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1.
Wound Repair Regen ; 24(2): 373-83, 2016 03.
Artículo en Inglés | MEDLINE | ID: mdl-26748963

RESUMEN

Biofilms have been implicated in delayed wound healing, although the mechanisms by which biofilms impair wound healing are poorly understood. Many species of bacteria produce exotoxins and exoenzymes that may inhibit healing. In addition, oxygen consumption by biofilms and by the responding leukocytes, may impede wound healing by depleting the oxygen that is required for healing. In this study, oxygen microsensors to measure oxygen transects through in vitro cultured biofilms, biofilms formed in vivo within scabs from a diabetic (db/db) mouse wound model, and ex vivo human chronic wound specimens was used. The results showed that oxygen levels within mouse scabs had steep gradients that reached minima ranging from 17 to 72 mmHg on live mice and from 6.4 to 1.1 mmHg on euthanized mice. The oxygen gradients in the mouse scabs were similar to those observed for clinical isolates cultured in vitro and for human ex vivo specimens. To characterize the metabolic activities of the bacteria in the mouse scabs, transcriptomics analyses of Pseudomonas aeruginosa biofilms associated with the db/db mice wounds was performed. The results demonstrated that the bacteria expressed genes for metabolic activities associated with cell growth. Interestingly, the transcriptome results also indicated that the bacteria within the wounds experienced oxygen-limitation stress. Among the bacterial genes that were expressed in vivo were genes associated with the Anr-mediated hypoxia-stress response. Other bacterial stress response genes highly expressed in vivo were genes associated with stationary-phase growth, osmotic stress, and RpoH-mediated heat shock stress. Overall, the results supported the hypothesis that bacterial biofilms in chronic wounds promote chronicity by contributing to the maintenance of localized low oxygen tensions, through their metabolic activities and through their recruitment of cells that consume oxygen for host defensive processes.


Asunto(s)
Biopelículas/crecimiento & desarrollo , Técnicas Biosensibles , Diabetes Mellitus Experimental/metabolismo , Oxígeno/metabolismo , Infecciones por Pseudomonas/microbiología , Transcriptoma/fisiología , Infección de Heridas/metabolismo , Animales , Diabetes Mellitus Experimental/patología , Modelos Animales de Enfermedad , Humanos , Ratones , Presión Osmótica , Infecciones por Pseudomonas/patología , Cicatrización de Heridas/fisiología , Infección de Heridas/patología
2.
Wound Repair Regen ; 20(3): 342-52, 2012.
Artículo en Inglés | MEDLINE | ID: mdl-22564229

RESUMEN

Bacterial biofilm has been shown to play a role in delaying wound healing of chronic wounds, a major medical problem that results in significant health care burden. A reproducible animal model could be very valuable for studying the mechanism and management of chronic wounds. Our previous work showed that Pseudomonas aeruginosa (PAO1) biofilm challenge on wounds in diabetic (db/db) mice significantly delayed wound healing. In this wound time course study, we further characterize the bacterial burden, delayed wound healing, and certain aspects of the host inflammatory response in the PAO1 biofilm-challenged db/db mouse model. PAO1 biofilms were transferred onto 2-day-old wounds created on the dorsal surface of db/db mice. Control wounds without biofilm challenge healed by 4 weeks, consistent with previous studies; none of the biofilm-challenged wounds healed by 4 weeks. Of the biofilm-challenged wounds, 64% healed by 6 weeks, and all of the biofilm-challenged wounds healed by 8 weeks. During the wound-healing process, P. aeruginosa was gradually cleared from the wounds while the presence of Staphylococcus aureus (part of the normal mouse skin flora) increased. Scabs from all unhealed wounds contained 10(7) P. aeruginosa, which was 100-fold higher than the counts isolated from wound beds (i.e., 99% of the P. aeruginosa was in the scab). Histology and genetic analysis showed proliferative epidermis, deficient vascularization, and increased inflammatory cytokines. Hypoxia inducible factor expression increased threefold in 4-week wounds. In summary, our study shows that biofilm-challenged wounds typically heal in approximately 6 weeks, at least 2 weeks longer than nonbiofilm-challenged normal wounds. These data suggest that this delayed wound healing model enables the in vivo study of bacterial biofilm responses to host defenses and the effects of biofilms on host wound healing pathways. It may also be used to test antibiofilm strategies for treating chronic wounds.


