RESUMO
PDGF has been implicated as one of the principal mitogens involved in cutaneous wound healing. While it has been previously reported that both platelets and monocytes are a source of PDGF in human dermal wound repair, the production of PDGF by human keratinocytes has not yet been described. In this manuscript, we report the production of PDGF by cultured human keratinocytes. Both PDGF A and B chain mRNA can be detected in cultured cells. While only PDGF-AA polypeptide is found in significant levels in keratinocyte-conditioned culture media, all three PDGF isoforms (AA, AB, and BB) are present in detergent-solubilized cell extracts. No evidence of PDGF receptor expression was observed in cultured keratinocytes when analyzed for either mRNA levels or polypeptide expression, suggesting that PDGF does not play an autocrine role in keratinocyte growth. Analysis of cryosections of human cutaneous wounds by immunostaining for PDGF showed that both PDGF A and B chain is constitutively expressed in normal epidermis, as well as in newly reconstituted wound epidermis. No evidence for PDGF receptor polypeptide expression in the epidermis was detected by immunostaining of cryosections.
Assuntos
Queratinócitos/metabolismo , Fator de Crescimento Derivado de Plaquetas/biossíntese , Pele/metabolismo , Ferimentos e Lesões/fisiopatologia , Anticorpos Monoclonais , Biópsia , Células Cultivadas , Meios de Cultivo Condicionados , Ensaio de Imunoadsorção Enzimática , Expressão Gênica , Humanos , Técnicas Imunoenzimáticas , Imuno-Histoquímica , Recém-Nascido , Cinética , Substâncias Macromoleculares , Masculino , Fator de Crescimento Derivado de Plaquetas/análise , RNA Mensageiro/análise , RNA Mensageiro/biossíntese , Receptores do Fator de Crescimento Derivado de Plaquetas/análise , Receptores do Fator de Crescimento Derivado de Plaquetas/biossíntese , Receptores do Fator de Crescimento Derivado de Plaquetas/metabolismo , Ferimentos e Lesões/patologiaRESUMO
Percutaneous medical devices are integral in the management and treatment of disease. The space created between the skin and the device becomes a haven for bacterial invasion and biofilm formation and results in infection. We hypothesize that sealing this space via integration of the skin into the device will create a barrier against bacterial invasion. The purpose of this study was to develop an animal model in which the interaction between skin and biomaterials can be evaluated. Porous poly(2-hydroxyethyl methacrylate) [poly(HEMA)] rods were implanted for 7 days in the dorsal skin of C57 BL/6 mice. The porous poly(HEMA) rods were surface-modified with carbonyldiimidazole (CDI) or CDI plus laminin 5; unmodified rods served as control. Implant sites were sealed with 2-octyl cyanoacrylate; corn pads and adhesive dressings were tested for stabilization of implants. All rods remained intact for the duration of the study. There was histological evidence of both epidermal and dermal integration into all poly(HEMA) rods regardless of treatment. This in vivo model permits examination of the implant/skin interface and will be useful for future studies designed to facilitate skin cell attachment where percutaneous devices penetrate the skin.
Assuntos
Materiais Biocompatíveis , Modelos Animais , Pele , Animais , Camundongos , Poli-Hidroxietil Metacrilato , Absorção CutâneaRESUMO
In a study initially designed to evaluate reinnervation of human cutaneous wounds using an antibody to the neuroneal marker protein gene product (PGP) 9.5, we observed marked immunostaining of cells with morphologic features of fibroblasts in the wounds. PGP 9.5 has recently been shown to be an important enzyme in the highly conserved ubiquitin system of proteolysis. Because the ubiquitin system is known to play an important role in regulating the cell cycle, the presence of PGP 9.5 in cells at a wound site was of considerable interest. Our objectives were to clarify the time frame for the appearance of PGP 9.5 and ubiquitin in wounds, to verify that PGP 9.5 is produced by wound fibroblasts, and to evaluate a potential role for these proteins in the tissue repair process. Standard incisional human wounds were stained with antibodies specific for PGP 9.5 and ubiquitin. At 7 d, stellate cells with morphologic features of fibroblasts stained for PGP 9.5, whereas earlier wounds were generally negative. In 14 and 21 d incised wounds and in chronic granulation tissue from nonhealing ulcers there was strong cellular staining for PGP 9.5 and for ubiquitin. These stellate cells also showed expression of mRNA for PGP 9.5 by reverse transcriptase-polymerase chain reaction in situ hybridization. PGP 9.5 was detected in cultured fibroblasts both by reverse transcriptase-polymerase chain reaction and by northern blot analysis. Confocal microscopy showed colocalization of antibodies to PGP 9.5 and prolyl-4-hydroxylase (a fibroblast marker) as well as colocalization of PGP 9.5 and the platelet derived growth factor beta receptor. We conclude that ubiquitin and PGP 9.5 were expressed by fibroblasts during the granulation tissue and remodeling phases wound healing. The mRNA for PGP 9.5 was demonstrated in stellate cells in chronic wounds and in fibroblasts in culture. The appearance of these degradative proteins in later wounds suggests a downregulation function in the wound healing response.
