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.

Introduction: Emerging therapies for regenerating skeletal tissues are focused on the repair of pathologically altered tissue by the transplantation of functionally competent cells and supportive matrices. Stem cells have the potential to differentiate into musculoskeletal tissue and may be the optimal cell source for such therapies. In vitro studies have demonstrated the ability of adult bone marrow stromal cells (MSC) and human embryonic stem cells (hES) to generate bone, but little is known regarding their potential to repair bone in vivo. Preclinical studies in animal models will allow investigation into the extent that regenerated tissue resembles functional and healthy tissue, and its potential clinical application.

Aim: To assess whether adult and embryonic stem cells maintained their ability to form musculoskeletal tissues in vivo using diffusion chambers implanted into the peritoneal cavity of nude mice. Currently, ongoing experiments are assessing the use of MSCs and hES cells to regenerate bone in a rodent preclinical model.

Methods: MSC cells and embryoid body-derived H9 hES cells were prepared as previously described (Haynesworth et al Bone 1992; Sottile et al Cloning Stem Cells 2003). Groups of cells were left untreated or pre-treated with osteogenic (OS) media for 5 days. Study 1: Single cell suspensions of untreated or pre-treated cells were injected into diffusion chambers which were implanted intraperitonealy into nude mice and left for 79 days. Study 2: OS pre-treated cells were implanted into an experimentally created full thickness calvarial defect in adult male Wistar rats. The defect area was left empty or filled with demineralised bone matrix (DBM: Allosource®) alone or with DBM/MSCs or DBM/hES composite. Tissues were collected 4 weeks after surgery.

Analysis: Histological and immunochemical techniques were used to evaluate cell phenotypes and the contribution of transplanted cells to tissue repair.

Results: Study 1: Both hES (in 2/3 chambers) and MSC (3/3) cells pre-treated with OS media formed only mineralised bone. No cartilage was detected in these OS pre-treated cells. Untreated hES cells formed both mineralised bone and cartilage within the chambers (2/3). In contrast, untreated MSC cells (3/3) produced no mineralised bone or cartilage. Preliminary analysis demonstrated the absence of any other tissue type in the diffusion chambers. Study 2: Active bone regeneration was observed at the edges of the calvarial defect after 4 weeks, with a high density of cells present within the MSC or hES/DBM composite. No signs of local cellular immunological response were seen.

Summary: OS pre-treatment restricted differentiation towards the osteoblast lineage in both hES and MSC cells indicating successful directed differentiation in vivo. Untreated hES and MSC cells produce a different range of cell phenotypes suggesting that the two cell sources represent cells at a different stage of commitment in a common cell lineage or cells derived from two distinct cell lineages. New bone formation was seen at the site of the calvarial defect in the presence OS pre-treated MSC and hES cells suggesting that these cells may support in vivo bone repair in a preclinical model. Funded by Geron Corporation