Scaffold-based developmental tissue engineering strategies for ectodermal organ regeneration.

Tissue engineering (TE) is a multidisciplinary research field aiming at the regeneration, restoration, or replacement of damaged tissues and organs. Classical TE approaches combine scaffolds, cells and soluble factors to fabricate constructs mimicking the native tissue to be regenerated. However, to date, limited success in clinical translations has been achieved by classical TE approaches, because of the lack of satisfactory biomorphological and biofunctional features of the obtained constructs. Developmental TE has emerged as a novel TE paradigm to obtain tissues and organs with correct biomorphology and biofunctionality by mimicking the morphogenetic processes leading to the tissue/organ generation in the embryo. Ectodermal appendages, for instance, develop in vivo by sequential interactions between epithelium and mesenchyme, in a process known as secondary induction. A fine artificial replication of these complex interactions can potentially lead to the fabrication of the tissues/organs to be regenerated. Successful developmental TE applications have been reported, in vitro and in vivo, for ectodermal appendages such as teeth, hair follicles and glands. Developmental TE strategies require an accurate selection of cell sources, scaffolds and cell culture configurations to allow for the correct replication of the in vivo morphogenetic cues. Herein, we describe and discuss the emergence of this TE paradigm by reviewing the achievements obtained so far in developmental TE 3D scaffolds for teeth, hair follicles, and salivary and lacrimal glands, with particular focus on the selection of biomaterials and cell culture configurations.

Subpopulations of dermal skin fibroblasts secrete distinct extracellular matrix: implications for using skin substitutes in the clinic.

BACKGROUND: While several commercial dermoepidermal scaffolds can promote wound healing of the skin, the achievement of complete skin regeneration still represents a major challenge. OBJECTIVES: To perform biological characterization of self-assembled extracellular matrices (ECMs) from three different subpopulations of fibroblasts found in human skin: papillary fibroblasts (Pfi), reticular fibroblasts (Rfi) and dermal papilla fibroblasts (DPfi). METHODS: Fibroblast subpopulations were cultured with ascorbic acid to promote cell-assembled matrix production for 10 days. Subsequently, cells were removed and the remaining matrices characterized. Additionally, in another experiment, keratinocytes were seeded on the top of cell-depleted ECMs to generate epidermal-only skin constructs. RESULTS: We found that the ECM self-assembled by Pfi exhibited randomly oriented fibres associated with the highest interfibrillar space, reflecting ECM characteristics that are physiologically present within the papillary dermis. Mass spectrometry followed by validation with immunofluorescence analysis showed that thrombospondin 1 is preferentially expressed within the DPfi-derived matrix. Moreover, we observed that epidermal constructs grown on DPfi or Pfi matrices exhibited normal basement membrane formation, whereas Rfi matrices were unable to support membrane formation. CONCLUSIONS: We argue that inspiration can be taken from these different ECMs, to improve the design of therapeutic biomaterials in skin engineering applications.

Multifaceted role of hair follicle dermal cells in bioengineered skins.

BACKGROUND: The method of generating bioengineered skin constructs was pioneered several decades ago; nowadays these constructs are used regularly for the treatment of severe burns and nonhealing wounds. Commonly, these constructs are comprised of skin fibroblasts within a collagen scaffold, forming the skin dermis, and stratified keratinocytes overlying this, forming the skin epidermis. In the past decade there has been a surge of interest in bioengineered skins, with researchers seeking alternative cell sources, or scaffolds, from which constructs can be established, and for more biomimetic equivalents with skin appendages. OBJECTIVES: To evaluate whether human hair follicle dermal cells can act as an alternative cell source for engineering the dermal component of engineered skin constructs. METHODS: We established in vitro skin constructs by incorporating into the collagenous dermal compartment: (i) primary interfollicular dermal fibroblasts, (ii) hair follicle dermal papilla cells or (iii) hair follicle dermal sheath cells. In vivo skins were established by mixing dermal cells and keratinocytes in chambers on top of immunologically compromised mice. RESULTS: All fibroblast subtypes were capable of supporting growth of overlying epithelial cells, both in vitro and in vivo. However, we found hair follicle dermal sheath cells to be superior to fibroblasts in their capacity to influence the establishment of a basal lamina. CONCLUSIONS: Human hair follicle dermal cells can be readily interchanged with interfollicular fibroblasts and used as an alternative cell source for establishing the dermal component of engineered skin both in vitro and in vivo.

Differential affinity of FLIP and procaspase 8 for FADD's DED binding surfaces regulates DISC assembly.

