Pernilla Eliasson

Tendon injuries are among the most prevalent disorders affecting the musculoskeletal system. Their limited regenerative capacity often results in the formation of fibrovascular scar tissue with inferior properties and an increased risk of reinjury. In vivo animal models, clinical studies, and in vitro 2D culture systems each have limitations in reproducing the coordinated sequence of tendon healing (cell invasion, provisional collagen deposition, and matrix remodeling) under controlled conditions. Here, we present a 3D in vitro tendon injury model for studies of tendon healing. Primary human tenocytes embedded in a fibrin hydrogel self-organize into an aligned, tendon-like construct within 14 days. A reproducible and defined injury is then created and filled with an acellular fibrin hydrogel, generating a defined wound space that cells must invade to restore tissue continuity. Cells migrated into the injured area of the 3D constructs, achieving 96.5 % wound closure within 72 h. The COLIII/COLI ratio increased transiently at day 15 and returned to uninjured levels by day 21. At day 21 post-injury, the injured zone did not differ significantly from the uninjured constructs in histological matrix organization, collagen fibril diameter, COLIII/COLI ratio, or the expression of most analyzed genes, although higher order matrix organization remained incomplete. The model also captured early molecular response to injury, including transient increases in secreted MMP1 and COLI at 24 h, a reduction in secreted PINP at 24 h and 48 h, and a transient increase in ACTA2 expression at day 15. Taken together, this model provides access to the early healing phase, which is difficult in vivo, and offers a biological relevant tool for mechanistic studies and therapeutic screening in the field of tendon healing.

Pathological ossification is seen affecting different organs across the body. This includes the Achilles tendon, where the healing process after rupture can be impaired by heterotopic ossifications (HO). The formation pathways for HO in tendons are still debated, and one reason for this is our limited knowledge of their structure and composition in 3D. This study utilizes a multimodal 3D characterization approach to study HO and the surrounding healing tissue at 3‐, 6‐, and 12‐week post‐transection in rat Achilles tendons. Microcomputed tomography provided 3D microstructural information. Small‐ and wide‐angle scattering tensor tomography contributed with combined collagen and hydroxyapatite (HA) 3D nanostructural information. Combined 3D X‐ray diffraction and fluorescence provided high‐resolution structural dimensions of HA crystallites, and chemical composition of both the deposits and the surrounding healing tendon tissue. Morphologically, dense mineralized tissue appeared more fibrous, with higher co‐localized quantities of Zn present internally, while porous regions of HO deposits had Zn localization primarily at the boundary. Collagen d‐spacing was higher within HO deposits than in the surrounding collagenous tendon tissue. Collagen misalignment close to the HO boundary supports the notion of tissue mineralization occurring prior to the full assembly of the healing tendon network. This methodology can be extrapolated to investigate pathological ossification in other tissues at the nanoscale while capturing the heterogeneity of entire specimens.

Purpose: Incomplete recovery with persistent muscle weakness is frequently observed following Achilles tendon rupture. The mechanisms for this weakness remain unclear, but tendon elongation has been suggested as a contributing factor. The aim of this study was to compare tendon and muscle morphology in high‐ and low‐functioning patients more than 2 years after non‐surgical treatment of a total Achilles tendon rupture. Methods: Forty‐six patients underwent screening, including a standardized heel‐rise work test on both legs. Based on this test, a heel‐rise index (HRI) for total muscle work, categorized 29 patients into a low‐ (HRI < 33%) or high‐functioning (HRI > 67%) group. Both groups underwent bilateral magnetic resonance imaging to assess tendon and muscle morphology, and the main variable was tendon elongation. Results: High‐functioning patients were on average 13 years younger than the low‐functioning patients ( p < 0.001). Free tendon length was longer on the injured side in both groups, 3.59 and 5.19 cm in the high‐ and low‐functioning group ( p = 0.053). Tendon cross‐sectional area was significantly larger in the high‐functioning group compared to the low‐functioning group (383% vs. 256% after normalization to the uninjured side, p = 0.005). The soleus muscle had notable differences between the groups, as low‐functioning patients had a smaller mediolateral diameter ( p = 0.002), a more pronounced muscle length difference ( p = 0.009) and a higher atrophy grading. Additionally, there were significant correlations between age, HRI and tendon size. Conclusion: Free tendon length after rupture may play a role in muscle weakness. However, tendon elongation does not necessarily lead to low function, as it was also evident in the high‐functioning group. These findings may be important, as they suggest that tendon elongation is not the sole determinant of functional outcome and that other factors may contribute to muscle performance after rupture. Further research is needed on the role of age in muscle function following Achilles tendon ruptures. Level of Evidence: Level III.

