Development of 2-D and 3-D culture platforms derived from decellularized nucleus pulposus.

Bioscaffolds derived from the extracellular matrix (ECM) have shown the capacity to promote regeneration by providing tissue-specific biological instructive cues that can enhance cell survival and direct lineage-specific differentiation. This study focused on the development and characterization of two-dimensional (2-D) and three-dimensional (3-D) cell culture platforms incorporating decellularized nucleus pulposus (DNP). First, a detergent-free protocol was developed for decellularizing bovine nucleus pulposus (NP) tissues that was effective at removing cellular content while preserving key ECM constituents including collagens, glycosaminoglycans, and the cell-adhesive glycoproteins laminin and fibronectin. Next, novel 2-D coatings were generated using the DNP or commercially-sourced bovine collagen type I (COL) as a non-tissue-specific control. In addition, cryo-milled DNP or COL particles were incorporated within methacrylated chondroitin sulphate (MCS) hydrogels as a 3-D cell culture platform for exploring the effects of ECM particle composition. Culture studies showed that the 2-D coatings derived from the DNP could support cell attachment and growth, but did not maintain or rescue the phenotype of primary bovine NP cells, which de-differentiated when serially passaged in monolayer culture. Similarly, while bovine NP cells remained highly viable following encapsulation and 14 days of culture within the hydrogel composites, the incorporation of DNP particles within the MCS hydrogels was insufficient to maintain or rescue changes in NP phenotype associated with extended in vitro culture based on gene expression patterns. Overall, DNP produced with our new decellularization protocol was successfully applied to generate both 2-D and 3-D bioscaffolds; however, further studies are required to assess if these platforms can be combined with additional components of the endogenous NP microenvironment to stimulate regeneration or lineage-specific cell differentiation.

Modular cell-assembled adipose matrix-derived bead foams as a mesenchymal stromal cell delivery platform for soft tissue regeneration.

With the goal of establishing a new clinically-relevant bioscaffold format to enable the delivery of high densities of human adipose-derived stromal cells (ASCs) for applications in soft tissue regeneration, a novel "cell-assembly" method was developed to generate robust 3-D scaffolds comprised of fused networks of decellularized adipose tissue (DAT)-derived beads. In vitro studies confirmed that the assembly process was mediated by remodelling of the extracellular matrix by the seeded ASCs, which were well distributed throughout the scaffolds and remained highly viable after 8 days in culture. The ASC density, sulphated glycosaminoglycan content and scaffold stability were enhanced under culture conditions that included growth factor preconditioning. In vivo testing was performed to compare ASCs delivered within the cell-assembled DAT bead foams to an equivalent number of ASCs delivered on a previously-established pre-assembled DAT bead foam platform in a subcutaneous implant model in athymic nude mice. Scaffolds were fabricated with human ASCs engineered to stably co-express firefly luciferase and tdTomato to enable long-term cell tracking. Longitudinal bioluminescence imaging showed a significantly stronger signal associated with viable human ASCs at timepoints up to 7 days in the cell-assembled scaffolds, although both implant groups were found to retain similar densities of human ASCs at 28 days. Notably, the infiltration of CD31+ murine endothelial cells was enhanced in the cell-assembled implants at 28 days. Moreover, microcomputed tomography angiography revealed that there was a marked reduction in vascular permeability in the cell-assembled group, indicating that the developing vascular network was more stable in the new scaffold format. Overall, the novel cell-assembled DAT bead foams represent a promising platform to harness the pro-regenerative paracrine functionality of human ASCs and warrant further investigation as a clinically-translational approach for volume augmentation.

Adipose Stromal Cells Enhance Decellularized Adipose Tissue Remodeling Through Multimodal Mechanisms.

Decellularized adipose tissue (DAT) scaffolds represent a promising cell-instructive platform for soft tissue engineering. While recent work has highlighted that mesenchymal stromal cells, including adipose-derived stromal cells (ASCs), can be combined with decellularized scaffolds to augment tissue regeneration, the mechanisms involved require further study. The objective of this work was to probe the roles of syngeneic donor ASCs and host-derived macrophages in tissue remodeling of DAT scaffolds within an immunocompetent mouse model. Dual transgenic reporter mouse strains were employed to track and characterize the donor ASCs and host macrophages within the DAT implants. More specifically, ASCs isolated from dsRed mice were seeded on DAT scaffolds, and the seeded and unseeded control scaffolds were implanted subcutaneously into MacGreen transgenic mice for up to 8 weeks. ASC seeding was shown to augment cell infiltration into the DAT implants at 8 weeks, and this was linked to significantly enhanced angiogenesis relative to the unseeded controls. Immunohistochemical staining demonstrated long-term retention of the syngeneic donor ASCs over the duration of the 8-week study, providing evidence that the DAT scaffolds are a cell-supportive delivery platform. Notably, newly formed adipocytes within the DAT implants were not dsRed+, indicating that the donor ASCs supported fat formation through indirect mechanisms. Immunohistochemical tracking of host macrophages through costaining for enhanced green fluorescent protein with the macrophage marker Iba1 revealed that ASC seeding significantly increased the number of infiltrating macrophages within the DAT implants at 3 weeks, while the fraction of macrophages relative to the total cellular infiltrate was similar between the groups at 1, 3, and 8 weeks. Consistent with the tissue remodeling response that was observed, western blotting demonstrated that there was significantly augmented expression of CD163 and CD206, markers of constructive M2-like macrophages, within the ASC-seeded DAT implants. Overall, our results demonstrate that exogenous ASCs enhance tissue regeneration within DAT scaffolds indirectly through multimodal mechanisms that include host cell recruitment and immunomodulation. These data provide further evidence to support the use of decellularized scaffolds as a delivery platform for ASCs in tissue engineering.

