Isolation of connective tissue cells from the P2 and P3 regions.

<p>A, B) Histological section of the P2 (A) and P3 (B) bone after dissection. Loose connective tissue is attached to the surface of the intact bone. C, D) After enzymatic digestion the majority of loose connective tissue was digested off the bone while tissues within the bone marrow are still present. E) DiI labeled cells (red) within the connective tissue (CT) dorsal to the P3 skeletal element (B) are clustered in the amputation stump 2 days post-injection. BM, bone marrow. F) Blastema stage regenerate at 13 DPA showing DiI labeled cells (red) scattered throughout the blastema but not overlapping with Osteocalcin immunohistochemical labeling (green) to identify regenerating osteoblasts. The blastema is contiguous with the connective tissue (CT) and bone (B) of the stump. Scale bars  = 200 µm.</p

P3 cells proliferate in the blastema.

<p>A–C) GFP and Ki67 co-immunohistochemical reveal P3 isolated cells engraft and proliferate. A) GFP labeled P3 cells are identified within the digit. B) Ki-67, a marker for proliferation, is expressed in engrafted and endogenous P3 cells. C) Merged GFP and Ki67 expression identifies a fraction of engrafted P3 cells that are proliferating. Double labeled cells shown in C are indicated with arrows in the image set. GFP- red, Ki67- green, DAPI nuclear stain- blue. Scale bar  = 50 µm. D) Quantification of the proliferation indices of P2 and P3 engrafted cells compared to endogenous neighboring cells. Left: In unamputated digits 17 days post engraftment, P3 cells proliferated at a rate similar to neighboring control cells, whereas P2 cells are non-proliferative. Right: P3 cells participating in blastema formation at 10 DPA display a proliferation index that was lower than neighboring endogenous cells but significantly higher than P2 cells within the blastema. All chart values are expressed as means ± SEM. The proliferative index of P3 cells is significantly greater than P2 cells in both studies (**, p-value <0.005).</p

Position-specific characterization of P2 and P3 cells.

<p>A) Hematoxylin and eosin stained sections of skin equivalents generated from P2 cells co-cultured with human keratinocytes showing the induction of stratified sheets of differentiating epidermis. B) Hematoxylin and eosin stained sections of skin equivalents generated from P3 cells co-cultured with human keratinocytes showing the induction of aggregate structures that display keratosis similar to that observed from cultures of nail matrix epidermis. C) Whole mount preparations of P3 skin equivalents stained with Rhodanile blue showing the aggregation of keratinocytes induced by P3 cells. D) Collagen gels seeded with P2 cells (top) resulted in a contracted gel phenotypes whereas gels seeded with P3 cells did not display a contraction response. E) Area measurements of collagen gels show that P2 cells induced a cell density dependent contraction response, whereas P3 cells failed to contract the gel at similar seeding densities. F) Co-cultures of P2 cells with human keratinocytes enhanced the contraction response which was measured by determining the width of the collagen gel. Keratinocyte co-cultured with P3 cells induced a contraction response that was of a similar magnitude. All chart measurements are mean ± SEM (n = 3). A and B, scale bar  = 100 µm; C, scale bar  = 300 µm.</p

P3 cells are regeneration competent after expansion in vitro.

<p>A) LacZ positive P3 cells were injected into the digit tip of SCID mice 1 day prior to amputation and collected at 10 DPA when the regenerate at the blastema stage. LacZ positive cells are present at the injection site in the dorsal connective tissue (*) and are scattered throughout the blastema. B) During the differentiation stage (16 DPA) LacZ positive P3 cells are primarily found in the regenerating connective tissue with small clusters of cells present within the trabeculae of the regenerating bone (arrows). C–E) GFP+ human breast cancer cells injected into P3 connective tissue prior to amputation remained aggregated in the regeneration stump and did not enter the blastema. GFP positive cells (C) shown aggregated in the stump of a 16 DPA regenerate indicating that they survive engraftment. Immunohistochemical co-staining for the endothelial marker vWF (D, E) indicates that these cells differentiate in situ without participating in the regenerative response. A and B, scale bar  = 200 µm; C–E, scale bar  = 100 µm.</p

Flow cytometric profiling and RT-PCR analysis of P2 and P3 cells.

<p>A) Flow cytometry analysis identify a similar signature for P2 (bottom panels) and P3 cells (top panels). Analyses were performed in series on the same samples, P1-P4 denote the order of the measurements. In the scatter plots, both cell lines are positive with a single peak for Sca-1, CD29, V-CAM and negative for CD45, C-kit, CD49f, CD104 and CD34. This signature is similar to other characterized MSC lines. B) RT-PCR results from both P3 and P2 cells expanded for 30 days compared with established cell lines. Digit regeneration marker genes <i>Bmp4</i> and <i>Msx1</i> are stably expressed in P3 cells and variably expressed in P2 cells. Stem cell marker genes are differentially expressed in P2 and P3 cells. <i>Oct4</i> is not expressed in P2 and P3 cells. <i>Rex1</i> is minimally expressed in P2 and P3 cells. <i>Sox2</i> is minimally expressed in P2 cells and in early passage P3 cells but strongly expressed in the P3 cell line.</p

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