Profiling native pulmonary basement membrane stiffness using atomic force microscopy.

Mammalian cells sense and react to the mechanics of their immediate microenvironment. Therefore, the characterization of the biomechanical properties of tissues with high spatial resolution provides valuable insights into a broad variety of developmental, homeostatic and pathological processes within living organisms. The biomechanical properties of the basement membrane (BM), an extracellular matrix (ECM) substructure measuring only ∼100-400 nm across, are, among other things, pivotal to tumor progression and metastasis formation. Although the precise assignment of the Young's modulus E of such a thin ECM substructure especially in between two cell layers is still challenging, biomechanical data of the BM can provide information of eminent diagnostic potential. Here we present a detailed protocol to quantify the elastic modulus of the BM in murine and human lung tissue, which is one of the major organs prone to metastasis. This protocol describes a streamlined workflow to determine the Young's modulus E of the BM between the endothelial and epithelial cell layers shaping the alveolar wall in lung tissues using atomic force microscopy (AFM). Our step-by-step protocol provides instructions for murine and human lung tissue extraction, inflation of these tissues with cryogenic cutting medium, freezing and cryosectioning of the tissue samples, and AFM force-map recording. In addition, it guides the reader through a semi-automatic data analysis procedure to identify the pulmonary BM and extract its Young's modulus E using an in-house tailored user-friendly AFM data analysis software, the Center for Applied Tissue Engineering and Regenerative Medicine processing toolbox, which enables automatic loading of the recorded force maps, conversion of the force versus piezo-extension curves to force versus indentation curves, calculation of Young's moduli and generation of Young's modulus maps, where the pulmonary BM can be identified using a semi-automatic spatial filtering tool. The entire protocol takes 1-2 d.

Fibroblast-derived matrix models desmoplastic properties and forms a prognostic signature in cancer progression.

The desmoplastic reaction observed in many cancers is a hallmark of disease progression and prognosis, particularly in breast and pancreatic cancer. Stromal-derived extracellular matrix (ECM) is significantly altered in desmoplasia, and as such plays a critical role in driving cancer progression. Using fibroblast-derived matrices (FDMs), we show that cancer cells have increased growth on cancer associated FDMs, when compared to FDMs derived from non-malignant tissue (normal) fibroblasts. We assess the changes in ECM characteristics from normal to cancer-associated stroma at the primary tumor site. Compositional, structural, and mechanical analyses reveal significant differences, with an increase in abundance of core ECM proteins, coupled with an increase in stiffness and density in cancer-associated FDMs. From compositional changes of FDM, we derived a 36-ECM protein signature, which we show matches in large part with the changes in pancreatic ductal adenocarcinoma (PDAC) tumor and metastases progression. Additionally, this signature also matches at the transcriptomic level in multiple cancer types in patients, prognostic of their survival. Together, our results show relevance of FDMs for cancer modelling and identification of desmoplastic ECM components for further mechanistic studies.

Dynamics of single cell femtosecond laser printing.

In the present study, we investigated the dynamics of a femtosecond (fs) laser induced bio-printing with cell-free and cell-laden jets under the variation of laser pulse energy and focus depth, by using time-resolved imaging. By increasing the laser pulse energy or decreasing the focus depth thresholds for a first and second jet are exceeded and more laser pulse energy is converted to kinetic jet energy. With increasing jet velocity, the jet behavior changes from a well-defined laminar jet, to a curved jet and further to an undesired splashing jet. We quantified the observed jet forms with the dimensionless hydrodynamic Weber and Rayleigh numbers and identified the Rayleigh breakup regime as the preferred process window for single cell bioprinting. Herein, the best spatial printing resolution of 42 ± 3 µm and single cell positioning precision of 12.4 µm are reached, which is less than one single cell diameter about 15 µm.

Aggrecan governs intervertebral discs development by providing critical mechanical cues of the extracellular matrix.

