Hyperbaric oxygen treatment of tissue-engineered mucosa enhances secretion of angiogenic factors in vitro.

The survival of tissue-engineered mucosa (TEM) after implantation is mostly dependent on the presence of blood vessels for continuous oxygen supply. Therefore the stimulation of vascularization of TEM is essential to improve survival in vivo. Hyperbaric oxygen (HBO) treatment, used to improve wound healing, stimulates the secretion of angiogenic factors. In this study we evaluated the effect of daily HBO treatments on TEM for 1, 3, or 5 consecutive days. Overall histology with hematoxylin-eosin staining showed no apparent changes after one treatment. After three and five HBO treatments, the basal layer became irregular and pyknotic cells were observed. Measurements of the viable epithelium showed significant thinning after one and five treatments, however, proliferation was not affected. The angiogenic factors keratinocyte growth factor (KGF), hepatocyte growth factor (HGF), basic fibroblast growth factor (FGFbasic), and placental growth factor (PlGF) were significantly increased after one HBO treatment, whereas after three treatments a significant decrease of FGFbasic and PlGF was seen. After five treatments KGF, PlGF, and vascular endothelial growth factor (VEGF) were significantly increased. One HBO treatment of TEM enhances the secretion of important angiogenic factors, hereby potentially improving the survival rate after in vivo implantation.

Characterization of a three-dimensional mucosal equivalent: similarities and differences with native oral mucosa.

The aim of this study was to create and characterize a tissue-engineered mucosal equivalent (TEM) that closely resembles native mucosa. TEM consists of human primary keratinocytes and fibroblasts isolated from biopsies taken from healthy donors and seeded onto a de-epidermized dermis and cultured for 14 days at the air/liquid interface. The structure of TEM was examined and compared with native nonkeratinizing oral mucosa (NNOM). The various components of the newly formed epidermal layer, basement membrane and underlying connective tissue were analyzed using immunohistochemistry. The mucosal substitute presented in this study showed a mature stratified squamous epithelium that was similar to that of native oral mucosa, as demonstrated by K19, desmoglein-3 and involucrin staining. In addition, the expression of basement membrane components collagen type IV, laminin-5 and integrin α6 and ß4 in TEM proved to be consistent with native oral mucosa. The expression of PAS, Ki67, K10 and K13, however, appeared to be different in TEM compared to NNOM. Nevertheless, the similarities with native oral mucosa makes TEM a promising tool for studying the biology of mucosal pathologies such as oral mucositis or fibrosis as well as the development of new therapies.

Hypoxia preconditioning of tissue-engineered mucosa enhances its angiogenic capacity in vitro.

Improving vascularization of tissue-engineered oral mucosa (TEM) is a major challenge in the field of plastic surgery. Hypoxia is a stimulator of angiogenesis through a number of mechanisms. Therefore, hypoxia is a critical parameter that can be controlled in an effort to improve angiogenesis. In the present study we studied the secretion of a number of angiogenic factors during hypoxia exposure and evaluated the effect of TEM conditioned medium on endothelial cells. TEM was constructed by seeding human oral mucosa keratinocytes and fibroblasts on acellular human donor skin. TEM was exposed to hypoxia during 6, 12, and 24 h. Cellular hypoxia was assessed by immunolocalization of the hypoxia-inducible factor-1α. Secretion of vascular endothelial growth factor, placental growth factor (PlGF), tissue inhibitors of matrix metalloproteinases-1 and -2, and the activity of matrix metalloproteinase-9 significantly increased during hypoxia exposure. Moreover, conditioned medium from hypoxic TEM strongly enhanced endothelial cell proliferation and migration. In vitro exposure of TEM to hypoxia improves its capacity to support endothelial cell proliferation and migration, which suggests that hypoxia preconditioning of TEM potentially improves angiogenic responses for in vivo implantation.

Delay of denervation atrophy by sensory protection in an end-to-side neurorrhaphy model: a pilot study.

OBJECT: Temporary sensory innervation delays the atrophy process. A major disadvantage of most experimental models is that sensory-protected muscles must be denervated a second time to allow reinnervation by the affected nerve. The aim of this study was to assess the effect of sensory protection on denervated gastrocnemius muscle in an end-to-side neurorrhaphy model, in which denervated muscles may be preserved until axons of the native nerve reach their target without the necessity for a second operation. METHODS: The tibial nerve of 24 female Lewis rats was transected. Twelve animals acted as the controls. In the other 12 animals, the end of the sural nerve was connected to the side of the distal tibial nerve stump (sensory protection group). At 5 and 10 weeks, wet gastrocnemius muscle weight was reported as a ratio of the operated to the unoperated side. For histological analysis, muscle samples were rapidly frozen and sections were stained with haematoxylin and eosin, Oil Red O stain and modified Gomori trichrome stain. RESULTS: The difference between the sensory protection group and the control group was statistically significant at 5 (0.36±0.01 and 0.29±0.01, respectively; p<0.001) and 10 weeks postoperatively (0.28±0.01 and 0.19±0.00, respectively; p<0.001). Histological observations revealed that sensory-protected muscles underwent less atrophy. CONCLUSION: Sensory protection delays atrophy in an end-to-side neurorrhaphy model.

Auto-crosslinked hyaluronic acid gel accelerates healing of rabbit flexor tendons in vivo.

