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Mech Dev
2018 Dec 01;154:153-161. doi: 10.1016/j.mod.2018.06.007.
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Bone regeneration after traumatic skull injury in Xenopus tropicalis.
Muñoz D
,
Castillo H
,
Henríquez JP
,
Marcellini S
.
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The main purpose of regenerative biology is to improve human health by exploiting cellular and molecular mechanisms favoring tissue repair. In recent years, non-mammalian vertebrates have emerged as powerful model organisms to tackle the problem of tissue regeneration. Here, we analyze the process of bone repair in metamorphosing Xenopus tropicalis tadpoles subjected to traumatic skull injury. Five days after skull perforation, a dense and highly vascularized mesenchymal is apparent over the injury site. Using an in vivo bone staining procedure based on independent pulses of Alizarin red and Calcein green, we show that the deposition of new bone matrix completely closes the wound in 15 days. The absence of cartilage implies that bone repair follows an intramembranous ossification route. Collagen second harmonic imaging reveals that while a well-organized lamellar type of bone is deposited during development, a woven type of bone is produced during the early-phase of the regeneration process. Osteoblasts lying against the regenerating bone robustly express fibrillar collagen 1a1, SPARC and Dlx5. These analyses establish Xenopus tropicalis as a new model system to improve traumatic skull injury recovery.
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Fig. 1. A novel traumatic skull injury assay in Xenopus tropicalis.
Stage NF66 Xenopus specimens were subjected to skull damage and examined at 0 (A, B, E and F) and 5 (C, D, Eâ² and Fâ²) days post-injury (dpi). A dorsal view of the head is shown before (A) and after (B) skinning. (C) Dorsal view showing that regenerating skin has covered the wound at 5 dpi. Arrows in (AâC) point at the impact site. The region delineated by a white square in (C) is shown at a higher magnification in (D). (E, Eâ²) Calvariae were dissected together with their surrounding tissue serving as an internal positive control for the presence of cartilage (Sc, skull cartilage), and double stained with Alizarin red and Alcian blue. (F, Fâ²) Three-dimensional reconstructions of dissected calvariae stained for bone mineral (Alizarin red, red) and cell bodies (Alexa Fluor 488-phalloidin, green). Note that the hollow lesion (Hl) converts into an Alcian blue-negative regeneration plug (Rp) highly irrigated by blood vessels (Bv) in five days. The dotted circles in F and F´ show the injured zone which contains remnants of âoldâ (non-regenerated) matrix (âoâ, located between the continuous and dotted lines). Anterior is left in all panels. Scale bars represent 1â¯mm in (AâE and E´) and 500â¯Î¼m in (F and Fâ²). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 2. Bone growth can be monitored in Xenopus tropicalis tadpoles.
A tadpole was subjected to an Alizarin red pulse at the NF56+ stage (an intermediate stage between NF56 and NF57) and to a Calcein green pulse at stage NF57. Hindlimbs (A–D, anterior up) and dissected calvaria (E–H, anterior left) were photographed at stage NF57 as indicated. The arrowheads in (D and H) point at Alizarin red-negative and Calcein green-positive matrix regions. In (H) the regions delineated by white squares are shown at higher magnification (insets). The scale bar represents 1 mm in all panels. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 3. Bone regeneration can be monitored in Xenopus tropicalis tadpoles.
Brightfield and fluorescent pictures of calvariae dissected from tadpoles having been subjected to an Alizarin red pulse 1â¯h before traumatic skull injury (at stage NF60), and subsequently exposed to a Calcein green pulse at 1 (AâD, Aâ²âDâ²), 7 (EâH, Eâ²âHâ²) or 15 (IâL, Iâ²âLâ²) dpi. The dotted circles in Aâ², Eâ² and Iâ² delineate the injured zone. A continuous line in Aâ²âDâ² and Eâ²âHâ² delineates a mineralized matrix-free region. Regions of the matrix located within the circular lesion that are either double positive (o, old bone), or Alizarin red-negative and Calcein green-positive (n, new bone), are indicated. Note that the Alizarin red signal in (Bâ²) is faint as the dislodged bone (o) is out of focus. The scale bar shown in (A) represents 1â¯mm in (AâD, EâH, IâL). The scale bar shown in (Aâ²) represents 500â¯Î¼m in (Aâ²âDâ², Eâ²âHâ², Iâ²âLâ²). Calvariae are shown in dorsal views with their anterior region towards the left. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 4. Structural analysis of the regenerating bone matrix.
(A, B) Collagen second harmonic imaging was performed at 15 (A) and 30 (B) dpi (dorsal upward). The impact site is delineated by a double arrow. Note that at 15â¯dpi the regenerated matrix is thin, disorganized and displays characteristics of woven bone. By contrast, matrix located outside of the damaged zone and within the 30â¯dpi regenerated bone exhibit collagen fibers that are tightly packed in parallel layers characteristic of lamellar bone. (CâF) A 30â¯dpi calvaria was examined by scanning electron microscopy (anterior to the left). The regions delineated by rectangles in (C) and (D) are shown at higher magnification in (D) and (E), respectively. (C) Whole calvaria. (D) Damaged site. Note the presence of irregular bone remnants (o) resulting from the drilling of the older matrix. (E) Circular cavities (â) are present within the regenerating region. (F) A superficial osteocyte lacuna from the regenerating region. Note the presence of collagen fibers and osteocytic canaliculi.
Fig. 5. Gene expression analysis during calvaria regeneration.
Calvariae were damaged at stage NF60 and were allowed to regenerate for 15 (A–F) and 30 (A′–F′) days. (A–A′) Hematoxylin eosin staining of histological sections located at the transition zone between the “old†and “new†(regenerated) bone. Schematic drawing of (A) and (A′) are shown in (B) and (B′) respectively. Note the presence of abundant mesenchyme irrigated by blood vessels (Bv). The newly formed bone (green in B, B′) contacts the matrix of the damaged calvaria (red in B and B′) where empty osteocyte lacunae lie (arrows in A, A′). Osteoblasts cover the surface of both the old and new bone matrix (arrowheads in A and A′). At 30 dpi, an irregular old bone matrix remnant (o in B′) is found embedded within the regenerated bone, and a cavity filled with mesenchyme can be observed within the regenerating region (* in B′). In situ hybridization was performed using a control probe (C, C′), or probes specific to the Collagen 1a1 (D, D′), SPARC (E, E′) and Dlx5 (F, F′) genes, revealing strong expression in osteoblastic cells at both stages. Dorsal is up in all panels. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
