material and BMSCs at 8 weeks

(A) DAPI-cells, (B) AP/DAPI-osteoblastic activity, (C) TRAP-osteoclastic activity.