Exercise is well known to promote tendon healing, an effect traditionally attributed to mechanical loading‐induced responses within the tendon itself. However, skeletal muscle also functions as a secretory organ, releasing bioactive factors (secretome) during exercise that influence various tissues. We hypothesized that muscle‐derived secretome released during exercise may also contribute to tendon healing. To test this, we applied mechanical loading to cultured muscle cells (myoblasts) using the FlexCell tension system to simulate exercise in vitro. Our previous studies, using 2D‐cultured tendon cells (tenocytes), have demonstrated that secretome from statically loaded myoblasts, particularly under 2% loading, enhanced tendon healing‐related responses. Building upon these findings, we employed a 3D tendon construct model to more closely mimic in vivo healing conditions. We found that secretome derived from statically loaded myoblasts, especially at 2% loading, promoted tendon healing‐related processes as compared with the control group, which received no secretome treatment (no conditioned media). These included increased cell‐covered area, expression of the tenocyte marker scleraxis ( SCX ), and elevated production of Type I and III collagens at an early stage (Day 7). Additionally, a reduction in type III collagen production was found at a later stage (Day 14), suggesting a potentially accelerated healing process. These findings highlight the therapeutic potential of the muscle‐derived secretome in promoting tendon healing and may inform future strategies for rehabilitation and regenerative medicine.

Purpose: The aim of this study was to investigate how fear of reinjury to the Achilles tendon affects return to previous levels of physical activity and self‐reported Achilles tendon Total Rupture Score (ATRS) outcomes.Methods: Data were collected from a large cohort of patients treated for an acute Achilles tendon rupture at Sahlgrenska University Hospital Mölndal between 2015 and 2020. The ATRS and additional questions concerning fear of reinjury, treatment modality, satisfaction of treatment and recovery were analyzed 1–6 years postinjury. Analysis was performed to determine the impact of fear of reinjury on patient‐reported recovery and physical activity.Results: Of a total of 856 eligible patients, 550 (64%) answered the self‐reported questionnaire and participated in the follow‐up. Of the participants, 425 (77%) were men and 125 (23%) were women. ATRS, recovery in percentage, satisfaction of treatment, recovery on a 5‐point scale and physical activity level post‐ versus preinjury were significantly related to fear of reinjury (p < 0.001). Of the nonsurgically treated patients, 59% reported fear of reinjury compared to 48% of the surgically treated patients (p = 0.024) Patients that reported fear of reinjury had a 15‐point lower median ATRS score than those who did not (p < 0.001).Conclusion: More than half of patients who have suffered an Achilles tendon rupture are afraid of reinjuring their tendon. Patients who reported fear of reinjury exhibited a significantly lower ATRS score. This indicates the importance of addressing psychological aspects in the treatment after this injury.Level of Evidence: Level II.

Tendon healing involves mechanosensitive cells that adapt to mechanical stimuli through mechanotransduction, resulting in increased tissue strength. However, detailed insights into this process in response to different loads remain limited. We aimed to investigate how different loading regimes impact the spatial composition of elastin and collagens during Achilles tendon healing. Histological analysis was conducted on healing rat Achilles tendons exposed to (1) full loading, (2) reduced loading, or (3) minimal loading. Histological analysis included Hematoxylin & Eosin and immunohistochemical staining targeting elastin, Collagen 1, Collagen 3, and CD31. Our results showed that the impact of mechanical stimuli on healing tendons varied with the degree of loading. Unexpectedly, minimal loading led to higher staining intensity for collagens and elastin. However, tendons exposed to minimal loading appeared thinner and exhibited a less organized matrix structure, with fewer, less aligned, and more rounded cells. Additionally, our findings indicated an inverse correlation between angiogenesis and load level, with more blood vessels in tendons subjected to less loading. Tissue integrity improved by 12 weeks post-injury, but the healing process continued and did not regain the structure seen in intact tendons even after 20 weeks. This study reveals a load-dependent effect on matrix alignment, cell density, and cell alignment.

