Harnessing the interplay between tendon stem/progenitor cell and microenvironment boosts tendon tissue regeneration

Background: Tendons have a limited blood supply and form inferior scar tissue during repair, which increases the risk of reruptures, causes complications, and limits regenerative capacity. Current methods to repair injured tendon tissue use solid scaffolds, which carry the risk of contamination (infections) and require open surgery for transplantation. Hypothesis: Alginate-cell cross-linked gels, which can be applied by a percutaneous injection and transmit mechanical stress to cells via direct cell interaction, could induce tendon tissue regeneration. Study Design: Controlled laboratory study. Methods: A cross-linked gel was prepared to suspend azide-modified mesenchymal stromal cells (MSCs) in a dibenzocyclooctyne-modified branched alginic acid solution. The cross-linked gel was cultured in a bioreactor. In vivo, the Achilles tendon defects of 104 Lewis rats were injected with saline (control group), alginate gel alone (alginate group), alginate gel with MSCs (MSC group), and cross-linked gel (cross-link group). At 2 and 4 weeks postoperatively, histological and biochemical evaluations were performed. The biomechanical properties of repaired tissue were assessed at 4 weeks. Results: In the bioreactor culture, the cell orientation in the cross-linked gel was parallel to the direction of tension. Histological analysis of the cross-link group showed significantly more repaired tendon tissue and improved collagen fiber orientation compared with the alginate group or MSC group. The biomechanical properties of the cross-link group included higher stiffness. Conclusion: The cross-linked gel was injectable at the injury site and was able to induce tissue regeneration with cell-oriented adaptability to the mechanical environment of tissue defects. Clinical Relevance: Intercellular cross-linking technology holds the potential for clinical application as a minimally invasive therapeutic approach that can contribute to the qualitative improvement of tendon tissue regeneration.

Tendon regeneration is difficult because detailed knowledge about tendon progenitor cells (TPCs), which produce tenocytes to repair tendon tissue, has not been revealed. Mohawk homeobox (MKX) is a marker of TPCs or tenocytes, but a human pluripotent stem cell (hPSC)-based reporter system that visualizes MKX+ cells has not been developed. Here, we established an hPSC-derived MKX-tdTomato reporter cell line and tested the induction ratio of MKX-tdTomato+ cells using our stepwise/xeno-free differentiation protocol. MKX-tdTomato+ cells were generated with high efficiency and expressed tendon-specific markers, including MKX, SCX, TNMD, and COL1A1. Our MKX-tdTomato hPSC line would be a useful tool for studying the development or regeneration of tendon tissue.

In recent years, with the application of natural extracellular matrix (ECM) components, tissue-induced scaffolds have become one of the hot topics in tissue engineering. As a natural source of material, ECM components are easily obtained from animals, and have good biocompatibility. Moreover, tendon ECM materials can well simulate the composition and structure of natural tendon ECM, and can provide mechanics fulcrum and stimulus signal for host cells so as to promote their tenogenic differentiation. Therefore, the tendon ECM components contained scaffold is expected to promote the repair and regeneration of tendon tissue. In this paper, the recent progress of tendon decellularization and preparation of different forms of scaffolds were reviewed, as well as the application of tendon ECM in the construction of tissue engineering tendons and mechanism of ECM induced tenogenic differentiation. Moreover, the optimized methods for preparing tendon ECM contained scaffolds were proposed to promote the application. Key words: Tendon tissue engineering; Scaffold; Extracellular matrix; Tendon differentiation