Asunto(s)
Biopelículas/crecimiento & desarrollo , Diabetes Mellitus Experimental/microbiología , Infecciones por Pseudomonas/microbiología , Pseudomonas aeruginosa/crecimiento & desarrollo , Cicatrización de Heridas , Animales , Diabetes Mellitus Experimental/patología , Masculino , Ratones , Ratones Endogámicos NOD , Infecciones por Pseudomonas/patología , Factores de Tiempo
3.
Wound Repair Regen ; 18(5): 467-77, 2010.
Artículo en Inglés | MEDLINE | ID: mdl-20731798

RESUMEN

Chronic wounds are a major clinical problem that lead to considerable morbidity and mortality. We hypothesized that an important factor in the failure of chronic wounds to heal was the presence of microbial biofilm resistant to antibiotics and protected from host defenses. A major difficulty in studying chronic wounds is the absence of suitable animal models. The goal of this study was to create a reproducible chronic wound model in diabetic mice by the application of bacterial biofilm. Six-millimeter punch biopsy wounds were created on the dorsal surface of diabetic (db/db) mice, subsequently challenged with Pseudomonas aeruginosa (PAO1) biofilms 2 days postwounding, and covered with semiocclusive dressings for 2 weeks. Most of the control wounds were epithelialized by 28 days postwounding. In contrast, none of biofilm-challenged wounds were closed. Histological analysis showed extensive inflammatory cell infiltration, tissue necrosis, and epidermal hyperplasia adjacent to challenged wounds-all indicators of an inflammatory nonhealing wound. Quantitative cultures and transmission electron microscopy demonstrated that the majority of bacteria were in the scab above the wound bed rather than in the wound tissue. The model was reproducible, allowed localized cutaneous wound infections without high mortality, and demonstrated delayed wound healing following a biofilm challenge. This model may provide an approach to study the role of microbial biofilms in chronic wounds as well as the effect of specific biofilm therapy on wound healing.


Asunto(s)
Biopelículas/crecimiento & desarrollo , Diabetes Mellitus Experimental/complicaciones , Infecciones por Pseudomonas/microbiología , Pseudomonas aeruginosa/fisiología , Cicatrización de Heridas/fisiología , Infección de Heridas/microbiología , Animales , Enfermedad Crónica , Femenino , Estudios de Seguimiento , Ratones , Proyectos Piloto , Infecciones por Pseudomonas/complicaciones , Infecciones por Pseudomonas/patología , Factores de Tiempo , Infección de Heridas/complicaciones , Infección de Heridas/patología
4.
J Histochem Cytochem ; 57(2): 123-42, 2009 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-18824633

RESUMEN

Subsequent to wounding, keratinocytes must quickly restore barrier function. In vitro wound models have served to elucidate mechanisms of epithelial closure and key roles for integrins alpha6beta4 and alpha3beta1. To extrapolate in vitro data to in vivo human tissues, we used ultrathin cryomicrotomy to simultaneously observe tissue ultrastructure and immunogold localization in unwounded skin and acute human cutaneous wounds. Localization of the beta4 integrin subunit in unwounded skin shows dominant hemidesmosomal association and minor basal keratinocyte lateral filopodic cell-cell expression. After wounding, beta4 dominantly localized to cytokeratin-rich regions (trailing edge hemidesmosomes) and minor association with lamellipodia (leading edge). beta4 colocalizes with alpha3 within filopodia juxtaposed to wound matrix, and increased concentrations of beta4 were found in cytoplasmic vesicles within basal keratinocytes of the migrating tongue. alpha3 integrin subunit dominantly localized to filopodia within basal keratinocyte lateral cell-cell interfaces in unwounded skin and both cell-cell and cell-matrix filopodic interactions in wounded skin. This study indicates that beta4 interacts with the extracellular environment through both stable and transient interactions and may be managed through a different endosomal trafficking pathway than alpha3. alpha3 integrin, despite its ability to respond to alternate ligands after wounding, does so through a single structure, the filopodia.


