Fibroblast Reticular Cells Engineer a Blastema Extracellular Network During Digit Tip Regeneration in Mice

The regeneration blastema which forms following amputation of the mouse digit tip is composed of undifferentiated cells bound together by an organized network of fibers. A monoclonal antibody (ER‐TR7) that identifies extracellular matrix (ECM) fibers produced by fibroblast reticular cells during lymphoid organogenesis was used to characterize the ECM of the digit, the blastema, and the regenerate. Digit fibroblast reticular cells produce an ER‐TR7+ ECM network associated with different tissues and represent a subset of loose connective tissue fibroblasts. During blastema formation there is an upregulation of matrix production that returns to its pre‐existing level and anatomical pattern in the endpoint regenerate. Co‐localization studies demonstrate a strong spatial correlation between the ER‐TR7 antigen and collagen type III (COL3) in histological sections. ER‐TR7 and COL3 are co‐induced in cultured digit fibroblasts following treatment with tumor necrosis factor alpha and a lymphotoxin beta receptor agonist. These results provide an initial characterization of the ECM during digit regeneration and identify a subpopulation of fibroblasts involved in producing the blastema provisional matrix that is remodeled during the regeneration response

Analogous Cellular Contribution and Healing Mechanisms Following Digit Amputation and Phalangeal Fracture in Mice

Regeneration of amputated structures is severely limited in humans and mice, with complete regeneration restricted to the distal portion of the terminal phalanx (P3). Here, we investigate the dynamic tissue repair response of the second phalangeal element (P2) post amputation in the adult mouse, and show that the repair response of the amputated bone is similar to the proximal P2 bone fragment in fracture healing. The regeneration-incompetent P2 amputation response is characterized by periosteal endochondral ossification resulting in the deposition of new trabecular bone, corresponding to a significant increase in bone volume; however, this response is not associated with bone lengthening. We show that cells of the periosteum respond to amputation and fracture by contributing both chondrocytes and osteoblasts to the endochondral ossification response. Based on our studies, we suggest that the amputation response represents an attempt at regeneration that ultimately fails due to the lack of a distal organizing influence that is present in fracture healing

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

Influence of FGF4 on Digit Morphogenesis during Limb Development in the Mouse

AbstractMuch of what we currently know about digit morphogenesis during limb development is deduced from embryonic studies in the chick. In this study, we used ex utero surgical procedures to study digit morphogenesis during mouse embryogenesis. Our studies reveal some similarities; however, we have found considerable differences in how the chick and the mouse autopods respond to experimentation. First, we are not able to induce ectopic digit formation from interdigital cells as a result of wounding or TGFβ-1 application in the mouse, in contrast to what is observed in the chick. Second, FGF4, which inhibits the formation of ectopic digits in the chick, induces a digit bifurcation response in the mouse. We demonstrate with cell marking studies that this bifurcation response results from a reorganization of the prechondrogenic tip of the digit rudiment. The FGF4 effect on digit morphogenesis correlates with changes in the expression of a number of genes, including Msx1, Igf2, and the posterior members of the HoxD cluster. In addition, the bifurcation response is digit-specific, being restricted to digit IV. We propose that FGF4 is an endogenous signal essential for skeletal branching morphogenesis in the mouse. This work stresses the existence of major differences between the chick and the mouse in how digit morphogenesis is regulated and is thus consistent with the view that vertebrate digit evolution is a relatively recent event. Finally, we discuss the relationship between the digit IV bifurcation restriction and the placement of the metapterygial axis in the evolution of the tetrapod limb