Tendon injury is common in sports and other rigorous activities, which may result in dysfunction and disability. Recently, scaffolds with a knitted structure have been widely applied for tendon tissue engineering. The purpose of this study was to fabricate a novel knitted tendon scaffold made of microfiber/nanofiber core-sheath yarns and evaluate the biocompatibility and the effect of tenogenic differentiation and tendon tissue regeneration in vitro and in vivo. Poly(ε-caprolactone) (PCL) microfibers, PCL microfibers-PCL nanofibers (PCL-PCL) and PCL microfiber-silk fibroin/poly(l-lactic acid-co-ε-caprolactone) nanofiber (SF/PLCL) core-sheath yarns were fabricated and then knitted with an automatic knitting machine to produce PCL, PCL-PCL and PCL-SF/PLCL fabric scaffolds. The characterization of the scaffolds was performed by using scanning electron microscopy, attenuated total reflectance Fourier transform infrared spectroscopy and an universal mechanical instrument. The in vitro experiment showed that rabbit bone marrow stem cells seeded on the scaffolds exhibited an elongated morphology and proliferated better in the PCL-SF/PLCL group, as compared to the PCL and PCL-PCL groups. Moreover, the PCL-SF/PLCL scaffold promoted the tenogenic differentiation of the cells for the highest expression levels of the tendon-related genes through down-regulating p-ERK1/2 expression among the three groups. Furthermore, the in vivo study in a rabbit patellar defect model demonstrated that the PCL-SF/PLCL scaffold could enhance the tissue regeneration and remodeling process as indicated by the better structural and biomechanical properties according to the results of histology, immunohistochemistry, transmission electron microscope examination and biomechanical tests. Therefore, the PCL-SF/PLCL scaffold is proved to be a promising biomaterial for tendon tissue engineering and a potential candidate for clinical treatment of tendon injury in the future.

Tendon stem/progenitor cells (TSPCs) are a potential cell source for tendon tissue engineering. The striking morphological and structural changes of tendon tissue during development indicate the complexity of TSPCs at different stages. This study aims to characterize and compare post-natal rat Achilles tendon tissue and TSPCs at different stages of development. The tendon tissue showed distinct differences during development: the tissue structure became denser and more regular, the nuclei became spindle-shaped and the cell number decreased with time. TSPCs derived from 7 day Achilles tendon tissue showed the highest self-renewal ability, cell proliferation, and differentiation potential towards mesenchymal lineage, compared to TSPCs derived from 1 day and 56 day tissue. Microarray data showed up-regulation of several groups of genes in TSPCs derived from 7 day Achilles tendon tissue, which may account for the unique cell characteristics during this specific stage of development. Our results indicate that TSPCs derived from 7 day Achilles tendon tissue is a superior cell source as compared to TSPCs derived from 1 day and 56 day tissue, demonstrating the importance of choosing a suitable stem cell source for effective tendon tissue engineering and regeneration.

Tendon regeneration has been one of the most challenging issues in orthopedics. Despite various surgical techniques and rehabilitation methods, tendon tears or ruptures cannot wholly regenerate and gain the load-bearing capacity the tendon tissue had before the injury. The enhancement of tendon regeneration mostly requires grafting or an artificial tendon-like tissue to replace the damaged tendon. Tendon tissue engineering offers promising regenerative effects with numerous techniques in the additive manufacturing context. 3D bioprinting is a widely used additive manufacturing method to produce tendon-like artificial tissues based on biocompatible substitutes. There are multiple techniques and bio-inks for fabricating innovative scaffolds for tendon applications. Nevertheless, there are still many drawbacks to overcome for the successful regeneration of injured tendon tissue. The most important target is to catch the highest similarity to the tissue requirements such as anisotropy, porosity, viscoelasticity, mechanical strength, and cell-compatible constructs. To achieve the best-designed artificial tendon-like structure, novel AI-based systems in the field of 3D bioprinting may unveil excellent final products to re-establish tendon integrity and functionality. AI-driven optimization can enhance bio-ink selection, scaffold architecture, and printing parameters, ensuring better alignment with the biomechanical properties of native tendons. Furthermore, AI algorithms facilitate real-time process monitoring and adaptive adjustments, improving reproducibility and precision in scaffold fabrication. Thus, in vitro biocompatibility and in vivo application-based experimental processes will make it possible to accelerate tendon healing and reach the required mechanical strength. Integrating AI-based predictive modeling can further refine these experimental processes to evaluate scaffold performance, cell viability, and mechanical durability, ultimately improving translation into clinical applications. Here in this review, 3D bioprinting approaches and AI-based technology incorporation were given in addition to in vivo models.