Aggrecan (ACAN) is localized in the intervertebral disc (IVD) in unique compartment-specific patterns where it contributes to the tissue structure and mechanical function together with collagens. The extracellular matrix (ECM) of the IVD undergoes degenerative changes during aging, misuse or trauma, which inevitably alter the biochemical and biomechanical properties of the tissue. A deeper understanding of these processes can be achieved in genetically engineered mouse models, taking into account the multifaceted aspects of IVD development. In this study, we generated aggrecan insertion mutant mice (Acan iE5/iE5 ) by interrupting exon 5 coding for the G1 domain of ACAN, and analyzed the morphological and mechanical properties of the different IVD compartments during embryonic development. Western blotting using an antibody against the total core protein failed to detect ACAN in cartilage extracts, whereas immunohistochemistry by a G1-specific antibody showed weak signals in vertebral tissues of Acan iE5/iE5 mice. Homozygous mutant mice are perinatally lethal and characterized by short snout, cleft palate and disproportionate dwarfism. Whole-mount skeletal staining and µ-CT analysis of Acan iE5/iE5 mice at embryonic day 18.5 revealed compressed vertebral bodies with accelerated mineralization compared to wild type controls. In Acan iE5/iE5 mice, histochemical staining revealed collapsed extracellular matrix with negligible sulfated glycosaminoglycan content accompanied by a high cellular density. Collagen type II deposition was not impaired in the IVD of Acan iE5/iE5 mice, as shown by immunohistochemistry. Mutant mice developed a severe IVD phenotype with deformed nucleus pulposus and thinned cartilaginous endplates accompanied by a disrupted growth plate structure in the vertebral body. Atomic force microscopy (AFM) imaging demonstrated a denser collagen network with thinner fibrils in the mutant IVD zones compared to wild type. Nanoscale AFM indentation revealed bimodal stiffness distribution attributable to the softer proteoglycan moiety and harder collagenous fibrils of the wild type IVD ECM. In Acan iE5/iE5 mice, loss of aggrecan resulted in a marked shift of the Young's modulus to higher values in all IVD zones. In conclusion, we demonstrated that aggrecan is pivotal for the determination and maintenance of the proper stiffness of IVD and vertebral tissues, which in turn could play an essential role in providing developmental biomechanical cues.

Angiogenic Potential of Co-Cultured Human Umbilical Vein Endothelial Cells and Adipose Stromal Cells in Customizable 3D Engineered Collagen Sheets.

The wound healing process is much more complex than just the four phases of hemostasis, inflammation, proliferation, and maturation. Three-dimensional (3D) scaffolds made of biopolymers or ECM molecules using bioprinting can be used to promote the wound healing process, especially for complex 3D tissue lesions like chronic wounds. Here, a 3D-printed mold has been designed to produce customizable collagen type-I sheets containing human umbilical vein endothelial cells (HUVECs) and adipose stromal cells (ASCs) for the first time. In these 3D collagen sheets, the cellular activity leads to a restructuring of the collagen matrix. The upregulation of the growth factors Serpin E1 and TIMP-1 could be demonstrated in the 3D scaffolds with ACSs and HUVECs in co-culture. Both growth factors play a key role in the wound healing process. The capillary-like tube formation of HUVECs treated with supernatant from the collagen sheets revealed the secretion of angiogenic growth factors. Altogether, this demonstrates that collagen type I combined with the co-cultivation of HUVECs and ACSs has the potential to accelerate the process of angiogenesis and, thereby, might promote wound healing.

Synthetic cell-based materials extract positional information from morphogen gradients.

Biomaterials composed of synthetic cells have the potential to adapt and differentiate guided by physicochemical environmental cues. Inspired by biological systems in development, which extract positional information (PI) from morphogen gradients in the presence of uncertainties, we here investigate how well synthetic cells can determine their position within a multicellular structure. To calculate PI, we created and analyzed a large number of synthetic cellular assemblies composed of emulsion droplets connected via lipid bilayer membranes. These droplets contained cell-free feedback gene circuits that responded to gradients of a genetic inducer acting as a morphogen. PI is found to be limited by gene expression noise and affected by the temporal evolution of the morphogen gradient and the cell-free expression system itself. The generation of PI can be rationalized by computational modeling of the system. We scale our approach using three-dimensional printing and demonstrate morphogen-based differentiation in larger tissue-like assemblies.

Extending Single Cell Bioprinting from Femtosecond to Picosecond Laser Pulse Durations.

Femtosecond laser pulses have been successfully used for film-free single-cell bioprinting, enabling precise and efficient selection and positioning of individual mammalian cells from a complex cell mixture (based on morphology or fluorescence) onto a 2D target substrate or a 3D pre-processed scaffold. In order to evaluate the effects of higher pulse durations on the bioprinting process, we investigated cavitation bubble and jet dynamics in the femto- and picosecond regime. By increasing the laser pulse duration from 600 fs to 14.1 ps, less energy is deposited in the hydrogel for the cavitation bubble expansion, resulting in less kinetic energy for the jet propagation with a slower jet velocity. Under appropriate conditions, single cells can be reliably transferred with a cell survival rate after transfer above 95% through the entire pulse duration range. More cost efficient and compact laser sources with pulse durations in the picosecond range could be used for film-free bioprinting and single-cell transfer.