Death receptor activation triggers recruitment of FADD, which via its death effector domain (DED) engages the DEDs of procaspase 8 and its inhibitor FLIP to form death-inducing signalling complexes (DISCs). The DEDs of FADD, FLIP and procaspase 8 interact with one another using two binding surfaces defined by α1/α4 and α2/α5 helices, respectively. Here we report that FLIP has preferential affinity for the α1/α4 surface of FADD, whereas procaspase 8 has preferential affinity for FADD's α2/α5 surface. These relative affinities contribute to FLIP being recruited to the DISC at comparable levels to procaspase 8 despite lower cellular expression. Additional studies, including assessment of DISC stoichiometry and functional assays, suggest that following death receptor recruitment, the FADD DED preferentially engages FLIP using its α1/α4 surface and procaspase 8 using its α2/α5 surface; these tripartite intermediates then interact via the α1/α4 surface of FLIP DED1 and the α2/α5 surface of procaspase 8 DED2.

Protease activity, localization and inhibition in the human hair follicle.

OBJECTIVE: In humans, the process of hair shedding, referred to as exogen, is believed to occur independently of the other hair cycle phases. Although the actual mechanisms involved in hair shedding are not fully known, it has been hypothesized that the processes leading to the final step of hair shedding may be driven by proteases and/or protease inhibitor activity. In this study, we investigated the presence of proteases and protease activity in naturally shed human hairs and assessed enzyme inhibition activity of test materials. METHODS: We measured enzyme activity using a fluorescence-based assay and protein localization by indirect immunohistochemistry (IHC). We also developed an ex vivo skin model for measuring the force required to pull hair fibres from skin. RESULTS: Our data demonstrate the presence of protease activity in the tissue material surrounding club roots. We also demonstrated the localization of specific serine protease protein expression in human hair follicle by IHC. These data provide evidence demonstrating the presence of proteases around the hair club roots, which may play a role during exogen. We further tested the hypothesis that a novel protease inhibitor system (combination of Trichogen) and climbazole) could inhibit protease activity in hair fibre club root extracts collected from a range of ethnic groups (U.K., Brazil, China, first-generation Mexicans in the U.S.A., Thailand and Turkey) in both males and females. Furthermore, we demonstrated that this combination is capable of increasing the force required to remove hair in an ex vivo skin model system. CONCLUSION: These studies indicate the presence of proteolytic activity in the tissue surrounding the human hair club root and show that it is possible to inhibit this activity with a combination of Trichogen and climbazole. This technology may have potential to reduce excessive hair shedding.

A population-based case-control study of aetiological factors associated with vulval lichen sclerosus.

We aimed to investigate the association between possible aetiological factors and the risk of developing vulval lichen sclerosus (VLS). A population-based case-control questionnaire study was performed comparing women with a diagnosis of VLS (n= 92), with those attending a general gynaecology clinic with no known anogenital dermatosis (n= 66). After adjustment for confounders, factors associated with VLS included a family history of diabetes mellitus (OR= 7.0, p= 0.012) and previous pelvic surgery (OR= 4.75, p= 0.007). The use of barrier and progesterone only methods of contraception (OR= 0.19, p= 0.045), hormone replacement therapy (OR= 0.209, p= 0.025) or hayfever (OR= 0.18, p= 0.008) appeared to be associated with a reduced risk of VLS. In conclusion, we were unable to confirm many proposed aetiological theories associated with the development of VLS, in particular those associated with autoimmunity.

Expression of oestrogen and progesterone receptors by mast cells alone, but not lymphocytes, macrophages or other immune cells in human upper airways.

BACKGROUND: Nasal polyposis often coexists with asthma in airway inflammatory conditions characterised by the infiltration of a range of immune cells. A potentially important role for ovarian hormones has been implicated in airway inflammation but the cellular target for such action is not known. METHODS: Expression of oestrogen receptors (ER) and progesterone receptors (PR) was examined using immunohistochemistry in formalin fixed nasal polyp tissues from 47 subjects. The cells positive for ER or PR were confirmed by spatial location, dual immunolabelling, and histochemical staining. RESULTS: Consistent with the known features of nasal polyps, CD4+ (T helper/inducer), CD8+ (cytotoxic/suppressor), CD68+ (macrophages), mast cells, eosinophils and neutrophils were all clearly detected by their relevant monoclonal antibodies or appropriate histochemical staining, but only mast cells tested positive for ER/PR labelling with their polyclonal and monoclonal antibodies. The frequencies for expression were 61.7% for ER positive and 59.6% for PR positive cells. The expression of ER/PR was independent of patient sex and age but was highly correlated with the numbers of mast cells (r = 0.973, p<0.001 for ER; r = 0.955, p<0.001 for PR). Fewer than 5% of mast cells were found to be negative for ER/PR expression. CONCLUSIONS: Mast cells alone, but not lymphocytes, macrophages, or other immune cells, express ER/PR in human upper airways. Numerous ER/PR positive mast cells exist in nasal polyps, indicating that this may be a major route for the involvement of sex hormones in airway inflammation when exposed to the higher and varying concentration of oestrogen and progesterone characteristic of females.