Asunto(s)
Integrina alfa3/metabolismo , Integrina beta4/metabolismo , Piel/lesiones , Piel/metabolismo , Movimiento Celular , Citoplasma/metabolismo , Epitelio/metabolismo , Epitelio/ultraestructura , Secciones por Congelación , Hemidesmosomas/metabolismo , Humanos , Inmunohistoquímica , Queratinas/metabolismo , Subunidades de Proteína/metabolismo , Seudópodos/metabolismo , Piel/ultraestructura
5.
Bone ; 41(4): 535-42, 2007 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-17693149

RESUMEN

COL27A1 is a member of the collagen fibrillar gene family and is expressed in cartilaginous tissues including the anlage of endochondral bone. To begin to understand its role in skeletogenesis, the temporospatial distributions of its RNA message and protein product, type XXVII collagen, were determined in developing human skeletal tissues. Laser capture microdissection and quantitative reverse-transcription polymerase chain reaction demonstrated that gene expression occurred throughout the growth plate and that it was higher in the resting and proliferative zones than in hypertrophic cartilage. Immunohistochemical analyses showed that type XXVII collagen was most evident in hypertrophic cartilage at the primary ossification center and at the growth plate and that it accumulated in the pericellular matrix. Synthesis of type XXVII collagen overlapped partly with that of type X collagen, a marker of chondrocyte hypertrophy, preceded the transition of cartilage to bone, and was associated with cartilage calcification. Immunogold electron microscopy of extracted ECM components from mouse growth plate showed that type XXVII collagen was a component of long non-banded fibrous structures, filamentous networks, and thin banded fibrils. The timing and location of synthesis suggest that type XXVII collagen plays a role during the calcification of cartilage and the transition of cartilage to bone.


Asunto(s)
Huesos/citología , Huesos/metabolismo , Cartílago/citología , Cartílago/metabolismo , Diferenciación Celular , Colágenos Fibrilares/metabolismo , Esqueleto , Animales , Colágenos Fibrilares/genética , Humanos , Ratones , Microscopía Inmunoelectrónica , ARN Mensajero/genética
6.
J Dermatol Sci ; 48(3): 177-88, 2007 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-17719208

RESUMEN

BACKGROUND: Keratinocyte migration is essential for wound healing and diabetic wound keratinocytes migrate poorly. Keratinocyte migration and anchorage appears to be mediated by laminin-332 (LM-332). Impaired diabetic wound healing may be due to defective LM-332 mediated keratinocyte migration. OBJECTIVE: To evaluate LM-332 expression in diabetic (db/db) and control (db/-) mice and to test LM-332 wound healing effects when applied to mouse wounds. METHODS: LM-332 expression in mouse wounds was evaluated using immunohistochemistry. LM-332 wound healing effects were evaluated by directly applying soluble LM-332, a LM-332 biomaterial, or a control to mouse wounds. Percent wound closure and histology score, based on healing extent, were measured. RESULTS: Precursor LM-332 expression was markedly reduced in db/db when compared to db/- mice. In vitro, soluble LM-332 and LM-332 biomaterial demonstrated significant keratinocyte adhesion. In vivo, soluble LM-332 treated wounds had the highest histology score, but significant differences were not found between wound treatments (p>0.05). No differences in percentage wound closure between treatment and control wounds were found (p>0.05). CONCLUSION: The db/db wounds express less precursor LM-332 when compared to db/-. However, LM-332 application did not improve db/db wound healing. LM-332 purified from keratinocytes was primarily physiologically cleaved LM-332 and may not regulate keratinocyte migration. Application of precursor LM-332 rather than cleaved LM-332 may be necessary to improve wound healing, but this isoform is not currently available in quantities sufficient for testing.


