Coherent movement of cell layers during wound healing by image correlation spectroscopy.

We have determined the complex sequence of events from the point of injury until reepithelialization in axolotl skin explant model and shown that cell layers move coherently driven by cell swelling after injury. We quantified three-dimensional cell migration using correlation spectroscopy and resolved complex dynamics such as the formation of dislocation points and concerted cell motion. We quantified relative behavior such as velocities and swelling of cells as a function of cell layer during healing. We propose that increased cell volume ( approximately 37% at the basal layer) is the driving impetus for the start of cell migration after injury where the enlarged cells produce a point of dislocation that foreshadows and dictates the initial direction of the migrating cells. Globally, the cells follow a concerted vortex motion that is maintained after wound closure. Our results suggest that cell volume changes the migration of the cells after injury

Coherent Movement of Cell Layers during Wound Healing by Image Correlation Spectroscopy

We have determined the complex sequence of events from the point of injury until reepithelialization in axolotl skin explant model and shown that cell layers move coherently driven by cell swelling after injury. We quantified three-dimensional cell migration using correlation spectroscopy and resolved complex dynamics such as the formation of dislocation points and concerted cell motion. We quantified relative behavior such as velocities and swelling of cells as a function of cell layer during healing. We propose that increased cell volume (∼37% at the basal layer) is the driving impetus for the start of cell migration after injury where the enlarged cells produce a point of dislocation that foreshadows and dictates the initial direction of the migrating cells. Globally, the cells follow a concerted vortex motion that is maintained after wound closure. Our results suggest that cell volume changes the migration of the cells after injury

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

Immunohistochemistry of angiogenesis mediators before and after pulsed dye laser treatment of angiomas.

Tissue effects of vascular lesion laser treatment are incompletely understood. Injury caused by pulsed dye laser (PDL) treatment may result in altered expression of mediators associated with angiogenesis.Eight human subjects had one angioma treated with PDL (7 mm, 1.5 millisecond pulse duration, 9 J/cm(2), cryogen spray cooling of 30 millisecond with a 30 millisecond delay). One week later, three biopsies were taken: normal skin, untreated angioma, angioma post-PDL. Tissue was frozen and sections processed for immunohistochemistry staining of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), matrix metalloproteinase 9 (MMP-9), and angiopoietin 2 (ANG-2). Images were graded in a blinded fashion by a board certified dermatopathologist.There were no clear trends in VEGF expression in the epidermis, dermis, or endothelial cells. As compared to normal skin, angiomas demonstrated the following: bFGF was decreased in the epidermis; MMP-9 was decreased or unchanged in the epidermis and increased in the endothelial cells; ANG-2 was increased in the endothelial cells. When comparing normal skin to angiomas + PDL, bFGF was decreased in the epidermis and increased in the dermis; MMP-9 was decreased or unchanged in the epidermis; ANG-2 was again increased in the endothelial cells. Comparison of staining in angioma to angioma + PDL samples revealed increased dermal bFGF expression.Alterations in angiogenesis mediators were noted after PDL. Angiogenesis mediator changes associated with PDL treatment differed from those previously reported for incisional biopsies. This pilot study can guide future work on laser-induced alterations in vascular lesions and such information may ultimately be used to optimize treatment outcomes

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