Development of secretome-based strategies to improve cell culture protocols in tissue engineering.

Advances in skin tissue engineering have promoted the development of artificial skin substitutes to treat large burns and other major skin loss conditions. However, one of the main drawbacks to bioengineered skin is the need to obtain a large amount of viable epithelial cells in short periods of time, making the skin biofabrication process challenging and slow. Enhancing skin epithelial cell cultures by using mesenchymal stem cells secretome can favor the scalability of manufacturing processes for bioengineered skin. The effects of three different types of secretome derived from human mesenchymal stem cells, e.g. hADSC-s (adipose cells), hDPSC-s (dental pulp) and hWJSC-s (umbilical cord), were evaluated on cultured skin epithelial cells during 24, 48, 72 and 120 h to determine the potential of this product to enhance cell proliferation and improve biofabrication strategies for tissue engineering. Then, secretomes were applied in vivo in preliminary analyses carried out on Wistar rats. Results showed that the use of secretomes derived from mesenchymal stem cells enhanced currently available cell culture protocols. Secretome was associated with increased viability, proliferation and migration of human skin epithelial cells, with hDPSC-s and hWJSC-s yielding greater inductive effects than hADSC-s. Animals treated with hWJSC-s and especially, hDPSC-s tended to show enhanced wound healing in vivo with no detectable side effects. Mesenchymal stem cells derived secretomes could be considered as a promising approach to cell-free therapy able to improve skin wound healing and regeneration.

Evaluation of myopic cornea lenticules. A histochemical and clinical correlation.

In this work, we have analyzed the main clinical and corneal histological parameters that may be associated to the spherical equivalent (SE), age and gender of individuals with myopic refractive errors. For this purpose, 108 cornea stroma lenticules were obtained from patients subjected to ReLEx-SMILE myopia correction. Histological analyses were carried out and histochemistry and immunohistochemistry were used to quantify key histological components of the cornea stroma, including mature collagen fibers, reticular and elastic fibers, glycoproteins, proteoglycans, type-V collagen and several crystallins. Clinical and histological data were analyzed to determine their association with SE, age and gender. Results showed a significant correlation between the age range of the patients and the expression of crystallins CRY-α-A, CRY-λ1 and type-V collagen and between CRY-λ1 and corneal thickness, spherical diopters (D) and SE, although correlation between CRY-λ1 and SE was non-significant when age was controlled. Comparison of cases with low myopia and high/moderate myopia found statistical differences for D and lenticule thickness and diameter. The binary logistic regression analysis allowed us to construct a model using two clinical parameters (D and lenticule thickness). Parameters showing significant correlation with the age were the corneal radius, keratometry reading (K), OZ, CRY-α-A and type-V collagen, whereas SE, lenticule thickness, OZ, CRY-λ1 and type-V collagen showed statistically significant differences between the youngest and the oldest patients. A binary logistic regression analysis model was generated including 3 variables (D, cornea radius and OZ). No gender differences were found. The specific clinical and histological modifications found to be associated to the SE and age could be useful for a better understanding of the mechanisms involved in the genesis or progression of myopia and could establish the basement for future therapeutic options.

Effective use of mesenchymal stem cells in human skin substitutes generated by tissue engineering.

Mesenchymal stem cells (MSCs) can differentiate toward epithelial cells and may be used as an alternative source for generation of heterotypical artificial human skin substitutes, thus, enhancing their development and translation potential to the clinic. The present study aimed at comparing four types of heterotypical human bioengineered skin generated using MSCs as an alternative epithelial cell source. Adipose-tissue-derived stem cells (ADSCs), dental pulp stem cells (DPSCs), Wharton's jelly stem cells (WJSCs) and bone marrow stem cells (BMSCs) were used for epidermal regeneration on top of dermal skin substitutes. Heterotypic human skin substitutes were evaluated before and after implantation in immune-deficient athymic mice for 30 d. Histological and genetic studies were performed to evaluate extracellular matrix synthesis, epidermal differentiation and human leukocyte antigen (HLA) molecule expression. The four cell types differentiated into keratinocytes, as shown by the expression of cytokeratin 10 and filaggrin 30 d post-grafting; also, they induced dermal fibroblasts responsible for the synthesis of extracellular fibrillar and non-fibrillar components, in a similar way among each other. WJSCs and BMSCs showed higher expression of cytokeratin 10 and filaggrin, suggesting these cells were more prone to epidermal regeneration. The absence of HLA molecules, even when the epithelial layer was differentiated, supports the future clinical use of these substitutes - especially ADSCs, DPSCs and WJSCs - with low rejection risk. MSCs allowed the generation of bioengineered human skin substitutes with potential clinical usefulness. According to their epidermal differentiation potential and lack of HLA antigens, WJSCs should preferentially be used.

