Amélie Lavoie

Irradiated Human Dermal Fibroblasts Are as Efficient as Mouse Fibroblasts as a Feeder Layer to Improve Human Epidermal Cell Culture Lifespan

A fibroblast feeder layer is currently the best option for large scale expansion of autologous skin keratinocytes that are to be used for the treatment of severely burned patients. In a clinical context, using a human rather than a mouse feeder layer is desirable to reduce the risk of introducing animal antigens and unknown viruses. This study was designed to evaluate if irradiated human fibroblasts can be used in keratinocyte cultures without affecting their morphological and physiological properties. Keratinocytes were grown either with or without a feeder layer in serum-containing medium. Our results showed that keratinocytes grown either on an irradiated human feeder layer or irradiated 3T3 cells (i3T3) can be cultured for a comparable number of passages. The average epithelial cell size and morphology were also similar. On the other hand, keratinocytes grown without a feeder layer showed heavily bloated cells at early passages and stop proliferating after only a few passages. On the molecular aspect, the expression level of the transcription factor Sp1, a useful marker of keratinocytes lifespan, was maintained and stabilized for a high number of passages in keratinocytes grown with feeder layers whereas Sp1 expression dropped quickly without a feeder layer. Furthermore, gene profiling on microarrays identified potential target genes whose expression is differentially regulated in the absence or presence of an i3T3 feeder layer and which may contribute at preserving the growth characteristics of these cells. Irradiated human dermal fibroblasts therefore provide a good human feeder layer for an effective expansion of keratinocytes in vitro that are to be used for clinical purposes.

Identification of epithelial stem cells in vivo and in vitro using keratin 19 and BrdU.

Progress in the identification of skin stem cells and the improvement of culture methods open the possibility to use stem cells in regenerative medicine. Based on their quiescent nature, the development of label retention assays allowed the localization of skin stem cells in the bulge region of the pilosebaceous units and in the bottom of rete ridges in glabrous skin. The development of markers such as keratin 19 also permits their study in human tissues. In this chapter, protocols to identify skin stem cells based on their slow-cycling property and their expression of keratin 19 will be described in detail. The methods include the labeling of skin stem cells within mouse or rat tissues in vivo, the labeling of proliferative human cells in vitro using 5-bromo-2-deoxyuridine (BrdU), and the detection of keratin 19 and BrdU by immunofluorescence or immunoperoxidase staining.

Considerations in the choice of a skin donor site for harvesting keratinocytes containing a high proportion of stem cells for culture in vitro

The treatment of severely burned patients has benefited from the grafting of skin substitutes obtained by expansion of epithelial cells in culture. The aim of this study was to evaluate whether the anatomic site chosen for harvesting skin had an impact on the quality of the derived cell cultures. Considering that hair follicles contain epithelial stem cells, we compared hairy skin sites featuring different densities and sizes of hair follicles for their capacity to generate high quality keratinocyte cultures. Three anatomic sites from adult subjects were compared: scalp, chest skin and p-auricular (comprising pre-auricular and post-auricular) skin. Keratin (K) 19 was used as a marker to evaluate the proportion of stem cells. Keratinocytes were isolated using the two-step thermolysin and trypsin cell extraction method, and cultured in vitro. The proportion of K19-positive cells harvested from p-auricular skin was about twice that of the scalp. This K19-positive cell content also remained higher during the first subcultures. In contrast to these in vitro results, the number of K19-positive cells estimated in situ on skin sections was about double in scalp as in p-auricular skin. Chest skin had the lowest number of K19-positive cells. These results indicate that in addition to the choice of an adult anatomic site featuring a high number of stem cells in situ, the quality of the cultures greatly depends on the ability to extract stem cells from the skin biopsy.

Improved Methods to Produce Tissue-Engineered Skin Substitutes Suitable for the Permanent Closure of Full-Thickness Skin Injuries

Abstract There is a clinical need for skin substitutes to replace full-thickness skin loss. Our group has developed a bilayered skin substitute produced from the patients own fibroblasts and keratinocytes referred to as Self-Assembled Skin Substitute (SASS). After cell isolation and expansion, the current time required to produce SASS is 45 days. We aimed to optimize the manufacturing process to standardize the production of SASS and to reduce production time. The new approach consisted in seeding keratinocytes on a fibroblast-derived tissue sheet before its detachment from the culture plate. Four days following keratinocyte seeding, the resulting tissue was stacked on two fibroblast-derived tissue sheets and cultured at the air–liquid interface for 10 days. The resulting total production time was 31 days. An alternative method adapted to more contractile fibroblasts was also developed. It consisted in adding a peripheral frame before seeding fibroblasts in the culture plate. SASSs produced by both new methods shared similar histology, contractile behavior in vitro and in vivo evolution after grafting onto mice when compared with SASSs produced by the 45-day standard method. In conclusion, the new approach for the production of high-quality human skin substitutes should allow an earlier autologous grafting for the treatment of severely burned patients.

