Electrospun Scaffolds of Polylactic Acid, Collagen, and Amorphous Calcium Phosphate for Bone Repair.

Most electrospun scaffolds for bone tissue engineering typically use hydroxyapatite (HA) or beta tricalcium phosphate (ß-TCP). However, the biological activity of these crystalline compounds can be limited due to their low solubility. Therefore, amorphous calcium phosphate (ACP) may be an alternative in bone repair scaffolds. This study analyzes the morphology, porosity, mechanical strength, and surface chemistry of electrospun scaffolds composed of polylactic acid and collagen integrated with hydroxyapatite (MHAP) or amorphous calcium phosphate (MACP). In addition, the in vitro biocompatibility, osteogenic differentiation, and growth factor production associated with bone repair using human Wharton's jelly-derived mesenchymal stem cells (hWJ-MSCs) are evaluated. The results show that the electrospun MHAP and MACP scaffolds exhibit a fibrous morphology with interconnected pores. Both scaffolds exhibit favorable biocompatibility and stimulate the proliferation and osteogenesis of hWJ-MSCs. However, cell adhesion and osteocalcin production are greater in the MACP scaffold compared to the MHAP scaffold. In addition, the MACP scaffold shows significant production of bone-repair-related growth factors such as transforming growth factor-beta 1 (TGF-ß1), providing a solid basis for its use in bone tissue engineering.

Bioengineered skin constructs based on mesenchymal stromal cells and acellular dermal matrix exposed to inflammatory microenvironment releasing growth factors involved in skin repair.

BACKGROUND: Skin tissue engineering is a rapidly evolving field of research that effectively combines stem cells and biological scaffolds to replace damaged tissues. Human Wharton's jelly mesenchymal stromal cells (hWJ-MSCs) are essential to generate tissue constructs, due to their potent immunomodulatory effects and release of paracrine factors for tissue repair. Here, we investigated whether hWJ-MSC grown on human acellular dermal matrix (hADM) scaffolds and exposed to a proinflammatory environment maintain their ability to produce in vitro growth factors involved in skin injury repair and promote in vivo wound healing. METHODS: We developed a novel method involving physicochemical and enzymatic treatment of cadaveric human skin to obtain hADM scaffold. Subsequently, skin bioengineered constructs were generated by seeding hWJ-MSCs on the hADM scaffold (construct 1) and coating it with human platelet lysate clot (hPL) (construct 2). Either construct 1 or 2 were then incubated with proinflammatory cytokines (IL-1α, IL-1ß, IL-6, TNF-α) for 12, 24, 48, 72 and 96 h. Supernatants from treated and untreated constructs and hWJ-MSCs on tissue culture plate (TCP) were collected, and concentration of the following growth factors, bFGF, EGF, HGF, PDGF, VEGF and Angiopoietin-I, was determined by immunoassay. We also asked whether hWJ-MSCs in the construct 1 have potential toward epithelial differentiation after being cultured in an epithelial induction stimulus using an air-liquid system. Immunostaining was used to analyze the synthesis of epithelial markers such as filaggrin, involucrin, plakoglobin and the mesenchymal marker vimentin. Finally, we evaluated the in vivo potential of hADM and construct 1 in a porcine full-thickness excisional wound model. RESULTS: We obtained and characterized the hADM and confirmed the viability of hWJ-MSCs on the scaffold. In both constructs without proinflammatory treatment, we reported high bFGF production. In contrast, the levels of other growth factors were similar to the control (hWJ-MSC/TCP) with or without proinflammatory treatment. Except for PDGF in the stimulated group. These results indicated that the hADM scaffold maintained or enhanced the production of these bioactive molecules by hWJ-MSCs. On the other hand, increased expression of filaggrin, involucrin, and plakoglobin and decreased expression of vimentin were observed in constructs cultured in an air-liquid system. In vivo experiments demonstrated the potential of both hADM and hADM/hWJ-MSCs constructs to repair skin wounds with the formation of stratified epithelium, basement membrane and dermal papillae, improving the appearance of the repaired tissue. CONCLUSIONS: hADM is viable to fabricate a tissue construct with hWJ-MSCs able to promote the in vitro synthesis of growth factors and differentiation of these cells toward epithelial lineage, as well as, promote in a full-thickness skin injury the new tissue formation. These results indicate that hADM 3D architecture and its natural composition improved or maintained the cell function supporting the potential therapeutic use of this matrix or the construct for wound repair and providing an effective tissue engineering strategy for skin repair.