Characterization of the human ridged and non-ridged skin: a comprehensive histological, histochemical and immunohistochemical analysis.

The structure of the human skin is directly dependent on its location and the mechanical forces to which it is subjected. In the present work, we have performed a comprehensive analysis of the human ridged and non-ridged skin to identify the differences and similarities between both skin types. For this purpose, human skin samples were obtained from dorsal hand skin (DHS), palmar hand skin (PHS), dorsal foot skin (DFS) and plantar foot skin (PFS) from the same cadaveric donors. Histological, histochemical and semiquantitative and quantitative immunohistochemical analyses were carried out to evaluate the epidermis, dermis and basement membrane. Results show that the epithelial layer of ridged skin had larger cell number and size than non-ridged skin for most strata. Melanocytes and Langerhans cells were more abundant in non-ridged skin, whereas Merkel cells were preferentially found in ridged skin. The expression pattern of CK5/6 was slightly differed between non-ridged and ridged skin. Involucrin expression was slightly more intense in non-ridged skin than in ridged skin. Collagen was more abundant in foot skin dermis than in hand skin, and in ridged skin as compared to non-ridged skin. Elastic fibers were more abundant in DHS. Biglycan was more abundant in foot skin than in hand skin. No differences were found for blood and lymphatic vessels. The basement membrane laminin was preferentially found in foot skin. These results revealed important differences at the epithelial, dermal and basement membrane levels that could contribute to a better knowledge of the human skin histology.

Membranes derived from human umbilical cord Wharton's jelly stem cells as novel bioengineered tissue-like constructs.

Cell-derived matrices were recently described as novel biomaterials generated by human cells allowed to grow and synthetize their own extracellular matrix in culture. In the present work, we generated and evaluated a novel tissue-like substitute (WDM) consisting of a membrane derived from cultured human Wharton's jelly stem cells. WDM were evaluated ex vivo and in vivo by histochemistry and immunohistochemistry for several mesenchymal cell markers and fibrillar and non-fibrillar extracellular matrix components. Results show that WDM were heterogeneous and consisted of dense cell-poor areas surrounded by cell-rich zones with abundant HWJSC. Histological analyses demonstrated that cell-poor areas were very rich in fibrillar and non-fibrillar extracellular matrix components such as collagen and proteoglycans, and cells in the WDM were highly viable and mostly PCNA-positive. HWJSC in the WDM expressed all markers of this cell type, including CD90, CD105, pan cytokeratin and CK8. In vivo analysis showed that the WDM was highly biocompatible and grafting this membrane in the muscle of laboratory rats was not associated to increased inflammation, necrosis, tumorigenesis or other side effects, while cells properly integrated at the damage site and showed high proliferation index. These results suggest that the structure and composition of the extracellular matrix of these novel WDM could reproduce the situation of native human tissues and that WDM implanted in vivo are highly biocompatible and rapidly integrate in the host tissues. For these reasons, we hypothesize that WDM could be used in regenerative medicine protocols.

Bioactive injectable aggregates with nanofibrous microspheres and human dental pulp stem cells: A translational strategy in dental endodontics.

Regeneration of the pulp-dentin complex with stem cells is a potential alternative to conventional root canal treatments. Human dental pulp stem cells (hDPSCs) have been extensively studied because of their ability to proliferate and differentiate into mineralized dental and non-dental tissues. Here we combined hDPSCs with two types of injectable poly-l-lactic acid (PLLA) microsphere with a nanofibrous or smooth surface to form bioactive injectable aggregates, and examined their ability to promote pulp regeneration in the root canal in an in vivo model. We investigated the biocompatibility, biosafety and odontogenic potential of fibrous (F-BIM) and smooth bioactive injectable microspheres (S-BIM) in vitro and in vivo. Our results demonstrated that PLLA microspheres and hDPSCs were able to form bioactive injectable aggregates that promoted dentin regeneration in both in vitro and in vivo models. Our results suggest that F-BIM and S-BIM may induce dentinogenesis upon in vivo grafting, and propose that the potential usefulness of the microsphere-hDPSC aggregates described here should be evaluated in clinical settings. Copyright © 2017 John Wiley & Sons, Ltd.

Development of a multilayered palate substitute in rabbits: a histochemical ex vivo and in vivo analysis.

