Wound healing and blastema formation in regenerating digit tips of adult mice

AbstractAmputation of the distal region of the terminal phalanx of mice causes an initial wound healing response followed by blastema formation and the regeneration of the digit tip. Thus far, most regeneration studies have focused in embryonic or neonatal models and few studies have examined adult digit regeneration. Here we report on studies that include morphological, immunohistological, and volumetric analyses of adult digit regeneration stages. The regenerated digit is grossly similar to the original, but is not a perfect replacement. Re-differentiation of the digit tip occurs by intramembranous ossification forming a trabecular bone network that replaces the amputated cortical bone. The digit blastema is comprised of proliferating cells that express vimentin, a general mesenchymal marker, and by comparison to mature tissues, contains fewer endothelial cells indicative of reduced vascularity. The majority of blastemal cells expressing the stem cell marker SCA-1, also co-express the endothelial marker CD31, suggesting the presence of endothelial progenitor cells. Epidermal closure during wound healing is very slow and is characterized by a failure of the wound epidermis to close across amputated bone. Instead, the wound healing phase is associated with an osteoclast response that degrades the stump bone allowing the wound epidermis to undercut the distal bone resulting in a novel re-amputation response. Thus, the regeneration process initiates from a level that is proximal to the original plane of amputation

High quality RNA isolation from Aedes aegypti midguts using laser microdissection microscopy

Background: Laser microdissection microscopy (LMM) has potential as a research tool because it allows precise excision of target tissues or cells from a complex biological specimen, and facilitates tissue-specific sample preparation. However, this method has not been used in mosquito vectors to date. To this end, we have developed an LMM method to isolate midgut RNA using Aedes aegypti

Development and regeneration of the neonatal digit tip in mice

AbstractThe digit tips of children and rodents are known to regenerate following amputation. The skeletal structure that regenerates is the distal region of the terminal phalangeal bone that is associated with the nail organ. The terminal phalanx forms late in gestation by endochondral ossification and continues to elongate until sexual maturity (8 weeks of age). Postnatal elongation at its distal end occurs by appositional ossification, i.e. direct ossification on the surface of the terminal phalanx, whereas proximal elongation results from an endochondral growth plate. Amputation through the middle of the terminal phalanx regenerates whereas regenerative failure is observed following amputation to remove the distal 2/3 of the bone. Regeneration is characterized by the formation of a blastema of proliferating cells that appear undifferentiated and express Bmp4. Using chondrogenic and osteogenic markers we show that redifferentiation does not occur by endochondral ossification but by the direct ossification of blastema cells that form the rudiment of the digit tip. Once formed the rudiment elongates by appositional ossification in parallel with unamputated control digits. Regenerated digits are consistently shorter than unamputated control digits. Finally, we present a case study of a child who suffered an amputation injury at a proximal level of the terminal phalanx, but failed to regenerate despite conservative treatment and the presence of the nail organ. These clinical and experimental findings expand on previously published observations and initiate a molecular assessment of a mammalian regeneration model

Connective Tissue Fibroblast Properties Are Position-Dependent during Mouse Digit Tip Regeneration

A key factor that contributes to the regenerative ability of regeneration-competent animals such as the salamander is their use of innate positional cues that guide the regeneration process. The limbs of mammals has severe regenerative limitations, however the distal most portion of the terminal phalange is regeneration competent. This regenerative ability of the adult mouse digit is level dependent: amputation through the distal half of the terminal phalanx (P3) leads to successful regeneration, whereas amputation through a more proximal location, e.g. the subterminal phalangeal element (P2), fails to regenerate. Do the connective tissue cells of the mammalian digit play a role similar to that of the salamander limb in controlling the regenerative response? To begin to address this question, we isolated and cultured cells of the connective tissue surrounding the phalangeal bones of regeneration competent (P3) and incompetent (P2) levels. Despite their close proximity and localization, these cells show very distinctive profiles when characterized in vitro and in vivo. In vitro studies comparing their proliferation and position-specific interactions reveal that cells isolated from the P3 and P2 are both capable of organizing and differentiating epithelial progenitors, but with different outcomes. The difference in interactions are further characterized with three-dimension cultures, in which P3 regenerative cells are shown to lack a contractile response that is seen in other fibroblast cultures, including the P2 cultures. In in vivo engraftment studies, the difference between these two cell lines is made more apparent. While both P2 and P3 cells participated in the regeneration of the terminal phalanx, their survival and proliferative indices were distinct, thus suggesting a key difference in their ability to interact within a regeneration permissive environment. These studies are the first to demonstrate distinct positional characteristics of connective tissue cells that are associated with their regenerative capabilities

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

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