Asunto(s)
Moléculas de Adhesión Celular/uso terapéutico , Complicaciones de la Diabetes/tratamiento farmacológico , Heridas y Lesiones/tratamiento farmacológico , Administración Tópica , Animales , Membrana Basal/metabolismo , Membrana Basal/patología , Moléculas de Adhesión Celular/administración & dosificación , Moléculas de Adhesión Celular/metabolismo , Movimiento Celular/efectos de los fármacos , Movimiento Celular/fisiología , Colágeno/metabolismo , Complicaciones de la Diabetes/metabolismo , Complicaciones de la Diabetes/patología , Integrinas/metabolismo , Queratinocitos/metabolismo , Queratinocitos/patología , Queratinocitos/fisiología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Ratones Mutantes , Cicatrización de Heridas/efectos de los fármacos , Heridas y Lesiones/metabolismo , Heridas y Lesiones/patología , Kalinina
7.
J Biomed Opt ; 11(6): 064002, 2006.
Artículo en Inglés | MEDLINE | ID: mdl-17212525

RESUMEN

Ultrahigh-resolution optical coherence tomography (OCT) was used for noninvasive in vivo evaluation of the wound healing process. Cutaneous wounds were induced by 2.5-mm diameter full-thickness punch biopsies on the dorsal surface of seven mice. OCT imaging was performed to assess the structural characteristics associated with the healing process. The OCT results were compared to corresponding histology. Two automated quantitative analysis routines were implemented to identify the dermal-epidermal junction and segment the OCT images. Hallmarks of cutaneous wound healing such as wound size, epidermal migration, dermal-epidermal junction formation, and differences in wound composition were readily identified on the OCT images. Blister formation was also observed. Preliminary findings suggest OCT is a viable tool to noninvasively monitor wound healing in vivo.


Asunto(s)
Inteligencia Artificial , Aumento de la Imagen/métodos , Interpretación de Imagen Asistida por Computador/métodos , Piel/lesiones , Piel/ultraestructura , Cicatrización de Heridas , Heridas Penetrantes/patología , Algoritmos , Animales , Estudios de Factibilidad , Masculino , Ratones , Reconocimiento de Normas Patrones Automatizadas/métodos , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Técnica de Sustracción
8.
J Biomed Mater Res A ; 74(3): 482-8, 2005 Sep 01.
Artículo en Inglés | MEDLINE | ID: mdl-15983994

RESUMEN

Percutaneous devices are indispensable in modern medicine, yet complications from their use result in significant morbidity, mortality, and cost. Bacterial biofilm at the device exit site accounts for most infections in short-term devices. We hypothesize that advanced biomaterials can be developed that facilitate attachment of skin cells to percutaneous devices, forming a seal to preclude bacterial invasion. To study the skin/biomaterial interface systematically, we first identified biomaterials with physical properties compatible with histological processing of skin. Second, we developed an organ culture system to study skin response to implants. Organ cultures implanted with porous poly(2-hydroxyethyl methacrylate) [poly(HEMA)] or polytetrafluoroethylene (PTFE) could easily be evaluated histologically with preservation of the skin/biomaterial interface. Epithelial cells migrated down the cut edges of the biomaterial in a pattern seen in marsupialization of percutaneous devices in vivo. This in vitro model maintains skin viability and allows histologic evaluation of the skin/biomaterial interface, making this a useful, inexpensive test-bed for studies of epidermal attachment to modified biomaterials.


Asunto(s)
Materiales Biocompatibles , Cateterismo/instrumentación , Adhesión Celular/fisiología , Modelos Biológicos , Piel/metabolismo , Administración Cutánea , Humanos , Recién Nacido , Masculino , Piel/citología
9.
Burns ; 30(1): 57-64, 2004 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-14693087

RESUMEN

A significant impediment to studying hypertrophic scar is the lack of an animal model. We have confirmed similarities between scarring in the female red Duroc pig (FRDP) and human hypertrophic scar and conclude that this model warrants validation. Reports have suggested that the cutaneous nervous system may play a role in hypertrophic scar development and several studies have shown nerve density in hypertrophic scar to be increased. The purpose of this study was to further validate the FRDP model of hypertrophic scar by quantifying nerves in FRDP tissue and comparing the findings to human hypertrophic scar. Wounds of varying depth were created on the backs of two FRDP and tissue samples were harvested at 10 days, 1 month and 5 months post-wounding. Human specimens were obtained from six burn patients. Immunohistochemistry was performed and digital images were captured. Color subtractive computer-assisted image analysis was used to quantify nerve density and nerve area fraction. The results demonstrate that nerve tissue is increased in FRDP scar tissue and is quite similar to that in human hypertrophic scar and to that described in the literature. These data provide additional evidence that the FRDP model may be useful for studying hypertrophic scarring.