Fourier Transform Infrared Microspectroscopy Combined with Principal Component Analysis and Artificial Neural Networks for the Study of the Effect of ß-Hydroxy-ß-Methylbutyrate (HMB) Supplementation on Articular Cartilage.

The potential of Fourier Transform infrared microspectroscopy (FTIR microspectroscopy) and multivariate analyses were applied for the classification of the frequency ranges responsible for the distribution changes of the main components of articular cartilage (AC) that occur during dietary ß-hydroxy-ß-methyl butyrate (HMB) supplementation. The FTIR imaging analysis of histological AC sections originating from 35-day old male piglets showed the change in the collagen and proteoglycan contents of the HMB-supplemented group compared to the control. The relative amount of collagen content in the superficial zone increased by more than 23% and in the middle zone by about 17%, while no changes in the deep zone were observed compared to the control group. Considering proteoglycans content, a significant increase was registered in the middle and deep zones, respectively; 62% and 52% compared to the control. AFM nanoindentation measurements collected from animals administered with HMB displayed an increase in AC tissue stiffness by detecting a higher value of Young's modulus in all investigated AC zones. We demonstrated that principal component analysis and artificial neural networks could be trained with spectral information to distinguish AC histological sections and the group under study accurately. This work may support the use and effectiveness of FTIR imaging combined with multivariate analyses as a quantitative alternative to traditional collagenous tissue-related histology.

Mitochondrial respiratory chain function promotes extracellular matrix integrity in cartilage.

Energy metabolism and extracellular matrix (ECM) function together orchestrate and maintain tissue organization, but crosstalk between these processes is poorly understood. Here, we used single-cell RNA-Seq (scRNA-Seq) analysis to uncover the importance of the mitochondrial respiratory chain for ECM homeostasis in mature cartilage. This tissue produces large amounts of a specialized ECM to promote skeletal growth during development and maintain mobility throughout life. A combined approach of high-resolution scRNA-Seq, mass spectrometry/matrisome analysis, and atomic force microscopy was applied to mutant mice with cartilage-specific inactivation of respiratory chain function. This genetic inhibition in cartilage results in the expansion of a central area of 1-month-old mouse femur head cartilage, showing disorganized chondrocytes and increased deposition of ECM material. scRNA-Seq analysis identified a cell cluster-specific decrease in mitochondrial DNA-encoded respiratory chain genes and a unique regulation of ECM-related genes in nonarticular chondrocytes. These changes were associated with alterations in ECM composition, a shift in collagen/noncollagen protein content, and an increase of collagen crosslinking and ECM stiffness. These results demonstrate that mitochondrial respiratory chain dysfunction is a key factor that can promote ECM integrity and mechanostability in cartilage and presumably also in many other tissues.

Basement membrane stiffness determines metastases formation.

The basement membrane (BM) is a special type of extracellular matrix and presents the major barrier cancer cells have to overcome multiple times to form metastases. Here we show that BM stiffness is a major determinant of metastases formation in several tissues and identify netrin-4 (Net4) as a key regulator of BM stiffness. Mechanistically, our biophysical and functional analyses in combination with mathematical simulations show that Net4 softens the mechanical properties of native BMs by opening laminin node complexes, decreasing cancer cell potential to transmigrate this barrier despite creating bigger pores. Our results therefore reveal that BM stiffness is dominant over pore size, and that the mechanical properties of 'normal' BMs determine metastases formation and patient survival independent of cancer-mediated alterations. Thus, identifying individual Net4 protein levels within native BMs in major metastatic organs may have the potential to define patient survival even before tumour formation. The ratio of Net4 to laminin molecules determines BM stiffness, such that the more Net4, the softer the BM, thereby decreasing cancer cell invasion activity.

ER Stress in ERp57 Knockout Knee Joint Chondrocytes Induces Osteoarthritic Cartilage Degradation and Osteophyte Formation.

