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.
An additional pathology should be considered for Achillodynia differentials – the intratendinous tear (ITT) – for which we describe symptoms, ultrasound findings and co-presenting pathology. Examinations of 740 Achillodynic patients in one specialist centre were reviewed. ITTs were defined as a clearly visualised echopoor area situated centrally and extending to, but not through the tendon periphery, with pain on palpation and no clinical findings consistent with Achilles rupture. Descriptive statistics were used to analyse differences between pathological sub-groups, and images described qualitatively.Introduction
Materials and Methods
Achilles tendinopathy (AT) is a highly prevalent injury in athletes and non-athletes with an unknown aetiology. Genetic risk factors have been a recent focus of investigation. The aim of this systematic review was to determine which loci have been linked with mid-portion AT and could potentially be used as biomarkers in tendinopathy risk models or as preventative or therapeutic targets. Eight electronic bibliographic databases were searched from inception to April 2015 for cross-sectional, prospective cohort and case-control studies that included empirical research investigating genes associated with mid-portion AT. Potential publications were assessed by two independent reviewers (AAC and PRJ) for inclusion and quality. Quality was evaluated using a validated scale.Introduction
Materials and Methods
Rotator cuff healing after an arthroscopic repair is discussible because of the high incidence of failures. Among biologic augmentations currently used, platelet-rich plasma (PRP) is one of the most applied, supposed to enhance and accelerate the healing process in different musculoskeletal disorders. However, the evidence supporting its successful administration is still lacking, especially in the field of the rotator cuff repair. Our purpose is to clarify if the recovery is accelerated and the integrity of repaired construct is increased in patients undergoing PRP injections after arthroscopic repair of the rotator cuff. Thirty-eight patients with full-thickness rotator cuff tears have been enrolled after they had been informed about the use of PRP and the timing of its application postoperatively. Seventeen patients underwent arthroscopic rotator cuff repair and PRP injections (3 injections at 10 days each other), 21 underwent arthroscopic rotator cuff repair without PRP injections. Outcomes were assessed preoperatively, at 3, 6, 12, and minimum 16 months after surgery (average 17.7 +/− 1.7 months). Constant system, the University of California at Los Angeles (UCLA) system and a Visual Analogue Scale (VAS) scale were used; range of motion and strength in all planes were also assessed. The healing of the repair was assessed at magnetic resonance imaging at a minimum follow up of 6 months from surgery. All patients had the same rehabilitation protocol.Introduction
Patients & Methods
recent studies recognised metabolic abnormalities as additional factors in the development of rotator cuff (RC) tendinopathy. It has been hypothesised that the insertional area of this tendon is susceptible to degenerative changes due to intrinsic hypovascularization. The mechanisms underlying this process are not yet clear. In this study we attempted to confirm if larger lesions of the RC are related to impaired vasodilatatory response of the local circulation in conditions of “hemodynamic stress”. it was assumed that impaired vasal reaction to “hemodynamic stress” was a systemic condition. This phenomenon should therefore be not limited to the critical area of the tendon tear. Given this assumption post-ischemic vasodilation of brachial artery was studied through an echo-doppler (US) evaluation. 50 patients (mean 61 ± 4, range 50–65) all scheduled for surgical rotator cuff repair following a tendon tear, were enrolled. Three preoperative measurements of the brachial artery diameter before and after application of an ischemic band were collected. The size of the lesions was later assessed at the time of surgery. A statistical analysis was carried on to investigate the correlation between US assessment of brachial artery diameter and the corresponding size of the RC lesions. UCLA and ASES scores were also measured to assess clinical and functional outcomes.Introduction
Patients & Methods
Ostochondral lesion of the knee is a common cause of chronic knee pain. Arthroscopic treatment with subcondral microfracture is a widespread technique leading to noticeable improvement of knee function and pain. To improve the effectiveness of this treatment options, we thought to add intra (PRF) or post-operative (PRP) growth factors. Platelet rich plasma (PRP) is obtained by centrifugation of the blood to produce a plasma with high concentration of platelets and growth factors. This latter represents a promising method to manage degenerative cartilage lesion and can be used postoperatively to improve clinical results of patients treated arthroscopically. Platelet Rich Fibrin (PRF) has been presented as a second-generation platelet concentrate, and it is used intraoperatively to cover the microfracuteres’ holes. No literature was found about using of PRF intraoperative in association with arthroscopic microfracture technique. The aim of this study is to compare clinical outcomes of the treatment of knee osteochondral lesion using arthroscopic microfracture technique alone or in association with PRF Intraoperative application using “Vivostat” system or with PRP “ReGen Lab” postoperative injection. 90 patients with clinical and radiographic evidence of osteochondral lesion of the medial or lateral compartment of the knee were enrolled. All patients received arthroscopic debridement and Microfractures and were randomised into 3 groups: 30 patients received microfractures and intraoperative PRF “Vivostat” injection(Group A), 30 patients received microfracture and 3 intra-articular injections of 5.5 mL PRP “Regen”(Group B), 30 patients received microfracture only. IKDC, KOOS and VAS score were administered to all patients before starting the treatment, at 1, 6 and 12 months from the end of the management.Introduction
Patients & Methods
