Dose-Response Tendon-Specific Markers Induction by Growth Differentiation Factor-5 in human bone marrow and umbilical cord Mesenchymal Stem Cells

Mesenchymal stem cells derived from human bone marrow (hBM-MSCs) are utilized in tendon tissue-engineering protocols while extra-embryonic cord-derived, including from Wharton's Jelly (hWJ-MSCs), are emerging as useful alternatives. To explore the tenogenic responsiveness of hBM-MSCs and hWJ-MSCs to human Growth Differentiation Factor 5 (hGDF-5) we supplemented each at doses of 1, 10, and 100 ng/mL of hGDF-5 and determined proliferation, morphology and time-dependent expression of tenogenic markers. We evaluated the expression of collagen types 1 (COL1A1) and 3 (COL3A1), Decorin (DCN), Scleraxis-A (SCX-A), Tenascin-C (TNC) and Tenomodulin (TNMD) noting the earliest and largest increase with 100 ng/mL. With 100 ng/mL, hBM-MSCs showed up-regulation of SCX-A (1.7-fold) at Day 1, TNC (1.3-fold) and TNMD (12-fold) at Day 8. hWJ-MSCs, at the same dose, showed up-regulation of COL1A1 (3-fold), DCN (2.7-fold), SCX-A (3.8-fold) and TNC (2.3-fold) after three days of culture. hWJ-MSCs also showed larger proliferation rate and marked aggregation into a tubular-shaped system at Day 7 (with 100 ng/mL of hGDF-5). Simultaneous to this, we explored the expression of pro-inflammatory (IL-6, TNF, IL-12A, IL-1β) and anti-inflammatory (IL-10, TGF-β1) cytokines across for both cell types. hBM-MSCs exhibited a better balance of pro-inflammatory and anti-inflammatory cytokines up-regulating IL-1β (11-fold) and IL-10 (10-fold) at Day 8; hWJ-MSCs, had a slight expression of IL-12A (1.5-fold), but a greater up-regulation of IL-10 (2.5-fold). Type 1 collagen and tenomodulin proteins, detected by immunofluorescence, confirming the greater protein expression when 100 ng/mL were supplemented. In the same conditions, both cell types showed specific alignment and shape modification with a length/width ratio increase, suggesting their response in activating tenogenic commitment events, and they both potential use in 3D in vitro tissue-engineering protocols

Chondrogenic Commitment of Human Bone Marrow Mesenchymal Stem Cells in a Perfused Collagen Hydrogel Functionalized with hTGF-β1-Releasing PLGA Microcarrier

Tissue engineering strategies can be relevant for cartilage repair and regeneration. A collagen matrix was functionalized with the addition of poly-lactic-co-glycolic acid microcarriers (PLGA-MCs) carrying a human Transforming Growth Factor β1 (hTFG-β1) payload, to provide a 3D biomimetic environment with the capacity to direct stem cell commitment towards a chondrogenic phenotype. PLGA-MCs (mean size 3 ± 0.9 μm) were prepared via supercritical emulsion extraction technology and tailored to sustain delivery of payload into the collagen hydrogel for 21 days. PLGA-MCs were coseeded with human Bone Marrow Mesenchymal Stem Cells (hBM-MSCs) in the collagen matrix. Chondrogenic induction was suggested when dynamic perfusion was applied as indicated by transcriptional upregulation of COL2A1 gene (5-fold; p < 0.01) and downregulation of COL1A1 (0.07-fold; p < 0.05) and COL3A1 (0.11-fold; p < 0.05) genes, at day 16, as monitored by qRT-PCR. Histological and quantitative-immunofluorescence (qIF) analysis confirmed cell activity by remodeling the synthetic extracellular matrix when cultured in perfused conditions. Static constructs lacked evidence of chondrogenic specific gene overexpression, which was probably due to a reduced mass exchange, as determined by 3D system Finite Element Modelling (FEM) analysis. Proinflammatory (IL-6, TNF, IL-12A, IL-1β) and anti-inflammatory (IL-10, TGF-β1) cytokine gene expression by hBM-MSC was observed only in dynamic culture (TNF and IL-1β 10-fold, p < 0.001; TGF-β1 4-fold, p < 0.01 at Day 16) confirming the cells' immunomodulatory activity mainly in relation to their commitment and not due to the synthetic environment. This study supports the use of 3D hydrogel scaffolds, equipped for growth factor controlled delivery, as tissue engineered models for the study of in vitro chondrogenic differentiation and opens clinical perspectives for injectable collagen-based advanced therapy systems

3D Biomimetic Scaffold for Growth Factor Controlled Delivery: An In-Vitro Study of Tenogenic Events on Wharton's Jelly Mesenchymal Stem Cells.

The present work described a bio-functionalized 3D fibrous construct, as an interactive teno-inductive graft model to study tenogenic potential events of human mesenchymal stem cells collected from Wharton's Jelly (hWJ-MSCs). The 3D-biomimetic and bioresorbable scaffold was functionalized with nanocarriers for the local controlled delivery of a teno-inductive factor, i.e., the human Growth Differentiation factor 5 (hGDF-5). Significant results in terms of gene expression were obtained. Namely, the up-regulation of Scleraxis (350-fold, p ≤ 0.05), type I Collagen (8-fold), Decorin (2.5-fold), and Tenascin-C (1.3-fold) was detected at day 14; on the other hand, when hGDF-5 was supplemented in the external medium only (in absence of nanocarriers), a limited effect on gene expression was evident. Teno-inductive environment also induced pro-inflammatory, (IL-6 (1.6-fold), TNF (45-fold, p ≤ 0.001), and IL-12A (1.4-fold)), and anti-inflammatory (IL-10 (120-fold) and TGF-β1 (1.8-fold)) cytokine expression upregulation at day 14. The presented 3D construct opens perspectives for the study of drug controlled delivery devices to promote teno-regenerative events