Ageing or obesity are risk factors for protein aggregation in the endoplasmic reticulum (ER) of chondrocytes. This condition is called ER stress and leads to induction of the unfolded protein response (UPR), which, depending on the stress level, restores normal cell function or initiates apoptotic cell death. Here the role of ER stress in knee osteoarthritis (OA) was evaluated. It was first tested in vitro and in vivo whether a knockout (KO) of the protein disulfide isomerase ERp57 in chondrocytes induces sufficient ER stress for such analyses. ER stress in ERp57 KO chondrocytes was confirmed by immunofluorescence, immunohistochemistry, and transmission electron microscopy. Knee joints of wildtype (WT) and cartilage-specific ERp57 KO mice (ERp57 cKO) were analyzed by indentation-type atomic force microscopy (IT-AFM), toluidine blue, and immunofluorescence/-histochemical staining. Apoptotic cell death was investigated by a TUNEL assay. Additionally, OA was induced via forced exercise on a treadmill. ER stress in chondrocytes resulted in a reduced compressive stiffness of knee cartilage. With ER stress, 18-month-old mice developed osteoarthritic cartilage degeneration with osteophyte formation in knee joints. These degenerative changes were preceded by apoptotic death in articular chondrocytes. Young mice were not susceptible to OA, even when subjected to forced exercise. This study demonstrates that ER stress induces the development of age-related knee osteoarthritis owing to a decreased protective function of the UPR in chondrocytes with increasing age, while apoptosis increases. Therefore, inhibition of ER stress appears to be an attractive therapeutic target for OA.

Imbalanced cellular metabolism compromises cartilage homeostasis and joint function in a mouse model of mucolipidosis type III gamma.

Mucolipidosis type III (MLIII) gamma is a rare inherited lysosomal storage disorder caused by mutations in GNPTG encoding the γ-subunit of GlcNAc-1-phosphotransferase, the key enzyme ensuring proper intracellular location of multiple lysosomal enzymes. Patients with MLIII gamma typically present with osteoarthritis and joint stiffness, suggesting cartilage involvement. Using Gnptg knockout (Gnptgko ) mice as a model of the human disease, we showed that missorting of a number of lysosomal enzymes is associated with intracellular accumulation of chondroitin sulfate in Gnptgko chondrocytes and their impaired differentiation, as well as with altered microstructure of the cartilage extracellular matrix (ECM). We also demonstrated distinct functional and structural properties of the Achilles tendons isolated from Gnptgko and Gnptab knock-in (Gnptabki ) mice, the latter displaying a more severe phenotype resembling mucolipidosis type II (MLII) in humans. Together with comparative analyses of joint mobility in MLII and MLIII patients, these findings provide a basis for better understanding of the molecular reasons leading to joint pathology in these patients. Our data suggest that lack of GlcNAc-1-phosphotransferase activity due to defects in the γ-subunit causes structural changes within the ECM of connective and mechanosensitive tissues, such as cartilage and tendon, and eventually results in functional joint abnormalities typically observed in MLIII gamma patients. This idea was supported by a deficit of the limb motor function in Gnptgko mice challenged on a rotarod under fatigue-associated conditions, suggesting that the impaired motor performance of Gnptgko mice was caused by fatigue and/or pain at the joint.This article has an associated First Person interview with the first author of the paper.

Precision 3D-Printed Cell Scaffolds Mimicking Native Tissue Composition and Mechanics.

Cellular dynamics are modeled by the 3D architecture and mechanics of the extracellular matrix (ECM) and vice versa. These bidirectional cell-ECM interactions are the basis for all vital tissues, many of which have been investigated in 2D environments over the last decades. Experimental approaches to mimic in vivo cell niches in 3D with the highest biological conformity and resolution can enable new insights into these cell-ECM interactions including proliferation, differentiation, migration, and invasion assays. Here, two-photon stereolithography is adopted to print up to mm-sized high-precision 3D cell scaffolds at micrometer resolution with defined mechanical properties from protein-based resins, such as bovine serum albumin or gelatin methacryloyl. By modifying the manufacturing process including two-pass printing or post-print crosslinking, high precision scaffolds with varying Young's moduli ranging from 7-300 kPa are printed and quantified through atomic force microscopy. The impact of varying scaffold topographies on the dynamics of colonizing cells is observed using mouse myoblast cells and a 3D-lung microtissue replica colonized with primary human lung fibroblast. This approach will allow for a systematic investigation of single-cell and tissue dynamics in response to defined mechanical and bio-molecular cues and is ultimately scalable to full organs.

Early Detection of Cartilage Degeneration: A Comparison of Histology, Fiber Bragg Grating-Based Micro-Indentation, and Atomic Force Microscopy-Based Nano-Indentation.