Current tissue engineering technology focuses on developing simple tissues, whereas multilayered structures comprising several tissue types have rarely been described. We developed a highly biomimetic multilayered palate substitute with bone and oral mucosa tissues using rabbit cells and biomaterials subjected to nanotechnological techniques based on plastic compression. This novel palate substitute was autologously grafted in vivo, and histological and histochemical analyses were used to evaluate biointegration, cell function, and cell differentiation in the multilayered palate substitute. The three-dimensional structure of the multilayered palate substitute was histologically similar to control tissues, but the ex vivo level of cell and tissue differentiation were low as determined by the absence of epithelial differentiation although cytokeratins 4 and 13 were expressed. In vivo grafting was associated with greater cell differentiation, epithelial stratification, and maturation, but the expression of cytokeratins 4, 13, 5, and 19 at did not reach control tissue levels. Histochemical analysis of the oral mucosa stroma and bone detected weak signals for proteoglycans, elastic and collagen fibers, mineralization deposits and osteocalcin in the multilayered palate substitute cultured ex vivo. However, in vivo grafting was able to induce cell and tissue differentiation, although the expression levels of these components were always significantly lower than those found in controls, except for collagen in the bone layer. These results suggest that generation of a full-thickness multilayered palate substitute is achievable and that tissues become partially differentiated upon in vivo grafting.

Human Dental Pulp Stem Cells. A promising epithelial-like cell source.

Several models of tissue-engineered human skin based on three-dimensional (3D) co-culture techniques have been proposed to the date. However, normal skin biopsies are not always available, especially in patients with a high percentage of skin affected by deep burning, and the generation of large amounts of cultured keratinocytes may take very long time, with an associated risk for the patients' survival. For those reasons, the search of alternative cell sources for tissue reconstruction is a clinical need. In this context, Human Dental Pulp Stem Cells (HDPSC) have the potential to differentiate into multiple cell lineages by the appropriate differentiation conditions, but skin epidermis differentiation has not been demonstrated so far. Here, we hypothesize that HDPSC may have pluripotent differentiation capability, and may be able to differentiate into skin epithelial keratinocytes in culture using organotypic 3D models based on the interaction with the subjacent dermal fibroblasts. By using HDPSC, the problems associated to the donor site availability and the proliferation capability of the epithelial cells could be solved. The rapid accessibility to these cells could be translated to a more immediate generation of a bioengineered human skin substitute for the future clinical treatment.

Sequential keratinocytic differentiation and maturation in a three-dimensional model of human artificial oral mucosa.

BACKGROUND AND OBJECTIVE: Oral mucosa shortage may limit or condition some clinical approaches in maxillofacial, periodontal and implant treatment. The availability of a human oral mucosa model generated by tissue engineering could help clinicians to address the lack of oral mucosa. In this work, we carried out a sequential maturation and differentiation study of the epithelial cell layer of an artificial human oral mucosa substitute based on fibrin-agarose biomaterials with fibroblasts and keratinocytes. MATERIAL AND METHODS: Histological, immunohistochemical and gene expression analyses were carried out in artificial human oral mucosa models developed and cultured for 1, 2 and 3 wk. RESULTS: Artificial oral mucosa models showed expression of tight junction proteins and cytokeratins from the first week of in vitro development. Mature samples of 3 wk of development subjected to air-liquid conditions showed signs of epithelial differentiation and expressed specific RNAs and proteins corresponding to adherent and gap junctions and basement lamina. Moreover, these mature samples overexpressed some desmosomal and tight junction transcripts, with gap junction components being downregulated. CONCLUSION: These results suggest that bioengineered human oral mucosa substitutes form a well-developed epithelial layer that was very similar to human native tissues. In consequence, the epithelial layer could be fully functional in these oral mucosa substitutes, thus implying that these tissues may have clinical usefulness.

Expression of epithelial markers by human umbilical cord stem cells. A topographical analysis.

INTRODUCTION: Human umbilical cord stem cells have inherent differentiation capabilities and potential usefulness in regenerative medicine. However, the epithelial differentiation capability and the heterogeneity of these cells have not been fully explored to the date. METHODS: We analyzed the expression of several undifferentiation and epithelial markers in cells located in situ in different zones of the umbilical cord -in situ analysis- and in primary ex vivo cell cultures of Wharton's jelly stem cells by microarray and immunofluorescence. RESULTS: Our results demonstrated that umbilical cord cells were heterogeneous and had intrinsic capability to express in situ stem cell markers, CD90 and CD105 and the epithelial markers cytokeratins 3, 4, 7, 8, 12, 13, 19, desmoplakin and zonula occludens 1 as determined by microarray and immunofluorescence, and most of these markers remained expressed after transferring the cells from the in situ to the ex vivo cell culture conditions. However, important differences were detected among some cell types in the umbilical cord, with subvascular zone cells showing less expression of stem cell markers and cells in Wharton's jelly and the amnioblastic zones showing the highest expression of stem cells and epithelial markers. CONCLUSIONS: These results suggest that umbilical cord mesenchymal cells have intrinsic potential to express relevant epithelial markers, and support the idea that they could be used as alternative cell sources for epithelial tissue engineering.