Spontaneous muscle regenerative potential is limited, as severe injuries incompletely recover and result in chronic inflammation. Current therapies are restricted to conservative management, not providing a complete restitutio ad integrum; therefore, alternative therapeutic strategies are welcome, such as cell-based therapies with stem cells or Peripheral Blood Mononuclear Cells (PBMCs). Here, we described two different in vitro myogenic models: a 2D perfused system and a 3D bioengineered scaffold within a perfusion bioreactor. Both models were assembled with human bone marrow-derived mesenchymal stem cells (hBM-MSCs) and human primary skeletal myoblasts (hSkMs) to study induction and maintenance of myogenic phenotype in presence of PBMCs. When hBM-MSCs were cultured with human primary skeletal myoblasts (hSkMs) in medium supplemented with 10 ng/mL of bFGF; cells showed increased expression of myogenic-related gene, such as Desmin and Myosin Heavy Chain II (MYH2) after 21 days, and a prevalent expression of anti-inflammatory cytokines (IL10, 15-fold). Next, PBMCs were added in an upper transwell chamber and hBM-MSCs significantly upregulated myogenic genes throughout the culture period, while pro-inflammatory cytokines (e.g., IL12A) were downregulated. In 3D, hBM-MSCs plus hSkMs embedded in fibrin-based scaffolds, cultured in dynamic conditions, showed that all myogenic-related genes tended to be upregulated in the presence of PBMCs, and Desmin and MYH2 were also detected at protein level, while pro-inflammatory cytokine genes were significantly downregulated in the presence of PBMCs. In conclusion, our works suggest that hBM-MSCs have a versatile myogenic potential, enhanced and modulated by PMBCs. Moreover, our 3D biomimetic approach seemed to better resemble the tissue architecture allowing an efficient in vitro cellular cross-talk.

Poor tendon repair is an unsolved issue in clinical practice, due to complex tendon structure. Tendon stem/progenitor cells (TSPCs) play key roles in homeostasis, regeneration, and inflammation regulation in acute tendon injuries, and rely on TGF-β signaling for recruitment into degenerative tendons. In this study, we aimed to develop an in vitro model for tenogenesis adopting a dynamic culture of a fibrin 3D scaffold, bioengineered with human TSPCs collected from both healthy and tendinopathic surgery explants (Review Board prot./SCCE n.151, 29 October 2020). 3D culture was maintained for 21 days under perfusion provided by a custom-made bioreactor, in a medium supplemented with hTGF-β1 at 20 ng/mL. The data collected suggested that the 3D in vitro model well supported survival of both pathological and healthy cells, and that hTGF-β signaling, coupled to a dynamic environment, promoted differentiation events. However, pathological hTSPCs showed a different expression pattern of tendon-related genes throughout the culture and an impaired balance of pro-inflammatory and anti-inflammatory cytokines, compared to healthy hTSPCs, as indicated by qRT-PCT and immunofluorescence analyses. Additionally, the expression of both tenogenic and cytokine genes in hTSPCs was influenced by hTGF-β1, indicating that the environment assembled was suitable for studying tendon stem cells differentiation. The study offers insights into the use of 3D cultures of hTSPCs as an in vitro model for investigating their behavior during tenogenic events and opens perspectives for following the potential impact on resident stem cells during regeneration and healing events.

Tendon injuries are a common problem that can significantly impact an individual's quality of life. While traditional surgical methods have been used to address this issue, Extracellular Vesicles (EVs) have emerged as a promising approach to promote tendon repair and regeneration mechanisms, as they deliver specific biological signals to neighbouring cells. In this study, we extracted human Tendon Progenitor Stem cells (hTPSCs) from surgery explants and isolated their EVs from perfused and static media.

hTPSCs were isolated from tendon surgery biopsy (Review Board prot./SCCE n.151, 29/10/2020) and cultured in both static and dynamic conditions, using a perfusion bioreactor (1ml/min). When cells reached 80% confluence, they were switched into a serum-free medium for 24 hours for EVs-production. Conditioned media was ultra-centrifuged for 90min (100000g). The recovered pellet was then characterized by size and concentration (Nanosight NS300), Zeta potential (Mastersizer S), morphology (SEM and TEM) and protein quantification.

hTPSCs stemness and multipotency were confirmed through CD73, CD90, and CD105 expression and confirmation of quad-lineage (adipo-osteo-chondro-teno) differentiation. After 7 days, hTPSCs were ready for EVs-production. Ultracentrifugation revealed the presence of particles with a concentration of 7×107 particles/mL consistent across both cultures. Further characterization indicated that EVs collected from perfused conditions displayed an elevated vesicle mean size (mean 143±6.5 nm) in comparison to static conditions (mean 112±7.4 nm). Consistent with, but not in proportion with, the above protein content was measured at 20 ng/ml (dynamic) and 7 ng/mL (static) indicating a nearly 3-fold increase in concentration associated with a ~22% increase in particle size.

Proposed data showed that sub-200 diameter vesicles were successfully collected from multipotent hTPSCs starvation, and the vesicle size and protein concentration were compatible with established EV literature; furthermore, dynamic culture conditions seemed more suitable for EVs-production. Further characterization will be required to better understand, EVs-compositions and their role in tendon regenerative events.