A Comparative Study of the Resorption and Immune Response for Two Starch-Based Hemostat Powders.

INTRODUCTION: Powder hemostats are valuable adjuncts to minimize intraoperative and postoperative complications. In addition to promotion of rapid coagulation, resorption, and biocompatibility are desirable attributes. Plant starch-based polysaccharide hemostat powders are effective and widely used hemostatic agents, however their source and/or processing can affect characteristics such as in vivo degradability. For example, Arista is a purified/hydrolyzed starch powder that is rapidly resorbed in vivo; whereas PerClot shows slow resorption and preservation of a crystalline form. MATERIALS AND METHODS: In the present study, we compared the cellular response to the hemostatic agents PerClot and Arista both in vitro and in vivo, and used potato starch and urinary bladder extracellular matrix (UBM-ECM) as high crystallinity/slowly resorbable and prohealing controls, respectively. RESULTS: All test articles and their degradation products were cytocompatible in vitro as measured by cell viability and metabolic activity of bone-marrow macrophages. PerClot induced a stronger proinflammatory, M1-like macrophage response in vitro (P < 0.001) than Arista, likely due to differences in source composition. Histologic examination of the in vivo surgical site showed the almost complete degradation of Arista after 12 h (day 0), whereas both PerClot and potato starch were still present at 28 d with crystals identifiable with polarized light microscopy and periodic acid Schiff (PAS) staining. Macrophage phenotype in vivo showed no differences between PerClot and Arista. Collagen deposition and mononuclear cell accumulation consistent with an early foreign body response were present around PerClot and potato starch crystals, whereas no such cell or connective tissue deposition was noted at the site of Arista or UBM-ECM placement.

Culture on Tissue-Specific Coatings Derived from α-Amylase-Digested Decellularized Adipose Tissue Enhances the Proliferation and Adipogenic Differentiation of Human Adipose-Derived Stromal Cells.

While extracellular matrix (ECM)-derived coatings have the potential to direct the response of cell populations in culture, there is a need to investigate the effects of ECM sourcing and processing on substrate bioactivity. To develop improved cell culture models for studying adipogenesis, the current study examines the proliferation and adipogenic differentiation of human adipose-derived stem/stromal cells (ASCs) on a range of ECM-derived coatings. Human decellularized adipose tissue (DAT) and commercially available bovine tendon collagen (COL) are digested with α-amylase or pepsin to prepare the coatings. Physical characterization demonstrates that α-amylase digestion generates softer, thicker, and more stable coatings, with a fibrous tissue-like ultrastructure that is lost in the pepsin-digested thin films. ASCs cultured on the α-amylase-digested ECM have a more spindle-shaped morphology, and proliferation is significantly enhanced on the α-amylase-digested DAT coatings. Further, the α-amylase-digested DAT provides a more pro-adipogenic microenvironment, based on higher levels of adipogenic gene expression, glycerol-3-phosphate dehydrogenase (GPDH) enzyme activity, and perilipin staining. Overall, this study supports α-amylase digestion as a new approach for generating bioactive ECM-derived coatings, and demonstrates tissue-specific bioactivity using adipose-derived ECM to enhance ASC proliferation and adipogenic differentiation.

Extracellular Matrix-Modified Fiber Scaffolds as a Proadipogenic Mesenchymal Stromal Cell Delivery Platform.

Melt electrowriting (MEW) is an additive manufacturing technology that produces readily handleable fibrous scaffolds with controlled geometry to support cell infiltration. Although MEW scaffolds have excellent potential for cell delivery in regenerative medicine applications, studies to date have primarily focused on polymers such as poly(ε-caprolactone) (PCL) that lack bioactive cues to affect cell function. To address this aspect, MEW scaffolds with extracellular matrix (ECM) coatings were developed as a proadipogenic platform for human mesenchymal stromal cells (hMSCs). More specifically, highly flexible PCL scaffolds fabricated through MEW were coated with a complex ECM suspension prepared from human decellularized adipose tissue (DAT), purified fibronectin, or laminin to determine the effects of two key bioactive proteins present within adipose-derived ECM. In vitro studies exploring the response of human bone marrow-derived mesenchymal stromal cells cultured under adipogenic differentiation conditions indicated a high level of differentiation on all substrates studied, including unmodified PCL scaffolds and two-dimensional controls. To more fully assess the intrinsic proadipogenic capacity of the composite biomaterials, a modified culture regime was established that involved a short-term adipogenic induction in differentiation medium, followed by continued culture in maintenance medium supplemented with insulin for up to 3 weeks. Under these conditions, adipogenic differentiation was enhanced on all fiber scaffolds as compared to the tissue culture controls. Notably, the highest adipogenic response was consistently observed on the PCL + DAT scaffolds, based on the analysis of multiple markers including adipogenic gene [lipoprotein lipase, fatty acid binding protein 4 (FABP4), adiponectin, perilipin 1] and protein (FABP4, leptin) expression and intracellular triglyceride accumulation. Taken together, the PCL scaffolds incorporating DAT provide an adipoinductive microenvironment for the hMSCs, with particular applicability of this cell-instructive delivery platform for applications in plastic and reconstructive surgery.

Investigating the Effects of Tissue-Specific Extracellular Matrix on the Adipogenic and Osteogenic Differentiation of Human Adipose-Derived Stromal Cells Within Composite Hydrogel Scaffolds.

