We conducted a systematic study on two-photon excited fluorescence (TPEF) of hemoglobin using the near transform-limited and Gaussian-shaped femtosecond pulse sources. We found that the two-photon action cross section of hemoglobin drops over 2 orders of magnitude in the wavelength range from 550 to 800 nm, while the spectral and temporal characteristics of hemoglobin TPEF are insensitive to the change of excitation wavelength. In particular, our new findings showed that the hemoglobin fluorescence could be excited with sufficient efficiency using a conventional Ti:sapphire laser tuned at the wavelength close to 700 nm. With the employment of a time-resolved detection method, we demonstrated that the TPEF signals of hemoglobin excited by a Ti:sapphire laser could be clearly differentiated from other nonlinear signals presented within the living biological tissues, indicating that a standard TPEF microscope can become a routine tool for in vivo label-free microangiography imaging.

BACKGROUND: The host response to tissue injury requires a complex interplay of diverse cellular, humoral, and connective tissue elements. Fibroblasts participate in this process by proliferating within injured sites and contributing to scar formation and the longterm remodeling of damaged tissue. Fibroblasts present in areas of tissue injury generally have been regarded to arise by recruitment from surrounding connective tissue; however this may not be the only source of these cells. MATERIALS AND METHODS: Long-term culture of adherent, human, and murine leukocyte subpopulations was combined with a variety of immunofluorescence and functional analyses to identify a blood-borne cell type with fibroblast-like properties. RESULTS: We describe for the first time a population of circulating cells with fibroblast properties that specifically enter sites of tissue injury. This novel cell type, termed a "fibrocyte," was characterized by its distinctive phenotype (collagen+/vimentin+/CD34+), by its rapid entry from blood into subcutaneously implanted wound chambers, and by its presence in connective tissue scars. CONCLUSIONS: Blood-borne fibrocytes contribute to scar formation and may play an important role both in normal wound repair and in pathological fibrotic responses.

The venous architecture of the integument and the underlying deep tissues was studied in six total-body human fresh cadavers and a series of isolated regional studies of the limbs and torso. A radiopaque lead oxide mixture was injected, and the integument and deep tissues were dissected and radiographed. The sites of the venous perforators were plotted and traced to their underlying parent veins that accompany the source (segmental) arteries. A series of cross-sectional studies were made in one subject to illustrate the course of the perforators between the integument and the deep tissues. The veins were dissected under magnification to identify the site and orientation of the valves. Results revealed a large number of valveless (oscillating) veins within the integument and deep tissues that link adjacent valved venous territories and allow equilibration of flow and pressure throughout the tissue. Where choke arteries define the arterial territories, they are matched by boundaries of oscillating veins in the venous studies. The venous architecture is a continuous network of arcades that follow the connective-tissue framework of the body. The veins converge from mobile to fixed areas, and they "hitchhike" with nerves. The venous drainage mirrors the arterial supply in the deep tissues and in most areas of the integument in the head, neck, and torso. In the limbs, the stellate pattern of the venous perforators is modified by longitudinal channels in the subdermal network. However, when an island flap is raised, these longitudinal channels are disconnected, and once again the arterial and venous patterns match. Our venous studies add strength to the angiosome concept. Where source arteries supply a composite block of tissue, we have demonstrated radiologically and by microdissection that the branches of these arteries are accompanied by veins that drain in the opposite direction and return to the same locus. Hence each angiosome consists of matching arteriosomes and venosomes. The clinical implications of these results are discussed with particular reference to the design of flaps, the delay phenomenon, venous free flaps, the pathogenesis of flap necrosis, the "muscle pump," varicose veins, and venous ulceration.

Human WM9 melanoma cells, previously shown to be devoid of PDGF expression, were stably transfected with a PDGF-B cDNA under the transcriptional control of a cytomegalovirus promoter. Northern blot analysis revealed high expression of an mRNA of the expected size in the PDGF-B-transfected cells. Synthesis and secretion of PDGF-BB was confirmed by immunoprecipitation. Furthermore, conditioned medium from PDGF-B-transfected cells contained a mitogenic activity for fibroblasts. For analysis of tumor growth in vivo, cells of each type were injected subcutaneously into BALB/c nu/nu mice. Tumors from mice injected with WM9 cells transfected with the vector only contained large necrotic areas; only scant blood vessels with narrow lumina were observed. No connective tissue was present. In the tumors from PDGF-B-transfected WM9 cells, nests of tumor were divided by connective tissue septa. An abundance of blood vessels was observed in the connective tissue septa and within the tumor cell nests. There was a complete absence of necrosis in these tumors. The present results suggest that tumor-derived PDGF-BB is a potent mediator of connective tissue stroma formation. The connective tissue framework that is generated in response to PDGF-BB may form a solid support for newly formed blood vessels and, thereby, facilitate the formation of a functional vascular system in the tumor.

If hyaline cartilage is explanted to the chick chorioallantoic membrane, it resists invasion by vascularized mesenchyme of the host. This resistance is diminished if the tissue is extracted with relatively low concentrations (1.0 M) of guanidine HCl. The extracts contain antiproteolytic activity. This molarity of guanidine HCl extracts only small amounts of the major structural components of cartilage extracellular matrix. It, therefore, seems reasonable to suggest that hyaline cartilage is both avascular and resistant to invasion because it contains extractable inhibitors of invasion, perhaps in the form of proteinase inhibitors.

