9 research outputs found
MMP1 treatment enhanced stem cell population.
<p>The localization of Sca-1 expressive cells at 10 days after amputation is compared between untreated (<b><i>Aa</i></b>) and MMP1 treated (<b><i>Bb</i></b>) regenerating digits. The number of expression of Sca-1 positive cells was enriched in MMP1 treated digits compared to non-treated digits, respectively (<b><i>C</i></b>). Arrows (red): Sca-1 positive cells; DAPI staining (blue): indicated cells’ nucleus. N = 4 for each group; *p<0.05 was considered as significant.</p
Cre/Lox-β-galactosidase cell transplantation and muscle injury resulted in β-gal/LacZ positive mononuclear cells.
<p>Three weeks after cell transplantation into MDX/SCID mice, many (β-gal/LacZ)+/dystrophin+ myofibers were observed (<b>A</b>, <b>B</b>). β-gal/LacZ staining and immunofluorescent Pax7/dystrophin staining are shown here; arrows indicate (β-gal/LacZ)+/dystrophin+ myofibers which are the result of the fusion of Cre-Lox myocytes, and arrowheads indicate (β-gal/LacZ)−/dystrophin− host myofiber (<b>A</b>, <b>B</b>). Three weeks after transplantation and four days after muscle injury in SCID mice, some (β-gal/LacZ)+ mononuclear cells were observed in the injured muscle (<b>E–G</b>), but not in the control non-injured muscle (<b>C</b>, <b>D</b>). Results of β-gal/LacZ staining and HE staining are shown here; arrows indicate (β-gal/LacZ)+ myofibers, and arrowheads indicate (β-gal/LacZ)+ mononuclear cells (<b>C–G</b>). Some of the (β-gal/LacZ)+ mononuclear cells were shown to be Pax7+ (<b>H–K</b>). Images H–J are of the same location, and result of β-gal/LacZ staining and immunofluorescent Pax7/dystrophin staining are shown here (<b>H–J</b>); arrows indicate β-gal+/Pax7+ mononuclear cells, and arrowheads indicate β-gal+ myofibers (<b>H–J</b>). Fluorescent co-staining of β-gal and Pax7 are also shown (K); arrowheads indicate β-gal+/Pax7+ mononuclear cells, and arrows indicate Pax7+ cells. Statistical quantification of the results is also shown (<b>L</b>).</p
β-gal/LacZ positive mononuclear cells from injured muscle contain different cell populations (i.e., myoblasts, satellite cells, and MDSCs).
<p>The result of flow cytometry analysis, β-gal/LacZ staining and immune-staining to MyoD or Pax7 showed that, the isolated PP2 (myoblasts) (<b>A–C</b>) and PP5 (satellite cells) (<b>D–F</b>) cell populations both contain (β-gal/LacZ)+ cells, which can be MyoD+ or Pax7+. The result of β-gal/LacZ staining and immune-staining to Sca-1 also showed that, the PP6 cells (MDSCs) also contain (β-gal/LacZ)+ cells, which were shown to be Sca-1+ (<b>G</b>, <b>H</b>). Flow cytometry analysis comparing PP6 cells from non-injured control muscles and the PP6 cells from injured muscle demonstrated a greater number of (β-gal/LacZ)+ cells which also expressed Sca-1 and CD34 in the injured muscle (I–J), indicating that adult stem cells can be dedifferentiated from “terminally” differentiated muscle fibers in injured skeletal muscle of mice. Statistical quantification of the flow cytometry results is shown (<b>K</b>).</p
MMP1 treatment improved angiogenesis and re-vascularization in the amputated digits.
<p>The localization of CD31 and utrophin proteins in the regenerating digits (<b><i>A</i></b>) was compared between MMP1 treated and non-treated digits (<b><i>B–C</i></b>, day 10, sections with bone; <b><i>E–F</i></b>, day 10, sections without bone; <b><i>H–I</i></b>, day 25, sections with bone). “<b>*</b>” in image <b>A</b> indicates the proximal end of middle phalanx. Arrows (red): CD31 positive blood vessels or capillary vasculature; arrowheads (green): utrophin positive cells; DAPI (blue): present cell nucleus. The level/position of sections in each comparison is demonstrated (<b><i>D</i></b><i>, </i><b><i>G &J</i></b>). The statistical analysis of CD31 positive signal (<b><i>K</i></b><i>,</i> day10; <b><i>L</i></b><i>,</i> day 25), and utrophin positive signal (<b><i>M</i></b>, day 10) in the amputated digits are also shown. N = 4 for each group; *p<0.05 was considered as significant.</p
Specificity of the Cre-Lox system in differentiated myotubes <i>in vitro</i>.
