Rapid treatment of full-thickness skin loss using ovine tendon collagen type I scaffold with skin cells.

The full-thickness skin wound is a common skin complication affecting millions of people worldwide. Delayed treatment of this condition causes the loss of skin function and integrity that could lead to the development of chronic wounds or even death. This study was aimed to develop a rapid wound treatment modality using ovine tendon collagen type I (OTC-I) bio-scaffold with or without noncultured skin cells. Genipin (GNP) and carbodiimide (EDC) were used to cross-link OTC-I scaffold to improve the mechanical strength of the bio-scaffold. The physicochemical, biomechanical, biodegradation, biocompatibility, and immunogenicity properties of OTC-I scaffolds were investigated. The efficacy of this treatment approach was evaluated in an in vivo skin wound model. The results demonstrated that GNP cross-linked OTC-I scaffold (OTC-I_GNP) had better physicochemical and mechanical properties compared with EDC cross-linked OTC-I scaffold (OTC-I_EDC) and noncross-link OTC-I scaffold (OTC-I_NC). OTC-I_GNP and OTC-I_NC demonstrated no toxic effect on cells as it promoted higher cell attachment and proliferation of both primary human epidermal keratinocytes and human dermal fibroblasts compared with OTC-I_EDC. Both OTC-I_GNP and OTC-I_NC exhibited spontaneous formation of bilayer structure in vitro. Immunogenic evaluation of OTC-I scaffolds, in vitro and in vivo, revealed no sign of immune response. Finally, implantation of OTC-I_NC and OTC-I_GNP scaffolds with noncultured skin cells demonstrated enhanced healing with superior skin maturity and microstructure features, resembling native skin in contrast to other treatment (without noncultured skin cells) and control group. The findings of this study, therefore, suggested that both OTC-I scaffolds with noncultured skin cells could be promising for the rapid treatment of full-thickness skin wound.

Expression of mesenchymal stem cell phenotype in human nasal respiratory epithelial cells / Expresión del fenotipo de células troncales mesenquimales en células epiteliales nasales humanas

The respiratory epithelium is the first line of contact with the external hazards. Thus it can be damaged and need to be replaced to avoid healing by fibrosis. Tracheal tissue engineering is an alternative promising treatment modality. Mesenchymal stem cell markers are surface proteins, which are responsible for some of these cells unique properties. The objective of this study was to detect the mesenchymal stem cell phenotype among the human nasal respiratory epithelial cells via two immunophenotyping techniques. Respiratory epithelial cells were cultured using co-culture technique, fibroblasts was removed at confluence leaving respiratory epithelial cells, which were passage further to passage 4. Cells were evaluated for mesenchymal stem cell markers that were CD73, CD90, CD105 and the hematopoietic stem cell marker CD45 at passage 1 (P1) and passage 4 (P4) using Flow cytometry and Immunocytochemistry techniques. Respiratory epithelial cells expressed the mesenchymal stem cell markers at P1 and maintain the expression these markers until P4. Using both techniques, to compare the values of mesenchymal stem cell markers expression at P1 to P4 there was no significant difference. This study indicates that respiratory epithelial cells derived from nasal turbinate retain some of mesenchymal stem cells properties even after serial passages. Both methods of Immunophenotyping are comparable.

El epitelio respiratorio es la primera línea de contacto con los peligros externos. Por lo tanto, puede ser dañado y necesita ser reemplazado para evitar uan cicatrización por fibrosis. La ingeniería de tejidos traqueales es una modalidad de tratamiento alternativo prometedora. Los marcadores de células troncales mesenquimales son proteínas de superficie, que son responsables de algunas propiedades únicas de estas células. El objetivo fue detectar el fenotipo de células troncales mesenquimales entre las células epiteliales respiratorias nasales humanas a través de dos técnicas de inmunofenotipaje. Fueron cultivadas las células epiteliales respiratorias utilizando la técnica de co-cultivo; los fibroblastos se eliminaron en la confluencia dejando solo células epiteliales respiratorias, resultantes de los 4 pasajes. Las células fueron evaluadas para encontrar marcadores de células troncales mesenquimales mediante CD73, CD90, CD105 y el marcador de células troncales hematopoyéticas CD45 en el paso 1 (P1) y el paso 4 (P4), usando citometría de flujo y técnicas de inmunocitoquímica. Las células epiteliales respiratorias expresaron los marcadores de células troncales mesenquimales en P1 y mantuvieron la expresión de estos marcadores hasta P4. No hubo diferencias significativas en el uso de ambas técnicas al comparar los valores de los marcadores de células troncales mesenquimales expresadas desde P1 a P4. Este estudio indica que las células epiteliales respiratorias derivadas de la concha nasal retienen algunas de las propiedades de células troncales mesenquimales, incluso después de pases seriados. Ambos métodos de inmunofenotipificación son comparables.