In vitro and in vivo evaluation of electrospun poly (ε-caprolactone)/collagen scaffolds and Wharton's jelly mesenchymal stromal cells (hWJ-MSCs) constructs as potential alternative for skin tissue engineering.

Dermal substitutes bear a high clinical demand because of their ability to promote the healing process of cutaneous wounds by reducing the healing time the appearance and improving the functionality of the repaired tissue. Despite the increasing development of dermal substitutes, most of them are only composed of biological or biosynthetic matrices. This demonstrates the need for new developments focused on using scaffolds with cells (tissue construct) that promote the production of factors for biological signaling, wound coverage, and general support of the tissue repair process. Here, we fabricate by electrospinning two scaffolds: poly(ε-caprolactone) (PCL) as a control and poly(ε-caprolactone)/collagen type I (PCol) in a ratio lower collagen than previously reported, 19:1, respectively. Then, characterize their physicochemical and mechanical properties. As we bear in mind the creation of a biologically functional construct, we characterize and assess in vitro the implications of seeding human Wharton's jelly mesenchymal stromal cells (hWJ-MSCs) on both scaffolds. Finally, to determine the potential functionality of the constructs in vivo, their efficiency was evaluated in a porcine biomodel. Our findings demonstrated that collagen incorporation in the scaffolds produces fibers with similar diameters to those in the human native extracellular matrix, increases wettability, and enhances the presence of nitrogen on the scaffold surface, improving cell adhesion and proliferation. These synthetic scaffolds improved the secretion of factors by hWJ-MSCs involved in skin repair processes such as b-FGF and Angiopoietin I and induced its differentiation towards epithelial lineage, as shown by the increased expression of Involucrin and JUP. In vivo experiments confirmed that lesions treated with the PCol/hWJ-MSCs constructs might reproduce a morphological organization that seems relatively equivalent to normal skin. These results suggest that the PCol/hWJ-MSCs construct is a promising alternative for skin lesions repair in the clinic.

Strategy for the Generation of Engineered Bone Constructs Based on Umbilical Cord Mesenchymal Stromal Cells Expanded with Human Platelet Lysate.

Umbilical cord mesenchymal stromal cells (UC-MSC) are promising candidates for cell therapy due to their potent multilineage differentiation, enhanced self-renewal capacity, and immediate availability for clinical use. Clinical experience has demonstrated satisfactory biosafety profiles and feasibility of UC-MSC application in the allogeneic setting. However, the use of UC-MSC for bone regeneration has not been fully established. A major challenge in the generation of successful therapeutic strategies for bone engineering lies on the combination of highly functional proosteogenic MSC populations and bioactive matrix scaffolds. To address that, in this study we proposed a new approach for the generation of bone-like constructs based on UC-MSC expanded in human platelet lysate (hPL) and evaluated its potential to induce bone structures in vivo. In order to obtain UC-MSC for potential clinical use, we first assessed parameters such as the isolation method, growth supplementation, microbiological monitoring, and cryopreservation and performed full characterization of the cell product including phenotype, growth performance, tree-lineage differentiation, and gene expression. Finally, we evaluated bone-like constructs based on the combination of stimulated UC-MSC and collagen microbeads for in vivo bone formation. UC-MSC were successfully cultured from 100% of processed UC donors, and efficient cell derivation was observed at day 14 ± 3 by the explant method. UC-MSC maintained mesenchymal cell morphology, phenotype, high cell growth performance, and probed multipotent differentiation capacity. No striking variations between donors were recorded. As expected, UC-MSC showed tree-lineage differentiation and gene expression profiles similar to bone marrow- and adipose-derived MSC. Importantly, upon osteogenic and endothelial induction, UC-MSC displayed strong proangiogenic and bone formation features. The combination of hPL-expanded MSC and collagen microbeads led to bone/vessel formation following implantation into an immune competent mouse model. Collectively, we developed a high-performance UC-MSC-based cell manufacturing bioprocess that fulfills the requirements for human application and triggers the potency and effectivity of cell-engineered scaffolds for bone regeneration.