Matrix composition in 3-D collagenous bioscaffolds modulates the survival and angiogenic phenotype of human chronic wound dermal fibroblasts.

There is a substantial need for new strategies to stimulate cutaneous tissue repair in the treatment of chronic wounds. To address this challenge, our team is developing modular biomaterials termed "bead foams", comprised of porous beads synthesized exclusively of extracellular matrix (ECM) and assembled into a cohesive three-dimensional (3-D) network. In the current study, bead foams were fabricated from human decellularized adipose tissue (DAT) or commercially-sourced bovine tendon collagen (COL) to explore the effects of ECM composition on human wound edge dermal fibroblasts (weDF) sourced from chronic wound tissues. The DAT and COL bead foams were shown to be structurally similar, but compositionally distinct, containing different levels of glycosaminoglycan content and collagen types IV, V, and VI. In vitro testing under conditions simulating stresses within the chronic wound microenvironment indicated that weDF survival and angiogenic marker expression were significantly enhanced in the DAT bead foams as compared to the COL bead foams. These findings were corroborated through in vivo assessment in a subcutaneous athymic mouse model. Taken together, the results demonstrate that weDF survival and paracrine function can be modulated by the matrix source applied in the design of ECM-derived scaffolds and that the DAT bead foams hold promise as cell-instructive biological wound dressings. STATEMENT OF SIGNIFICANCE: Biological wound dressings derived from the extracellular matrix (ECM) can be designed to promote the establishment of a more permissive microenvironment for healing in the treatment of chronic wounds. In the current work, we developed modular biomaterials comprised of fused networks of porous ECM-derived beads fabricated from human decellularized adipose tissue (DAT) or commercially-available bovine collagen. The bioscaffolds were designed to be structurally similar to provide a platform for investigating the effects of ECM composition on human dermal fibroblasts isolated from chronic wounds. Testing in in vitro and in vivo models demonstrated that cell survival and pro-angiogenic function were enhanced in the adipose-derived bioscaffolds, which contained higher levels of glycosaminoglycans and collagen types IV, V, and VI. Our findings support that the complex matrix composition within DAT can induce a more pro-regenerative cellular response for applications in wound healing.

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.

Enhancing repair of full-thickness excisional wounds in a murine model: Impact of tissue-engineered biological dressings featuring human differentiated adipocytes.

Promotion of skin repair for acute or chronic wounds through the use of tissue-engineered products is an active field of research. This study evaluates the effects mediated by tissue-engineered biological dressings containing human in vitro-differentiated adipocytes and adipose-derived stromal cells (ASCs). Re-epithelialization, granulation tissue formation and neovascularization of full-thickness cutaneous wounds were specifically assessed using a murine model featuring a fluorescent epidermis. In comparison with wounds that did not receive an adipocyte-containing biological dressing, treated wounds displayed a slight but significantly faster wound closure based on macroscopic observations over 18 days. Non-invasive imaging of GFP-expressing keratinocytes determined that the kinetics of re-epithelialization were similar for both groups. Treated wounds featured thicker granulation tissues (1.7-fold, P < 0.0001) enriched in collagens (1.3-fold, P < 0.0104). In addition, wound cryosections labeled for detection of CD31-expressing cells indicated a 2.2-fold (P < 0.0002) increased neovascularization for the treated wounds at the time of terminal biopsy. This is in accordance with the secretion of pro-angiogenic factors detected in media conditioned by the dressings. Taken together, these results establish that a new type of engineered substitutes featuring a mixture of adipocytes and ASCs can promote cutaneous healing when applied as temporary dressings, suggesting their potential relevance for chronic wound management studies.

Using human umbilical cord cells for tissue engineering: a comparison with skin cells.

The epithelial cells and Wharton׳s jelly cells (WJC) from the human umbilical cord have yet to be extensively studied in respect to their capacity to generate tissue-engineered substitutes for clinical applications. Our reconstruction strategy, based on the self-assembly approach of tissue engineering, allows the production of various types of living human tissues such as skin and cornea from a wide range of cell types originating from post-natal tissue sources. Here we placed epithelial cells and WJC from the umbilical cord in the context of a reconstructed skin substitute in combination with skin keratinocytes and fibroblasts. We compared the ability of the epithelial cells from both sources to generate a stratified, differentiated skin-like epithelium upon exposure to air when cultured on the two stromal cell types. Conversely, the ability of the WJC to behave as dermal fibroblasts, producing extracellular matrix and supporting the formation of a differentiated epithelium for both types of epithelial cells, was also investigated. Of the four types of constructs produced, the combination of WJC and keratinocytes was the most similar to skin engineered from dermal fibroblasts and keratinocytes. When cultured on dermal fibroblasts, the cord epithelial cells were able to differentiate in vitro into a stratified multilayered epithelium expressing molecules characteristic of keratinocyte differentiation after exposure to air, and maintaining the expression of keratins K18 and K19, typical of the umbilical cord epithelium. WJC were able to support the growth and differentiation of keratinocytes, especially at the early stages of air-liquid culture. In contrast, cord epithelial cells cultured on WJC did not form a differentiated epidermis when exposed to air. These results support the premise that the tissue from which cells originate can largely affect the properties and homoeostasis of reconstructed substitutes featuring both epithelial and stromal compartments.