Figure 4

<p>FAK inactivation specifically blocks mechanically induced osteogenesis <i>in vivo</i>. (A) <i>Col I</i> expression marks peri-implant cells, (B) including those juxtaposed to the implant (im). (C) The schematic indicates the genomic structure of floxed FAK mice; crossing these mice with Cre mice carrying a 2.3Kb osteoblast-specific <i>Col1a1</i> promoter resulted in Col1Cre^+/+;FAK^fl/fl (FAK mutant) mice. PCR was used to identify deletion of the <i>fak</i> allele in the animal. (D) In wildtype animals, seven days of stimulation result in abundant bone formation. (E) High magnification (Aniline blue) shows newly deposited bone matrix (blue) interlaced with blood vessels. (F) In FAK mutant mice, mechanical stimulation failed to induce osteogenesis. Note that FAK mutants were able to regenerate bone in unstimulated regions, as seen on the right periosteal surface. (G) Aniline blue staining shows complete absence of mineralized tissue in the peri-implant site. (H) Vascular ingrowth is not impeded by the deletion of FAK. (I,J,K,L) FAK mutant cells express <i>sox9, runx2, col I</i> and <i>osteocalcin</i> indicating that loss of FAK does not hamper the recruitment of osteochondroprogenitor cells to the peri-implant site. (M) Quantitative histomorphometric assessment of newly deposited bone matrix in unstimulated wild type (wt) bone marrow cavities (white), in stimulated wt bone marrow cavities (light gray), stationary FAK mutant bone marrow (gray), and in stimulated FAK mutant bone marrow cavities (black). * (P<0.1), # (p<0.001) indicates significant difference. Scale bar in A,D and F: 300 µm; in B,E and G–L: 100 µm.</p

Figure 1

<p><i>In vivo</i> implant device permits defined stimulation of the bone marrow tissue. (A) A motion device, consisting of an intra-osseous, pin-shaped implant (im), held in place by a subcutaneous fixation plate is secured to the mouse tibia by two screws (dotted line is approximate skin level). An O-ring placed between the head of the implant and the center column of the fixation plate acts as a spring to return the implant to its starting position after axial displacement. (B) <i>In vivo</i> setting of micromotion device on murine tibia. (C) A linear variable differential transducer (LVDT) and load cell connected to the implant head and fixation plate allows the application and recording of displacement (∼150 µm) and the force (∼1N) required to produce motion.</p

Figure 3

<p>Molecular and cellular response mirrors strain pattern. (A,B) PCNA staining reveals no differences in cell proliferation between unloaded and loaded samples. (C) In stimulated and (D) stationary implants <i>sox9</i> is diffusely expressed throughout the surrounding bone marrow cavity. (E) <i>Runx2</i> is broadly and strongly expressed in the peri-implant region in unstimulated samples, (F) whereas physical stimulation induces restriction of the <i>runx2</i> transcripts to the cells adjacent to the implant. (G) Finite element modeling shows strain concentrations (tensile strain (t), compressive strain (c)) at the circumferential ridges and at the bottom of the implant (for illustration purposes, tensile strains were plotted on the right and compressive strains on the left). (H,I) µCT was used to record displacement of Tantalum particles, and principal strains were calculated by digital image correlation. Implant displacement generated a range of strain fields concentrated around circumferential ridges (cr)(*). (J,K) Picrosirius red staining in conjunction with polarized light microscopy reveals that in loaded samples, the peri-implant collagen fibrils (yellow-red) are abundant, tightly packed, and aligned parallel to the displacement trajectory, (L) whereas in unloaded samples, the collagen fibrils are unorganized. Scale bar: 100 µm.</p

Figure 2

<p>Mechanical stimulation expands the pool of osteoprogenitor cells and accelerates their differentiation into osteoblasts. (A) On post-surgical d3, cells in the stimulated peri-implant space are densely packed within a proteoglycan-rich extracellular matrix (blue). (B) In the stationary environment, cells are loosely organized with no evidence of a mineralized extracellular matrix. (C) By d7, a thick (250 µm), fully mineralized bony sheath encapsulates the stimulated implant. (D) The tissue surrounding the stationary implant is absent of any bone matrix. (E) By d14, the bony encasement is more organized and still retains its original thickness. (F) The stationary tissue exhibits first sign of mineralization (90 µm thick) after 14 days. Scale bar: 100 µm.</p