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Bone Regeneration via a Mineral Substrate and Induced Angiogenesis

¹ Department of Biomedical Engineering, ² Department of Biologic and Materials Sciences, and ³ Department of Chemical Engineering, University of Michigan, 5213 Dental, 1011 North University Avenue, Ann Arbor, MI 48109-1078;

View larger version (77K): [in a new window]   Figure 1. Imaging of mineralized scaffolds and measurement of VEGF release kinetics. (A) Electron micrographs displaying 85:15 PLG scaffolds with an interconnected pore structure (250 < d < 425 µm). Scale bar = 1 mm. (A, inset) The surface of a single pore within the scaffold coated with a thin biomineral film (scale bar = 60 µm). (B) The biomineral film displays a platelike nanostructure similar to that of native bone mineral (scale bar = 20 µm). Biomineralization was achieved via incubation of pre-hydrolyzed scaffolds in mSBF for 5 days. (C) In vitro release kinetics of VEGF from mineralized scaffolds. Data in plot represent mean ± standard deviation (n = 5).

View larger version (64K): [in a new window]   Figure 2. VEGF release enhances blood vessel ingrowth 2 wks post-implantation. (A-D) vWF immunostaining of blood vessels within sections of implanted PLG (A), mineralized PLG (B), and VEGF-releasing, mineralized PLG (C) scaffolds. Positive vWF staining is brown, and circular vWF staining represents a blood vessel. (D) Higher-magnification image of a different region within a VEGF-releasing, mineralized PLG scaffold. (E) Quantification of blood vessel densities within the total scaffold area and a region in the center of the scaffold for each condition. *P < 0.01 relative to the PLG condition; **P < 0.01 relative to the mineralized PLG condition (n = 4 for MIN; n = 6 for PLG and MIN + VEGF). Data in plot represent mean ± standard deviation. Scale bars = 100 µm (in A-C) or 20 µm (in D). Sections were counterstained with hematoxylin.

View larger version (71K): [in a new window]   Figure 3. Mineralized scaffolds support enhanced bone tissue ingrowth, and biomineral presence significantly enhances osteoid matrix deposition. (A,B) Hematoxylin and eosin (H&E) staining of sections of a critical-sized defect without an implanted scaffold (A) and a defect after implantation of a mineralized, VEGF-releasing PLG scaffold (B). Bridging of the defect area with tissue was evident for all three experimental conditions (data not shown). (C-E) Higher-magnification H&E staining of implanted PLG (C), mineralized PLG (D), and VEGF-releasing, mineralized PLG (E) scaffolds 14 wks postimplantation. (F-H) Goldner’s trichrome staining of implanted PLG (F), mineralized PLG (G), and VEGF-releasing, mineralized PLG (H) scaffolds 14 wks post-implantation (red = osteoid matrix). (I) Quantification of osteoid matrix fractional area within the scaffold area for each condition. *P < 0.05 relative to the PLG condition (n = 5 for PLG and MIN; n = 6 for MIN + VEGF). Data in plot represent mean ± standard deviation. Scale bar = 1 mm (in A-B) or 100 µm (in C-H).

View larger version (68K): [in a new window]   Figure 4. Biomineral presence and induced angiogenesis concertedly enhance mineralized tissue regeneration. (A-C) von Kossa staining of implanted PLG (A), mineralized PLG (B), and VEGF-releasing, mineralized PLG (C) scaffolds 14 wks post-implantation (dark purple-black = mineralized tissue) displaying bone regeneration in the interior of the scaffold. (D) Quantification of mineralized tissue fractional area within the scaffold area for each condition. *P = 0.065 relative to the PLG condition; **P < 0.01 relative to the PLG condition; ***P < 0.01 relative to the mineralized PLG condition (n = 5 for PLG and MIN; n = 6 for MIN + VEGF). Data in plot represent mean ± standard deviation. Scale bars = 100 µm.