Proper development of tendons is crucial for the integration and function of the musculoskeletal system. Currently little is known about the molecular mechanisms controlling tendon development and tendon cell differentiation. The transcription factor Scleraxis (Scx) is expressed throughout tendon development and plays essential roles in both embryonic tendon development and adult tendon healing, but few direct target genes of Scx in tendon development have been reported and genome-wide identification of Scx direct target genes in vivo has been lacking. In this study, we have generated a ScxFlag knockin mouse strain, which produces fully functional endogenous Scx proteins containing a 2xFLAG epitope tag at the carboxy terminus. We mapped the genome-wide Scx binding sites in the developing limb tendon tissues, identifying 12,097 high quality Scx regulatory cis-elements in-around 7,520 genes. Comparative analysis with previously reported embryonic tendon cell RNA-seq data identified 490 candidate Scx direct target genes in early tendon development. Furthermore, we characterized a new Scx gene-knockout mouse line and performed whole transcriptome RNA sequencing analysis of E15.5 forelimb tendon cells from Scx–/– embryos and control littermates, identifying 68 genes whose expression in the developing tendon tissues significantly depended on Scx function. Combined analysis of the ChIP-seq and RNA-seq data yielded 32 direct target genes that required Scx for activation and an additional 17 target genes whose expression was suppressed by Scx during early tendon development. We further analyzed and validated Scx-dependent tendon-specific expression patterns of a subset of the target genes, including Fmod, Kera, Htra3, Ssc5d, Tnmd, and Zfp185, by in situ hybridization and real-time quantitative polymerase chain reaction assays. These results provide novel insights into the molecular mechanisms mediating Scx function in tendon development and homeostasis. The ChIP-seq and RNA-seq data provide a rich resource for aiding design of further studies of the mechanisms regulating tendon cell differentiation and tendon tissue regeneration. The ScxFlag mice provide a valuable new tool for unraveling the molecular mechanisms involving Scx in the protein interaction and gene-regulatory networks underlying many developmental and disease processes.

Native tissue structures possess elaborate extracellular matrix (ECM) architectures that inspire the design of fibrous structures in the field of regenerative medicine. We review the literature with respect to the successes and failures, as well as the future promise of biopolymer microthreads as scaffolds to promote endogenous and exogenous tissue regeneration. Biomimetic microthread tissue constructs have been proposed for the functional regeneration of tendon, ligament, skeletal muscle, and ventricular myocardial tissues. To date, biopolymer microthreads have demonstrated promising results as materials to recapitulate the hierarchical structure of simple and complex tissues and well as biochemical signaling cues to direct cell-mediated tissue regeneration. Biopolymer microthreads have also demonstrated exciting potential as a platform technology for the targeted delivery of stem cells and therapeutic molecules. Future studies will focus on the design of microthread-based tissue analogs that strategically integrate growth factors and progenitor cells to temporally direct cell-mediated processes that promote enhanced functional tissue regeneration.

Reconstituted collagen fibres are promising candidates for tendon and ligament tissue regeneration. The crosslinking procedure determines the fibres' mechanical properties, degradation rate, and cell-fibre interactions. We aimed to compare mechanical and biological properties of collagen fibres resulting from two different types of crosslinking chemistry based on 1-ethyl-3-(3-dimethyllaminopropyl)carbodiimide (EDC). Fibres were crosslinked with either EDC or with EDC and ethylene-glycol-diglycidyl-ether (EDC/EGDE). Single fibres were mechanically tested to failure and bundles of fibres were seeded with tendon fibroblasts (TFs) and cell attachment and proliferation were determined over 14 days in culture. Collagen type I and tenascin-C production were assessed by immunohistochemistry and dot-blotting. EDC chemistry resulted in fibres with average mechanical properties but the highest cell proliferation rate and matrix protein production. EDC/EGDE chemistry resulted in fibres with improved mechanical properties but with a lower biocompatibility profile. Both chemistries may provide useful structures for scaffolding regeneration of tendon and ligament tissue and will be evaluated for in vivo tendon regeneration in future experiments.

Importance of the field: Tendon injuries are common especially in sports activities, but tendon is a unique connective tissue with poor self-repair capability. With advances in stem cell biology, tissue engineering is becoming increasingly powerful for tissue regeneration. Stem cells with capacity of multipotency and self-renewal are an ideal cell source for tissue engineering.Areas covered in this review: This review focus on discussing the potential strategies including inductive growth factors, bio-scaffolds, mechanical stimulation, genetic modification and co-culture techniques to direct tendon-lineage differentiation of stem cells for complete tendon regeneration. Attempting to use embryonic stem cells as seed cells for tendon tissue engineering have achieved encouraging results. The combination of chemical and physical signals in stem cell microenvironment could be regulated to induce differentiation of the embryonic stem cells into tendon.What the reader will gain: We summarize fundamental questions, as well as future directions in tendon biology and tissue engineering.Take home message: Multifaceted technologies are increasingly required to control stem cell differentiation, to develop novel stem cell-based therapy, and, ultimately, to achieve more effective repair or regeneration of injured tendons.