Asunto(s)
Cicatriz Hipertrófica/patología , Modelos Animales de Enfermedad , Piel/inervación , Porcinos , Adolescente , Animales , Niño , Femenino , Humanos , Procesamiento de Imagen Asistido por Computador , Técnicas para Inmunoenzimas , Masculino , Persona de Mediana Edad , Fibras Nerviosas/patología , Factores de Tiempo
10.
Plast Reconstr Surg ; 113(3): 953-60, 2004 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-15108888

RESUMEN

The genetically diabetic db/db mouse exhibits symptoms that resemble human type 2 diabetes mellitus, demonstrates delayed wound healing, and has been used extensively as a model to study the role of therapeutic topical reagents in wound healing. The purpose of the authors' study was to validate an excisional wound model using a 6-mm biopsy punch to create four full-thickness dorsal wounds on a single db/db mouse. Factors considered in developing the db/db wound model include reproducibility of size and shape of wounds, the effect of semiocclusive dressings, comparison with littermate controls (db/-), clinical versus histologic evidence of wound closure, and cross-contamination of wounds with topically applied reagents. The size of wounds was larger, with less variation in the db/db mice (31.11 +/- 3.76 mm2) versus db/- mice (23.64 +/- 4.78 mm2). Wounds on db/db mice that were covered with a semiocclusive dressing healed significantly more slowly (mean, 27.75 days) than wounds not covered with the dressing (mean, 13 days; p < 0.001), suggesting the dressings may splint the wounds open. As expected, wounds healed more slowly on db/db mice than db/- mice (covered wounds, 27.75 days versus 11.86 days, p < 0.001; wounds not covered, 13 days versus 11.75 days, p = 0.39). Covered wounds, thought to be closed by clinical examination, were confirmed closed by histology only 62 percent of the time in the db/db and 100 percent of the time in the db/- mice. Topical application of blue histologic dye or soluble biotinylated laminin 5 to one of the four wounds did not spread locally and contaminate adjacent wounds. Multiple, uniform, 6-mm wounds in db/db mice heal in a relatively short time, decrease the number of animals needed for each study, and allow each animal to serve as its own control. The db/db diabetic mouse appears to be an excellent model of delayed wound healing, particularly for studying factors related to epithelial migration.


Asunto(s)
Vendajes , Diabetes Mellitus Experimental/complicaciones , Modelos Animales de Enfermedad , Piel/lesiones , Cicatrización de Heridas , Animales , Masculino , Ratones , Ratones Endogámicos C57BL , Piel/patología
11.
J Biomed Mater Res A ; 98(4): 499-508, 2011 Sep 15.
Artículo en Inglés | MEDLINE | ID: mdl-21681942

RESUMEN

The sinus between skin and a percutaneous medical device is often a portal for infection. Epidermal integration into an optimized porous biomaterial could seal this sinus. In this study, we measured epithelial ingrowth into rods of sphere-templated porous poly(2-hydroxyethyl methacrylate) implanted percutaneously in mice. The rods contained spherical 20-, 40-, or 60-µm pores with and without surface modification. Epithelial migration was measured 3, 7, and 14 days post-implantation utilizing immunohistochemistry for pankeratins and image analysis. Our global results showed average keratinocyte migration distances of 81 ± 16.85 µm (SD). Migration was shorter through 20-µm pores (69.32 ± 21.73) compared with 40 and 60 µm (87.04 ± 13.38 µm and 86.63 ± 8.31 µm, respectively). Migration was unaffected by 1,1' carbonyldiimidazole surface modification without considering factors of pore size and healing duration. Epithelial integration occurred quickly showing an average migration distance of 74.13 ± 12.54 µm after 3 days without significant progression over time. These data show that the epidermis closes the sinus within 3 days, migrates into the biomaterial (an average of 11% of total rod diameter), and stops. This process forms an integrated epithelial collar without evidence of marsupialization or permigration.


