Nanotopographic Regulation of Human Mesenchymal Stem Cell Osteogenesis

Mesenchymal stem cell (MSC) differentiation can be manipulated by nanotopographic interface providing a unique strategy to engineering stem cell therapy and circumventing complex cellular reprogramming. However, our understanding of the nanotopographic–mechanosensitive properties of MSCs and the underlying biophysical linkage of the nanotopography-engineered stem cell to directed commitment remains elusive. Here, we show that osteogenic differentiation of human MSCs (hMSCs) can be largely promoted using our nanoengineered topographic glass substrates in the absence of dexamethasone, a key exogenous factor for osteogenesis induction. We demonstrate that hMSCs sense and respond to surface nanotopography, through modulation of adhesion, cytoskeleton tension, and nuclear activation of TAZ (transcriptional coactivator with PDZ-binding motif), a transcriptional modulator of hMSCs. Our findings demonstrate the potential of nanotopographic surfaces as noninvasive tools to advance cell-based therapies for bone engineering and highlight the origin of biophysical response of hMSC to nanotopography

<i>Kif3a</i> deletion in osteoblasts and osteocytes has no effect on tibial midshaft geometry.

<p>Imin and Imax are maximum and minimum second moment of inertia, respectively. pMOI is polar moment of inertia. Cortical bone geometry in 16 week old skeletally mature <i>Colα1(I) 2.3-Cre;Kif3a^fl/fl</i> and control mice was assessed using as microCT. Data presented as mean±SEM. N.S. is not significant (p>0.15).</p

<i>Colα1(I) 2.3-Cre;Kif3a^fl/fl</i> were significantly less responsive to mechanical loading than control mice.

<p><i>Colα1(I) 2.3-Cre;Kif3a^fl/fl</i> and control mice responded to mechanical loading with increased mineralizing surface (MS/BS), mineral apposition rate (MAR), and bone formation rate (BFR/BS), however, <i>Colα1(I) 2.3-Cre;Kif3a^fl/fl</i> were significantly less responsive to mechanical loading than control mice. Data presented as mean±SEM.</p>+++<p>p<0.001 for loaded vs. non-loaded values.</p>*<p>p<0.05 for <i>Colα1(I) 2.3-Cre;Kif3a^fl/fl</i> vs. control mice.</p

Skeletally mature <i>Colα1(I) 2.3-Cre;Kif3a^fl/fl</i> mice exhibit less responsiveness to mechanical loading compared to control mice.

<p>(<b>A</b>) Representative images of non-loaded (left) and loaded (right) ulnae of <i>Colα1(I) 2.3-Cre;Kif3a^fl/fl</i> (top) and control (bottom) mice. Fluorochrome labels (Calcein-green and Alizarin Red-red) given on Days 5 and 12 after the onset of mechanical loading. (<b>B to D</b>) Relative mineralizing surface (rMS/BS, %, B), mineral apposition rate (rMAR, µm per day, C), and bone formation rate (rBFR/BS, µm³/µm² per year, D) of mechanically loaded mice. <i>Colα1(I) 2.3-Cre;Kif3a^fl/fl</i> mice exhibited a decrease of 32% in rMAR and 33% in rBFR/BS when compared to control mice. Data presented as mean ± SEM. * p<0.05.</p

<i>Kif3a</i> deletion in osteoblasts and osteocytes has minimal effect on trabecular bone architecture.

<p>Trabecular bone volume fraction (BV/TV, %), trabecular number (Tb.N, µm⁻¹), trabecular thickness (Tb.Th, µm), trabecular spacing (Tb.Sp, µm), and connectivity density (Conn.D, µm⁻³) of the proximal tibia, distal femur, and L5 vertebra were measured using microCT. Data presented as mean±SEM. N.S. is not significant (p>0.15).</p

Kif3a expression in osteoblasts and osteocytes is not critical for embryonic skeletal development.

