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.

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.

Human embryonic stem cells (hESCs) are conventionally expanded and maintained in vitro on biological substrates. Synthetic electrospun polymer nanofibers have the potential to act as non-biological substrates in the culture of hESCs. Three synthetic, FDA approved polymers: poly- ɛ -caprolactone (PCL), poly-L-lactic acid (PLLA) and poly lactic-co-glycolic acid (PLGA) were electrospun as nanofibers (random or aligned conformations) on glass coverslips and their supportive role in hESC culture examined. Clonogenicity experiments demonstrated that nanofibrous scaffolds (PCL aligned and random, PLLA aligned and PLGA aligned) supported hESC adhesion and expansion. A significantly greater number of colonies were observed on PCL-aligned nanofibrous scaffolds in comparison to PLLA-aligned and PLGA-aligned substrates ( p < 0.05). hESC colonies were significantly larger on PCL aligned nanofibrous substrates when compared to other polymer substrates ( p   < 0.05–0.001), where fiber diameter played a pivotal role in support of hESC clonogenicity (on PCL). Retention of pluripotentiality was confirmed by expression of Alkaline phosphatase, OCT-3/4 and Nanog expression on PCL scaffolds and the expression of transcripts representative of mesoderm (ACTC1), ectoderm (SOX1) and endoderm (AFP) during subsequent spontaneous in vitro differentiation. These results demonstrate the potential of nanofibers as xeno-free scaffolds supportive of hESC adhesion, self-renewal and differentiation in in vitro culture conditions.

Tendon tissue engineering draws on regenerative medicine principles, offering innovative solutions to address the challenges posed by tendon injuries and degenerative conditions. Tendons’ inherent limited regenerative capacity often hinders complete recovery from injuries, leading to chronic conditions and impaired functionality. Autologous mesenchymal/stromal stem cells (MSCs) and tendon‐derived stem cells (TSCs), combined with growth factors (GFs) like GDF‐5, GDF‐6 and GDF‐7, are emerging as potential therapies for tendinopathy. These GFs are crucial for tendon development and promoting tenogenic differentiation, though the exact pathways they activate remain unclear. For this reason, directly comparing all three pathways to assess their impact on both MSCs and TSCs is essential. This study examined the effects of GDF‐5, GDF‐6 and GDF‐7 on tenogenic differentiation in MSCs and TSCs, with a focus on how oxygen levels (21% O ₂ vs. physoxia at 2% O ₂ ) influence this process. The expression profiles of key tenogenic genes (Scleraxis [Scx], Tenomodulin [Tnmd], Thrombospondin‐4 [Thromb‐4] and Tenascin‐C [Tnc‐C]) were explored by quantitative reverse transcription PCR (RT‐qPCR) following supplementation with individual GFs. Transcriptional analysis was complemented by Tnmd immunofluorescence (IF) and image analysis to identify optimal differentiation parameters. The study highlighted GDF‐7 as a powerful inducer of tenocyte‐like cell differentiation in MSCs, showcasing sustained expression of tenogenic genes over time in 21% O ₂ . Moreover, TSCs in physoxia differentiate into tenocytes without an additional GF requirement. In conclusion, the study lays a foundation for understanding the complex interplay of GFs, oxygen levels and cellular responses in the quest for tendon regeneration. In doing so, it establishes that different cell types have differing biochemical requirements for induction of tenogenic differentiation. While offering promising avenues for tissue engineering platforms, it underscores the need for further research to fully harness the potential of MSCs and TSCs in vivo for tendon regeneration.

Tendon injuries caused by overuse or age-related deterioration are frequent. Incomplete knowledge of somatic tendon cell biology and their progenitors has hindered interventions for the effective repair of injured tendons. Here, we sought to compare and contrast distinct tendon-derived cell populations: type I and II tendon stem cells (TSCs) and tenocytes (TNCs). Porcine type I and II TSCs were isolated via the enzymatic digestion of distinct membranes (paratenon and endotenon, respectively), while tenocytes were isolated through an explant method. Resultant cell populations were characterized by morphology, differentiation, molecular, flow cytometry, and immunofluorescence analysis. Cells were isolated, cultured, and evaluated in two alternate oxygen concentrations (physiological (2%) and air (21%)) to determine the role of oxygen in cell biology determination within this relatively avascular tissue. The different cell populations demonstrated distinct proliferative potential, morphology, and transcript levels (both for tenogenic and stem cell markers). In contrast, all tendon-derived cell populations displayed multipotent differentiation potential and immunophenotypes (positive for CD90 and CD44). Type II TSCs emerged as the most promising tendon-derived cell population for expansion, given their enhanced proliferative potential, multipotency, and maintenance of a tenogenic profile at early and late passage. Moreover, in all cases, physoxia promoted the enhanced proliferation and maintenance of a tenogenic profile. These observations help shed light on the biological mechanisms of tendon cells, with the potential to aid in the development of novel therapeutic approaches for tendon disorders.

Tendon injuries significantly impact quality of life, prompting the exploration of innovative solutions beyond conventional surgery. Extracellular Vesicles (EVs) have emerged as a promising strategy to enhance tendon regeneration. In this study, human Tendon Stem/Progenitor Cells (TSPCs) were isolated from surgical biopsies and cultured in a Growth-Differentiation Factor-5-supplemented medium to promote tenogenic differentiation under static and dynamic conditions using a custom-made perfusion bioreactor. Once at 80% confluence, cells were transitioned to a serum-free medium for conditioned media collection. Ultracentrifugation revealed the presence of vesicles with a 10⁶ particles/mL concentration and sub-200nm diameter size. Dynamic culture yielded a 3-fold increase in EV protein content compared to static culture, as confirmed by Western-blot analysis. Differences in surface marker expression were also shown by flow cytometric analysis. Data suggest that we efficiently developed a protocol for extracting EVs from human TSPCs, particularly under dynamic conditions. This approach enhances EV protein content, offering potential therapeutic benefits for tendon regeneration. However, further research is needed to fully understand the role of EVs in tendon regeneration.