Asunto(s)
Materiales Biocompatibles/química , Epidermis/metabolismo , Implantes Experimentales , Animales , Materiales Biocompatibles/metabolismo , Movimiento Celular , Células Epidérmicas , Queratinocitos/citología , Queratinocitos/fisiología , Masculino , Ensayo de Materiales , Metacrilatos/química , Ratones , Ratones Endogámicos C57BL , Porosidad , Propiedades de Superficie
12.
J Am Assoc Lab Anim Sci ; 49(5): 588-91, 2010 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-20858360

RESUMEN

Research in cutaneous biology frequently involves models that use mice housed in SPF conditions. Little information is available concerning the species of bacteria that normally inhabit the skin of these mice. The aim of this study was to characterize the bacterial skin flora of mice housed under SPF conditions. Skin biopsies from C57BL/6 mice under normal and surgically prepped conditions were both cultured and analyzed by using DNA extraction and sequencing. The species isolated most commonly from culture were staphylococci. Coagulase-negative staphylococci were isolated more frequently than was Staphylococcus aureus. Molecular sequencing yielded several additional organisms not found by culture. Overall, culturing of isolates yielded 14 species of bacteria, and molecular sequencing identified another 6 species. Investigators conducting cutaneous research in mouse models should aware of the cutaneous bacterial flora present on these mice.


Asunto(s)
Bacterias/aislamiento & purificación , Ratones/microbiología , Piel/microbiología , Animales , Bacterias/clasificación , Bacterias/genética , ADN Bacteriano/análisis , Vivienda para Animales , Ratones Endogámicos C57BL , ARN Ribosómico 16S/análisis , Análisis de Secuencia , Organismos Libres de Patógenos Específicos
14.
Wound Repair Regen ; 14(4): 398-404, 2006.
Artículo en Inglés | MEDLINE | ID: mdl-16939566

RESUMEN

Regeneration of mammalian digit tips is well described; however, associated cellular or molecular events have not been studied in humans. We describe an in vitro human fetal model of response to digit tip amputation, and report expression of the transcription repressor Msx1 in the developing and regrowing human digit tip. Human fetal digits from specimens ranging from 53 to 117 days' estimated gestational age (EGA) were cultured in a defined serum-free medium with supplemented oxygen for time periods from 4 days to 4 weeks. Histology and immunohistochemistry were performed on paired control and tip-amputated digits. Regrowing tissue covered the cut end of the distal phalanx in digits up to 80 days' EGA. Msx1 expression was detected beneath the nail field in control digits to at least 70 days' EGA and at the regrowing tip of 57-day digits at 4 and 7 days post-amputation. Our results show that human fetal digits regrow tissue in vitro in response to tip amputation. This process appears spatially associated with Msx1 expression. Msx1 expression appears increased at the regrowing tip of 57-day digits by 4 days after amputation.


Asunto(s)
Amputación Traumática/metabolismo , Amputación Traumática/fisiopatología , Dedos/fisiopatología , Factor de Transcripción MSX1/metabolismo , Regeneración/fisiología , Feto , Dedos/embriología , Edad Gestacional , Humanos , Factores de Tiempo , Técnicas de Cultivo de Tejidos , Cicatrización de Heridas/fisiología
15.
Wound Repair Regen ; 14(4): 484-91, 2006.
Artículo en Inglés | MEDLINE | ID: mdl-16939578

RESUMEN

Percutaneous devices play an essential role in medicine; however, they are often associated with a significant risk of infection. One approach to circumvent infection would be to heal the wound around the devices by promoting skin cell attachment. We used two in vitro assay models to evaluate cutaneous response to poly(2-hydoxyethyl methacrylate) (poly(HEMA)). One approach was to use a cell adhesion assay to test the effects of surface modification of poly(HEMA), and the second used an organ culture system of newborn foreskin biopsies implanted with porous poly(HEMA) rods (20 microm pores) to evaluate the skin/poly(HEMA) interface. Surface modification of poly(HEMA) using 1,1'-carbonyldiimidazole (CDI) enhanced keratinocyte, fibroblast, and endothelial cell adhesion. Keratinocytes in the organ culture model not only remained functionally and structurally viable as observed by immunohistochemistry and electron microscopy, but migrated into the pores of CDI-modified poly(HEMA) rods. No biointegration was seen in the non-CDI-modified poly(HEMA). Laminin 5 immunostaining was seen along the poly(HEMA)/skin interface in a pattern resembling the junctional epithelium of the tooth, the unique natural interface between the skin and tooth that serves as a barrier to bacteria. In vitro systematic evaluation of biomaterials for use in animal implant studies is both cost effective and time efficient.


