Nicholas Robert Forsyth

Tendon injuries affect millions each year, yet effective repair remains limited by the tissue's avascular nature and complex mechanobiology. An anisotropic electrospun scaffold functionalized with GDF‐7‐loaded mesoporous silica nanoparticles (MSNs) is developed to address these challenges. GDF‐7 is efficiently encapsulated (99.9%) in MSNs and incorporated into aligned poly(ε‐caprolactone) (PCL) fibers, achieving controlled release over 46 days following non‐Fickian kinetics. MSN incorporation initially reduces scaffold stiffness and tensile strength; however, dynamic bioreactor culture of human mesenchymal stromal cell (MSCs) and porcine tendon progenitor stem cell (TPSCs) seeded scaffolds, under physiological axial strain, enhances cell alignment, proliferation, and extracellular matrix deposition. Culture under physiological oxygen (2%) conditions further promotes cell viability, proliferation, and tenogenic gene expression. Notably, the combination of topographical guidance, mechanical loading, and physoxia synergistically enhances the expression of terminal tenogenic markers, such as tenomodulin (Tnmd), in both MSCs and TPSCs, as evidenced by increased protein levels detected via immunofluorescence and elevated gene expression confirmed by RT‐qPCR analysis. This integration of topographical, biochemical, and mechanical cues in a tissue engineering approach supports tendon‐specific lineage commitment, offering a promising strategy for functional tendon regeneration.

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.

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.

Telomere repeats protect linear chromosomes from degradation, and telomerase has a prominent role in their maintenance. Telomerase has telomere-independent effects on cell proliferation, DNA replication, differentiation, and tumorigenesis. TERT (telomerase reverse transcriptase enzyme), the catalytic subunit of telomerase, is required for enzyme activity. TERT promoter mutation and methylation are strongly associated with increased telomerase activation in cancer cells. TERT levels and telomerase activity are downregulated in stem cells during differentiation. The link between differentiation and telomerase can provide a valuable tool for the study of the epigenetic regulation of TERT. Oxygen levels can affect cellular behaviors including proliferation, metabolic activity, stemness, and differentiation. The role of oxygen in driving TERT promoter modifications in embryonic stem cells (ESCs) is poorly understood. We adopted a monolayer ESC differentiation model to explore the role of physiological oxygen (physoxia) in the epigenetic regulation of telomerase and TERT. We further hypothesized that DNMTs played a role in physoxia-driven epigenetic modification. ESCs were cultured in either air or a 2% O₂ environment. Physoxia culture increased the proliferation rate and stemness of the ESCs and induced a slower onset of differentiation than in ambient air. As anticipated, downregulated TERT expression correlated with reduced telomerase activity during differentiation. Consistent with the slower onset of differentiation in physoxia, the TERT expression and telomerase activity were elevated in comparison to the air-oxygen-cultured ESCs. The TERT promoter methylation levels increased during differentiation in ambient air to a greater extent than in physoxia. The chemical inhibition of DNMT3B reduced TERT promoter methylation and was associated with increased TERT gene and telomerase activity during differentiation. DNMT3B ChIP (Chromatin immunoprecipitation) demonstrated that downregulated TERT expression and increased proximal promoter methylation were associated with DNMT3B promoter binding. In conclusion, we have demonstrated that DNMT3B directly associates with TERT promoter, is associated with differentiation-linked TERT downregulation, and displays oxygen sensitivity. Taken together, these findings help identify novel aspects of telomerase regulation that may play a role in better understanding developmental regulation and potential targets for therapeutic intervention.

Persistent air leaks caused by thoracic surgery, physical trauma, or spontaneous pneumothoraces are a cause of patient morbidity with need for extended chest tube durations and surgical interventions. Current treatment measures involve mechanical closure of air leaks in the compromised pleura. Organ and membrane decellularisation offers a broad range of biomimetic scaffolds of allogeneic and xenogeneic origins, exhibiting innate tissue‐specific characteristics. We explored a physicochemical method for decellularising porcine pleural membranes (PPM) as potential tissue‐engineered surrogates for lung tissue repair. Decellularised PPM (dPPM) was characterised with histology, quantitative assays, mechanical testing, and sterility evaluation. Cytotoxicity and recellularisation assays assessed biocompatibility of decellularised PPM (dPPM). Haematoxylin and Eosin (H&E) staining showed an evident reduction in stained nuclei in the dPPM, confirmed with nuclear staining and analysis ( ^∗∗∗∗p < 0.0001). Sulphated glycosaminoglycans (sGAG) and collagen histology demonstrated minimal disruption to the gross structural assembly of core extracellular matrix (ECM) in dPPM. Confocal imaging demonstrated realignment of ECM fibres in dPPM against native control. Quantitative analysis defined a significant change in the angular distribution ( ^∗∗∗∗p < 0.0001) and coherence ( ^∗∗∗p < 0.001) of fibre orientations in dPPM versus native ECM. DNA quantification indicated ≥85% reduction in native nuclear dsDNA in dPPM ( ^∗∗p < 0.01). Collagen and sGAG quantification indicated reductions of both ( ^∗∗p < 0.01). dPPM displayed increased membrane thickness ( ^∗∗∗p < 0.001). However, Young’s modulus (459.67 ± 10.36 kPa) and ultimate tensile strength (4036.22 ± 155.1 kPa) of dPPM were comparable with those of native controls at (465.82 ± 10.51 kPa) and (3912.9 ± 247.42 kPa), respectively. In vitro cytotoxicity and scaffold biocompatibility assays demonstrated robust human mesothelial cell line (MeT‐5A) attachment and viability. DNA quantification in reseeded dPPM with MeT‐5A cells exhibited significant increase in DNA content at day 7 ( ^∗∗p < 0.01) and day 15 ( ^∗∗∗∗p < 0.0001) against unseeded dPPM. Here, we define a decellularisation protocol for porcine pleura that represents a step forward in their potential tissue engineering applications as bioscaffolds.