<p>Schematic representation of the Cre/Lox-β-galactosidase system used in the current study (<b>A</b>). (β-gal/LacZ)+ cells were not observed with homogenous fusion of MDC-Lox cells (<b>B</b>). In co-cultured MDC-Cre and MDC-Lox cells, (β-gal/LacZ)+ cells can be either nascent myotubes with 2 nuclei (<b>C</b>, nuclei marked by arrows; 2 days after myogenic differentiation), or mature myotubes with multiple nuclei (<b>D</b>, nuclei marked by arrows; 4 days after myogenic differentiation). No (β-gal/LacZ)+ mononuclear cells were observed. Statistical quantification of (β-gal/LacZ)+ myotubes is shown (<b>E</b>). It is proposed that the presence of any mononuclear cells positive for β-gal expression in the culture have to be released from (β-gal/LacZ)+ myotubes, that is, after some type of stimulation, myotubes formed through the fusion of MDC-Cre and MDC-Lox cells, dedifferentiate and release the mononuclear (β-gal/LacZ)+ cells (<b>F</b>).</p
Process of digit amputation and regeneration.
<p>Digits were amputated at the central position of the middle phalanges (<b><i>A–B</i></b>, day 0) (n = 4 for each group). At the early stage of digit regeneration, the wound closure was shown to be faster in MMP1 treated digits (<b><i>C–D</i></b><i>,</i> day 10). However, during the whole process of digit regeneration, there was no significant difference in the length of the regenerating digits (<b><i>C–D</i></b><i>,</i> day 10; <b><i>E–F</i></b><i>,</i> day 25). The site of the amputations is demonstrated (<b><i>G</i></b>). The lengths of the regenerating digits from day 0 to day 30 after amputation are also plotted as the percentage of the length of normal digits (<b><i>H</i></b>).</p
β-gal/LacZ positive cells can proliferate and contribute to myotube formation <i>in vitro</i>, and blood vessel formation <i>in vivo</i>.
<p>BrdU incorporation assay showed that (β-gal/LacZ)+ cells can proliferate (<b>A–C</b>). A myogenic differentiation assay, which deprives the cultures of serum, showed that (β-gal/LacZ)+ cells, in both cell populations without purification [ around 6% of cells were (β-gal/LacZ)+] (<b>D</b>) and after purification with Fluorescence-activated cell sorting (<b>E</b>), can participate in myotube formation (<b>F</b>). <i>In vivo</i>, ten days after laceration-injury of GM muscles that were transplanted with Cre-cells and Lox-cells for 3 weeks, some (β-gal/LacZ)+ signal was also found to co-localize with CD31+ signal in the blood vasculature (<b>G–L</b>). Images G–I or J–L are of the same location in tissue, and result of β-gal/LacZ staining and immunofluorescent CD31/Utrophin staining are shown here (<b>G–I</b>); arrowheads indicate β-gal+/CD31+ cells, and arrows indicate β-gal+ myofibers (<b>G–I</b>). Fluorescent co-staining of β-gal and Pax7 are also shown (<b>J–L</b>); arrowheads indicate β-gal+/Pax7+ cells, and arrows indicate CD31+ cells (<b>J–L</b>).</p
MMP1 treatment improved nerve regeneration and reduced fibrosis formation in the amputated digits.
<p>The localization of NCAM and dystrophin proteins in regenerating digits was compared between MMP1 treated and non-treated digits at day 25 (<b><i>A–D</i></b>). The sub-image in <b>D</b> shows the cross-section view of the nerve-related structure (<b><i>D</i></b><b>,</b> marked area with red squared line). Arrows: NCAM positive peripheral nerves (red); arrowheads: NCAM positive nerve-related structure (yellow). Dystrophin expression is shown in green and cell nucleus blue (DAPI). The level/position of sections in this comparison is demonstrated (<b>E</b>). NCAM expression in the amputated digits was significantly higher in MMP1 treated digits (<b>F</b>). To compare fibrosis formation in the differentially treated digits, trichrome staining was conducted with tissue sections of non-treated digits (<b><i>G&I</i></b>) and MMP1 treated digits (<b><i>H&J</i></b>). The level/position of sections in this comparison is demonstrated (<b>K</b>) and the quantification of fibrotic structure (collagen deposition in ECM) at day 25 shown (<b>L</b>). N = 4 for each group; *p<0.05 was considered as significant.</p
MMP1 treatment accelerated wound closure and healing of soft tissue but not the hard tissue.
<p>With hematoxylin and eosin (H&E) staining of tissue sections, soft tissues and bones can be observed in normal digits (<b>A</b>) and regenerating digits 10 days (<b><i>B&C</i></b>) or 25 days after amputation (<b><i>E&F</i></b>). MMP1 treated digits showed improved regeneration of soft tissues (faster wound closure) (<b><i>B–D</i></b>), but not significant improvement in the growth or elongation (<b><i>E–G</i></b>). N = 4 for each group; *p<0.05 was considered as significant.</p