Asunto(s)
Materiales Biocompatibles/farmacología , Prepucio/efectos de los fármacos , Queratinocitos/efectos de los fármacos , Poliaminas/farmacología , Polihidroxietil Metacrilato/análogos & derivados , Piel/efectos de los fármacos , Heridas Penetrantes/fisiopatología , Anciano , Adhesión Celular/efectos de los fármacos , Técnicas de Cultivo de Célula , Femenino , Humanos , Recién Nacido , Queratinocitos/fisiología , Masculino , Persona de Mediana Edad , Técnicas de Cultivo de Órganos , Polihidroxietil Metacrilato/farmacología , Piel/lesiones , Piel/fisiopatología , Cicatrización de Heridas/efectos de los fármacos
16.
Wound Repair Regen ; 13(5): 468-79, 2005.
Artículo en Inglés | MEDLINE | ID: mdl-16176455

RESUMEN

The process by which wounds reepithelialize remains controversial. Two models have been proposed to describe reepithelialization: the "sliding" model and the "rolling" model. In the "sliding" model, basal keratinocytes are the principal cells responsible for migration and wound closure. In this model, basal and suprabasal keratinocytes remain strongly attached to leading edge basal keratinocytes and are then passively dragged along as a sheet. The "rolling" model postulates that basal keratinocytes remain strongly attached to the basement membrane zone while suprabasal keratinocytes at the wound margin are activated to roll into the wound site. The purpose of this study was to determine which populations of keratinocytes are actively involved in reepithelialization. We evaluated expression of keratins K14, K15, K10, K2e, and K16 as well as the proliferation marker Ki67 in the migrating tongue of normal human incisional 1-hour to 28-day wounds and normal human 3 mm diameter excisional 1- to 7-day wounds. Our results show dramatic changes in phenotype and protein expression of keratins K10, K2e, K14, K15, and K16 in suprabasal keratinocytes in response to injury. We conclude that this large population of suprabasal keratinocytes actively participates in wound closure.


Asunto(s)
Proteínas de Filamentos Intermediarios/fisiología , Queratinocitos/fisiología , Queratinas/fisiología , Piel/fisiopatología , Cicatrización de Heridas/fisiología , Anciano , Movimiento Celular/fisiología , Proliferación Celular , Femenino , Humanos , Antígeno Ki-67 , Masculino , Persona de Mediana Edad , Piel/lesiones
17.
Wound Repair Regen ; 12(1): 44-52, 2004.
Artículo en Inglés | MEDLINE | ID: mdl-14974964

RESUMEN

Patients with diabetic neuropathy have reduced numbers of cutaneous nerves, which may contribute to an increased incidence of nonhealing wounds. Nerve growth factor (NGF) has been reported to augment wound closure. We hypothesized that topical 2.5S NGF, a biologically active subunit of the NGF polymer, would accelerate wound repair, augment nerve regeneration, and increase inflammation in excisional wounds in diabetic mice. A full-thickness 6-mm punch biopsy wound was created on the dorsum of C57BL/6J-m+ Leprdb mice (db/db) and heterozygous (db/-) littermates and treated daily with normal saline or 2.5S NGF (1 microg/day or 10 microg/day) on post-injury days 0-6. Time to closure, wound epithelialization, and degree of inflammation were compared using a Student's t-test. Color subtractive-computer-assisted image analysis was used to quantify immunolocalized nerves in wounds. Non-overlapping (20x) digital images of the wound were analyzed for nerve profile counts, area density (number of protein gene product 9.5 positive profiles per unit dermal area) and area fraction (protein gene product 9.5 positive area per unit dermal area). Healing times in db/db mice decreased from 30 days in normal saline-treated mice to 26 days in mice treated with 1 microg/day NGF (p<0.05) and 24 days in mice treated with 10 microg/day NGF (p<0.02). A similar trend in db/- mice was not significant. NGF treatment augmented epithelialization in the db/db mice (p<0.05). Histological evaluation of inflammation in healed wounds showed no statistical difference between treatment groups. Total nerve number, area density, and area fraction were increased in NGF-treated wounds at 14, 21, and 35 days (p<0.05). The 2.5 NGF subunit may improve wound closure kinetics by promoting epithelialization and nerve regeneration. Further studies to determine the role of nerves in wound repair are warranted.


