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Engineered dermal templates have revolutionised the repair and reconstruction of skin defects. Their interaction with the wound microenvironment and linked molecular mediators of wound repair is still not clear. This study investigated the wound bed and acellular &ldquo, off the shelf&rdquo, dermal template interaction in a mouse model. Full-thickness wounds in nude mice were grafted with allogenic skin, and either collagen-based or fully synthetic dermal templates. Changes in the wound bed showed significantly higher vascularisation and fibroblast infiltration in synthetic grafts when compared to collagen-based grafts (P &le, 0.05). Greater tissue growth was associated with higher prostaglandin-endoperoxide synthase 2 (Ptgs2) RNA and cyclooxygenase-2 (COX-2) protein levels in fully synthetic grafts. Collagen-based grafts had higher levels of collagen III and matrix metallopeptidase 2. To compare the capacity to form a double layer skin substitute, both templates were seeded with human fibroblasts and keratinocytes (so-called human skin equivalent or HSE). Mice were grafted with HSEs to test permanent wound closure with no further treatment required. We found the synthetic dermal template to have a significantly greater capacity to support human epidermal cells. In conclusion, the synthetic template showed advantages over the collagen-based template in a short-term mouse model of wound repair.

Cultured epithelial autograft (CEA) was the birth of skin tissue engineering and encompassed methodologies for the isolation and expansion of autologous basal keratinocytes for burn treatment that are still practiced at some specialised units around the world. One of the limitations of CEA, however, is the reliance on animal-derived material during the manufacturing process and despite all efforts to date, no xeno-free alternative with proven efficacy has been reported. Here, we investigate whether human-derived fibroblast feeder cells and human serum can sufficiently and effectively provide a suitable microenvironment for adult keratinocyte isolation and expansion. Human dermal fibroblasts and epidermal keratinocytes were isolated from discarded skin during abdominoplasty and breast reduction procedures and cultured in xeno-free conditions. We report that these xeno-free adult keratinocytes form similar numbers of colony-forming units as those cultured using the Green's methods; however, xeno-free keratinocytes express lower levels of α6 integrin (CD49f; a progenitor and stem cell marker). We identified IL-8 as a potential growth factor secreted by adult human fibroblasts that may enhance keratinocyte colony formation in human serum. Finally, we propose a step-by-step xeno-free isolation and cultivation methodology for adult keratinocytes that can be tested further in serial cultivation for clinical application.

Background Artificial dermal templates play an important role in physiologic wound closure after injury. In addition to contributing to stable, durable and flexible wound closure, they provide a scaffold for tissue repair. Several dermal templates are commercially available, with animal-derived Integra® dermal regeneration template perhaps the most widely used. NovoSorb™ Biodegradable Temporising Matrix (BTM) is a fully synthetic alternative that eliminates any risk of cross-species residual antigenicity. In this study, we aimed to compare early response after application of NovoSorb™ BTM with Integra® in terms of temporary wound closure, host cell infiltration, neovascularisation and collagen deposition in a mouse model. Methods Twenty athymic nude mice received full-thickness skin excision followed by grafting of the dermal template (n = 10 NovoSorb™ BTM, n = 10 Integra®), with the grafts excised and assessed after two weeks. Results All twenty mice achieved temporary wound closure with no evidence of wound contracture. Microscopically, all twenty grafts became infiltrated with host cells along the entire length of the template, with NovoSorb™ BTM demonstrating a particular abundance of host inflammatory cells. Evidence of new collagen deposition and neovascularisation was observed in both templates, with NovoSorb™ BTM demonstrating a more extensive vascular network at this time point. However, a greater inflammatory response was also observed in the NovoSorb™ BTM grafts at this time point. Conclusions In this study, NovoSorb™ BTM demonstrates favourable properties as a dermal template, but further investigation is required to assess the significance of the differing inflammatory and vascular response to its implantation compared with Integra®.