<p>(<b>A,B</b>) Whole mount Alizarin Red (bone) and Alcian Blue (cartilage) staining of E18.5 <i>Colα1(I) 2.3-Cre;Kif3a^fl/f</i>^l (A) and control (B) embryos. The size and limb patterning of <i>Colα1(I) 2.3-Cre;Kif3a^fl/fl</i> mice was similar to that of the control mice. (<b>C,D</b>) Movat's pentachrome staining of cross-sections of the radial/ulnar growth plates (cartilage-blue) in E16.5 <i>Colα1(I) 2.3-Cre;Kif3a^fl/f</i>^l (C) and control (D) mice. (<b>E–J</b>) Cross-sections of E16.5 long bones stained with Picrosirius red (E,F-bright field; G,H-polarized light) to illuminate collagen and Safranin O (I,J) to demarcate cartilage. Both control and <i>Colα1(I) 2.3-Cre;Kif3a^fl/f</i>^l mice have similar patterns of osteogenic and chondrogenic differentiation. Scale bar: 100 µm.</p

Axial ulnar loading leads to similar strain at the ulnar midshaft of <i>Colα1(I) 2.3-Cre;Kif3a^fl/fl</i> and control mice.

<p>(<b>A</b>) Image of strain gaging and axial ulnar loading experimental set-up. The right forearms of 16 week old skeletal mature mice were axially loaded for 120 cycles per day for 3 consecutive days with a 2 Hz sine wave using an electromagnetic loading system with feedback control. The left forearms were not loaded and used as non-loaded internal controls. (<b>B</b>) Strain in cortical bone at given mechanical loading levels. Open and closed circles indicate <i>Colα1(I) 2.3-Cre;Kif3a^fl/fl</i> (n = 35) and control (n = 27) mice, respectively. Data presented as mean ± SEM. * p<0.05.</p

Generation and confirmation of bone-specific Kif3a conditional knockout mice.

<p>(<b>A</b>) A typical agarose gel resulting from PCR genotyping of genomic DNA from tail biopsies of transgenic mice. Bands indicate floxed (490 bp) and wild-type (360 bp) <i>Kif3a</i> and <i>Cre</i> recombinase (650 bp). 18S (870 bp) used as a positive control in the <i>Cre</i> PCR reactions. Floxed and recombined <i>Kif3a</i> allele present in <i>Colα1(I) 2.3-Cre;Kif3a^fl/fl</i> mice due to heterogeneous tissue in tail biopsies. (<b>B</b>) To assess <i>Cre</i> specificity, <i>Colα1(I) 2.3-Cre</i> mice were crossed with <i>Rosa26R</i> reporter mice. Effective <i>Cre</i> recombination was detected by LacZ staining in osteoblasts and osteocytes of <i>Colα1(I) 2.3-Cre;R26R</i> mice (right) but not in littermates lacking <i>Cre</i> (left). LacZ staining was not visible in muscle tissue of <i>Colα1(I) 2.3-Cre;R26R</i> mice. (b- bone, bm- bone marrow, m- muscle).</p

Skeletal morphology of adult <i>Colα1(I) 2.3-Cre;Kif3a^fl/fl</i> mice is similar to control mice.

<p>(<b>A,B</b>) Comparison of 16 week old <i>Colα1(I) 2.3-Cre;Kif3a^fl/fl</i> (A) and control (B) mice stained with Alizarin Red (bone) and Alcian Blue (cartilage) revealed no differences in size or morphology of the axial or appendicular skeleton.</p

Transverse cross-sections at ulnar midshaft in nonloaded (left) and loaded (right) forearms in WT (top) and FAK−/− (bottom) mice given calcein (green) and alizarin (red) fluorochrome bone labels at 4 and 11 days, respectively, after the first day of loading.

<p>In response to applied mechanical loading, most new bone is formed on the medial and lateral surfaces where bending strains are highest. Note the appearance of double bone labels (green and red) on the medial and lateral surfaces of the ulna, as well as on the rostral surface, in the loaded ulna (top, right) compared to the nonloaded internal control (top, left) where very little bone formation is observed. The loaded ulna (bottom, right) in FAK−/− mice exhibit bone formation on the medial and lateral ulnar surfaces, but much less new bone formation, in terms of percent mineralizing surface, is observed relative to the nonloaded ulna (bottom, left). Sections are representative of the response observed for WT and FAK−/− mice. Magnification = 10×.</p