We have determined the sensitivity and detection limit of a new fiber Bragg grating (FBG)-based optoelectronic micro-indenter for biomechanical testing of cartilage and compared the results to indentation-type atomic force microscopy (IT-AFM) and histological staining. As test samples, we used bovine articular cartilage, which was enzymatically degraded ex vivo for five minutes using different concentrations of collagenase (5, 50, 100 and 500 µg/mL) to mimic moderate extracellular matrix deterioration seen in early-stage osteoarthritis (OA). Picrosirius Red staining and polarization microscopy demonstrated gradual, concentration-dependent disorganization of the collagen fibrillar network in the superficial zone of the explants. Osteoarthritis Research Society International (OARSI) grading of histopathological changes did not discriminate between undigested and enzymatically degraded explants. IT-AFM was the most sensitive method for detecting minute changes in cartilage biomechanics induced by the lowest collagenase concentration, however, it did not distinguish different levels of cartilage degeneration for collagenase concentrations higher than 5 µg/mL. The FBG micro-indenter provided a better and more precise assessment of the level of cartilage degeneration than the OARSI histological grading system but it was less sensitive at detecting mechanical changes than IT-AFM. The FBG-sensor allowed us to observe differences in cartilage biomechanics for collagenase concentrations of 100 and 500 µg/mL. Our results confirm that the FBG sensor is capable of detecting small changes in articular cartilage stiffness, which may be associated with initial cartilage degeneration caused by early OA.

Inadequate tissue mineralization promotes cancer cell attachment.

Bone metastases are a frequent complication in prostate cancer, and several studies have shown that vitamin D deficiency promotes bone metastases. However, while many studies focus on vitamin D's role in cell metabolism, the effect of chronically low vitamin D levels on bone tissue, i.e. insufficient mineralization of the tissue, has largely been ignored. To investigate, whether poor tissue mineralization promotes cancer cell attachment, we used a fluorescence based adhesion assay and single cell force spectroscopy to quantify the adhesion of two prostate cancer cell lines to well-mineralized and demineralized dentin, serving as biomimetic bone model system. Adhesion rates of bone metastases-derived PC3 cells increased significantly on demineralized dentin. Additionally, on mineralized dentin, PC3 cells adhered mainly via membrane anchored surface receptors, while on demineralized dentin, they adhered via cytoskeleton-anchored transmembrane receptors, pointing to an interaction via exposed collagen fibrils. The adhesion rate of lymph node derived LNCaP cells on the other hand is significantly lower than that of PC3 and not predominately mediated by cytoskeleton-linked receptors. This indicates that poor tissue mineralization facilitates the adhesion of invasive cancer cells by the exposure of collagen and emphasizes the disease modifying effect of sufficient vitamin D for cancer patients.

Adhesive Properties of the Hyaluronan Pericellular Coat in Hyaluronan Synthases Overexpressing Mesenchymal Stem Cells.

Hyaluronan (HA), a natural component of the extracellular matrix, is supposed to have a regulatory function in the stem cell niche. Bone marrow-derived human mesenchymal stem cells (hMSCs) are known to express all three hyaluronan synthases (HASes), which are responsible for HA production. HA is extruded into the extracellular matrix, but also stays bound to the plasma membrane forming a pericellular coat, which plays a key role during early cell adhesion. Since HAS isoenzymes, HAS1, HAS2 and HAS3, produce HA with different molecular weights, a difference in their role for cell adhesion is expected. Here, we transduced the immortalized hMSC cell line SCP1 to constitutively express eGFP-tagged HASes (SCP1-HAS-eGFP) by lentiviral gene transfer. The overexpression of the HAS-eGFP was shown on RNA and protein levels, HA was determined by ELISA and the stained HA-coat was analyzed using confocal microscopy. Time-lapse microscopy, spreading assay and single cell force spectroscopy using atomic force microscopy were applied to characterize adhesion of the different HAS transduced SCP1 cells. We showed in this study that HAS3 overexpressing cells formed the thickest pericellular coat compared with control or HAS1 and HAS2 transduced cells. Furthermore, SCP1-HAS3-eGFP displayed faster and stronger adhesion compared to cells overexpressing the other synthases or control cells. We conclude that overexpression of HASes in hMSCs differentially modulates their initial adhesive interactions with the substrate. This observation might be helpful in regenerative medicine goals.

Osteoidosis leads to altered differentiation and function of osteoclasts.