Assuntos
Fibroblastos/química , Tioléster Hidrolases/biossíntese , Ferimentos e Lesões/patologia , Apoptose , Northern Blotting , DNA Complementar/análise , Fibroblastos/ultraestrutura , Expressão Gênica , Técnicas Genéticas , Humanos , Hibridização In Situ , Microscopia Confocal , Microscopia Eletrônica , Proteínas do Tecido Nervoso/biossíntese , Reação em Cadeia da Polimerase , RNA Mensageiro/análise , Receptores do Fator de Crescimento Derivado de Plaquetas , Tioléster Hidrolases/genética , Fatores de Tempo , Ubiquitina Tiolesterase , Ubiquitinas/análiseRESUMO
Cutaneous sensory nerves mediate inflammation and wound healing by the release of neuropeptides such as substance P. Neutral endopeptidase is a cell surface enzyme that degrades substance P and thereby terminates its biologic actions. The distribution of neutral endopeptidase in normal skin and wounded human skin, however, has not been examined. The objectives of this study were to evaluate neutral endopeptidase expression in wounded and unwounded skin as well as in cells derived from human skin. Neutral endopeptidase was strikingly localized in normal skin by immunohistochemistry to keratinocytes of the epidermal basal layer, to hair follicles, eccrine and sebaceous glands as well as to endothelium of blood vessels and to large nerves. Standard incisional human wounds were studied at several time points between 1 h and 28 d after wounding. Staining for neutral endopeptidase was noted in the wound bed 6 h after wounding. In contrast to normal skin, staining of all the epidermal cell layers was noted in the migrating tongue of epithelium in l d wounds. Similar full-thickness staining was noted in 3 d and 7 d wounds in all layers of the new wound epithelium and in a "transition epithelium" near the wound edge. By 28 d post wounding neutral endopeptidase staining again was detected only in the basal layer of the epidermis. Neutral endopeptidase mRNA was detected in normal skin and wounds as well as cultured keratinocytes, fibroblasts and endothelial cells. Neutral endopeptidase enzymatic bioactivity was demonstrated in cultured keratinocytes. While it is known that several metalloproteinases important to tissue repair are produced by keratinocytes, this is the first evidence that keratinocytes produce neutral endopeptidase. Neutral endopeptidase may terminate the proinflammatory and mitogenic actions of neuropeptides in normal skin and wounds.
Assuntos
Neprilisina/biossíntese , Pele/enzimologia , Ferimentos e Lesões/enzimologia , Idoso , Anticorpos/imunologia , Especificidade de Anticorpos , Western Blotting , Corantes , Contaminação de Medicamentos , Endotélio Vascular/citologia , Feminino , Fibroblastos/enzimologia , Humanos , Imuno-Histoquímica , Queratinócitos/enzimologia , Queratinócitos/metabolismo , Queratinas/imunologia , Masculino , Microcirculação , Pessoa de Meia-Idade , Neprilisina/genética , Neprilisina/imunologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Pele/químicaRESUMO
Immunohistochemistry (IHC) is a valuable tool for labeling structures in tissue samples. Quantification of immunolabeled structures using traditional approaches has proved to be difficult. Manual counts of IHC-stained structures are inherently biased, require multiple observers, and generate qualitative data. Stereological methods provide accurate quantification but are complex and labor-intensive when staining must be compared among large numbers of samples. In an effort to quickly, objectively, and reproducibly quantify cutaneous innervation in a large number of counterstained tissue sections, we developed a color subtractive-computer-assisted image analysis (CS-CAIA) system. To develop and test the CS-CAIA method, tissue sections of diabetic (db/db) mouse skin and their wild-type (db/-) littermates were stained by IHC for the neural marker PGP 9.5. The brown-red PGP 9.5 peroxidase stain was colorimetrically isolated through a scripted process of color background removal. The remaining stain was thresholded and binarized for computer determination of nerve profile counts (number of stained regions), area fraction (total area of nerve profiles per unit area of tissue), and area density (total number of nerve profiles per unit area of tissue). Using CS-CAIA, epidermal nerve profile counts, area fraction, and area density were significantly lower in db/db compared to db/- mice.