Tissue-engineered composite skin is a promising therapy for the treatment of chronic and acute wounds, including burns. Providing the wound bed with a dermal scaffold populated by autologous dermal and epidermal cellular components can further entice host cell infiltration and vascularization to achieve permanent wound closure in a single stage. However, the high porosity and the lack of a supportive basement membrane in most commercially available dermal scaffolds hinders organized keratinocyte proliferation and stratification in vitro and may delay re-epithelization in vivo. The objective of this study was to develop a method to enable the in vitro production of a human skin equivalent (HSE) that included a porous scaffold and dermal and epidermal cells expanded ex vivo, with the potential to be used for definitive treatment of skin defects in a single procedure. A collagen-glycosaminoglycan dermal scaffold (Integra(®)) was populated with adult fibroblasts. A near-normal skin architecture was achieved by the addition of coagulated human plasma to the fibroblast-populated scaffold before seeding cultured keratinocytes. This resulted in reducing scaffold pore size and improving contact surfaces. Skin architecture and basement membrane formation was further improved by the addition of aprotinin (a serine protease inhibitor) to the culture media to inhibit premature clot digestion. Histological assessment of the novel HSE revealed expression of keratin 14 and keratin 10 similar to native skin, with a multilayered neoepidermis morphologically comparable to human skin. Furthermore, deposition of collagen IV and laminin-511 were detected by immunofluorescence, indicating the formation of a continuous basement membrane at the dermal-epidermal junction. The proposed method was efficient in producing an in vitro near native HSE using the chosen off-the-shelf porous scaffold (Integra). The same principles and promising outcomes should be applicable to other biodegradable porous scaffolds, combined with autologous cells, for use in wound treatment.

Preclinical animal models implicate serotonin neurons in the pathophysiology of levodopa (L- dopa)-induced dyskinesias in Parkinson's disease (PD), but effective treatment remains elusive. We examined the relationship between serotonin and L-dopa-induced dys- kinesias in a pathologically confirmed cohort of PD patients. We obtained brain tissue from 44 PD cases and 17 age-matched controls and assessed monoamine lev- els and the serotonin and dopamine transporters in the striatum, and the extent of dopaminergic and serotoner- gic cell preservation in the substantia nigra (SN) and the dorsal raphe nuclei (DRN), respectively. As expected, PD patients demonstrated a severe loss of all dopaminergic markers, including dopamine (P < 0.0001) and the dopa- mine transporter (P < 0.0001) in the striatum, and dopa- minergic neurons (P < 0.001) in the SN, compared with controls. Marked serotonin loss was observed in the cau- date (but not putamen) in PD patients compared with controls (P < 0.001), but no difference was found in the levels of the serotonin transporter in the striatum or den- sity of serotonergic neurons in the DRN between these groups, suggesting a functional but not structural change in the serotonergic system in PD. No difference was seen in levels of serotonergic and dopaminergic markers in the striatum between PD patients with and without dyskine- sias, or between cases separated according to the clini- cal severity of their dyskinesias. The absence of a correlation between striatal serotonin markers and the incidence and severity of L-dopa-induced dyskinesias suggests that an intact and functioning serotonergic sys- tem is not a risk factor for developing dyskinesias in PD. V C 2015 International Parkinson and Movement Disorder

Characteristic tau isoform composition of the insoluble fibrillar tau inclusions define tauopathies, including Alzheimer's disease (AD), progressive supranuclear palsy (PSP) and frontotemporal dementia with parkinsonism linked to chromosome 17/frontotemporal lobar degeneration-tau (FTDP-17/FTLD-tau). Exon 10 splicing mutations in the tau gene, MAPT, in familial FTDP-17 cause elevation of tau isoforms with four microtubule-binding repeat domains (4R-tau) compared to those with three repeats (3R-tau). On the basis of two well-characterised monoclonal antibodies against 3R- and 4R-tau, we developed novel, sensitive immuno-PCR assays for measuring the trace amounts of these isoforms in CSF. This was with the aim of assessing if CSF tau isoform changes reflect the pathological changes in tau isoform homeostasis in the degenerative brain and if these would be relevant for differential clinical diagnosis. Initial analysis of clinical CSF samples of PSP (n = 46), corticobasal syndrome (CBS; n = 22), AD (n = 11), Parkinson's disease with dementia (PDD; n = 16) and 35 controls revealed selective decreases of immunoreactive 4R-tau in CSF of PSP and AD patients compared with controls, and lower 4R-tau levels in AD compared with PDD. These decreases could be related to the disease-specific conformational masking of the RD4-binding epitope because of abnormal folding and/or aggregation of the 4R-tau isoforms in tauopathies or increased sequestration of the 4R-tau isoforms in brain tau pathology.

Levodopa-induced dyskinesias (LID) represent a substantial barrier to effective symptomatic management of Parkinson's disease, but current treatment options for this debilitating side effect are limited, despite an increasing understanding of their pathophysiology from animal models. Increasing evidence suggests that serotonin neurons have a pivotal role in the induction and maintenance of dyskinesias, and provide a promising target for anti-dyskinetic therapies. Here, we review the evidence for serotonergic involvement in dyskinesias from animal and human data, and highlight some of the translational gaps which may explain why the success of serotonin autoreceptor agonists as anti-dyskinetic agents in experimental models has failed to be replicated in clinical trials.