Inflammation, corticosteroids, and loading all affect tendon healing, with an interaction between them. However, underlying mechanisms behind the effect of corticosteroids and the interaction with loading remain unclear. The aim of this study was to investigate the role of dexamethasone during tendon healing, including specific effects on tendon cells. Rats (n = 36) were randomized to heavy loading or mild loading, the Achilles tendon was transected, and animals were treated with dexamethasone or saline. Gene and protein analyses of the healing tendon were performed for extracellular matrix-, inflammation-, and tendon cell markers. We further tested specific effects of dexamethasone on tendon cells in vitro. Dexamethasone increased mRNA levels of S100A4 and decreased levels of ACTA2/α-SMA, irrespective of load level. Heavy loading + dexamethasone reduced mRNA levels of FN1 and TenC (p < 0.05), while resolution-related genes were unaltered (p > 0.05). In contrast, mild loading + dexamethasone increased mRNA levels of resolution-related genes ANXA1, MRC1, PDPN, and PTGES (p < 0.03). Altered protein levels were confirmed in tendons with mild loading. Dexamethasone treatment in vitro prevented tendon construct formation, increased mRNA levels of S100A4 and decreased levels of SCX and collagens. Dexamethasone during tendon healing appears to act through immunomodulation by promoting resolution, but also through an effect on tendon cells.

Inflammation, corticosteroids, and loading all affect tendon healing, with an interaction between them. However, underlying mechanisms behind the effect of corticosteroids and the interaction with loading remain unclear. The aim of this study was to investigate the role of dexamethasone during tendon healing, including specific effects on tendon cells. Rats (n = 36) were randomized to strong or moderate loading, the Achilles tendon was transected, and animals were treated with dexamethasone or saline. Gene and protein analyses of the healing tendon were performed for extracellular matrix-, inflammation-, and tendon cell markers. We further tested specific effects of dexamethasone on tendon cells in vitro. Dexamethasone increased mRNA levels of S100A4 and decreased levels of ACTA2/α-SMA, irrespective of load level. Strong loading + dexamethasone reduced mRNA levels of FN1 and TenC (p < 0.05), while resolution-related genes were unaltered (p > 0.05). In contrast, moderate loading + dexamethasone increased mRNA levels of resolution-related genes ANXA1, MRC1, PDPN, and PTGES (p < 0.03). Altered protein levels were confirmed in moderately loaded tendons. Dexamethasone treatment in vitro prevented tendon construct formation, increased mRNA levels of S100A4 and decreased levels of SCX and collagens. Dexamethasone during tendon healing appear to act through immunomodulation by promoting resolution, but also through a direct effect on tendon cells.

Exercise is widely recognized as beneficial for tendon healing. Recently, it has been described that muscle‐derived molecules secreted in response to static exercise influence tendon healing. In this study, the optimal static loading intensity for tendon healing and the composition of secretome released by myoblasts in response to different intensities of static strain were investigated. In an in vitro coculture model, myoblasts were mechanically loaded using a Flexcell Tension System. Tenocytes were seeded on transwell inserts that allowed communication between the tenocytes and myoblasts without direct contact. Proliferation and migration assays, together with RNA sequencing, were used to determine potential cellular signaling pathways. The secretome from myoblasts exposed to 2% static loading increased the proliferation and migration of the cocultured tenocytes. RNA‐seq analysis revealed that this loading condition upregulated the expression of numerous genes encoding secretory proteins, including insulin‐like growth factor‐1 (IGF‐1). Confirmation of IGF‐1 expression and secretion was carried out using qPCR and enzyme‐linked immunosorbt assay (ELISA), revealing a statistically significant upregulation in response to 2% static loading in comparison to both control conditions and higher loading intensities of 5% and 10%. Addition of an inhibitor of the IGF‐1 receptor (PQ401) to the tenocytes significantly reduced myoblast secretome‐induced tenocyte proliferation. In conclusion, IGF‐1 may be an important molecule in the statically loaded myoblast secretome, which is responsible for influencing tenocytes during exercise‐induced healing.