Isolation and Culture of Human Keratinocytes.

Culturing keratinocytes to form coherent epithelial tissue sheets has improved the treatment of extensively burned patients. Keratinocyte culture is also used to investigate various cellular and molecular mechanisms involved in different skin pathologies. To preserve stem cells during epithelial cell culture, reliable methods and conditions are of the utmost importance. Properly cultured keratinocytes will exhibit a consistent cuboid morphology and can proliferate for many passages. This chapter details materials needed and methods for all aspects of efficient keratinocyte culture for clinical applications, namely tissue sampling and transportation, isolation, routine culture, subculture, and cryopreservation.

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.

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

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.

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

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 patient's 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.

Production of a Bilayered Self-Assembled Skin Substitute Using a Tissue-Engineered Acellular Dermal Matrix.

Our bilayered self-assembled skin substitutes (SASS) are skin substitutes showing a structure and functionality very similar to native human skin. These constructs are used, in life-threatening burn wounds, as permanent autologous grafts for the treatment of such affected patients even though their production is exacting. We thus intended to shorten their current production time to improve their clinical applicability. A self-assembled decellularized dermal matrix (DM) was used. It allowed the production of an autologous skin substitute from patient's cells. The characterization of SASS reconstructed using a decellularized dermal matrix (SASS-DM) was performed by histology, immunofluorescence, transmission electron microscopy, and uniaxial tensile analysis. Using the SASS-DM, it was possible to reduce the standard production time from about 8 to 4 and a half weeks. The structure, cell differentiation, and mechanical properties of the new skin substitutes were shown to be similar to the SASS. The decellularization process had no influence on the final microstructure and mechanical properties of the DM. This model, by enabling the production of a skin substitute in a shorter time frame without compromising its intrinsic tissue properties, represents a promising addition to the currently available burn and wound treatments.

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 µm). 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.

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.

Human epithelial stem cells persist within tissue-engineered skin produced by the self-assembly approach.

To adequately and permanently restore organ function after grafting, human tissue-engineered skin substitutes (TESs) must ultimately contain and preserve functional epithelial stem cells (SCs). It is therefore essential that a maximum of SCs be preserved during each in vitro step leading to the production of TESs such as the culture process and the elaboration of a skin cell bank by cryopreservation. To investigate the presence and functionality of epithelial SCs within the human TESs made by the self-assembly approach, slow-cycling cells were identified using 5'-bromo-2'-deoxyuridine (BrdU) in the three-dimensional construct. A subset of basal epithelial cells retained the BrdU label and was positive for the SC-associated marker keratin 19 within TESs after a chase of 21 days in culture post-BrdU labeling. Moreover, keratinocytes harvested from TESs gave rise to SC-like colonies in secondary monolayer subcultures, indicating that SCs were preserved within TESs. To evaluate the effect of cryopreservation with dimethyl sulfoxide and storage in liquid nitrogen on SCs, human epithelial cells were extracted from skin samples, amplified in culture, and used to produce TESs, before cryopreservation as well as after thawing. We found that the proportion and the growth potential of epithelial SCs in monolayer culture and in TESs remained constant before and after cryopreservation. Further, the functionality of these substitutes was demonstrated by successfully grafting human TESs on athymic mice for 6 months. We conclude that human epithelial skin SCs are adequately preserved upon human tissue reconstruction. Thus, these TESs produced by the self-assembly approach are suitable for clinical applications.

Human fibroblast-derived ECM as a scaffold for vascular tissue engineering.

The self-assembly approach is based on the capability of mesenchymal cells to secrete and organize their own extracellular matrix (ECM). This tissue engineering method allows for the fabrication of autologous living tissues, such as tissue-engineered blood vessels (TEBV) and skin. However, the secretion of ECM by smooth muscle cells (SMCs), required to produce the vascular media, may represent a long process in vitro. The aim of this work was to reduce the time required to produce a tissue-engineered vascular media (TEVM) and extend the production of TEVM with SMCs from all patients without compromising its mechanical and functional properties. Therefore, we developed a decellularized matrix scaffold (dMS) produced from dermal fibroblasts (DF) or saphenous vein fibroblasts (SVF), in which SMCs were seeded to produce a TEVM. Mechanical and contractile properties of these TEVM (referred to as nTEVM) were compared to standard self-assembled TEVM (sTEVM). This approach reduced the production time from 6 to 4 weeks. Moreover, nTEVM were more resistant to tensile load than sTEVM and their vascular reactivity was also improved. This new fabrication technique allows for the production of a vascular media using SMCs isolated from any patient, regardless of their capacity to synthesize ECM. Moreover, these scaffolds can be stored to be available when needed, in order to accelerate the production of the vascular substitute using autologous vascular cells.

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.

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.