This study's purpose was to assess the in vivo effect of auto-crosslinked hyaluronic acid (HA) gel, a natural HA derivative with increased viscosity and tissue residence time, on adhesions and healing of injured and surgically repaired rabbit digital flexor tendons. The second and third right deep digital flexor tendons from 48 rabbits (n = 96 tendons) were cut and repaired with a modified Kessler and running peripheral suture. Animals were randomized to two groups, receiving either HA gel or saline injected around both freshly repaired tendons. After 2, 3, 6, and 12 weeks, six rabbits in each group were euthanized. Tendon pull-out force and breaking strength were measured as a value for adhesion formation and tendon healing, respectively. A histological assessment of adhesions and healing was related to the mechanical results. A significantly faster increase in breaking strength was found in HA gel-treated compared to saline-treated tendons; this coincided with a significantly accelerated tissue repair response after injury. No significant difference in adhesion formation was found between the two groups at any time. Our results indicate a significant acceleration of in vivo healing of tendons treated with HA gel. Adhesion formation was unaffected. These results could have important clinical value in promoting rehabilitation after tendon injury.

Fibroblasts accelerate culturing of mucosal substitutes.

Reconstruction of large mucosal defects of the floor of the mouth is typically performed with keratinizing skin. Drawbacks include donor site defects and hair bearing of the flaps. Cultured mucosal substitutes (CMSs) have been developed for clinical use to replace keratinizing skin. Acellular dermis is often used as a dermal carrier for autologous cells, because it reduces wound contraction and is easier for the surgeon to handle than, for example, collagen gels. A major problem of CMSs using acellular dermis is variation in epidermal quality. To improve the quality of the CMSs, human fibroblasts were incorporated into the acellular dermis and seeded with human keratinocytes. To study the role of the fibroblasts in epidermal morphology and basement membrane formation, CMSs were stained for differentiation markers beta1 integrin, cytokeratin 10, and involucrin after 1 and 2 weeks in culture. Basement membrane formation was analyzed using laminin 5 and collagen IV and VII staining; proliferation was analyzed using Ki-67 staining. The epidermises of fibroblast-containing CMSs matured faster into a well-organized epithelium than did those that did not contain CMSs. A 52.7% increase in basal cells, a 53.5% increase in mitosis index, and a 78.0% increase in keratinocyte cell layers were observed. Addition of fibroblasts reduced culturing time and enhanced proliferation, maturation, and quality of the epidermis.

Quantitative analysis of atherosclerotic lesion composition in mice.

Comparative quantitation has become an increasingly desirable tool in determining compositional differences of aortic plaque lesion in transgenically altered mice. To this end, methodology has been developed to identify lipid, cellularity, collagen, and elastin components using traditional bright-field microscopy, fluorescence, and polarized light microscopy, employing both confocal and wide-field imaging systems. Subsequent imaging processing and analysis on the digitally captured images reveals differences in compositional components as influenced by diet, age and gender. This method can be expanded to employ a rich variety of histochemical and immunohistochemical staining protocols.

Quantitative analysis of radiation-induced DNA break repair in a cultured oral mucosal model.

Oral mucositis is a major side effect of radiation therapy. Development of strategies for reduction of this problem calls for quantitative models. The goal of the present study was to test the feasibility of detecting double-strand breaks (DSBs) and DSB repair proteins upon radiation of mucosa in a 3-dimensional culture system using morphology and immunohistochemistry. Human oral keratinocytes and fibroblasts were seeded onto and into an acellular dermal carrier to produce a cultured mucosal substitute (CMS). CMSs were gamma-irradiated with 0, 2, and 12 Gy. One group received 4 Gy through 2 Gy fractions with a 24-h interval. Radiation-induced damage was quantified using hematoxylin and eosin (H&E). DSBs and DSB repair proteins were visualized and quantified using antibodies against P53 binding protein 1 (53BP1), MRE11, and RAD51. As in cell culture, CMSs showed intranuclear loci of damage and repair, mostly in the proliferative basal cell layers. Maximum percentages of damaged basal layer keratinocytes were 54.8% using H&E (12 Gy) up to 78.9% (12 Gy) for 53BP1. This study shows the feasibility of DNA repair markers to quantify radiation damage. This is an important step forward in the study of mucositis and the development of treatment and prevention strategies, proving once more the power and clinical importance of tissue engineering.

Barrett's oesophagus is characterized by a predominantly humoral inflammatory response.

Barrett's oesophagus (BO) is thought to be an intermediate step in the progression from reflux oesophagitis (RO) to oesophageal adenocarcinoma. Premalignant conditions that develop in the presence of chronic inflammation are often associated with the development of a more pronounced humoral immune response during progression of the disease. The aim of this study was to determine whether BO is also associated with a more pronounced humoral immune response when compared to RO. Immunohistochemical studies were performed to quantify the mean numbers of Th2 effector cells (plasma cells and mast cells) and Th1 effector cells (macrophages and CD8(+) T cells) to detect the antibody classes produced by plasma cells (IgA, IgG, IgM or IgE) and to determine the presence of isolated lymph follicles [segregated B and T cell areas, follicular dendritic cells (CD23) and expression of CXCL13] in 124 oesophageal biopsies from 20 patients with BO and 20 patients with RO. The proportion of Th2 effector cells was higher in BO than in RO, mainly due to higher numbers of plasma cells and mast cells in BO (p < 0.001). Most plasma cells in BO and RO expressed IgG, but several IgE(+) plasma cells were detected in BO: these were rare in RO. In line with this, isolated lymph follicles were observed in 4/20 (20%) patients with BO, but not in RO. We therefore conclude that the inflammatory response is skewed towards a more pronounced humoral immune response when RO progresses to BO. It may be that this shift, which is similar to that found in other chronic inflammatory conditions, contributes to an increased cancer risk in BO.