In patients with osteomalacia, a defect in bone mineralization leads to changed characteristics of the bone surface. Considering that the properties of the surrounding matrix influence function and differentiation of cells, we aimed to investigate the effect of osteoidosis on differentiation and function of osteoclasts. Based on osteomalacic bone biopsies, a model for osteoidosis in vitro (OIV) was established. Peripheral blood mononuclear cells were differentiated to osteoclasts on mineralized surfaces (MS) as internal control and on OIV. We observed a significantly reduced number of osteoclasts and surface resorption on OIV. Atomic force microscopy revealed a significant effect of the altered degree of mineralization on surface mechanics and an unmasking of collagen fibres on the surface. Indeed, coating of MS with RGD peptides mimicked the resorption phenotype observed in OIV, suggesting that the altered differentiation of osteoclasts on OIV might be associated with an interaction of the cells with amino acid sequences of unmasked extracellular matrix proteins containing RGD sequences. Transcriptome analysis uncovered a strong significant up-regulation of transmembrane glycoprotein TROP2 in osteoclastic cultures on OIV. TROP2 expression on OIV was also confirmed on the protein level and found on the bone surface of patients with osteomalacia. Taken together, our results show a direct influence of the mineralization state of the extracellular matrix surface on differentiation and function of osteoclasts on this surface which may be important for the pathophysiology of osteomalacia and other bone disorders with changed ratio of osteoid to bone.

Loss of tenomodulin expression is a risk factor for age-related intervertebral disc degeneration.

The intervertebral disc (IVD) degeneration is thought to be closely related to ingrowth of new blood vessels. However, the impact of anti-angiogenic factors in the maintenance of IVD avascularity remains unknown. Tenomodulin (Tnmd) is a tendon/ligament-specific marker and anti-angiogenic factor with abundant expression in the IVD. It is still unclear whether Tnmd contributes to the maintenance of IVD homeostasis, acting to inhibit vascular ingrowth into this normally avascular tissue. Herein, we investigated whether IVD degeneration could be induced spontaneously by the absence of Tnmd. Our results showed that Tnmd was expressed in an age-dependent manner primarily in the outer annulus fibrous (OAF) and it was downregulated at 6 months of age corresponding to the early IVD degeneration stage in mice. Tnmd knockout (Tnmd-/- ) mice exhibited more rapid progression of age-related IVD degeneration. These signs include smaller collagen fibril diameter, markedly lower compressive stiffness, reduced multiple IVD- and tendon/ligament-related gene expression, induced angiogenesis, and macrophage infiltration in OAF, as well as more hypertrophic-like chondrocytes in the nucleus pulposus. In addition, Tnmd and chondromodulin I (Chm1, the only homologous gene to Tnmd) double knockout (Tnmd-/- Chm1-/- ) mice displayed not only accelerated IVD degeneration, but also ectopic bone formation of IVD. Lastly, the absence of Tnmd in OAF-derived cells promoted p65 and matrix metalloproteinases upregulation, and increased migratory capacity of human umbilical vein endothelial cells. In sum, our data provide clear evidences that Tnmd acts as an angiogenic inhibitor in the IVD homeostasis and protects against age-related IVD degeneration. Targeting Tnmd may represent a novel therapeutic strategy for attenuating age-related IVD degeneration.

Three-dimensional self-assembling nanofiber matrix rejuvenates aged/degenerative human tendon stem/progenitor cells.

The poor healing capacity of tendons is known to worsen in the elderly. During tendon aging and degeneration, endogenous human tendon stem/progenitor cells (hTSPCs) experience profound pathological changes. Here, we explored a rejuvenation strategy for hTSPCs derived from aged/degenerated Achilles tendons (A-TSPCs) by providing three-dimensional (3D) nanofiber hydrogels and comparing them to young/healthy TSPCs (Y-TSPCs). RADA peptide hydrogel has a self-assembling ability, forms a nanofibrous 3D niche and can be further functionalized by adding RGD motifs. Cell survival, apoptosis, and proliferation assays demonstrated that RADA and RADA/RGD hydrogels support A-TSPCs in a comparable manner to Y-TSPCs. Moreover, they rejuvenated A-TSPCs to a phenotype similar to that of Y-TSPCs, as evidenced by restored cell morphology and cytoskeletal architecture. Transmission electron, confocal laser scanning and atomic force microscopies demonstrated comparable ultrastructure, surface roughness and elastic modulus of A- and Y-TSPC-loaded hydrogels. Lastly, quantitative PCR revealed similar expression profiles, as well a significant upregulation of genes related to tenogenesis and multipotency. Taken together, the RADA-based hydrogels exert a rejuvenating effect by recapitulating in vitro specific features of the natural microenvironment of human TSPCs, which strongly indicates their potential to direct cell behaviour and overcome the challenge of cell aging and degeneration in tendon repair.