HBO treatment delays wound closure and blastema formation.

<p>H&E staining of 7, 10, and 14 DPA sections of control (A-C) and HBO-treated (A'-C') digits show cellular changes in the marrow and delayed wound closure and blastema formation in HBO-treated digits. Arrows indicate areas of vasculature on control 7 DPA (A) and HBO treated 7 DPA (A') samples. (B') Epidermis remains open at 10 DPA in samples treated with hyperbaric oxygen. (C') Blastema formation is delayed until 14 DPA in HBO-treated samples when control samples (C) are already forming bone. Higher magnification analysis of the proximal endosteum show activated osteoblasts in HBO-treated samples (A'-C') while control samples return to an inactivated morphology at 10 DPA (B). Scale bars = 100 μm in panels and 5 μm in insets. (N = 8 HBO and N = 7 control with representative figure shown).</p

HBO treatment alters the morphology of regenerated bone.

<p>Polarized light micrographs of control and HBO treated digits at 7, 10, 14, and 21 DPA show differences in collagen fiber composition at distinct time points during regeneration. Collagen fiber alignment and thickness is readily identifiable under polarized light. Thinner fibers (a general indication of either thin collagen I fibrils, <50 nm in width, or collagen III fibers) appear green. Thick, more mature and aligned collagen I fibers appear red or yellow [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140156#pone.0140156.ref022" target="_blank">22</a>]. Photomicrographs are presented in transmitted light (A—F) and polarized light (A'-F'). Digit orientation is portrayed by black cartoon on the upper right corner of each image. Scale bars = 100 μm. N = 3 with representative sample shown.</p

Continuous HBO treatment enhances bone degradation.

<p>(A) Bone regeneration during HBO application shows a similar rate of bone degradation before beginning to grow bone at 12 DPA (N = 4 mice, N = 16 digits). Samples were analyzed for bone growth using μCT. Data are normalized to initial 0 DPA bone volume and analyzed using a SS ANOVA algorithm accounting for variation between the individual mice (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140156#sec006" target="_blank">methods</a>). (B) Representative X-ray images of a regenerating P3 digit at 0, 7, 10, 17, and 28 DPA. (C) 14 DPA time point capture showing the P3 regenerative response in a control digit, (from A). (D) 14 DPA time point capture of a representative digit (from A) treated with continuous HBO twice daily showing increased bone degradation. Full time lapse sequence available in supplemental materials. (E) Effect of daily HBO application on osteoclast numbers at 7, 10, and 14 DPA. Results are expressed as mean ± SEM. ^# P<0.05, comparison of control to HBO. NOc/BPm; number of osteoclasts/bone perimeter. (F) Micro-CT scans of the same control sample seen in C and (G) same HBO sample seen in D are pseudo-colored according to trabecular thickness. (*) Asterisk indicates degradation through the proximal os-hole. Color changes indicate bone thickness in μm.</p

Hyperbaric oxygen extends the level of regenerative competency.

<p>A) Mallory trichrome staining at 14 and (B) 28 DPA of a HBO treated digit show proximal degradation of P3 bone and subsequent patterned bone and regeneration of the marrow cavity. Scale bar = 100 μm. Cross-sections of μCT-generated 3D renderings of the sample shown in 4B at 14 DPA (C) and 28 DPA (D) show regeneration of the marrow cavity and patterned distal outgrowth of bone. (E) μCT-generated 3D renderings show exacerbated bone degradation through the P3 joint area that regenerates along with the distal bone. The regenerated P3 shows a defect in the joint at DPA 37 (asterisk), which coincides with an imperfect joint lining in this area (G). (F) Mallory trichrome staining of the regenerated digit at 36 DPA shows imperfect joint patterning in the dorsal region of the joint (G, box 1 in F) when compared to a control P2/P3 joint (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140156#pone.0140156.s004" target="_blank">S4 Fig</a>). Scale bar = 100 μm. Higher magnification images show regenerated, but discontinuous staining of the cartilage joint region when compared to the non-degraded ventral region (H, box 2 in F). Scale bar = 25 μm. (I and J) Picro-Sirius red staining of control digits at 28 DPA shows regenerated woven bone, that (K) do not resolve trabecular woven bone to lamellar bone even after 77 days. Scale bar = 50 μm. (L) In contrast, 28 DPA HBO treated digits show parallel collagen I fibers that are more akin to the lamellar bone seen in the cortical bone stump. Scale bar = 50 μm. Original lamellar bone stump is outlined and indicated by an asterisk, highlighting the boundary with the newly regenerated bone. (N = 3, representative figure shown).</p