A community-wide infant mortality review: findings and implications.

The authors present the results of a community-wide infant mortality review, describe implications for the delivery of maternal and child health services, and discuss the value of such reviews in addressing local public health concerns. The review included an analysis of birth and death certificates and medical record data; maternal interviews; review of cases and development of recommendations by provider panels; and convening of community groups to develop strategies to improve the health and health care of women and infants. The review focused on 287 infant deaths during 1990-1993. More than half of all neonatal deaths were attributable to "previable" or "borderline viable" births. Sexually transmitted infections were the most frequently identified underlying risk, and smoking was the most frequently identified prenatal risk. Homelessness, physical and sexual abuse, and alcohol use were at least twice as likely among women whose babies died than among a high risk comparison group. Panelists identified fragmented health care over the course of women's reproductive lives as a predominant theme. The authors conclude that: (a) The focus of maternal and child health care should shift to a model of women's health care that addresses the chronicity of social and clinical risks. (b) Infant mortality reviews are a valuable tool for community education, systems review, and policy development and can be applied to other public health issues with local significance. (c) Expectations about the review process's ability to produce conclusions about causality or recommendations narrowly geared to reducing infant mortality rates need to be reframed. (d) The model will be strengthened by greater participation of families affected by infant death.

Outcome measurement in home health.

Home health occupational therapists, as well as their colleagues in other settings, cannot afford to ignore the need for clear, objective, and reliable outcome measurements. Such measurement will provide patients and payers with information on the value and quality of specific occupational therapy services and their overall value to various patient populations.

Macrophages and apoptotic cells in human colorectal tumours.

The numerical relationship between tumour associated macrophages (TAM) and apoptotic cells in 12 human colorectal tumours was evaluated. TAM were labelled immunohistochemically and apoptotic cells were visualized by counterstaining with haematoxylin and eosin (H&E). The stereological techniques, Cavalieri's estimator of volume and the Disector were used to estimate both tumour volume and numerical density of both cell types. The occurrence of TAM per unit volume of tissue increased with increasing tumour volume to a maximum in a tumour of 110.5 cm3, after which numbers declined. Levels of apoptosis also increased with tumour volume though more erratically than levels of TAM and declined for tumour volumes greater than 80 cm3. This is the first report of an attempt to assess the relationship between apoptotic cells and TAM in human tumours.

Health problems encountered by three levels of providers in a remote setting.

A study conducted in the Sioux Lookout Health Zone in northwestern Ontario, Canada analyzed the diagnoses and managements for 139,618 patient visits to three levels of practitioners: physicians, nurse practitioners, and minimally trained health aides. There were major differences between providers in their diagnostic and management patterns. Some of these differences were the result of administrative policy (e.g., physicians and nurses do preventive medicine) but even when adjustment had been made for these differences there was still considerable variation. The minimally trained practitioners made many more signs and symptoms diagnoses and asked for help more frequently. The nurses did much of the preventive measures and made more diagnoses in the supplementary diagnostic class. Physicians diagnosed medically sophisticated conditions more frequently. The physicians did considerable reassuring, suggesting that many cases referred to them were adequately handled. They were also more likely to order investigations.

Provider acceptance of telemedicine systems in remote areas of Ontario.

Telemedicine, the use of telecommunications technology to assist in the delivery of health care, is an increasingly popular solution to some of the problems faced by rural residents in obtaining health care. Since September 1, 1977, the Sioux Lookout Zone in northwestern Ontario has been experimenting with slow-scan video equipment as part of its health care delivery system. The attitudes of the providers who use the system were surveyed. The nurses were positive about slow-scan video as an aid in the delivery of health care; however, the physicians were less enthusiastic. This difference can be explained by physicians' having had more extensive training than the nurses, and therefore not feeling the same need for medical backup and support. Both nurses and physicians had more positive attitudes toward the system after experience with it.