“…[25][26][27][28][29] A key choice in the development of a tissue-engineered implant is whether to engineer a cellularized construct. [29][30][31][32][33][34][35][36] Cellularized scaffolds may be particularly important when the cells are necessary for maintenance of the surrounding matrix or have an important function, for example, preservation of the antithrombogenicity of a vascular conduit via endothelization or simply to aid continual growth in young recipients. 30,31 Acellular scaffolds have the advantage of delivery as medical devices and avoid the regulatory issues associated with the delivery of living constructs, which require long-term culture in bioreactors.…”
Previously, we have described the development of an acellular porcine meniscal scaffold. The aims of this study were to determine the immunocompatibility of the scaffold and capacity for cellular attachment and infiltration to gain insight into its potential for meniscal repair and replacement. Porcine menisci were decellularized by exposing the tissue to freeze-thaw cycles, incubation in hypotonic tris buffer, 0.1% (w/v) sodium dodecyl sulfate in hypotonic buffer plus protease inhibitors, nucleases, hypertonic buffer followed by disinfection using 0.1% (v/v) peracetic, and final washing in phosphate-buffered saline. In vivo immunocompatibility was assessed after implantation of the acellular meniscal scaffold subcutaneously into galactosyltransferase knockout mice for 3 months in comparison to fresh and acellular tissue treated with a-galactosidase (negative control). The cellular infiltrates in the explants were assessed by histology and characterized using monoclonal antibodies against: CD3, CD4, CD34, F4/80, and C3c. Static culture was used to assess the potential of acellular porcine meniscal scaffold to support the attachment and infiltration of primary human dermal fibroblasts and primary porcine meniscal cells in vitro. The explants were surrounded by capsules that were more pronounced for the fresh meniscal tissue compared to the acellular tissues. Cellular infiltrates compromised mononuclear phagocytes, CD34-positive cells, and nonlabeled fibroblastic cells. T-lymphocytes were sparse in all explanted tissue types and there was no evidence of C3c deposition. The analysis revealed an absence of a specific immune response to all of the implanted tissues. Acellular porcine meniscus was shown to be capable of supporting the attachment and infiltration of primary human fibroblasts and primary porcine meniscal cells. In conclusion, acellular porcine meniscal tissue exhibits excellent immunocompatibility and potential for cellular regeneration in the longer term.
“…[25][26][27][28][29] A key choice in the development of a tissue-engineered implant is whether to engineer a cellularized construct. [29][30][31][32][33][34][35][36] Cellularized scaffolds may be particularly important when the cells are necessary for maintenance of the surrounding matrix or have an important function, for example, preservation of the antithrombogenicity of a vascular conduit via endothelization or simply to aid continual growth in young recipients. 30,31 Acellular scaffolds have the advantage of delivery as medical devices and avoid the regulatory issues associated with the delivery of living constructs, which require long-term culture in bioreactors.…”
Previously, we have described the development of an acellular porcine meniscal scaffold. The aims of this study were to determine the immunocompatibility of the scaffold and capacity for cellular attachment and infiltration to gain insight into its potential for meniscal repair and replacement. Porcine menisci were decellularized by exposing the tissue to freeze-thaw cycles, incubation in hypotonic tris buffer, 0.1% (w/v) sodium dodecyl sulfate in hypotonic buffer plus protease inhibitors, nucleases, hypertonic buffer followed by disinfection using 0.1% (v/v) peracetic, and final washing in phosphate-buffered saline. In vivo immunocompatibility was assessed after implantation of the acellular meniscal scaffold subcutaneously into galactosyltransferase knockout mice for 3 months in comparison to fresh and acellular tissue treated with a-galactosidase (negative control). The cellular infiltrates in the explants were assessed by histology and characterized using monoclonal antibodies against: CD3, CD4, CD34, F4/80, and C3c. Static culture was used to assess the potential of acellular porcine meniscal scaffold to support the attachment and infiltration of primary human dermal fibroblasts and primary porcine meniscal cells in vitro. The explants were surrounded by capsules that were more pronounced for the fresh meniscal tissue compared to the acellular tissues. Cellular infiltrates compromised mononuclear phagocytes, CD34-positive cells, and nonlabeled fibroblastic cells. T-lymphocytes were sparse in all explanted tissue types and there was no evidence of C3c deposition. The analysis revealed an absence of a specific immune response to all of the implanted tissues. Acellular porcine meniscus was shown to be capable of supporting the attachment and infiltration of primary human fibroblasts and primary porcine meniscal cells. In conclusion, acellular porcine meniscal tissue exhibits excellent immunocompatibility and potential for cellular regeneration in the longer term.
“…7). 132,133,140,205 Removed skin was placed over a transilluminator and digital pictures were taken to calculate vessel area and length in a semi-automated way. Altman et al used a comparable method, but did not quantify the vasculature.…”
Section: Transillumination For Observation Of Vascularizationmentioning
Cutaneous wounding often leads to contraction and scarring, which may result in a range of functional, cosmetic, and psychological complications. Tissue-engineered skin substitutes are being developed to enhance restoration of the skin and improve the quality of wound healing. The aim of this review is to provide researchers in the field of tissue engineering an overview of the methods that are currently used to clinically evaluate skin wound healing, and methods that are used to evaluate tissue-engineered constructs in animal models. Clinically, the quality of wound healing is assessed by noninvasive subjective scar assessment scales and objective techniques to measure individual scar features. Alternatively, invasive technologies are used. In animal models, most tissue-engineered skin constructs studied are at least evaluated macroscopically and by using conventional histology (hematoxylin-eosin staining). Planimetry and immunohistochemistry are also often applied. An overview of antibodies used is provided. In addition, some studies used methods to assess gene expression levels and mRNA location, transillumination for blood vessel observation, in situ/in vivo imaging, electron microscopy, mechanical strength assessment, and microbiological sampling. A more systematic evaluation of tissue-engineered skin constructs in animal models is recommended to enhance the comparison of different constructs, thereby accelerating the trajectory to application in human patients. This would be further enhanced by the embracement of more clinically relevant objective evaluation methods. In addition, fundamental knowledge on construct-mediated wound healing may be increased by new developments in, for example, gene expression analysis and noninvasive imaging.
“…[34][35][36] Vasculature did weakly correlate to BLI signal behavior in the alginate constructs over the 30-min imaging window, suggesting an impact of vascular volume on luciferin availability within the construct. Interestingly, greater vasculature correlated to a delayed rise in BLI signal following luciferin delivery initially, but then accelerated BLI signal increase between 10 and 30 min.…”
The use of multicomponent scaffolds for cell implantation has necessitated sophisticated techniques for tracking of cell survival in vivo. Bioluminescent imaging (BLI) has emerged as a noninvasive tool for evaluating the therapeutic potential of cell-based tissue engineering strategies. However, the ability to use BLI measurements to longitudinally assess large 3D cellular constructs in vivo and the effects of potential confounding factors are poorly understood. In this study, luciferase-expressing human mesenchymal stem cells (hMSCs) were delivered subcutaneously within agarose and RGD-functionalized alginate hydrogel vehicles to investigate the impact of construct composition and tissue formation on BLI signal. Results showed that alginate constructs exhibited twofold greater BLI counts than agarose constructs at comparable hMSC doses. However, each hydrogel type produced a linear correlation between BLI counts and live cell number, indicating that within a given material, relative differences in cell number could be accurately assessed at early time points. The survival efficiency of delivered hMSCs was highest for the lower cell doses embedded within alginate matrix. BLI signal remained predictive of live cell number through 1 week in vivo, although the strength of correlation decreased over time.
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