Liver sinusoidal endothelial cells promote lymphohematopoietic differentiation from murine embryonic stem cells: role of soluble factors.

Liver sinusoid endothelial cells (LSEC) constitute an in vitro and in vivo microenvironment for the proliferation and differentiation of hematopoietic stem cells (HSC). Previously, we have shown that LSEC support the survival and growth of murine embryonic stem cells (ESC). In this study, we investigated the capacity of LSEC to promote hematopoietic differentiation from the murine ESC cell line, CGR8. Undifferentiated ESC were cultured on LSEC monolayers in the absence of exogenous cytokines. After 10 and 20 days, cells were harvested and examined by their morphology, phenotype and capacity of hematopoietic colony formation. Microscopic observation of LSEC/ESC cocultures showed the presence of cobblestone areas formation, which indicates active hematopoiesis. Morphological analysis of cell from these foci showed the presence of hematopoietic cells at different stages of differentiation. Cells expressing B lymphoid markers (B220 and CD19) were detected by flow cytometry, and clonogenic assays showed the formation of CFU-pre B colonies. Similar results were observed when ESC were cultured with LSEC conditioned media. Myeloid precursors were also detected by the presence of CFU-GM colonies and cells expressing myeloid markers. These results indicate that LSEC provided an in vitro microenvironment mainly for B cell development, but also myeloid differentiation from ESC. Coculture of ESC with LSEC may constitute a very powerful tool to study the mechanisms involved in B cell generation from ESC.

Liver sinusoidal endothelial cells support the survival and undifferentiated growth of the CGR8 mouse embryonic stem cell line: possible role of leukemia inhibitory factor (LIF).

Murine embryonic stem cells (muESC) are maintained and expanded in vitro by culturing in the presence of leukemia inhibitory factor (LIF) or by coculturing on murine embryonic fibroblast (MEF). Previously we have shown that liver sinusoidal endothelial cells (LSEC) promote the survival, proliferation and differentiation of hematopoietic stem cells. In the present study we investigated whether LSEC might promote the survival and undifferentiated growth of muESC. For these purposes, muESC (CGR8 cell line) were cultured on LSEC monolayers (muESC/LSEC) or in the presence of conditioned medium from LSEC cultures (muESC/LSEC-CM), both in the absence of LIF. Microscopic observation showed the growth of undifferentiated ESC colonies in both muESC/LSEC or muESC/LSEC-CM cultures. A significant reduction in the growth of undifferentiated ESC colonies was observed when ESC were cultured in LSEC-CM previously incubated with anti-LIF. RT-PCR and Western blot analysis showed that LSEC constitutively express LIF at the mRNA and protein level. At different times of culture, muESC were harvested and analyzed for the expression of embryonic markers (SSEA-1 and Oct-4) and differentiation capacity. Flow cytometry analysis showed the presence of a higher percentage of muESC (>90%) expressing SSEA-1 in muESC/LSEC-CM, as compared with muESC/LSEC cocultures. muESC obtained from both types of cultures formed embryoid bodies in vitro, and form teratomas in testicles of mice. These results provide the first evidence that LSEC support the in vitro survival, self-renewal, undifferentiated growth and differentiation capacity of the muESC CGR8 cell line.