Cartilage regeneration by chondrogenic induced adult stem cells in osteoarthritic sheep model.

OBJECTIVES: In this study, Adipose stem cells (ADSC) and bone marrow stem cells (BMSC), multipotent adult cells with the potentials for cartilage regenerations were induced to chondrogenic lineage and used for cartilage regenerations in surgically induced osteoarthritis in sheep model. METHODS: Osteoarthritis was induced at the right knee of sheep by complete resection of the anterior cruciate ligament and medial meniscus following a 3-weeks exercise regimen. Stem cells from experimental sheep were culture expanded and induced to chondrogenic lineage. Test sheep received a single dose of 2 × 10(7) autologous PKH26-labelled, chondrogenically induced ADSCs or BMSCs as 5 mls injection, while controls received 5 mls culture medium. RESULTS: The proliferation rate of ADSCs 34.4 ± 1.6 hr was significantly higher than that of the BMSCs 48.8 ± 5.3 hr (P = 0.008). Chondrogenic induced BMSCs had significantly higher expressions of chondrogenic specific genes (Collagen II, SOX9 and Aggrecan) compared to chondrogenic ADSCs (P = 0.031, 0.010 and 0.013). Grossly, the treated knee joints showed regenerated de novo cartilages within 6 weeks post-treatment. On the International Cartilage Repair Society grade scores, chondrogenically induced ADSCs and BMSCs groups had significantly lower scores than controls (P = 0.0001 and 0.0001). Fluorescence of the tracking dye (PKH26) in the injected cells showed that they had populated the damaged area of cartilage. Histological staining revealed loosely packed matrixes of de novo cartilages and immunostaining demonstrated the presence of cartilage specific proteins, Collagen II and SOX9. CONCLUSION: Autologous chondrogenically induced ADSCs and BMSCs could be promising cell sources for cartilage regeneration in osteoarthritis.

Human nasal turbinates as a viable source of respiratory epithelial cells using co-culture system versus dispase-dissociation technique.

OBJECTIVE: To compare a co-culture system with a conventional dispase-dissociation method for obtaining functional human respiratory epithelial cells from the nasal turbinates for tissue engineering application. METHODS: Human respiratory epithelial cells were serially passaged using a co-culture system and a conventional dispase-dissociation technique. The growth kinetics and gene expression levels of the cultured respiratory epithelial cells were compared. Four genes were investigated, namely cytokeratin-18, a marker for ciliated and secretory epithelial cells; cytokeratin-14, a marker for basal epithelial cells; MKI67, a proliferation marker; and MUC5B, a marker for mucin secretion. Immunocytochemical analysis was performed using monoclonal antibodies against the high molecular-weight cytokeratin 34 beta E12, cytokeratin 18, and MUC5A to investigate the protein expression from cultured respiratory epithelial cells. RESULTS: Respiratory epithelial cells cultured using both methods maintained polygonal morphology throughout the passages. At passage 1, co-cultured respiratory epithelial showed a 2.6-times higher growth rate compared to conventional dispase dissociation technique, and 7.8 times higher at passage 2. Better basal gene expression was observed by co-cultured respiratory epithelial cells compared to dispase dissociated cells. Immunocytochemical analyses were positive for the respiratory epithelial cells cultured using both techniques. CONCLUSION: Co-culture system produced superior quality of cultured human respiratory epithelial cells from the nasal turbinates as compared to dispase dissociation technique.