Tendon and ligament injuries are common, and repair slowly with reduced biomechanical properties. With increasing financial demands on the health service and patients to recover from tendon and ligament injuries faster, and with less morbidity, health professionals are exploring new treatment options. Tissue engineering may provide the answer, with its unlimited source of natural cells that in the correct environment may improve repair and regeneration of tendon and ligament tissue. Mesenchymal stem cells have demonstrated the ability to self renew and have multilineage differentiation potential. The use of bone marrow-derived mesenchymal stem cells has been reported, however significant in vitro culture expansion is required due to the low yield of cells, which has financial implications. Harvesting of bone marrow cells also has associated morbidity. Several studies have looked at alternative sources for mesenchymal stem cells. Reports in literature from animal studies have been encouraging, however further work is required. This review assesses the potential sources of mesenchymal stem cells for tissue engineering in tendons and ligaments.

Tendon-bone injuries are a prevalent health concern associated with sports and other physically demanding activities. These injuries have a limited innate healing ability, often leading to the formation of scar tissue rather than the regeneration of healthy tendon tissue. This scar tissue results from excessive fibrosis during the early healing process and often leads to reduced tendon function and an increased risk of reinjury. Traditionally, surgical reconstruction has been the primary treatment for tendon-bone injuries. However, restoring the natural structure and mechanical properties of tendons after surgical reconstruction presents a considerable challenge. Recently, the potential of stem cell therapy has been explored as an alternative treatment approach. In particular, a new type of pluripotent stem cell known as tendon stem cells (TDSCs) has been identified within tendon tissue. These cells exhibit the potential for self-renewal and multidirectional differentiation, meaning they can differentiate into fibroblasts and chondrocytes. These differentiated cells can aid in the repair and regeneration of new tissues by producing collagen and other matrix molecules that provide structural support. TDSCs have become a focal point in research for treating tendon-bone injuries and related conditions. The potential use of these cells provides a basis for both basic research and clinical applications, particularly in understanding the tendon-bone healing process and identifying factors that affect the ability of TDSCs to promote this healing. This review article aims to analyze the role of TDSCs in tendon-bone healing, understanding their therapeutic potential and contributing to the development of effective treatment strategies for tendon-bone injuries.

The development of bio-devices for complete regeneration of ligament and tendon tissues is presently one of the biggest challenges in tissue engineering. Such device must simultaneously possess optimal mechanical performance, suitable porous structure, and biocompatible microenvironment. This study proposes a novel collagen-BDDGE-elastin (CBE)-based device for tendon tissue engineering, by the combination of two different modules: (i) a load-bearing, non-porous, “core scaffold” developed by braiding CBE membranes fabricated via an evaporative process and (ii) a hollow, highly porous, “shell scaffold” obtained by uniaxial freezing followed by freeze-drying of CBE suspension, designed to function as a physical guide and reservoir of cells to promote the regenerative process. Both core and shell materials demonstrated good cytocompatibility in vitro, and notably, the porous shell architecture directed cell alignment and population within the sample. Finally, a prototype of the core module was implanted in a rat tendon lesion model, and histological analysis demonstrated its safety, biocompatibility, and ability to induce tendon regeneration. Overall, our results indicate that such device may have the potential to support and induce in situ tendon regeneration.

Introduction Chronic musculoskeletal pain is very prevalent, and accounts for major health-care expenses. Many of the present therapeutic modalities are only partially effective, and great interest is now posed on regenerative medicine. Areas covered The authors discuss the role of a variety of regenerative medicine options to induce and favor regeneration and healing of tendon tissue, focusing on the role of mesenchymal stem cell therapy and their derivatives. Expert opinion Stem cells, tissue engineering, and growth factors are new strategies for tendon repair and regeneration. MSCs not only can differentiate in tendon cells, but also secrete several cytokines that modulate inflammation and tissue healing. Future studies should be undertaken to overcome current obstacles to clinical translation. Further investigation of cell source, isolation, expansion, and differentiation methods, characterization of the tenogenic differentiation pathways, and clarifications of tendon-specific molecular markers are required. The role of donor variability, tendon type, and anatomic location also requires further understanding and research.

Enhancement of tenogenic differentiation of human adipose stem cells by tendon-derived extracellular matrix