While it has been postulated that tissue-specific bioscaffolds derived from the extracellular matrix (ECM) can direct stem cell differentiation, systematic comparisons of multiple ECM sources are needed to more fully assess the benefits of incorporating tissue-specific ECM in stem cell culture and delivery platforms. To probe the effects of ECM sourced from decellularized adipose tissue (DAT) or decellularized trabecular bone (DTB) on the adipogenic and osteogenic differentiation of human adipose-derived stem/stromal cells (ASCs), a novel detergent-free decellularization protocol was developed for bovine trabecular bone that complemented our established detergent-free decellularization protocol for human adipose tissue and did not require specialized equipment or prolonged incubation times. Immunohistochemical and biochemical characterization revealed enhanced sulphated glycosaminoglycan content in the DTB, while the DAT contained higher levels of collagen IV, collagen VI and laminin. To generate platforms with similar structural and biomechanical properties to enable assessment of the compositional effects of the ECM on ASC differentiation, micronized DAT and DTB were encapsulated with human ASCs within methacrylated chondroitin sulfate (MCS) hydrogels through UV-initiated crosslinking. High ASC viability (>90%) was observed over 14 days in culture. Adipogenic differentiation was enhanced in the MCS+DAT composites relative to the MCS+DTB composites and MCS controls after 14 days of culture in adipogenic medium. Osteogenic differentiation studies revealed a peak in alkaline phosphatase (ALP) enzyme activity at 7 days in the MCS+DTB group cultured in osteogenic medium, suggesting that the DTB had bioactive effects on osteogenic protein expression. Overall, the current study suggests that tissue-specific ECM sourced from DAT or DTB can act synergistically with soluble differentiation factors to enhance the lineage-specific differentiation of human ASCs within 3-D hydrogel systems.

Decellularized Adipose Tissue Scaffolds for Soft Tissue Regeneration and Adipose-Derived Stem/Stromal Cell Delivery.

Surgically discarded adipose tissue is not only an abundant source of multipotent adipose-derived stem/stromal cells (ASCs) but can also be decellularized to obtain a biomimetic microenvironment for tissue engineering applications. The decellularization methods involve processing excised fat through a series of chemical, mechanical, and enzymatic treatment stages designed to extract cells, cellular components, and lipid from the tissues. This process yields a complex 3D bioscaffold enriched in collagens that mimics the biochemical and biomechanical properties of the native extracellular matrix (ECM). For ASC culture and delivery, decellularized adipose tissue (DAT) provides a cell-supportive platform that is conducive to adipogenesis. While DAT can be applied in its intact form as an off-the-shelf adipogenic matrix, it can also be used as an ECM source for the fabrication of an array of other scaffold formats including adipose ECM-derived microcarriers and porous foams. In this chapter, we describe the methods developed in our lab to decellularize human adipose tissue and to further process it into a variety of scaffolding materials for a range of applications in soft tissue regeneration, wound healing, and cell culture.

Composite Bioscaffolds Incorporating Decellularized ECM as a Cell-Instructive Component Within Hydrogels as In Vitro Models and Cell Delivery Systems.

Decellularized tissues represent promising biomaterials, which harness the innate capacity of the tissue-specific extracellular matrix (ECM) to direct cell functions including stem cell proliferation and lineage-specific differentiation. However, bioscaffolds derived exclusively from decellularized ECM offer limited versatility in terms of tuning biomechanical properties, as well as cell-cell and cell-ECM interactions that are important mediators of the cellular response. As an alternative approach, in the current chapter we describe methods for incorporating cryo-milled decellularized tissues as a cell-instructive component within a hydrogel carrier designed to crosslink under mild conditions. This composite strategy can enable in situ cell encapsulation with high cell viability, allowing efficient seeding with a homogeneous distribution of cells and ECM. Detailed protocols are provided for the effective decellularization of human adipose tissue and porcine auricular cartilage, as well as the cryo-milling process used to generate the ECM particles. Further, we describe methods for synthesizing methacrylated chondroitin sulphate (MCS) and for performing UV-initiated and thermally induced crosslinking to form hydrogel carriers for adipose and cartilage regeneration. The hydrogel composites offer great flexibility, and the hydrogel phase, ECM source, particle size, cell type(s) and seeding density can be tuned to promote the desired cellular response. Overall, these systems represent promising platforms for the development of tissue-specific 3-D in vitro cell culture models and in vivo cell delivery systems.

Decellularized Matrices As Cell-Instructive Scaffolds to Guide Tissue-Specific Regeneration.

Decellularized scaffolds are promising clinically translational biomaterials that can be applied to direct cell responses and promote tissue regeneration. Bioscaffolds derived from the extracellular matrix (ECM) of decellularized tissues can naturally mimic the complex extracellular microenvironment through the retention of compositional, biomechanical, and structural properties specific to the native ECM. Increasingly, studies have investigated the use of ECM-derived scaffolds as instructive substrates to recapitulate properties of the stem cell niche and guide cell proliferation, paracrine factor production, and differentiation in a tissue-specific manner. Here, we review the application of decellularized tissue scaffolds as instructive matrices for stem or progenitor cells, with a focus on the mechanisms through which ECM-derived scaffolds can mediate cell behavior to promote tissue-specific regeneration. We conclude that although additional preclinical studies are required, ECM-derived scaffolds are a promising platform to guide cell behavior and may have widespread clinical applications in the field of regenerative medicine.