La médecine régénératrice : les cellules souches, les interactions cellulaires et matricielles dans la reconstruction cutanée et cornéenne par génie tissulaire

Considering that there is a shortage of organ donor, the aim of tissue engineering is to develop substitutes for the replacement of wounded or diseased tissues. Autologous tissue is evidently a preferable transplant material for long-term graft persistence because of the unavoidable rejection reaction occuring against allogeneic transplant. For the production of such substitutes, it is essential to control the culture conditions for post-natal human stem cells. Furthermore, histological organization and functionality of reconstructed tissues must approach those of native organs. For self-renewing tissues such as skin and cornea, tissue engineering strategies must include the preservation of stem cells during the in vitro process as well as after grafting to ensure the long-term regeneration of the transplants. We described a tissue engineering method named the self-assembly approach allowing the production of autologous living organs from human cells without any exogenous biomaterial. This approach is based on the capacity of mesenchymal cells to create in vitro their own extracellular matrix and then reform a tissue. Thereafter, various techniques allow the reorganization of such tissues in more complex organ such as valve leaflets, blood vessels, skin or cornea. These tissues offer the hope of new alternatives for organ transplantation in the future. In this review, the importance of preserving stem cells during in vitro expansion and controlling cell differentiation as well as tissue organization to ensure quality and functionality of tissue-engineered organs will be discussed, while focusing on skin and cornea.

Access to publicly funded outpatient physiotherapy services in Quebec: waiting lists and management strategies

Abstract Purpose: Problems with access to outpatient physiotherapy services have been reported in publicly funded healthcare systems worldwide. A few studies have reported management strategies aimed at reducing extensive waiting lists, but their association with waiting times is not fully understood. The purpose of this study was to document access to public outpatient physiotherapy services for persons with musculoskeletal disorders in hospitals and explore organizational factors associated with waiting time. Methods: We surveyed outpatient physiotherapy services in publicly funded hospitals in the province of Quebec (Canada). Results: A total of 97 sites responded (99%) to the survey. The median waiting time was more than six months for 41% of outpatient physiotherapy services. The waiting time management strategies most frequently used were attendance and cancelation policies (99.0%) and referral prioritization (95.9%). Based on multivariate analyses, the use of a prioritization process with an initial evaluation and intervention was associated with shorter waiting times (p = 0.008). Conclusions: Our findings provide evidence that a large number of persons wait a long time for publicly funded physiotherapy services in Quebec. Based on our results, implementation of a prioritization process with an initial evaluation and intervention could help improve timely access to outpatient physiotherapy services. Implications for Rehabilitation Access to publicly funded outpatient physiotherapy services is limited by long waiting times in a great proportion of Quebec’s hospitals. The use of a specific prioritization process that combines an evaluation and an intervention could possibly help improve timely access to services. Policy-makers, managers, and other stakeholders should work together to address the issue of limited access to publicly funded outpatient physiotherapy services.

Effect of 27-MHz radiofrequency on hair follicles: histological evaluation of skin treated ex vivo.

BACKGROUND A multitude of methods and treatments exist for cosmetic hair removal. Electroepilation is a commonly performed method of hair removal that is so-called “permanent”; however, there is a paucity of histological studies of the effects of radiofrequency (RF) on hair follicles. OBJECTIVE This study aimed to observe the destruction of human hair follicles and surrounding tissue after the treatment with 27.12-MHz RF, with more attention paid to the thermal destruction of bulge and bulb/dermal papilla. METHODS Human scalp specimens obtained during face-lift surgery were treated with 27.12-MHz RF. The probe tip was inserted into hair follicle, RF current was applied, and treated specimens were processed for histological analysis. RESULTS Significant damages were observed on treated hair follicles. Thermal damage was lance-shaped and extended over several hundred micrometers (100–400 &mgr;m). The location of destruction areas varied, likely depending on the point of insertion of the probe. The epidermis remained intact. CONCLUSION This study shows that the general mechanism of thermolysis is to generate damage to cells and tissues surrounding the insertion point of the filament. The results suggest that if the insertion point is close to the bulge region, there is a risk to destroy hair follicle epithelial stem cells.

Variations in demand and provision for publicly funded outpatient musculoskeletal physiotherapy services across Quebec, Canada

RATIONALE The prevalence of musculoskeletal disorders is high and expected to increase in the next decade. Persons suffering from musculoskeletal disorders benefit from early physiotherapy services. However, access to publicly funded physiotherapy services has been shown to be compromised by long waiting times and limited availability of resources in many countries around the world. Decisions on resource allocation may create geographic disparities in provision and access to services, which may result in inequity in access. AIMS AND OBJECTIVES This study aimed to assess variations in demand and provision of publicly funded outpatient physiotherapy services across the province of Quebec, Canada, as well as to assess the demand to provision relationship. METHODS We conducted a secondary analysis of data retrieved from the 2008 Quebec Health Survey and data obtained from a survey of hospitals in the province of Quebec in 2015. We used geographic information systems analyses and descriptive analyses to assess geographic variations and the relationship between demand and provision. RESULTS Our results indicate substantial variations in the provision and demand for physiotherapy services in the province of Quebec. The variations in service provision did not follow the variations in demand. Long waiting times and insufficient provision of services were found in many regions. CONCLUSIONS The variations in provision of physiotherapy services between regions reported in our study did not correspond to the variations in demand. Such geographic variations and demand to provision mismatches may create inequity in access to services, especially for those unable to afford private services.