Asunto(s)
Diabetes Mellitus/fisiopatología , Sustancias de Crecimiento/administración & dosificación , Factores de Crecimiento Nervioso/administración & dosificación , Regeneración Nerviosa/efectos de los fármacos , Cicatrización de Heridas/efectos de los fármacos , Administración Tópica , Animales , Complicaciones de la Diabetes , Ratones , Ratones Endogámicos C57BL , Modelos Animales , Factores de Tiempo , Heridas y Lesiones/complicaciones , Heridas y Lesiones/fisiopatología
18.
Science ; 303(5661): 1198-201, 2004 Feb 20.
Artículo en Inglés | MEDLINE | ID: mdl-14976317

RESUMEN

Adult stem cells offer the potential to treat many diseases through a combination of ex vivo genetic manipulation and autologous transplantation. Mesenchymal stem cells (MSCs, also referred to as marrow stromal cells) are adult stem cells that can be isolated as proliferating, adherent cells from bones. MSCs can differentiate into multiple cell types present in several tissues, including bone, fat, cartilage, and muscle, making them ideal candidates for a variety of cell-based therapies. Here, we have used adeno-associated virus vectors to disrupt dominant-negative mutant COL1A1 collagen genes in MSCs from individuals with the brittle bone disorder osteogenesis imperfecta, demonstrating successful gene targeting in adult human stem cells.


Asunto(s)
Colágeno Tipo I/genética , Marcación de Gen , Células Madre Mesenquimatosas/fisiología , Osteogénesis Imperfecta/genética , Osteogénesis Imperfecta/terapia , Alelos , Animales , Células de la Médula Ósea/fisiología , Diferenciación Celular , Células Cultivadas , Colágeno Tipo I/química , Colágeno Tipo I/metabolismo , Dependovirus/genética , Terapia Genética , Vectores Genéticos , Humanos , Kanamicina Quinasa/genética , Masculino , Ratones , Osteogénesis , Mutación Puntual , Recombinación Genética , Trasplante de Células Madre
19.
J Surg Res ; 108(1): 122-8, 2002 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-12443724

RESUMEN

Background. Patients with diabetic sensory neuropathy have significant risk of chronic ulcers. Insufficient nerve-derived mediators such as substance P (SP) may contribute to the impaired response to injury. Mutant diabetic mice (db/db), which develop neuropathy and have delayed healing, may provide a model to study the role of nerves in cutaneous injury.Methods. Skin from human chronic nonhealing ulcers and age-matched control skin was immunohistochemically evaluated for nerves. Nerve counts were also compared in murine diabetic (C57BL/KsJ-m+/+ Lepr(db); db/db) and nondiabetic (db/-) skin. Excisional wounds on the backs of db/db and db/- mice were grouped as: (a) untreated db/- mice; (b) untreated db/db mice; (c) db/db mice with polyethylene glycol (PEG); (d) db/db mice with PEG and SP 10(-9) M; or (e) db/db mice with PEG and SP 10(-6) M.Results. We demonstrated fewer nerves in the epidermis and papillary dermis of skin from human subjects with diabetes. Likewise, db/db murine skin had significantly fewer epidermal nerves than nondiabetic littermates. We confirmed increased healing times in db/db mice (51.7 days) compared to db/- littermates (19.8 days; P

Asunto(s)
Neuropatías Diabéticas/metabolismo , Piel/inervación , Sustancia P/metabolismo , Cicatrización de Heridas/fisiología , Anciano , Animales , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes , Persona de Mediana Edad , Terminaciones Nerviosas/patología , Piel/metabolismo , Piel/patología , Sustancia P/farmacología , Tioléster Hidrolasas/metabolismo , Ubiquitina Tiolesterasa , Cicatrización de Heridas/efectos de los fármacos
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