Assuntos
Diabetes Mellitus/patologia , Pele/inervação , Animais , Diabetes Mellitus/genética , Processamento de Imagem Assistida por Computador , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos C57BL , Camundongos MutantesRESUMO
Percutaneous medical devices remain susceptible to infection and failure. We hypothesize that healing of the skin into the percutaneous device will provide a seal, preventing bacterial attachment, biofilm formation, and subsequent device failure. Porous poly(2-hydroxyethyl methacrylate) [poly(HEMA)] with sphere-templated pores (40 microm) and interconnecting throats (16 microm) were implanted in normal C57BL/6 mice for 7, 14, and 28 days. Poly(HEMA) was either untreated, keeping the surface nonadhesive for cells and proteins, or modified with carbonyldiimidazole (CDI) or CDI reacted with laminin 332 to enhance adhesion. No clinical signs of infection were observed. Epidermal and dermal response within the poly(HEMA) pores was evaluated using light and transmission electron microscopy. Cells (keratinocytes, fibroblasts, endothelial cells, inflammatory cells) and basement membrane proteins (laminin 332, beta4 integrin, type VII collagen) could be demonstrated within the poly(HEMA) pores of all implants. Blood vessels and dermal collagen bundles were evident in all of the 14- and 28-day implants. Fibrous capsule formation and permigration were not observed. Sphere-templated polymers with 40 microm pores demonstrate an ability to recapitulate key elements of both the dermal and the epidermal layers of skin. Our morphological findings indicate that the implant model can be used to study the effects of biomaterial pore size, pore interconnect (throat) size, and surface treatments on cutaneous biointegration. Further, this model may be used for bacterial challenge studies.
Assuntos
Derme/efeitos dos fármacos , Derme/fisiologia , Epiderme/efeitos dos fármacos , Epiderme/fisiologia , Implantes Experimentais , Metacrilatos/química , Metacrilatos/farmacologia , Animais , Derme/citologia , Derme/ultraestrutura , Células Epidérmicas , Epiderme/ultraestrutura , Imuno-Histoquímica , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Macrófagos/ultraestrutura , Camundongos , Camundongos Endogâmicos C57BL , Fenótipo , Porosidade/efeitos dos fármacos , Fixação de TecidosRESUMO
Platelet-derived growth factor (PDGF) is a polypeptide growth factor which has been implicated as a major mitogen involved in wound healing. The PDGF appears to promote periodontal regeneration; however, its distribution in gingival tissues is not known and how it participates in gingival wound healing is unclear. Using highly specific antibodies we have studied the distribution of PDGF A and B chains and alpha- and beta-PDGF receptors in healing human gingival wounds. Wounds were created by making a 0.75 mm deep incision in the papilla and healthy gingiva and biopsies were obtained from the same site after 8 h and 1, 3, 7, 14 and 21 d. Frozen sections were immunostained with affinity purified antibodies. The results showed that both epithelium and fibrin clot manifested positive immunostaining for anti-PDGF-A and B-chain antibodies. Staining was present in unwounded and wounded epithelia, and in the fibrin clot it appeared to be more intense for the PDGF-A chain. Blood vessels in connective tissue were also positive while other areas were largely negative. No significant staining was detectable in healthy tissues for anti-PDGF-alpha or -beta receptor antibodies. However, the wound site began to manifest positive immunostaining fro anti-beta-receptor antibody after 3 d of healing, became maximal at 7 d, and then decreased. Our data indicate, but do not prove, that gingival epithelium may be a source of PDGF A and B chains and that the A chain may have a more prominent role to play during early stages of healing. Expression of PDGF beta-receptor appears later at the wound site, indicating that the PDGF B isomer may regulate later wound healing events.