Early identification of patients at risk for impaired tendon healing and corresponding novel therapeutic approaches are urgent medical needs. This study aimed to clarify the role of CD3+ T-cells during acute Achilles tendon (AT) healing. Blood and hematoma aspirate were taken from 26 patients during AT reconstruction, and additional blood samples were obtained during clinical follow-up at 6, 26 and 52 weeks after surgery. T-cell subsets were analyzed by flow cytometry using CD3, CD4, CD8, CD11a, CD57 and CD28 antibodies. Clinical follow-up included functional tests, MRI assessments, and subjective questionnaires. In vitro, the functional behavior of patient-derived tenocytes was investigated in co-cultures with autologous unpolarized CD4+ or CD8+ T-cells, or IFNy-polarized CD8+ or IL17-polarized CD4+ Tcells (n=5-6). This included alterations in gene expression (qPCR), MMP secretion (ELISA), migration rate (scratch wound healing assay) or contractility (collagen gels). Analysis revealed that elevated CD4+ T-cell levels and reduced CD8+ T-cell levels (increased CD4/CD8 ratio) in hematoma aspirate and pre-operative blood were associated with inferior clinical outcomes regarding pain and function at 26 and 52 weeks. Increased levels of CD8+ -memory T-cell subpopulations in blood 6 weeks after surgery were associated with less tendon elongation. In vitro, tenocytes showed increased MMP1/2/3 levels and collagen III/I ratio in co-culture with unpolarized and/or IL17-polarized CD4+ T-cells compared to unpolarized CD8+ T-cells. This coincided with increased IL17 receptor expression in tenocytes co-cultured with CD4+ T-cells. Exposure of tenocytes to IL17-polarized CD4+ T-cells decreased their migration rate and increased their matrix contractility, especially compared to IFNy-polarized CD8+ T-cells. The CD4+ /CD8+ T-cell ratio could serve as prognostic marker for early identification of patients with impaired AT healing potential. Local reduction of CD4+ T-cell levels or their IL17 secretion represent a potential therapeutic approach to improve AT healing and to prevent weakening of the tendon ECM.

Tendon healing is a complex process that often results in compromised healing of the tendon tissue. It has recently been shown that temporal changes in the expression profile and the histological tissue quality of the tendons occur during the early healing process after acute Achilles tendon rupture. Whether these changes are accompanied by an altered healing process, is not yet known and was the aim of the present study.

Tendon biopsies were obtained from 24 patients with acute Achilles tendon rupture at the time of surgery (2–9 days after rupture) and examined histologically as well as on RNA level. Histologically, the tendon architecture, the amount of aligned collagen, glycosaminoglycan and fat as well as the cellularity, vascularity and immune cell infiltration were determined. On RNA level the expression of markers for the modeling/remodeling (MMPs and TIMPs), collagens (1, 3, 5), tendon markers (scleraxis, tenomodulin), pro- and anti-inflammatory markers (IL-1beta, IL6, IL10, IL33, TNFa, TGF-beta1, COX2) and immune cell markers (CD3, CD68, CD80, CD206) were analyzed by Real-Time PCR. To determine the clinical outcome, the patients were followed up 12 months after the operation and the following scores were recorded: Subjective score, Tegner score, Visual Analog Scale (VAS) pain, VAS function, Matles Test, Achilles tendon total rupture score (ATRS), Therman 100-points score, Heel rise test. Statistics: Spearman correlation analysis.

Correlation analysis shows that early post-rupture surgery is associated with better clinical outcome (ATRS Score: p=0.022). Histologically, a good functional healing outcome shows a positive correlation to the amount of aligned collagen (Heel Rise Test: p = 0.009) and glycosaminoglycans in the tendon (Heel Rise Test: p = 0.026, Matles difference: p = 0.029), as well as a negative correlation to the fat content (Thermann score: p = 0.018, subjective score: p = 0.027, VAS function: p = 0.031). On RNA level, a good healing outcome correlates with increased expression of MMP13, collagen 1, 3, 5 (Heel Rise Test: p = 0.019, p = 0.048, p = 0.030), and TIMP2 (Tegner Score: p = 0.040), TGF-beta1 (Thermann Score: p = 0.032) and CD80 (ATRS: p = 0.025, Thermann score:, p = 0.032). Whereas a limited healing outcome is associated with an increased expression of MMP2 (Heel Rise Test: p = 0.033), MMP3 (Matles Test: p=0.001, Heal Rise test p = 0.017), and IL33 (Tegner Score: p = 0.047).

The results of the study show a clear relationship between the tendon biology at the time of the surgery and the clinical and functional healing outcome 12 months after the operation. Especially matrix formation and remodeling play a crucial role, while the examined immunological factors seem to influence the tendon healing to a lesser extent. The modulation of matrix formation could potentially lead to improved treatment options in the future.

Tendon injuries are associated with the formation of inferior, disorganized scar tissue at the tendon bone insertion site and high failure rates. Two major processes are discussed being key players: the inflammatory reaction upon tear and the remodeling process of the tendon. In a previous study we demonstrated that the profile of MMPs and TIMPs, being key factors of tendon modeling and remodeling, is altered in tenocytes of rotator cuff tears from donors with higher age (>65 years) and degenerative status (high degree of muscle fatty infiltration)[1]. But do these cells also show different expression of inflammatory cytokines or react different upon cytokine stimulation? The aim of our project was to analyze the expression of inflammatory cytokines in human tenocyte-like cells (hTLCs) on mRNA-level and the responsiveness to cytokine stimulation regarding differences between varying donor characteristics such as age, sex and the degenerative status of the tendon.

TLCs were isolated from SSP tendon biopsies from 16 male and 14 female donors undergoing arthroscopic or open shoulder surgery. Cells from each donor (passage 1 or 2) were seeded in a 6-well plate and RNA was isolated after 7 days of culture. Quantitative Real-Time PCR was performed to analyze the expression of IL-6, IL-1β, TNF-α, IL-10, IL-33, TGF-β1 and COX-2. Furthermore, hTLCs of 12 male donors were stimulated for 3 days with a combination of TNF-α and IFN-γ (10ng/ml). The effect of the cytokines was analyzed by flow cytometry regarding surface marker expression: ICAM (CD54), VCAM (CD106), and Major Histocompatibility Complex (MHC)-class I and MHC-class II. Statistics: Mann-Whitney-U-Test, Spearman´s-Rho-correlation, p≤0.05.

Gene expression analysis revealed high levels of IL-6, TGF-β1 and COX-2 in hTLCs but low expression of TNF-α and IL-10. No differences in the expression of the inflammatory cytokines were found between low and high fatty infiltration or with respect to age. The stimulation of the hTLCs with TNF-α and IFN-γ increased the number of ICAM and VCAM positive cells up to 100% and 97±5%, respectively. MHC-class II was not expressed on unstimulated cells but 77±17% MHC-class II positive cells were present after stimulation. All unstimulated cells were positive for MHC-class I, but the MFI (Mean Fluorescent Intensity) increased after stimulation. No significant difference in the expression of surface markers was detected when comparing tenocytes of donors with low and high muscle fatty infiltration.

In contrast to the significant changes in expression levels of MMPs and TIMPs in tenocytes of donors with different age and degenerative status[1], we could not detect any significant changes in the expression of inflammatory cytokines or in the responsiveness of these tenocytes upon cytokine stimulation. All tenocytes showed the potential to respond to inflammatory processes. This indicates that the response of the tenocytes to inflammatory stimuli seems to be independent of donor characteristics, whereas the tendon remodeling might depend on age and degenerative status of the donor.