The behavior and role of blood vessels and blood-borne cells in the process of the remodeling of the periodontal ligament (PDL) incident to experimental tooth movement was studied in rats. Particular interest was focused on areas of tension and of pressure with frontal bone resorption but without overt hyalinization. An increase of vascular activity occurred in the above mentioned situations. Extensive breakdown of collagen was observed in pressure areas with frontal resorption and in areas of tension concomitant with vascular invasion. Two patterns of fiber and bone remodeling were seen in areas of tension: intense vascular activity within the periodontal membrane and intense vascular activity inside the alveolar bone. Macrophages occurred consistently near blood vessels both in areas of tension and in areas of resorption. These are multipotent cells that obviously influence the remodeling process.

An adequate revascularization is crucial for islet survival and function after transplantation. Previous studies have suggested that islet revascularization is concluded within 14 days after transplantation. We investigated if the vascular density of transplanted islets and endogenous pancreatic islets differs. Cultured islets were syngeneically transplanted into the kidney, liver, or spleen of C57BL/6 mice. One month later, the graft-bearing organ was removed, and histological specimens were prepared and stained for endothelium with the lectin Bandeiraea simplicifolia. Pancreata from nontransplanted control animals were prepared similarly. Uniform staining of endothelium within the grafts and endogenous islets was obtained. The vascular density was markedly decreased in transplanted islets at all implantation sites, but preferentially in islets implanted into the spleen. The vascular density in the connective tissue surrounding the transplanted islets was very high compared with that of graft intra-islet capillaries. A much lower vascular density was detected in connective tissue surrounding implanted microspheres of a size similar to the islets, which suggests that the islets per se induced blood vessel formation in their vicinity. We conclude that the vascular density in revascularized transplanted islets is markedly decreased compared with endogenous islets. This has potential implications for islet graft metabolism and function.

We investigated the hypothesis that keratinocyte-produced platelet-derived growth factor-AA (PDGF-AA) is involved in epidermal-dermal interactions and that PDGF-AA is an important mediator of the temporal and spatial events of tissue repair. Retroviral-mediated gene transfer was used to introduce the gene encoding human PDGF-A into cultures of human diploid keratinocytes. Genetic modification boosted the endogenous in vitro level of PDGF-AA secretion by over 300 fold. When PDGF-secreting cells were transplanted as epithelial sheets to athymic mice, modified keratinocytes underwent terminal differentiation and generated a stratified epithelium comparable to unmodified cells. Seven days after grafting the newly synthesized connective tissue layer subjacent to the PDGF-A-modified grafts was significantly thicker, was rich in mononuclear cells and fibroblasts, and had increased numbers of blood vessels when compared to control grafts of unmodified cells. These results suggest that PDGF-AA secreted by the epidermis is an important mediator of epithelial-mesenchymal interactions and helps to promote growth and vascularization of the underlying dermal tissue. Further, these data demonstrate the feasibility of using genetically modified cells to modulate tissue regeneration.

Endoneurial capillary abnormalities have been assessed quantitatively in sural nerve biopsies from diabetic patients with different syndromes of sensory polyneuropathy: chronic painful neuropathy, newly presenting painful neuropathy, and painless neuropathy associated with neurotrophic foot ulceration. Comparisons were made with age-matched nondiabetic control subjects. The diabetic groups showed no abnormality in capillary density or mean endoneurial area per fascicle. Compared with control subjects, all diabetic patients had an increase in mean capillary diameter, capillary wall thickness, and outer tunic (basement membrane and pericytes) thickness. The increase in wall thickness was most pronounced in patients with painless neuropathy (200%) and less marked in similar patients with painful neuropathy (100%). The pericyte volume fraction of the outer tunic was reduced in all diabetic patients, implying that basement membrane hypertrophy and reduplication were responsible for outer tunic thickening. There was evidence of endothelial cell hyperplasia rather than hypertrophy. There was a correlation between the degree of basement membrane thickening and the severity of myelinated fiber abnormality assessed neurophysiologically and morphologically. This study shows a link between the degree of endoneurial capillary basement membrane thickening, the type of neuropathology, and the clinical expression of neuropathy in diabetes mellitus.

The authors compared the clinical results obtained in gingival recession correction treatment using free gingival and bilaminar connective subpedicle grafts. 35 patients were treated with free gingival grafts (Group A) and 35 with subpedicle grafts (Group B). Class I and II Miller gingival recessions were chosen for treatment by the 2 procedures. The degree of gingival recession (GR), keratinized tissue (KT) and the exposed root surface area (ERSA) were measured preoperatively and again 5 years post-surgery. Bilaminar connective grafting showed better results in reducing the amount of GR while both techniques significantly increased the width of KT (p > 0.05). The mean % of root coverage obtained in patients in group A was 53.19%+/- 21.48, whereas for the group B, 85.23%+/- 17.86 of exposed root surface was covered post surgical intervention (p < 0.001). In group A, only 3 patients (8.75%) showed a complete resolution of gingival recession after treatment, whereas in group B, 17 subjects (48.57%) presented with complete coverage. On the basis of these results, the authors conclude that the subpedicle graft promises better results in the coverage of exposed root surfaces when compared with the free gingival graft.